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Guan X, Liu Y, An Y, Wang X, Wei L, Qi X. FAK Family Kinases: A Potential Therapeutic Target for Atherosclerosis. Diabetes Metab Syndr Obes 2024; 17:3151-3161. [PMID: 39220801 PMCID: PMC11363942 DOI: 10.2147/dmso.s465755] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/24/2024] [Accepted: 08/13/2024] [Indexed: 09/04/2024] Open
Abstract
Atherosclerosis (AS) is a chronic progressive inflammatory disease of the vascular wall and the primary pathological basis of cardiovascular and cerebrovascular disease. Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2), two highly homologous members of the FAK family kinases, play critical roles in integrin signaling. They also serve as scaffolding proteins that contribute to the assembly of cellular signaling complexes that regulate cell survival, cell cycle progression, and cell motility. Research indicates that the FAK family kinases is involved in the gene regulation of vascular cells and that aberrant expression of this family is associated with pathological changes in vascular disease. These findings establish the FAK family kinases as a critical signaling mediator in atherosclerotic lesions and inhibition of its activity has the potential to attenuate the pathological progression of AS. This review highlights the indispensable role of the FAK family kinases in abnormal vascular smooth muscle cell proliferation, endothelial cell dysfunction, inflammation, and lipid metabolism associated with AS. We also summarize therapeutic targets against the FAK family kinases, providing valuable insights into therapeutic strategies for AS.
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Affiliation(s)
- Xiuju Guan
- School of Graduate Studies, Tianjin University of Traditional Chinese Medicine, Tianjin, People’s Republic of China
| | - Yue Liu
- Department of Cardiology, Tianjin Union Medical Center, Tianjin, People’s Republic of China
| | - Yajuan An
- School of Graduate Studies, Tianjin University of Traditional Chinese Medicine, Tianjin, People’s Republic of China
| | - Xinshuang Wang
- School of Graduate Studies, Tianjin University of Traditional Chinese Medicine, Tianjin, People’s Republic of China
| | - Liping Wei
- Department of Cardiology, Tianjin Union Medical Center, Tianjin, People’s Republic of China
| | - Xin Qi
- Department of Cardiology, Tianjin Union Medical Center, Tianjin, People’s Republic of China
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Tsujioka M, Miyazawa K, Ohmuraya M, Nibe Y, Shirokawa T, Hayasaka H, Mizushima T, Fukuma T, Shimizu S. Identification of a novel type of focal adhesion remodelling via FAK/FRNK replacement, and its contribution to cancer progression. Cell Death Dis 2023; 14:256. [PMID: 37031228 PMCID: PMC10082854 DOI: 10.1038/s41419-023-05774-4] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2022] [Revised: 03/22/2023] [Accepted: 03/24/2023] [Indexed: 04/10/2023]
Abstract
Numerous studies have investigated the various cellular responses against genotoxic stress, including those mediated by focal adhesions. We here identified a novel type of focal adhesion remodelling that occurs under genotoxic stress conditions, which involves the replacement of active focal adhesion kinase (FAK) with FAK-related non-kinase (FRNK). FRNK stabilized focal adhesions, leading to strong cell-matrix adhesion, and FRNK-depleted cells were easily detached from extracellular matrix upon genotoxic stress. This remodelling occurred in a wide variety of cells. In vivo, the stomachs of Frnk-knockout mice were severely damaged by genotoxic stress, highlighting the protective role of FRNK against genotoxic stress. FRNK was also found to play a vital role in cancer progression, because FRNK depletion significantly inhibited cancer dissemination and progression in a mouse cancer model. Furthermore, in human cancers, FRNK was predominantly expressed in metastatic tissues and not in primary tissues. We hence conclude that this novel type of focal adhesion remodelling reinforces cell adhesion and acts against genotoxic stress, which results in the protection of normal tissues, but in turn facilitates cancer progression.
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Affiliation(s)
- Masatsune Tsujioka
- Department of Pathological Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan.
| | - Keisuke Miyazawa
- Division of Electrical Engineering and Computer Science, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192, Japan
- Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kakuma-machi, Kanazawa, 920-1192, Japan
| | - Masaki Ohmuraya
- Department of Genetics, Hyogo College of Medicine, Nishinomiya, Hyogo, 663-8501, Japan
| | - Yoichi Nibe
- Department of Pathological Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan
| | - Tetsuya Shirokawa
- Division of Electrical Engineering and Computer Science, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192, Japan
| | - Haruko Hayasaka
- Department of Life Science, Faculty of Science & Engineering, Kindai University, Higashi-osaka, Osaka, 577-8502, Japan
| | - Tsunekazu Mizushima
- Department of Gastroenterological Surgery, Graduate School of Medicine, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan
| | - Takeshi Fukuma
- Division of Electrical Engineering and Computer Science, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192, Japan
- Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kakuma-machi, Kanazawa, 920-1192, Japan
| | - Shigeomi Shimizu
- Department of Pathological Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan.
