|
1
|
Li HB, Liu C, Mao XD, Yuan SZ, Li L, Cong X. Identifying HIF1A and HGF as two hub genes in aortic dissection and function analysis by integrating RNA sequencing and single-cell RNA sequencing data. Front Cardiovasc Med 2024; 11:1475991. [PMID: 39479394 PMCID: PMC11521845 DOI: 10.3389/fcvm.2024.1475991] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2024] [Accepted: 09/29/2024] [Indexed: 11/02/2024] Open
Abstract
Objective Aortic dissection (AD) is a severe aortic disease with high mortality, and its pathogenesis remains elusive. To explore the regulatory mechanisms of AD, we integrated public RNA sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq) datasets to screen the hub genes of AD and further analyzed their functions, which may provide references to the diagnosis and treatment of AD. Methods Four AD-related datasets were obtained from the Gene Expression Omnibus (GEO) database. Weighted gene co-expression network analysis and differential expression analysis were applied to identify overlapping genes in dataset GSE153434. Protein-protein interaction (PPI) network was constructed based on overlapping genes. Five methods (closeness, degree, EPC, MCC, and MNN) were used to pick hub genes. The receiver operating characteristic curve was used to evaluate the diagnostic efficiency of the hub genes in extra datasets GSE98770 and GSE52093. scRNA-seq dataset GSE213740 was used to explore the expression and function of the hub genes at the single-cell level. Quantitative real-time polymerase chain reaction was used to verify the expression of hub genes in beta-aminopropionitrile (BAPN)-induced mouse thoracic aortic aneurysm and dissection (TAAD) model. Results A total of 71 overlapping genes were screened by intersecting the significant genes in the pink module and the differentially expressed genes. A PPI network with 45 nodes and 74 edges was generated, and five top hub genes (HIF1A, HGF, HMOX1, ITGA5, and ITGB3) were identified. All the hub genes had area under the curve values above 0.55. scRNA-seq data analysis showed that HIF1A was significantly upregulated in macrophages and HGF was significantly upregulated in vascular smooth muscle cells (SMCs) of the ascending aortas in AD patients. HIF1A may transcriptionally regulate multiple downstream target genes involving inflammation (TLR2, ALOX5AP, and MIF), glycolysis (ENO1, LDHA, and GAPDH), tissue remodeling (PLAU), and angiogenesis (SERPIN and VEGFA). HGF may participate in the signaling among SMCs, fibroblasts, and endothelial cells through binding to different receptors (MET, EGFR, IGF1R, and KDR). The mRNA expression of Hif1a, Hgf, and their target genes, including Alox5ap, Serpine1, Tlr2, Plau, Egfr, and Igf1r, was significantly upregulated in aortic tissues of BAPN-treated mice. Conclusion By integrating RNA-seq and scRNA-seq data, we identified HIF1A and HGF as two hub genes with good diagnostic efficiency for AD. HIF1A in macrophages may promote AD formation by promoting inflammation, glycolysis, tissue remodeling, and angiogenesis, and HGF may mediate signaling among SMCs, fibroblasts, and endothelial cells in the development of AD.
Collapse
Affiliation(s)
| | | | | | | | | | - Xin Cong
- Department of Physiology and Pathophysiology, State Key Laboratory of Vascular Homeostasis and Remodeling, Peking University School of Basic Medical Sciences, Beijing, China
| |
Collapse
|
|
2
|
Yang CM, Yang CC, Hsu WH, Hsiao LD, Tseng HC, Shih YF. Tumor Necrosis Factor-α-Induced C-C Motif Chemokine Ligand 20 Expression through TNF Receptor 1-Dependent Activation of EGFR/p38 MAPK and JNK1/2/FoxO1 or the NF-κB Pathway in Human Cardiac Fibroblasts. Int J Mol Sci 2022; 23:ijms23169086. [PMID: 36012347 PMCID: PMC9409325 DOI: 10.3390/ijms23169086] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2022] [Revised: 08/09/2022] [Accepted: 08/11/2022] [Indexed: 11/16/2022] Open
Abstract
Tumor necrosis factor (TNF)-α is involved in the pathogenesis of cardiac injury, inflammation, and apoptosis. It is a crucial pro-inflammatory cytokine in many heart disorders, including chronic heart failure and ischemic heart disease, contributing to cardiac remodeling and dysfunction. The implication of TNF-α in inflammatory responses in the heart has been indicated to be mediated through the induction of C-C Motif Chemokine Ligand 20 (CCL20). However, the detailed mechanisms of TNF-α-induced CCL20 upregulation in human cardiac fibroblasts (HCFs) are not completely defined. We demonstrated that in HCFs, TNF-α induced CCL20 mRNA expression and promoter activity leading to an increase in the secretion of CCL20. TNF-α-mediated responses were attenuated by pretreatment with TNFR1 antibody, the inhibitor of epidermal growth factor receptor (EGFR) (AG1478), p38 mitogen-activated protein kinase (MAPK) (p38 inhibitor VIII, p38i VIII), c-Jun amino N-terminal kinase (JNK)1/2 (SP600125), nuclear factor kappaB (NF-κB) (helenalin), or forkhead box O (FoxO)1 (AS1841856) and transfection with siRNA of TNFR1, EGFR, p38α, JNK2, p65, or FoxO1. Moreover, TNF-α markedly induced EGFR, p38 MAPK, JNK1/2, FoxO1, and NF-κB p65 phosphorylation which was inhibited by their respective inhibitors in these cells. In addition, TNF-α-enhanced binding of FoxO1 or p65 to the CCL20 promoter was inhibited by p38i VIII, SP600125, and AS1841856, or helenalin, respectively. Accordingly, in HCFs, our findings are the first to clarify that TNF-α-induced CCL20 secretion is mediated through a TNFR1-dependent EGFR/p38 MAPK and JNK1/2/FoxO1 or NF-κB cascade. We demonstrated that TNFR1-derived EGFR transactivation is involved in the TNF-α-induced responses in these cells. Understanding the regulation of CCL20 expression by TNF-α on HCFs may provide a potential therapeutic strategy in cardiac inflammatory disorders.
Collapse
Affiliation(s)
- Chuen-Mao Yang
- Department of Pharmacology, College of Medicine, China Medical University, Taichung 40402, Taiwan
- Ph.D. Program for Biotech Pharmaceutical Industry, China Medical University, Taichung 40402, Taiwan
- Department of Post-Baccalaureate Veterinary Medicine, College of Medical and Health Science, Asia University, Wufeng, Taichung 41354, Taiwan
- Correspondence: ; Tel.: +886-4-22053366 (ext. 2229)
| | - Chien-Chung Yang
- Department of Traditional Chinese Medicine, Chang Gung Memorial Hospital at Tao-Yuan, Kwei-San, Tao-Yuan 33302, Taiwan
- School of Traditional Chinese Medicine, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan 33302, Taiwan
| | - Wun-Hsin Hsu
- Department of Pharmacology, College of Medicine, China Medical University, Taichung 40402, Taiwan
| | - Li-Der Hsiao
- Department of Pharmacology, College of Medicine, China Medical University, Taichung 40402, Taiwan
| | - Hui-Ching Tseng
- Department of Pharmacology, College of Medicine, China Medical University, Taichung 40402, Taiwan
| | - Ya-Fang Shih
- Department of Pharmacology, College of Medicine, China Medical University, Taichung 40402, Taiwan
| |
Collapse
|
|
3
|
Peypoch O, Paüls-Vergés F, Vázquez-Santiago M, Dilme J, Romero J, Giner J, Plaza V, Escudero JR, Soria JM, Camacho M, Sabater-Lleal M. The TAGA Study: A Study of Factors Determining Aortic Diameter in Families at High Risk of Abdominal Aortic Aneurysm Reveal Two New Candidate Genes. J Clin Med 2020; 9:jcm9041242. [PMID: 32344696 PMCID: PMC7231034 DOI: 10.3390/jcm9041242] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2020] [Revised: 04/17/2020] [Accepted: 04/21/2020] [Indexed: 01/11/2023] Open
Abstract
A variety of disorders are known to be related with aortic geometry, among them abdominal aortic aneurysm (AAA). This work aims to present the main determinants of abdominal aortic diameter in a new cohort of families at high risk of AAA. The Triple-A Genomic Analysis (TAGA) study comprises 407 individuals related in 12 families. Each family was collected through a proband with AAA. We calculated heritability and genetic correlations between abdominal aortic diameter and clinical parameters. A genome-wide linkage scan was performed based on 4.6 million variants. A predictive model was calculated with conditional forest. Heritability of the abdominal aortic diameter was 34%. Old age, male sex, higher height, weight, creatinine levels in serum, and better lung capacity were the best predictors of aortic diameter. Linkage analyses suggested the implication of Epidermal Growth Factor Receptor (EGFR) and Betacellulin (BTC) genes with aortic diameter. This is the first study to evaluate genetic components of variation of the aortic diameter in a population of AAA high-risk individuals. These results reveal EGFR, a gene that had been previously implicated in AAA, as a determinant of aortic diameter variation in healthy genetically enriched individuals, and might indicate that a common genetic background could determine the diameter of the aorta and future risk of AAA.
Collapse
Affiliation(s)
- Olga Peypoch
- Servicios Mancomunados de Angiología, Cirugía Vascular y Endovascular, Hospitales de la Santa Creu i Sant Pau/Dos de Mayo, 08025 Barcelona, Spain; (O.P.); (J.D.); (J.R.); (J.R.E.)
- Genomics of Complex Diseases, Research Institute of Hospital de la Santa Creu i Sant Pau (IIB Sant Pau), 08041 Barcelona, Spain; (F.P.-V.); (M.V.-S.); (J.M.S.); (M.C.)
- Universitat Autonoma de Barcelona, (IIB Sant Pau), 08025 Barcelona, Spain; (J.G.); (V.P.)
| | - Ferran Paüls-Vergés
- Genomics of Complex Diseases, Research Institute of Hospital de la Santa Creu i Sant Pau (IIB Sant Pau), 08041 Barcelona, Spain; (F.P.-V.); (M.V.-S.); (J.M.S.); (M.C.)
| | - Miquel Vázquez-Santiago
- Genomics of Complex Diseases, Research Institute of Hospital de la Santa Creu i Sant Pau (IIB Sant Pau), 08041 Barcelona, Spain; (F.P.-V.); (M.V.-S.); (J.M.S.); (M.C.)
- ISGlobal, Hospital Clínic-Universitat de Barcelona, 08036 Barcelona, Spain
| | - Jaime Dilme
- Servicios Mancomunados de Angiología, Cirugía Vascular y Endovascular, Hospitales de la Santa Creu i Sant Pau/Dos de Mayo, 08025 Barcelona, Spain; (O.P.); (J.D.); (J.R.); (J.R.E.)
- Genomics of Complex Diseases, Research Institute of Hospital de la Santa Creu i Sant Pau (IIB Sant Pau), 08041 Barcelona, Spain; (F.P.-V.); (M.V.-S.); (J.M.S.); (M.C.)
- Universitat Autonoma de Barcelona, (IIB Sant Pau), 08025 Barcelona, Spain; (J.G.); (V.P.)
| | - Jose Romero
- Servicios Mancomunados de Angiología, Cirugía Vascular y Endovascular, Hospitales de la Santa Creu i Sant Pau/Dos de Mayo, 08025 Barcelona, Spain; (O.P.); (J.D.); (J.R.); (J.R.E.)
- Genomics of Complex Diseases, Research Institute of Hospital de la Santa Creu i Sant Pau (IIB Sant Pau), 08041 Barcelona, Spain; (F.P.-V.); (M.V.-S.); (J.M.S.); (M.C.)
- Universitat Autonoma de Barcelona, (IIB Sant Pau), 08025 Barcelona, Spain; (J.G.); (V.P.)
| | - Jordi Giner
- Universitat Autonoma de Barcelona, (IIB Sant Pau), 08025 Barcelona, Spain; (J.G.); (V.P.)
- Department of Respiratory Medicine, Hospital de la Santa Creu i Sant Pau, 08025 Barcelona, Spain
| | - Vicente Plaza
- Universitat Autonoma de Barcelona, (IIB Sant Pau), 08025 Barcelona, Spain; (J.G.); (V.P.)
- Department of Respiratory Medicine, Hospital de la Santa Creu i Sant Pau, 08025 Barcelona, Spain
| | - Jose Roman Escudero
- Servicios Mancomunados de Angiología, Cirugía Vascular y Endovascular, Hospitales de la Santa Creu i Sant Pau/Dos de Mayo, 08025 Barcelona, Spain; (O.P.); (J.D.); (J.R.); (J.R.E.)
- Genomics of Complex Diseases, Research Institute of Hospital de la Santa Creu i Sant Pau (IIB Sant Pau), 08041 Barcelona, Spain; (F.P.-V.); (M.V.-S.); (J.M.S.); (M.C.)
- Universitat Autonoma de Barcelona, (IIB Sant Pau), 08025 Barcelona, Spain; (J.G.); (V.P.)
| | - Jose Manuel Soria
- Genomics of Complex Diseases, Research Institute of Hospital de la Santa Creu i Sant Pau (IIB Sant Pau), 08041 Barcelona, Spain; (F.P.-V.); (M.V.-S.); (J.M.S.); (M.C.)
| | - Mercedes Camacho
- Genomics of Complex Diseases, Research Institute of Hospital de la Santa Creu i Sant Pau (IIB Sant Pau), 08041 Barcelona, Spain; (F.P.-V.); (M.V.-S.); (J.M.S.); (M.C.)
- Angiology, Vascular Biology and Inflammation Laboratory, Research Institute of Hospital de la Santa Creu i Sant Pau (IIB Sant Pau), 08041 Barcelona, Spain
| | - Maria Sabater-Lleal
- Genomics of Complex Diseases, Research Institute of Hospital de la Santa Creu i Sant Pau (IIB Sant Pau), 08041 Barcelona, Spain; (F.P.-V.); (M.V.-S.); (J.M.S.); (M.C.)
- Cardiovascular Medicine Unit, Department of Medicine, Karolinska Institutet, Center for Molecular Medicine, Karolinska University Hospital, 17176 Stockholm, Sweden
- Correspondence: ; Tel.: +93-291-9000 (ext. 8167)
| |
Collapse
|
|
4
|
Kim S, Subramanian V, Abdel-Latif A, Lee S. Role of Heparin-Binding Epidermal Growth Factor-Like Growth Factor in Oxidative Stress-Associated Metabolic Diseases. Metab Syndr Relat Disord 2020; 18:186-196. [PMID: 32077785 DOI: 10.1089/met.2019.0120] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
Heparin-binding EGF-like growth factor (HB-EGF) is an EGF family member that interacts with epidermal growth factor receptor (EGFR) and ERBB4. Since HB-EGF was first identified as a novel growth factor secreted from a human macrophage cell line, numerous pathological and physiological functions related to cell proliferation, migration, and inflammation have been reported. Notably, the expression of HB-EGF is sensitively upregulated by oxidative stress in the endothelial cells and functions for auto- and paracrine-EGFR signaling. Overnutrition and obesity cause elevation of HB-EGF expression and EGFR signaling in the hepatic and vascular systems. Modulations of HB-EGF signaling showed a series of protections against phenotypes related to metabolic syndrome and advanced metabolic diseases, suggesting HB-EGF as a potential target against metabolic diseases.
Collapse
Affiliation(s)
- Seonwook Kim
- Saha Cardiovascular Research Center, University of Kentucky College of Medicine, Lexington, Kentucky, USA
| | - Venkateswaran Subramanian
- Saha Cardiovascular Research Center, University of Kentucky College of Medicine, Lexington, Kentucky, USA.,Department of Physiology, University of Kentucky College of Medicine, Lexington, Kentucky, USA
| | - Ahmed Abdel-Latif
- Saha Cardiovascular Research Center, University of Kentucky College of Medicine, Lexington, Kentucky, USA.,Department of Medicine-Cardiology, University of Kentucky College of Medicine, Lexington, Kentucky, USA
| | - Sangderk Lee
- Saha Cardiovascular Research Center, University of Kentucky College of Medicine, Lexington, Kentucky, USA.,Department of Pharmacology and Nutritional Sciences, University of Kentucky College of Medicine, Lexington, Kentucky, USA
| |
Collapse
|
|
5
|
Han S, Bal NB, Sadi G, Usanmaz SE, Tuglu MM, Uludag MO, Demirel-Yilmaz E. Inhibition of endoplasmic reticulum stress protected DOCA-salt hypertension-induced vascular dysfunction. Vascul Pharmacol 2019; 113:38-46. [PMID: 30458302 DOI: 10.1016/j.vph.2018.11.004] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2018] [Revised: 09/21/2018] [Accepted: 11/16/2018] [Indexed: 01/08/2023]
Abstract
Hypertension has complex vascular pathogenesis and therefore the molecular etiology remains poorly elucidated. Endoplasmic reticulum stress (ERS), which is a condition of the unfolded/misfolded protein accumulation in the endoplasmic reticulum, has been defined as a potential target for cardiovascular disease. In the present study, the effects of ERS inhibition on hypertension-induced alterations in the vessels were investigated. In male Wistar albino rats, hypertension was induced through unilateral nephrectomy, deoxycorticosterone-acetate (DOCA) injection (20 mg/kg, twice a week) and 1% NaCl with 0.2% KCI added to drinking water for 12 weeks. An ERS inhibitor, tauroursodeoxycolic acid (TUDCA) (150 mg/kg/day, i.p.), was administered for the final four weeks. ERS inhibition in DOCA-salt induced hypertension was observed to have reduced systolic blood pressure, improved endothelial dysfunction, enhanced plasma nitric oxide (NO) level, reduced protein expressions of phosphorylated-double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (pPERK), 78 kDa glucose-regulated protein (GRP78), Inositol trisphosphate receptor1 (IP3R1) and Epidermal growth factor receptor (EGFR), increased expressions of endoplasmic reticulum Ca2+-ATPase2 (SERCA2) and B cell lymphoma2 (Bcl2) in vessels. These findings suggest that the beneficial effects of ERS inhibition on hypertension may be related to protection of vessel functions through restoration of endoplasmic reticulum calcium homeostasis, and apoptotic and mitotic pathways.
Collapse
Affiliation(s)
- Sevtap Han
- Gazi University, Faculty of Pharmacy, Department of Pharmacology, Etiler, 06330 Ankara, Turkey.
| | - Nur Banu Bal
- Gazi University, Faculty of Pharmacy, Department of Pharmacology, Etiler, 06330 Ankara, Turkey
| | - Gökhan Sadi
- Karamanoglu Mehmetbey University, K.Ö. Faculty of Science, Department of Biology, Karaman, Turkey
| | - Suzan Emel Usanmaz
- Ankara University, Faculty of Medicine, Department of Medical Pharmacology, Sihhiye, 06100 Ankara, Turkey
| | - Merve Matilda Tuglu
- Ankara University, Faculty of Medicine, Department of Medical Pharmacology, Sihhiye, 06100 Ankara, Turkey
| | - Mecit Orhan Uludag
- Gazi University, Faculty of Pharmacy, Department of Pharmacology, Etiler, 06330 Ankara, Turkey
| | - Emine Demirel-Yilmaz
- Ankara University, Faculty of Medicine, Department of Medical Pharmacology, Sihhiye, 06100 Ankara, Turkey
| |
Collapse
|
|
6
|
Anti-vasospastic Effects of Epidermal Growth Factor Receptor Inhibitors After Subarachnoid Hemorrhage in Mice. Mol Neurobiol 2018; 56:4730-4740. [PMID: 30382533 DOI: 10.1007/s12035-018-1400-6] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2018] [Accepted: 10/17/2018] [Indexed: 01/10/2023]
Abstract
Subarachnoid hemorrhage (SAH) is a devastating disease. Cerebral vasospasm is still an important cause of post-SAH poor outcomes, but its mechanisms remain unveiled. Activation of epidermal growth factor receptor (EGFR) is suggested to cause vasoconstriction in vitro, but no report has demonstrated the involvement of EGFR in vasospasm development after SAH in vivo. Cross-talk of EGFR and vascular endothelial growth factor (VEGF) receptor, which may affect post-SAH vasospasm, was also reported in cancer cells, but has not been demonstrated in post-SAH vasospasm. The aim of this study was to investigate whether EGFR as well as EGFR-VEGF receptor cross-talk engage in the development of cerebral vasospasm in a mouse SAH model. C57BL6 mice underwent endovascular perforation SAH or sham modeling. At 30 min post-modeling, mice were randomly administrated vehicle or 2 doses of selective EGFR inhibitors intracerebroventricularly. A higher dose of the inhibitor significantly prevented post-SAH neurological impairments at 72 h and vasospasm at 24 h associated with suppression of post-SAH activation of EGFR and extracellular signal-regulated kinase (ERK) 1/2 in the cerebral artery wall, especially in the smooth muscle cell layers. Anti-EGFR neutralizing antibody also showed similar effects. However, neither expression levels of VEGF nor activation levels of a major receptor of VEGF, VEGF receptor-2, were affected by SAH and two kinds of EGFR inactivation. Thus, this study first showed that EGFR-ERK1/2 pathways may be involved in post-SAH vasospasm development, and that EGFR-VEGF receptor cross-talk may not play a significant role in the development of vasospasm in mice.
Collapse
|
|
7
|
Rebaї M, Kallel I, Abdelhedi R, kharrat N, Abdemoula Bouayed N, Abid L, Rebaї A. Association analysis of polymorphisms in EGFR , HER2 , ESR1 and THRA genes with coronary artery diseases. EGYPTIAN JOURNAL OF MEDICAL HUMAN GENETICS 2017. [DOI: 10.1016/j.ejmhg.2016.08.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022] Open
|
|
8
|
Sharifat N, Mohammad Zadeh G, Ghaffari MA, Dayati P, Kamato D, Little PJ, Babaahmadi-Rezaei H. Endothelin-1 (ET-1) stimulates carboxy terminal Smad2 phosphorylation in vascular endothelial cells by a mechanism dependent on ET receptors and de novo protein synthesis. ACTA ACUST UNITED AC 2016; 69:66-72. [PMID: 27905105 DOI: 10.1111/jphp.12654] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2016] [Accepted: 09/18/2016] [Indexed: 12/11/2022]
Abstract
OBJECTIVE G protein-coupled receptor (GPCR) agonists through their receptors can transactivate protein tyrosine kinase receptors such as epidermal growth factor receptor and serine/threonine kinase receptors most notably transforming growth factor (TGF)-β receptor (TβRI). This signalling mechanism represents a major expansion in the cellular outcomes attributable to GPCR signalling. This study addressed the role and mechanisms involved in GPCR agonist, endothelin-1 (ET-1)-mediated transactivation of the TβRI in bovine aortic endothelial cells (BAECs). METHOD The in-vitro model used BAECs. Signalling intermediate phospho-Smad2 in the carboxy terminal was detected and quantified by Western blotting. KEY FINDING ET-1 treatment of BAECs resulted in a time and concentration-dependent increase in pSmad2C. Peak phosphorylation was evident with 100 nm treatment of ET-1 at 4-6 h. TβRI antagonist, SB431542 inhibited ET-1-mediated pSmad2C. In the presence of bosentan, a mixed ETA and ETB receptor antagonist ET-1-mediated pSmad2C levels were inhibited. The ET-mediated pSmad2C was blocked by the protein synthesis inhibitor, cycloheximide. CONCLUSION In BAECs, ET-1 via the ETB receptor is involved in transactivation of the TβRI. The transactivation-dependent response is dependent upon de novo protein synthesis.
Collapse
Affiliation(s)
- Narges Sharifat
- Student Research Committee, Department of Clinical Biochemistry, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Ghorban Mohammad Zadeh
- Hyperlipidemia Research Center, Department of Clinical Biochemistry, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Mohammad-Ali Ghaffari
- Cellular and Molecular Research Center, Ahvaz Jundishapour University of Medical Sciences, Ahvaz, Iran
| | - Parisa Dayati
- Student Research Committee, Department of Clinical Biochemistry, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Danielle Kamato
- Pharmacy Australia Centre of Excellence, The University of Queensland, Woolloongabba, Qld, Australia
| | - Peter J Little
- Pharmacy Australia Centre of Excellence, The University of Queensland, Woolloongabba, Qld, Australia
| | - Hossein Babaahmadi-Rezaei
- Department of Clinical Biochemistry, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Atherosclerosis Research Center, Ahvaz, Iran
| |
Collapse
|
|
9
|
Abstract
OPINION STATEMENT New neuroprotective treatments aimed at preventing or minimizing "delayed brain injury" are attractive areas of investigation and hold the potential to have substantial beneficial effects on aneurysmal subarachnoid hemorrhage (aSAH) survivors. The underlying mechanisms for this "delayed brain injury" are multi-factorial and not fully understood. The most ideal treatment strategies would have the potential for a pleotropic effect positively modulating multiple implicated pathophysiological mechanisms at once. My personal management (RFJ) of patients with aneurysmal subarachnoid hemorrhage closely follows those treatment recommendations contained in modern published guidelines. However, over the last 5 years, I have also utilized a novel treatment strategy, originally developed at the University of Maryland, which consists of a 14-day continuous low-dose intravenous heparin infusion (LDIVH) beginning 12 h after securing the ruptured aneurysm. In addition to its well-known anti-coagulant properties, unfractionated heparin has potent anti-inflammatory effects and through multiple mechanisms may favorably modulate the neurotoxic and neuroinflammatory processes prominent in aneurysmal subarachnoid hemorrhage. In my personal series of patients treated with LDIVH, I have found significant preservation of neurocognitive function as measured by the Montreal Cognitive Assessment (MoCA) compared to a control cohort of my patients treated without LDIVH (RFJ unpublished data presented at the 2015 AHA/ASA International Stroke Conference symposium on neuroinflammation in aSAH and in abstract format at the 2015 AANS/CNS Joint Cerebrovascular Section Annual Meeting). It is important for academic physicians involved in the management of these complex patients to continue to explore new treatment options that may be protective against the potentially devastating "delayed brain injury" following cerebral aneurysm rupture. Several of the treatment options included in this review show promise and could be carefully adopted as the level of evidence for each improves. Other proposed neuroprotective treatments like statins and magnesium sulfate were previously thought to be very promising and to varying degrees were adopted at numerous institutions based on somewhat limited human evidence. Recent clinical trials and meta-analysis have shown no benefit for these treatments, and I currently no longer utilize either treatment as prophylaxis in my practice.
Collapse
|
|
10
|
Pugh RJ, Slee JB, Farwell SLN, Li Y, Barthol T, Patton WA, Lowe-Krentz LJ. Transmembrane Protein 184A Is a Receptor Required for Vascular Smooth Muscle Cell Responses to Heparin. J Biol Chem 2016; 291:5326-41. [PMID: 26769966 DOI: 10.1074/jbc.m115.681122] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2015] [Indexed: 11/06/2022] Open
Abstract
Vascular cell responses to exogenous heparin have been documented to include decreased vascular smooth muscle cell proliferation following decreased ERK pathway signaling. However, the molecular mechanism(s) by which heparin interacts with cells to induce those responses has remained unclear. Previously characterized monoclonal antibodies that block heparin binding to vascular cells have been found to mimic heparin effects. In this study, those antibodies were employed to isolate a heparin binding protein. MALDI mass spectrometry data provide evidence that the protein isolated is transmembrane protein 184A (TMEM184A). Commercial antibodies against three separate regions of the TMEM184A human protein were used to identify the TMEM184A protein in vascular smooth muscle cells and endothelial cells. A GFP-TMEM184A construct was employed to determine colocalization with heparin after endocytosis. Knockdown of TMEM184A eliminated the physiological responses to heparin, including effects on ERK pathway activity and BrdU incorporation. Isolated GFP-TMEM184A binds heparin, and overexpression results in additional heparin uptake. Together, these data support the identification of TMEM184A as a heparin receptor in vascular cells.