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Huang T, Li YQX, Zhou MY, Hu RH, Zou GL, Li JC, Feng S, Liu YM, Xin CQ, Zhao XK. Focal adhesion kinase-related non-kinase ameliorates liver fibrosis by inhibiting aerobic glycolysis via the FAK/Ras/c-myc/ENO1 pathway. World J Gastroenterol 2022; 28:123-139. [PMID: 35125823 PMCID: PMC8793014 DOI: 10.3748/wjg.v28.i1.123] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/14/2021] [Revised: 10/22/2021] [Accepted: 12/22/2021] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Hepatic stellate cell (HSC) hyperactivation is a central link in liver fibrosis development. HSCs perform aerobic glycolysis to provide energy for their activation. Focal adhesion kinase (FAK) promotes aerobic glycolysis in cancer cells or fibroblasts, while FAK-related non-kinase (FRNK) inhibits FAK phosphorylation and biological functions.
AIM To elucidate the effect of FRNK on liver fibrosis at the level of aerobic glycolytic metabolism in HSCs.
METHODS Mouse liver fibrosis models were established by administering CCl4, and the effect of FRNK on the degree of liver fibrosis in the model was evaluated. Transforming growth factor-β1 was used to activate LX-2 cells. Tyrosine phosphorylation at position 397 (pY397-FAK) was detected to identify activated FAK, and the expression of the glycolysis-related proteins monocarboxylate transporter 1 (MCT-1) and enolase1 (ENO1) was assessed. Bioinformatics analysis was performed to predict putative binding sites for c-myc in the ENO1 promoter region, which were validated with chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays.
RESULTS The pY397-FAK level was increased in human fibrotic liver tissue. FRNK knockout promoted liver fibrosis in mouse models. It also increased the activation, migration, proliferation and aerobic glycolysis of primary hepatic stellate cells (pHSCs) but inhibited pHSC apoptosis. Nevertheless, opposite trends for these phenomena were observed after exogenous FRNK treatment in LX-2 cells. Mechanistically, the FAK/Ras/c-myc/ENO1 pathway promoted aerobic glycolysis, which was inhibited by exogenous FRNK.
CONCLUSION FRNK inhibits aerobic glycolysis in HSCs by inhibiting the FAK/Ras/c-myc/ ENO1 pathway, thereby improving liver fibrosis. FRNK might be a potential target for liver fibrosis treatment.
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Affiliation(s)
- Tao Huang
- Department of Infectious Diseases, Affiliated Hospital of Guizhou Medical University, Guizhou Medical University, Guiyang 550004, Guizhou Province, China
| | - Yuan-Qing-Xiao Li
- Department of Infectious Diseases, Affiliated Hospital of Guizhou Medical University, Guizhou Medical University, Guiyang 550004, Guizhou Province, China
| | - Ming-Yu Zhou
- Department of Infectious Diseases, Affiliated Hospital of Guizhou Medical University, Guizhou Medical University, Guiyang 550004, Guizhou Province, China
| | - Rui-Han Hu
- Department of Infectious Diseases, Affiliated Hospital of Guizhou Medical University, Guizhou Medical University, Guiyang 550004, Guizhou Province, China
| | - Gao-Liang Zou
- Department of Infectious Diseases, Affiliated Hospital of Guizhou Medical University, Guizhou Medical University, Guiyang 550004, Guizhou Province, China
| | - Jian-Chao Li
- Department of Infectious Diseases, Affiliated Hospital of Guizhou Medical University, Guizhou Medical University, Guiyang 550004, Guizhou Province, China
| | - Shu Feng
- Department of Infectious Diseases, Affiliated Hospital of Guizhou Medical University, Guizhou Medical University, Guiyang 550004, Guizhou Province, China
| | - Yong-Mei Liu
- Clinical Laboratory Center, Affiliated Hospital of Guizhou Medical University, Guiyang 550004, Guizhou Province, China
| | - Chang-Qin Xin
- Department of Infectious Diseases, People’s Hospital of Weining Yi, Hui and Miao Autonomous County, Weining 553100, Guizhou Province, China
| | - Xue-Ke Zhao
- Department of Infectious Diseases, Affiliated Hospital of Guizhou Medical University, Guizhou Medical University, Guiyang 550004, Guizhou Province, China
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Jeong K, Murphy JM, Kim JH, Campbell PM, Park H, Rodriguez Y, Choi C, Kim JS, Park S, Kim HJ, Scammell JG, Weber DS, Honkanen RE, Schlaepfer DD, Ahn EYE, Lim STS. FAK Activation Promotes SMC Dedifferentiation via Increased DNA Methylation in Contractile Genes. Circ Res 2021; 129:e215-e233. [PMID: 34702049 DOI: 10.1161/circresaha.121.319066] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Rationale: Vascular smooth muscle cells (SMCs) exhibit remarkable plasticity and can undergo dedifferentiation upon pathological stimuli associated with disease and interventions. Objective: Although epigenetic changes are critical in SMC phenotype switching, a fundamental regulator that governs the epigenetic machineries regulating the fate of SMC phenotype