Collapse
Affiliation(s)
- Raymond J Pugh
- Chemistry, Lehigh University, Bethlehem, Pennsylvania 18015
| | - Joshua B Slee
- From the Departments of Biological Sciences and the Department of Natural Sciences, DeSales University, Center Valley, Pennsylvania 18034
| | | | - Yaqiu Li
- From the Departments of Biological Sciences and
| | | | - Walter A Patton
- the Department of Chemistry, Lebanon Valley College, Annville, Pennsylvania 17003, and
| | | |
Collapse
|
|
11
|
Protease activated receptor-1 mediated dual kinase receptor transactivation stimulates the expression of glycosaminoglycan synthesizing genes. Cell Signal 2016; 28:110-9. [DOI: 10.1016/j.cellsig.2015.11.003] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2015] [Accepted: 11/04/2015] [Indexed: 11/21/2022]
|
|
12
|
Gradinaru I, Babaeva E, Schwinn DA, Oganesian A. Alpha1a-Adrenoceptor Genetic Variant Triggers Vascular Smooth Muscle Cell Hyperproliferation and Agonist Induced Hypertrophy via EGFR Transactivation Pathway. PLoS One 2015; 10:e0142787. [PMID: 26571308 PMCID: PMC4646490 DOI: 10.1371/journal.pone.0142787] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2015] [Accepted: 10/27/2015] [Indexed: 01/06/2023] Open
Abstract
α1a Adrenergic receptors (α1aARs) are the predominant AR subtype in human vascular smooth muscle cells (SMCs). α1aARs in resistance vessels are crucial in the control of blood pressure, yet the impact of naturally occurring human α1aAR genetic variants in cardiovascular disorders remains poorly understood. To this end, we present novel findings demonstrating that 3D cultures of vascular SMCs expressing human α1aAR-247R (247R) genetic variant demonstrate significantly increased SMC contractility compared with cells expressing the α1aAR-WT (WT) receptor. Stable expression of 247R genetic variant also triggers MMP/EGFR-transactivation dependent serum- and agonist-independent (constitutive) hyperproliferation and agonist-dependent hypertrophy of SMCs. Agonist stimulation reduces contractility Using pathway-specific inhibitors we determined that the observed hyperproliferation of 247R-expressing cells is triggered via β-arrestin1/Src/MMP-2/EGFR/ERK-dependent mechanism. MMP-2-specific siRNA inhibited 247R-triggered hyperproliferation indicating MMP-2 involvement in 247R-triggered hyperproliferation in SMCs. β-arrestin1-specific shRNA also inhibited 247R-triggered hyperproliferation but did not affect hypertrophy in 247R-expressing SMCs, indicating that agonist-dependent hypertrophy is independent of β-arrestin1. Our data reveal that in different cardiovascular cells the same human receptor genetic variant can activate alternative modulators of the same signaling pathway. Thus, our findings in SMCs demonstrate that depending on the type of cells expressing the same receptor (or receptor variant), different target-specific inhibitors could be used to modulate aberrant hyperproliferative or hypertrophic pathways in order to restore normal phenotype.
Collapse
Affiliation(s)
- Irina Gradinaru
- Department of Anesthesiology & Pain Medicine, University of Washington, Seattle, Washington, United States of America
| | - Ekaterina Babaeva
- Department of Anesthesiology & Pain Medicine, University of Washington, Seattle, Washington, United States of America
| | - Debra A. Schwinn
- Department of Anesthesiology, University of Iowa, Iowa City, Iowa, United States of America
- Department of Pharmacology, University of Iowa, Iowa City, Iowa, United States of America
- Department of Biochemistry, University of Iowa, Iowa City, Iowa, United States of America
| | - Anush Oganesian
- Department of Anesthesiology & Pain Medicine, University of Washington, Seattle, Washington, United States of America
- * E-mail:
| |
Collapse
|
|
13
|
Kamato D, Rostam MA, Bernard R, Piva TJ, Mantri N, Guidone D, Zheng W, Osman N, Little PJ. The expansion of GPCR transactivation-dependent signalling to include serine/threonine kinase receptors represents a new cell signalling frontier. Cell Mol Life Sci 2015; 72:799-808. [PMID: 25384733 PMCID: PMC11113717 DOI: 10.1007/s00018-014-1775-0] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2014] [Revised: 10/14/2014] [Accepted: 11/03/2014] [Indexed: 01/19/2023]
Abstract
G protein-coupled receptor (GPCR) signalling is mediated through transactivation-independent signalling pathways or the transactivation of protein tyrosine kinase receptors and the recently reported activation of the serine/threonine kinase receptors, most notably the transforming growth factor-β receptor family. Since the original observation of GPCR transactivation of protein tyrosine kinase receptors, there has been considerable work on the mechanism of transactivation and several pathways are prominent. These pathways include the "triple membrane bypass" pathway and the generation of reactive oxygen species. The recent recognition of GPCR transactivation of serine/threonine kinase receptors enormously broadens the GPCR signalling paradigm. It may be predicted that the transactivation of serine/threonine kinase receptors would have mechanistic similarities with transactivation of tyrosine kinase pathways; however, initial studies suggest that these two transactivation pathways are mechanistically distinct. Important questions are the relative importance of tyrosine and serine/threonine transactivation pathways, the contribution of transactivation to overall GPCR signalling, mechanisms of transactivation and the range of cell types in which this phenomenon occurs. The ultimate significance of transactivation-dependent signalling remains to be defined but it appears to be prominent and if so will represent a new cell signalling frontier.
Collapse
Affiliation(s)
- Danielle Kamato
- Diabetes Complications Laboratory, Discipline of Pharmacy, School of Medical Sciences and Diabetes Complications Group, RMIT University, Bundoora, VIC 3083 Australia
| | - Muhamad Ashraf Rostam
- Diabetes Complications Laboratory, Discipline of Pharmacy, School of Medical Sciences and Diabetes Complications Group, RMIT University, Bundoora, VIC 3083 Australia
| | - Rebekah Bernard
- Diabetes Complications Laboratory, Discipline of Pharmacy, School of Medical Sciences and Diabetes Complications Group, RMIT University, Bundoora, VIC 3083 Australia
| | - Terrence J. Piva
- Discipline of Cell Biology and Anatomy, School of Medical Sciences and Health Innovations Research Institute, Bundoora, VIC 3083 Australia
| | - Nitin Mantri
- School of Applied Sciences, RMIT University, Bundoora, VIC 3083 Australia
| | - Daniel Guidone
- Diabetes Complications Laboratory, Discipline of Pharmacy, School of Medical Sciences and Diabetes Complications Group, RMIT University, Bundoora, VIC 3083 Australia
| | - Wenhua Zheng
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Centre and School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, People’s Republic of China
| | - Narin Osman
- Diabetes Complications Laboratory, Discipline of Pharmacy, School of Medical Sciences and Diabetes Complications Group, RMIT University, Bundoora, VIC 3083 Australia
- Department of Medicine, Nursing and Health Sciences and Immunology, Monash University School of Medicine (Central and Eastern Clinical School, Alfred Health), Prahran, VIC 3004 Australia
| | - Peter J. Little
- Diabetes Complications Laboratory, Discipline of Pharmacy, School of Medical Sciences and Diabetes Complications Group, RMIT University, Bundoora, VIC 3083 Australia
- Department of Medicine, Nursing and Health Sciences and Immunology, Monash University School of Medicine (Central and Eastern Clinical School, Alfred Health), Prahran, VIC 3004 Australia
| |
Collapse
|
|
14
|
Pincas H, Choi SG, Wang Q, Jia J, Turgeon JL, Sealfon SC. Outside the box signaling: secreted factors modulate GnRH receptor-mediated gonadotropin regulation. Mol Cell Endocrinol 2014; 385:56-61. [PMID: 23994024 PMCID: PMC3964483 DOI: 10.1016/j.mce.2013.08.015] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/02/2013] [Revised: 08/13/2013] [Accepted: 08/19/2013] [Indexed: 12/24/2022]
Abstract
Control of gene expression following activation of membrane receptors results from the regulation of intracellular signaling pathways and transcription factors. Accordingly, research to elucidate the regulatory control circuits and cellular data processing mechanisms focuses on intracellular mechanisms. While autocrine and paracrine signaling are acknowledged in endocrinology, secreted factors are not typically recognized as fundamental components of the pathways connecting cell surface receptors to gene control in the nucleus. Studies of the gonadotrope suggest that extracellular regulatory loops may play a central role in the regulation of gonadotropin gene expression by gonadotropin-releasing hormone (GnRH) receptor activation. We review emerging evidence for this phenomenon, which we refer to as exosignaling, in gonadotropin gene control and in other receptor-mediated signaling systems. We propose that basic signaling circuit modules controlling gene expression can be seamlessly distributed across intracellular and exosignaling components that together orchestrate the precise physiological control of gene expression.
Collapse
Affiliation(s)
- Hanna Pincas
- Department of Neurology, Center for Translational Systems Biology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, United States.
| | - Soon Gang Choi
- Department of Neurology, Center for Translational Systems Biology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, United States.
| | - Qian Wang
- Department of Neurology, Center for Translational Systems Biology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, United States.
| | - Jingjing Jia
- Department of Neurology, Center for Translational Systems Biology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, United States.
| | - Judith L Turgeon
- Division of Endocrinology, Department of Internal Medicine, School of Medicine, University of California, Davis, CA 95616, United States.
| | - Stuart C Sealfon
- Department of Neurology, Center for Translational Systems Biology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, United States.
| |
Collapse
|
|
15
|
Chung H, Ramachandran R, Hollenberg MD, Muruve DA. Proteinase-activated receptor-2 transactivation of epidermal growth factor receptor and transforming growth factor-β receptor signaling pathways contributes to renal fibrosis. J Biol Chem 2013; 288:37319-31. [PMID: 24253040 DOI: 10.1074/jbc.m113.492793] [Citation(s) in RCA: 73] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Chronic kidney diseases cause significant morbidity and mortality in the population. During renal injury, kidney-localized proteinases can signal by cleaving and activating proteinase-activated receptor-2 (PAR2), a G-protein-coupled receptor involved in inflammation and fibrosis that is highly expressed in renal tubular cells. Following unilateral ureteric obstruction, PAR2-deficient mice displayed reduced renal tubular injury, fibrosis, collagen synthesis, connective tissue growth factor (CTGF), and α-smooth muscle actin gene expression at 7 days, compared with wild-type controls. In human proximal tubular epithelial cells in vitro, PAR2 stimulation with PAR2-activating peptide (PAR2-AP) alone significantly up-regulated the expression of CTGF, a potent profibrotic cytokine. The induction of CTGF by PAR2-AP was synergistically increased when combined with transforming growth factor-β (TGF-β). Consistent with these findings, treating human proximal tubular epithelial cells with PAR2-AP induced Smad2/3 phosphorylation in the canonical TGF-β signaling pathway. The Smad2 phosphorylation and CTGF induction required signaling via both the TGFβ-receptor and EGF receptor suggesting that PAR2 utilizes transactivation mechanisms to initiate fibrogenic signaling. Taken together, our data support the hypothesis that PAR2 synergizes with the TGFβ signaling pathway to contribute to renal injury and fibrosis.
Collapse
|
|
16
|
Gieseler F, Ungefroren H, Settmacher U, Hollenberg MD, Kaufmann R. Proteinase-activated receptors (PARs) - focus on receptor-receptor-interactions and their physiological and pathophysiological impact. Cell Commun Signal 2013; 11:86. [PMID: 24215724 PMCID: PMC3842752 DOI: 10.1186/1478-811x-11-86] [Citation(s) in RCA: 139] [Impact Index Per Article: 11.6] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2013] [Accepted: 10/25/2013] [Indexed: 02/07/2023] Open
Abstract
Proteinase-activated receptors (PARs) are a subfamily of G protein-coupled receptors (GPCRs) with four members, PAR1, PAR2, PAR3 and PAR4, playing critical functions in hemostasis, thrombosis, embryonic development, wound healing, inflammation and cancer progression. PARs are characterized by a unique activation mechanism involving receptor cleavage by different proteinases at specific sites within the extracellular amino-terminus and the exposure of amino-terminal “tethered ligand“ domains that bind to and activate the cleaved receptors. After activation, the PAR family members are able to stimulate complex intracellular signalling networks via classical G protein-mediated pathways and beta-arrestin signalling. In addition, different receptor crosstalk mechanisms critically contribute to a high diversity of PAR signal transduction and receptor-trafficking processes that result in multiple physiological effects. In this review, we summarize current information about PAR-initiated physical and functional receptor interactions and their physiological and pathological roles. We focus especially on PAR homo- and heterodimerization, transactivation of receptor tyrosine kinases (RTKs) and receptor serine/threonine kinases (RSTKs), communication with other GPCRs, toll-like receptors and NOD-like receptors, ion channel receptors, and on PAR association with cargo receptors. In addition, we discuss the suitability of these receptor interaction mechanisms as targets for modulating PAR signalling in disease.
Collapse
Affiliation(s)
| | | | | | | | - Roland Kaufmann
- Department of General, Visceral and Vascular Surgery, Experimental Transplantation Surgery, Jena University Hospital, Drackendorfer Str, 1, D-07747, Jena, Germany.
| |
Collapse
|
|
17
|
Simard JM, Aldrich EF, Schreibman D, James RF, Polifka A, Beaty N. Low-dose intravenous heparin infusion in patients with aneurysmal subarachnoid hemorrhage: a preliminary assessment. J Neurosurg 2013; 119:1611-9. [PMID: 24032706 DOI: 10.3171/2013.8.jns1337] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
OBJECT Aneurysmal subarachnoid hemorrhage (aSAH) predisposes to delayed neurological deficits, including stroke and cognitive and neuropsychological abnormalities. Heparin is a pleiotropic drug that antagonizes many of the pathophysiological mechanisms implicated in secondary brain injury after aSAH. METHODS The authors performed a retrospective analysis in 86 consecutive patients with Fisher Grade 3 aSAH due to rupture of a supratentorial aneurysm who presented within 36 hours and were treated by surgical clipping within 48 hours of their ictus. Forty-three patients were managed postoperatively with a low-dose intravenous heparin infusion (Maryland low-dose intravenous heparin infusion protocol: 8 U/kg/hr progressing over 36 hours to 10 U/kg/hr) beginning 12 hours after surgery and continuing until Day 14 after the ictus. Forty-three control patients received conventional subcutaneous heparin twice daily as deep vein thrombosis prophylaxis. RESULTS Patients in the 2 groups were balanced in terms of baseline characteristics. In the heparin group, activated partial thromboplastin times were normal to mildly elevated; no clinically significant hemorrhages or instances of heparin-induced thrombocytopenia or deep vein thrombosis were encountered. In the control group, the incidence of clinical vasospasm requiring rescue therapy (induced hypertension, selective intraarterial verapamil, and angioplasty) was 20 (47%) of 43 patients, and 9 (21%) of 43 patients experienced a delayed infarct on CT scanning. In the heparin group, the incidence of clinical vasospasm requiring rescue therapy was 9% (4 of 43, p = 0.0002), and no patient suffered a delayed infarct (p = 0.003). CONCLUSIONS In patients with Fisher Grade 3 aSAH whose aneurysm is secured, postprocedure use of a low-dose intravenous heparin infusion may be safe and beneficial.