has not been elucidated. Methods and Results: Using SMCs, mouse models, and human atherosclerosis specimens, we found that focal adhesion kinase (FAK) activation elicits SMC dedifferentiation by stabilizing DNA methyltransferase 3A (DNMT3A). FAK in SMCs is activated in the cytoplasm upon serum stimulation in vitro or vessel injury and active FAK prevents DNMT3A from nuclear FAK-mediated degradation. However, pharmacological or genetic FAK catalytic inhibition forced FAK nuclear localization, which reduced DNMT3A protein via enhanced ubiquitination and proteasomal degradation. Reduced DNMT3A protein led to DNA hypomethylation in contractile gene promoters, which increased SMC contractile protein expression. RNA sequencing identified SMC contractile genes as a foremost upregulated group by FAK inhibition from injured femoral artery samples compared to vehicle group. DNMT3A knockdown in injured arteries reduced DNA methylation and enhanced contractile gene expression supports the notion that nuclear FAK-mediated DNMT3A degradation via E3 ligase TRAF6 drives differentiation of SMCs. Furthermore, we observed that SMCs of human atherosclerotic lesions exhibited decreased nuclear FAK, which was associated with increased DNMT3A levels and decreased contractile gene expression. Conclusions: This study reveals that nuclear FAK induced by FAK catalytic inhibition specifically suppresses DNMT3A expression in injured vessels resulting in maintaining SMC differentiation by promoting the contractile gene expression. Thus, FAK inhibitors may provide a new treatment option to block SMC phenotypic switching during vascular remodeling and atherosclerosis.
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Affiliation(s)
- Kyuho Jeong
- Biochemistry and Molecular Biology, University of South Alabama College of Medicine, UNITED STATES
| | - James M Murphy
- Biochemistry and Molecular Biology, University of South Alabama College of Medicine, UNITED STATES
| | - Jung-Hyun Kim
- Biochemistry and Molecular Biology, University of South Alabama College of Medicine, UNITED STATES
| | | | - Hyeonsoo Park
- Biochemistry and Molecular Biology, University of South Alabama College of Medicine, KOREA, REPUBLIC OF
| | - Yelitza Rodriguez
- Biochemistry and Molecular Biology, University of South Alabama College of Medicine, UNITED STATES
| | - Chungsik Choi
- Physiology, University of South Alabama College of Medicine, UNITED STATES
| | - Jun-Sub Kim
- Biotechnology, Korea National University of Transportation, KOREA, REPUBLIC OF
| | - Sangwon Park
- Pharmacology, Gyeongsang National University, KOREA, REPUBLIC OF
| | - Hyun Joon Kim
- Anatomy and Convergence Medical Sciences, Gyeongsang National University
| | - Jonathan G Scammell
- Comparative Medicine, University of South Alabama College of Medicine, UNITED STATES
| | - David S Weber
- Physiology and Cell Biology, University of South Alabama College of Medicine, UNITED STATES
| | - Richard E Honkanen
- Biochemistry and Molecualr Biology, University of South Alabama College of Medicine, UNITED STATES
| | - David D Schlaepfer
- Obstetrics, Gynecology, and Reproductive Medicine, University of California, San Diego Moores Cancer Center, UNITED STATES
| | | | - Ssang-Taek Steve Lim
- Biochemistry and Molecular Biology, University of South Alabama College of Medicine, UNITED STATES
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Murphy JM, Jeong K, Lim STS. FAK Family Kinases in Vascular Diseases. Int J Mol Sci 2020; 21:ijms21103630. [PMID: 32455571 PMCID: PMC7279255 DOI: 10.3390/ijms21103630] [Citation(s) in RCA: 36] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2020] [Revised: 05/10/2020] [Accepted: 05/19/2020] [Indexed: 12/11/2022] Open
Abstract
In various vascular diseases, extracellular matrix (ECM) and integrin expression are frequently altered, leading to focal adhesion kinase (FAK) or proline-rich tyrosine kinase 2 (Pyk2) activation. In addition to the major roles of FAK and Pyk2 in regulating adhesion dynamics via integrins, recent studies have shown a new role for nuclear FAK in gene regulation in various vascular cells. In particular, FAK primarily localizes within the nuclei of vascular smooth muscle cells (VSMCs) of healthy arteries. However, vessel injury increased FAK localization back to adhesions and elevated FAK activity, leading to VSMC hyperplasia. The study suggested that abnormal FAK or Pyk2 activation in vascular cells may cause pathology in vascular diseases. Here we will review several studies of FAK and Pyk2 associated with integrin signaling in vascular diseases including restenosis, atherosclerosis, heart failure, pulmonary arterial hypertension, aneurysm, and thrombosis. Despite the importance of FAK family kinases in vascular diseases, comprehensive reviews are scarce. Therefore, we summarized animal models involving FAK family kinases in vascular diseases.