Collapse
|
|
18
|
Bartolini B, Thelin MA, Svensson L, Ghiselli G, van Kuppevelt TH, Malmström A, Maccarana M. Iduronic acid in chondroitin/dermatan sulfate affects directional migration of aortic smooth muscle cells. PLoS One 2013; 8:e66704. [PMID: 23843960 PMCID: PMC3699603 DOI: 10.1371/journal.pone.0066704] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2013] [Accepted: 05/08/2013] [Indexed: 11/18/2022] Open
Abstract
Aortic smooth muscle cells produce chondroitin/dermatan sulfate (CS/DS) proteoglycans that regulate extracellular matrix organization and cell behavior in normal and pathological conditions. A unique feature of CS/DS proteoglycans is the presence of iduronic acid (IdoA), catalyzed by two DS epimerases. Functional ablation of DS-epi1, the main epimerase in these cells, resulted in a major reduction of IdoA both on cell surface and in secreted CS/DS proteoglycans. Downregulation of IdoA led to delayed ability to re-populate wounded areas due to loss of directional persistence of migration. DS-epi1-/- aortic smooth muscle cells, however, had not lost the general property of migration showing even increased speed of movement compared to wild type cells. Where the cell membrane adheres to the substratum, stress fibers were denser whereas focal adhesion sites were fewer. Total cellular expression of focal adhesion kinase (FAK) and phospho-FAK (pFAK) was decreased in mutant cells compared to control cells. As many pathological conditions are dependent on migration, modulation of IdoA content may point to therapeutic strategies for diseases such as cancer and atherosclerosis.
Collapse
Affiliation(s)
- Barbara Bartolini
- Department of Experimental Medical Science, Lund University, Lund, Sweden.
| | | | | | | | | | | | | |
Collapse
|
|
19
|
Light A, Hammes SR. Membrane receptor cross talk in steroidogenesis: recent insights and clinical implications. Steroids 2013; 78:633-8. [PMID: 23380369 DOI: 10.1016/j.steroids.2012.12.016] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/01/2012] [Revised: 12/17/2012] [Accepted: 12/31/2012] [Indexed: 11/19/2022]
Abstract
Steroid production by all three major steroidogenic tissues, the adrenals, testes, and ovaries, is critical for survival and reproduction of all animals. As such, the pathways that regulate steroidogenesis are conserved between these tissues, from the steroidogenic enzymes and cofactors that synthesize steroids, to the intracellular signaling molecules and Gαs-coupled receptors that mediate the activity of these enzymes. Recent work has revealed another important conserved pathway in steroidogenesis: crosstalk between membrane G protein-coupled receptors and membrane receptor tyrosine kinases. Luteinizing hormone (LH) or adrencorticotropic hormone (ACTH) binding to their cognate Gαs-coupled membrane receptors in the gonads and adrenals, respectively, leads to cAMP-induced trans-activation of the epidermal growth factor (EGF) receptor, followed by activation of Akt and Erk signaling. These kinase signals then activate Steroidogenic Acute Regulatory (StAR) protein, which promotes steroid production. Inhibition of this pathway abrogates both LH- and ACTH-induced steroidogenesis. Interestingly, LH-induced transactivation of the EGF receptor in the ovary uniquely requires matrix metalloproteinase-mediated release of EGF receptor ligands, and inhibition of these proteases blocks LH-induced steroidogenesis. Given this unique need for matrix metalloproteinases in ovarian steroidogenesis, MMP inhibition may prove to be useful when treating diseases of excess ovarian steroid production, such as polycystic ovary syndrome.
Collapse
Affiliation(s)
- Allison Light
- Division of Endocrinology and Metabolism, Department of Medicine, University of Rochester School of Medicine, 601 Elmwood Ave., Rochester, NY 14642, USA
| | | |
Collapse
|
|
20
|
Fougerat A, Smirnova NF, Gayral S, Malet N, Hirsch E, Wymann MP, Perret B, Martinez LO, Douillon M, Laffargue M. Key role of PI3Kγ in monocyte chemotactic protein-1-mediated amplification of PDGF-induced aortic smooth muscle cell migration. Br J Pharmacol 2012; 166:1643-53. [PMID: 22251152 DOI: 10.1111/j.1476-5381.2012.01866.x] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
BACKGROUND AND PURPOSE Vascular smooth muscle cell (SMC) migration within the arterial wall is a crucial event in atherogenesis and restenosis. Monocyte chemotactic protein-1/CC-chemokine receptor 2 (MCP-1/CCR2) signalling is involved in SMC migration processes but the molecular mechanisms have not been well characterized. We investigated the role of PI3Kγ in SMC migration induced by MCP-1. EXPERIMENTAL APPROACHES A pharmacological PI3Kγ inhibitor, adenovirus encoding inactive forms of PI3Kγ and genetic deletion of PI3Kγ were used to investigate PI3Kγ functions in the MCP-1 and platelet-derived growth factor (PDGF) signalling pathway and migration process in primary aortic SMC. KEY RESULTS The γ isoform of PI3K was shown to be the major signalling molecule mediating PKB phosphorylation in MCP-1-stimulated SMC. Using a PI3Kγ inhibitor and an adenovirus encoding a dominant negative form of PI3Kγ, we demonstrated that PI3Kγ is essential for SMC migration triggered by MCP-1. PDGF receptor stimulation induced MCP-1 mRNA and protein accumulation in SMCs. Blockade of the MCP-1/CCR2 pathway or pharmacological inhibition of PI3Kγ reduced PDGF-stimulated aortic SMC migration by 50%. Thus PDGF promotes an autocrine loop involving MCP-1/CCR2 signalling which is required for PDGF-mediated SMC migration. Furthermore, SMCs isolated from PI3Kγ-deficient mice (PI3Kγ(-/-)), or mice expressing an inactive PI3Kγ (PI3Kγ(KD/KD)), migrated less than control cells in response to MCP-1 and PDGF. CONCLUSIONS AND IMPLICATIONS PI3Kγ is essential for MCP-1-stimulated aortic SMC migration and amplifies cell migration induced by PDGF by an autocrine/paracrine loop involving MCP-1 secretion and CCR2 activation. PI3Kγ is a promising target for the treatment of aortic fibroproliferative pathologies.
Collapse
|
|
21
|
Hodges RR, Bair JA, Carozza RB, Li D, Shatos MA, Dartt DA. Signaling pathways used by EGF to stimulate conjunctival goblet cell secretion. Exp Eye Res 2012; 103:99-113. [PMID: 22975404 DOI: 10.1016/j.exer.2012.08.010] [Citation(s) in RCA: 45] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2012] [Revised: 07/19/2012] [Accepted: 08/26/2012] [Indexed: 01/26/2023]
Abstract
The purpose of this study was to identify the signaling pathways that epidermal growth factor (EGF) uses to stimulate mucin secretion from cultured rat conjunctival goblet cells and to compare the pathways used by EGF with those used by the known secretagogue muscarinic, cholinergic agonists. To this end, goblet cells from rat conjunctiva were grown in culture using RPMI media. For immunofluorescence experiments, antibodies against EGF receptor (EGFR) and ERK 2 as well as muscarinic receptors (M(1)AchR, M(2)AchR, and M(3)AchR) were used, and the cells viewed by fluorescence microscopy. Intracellular [Ca(2+)] ([Ca(2+)](i)) was measured using fura 2/AM. Glycoconjugate secretion was determined after cultured goblet cells were preincubated with inhibitors, and then stimulated with EGF or the cholinergic agonist carbachol (Cch). Goblet cell secretion was measured using an enzyme-linked lectin assay with UEA-I or ELISA for MUC5AC. In cultured goblet cells EGF stimulated an increase in [Ca(2+)](i) in a concentration-dependent manner. EGF-stimulated increase in [Ca(2+)](i) was blocked by inhibitors of the EGF receptor and removal of extracellular Ca(2+). Inhibitors against the EGFR and ERK 1/2 blocked EGF-stimulated mucin secretion. In addition, cultured goblet cells expressed M(1)AchR, M(2)AchR, and M(3)AchRs. Cch-stimulated increase in [Ca(2+)](i) was blocked by inhibitors for the M(1)AchRs, matrix metalloproteinases, and EGF receptors. Inhibitors against the EGF receptor and ERK 1/2 also blocked Cch-stimulated mucin secretion. We conclude that in conjunctival goblet cells, EGF itself increases [Ca(2+)](i) and activates ERK 1/2 to stimulate mucin secretion. EGF-stimulated secretion is dependent on extracellular Ca(2+). This mechanism of action is similar to cholinergic agonists that use muscarinic receptors to transactivate the EGF receptor, increase [Ca(2+)](i), and activate ERK 1/2 leading to an increase in mucin secretion.
Collapse
Affiliation(s)
- Robin R Hodges
- Schepens Eye Research Institute, Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, 20 Staniford Street, Boston, MA 02114, USA
| | | | | | | | | | | |
Collapse
|
|
22
|
Kamato D, Burch ML, Osman N, Zheng W, Little PJ. Therapeutic implications of endothelin and thrombin G-protein-coupled receptor transactivation of tyrosine and serine/threonine kinase cell surface receptors. J Pharm Pharmacol 2012; 65:465-73. [DOI: 10.1111/j.2042-7158.2012.01577.x] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Abstract
Objectives
This review discusses the latest developments in G protein coupled receptor (GPCR) signalling related to the transactivation of cell surface protein kinase receptors and the therapeutic implications.
Key findings
Multiple GPCRs have been known to transactivate protein tyrosine kinase receptors for almost two decades. More recently it has been discovered that GPCRs can also transactivate protein serine/threonine kinase receptors such as that for transforming growth factor (TGF)-β. Using the model of proteoglycan synthesis and glycosaminoglycan elongation in human vascular smooth muscle cells which is a component of an in vitro model of atherosclerosis, the dual tyrosine and serine/threonine kinase receptor transactivation pathways appear to account for all of the response to the agonists, endothelin and thrombin.
Summary
The broadening of the paradigm of GPCR receptor transactivation explains the broad range of activities of these receptors and also the efficacy of GPCR antagonists in cardiovascular therapeutics. Deciphering the mechanisms of transactivation with the aim of identifying a common therapeutic target remains the next challenge.
Collapse
Affiliation(s)
- Danielle Kamato
- Discipline of Pharmacy, School of Medical Sciences, Australia
- Diabetes Complications Group, Metabolism, Exercise and Disease Program, Health Innovations Research Institute, RMIT University, Melbourne, Australia
| | - Micah L Burch
- Diabetes Complications Group, Metabolism, Exercise and Disease Program, Health Innovations Research Institute, RMIT University, Melbourne, Australia
- Department of Medicine, Monash University School of Medicine (Central and Eastern Clinical School, Alfred Health), Prahran VIC, Australia
| | - Narin Osman
- Discipline of Pharmacy, School of Medical Sciences, Australia
- Diabetes Complications Group, Metabolism, Exercise and Disease Program, Health Innovations Research Institute, RMIT University, Melbourne, Australia
| | - Wenhua Zheng
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Centre and School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, China
| | - Peter J Little
- Discipline of Pharmacy, School of Medical Sciences, Australia
- Diabetes Complications Group, Metabolism, Exercise and Disease Program, Health Innovations Research Institute, RMIT University, Melbourne, Australia
- Department of Medicine, Monash University School of Medicine (Central and Eastern Clinical School, Alfred Health), Prahran VIC, Australia
| |
Collapse
|
|
23
|
Simard JM, Tosun C, Ivanova S, Kurland DB, Hong C, Radecki L, Gisriel C, Mehta R, Schreibman D, Gerzanich V. Heparin reduces neuroinflammation and transsynaptic neuronal apoptosis in a model of subarachnoid hemorrhage. Transl Stroke Res 2012; 3:155-65. [PMID: 22707992 PMCID: PMC3372778 DOI: 10.1007/s12975-012-0166-9] [Citation(s) in RCA: 57] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2012] [Revised: 03/27/2012] [Accepted: 03/29/2012] [Indexed: 01/01/2023]
Abstract
Subarachnoid hemorrhage (SAH) can lead to disabling motor, cognitive, and neuropsychological abnormalities. Part of the secondary injury to cerebral tissues associated with SAH is attributable to the neuroinflammatory response induced by blood. Heparin is a pleiotropic compound that reduces inflammatory responses in conditions outside the central nervous system. Using a model of SAH devoid of global insult, we evaluated the effect of delayed intravenous (IV) infusion of heparin, at a dose that does not produce therapeutic anticoagulation, on neuroinflammation, myelin preservation, and apoptosis. Adult male rats underwent bilateral stereotactic injections of autologous blood (50 μL) into the subarachnoid space of the entorhinal cortex. The rats were implanted with mini-osmotic pumps that delivered either vehicle or unfractionated heparin (10 U/kg/h IV) beginning 12 h after SAH. No mechanical or hemorrhagic injury was observed in the hippocampus. In vehicle controls assessed at 48 h, SAH was associated with robust neuroinflammation in the adjacent cortex [neutrophils, activated phagocytic microglia, nuclear factor-kappa B, tumor necrosis factor-alpha, and interleukin-1beta] and neurodegeneration (Fluoro-Jade C staining and loss of NeuN). In the hippocampus, a muted neuroinflammatory response was indicated by Iba1-positive, ED1-negative microglia exhibiting an activated morphology. The perforant pathway showed Fluoro-Jade C staining and demyelination, and granule cells of the dentate gyrus had pyknotic nuclei, labeled with Fluoro-Jade C and showed upregulation of cleaved caspase-3, consistent with transsynaptic apoptosis. Administration of heparin significantly reduced neuroinflammation, demyelination, and transsynaptic apoptosis. We conclude that delayed IV infusion of low-dose unfractionated heparin may attenuate adverse neuroinflammatory effects of SAH.
Collapse
Affiliation(s)
- J. Marc Simard
- Department of Neurosurgery, University of Maryland School of Medicine, 22 S. Greene St., Suite S12D, Baltimore, MD 21201-1595 USA
- Department of Pathology, University of Maryland School of Medicine, Baltimore, MD USA
- Department of Physiology, University of Maryland School of Medicine, Baltimore, MD USA
| | - Cigdem Tosun
- Department of Neurosurgery, University of Maryland School of Medicine, 22 S. Greene St., Suite S12D, Baltimore, MD 21201-1595 USA
| | - Svetlana Ivanova
- Department of Neurosurgery, University of Maryland School of Medicine, 22 S. Greene St., Suite S12D, Baltimore, MD 21201-1595 USA
| | - David B. Kurland
- Department of Neurosurgery, University of Maryland School of Medicine, 22 S. Greene St., Suite S12D, Baltimore, MD 21201-1595 USA
| | - Caron Hong
- Department of Anesthesiology, University of Maryland School of Medicine, Baltimore, MD USA
| | - Leanne Radecki
- Department of Neurosurgery, University of Maryland School of Medicine, 22 S. Greene St., Suite S12D, Baltimore, MD 21201-1595 USA
| | - Carter Gisriel
- Department of Neurosurgery, University of Maryland School of Medicine, 22 S. Greene St., Suite S12D, Baltimore, MD 21201-1595 USA
| | - Rupal Mehta
- Department of Pathology, University of Maryland School of Medicine, Baltimore, MD USA
| | - David Schreibman
- Department of Anesthesiology, University of Maryland School of Medicine, Baltimore, MD USA
| | - Volodymyr Gerzanich
- Department of Neurosurgery, University of Maryland School of Medicine, 22 S. Greene St., Suite S12D, Baltimore, MD 21201-1595 USA
| |
Collapse
|
|
24
|
Chao JT, Davis MJ. The roles of integrins in mediating the effects of mechanical force and growth factors on blood vessels in hypertension. Curr Hypertens Rep 2012; 13:421-9. [PMID: 21879361 DOI: 10.1007/s11906-011-0227-6] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/17/2022]
Abstract
Hypertension is characterized by a sustained increase in vasoconstriction and attenuated vasodilation in the face of elevated mechanical stress in the blood vessel wall. To adapt to the increased stress, the vascular smooth muscle cell and its surrounding environment undergo structural and functional changes known as vascular remodeling. Multiple mechanisms underlie the remodeling process, including increased expression of humoral factors and their receptors as well as adhesion molecules and their receptors, all of which appear to collaborate and interact in the response to pressure elevation. In this review, we focus on the interactions between integrin signaling pathways and the activation of growth factor receptors in the response to the increased mechanical stress experienced by blood vessels in hypertension.
Collapse
Affiliation(s)
- Jun-Tzu Chao
- Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, 1 Hospital Drive, Columbia, MO 65212, USA
| | | |
Collapse
|
|
25
|
Gunaje JJ, Bahrami AJ, Schwartz SM, Daum G, Mahoney WM. PDGF-dependent regulation of regulator of G protein signaling-5 expression and vascular smooth muscle cell functionality. Am J Physiol Cell Physiol 2011; 301:C478-89. [PMID: 21593453 DOI: 10.1152/ajpcell.00348.2010] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Regulator of G protein signaling (RGS) proteins, and notably members of the RGS-R4 subfamily, control vasocontractility by accelerating the inactivation of Gα-dependent signaling. RGS5 is the most highly and differently expressed RGS-R4 subfamily member in arterial smooth muscle. Expression of RGS5 first appears in pericytes during development of the afferent vascular tree, suggesting that RGS5 is a good candidate for a regulator of arterial contractility and, perhaps, for determining the mass of the smooth muscle coats required to regulate blood flow in the branches of the arterial tree. Consistent with this hypothesis, using cultured vascular smooth muscle cells (VSMCs), we demonstrate RGS5 overexpression inhibits G protein-coupled receptor (GPCR)-mediated hypertrophic responses. The next objective was to determine which physiological agonists directly control RGS5 expression in VSMCs. GPCR agonists failed to directly regulate RGS5 mRNA expression; however, platelet-derived growth factor (PDGF) acutely represses expression. Downregulation of RGS5 results in the induction of migration and the activation of the GPCR-mediated signaling pathways. This stimulation leads to the activation of mitogen-activated protein kinases directly downstream of receptor stimulation, and ultimately VSMC hypertrophy. These results demonstrate that RGS5 expression is a critical mediator of both VSMC contraction and potentially, arterial remodeling.
Collapse
Affiliation(s)
- Jagadambika J Gunaje
- Department of Pathology and Center for Cardiovascular Biology, University of Washington, Seattle, WA 98109, USA
| | | | | | | | | |
Collapse
|
|
26
|
Simard JM, Schreibman D, Aldrich EF, Stallmeyer B, Le B, James RF, Beaty N. Unfractionated heparin: multitargeted therapy for delayed neurological deficits induced by subarachnoid hemorrhage. Neurocrit Care 2011; 13:439-49. [PMID: 20809188 DOI: 10.1007/s12028-010-9435-1] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Aneurysmal subarachnoid hemorrhage (SAH) is associated with numerous "delayed neurological deficits" (DNDs) that have been attributed to multiple pathophysiological mechanisms, including ischemia, microthrombosis, free radical damage, inflammation, and vascular remodeling. To date, effective prophylactic therapy for SAH-induced DNDs has been elusive, due perhaps to the multiplicity of mechanisms involved that render typical, single-agent therapy seemingly futile. We hypothesized that heparin, which has multiple underappreciated salutary effects, might be useful as a multitargeted prophylactic agent against SAH-induced DNDs. We performed a comprehensive review of the literature to evaluate the potential utility of heparin in targeting the multiple pathophysiological mechanisms that have been identified as contributing to SAH-induced DNDs. Our literature review revealed that unfractionated heparin can potentially antagonize essentially all of the pathophysiological mechanisms known to be activated following SAH. Heparin binds >100 proteins, including plasma proteins, proteins released from platelets, cytokines, and chemokines. Also, heparin complexes with oxyhemoglobin, blocks the activity of free radicals including reactive oxygen species, antagonizes endothelin-mediated vasoconstriction, smooth muscle depolarization, and inflammatory, growth and fibrogenic responses. Our review suggests that the use of prophylactic heparin following SAH may warrant formal study.
Collapse
Affiliation(s)
- J Marc Simard
- Department of Neurosurgery, University of Maryland School of Medicine, 22 S. Greene St., Suite S12D, Baltimore, MD 21201-1595, USA.
| | | | | | | | | | | | | |
Collapse
|
|
27
|
Chieng-Yane P, Bocquet A, Létienne R, Bourbon T, Sablayrolles S, Perez M, Hatem SN, Lompré AM, Le Grand B, David-Dufilho M. Protease-activated receptor-1 antagonist F 16618 reduces arterial restenosis by down-regulation of tumor necrosis factor α and matrix metalloproteinase 7 expression, migration, and proliferation of vascular smooth muscle cells. J Pharmacol Exp Ther 2011; 336:643-51. [PMID: 21139058 DOI: 10.1124/jpet.110.175182] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/07/2025] Open
Abstract
Wound healing after angioplasty or stenting is associated with increased production of thrombin and the activation of protease-activated receptor 1 (PAR1). The aim of the present study was to examine the effects of a new selective PAR1 antagonist, 2-[5-oxo-5-(4-pyridin-2-ylpiperazin-1-yl)-penta-1,3-dienyl]-benzonitrile (F 16618), in restenosis and vascular smooth muscle cell (SMC) proliferation and migration using both in vivo and in vitro approaches. Daily oral administration of F 16618 inhibited the restenosis induced by balloon angioplasty on rat carotid artery in a dose-dependent manner. Furthermore, single intravenous administration of F 16618 during the angioplasty procedure was sufficient to protect the carotid artery against restenosis. In vitro, F 16618 inhibited the growth of human aortic SMCs in a concentration-dependent manner with maximal effects at 10 μM. At that concentration, F 16618 also prevented thrombin-mediated SMC migration. In vivo, oral and intravenous F 16618 treatments reduced by 30 and 50% the expression of the inflammatory cytokine tumor necrosis factor α (TNFα) 24 h after angioplasty. However, only acute intravenous administration prevented the induction of matrix metalloproteinase 7 expression. In contrast, F 16618 treatments had no effect on early SMC de-differentiation and transcription of monocyte chemoattractant protein-1 and interleukin-6 and late re-endothelialization of injured arteries. Furthermore, F 16618 compensated for the carotid endothelium loss by inhibiting PAR1-mediated contraction. Altogether, these data demonstrate that PAR1 antagonists such as F 16618 are a highly effective treatment of restenosis after vascular injury, by inhibition of TNFα, matrix metalloproteinase 7, and SMC migration and proliferation in addition to an antithrombotic effect.
Collapse
MESH Headings
- Animals
- Carotid Stenosis/metabolism
- Carotid Stenosis/prevention & control
- Cell Movement/drug effects
- Cell Movement/physiology
- Cell Proliferation/drug effects
- Cells, Cultured
- Coronary Restenosis/drug therapy
- Coronary Restenosis/metabolism
- Down-Regulation/drug effects
- Down-Regulation/physiology
- Gene Expression Regulation, Enzymologic
- Humans
- Male
- Matrix Metalloproteinase 7/biosynthesis
- Matrix Metalloproteinase Inhibitors
- Muscle, Smooth, Vascular/drug effects
- Muscle, Smooth, Vascular/enzymology
- Muscle, Smooth, Vascular/metabolism
- Myocytes, Smooth Muscle/drug effects
- Myocytes, Smooth Muscle/enzymology
- Myocytes, Smooth Muscle/metabolism
- Piperazines/pharmacology
- Piperazines/therapeutic use
- Pyridines/pharmacology
- Pyridines/therapeutic use
- Rats
- Rats, Sprague-Dawley
- Receptor, PAR-1/physiology
- Tumor Necrosis Factor-alpha/antagonists & inhibitors
- Tumor Necrosis Factor-alpha/metabolism
- Platelet Aggregation Inhibitors
Collapse
|
|
28
|
Gao L, Smith RS, Chen LM, Chai KX, Chao L, Chao J. Tissue kallikrein promotes prostate cancer cell migration and invasion via a protease-activated receptor-1-dependent signaling pathway. Biol Chem 2011; 391:803-12. [PMID: 20482314 DOI: 10.1515/bc.2010.084] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
We recently demonstrated that tissue kallikrein (TK) promotes keratinocyte migration through activation of protease-activated receptor-1 (PAR(1)) and transactivation of the epi-dermal growth factor receptor (EGFR). In this study, we investigated the potential role of PAR(1) in mediating the effect of TK on cancer cell migration, invasion and proliferation. Our results show that TK promotes DU145 prostate cancer cell migration in a concentration-dependent manner, but has no effect on A549 lung cancer cells. Active TK markedly increases DU145 cell migration and invasion, which are blocked by aprotinin but minimally affected by icatibant; kinin treatment has little effect. TK-induced cell migration and invasion are abolished by inhibition of PAR(1) using a pharmacological inhibitor or RNA interference. The effect of TK on cell migration and invasion are also blocked by inhibitors of protein kinase C, c-Src, matrix metalloproteinase, EGFR and extracellular signal-regulated kinase (ERK). Moreover, TK stimulates ERK phosphorylation, which is inhibited by an EGFR antagonist. Additionally, TK but not kinin stimulates DU145 cell proliferation through activation of the kinin B2 receptor, but not PAR(1) and EGFR. These results indicate differential signaling pathways mediated by TK in promoting prostate cancer cell migration and invasion via PAR(1) activation, and proliferation via kinin B2 receptor stimulation.
Collapse
Affiliation(s)
- Lin Gao
- Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA
| | | | | | | | | | | |
Collapse
|
|
29
|
Delekta PC, Apel IJ, Gu S, Siu K, Hattori Y, McAllister-Lucas LM, Lucas PC. Thrombin-dependent NF-{kappa}B activation and monocyte/endothelial adhesion are mediated by the CARMA3·Bcl10·MALT1 signalosome. J Biol Chem 2010; 285:41432-42. [PMID: 21041303 DOI: 10.1074/jbc.m110.158949] [Citation(s) in RCA: 62] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
Thrombin is a potent modulator of endothelial function and, through stimulation of NF-κB, induces endothelial expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). These cell surface adhesion molecules recruit inflammatory cells to the vessel wall and thereby participate in the development of atherosclerosis, which is increasingly recognized as an inflammatory condition. The principal receptor for thrombin on endothelial cells is protease-activated receptor-1 (PAR-1), a member of the G protein-coupled receptor superfamily. Although it is known that PAR-1 signaling to NF-κB depends on initial PKC activation, the subsequent steps leading to stimulation of the canonical NF-κB machinery have remained unclear. Here, we demonstrate that a complex of proteins containing CARMA3, Bcl10, and MALT1 links PAR-1 activation to stimulation of the IκB kinase complex. IκB kinase in turn phosphorylates IκB, leading to its degradation and the release of active NF-κB. Further, we find that although this CARMA3·Bcl10·MALT1 signalosome shares features with a CARMA1-containing signalosome found in lymphocytes, there are significant differences in how the signalosomes communicate with their cognate receptors. Specifically, whereas the CARMA1-containing lymphocyte complex relies on 3-phosphoinositide-dependent protein kinase 1 for assembly and activation, the CARMA3-containing endothelial signalosome functions completely independent of 3-phosphoinositide-dependent protein kinase 1 and instead relies on β-arrestin 2 for assembly. Finally, we show that thrombin-dependent adhesion of monocytes to endothelial cells requires an intact endothelial CARMA3·Bcl10·MALT1 signalosome, underscoring the importance of the signalosome in mediating one of the most significant pro-atherogenic effects of thrombin.
Collapse
Affiliation(s)
- Phillip C Delekta
- Cellular and Molecular Biology Graduate Program, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA
| | | | | | | | | | | | | |
Collapse
|
|
30
|
Abdallah RT, Keum JS, El-Shewy HM, Lee MH, Wang B, Gooz M, Luttrell DK, Luttrell LM, Jaffa AA. Plasma kallikrein promotes epidermal growth factor receptor transactivation and signaling in vascular smooth muscle through direct activation of protease-activated receptors. J Biol Chem 2010; 285:35206-15. [PMID: 20826789 DOI: 10.1074/jbc.m110.171769] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The kallikrein-kinin system, along with the interlocking renin-angiotensin system, is a key regulator of vascular contractility and injury response. The principal effectors of the kallikrein-kinin system are plasma and tissue kallikreins, proteases that cleave high molecular weight kininogen to produce bradykinin. Most of the cellular actions of kallikrein (KK) are thought to be mediated by bradykinin, which acts via G protein-coupled B1 and B2 bradykinin receptors on VSMCs and endothelial cells. Here, we find that primary aortic vascular smooth muscle but not endothelial cells possess the ability to activate plasma prekallikrein. Surprisingly, exposing VSMCs to prekallikrein leads to activation of the ERK1/2 mitogen-activated protein kinase cascade via a mechanism that requires kallikrein activity but does not involve bradykinin receptors. In transfected HEK293 cells, we find that plasma kallikrein directly activates G protein-coupled protease-activated receptors (PARs) 1 and 2, which possess consensus kallikrein cleavage sites, but not PAR4. In vascular smooth muscles, KK stimulates ADAM (a disintegrin and metalloprotease) 17 activity via a PAR1/2 receptor-dependent mechanism, leading sequentially to release of the endogenous ADAM17 substrates, amphiregulin and tumor necrosis factor-α, metalloprotease-dependent transactivation of epidermal growth factor receptors, and metalloprotease and epidermal growth factor receptor-dependent ERK1/2 activation. These results suggest a novel mechanism of bradykinin-independent kallikrein action that may contribute to the regulation of vascular responses in pathophysiologic states, such as diabetes mellitus.
Collapse
Affiliation(s)
- Rany T Abdallah
- Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425, USA
| | | | | | | | | | | | | | | | | |
Collapse
|
|
31
|
Stanic B, Katsuyama M, Miller FJ. An oxidized extracellular oxidation-reduction state increases Nox1 expression and proliferation in vascular smooth muscle cells via epidermal growth factor receptor activation. Arterioscler Thromb Vasc Biol 2010; 30:2234-41. [PMID: 20814013 DOI: 10.1161/atvbaha.110.207639] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
OBJECTIVE To examine the effect of an oxidized extracellular oxidation-reduction (redox) state (E(h)) on the expression of NADPH oxidases in vascular cells. METHODS AND RESULTS The generation of reactive oxygen species by NADPH oxidase (Nox)-based NADPH oxidases activates redox-dependent signaling pathways and contributes to the development of "oxidative stress" in vascular disease. An oxidized plasma redox state is associated with cardiovascular disease in humans; however, the cellular mechanisms by which the extracellular redox state may cause disease are not known. Aortic segments and cultured aortic smooth muscle cells were exposed to E(h) between -150 mV (reduced) and 0 mV (oxidized) by altering the concentration of cysteine and its disulfide, cystine, the predominant redox couple in plasma. A more oxidized E(h) increased the expression of Nox1 and resulted in Nox1-dependent proliferation of smooth muscle cells. Oxidized E(h) rapidly induced epidermal growth factor receptor phosphorylation via shedding of epidermal growth factor-like ligands from the plasma membrane and caused extracellular signal-regulated kinase 1/2-dependent phosphorylation of the transcription factors activating transcription factor-1 and cAMP-response element-binding protein. Inhibition of epidermal growth factor receptor or extracellular signal-regulated kinase 1/2 activation, or addition of small interference RNA to activating transcription factor-1, prevented the increase in Nox1 expression. CONCLUSIONS Our results identify a novel mechanism by which extracellular oxidative stress increases expression and activity of Nox1 NADPH oxidase and contributes to vascular disease.
Collapse
Affiliation(s)
- Bojana Stanic
- Department of Internal Medicine, The University of Iowa, Iowa City, IA 52242, USA
| | | | | |
Collapse
|
|
32
|
Tsuchiya K, Jo T, Takeda N, Al Heialy S, Siddiqui S, Shalaby KH, Risse PA, Maghni K, Martin JG. EGF receptor activation during allergic sensitization affects IL-6-induced T-cell influx to airways in a rat model of asthma. Eur J Immunol 2010; 40:1590-602. [PMID: 20373517 DOI: 10.1002/eji.200939907] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
EGF receptor (EGFR) is involved in cell differentiation and proliferation in airways and may trigger cytokine production by T cells. We hypothesized that EGFR inhibition at the time of allergic sensitization may affect subsequent immune reactions. Brown Norway rats were sensitized with OVA, received the EGFR tyrosine kinase inhibitor, AG1478 from days 0 to 7 and OVA challenge on day 14. OVA-specific IgE in serum and cytokines and chemokines in BAL were measured 24 h after challenge. To evaluate effects on airway hyperresponsiveness (AHR), rats were sensitized, treated with AG1478, intranasally challenged, and then AHR was assessed. Furthermore chemotactic activity of BALF for CD4(+) T cells was examined. The eosinophils, neutrophils and lymphocytes in BAL were increased by OVA and only the lymphocytes were reduced by AG1478. OVA significantly enhanced IL-6 concentration in BAL, which was inhibited by AG1478. However AHR, OVA-specific IgE and IL-4 mRNA expression in CD4(+) T cells were not affected by AG1478. BALF from OVA-sensitized/challenged rats induced CD4(+) T-cell migration, which was inhibited by both AG1478 treatment in vivo and neutralization of IL-6 in vitro. EGFR activation during sensitization may affect the subsequent influx of CD4(+) T cells to airways, mainly mediated through IL-6.
Collapse
Affiliation(s)
- Kimitake Tsuchiya
- Meakins-Christie Laboratories, McGill University, Montreal, QC, Canada
| | | | | | | | | | | | | | | | | |
Collapse
|
|
33
|
Zhang F, Yang H, Pan Z, Wang Z, Wolosin JM, Gjorstrup P, Reinach PS. Dependence of resolvin-induced increases in corneal epithelial cell migration on EGF receptor transactivation. Invest Ophthalmol Vis Sci 2010; 51:5601-9. [PMID: 20538990 DOI: 10.1167/iovs.09-4468] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
PURPOSE To determine whether resolvin E1 (RvE1), an endogenous oxygenation product of eicosapentaenoic acid (EPA), induces increases in migration in human corneal epithelial cells (HCECs) and to identify signal pathways mediating this response. METHODS Migration was measured with the scratch wound assay. Western blot analysis identified changes in the phosphorylation status of prospective intracellular signal transduction mediators. Immunocytochemistry probed for intracellular paxillin localization and actin reorganization. RESULTS RvE1 enhanced HCEC migratory rates to levels comparable to those induced by epidermal growth factor (EGF). These increases were accompanied by increases in the phosphorylation status of epidermal growth factor receptor (EGFR), Akt, p38 MAPK, GSK-3α/β, and paxillin, which essentially persisted for up to 60 minutes. The EGFR inhibitor AG1478 blocked the subsequent effects of RvE1 to induce increases in phosphorylation status and cell migration. The PI3-K inhibitor LY294002 or wortmannin or the p38 inhibitor BIRB796 blocked resolvin-induced increases in cell migration. Either the matrix metalloproteinase (MMP) inhibitor GM6001 or the specific heparin-bound EGF-like growth factor inhibitor CRM197 suppressed RvE1-induced stimulation of EGFR/PI3-K/Akt phosphorylation and cell migration. CONCLUSIONS RvE1 enhances HCEC migration through MMP and sheddase-mediated EGFR transactivation. This response is dependent on PI3-K and p38-linked signaling eliciting paxillin (Tyr118) phosphorylation.
Collapse
Affiliation(s)
- Fan Zhang
- Department of Biological Sciences, State University of New York, College of Optometry, New York, New York 10036, USA.
| | | | | | | | | | | | | |
Collapse
|
|
34
|
Song L, Xiao W, Templeton DM. Low-concentration heparin suppresses ionomycin-activated CAMK-II/EGF receptor- and ERK-mediated signaling in mesangial cells. J Cell Physiol 2010; 224:484-90. [PMID: 20432446 DOI: 10.1002/jcp.22147] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Heparin and endogenous heparinoids inhibit the proliferation of smooth muscle cells, including renal mesangial cells; multiple effects on signaling pathways are well established, including effects on PKC, Erk, and CaMK-II. Many studies have used heparin at concentrations of 100 microg/ml or higher, whereas endogenous concentrations of heparinoids are much lower. Here we report the effects of low-concentration (1 microg/ml) heparin on activation of several kinases and subsequent induction of the c-fos gene in mesangial cells in response to the calcium ionophore, ionomycin, in the absence of serum factors. Ionomycin rapidly increases the phosphorylation of CaMK-II (by 30 s), and subsequently of the EGF receptor (EGFR), c-Src, and Erk 1/2. Low-dose heparin suppresses the ionomycin-dependent phosphorylation of EGFR, c-Src, and Erk 1/2, but not of CaMK-II, whereas inhibition of activated CaMK-II reduces phosphorylation of EGFR, c-Src, and Erk. Our data support a mechanism whereby heparin acts at the cell surface to suppress downstream targets of CaMK-II, including EGFR, leading in turn to a decrease in Erk- (but not c-Src-) dependent induction of c-fos.
Collapse
Affiliation(s)
- Lifang Song
- Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada
| | | | | |
Collapse
|
|
35
|
Isenovic ER, Kedees MH, Haidara MA, Trpkovic A, Mikhailidis DP, Marche P. Involvement of ERK1/2 kinase in insulin-and thrombin-stimulated vascular smooth muscle cell proliferation. Angiology 2010; 61:357-364. [PMID: 20304866 DOI: 10.1177/0003319709358693] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
It is well recognized that the proliferation of vascular smooth muscle cells (VSMCs) is a key event in the pathogenesis of various vascular diseases, including atherosclerosis and hypertension. We have previously shown that among extracellular signal-regulated protein kinases (ERKs), the 42- and 44-kDa isoforms (ERK1/2) participate in the cellular mitogenic machinery triggered by several VSMCs activators, including insulin (INS) and thrombin (Thr). However, understanding of the intracellular signal transduction pathways involved is incomplete. This review considers the recent findings in INS and Thr signaling mechanisms that modulate the proliferation of VSMCs with particular emphasis on the ERK1/2 signaling pathway, an important mediator of VSMCs hypertrophy and vascular disease. Moreover, because the ERK1/2 pathway have been acknowledged as an important mediator of VSMCs hypertrophy, ERK1/2 is identified as a key target for novel therapeutic interventions to minimize irreversible tissue damage associated with hypertension and atherosclerosis.
Collapse
Affiliation(s)
- Esma R Isenovic
- Vinca Institute of Nuclear Sciences, University of Belgrade, Laboratory for Molecular Genetics and Radiobiology, Belgrade, Serbia.
| | | | | | | | | | | |
Collapse
|
|
36
|
Nagareddy PR, MacLeod KM, McNeill JH. GPCR agonist-induced transactivation of the EGFR upregulates MLC II expression and promotes hypertension in insulin-resistant rats. Cardiovasc Res 2010; 87:177-86. [PMID: 20110336 DOI: 10.1093/cvr/cvq030] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
AIMS The presence of metabolic abnormalities such as insulin resistance and elevated levels of various vasoconstrictor G-protein-coupled receptor (GPCR) agonists contributes to the development of hypertension. Recent studies have suggested a link between disease progression and activation of growth factor receptor signalling pathways such as the epidermal growth factor receptor (EGFR) by matrix metalloproteinases (MMPs). We hypothesized that excessive stimulation of GPCRs such as alpha(1)-adrenergic receptors activates MMP-dependent EGFR transactivation and contributes to the development of hypertension by promoting increased synthesis of contractile proteins in vascular smooth muscle (VSM). METHODS AND RESULTS We tested this concept in experiments using insulin-resistant VSM cells (VSMCs) and fructose hypertensive rats (FHRs), a model of acquired systolic hypertension and insulin resistance. We found that insulin resistance and agonist stimulation increased the expression and activity of MMPs (MMP-2 and MMP-7), the EGFR, contractile proteins such as myosin light chain kinase and MLC II, and their transcriptional activators including P90 ribosomal kinase (P90RSK) and serum response factor, possibly via the activation of extracellular signal-regulated kinase (ERK1/2) in VSMCs. Further, in insulin-resistant VSMCs and arteries from FHRs, disruption of MMP-EGFR signalling either by a pharmacological or small interfering RNA approach normalized the increased expression and activity of contractile proteins and their transcriptional activators and prevented the development of hypertension in FHRs. CONCLUSION Our data suggest that the MMP-EGFR pathway could be a potential target in the treatment of hypertension in insulin resistance and/or hyperglycaemic conditions such as type 2 diabetes.
Collapse
Affiliation(s)
- Prabhakara Reddy Nagareddy
- Faculty of Pharmaceutical Sciences, The University of British Columbia, 2146 East Mall, Vancouver, BC, Canada, V6T 1Z3
| | | | | |
Collapse
|
|
37
|
A novel signaling pathway of tissue kallikrein in promoting keratinocyte migration: activation of proteinase-activated receptor 1 and epidermal growth factor receptor. Exp Cell Res 2009; 316:376-89. [PMID: 19879874 DOI: 10.1016/j.yexcr.2009.10.022] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2009] [Revised: 10/23/2009] [Accepted: 10/26/2009] [Indexed: 11/21/2022]
Abstract
Biological functions of tissue kallikrein (TK, KLK1) are mainly mediated by kinin generation and subsequent kinin B2 receptor activation. In this study, we investigated the potential role of TK and its signaling pathways in cultured human keratinocyte migration and in a rat skin wound healing model. Herein, we show that TK promoted cell migration and proliferation in a concentration- and time-dependent manner. Inactive TK or kinin had no significant effect on cell migration. Interestingly, cell migration induced by active TK was not blocked by icatibant or L-NAME, indicating an event independent of kinin B2 receptor and nitric oxide formation. TK's stimulatory effect on cell migration was inhibited by small interfering RNA for proteinase-activated receptor 1 (PAR(1)), and by PAR(1) inhibitor. TK-induced migration was associated with increased phosphorylation of epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK), which was blocked by inhibition of protein kinase C (PKC), Src, EGFR and ERK. TK-induced cell migration and EGFR phosphorylation were blocked by metalloproteinase (MMP) inhibitor, heparin, and antibodies against EGFR external domain, heparin-binding EGF-like growth factor (HB-EGF) and amphiregulin (AR). Local application of TK promoted skin wound healing in rats, whereas icatibant and EGFR inhibitor blocked TK's effect. Skin wound healing was further delayed by aprotinin and neutralizing TK antibody. This study demonstrates a novel role of TK in skin wound healing and uncovers new signaling pathways mediated by TK in promoting keratinocyte migration through activation of the PAR(1)-PKC-Src-MMP pathway and HB-EGF/AR shedding-dependent EGFR transactivation.
Collapse
|
|
38
|
Nagareddy PR, Chow FL, Hao L, Wang X, Nishimura T, MacLeod KM, McNeill JH, Fernandez-Patron C. Maintenance of adrenergic vascular tone by MMP transactivation of the EGFR requires PI3K and mitochondrial ATP synthesis. Cardiovasc Res 2009; 84:368-77. [PMID: 19578070 DOI: 10.1093/cvr/cvp230] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
AIMS G-protein-coupled receptors (GPCRs) modulate vascular tone, at least in part, via matrix metalloproteinase (MMP) transactivation of the epidermal growth factor receptor (EGFR). We previously have identified novel signalling pathways downstream of the EGFR suggestive of mitogen-activated protein kinase and mitochondrial redox control of vascular tone. In the present study, we examined whether MMP modulation of vascular tone involves phosphoinositide 3-kinase (PI3K) and mitochondrial ATP synthesis. METHODS AND RESULTS To determine whether PI3K is required for the maintenance of adrenergic vascular tone, we first constricted rat small mesenteric arteries with phenylephrine (PE) and then perfused with PI3K inhibitors, LY294002 and wortmannin, both of which produced a dose-dependent vasodilatation. Next, to investigate whether MMPs modulate PI3K activity, we cultured rat aortic vascular smooth muscle cells (VSMCs) and stimulated them with GPCR agonists such as PE and angiotensin II. Inhibition of MMPs (by GM6001) or EGFR (by AG1478) or suppressing the expression of MMP-2 or MMP-7 or the EGFR by small interfering RNA blunted the PI3K phosphorylation of Akt induced by PE. Further, in VSMCs, PI3K inhibitors reduced the PE-induced increase in ATP synthesis and glucose transporter-4 translocation, an effect that was also observed with MMP and the EGFR inhibitors. Further, the PE-induced increase in ATP synthesis activated MMP-7 by mechanisms involving purinergic (P2X) receptors and calcium. CONCLUSION These data suggest that the maintenance of adrenergic vascular tone by the MMP-EGFR pathway requires PI3K activation and ATP synthesis. Further, our data support the view that elevated levels of GPCR agonists exaggerate the MMP transactivation of EGFR response and contribute to enhanced vascular tone and development of cardiovascular disease such as hypertension.
Collapse
Affiliation(s)
- Prabhakara Reddy Nagareddy
- Department of Biochemistry, Faculty of Medicine and Dentistry, University of Alberta, Alberta, Canada T6G 2H7
| | | | | | | | | | | | | | | |
Collapse
|
|
39
|
Schauwienold D, Sastre AP, Genzel N, Schaefer M, Reusch HP. The transactivated epidermal growth factor receptor recruits Pyk2 to regulate Src kinase activity. J Biol Chem 2008; 283:27748-27756. [PMID: 18667434 DOI: 10.1074/jbc.m801431200] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
G protein-coupled receptors such as proteinase-activated receptor 1 induce phosphorylation of mitogen-activated protein kinases through multiple pathways including transactivation of receptor tyrosine kinases. In vascular smooth muscle cells, both matrix-metalloproteinase-dependent extracellular shedding of membrane-bound epidermal growth factor (EGF) receptor ligands and activation of the nonreceptor tyrosine kinases Pyk2 and Src contributed to the thrombin-induced ERK1/2 phosphorylation. Surprisingly, disruption of the HB-EGF-mediated extracellular mode of EGF receptor transactivation also prevented the phosphorylation of the nonreceptor tyrosine kinases Pyk2 and Src, locating these kinases downstream of the transactivated EGF receptor. The ionomycin-induced Pyk2 phosphorylation was partially sensitive to AG1478, heparin, or the matrix-metalloproteinase inhibitor BB2116, and the ionomycin-induced EGF receptor phosphorylation was almost completely blocked by these inhibitors of extracellular transactivation. Coimmunoprecipitation experiments revealed that, upon thrombin stimulation, a signaling complex consisting of Pyk2 and Src assembles at the EGF receptor. Reconstitution of the signaling molecules in HEK293 or vascular smooth muscle cells and subsequent determination of the EGF-induced Src kinase activity applying fluorescent sensor proteins demonstrated that a Ca(2+)-independent mode of Pyk2 activation is critical for the activation of Src downstream of the EGF receptor.
Collapse
Affiliation(s)
- Dag Schauwienold
- Abteilung Klinische Pharmakologie, Ruhr-Universität Bochum, 44780 Bochum, Germany
| | - Alejandra Pérez Sastre
- Neurowissenschaftliches Forschungszentrum, Molecular Pharmacology and Cell Biology, Charité-Universitätsmedizin Berlin, 14195 Berlin, Germany
| | - Nadine Genzel
- Neurowissenschaftliches Forschungszentrum, Molecular Pharmacology and Cell Biology, Charité-Universitätsmedizin Berlin, 14195 Berlin, Germany; ImaGenes GmbH, 13125 Berlin, Germany
| | - Michael Schaefer
- Neurowissenschaftliches Forschungszentrum, Molecular Pharmacology and Cell Biology, Charité-Universitätsmedizin Berlin, 14195 Berlin, Germany.
| | - H Peter Reusch
- Abteilung Klinische Pharmakologie, Ruhr-Universität Bochum, 44780 Bochum, Germany
| |
Collapse
|
|
40
|
Gao LB, Zhou B, Zhang L, Wei YS, Wang YY, Liang WB, Lv ML, Pan XM, Chen YC, Rao L. R497K polymorphism in epidermal growth factor receptor gene is associated with the risk of acute coronary syndrome. BMC MEDICAL GENETICS 2008; 9:74. [PMID: 18664296 PMCID: PMC2515827 DOI: 10.1186/1471-2350-9-74] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Received: 03/03/2008] [Accepted: 07/30/2008] [Indexed: 11/30/2022]
Abstract
Background Previous studies suggested that genetic polymorphisms in the epidermal growth factor receptor (EGFR) gene had been implicated in the susceptibility to some tumors and inflammatory diseases. EGFR has been recently implicated in vascular pathophysiological processes associated with excessive remodeling and atherosclerosis. Acute coronary syndrome (ACS) is a clinical manifestation of preceding atherosclerosis. Our purpose was to investigate the association of the EGFR polymorphism with the risk of ACS. In this context, we analyzed the HER-1 R497K and EGFR intron 1 (CA)n repeat polymorphisms in 191 patients with ACS and 210 age- and sex-matched controls in a Chinese population, using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) strategy and direct sequencing. Results There were significant differences in the genotype and allele distribution of R497K polymorphism of the EGFR gene between cases and controls. The Lys allele had a significantly increased risk of ACS compared with the Arg allele (adjusted OR = 1.49, 95% CI: 1.12–1.98, adjusted P = 0.006). However, no significant relationship between the number of (CA)n repeats of EGFR intron 1 (both alleles < 20 or any allele ≥ 20) and the risk of ACS was observed (adjusted OR = 0.97, 95% CI: 0.58–1.64, adjusted P = 0.911). Considering these two polymorphisms together, there was no statistically significant difference between the two groups. Conclusion R497K polymorphism of the EGFR gene is significantly associated with the risk of ACS. Our data suggests that R497K polymorphism may be used as a genetic susceptibility marker of the ACS.
Collapse
Affiliation(s)
- Lin-Bo Gao
- Department of Forensic Biology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, Sichuan, PR China.
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
41
|
Gao J, Crapo P, Nerem R, Wang Y. Co-expression of elastin and collagen leads to highly compliant engineered blood vessels. J Biomed Mater Res A 2008; 85:1120-8. [PMID: 18412137 DOI: 10.1002/jbm.a.32028] [Citation(s) in RCA: 56] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Abstract
Elastin synthesis and physiologic compliance are significant challenges in blood vessel tissue engineering. Here, we report that a biocompatible elastomeric scaffold can support the co-expression of elastin and collagen, which likely yielded the physiologic compliance in the constructs. A biodegradable elastomer, poly(glycerol sebacate), was fabricated into highly porous tubular scaffolds. Primary baboon arterial smooth muscle cells (SMCs) were seeded in the lumen of the scaffolds followed by a 1-week culture under gentle perfusion. Circulating endothelial progenitor cells (EPCs) isolated from baboon peripheral blood was seeded directly on the smooth muscle layer in the lumen on day 8. The constructs were perfused using a pulsatile flow system for another 2 weeks before characterization. In another set of experiments, the SMCs were cultured for 7 weeks and were co-cultured for 1 week with the EPCs. Constructs obtained using either set of culture conditions contained elastin and collagen: Masson's trichrome stain showed a circumferential collagen band in the constructs, and elastin was evident from its characteristic autofluorescence, Verhoff's stain, and amino acid analysis of insoluble remnants after hot alkali digestion. All constructs had a confluent cellular lumen with cells well-dispersed throughout the scaffolds. At physiologic pressures, the compliance of the 8-week construct was comparable to human arteries as observed in pressure-diameter testing. Combination of elastomeric scaffolds, co-culture of EPC and SMC, and mechanical conditioning appears to encourage the expression of a more natural extracellular matrix and lead to physiologically-relevant compliance; both are major challenges in blood vessel tissue engineering.
Collapse
Affiliation(s)
- Jin Gao
- Department of Biomedical Engineering and Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332, USA
| | | | | | | |
Collapse
|
|
42
|
Gao J, Ensley AE, Nerem RM, Wang Y. Poly(glycerol sebacate) supports the proliferation and phenotypic protein expression of primary baboon vascular cells. J Biomed Mater Res A 2008; 83:1070-1075. [PMID: 17584900 DOI: 10.1002/jbm.a.31434] [Citation(s) in RCA: 66] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
Poly(glycerol sebacate) (PGS) is a biodegradable and biocompatible elastomer specifically developed for soft tissue engineering. Vascular cells adhered to an elastomer may exhibit more physiological behavior because the substrate's mechanical properties more closely match those of the tissue. To investigate the feasibility of using PGS as a scaffold material for vascular tissue engineering, the authors examined the adhesion, proliferation, and phenotypic and morphologic properties of primary baboon endothelial progenitor cells (BaEPCs) and baboon smooth muscle cells (BaSMCs) cultured on PGS films and scaffolds. Tissue culture-treated polystyrene plates were used as controls. Phase contrast microscopy indicated that both types of cells showed normal morphology on PGS films. Immuofluorescent staining revealed that von Willebrand factor and alpha-smooth muscle actin were expressed by BaEPCs and BaSMCs, respectively. Both types of cells proliferated well on PGS surfaces. When cultured in PGS scaffolds, BaSMCs were distributed throughout the scaffolds and synthesized extracellular matrix, as indicated by histological evaluations. The distribution of the BaSMCs in the constructs was confirmed by scanning electron microscopy. Immunofluorescent staining of cocultured constructs indicated that the BaSMC-seeded constructs provided suitable surfaces for BaEPC adhesion, and both types of cells maintained their specific phenotypes. These results suggest that PGS is an appropriate scaffold material for blood vessel tissue engineering.
Collapse
Affiliation(s)
- Jin Gao
- Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332
- Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332
| | - Ann E Ensley
- Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332
- Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332
| | - Robert M Nerem
- Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332
- Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332
| | - Yadong Wang
- Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332
- Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332
| |
Collapse
|
|
43
|
Ivey ME, Osman N, Little PJ. Endothelin-1 signalling in vascular smooth muscle: pathways controlling cellular functions associated with atherosclerosis. Atherosclerosis 2008; 199:237-47. [PMID: 18436225 DOI: 10.1016/j.atherosclerosis.2008.03.006] [Citation(s) in RCA: 103] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/22/2007] [Revised: 03/04/2008] [Accepted: 03/10/2008] [Indexed: 12/16/2022]
Abstract
Atherosclerosis is the primary ischaemic vascular condition underlying a majority of cardiovascular disease related deaths. Endothelin-1 is a vasoactive peptide agent upregulated in atherosclerosis and in conjunction with its G protein-coupled receptors exerts diverse actions on all cells of the vasculature in particular vascular smooth muscle cells (VSMC). The effects of endothelin-1 include cell proliferation, migration and contraction, and the induction of extracellular matrix components and growth factors. VSMC as the major component of the neointima in atherosclerotic plaques accordingly play a key role in atherogenesis. In this review we examine classic and novel signalling pathways activated by endothelin-1 in VSMC (including phospholipase C, adenylate cyclase, Rho kinase, transactivation of receptor tyrosine kinases, mitogen activated protein kinase cascades and beta-arrestin) and their likely impact on the development and progression of atherosclerosis.
Collapse
Affiliation(s)
- Melanie E Ivey
- Cell Biology of Diabetes Laboratory, Baker Heart Research Institute, Melbourne, Victoria, Australia
| | | | | |
Collapse
|
|
44
|
Fernandez-Patron C. Therapeutic potential of the epidermal growth factor receptor transactivation in hypertension: a convergent signaling pathway of vascular tone, oxidative stress, and hypertrophic growth downstream of vasoactive G-protein-coupled receptors? Can J Physiol Pharmacol 2007; 85:97-104. [PMID: 17487249 DOI: 10.1139/y06-097] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
The concurrence of enhanced vascular tone, oxidative stress, and hypertrophic growth is a hallmark of hypertension, the condition characterized by sustained elevated blood pressure. However, it is unclear how and why such apparently distinct processes coincide in hypertension. Elevated levels of certain vasoactive G-protein-coupled receptor agonists (such as catecholamines, endothelin-1, and angiotensin II) can explain, at least in part, the development and progression of many hypertensive disorders. Here, we review findings made by other investigators and ourselves suggesting that enhanced vascular tone, oxidative stress, and hypertrophic growth characteristically induced by these agonists involve the transactivation of growth factor receptors. The first step in this transactivation mechanism is agonist-induced activation of metalloproteinase-dependent shedding of growth factors. Shed growth factors then trigger intracellular signaling cascades necessary for growth, production of reactive oxygen species, and maintenance of vascular tone. If this hypothesis is proven generally correct, then transactivation blockers have general therapeutic potential in hypertension regardless of the causative agonist.
Collapse
|
|
45
|
Zhou W, Ibe BO, Raj JU. Platelet-activating factor induces ovine fetal pulmonary venous smooth muscle cell proliferation: role of epidermal growth factor receptor transactivation. Am J Physiol Heart Circ Physiol 2007; 292:H2773-81. [PMID: 17322418 DOI: 10.1152/ajpheart.01018.2006] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
We have previously reported that platelet-activating factor (PAF) is present in very high levels in the ovine fetal lung and circulation and that PAF serves as an important physiological vasoconstrictor of the pulmonary circulation in utero. However, it is not known whether PAF stimulates pulmonary vascular smooth muscle cell (SMC) proliferation. In this study, we used ovine fetal pulmonary venous SMCs as our model system to study the effects and mechanisms of action of PAF on SMC proliferation. We found that PAF induced SMC proliferation in a dose-dependent manner. PAF also stimulated activation of both ERK and p38 but not c-Jun NH(2) terminal kinase (JNK) mitogen-activated protein (MAP) kinase pathways. PAF (10 nM) induced phosphorylation of epidermal growth factor receptor (EGFR). Specific inhibition of EGFR by AG-1478 and by the expression of a dominant-negative EGFR mutant in SMCs attenuated PAF-stimulated cell proliferation. Inhibition of heparin-binding EGF-like growth factor (HB-EGF) release by CRM-197 and inhibition of matrix metalloproteinases (MMP) by GM-6001 abolished PAF-induced MAP kinase activation and cell proliferation. Increased alkaline phosphatase (AP) activity after PAF treatment in AP-HB-EGF fusion construct-transfected SMCs indicated that PAF induced the release of HB-EGF within 1 min. Gelatin zymography data showed that PAF stimulated MMP-2 activity and MMP-9 activity within 1 min. These results suggest that PAF promotes pulmonary vascular SMC proliferation via transactivation of EGFR through MMP activation and HB-EGF, resulting in p38 and ERK activation and that EGFR transactivation is essential for the mitogenic effect of PAF in pulmonary venous SMC.
Collapse
MESH Headings
- Animals
- Bacterial Proteins/pharmacology
- Cell Proliferation/drug effects
- Cells, Cultured
- Dipeptides/pharmacology
- Dose-Response Relationship, Drug
- Enzyme Activation
- Enzyme Inhibitors/pharmacology
- Epidermal Growth Factor/genetics
- Epidermal Growth Factor/metabolism
- ErbB Receptors/drug effects
- ErbB Receptors/genetics
- ErbB Receptors/metabolism
- Extracellular Signal-Regulated MAP Kinases/metabolism
- Heparin-binding EGF-like Growth Factor
- Intercellular Signaling Peptides and Proteins
- Matrix Metalloproteinase 2/metabolism
- Matrix Metalloproteinase 9/metabolism
- Matrix Metalloproteinase Inhibitors
- Muscle, Smooth, Vascular/cytology
- Muscle, Smooth, Vascular/drug effects
- Muscle, Smooth, Vascular/embryology
- Muscle, Smooth, Vascular/metabolism
- Mutation
- Myocytes, Smooth Muscle/drug effects
- Myocytes, Smooth Muscle/metabolism
- Phosphorylation
- Platelet Activating Factor/metabolism
- Platelet Activating Factor/pharmacology
- Pulmonary Veins/cytology
- Pulmonary Veins/embryology
- Pulmonary Veins/metabolism
- Quinazolines
- Sheep
- Signal Transduction/drug effects
- Time Factors
- Transfection
- Tyrphostins/pharmacology
- p38 Mitogen-Activated Protein Kinases/metabolism
Collapse
Affiliation(s)
- Weilin Zhou
- Division of Neonatology, Harbor-University of California, Los Angeles (UCLA) Medical Center, Los Angeles Biomedical Institute, 1124 West Carson St., Torrance, CA 90502, USA.
| | | | | |
Collapse
|
|
46
|
Matsuo M, Sakurai H, Koizumi K, Saiki I. Curcumin inhibits the formation of capillary-like tubes by rat lymphatic endothelial cells. Cancer Lett 2007; 251:288-95. [PMID: 17197075 DOI: 10.1016/j.canlet.2006.11.027] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2006] [Accepted: 11/27/2006] [Indexed: 02/08/2023]
Abstract
The natural pigments curcumin and berberine have been shown to exhibit a variety of pharmacologic effects including anti-inflammatory, anti-cancer, and anti-metastatic properties. Here, we investigated the anti-lymphangiogenic effect with an in vitro tube-forming model using conditionally immortalized lymphatic endothelial TR-LE cells, a newly established cell line originating from the thoracic duct of a transgenic rat expressing the temperature-sensitive SV40 large T-antigen. Curcumin, but not berberine, exhibited a dose-dependent inhibition of the formation of capillary-like tubes by TR-LE cells without affecting cell viability and adhesion to Matrigel. To address the molecular mechanisms involved, we performed experiments with specific inhibitors against putative targets of curcumin, including IkappaB kinase (IKK), epidermal growth factor receptor (EGFR), phosphatidylinositol-3 kinase (PI3K)/Akt, and matrix metalloproteinases (MMPs). While the IKK-2 inhibitor VI and EGFR tyrosine kinase inhibitors gefitinib and PD153035 had no effect, both the PI3K inhibitor LY294002 and the MMP inhibitor GM6001 shortened the tubes by approximately 50%. Western blot analysis and gelatin zymography revealed that curcumin, but not berberine, has an inhibitory effect on the phosphorylation of Akt and enzymatic activity of MMP-2 in TR-LE cells. These results suggest that curcumin exerts its inhibitory effect on lymphangiogenesis partly through Akt and MMP-2.
Collapse
Affiliation(s)
- Mitsuhiro Matsuo
- Department of Anatomy, Faculty of Medicine, University of Toyama, Toyama 930-0194, Japan
| | | | | | | |
Collapse
|
|
47
|
Estrada CR, Adam RM, Eaton SH, Bägli DJ, Freeman MR. Inhibition of EGFR signaling abrogates smooth muscle proliferation resulting from sustained distension of the urinary bladder. J Transl Med 2006; 86:1293-302. [PMID: 17043666 DOI: 10.1038/labinvest.3700483] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022] Open
Abstract
Urinary bladder outlet obstruction results in sustained stretch of the detrusor muscle and can lead to pathological smooth muscle hyperplasia and hypertrophy. The epidermal growth factor receptor (EGFR) is a cognate receptor for mitogens implicated in bladder hyperplasia/hypertrophy. Here, we investigated the potential for modulation of this pathway by pharmacologic targeting with a clinically available EGFR antagonist using an organ culture model of bladder stretch injury as a test system. Urinary bladders from adult female rats were distended in vivo with medium containing the EGFR inhibitor ZD1839 (gefitinib, Iressa). The bladders were excised and incubated in ex vivo organ culture for 4-24 h. EGFR phosphorylation, DNA proliferation, and the extent of apoptosis in the cultured tissues were assessed. To verify that the smooth muscle cells (SMC) are a target of the EGFR inhibitor, primary culture human and rat bladder SMC were subjected to cyclic mechanical stretch in vitro in the presence of ZD1839. Levels of phosphorylated EGFR were significantly increased in the detrusor muscle with 12 h of stretch in the organ cultures. This activation coincided with a subsequent 23-fold increase in DNA synthesis and a 30-fold decrease in apoptosis in the muscle compartment at 24 h. In the presence of ZD1839, DNA synthesis was reduced to basal levels without an increase in the rate of apoptosis under ex vivo conditions. Mechanical stretch of bladder SMC in vitro resulted in a significant increase in DNA synthesis, which was completely abrogated by treatment with ZD1839 but not by AG825, an inhibitor of the related receptor, ErbB2. Our results indicate that the EGFR pathway is a physiologically relevant signaling mechanism in hypertrophic bladder disease resulting from mechanical distension and may be amenable to pharmacologic intervention.
Collapse
Affiliation(s)
- Carlos R Estrada
- Urological Diseases Research Center, Department of Urology, Children's Hospital Boston, Boston, MA 02115, USA
| | | | | | | | | |
Collapse
|
|
48
|
Luo L, Yano N, Luo JZQ. The molecular mechanism of EGF receptor activation in pancreatic beta-cells by thyrotropin-releasing hormone. Am J Physiol Endocrinol Metab 2006; 290:E889-99. [PMID: 16603724 DOI: 10.1152/ajpendo.00466.2005] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Thyrotropin-releasing hormone (TRH) and its receptor subtype TRH receptor-1 (TRHR1) are found in pancreatic beta-cells, and it has been shown that TRH might have potential for autocrine/paracrine regulation through the TRHR1 receptor. In this paper, TRHR1 is studied to find whether it can initiate multiple signal transduction pathways to activate the epidermal growth factor (EGF) receptor in pancreatic beta-cells. By initiating TRHR1 G protein-coupled receptor (GPCR) and dissociated alphabetagamma-complex, TRH (200 nM) activates tyrosine residues at Tyr845 (a known target for Src) and Tyr1068 in the EGF receptor complex of an immortalized mouse beta-cell line, betaTC-6. Through manipulating the activation of Src, PKC, and heparin-binding EGF-like growth factor (HB-EGF), with corresponding individual inhibitors and activators, multiple signal transduction pathways linking TRH to EGF receptors in betaTC-6 cell line have been revealed. The pathways include the activation of Src kinase and the release of HB-EGF as a consequence of matrix metalloproteinase (MMP)-3 activation. Alternatively, TRH inhibited PKC activity by reducing the EGF receptor serine/threonine phosphorylation, thereby enhancing tyrosine phosphorylation. TRH receptor activation of Src may have a central role in mediating the effects of TRH on the EGF receptor. The activation of the EGF receptor by TRH in multiple circumstances may have important implications for pancreatic beta-cell biology.
Collapse
Affiliation(s)
- LuGuang Luo
- The Center for Stem Cell Biology, Department of Research, Roger Williams Hospital, 825 Chalkstone Ave., Providence, RI 02908, USA
| | | | | |
Collapse
|
|
49
|
Faber JE, Yang N. Balloon injury alters alpha-adrenoceptor expression across rat carotid artery wall. Clin Exp Pharmacol Physiol 2006; 33:204-10. [PMID: 16487263 DOI: 10.1111/j.1440-1681.2006.04347.x] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
alpha(1)-Adrenoceptors (AR) mediate growth factor-like activity of catecholamines on vascular smooth muscle cells (SMC) and adventitial fibroblasts. This trophic activity is strongly augmented by balloon injury, contributes significantly to subsequent proliferation, wall hypertrophy and lumen loss and is mediated by alpha(1A)- and alpha(1B)-AR. However, it is not known how injury augments adrenergic trophic activity. The aim of the present study was to examine alpha-AR expression in rat carotid artery and to test the hypothesis that balloon injury augments a(1)-AR expression. 2. Neointima, media and adventitia were isolated at various days after balloon injury of rat carotid artery and subjected to quantitative reverse transcription-polymerase chain reaction and radioligand binding. Cultured SMC were also studied. 3. Transcripts for alpha(1A)-, alpha(1B)-, alpha(1D)- and alpha(2D)-AR were expressed in different proportions in media and adventitia from uninjured carotid artery. Injury caused a reduction by as much as 85% at day 4 in all alpha-AR mRNA (but not cyclophilin) in both the media and adventitia. In both layers, expression returned to control by day 21 for alpha(2D)-AR and by day 42 for alpha(1A)-AR, but remained reduced by 25-50% for alpha(1B)- and alpha(1D)-AR at 42 days. alpha(1)-Adrenoceptor transcripts in the neointima at 21 and 42 days after injury were expressed at levels more than 80% lower than in the media or adventitia of uninjured carotid; alpha(2D)-AR mRNA was undetectable. The density of total alpha(1)-AR binding sites was similar in the media and adventitia of uninjured carotid. Density was reduced by approximately 60% in the intima-media and adventitia 21 days after injury. To examine possible mechanisms, early passaged cultured SMC were studied that express alpha(1D)- and alpha(1B)-AR at levels similar to in vivo but that do not express other alpha-AR. Basic fibroblast growth factor caused downregulation of alpha(1D)-AR mRNA and alpha(1)-AR density, without affecting mRNA half-life, whereas transforming growth factor-beta1 had no effect. Neither growth factor altered alpha(1B)-AR message expression. 4. These data demonstrate that: (i) carotid artery expresses the same four alpha-AR genes and similar total alpha(1)-AR density in the SMC media and fibroblast-rich adventitia; and (ii) injury induced enhancement of adrenergic trophic activity is not caused by upregulation of alpha(1)-AR, but, instead, is associated with a generalized reduction in alpha-AR expression.
Collapse
Affiliation(s)
- James E Faber
- Department of Cell and Molecular Physiology, University of North Carolina, Chapel Hill, North Carolina, USA.
| | | |
Collapse
|
|
50
|
Matsuo M, Sakurai H, Ueno Y, Ohtani O, Saiki I. Activation of MEK/ERK and PI3K/Akt pathways by fibronectin requires integrin alphav-mediated ADAM activity in hepatocellular carcinoma: a novel functional target for gefitinib. Cancer Sci 2006; 97:155-62. [PMID: 16441427 PMCID: PMC11159791 DOI: 10.1111/j.1349-7006.2006.00152.x] [Citation(s) in RCA: 55] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023] Open
Abstract
We have shown that the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib ('Iressa', ZD1839) inhibits the development of intrahepatic metastases of hepatocellular carcinoma CBO140C12, and EGFR transactivation by tumor necrosis factor-alpha is a possible target of gefitinib. In the present study, we focused on the fibronectin (FN)-dependent signaling pathway to further elucidate the antimetastatic activity of gefitinib in CBO140C12 cells. We initially observed that FN induced activation of extracellular signal-regulated kinase (ERK), p38 and Akt, as well as cell proliferation and CBO140C12 cell invasion. These responses were mediated by EGFR tyrosine kinase, because gefitinib inhibited these effects of FN. FN-induced ERK, p38 and Akt activation was partly blocked by the Arg-Gly-Asp (RGD)-pseudo-peptide FC-336, anti-alphav integrin antibody RMV-7, the broad-spectrum matrix metalloprotease inhibitor GM6001 and the broad spectrum a disintegrin and metalloprotease (ADAM) inhibitor TAPI-1. But these inhibitors had no effect on EGF-induced signaling pathways, suggesting that integrins and ADAM may be upstream components of EGFR in these responses. These results suggest that FN-induced activation of ERK, p38, Akt, cell proliferation and invasion was mediated, at least in part, via integrins, ADAM and EGFR, and that this FN-induced signaling pathway might be involved in the antimetastatic activity of gefitinib.
Collapse
Affiliation(s)
- Mitsuhiro Matsuo
- Department of Anatomy, Faculty of Medicine, University of Toyama, Toyama 930-0194, Japan
| | | | | | | | | |
Collapse
|