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Khosravi M, Kakavandi N, Rezaee S, Shabani M, Najafi M. A Peptide Construct Mediates Focal Adhesion Pathway Through the Activation of Integrin Receptor. Curr Pharm Des 2020; 26:1749-1755. [PMID: 32160840 DOI: 10.2174/1381612826666200311125325] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2019] [Accepted: 02/22/2020] [Indexed: 01/06/2023]
Abstract
BACKGROUND The integrin family receptors stimulate the cellular proliferation and migration through the focal adhesion pathway by the activation of PTK2, VASP and TSP1 proteins. The purpose of this study was to investigate the integrin-ligated motifs through the activation of focal adhesion pathway. METHODS A chimeric peptide was predicted from the integrin-mediated ligands by bioinformatics tools. The VSMCs were treated with the chimeric peptide and simvastatin. The PTK2, VASP and TSP1 protein and gene expression levels were measured by RT-qPCR and Western Blotting techniques, respectively. AutoDock Tools were used for the docking technique. RESULTS The PTK2, VASP and TSP1 protein expression levels increased significantly in the VSMCs treated with chimeric peptide in conversely with the effects of simvastatin. The docking results suggested two motifs in the chimeric peptide. CONCLUSION In conclusion, the chimeric peptide activated the focal adhesion pathway. The motifs 1 and 2 may be directly involved in the transduction of signal by integrin family receptors.
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Affiliation(s)
- Mohsen Khosravi
- Medicine Biochemistry, Qom Branch, Islamic Azad University, Qom, Iran
| | - Naser Kakavandi
- Biochemistry Department, Iran University of Medical Sciences, Tehran, Iran
| | - Shima Rezaee
- Biochemistry Department, Iran University of Medical Sciences, Tehran, Iran
| | - Mohammad Shabani
- Biochemistry Department, Iran University of Medical Sciences, Tehran, Iran
| | - Mohammad Najafi
- Biochemistry Department, Iran University of Medical Sciences, Tehran, Iran.,Microbial Biotechnology Research Center, Iran University of Medical Sciences, Tehran, Iran
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Jeong K, Kim JH, Murphy JM, Park H, Kim SJ, Rodriguez YAR, Kong H, Choi C, Guan JL, Taylor JM, Lincoln TM, Gerthoffer WT, Kim JS, Ahn EYE, Schlaepfer DD, Lim STS. Nuclear Focal Adhesion Kinase Controls Vascular Smooth Muscle Cell Proliferation and Neointimal Hyperplasia Through GATA4-Mediated Cyclin D1 Transcription. Circ Res 2019; 125:152-166. [PMID: 31096851 DOI: 10.1161/circresaha.118.314344] [Citation(s) in RCA: 53] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
RATIONALE Neointimal hyperplasia is characterized by excessive accumulation of vascular smooth muscle cells (SMCs) leading to occlusive disorders, such as atherosclerosis and stenosis. Blood vessel injury increases growth factor secretion and matrix synthesis, which promotes SMC proliferation and neointimal hyperplasia via FAK (focal adhesion kinase). OBJECTIVE To understand the mechanism of FAK action in SMC proliferation and neointimal hyperplasia. METHODS AND RESULTS Using combined pharmacological FAK catalytic inhibition (VS-4718) and SMC-specific FAK kinase-dead (Myh11-Cre-ERT2) mouse models, we report that FAK regulates SMC proliferation and neointimal hyperplasia in part by governing GATA4- (GATA-binding protein 4) cyclin D1 signaling. Inhibition of FAK catalytic activity facilitates FAK nuclear localization, which is required for proteasome-mediated GATA4 degradation in the cytoplasm. Chromatin immunoprecipitation identified GATA4 binding to the mouse cyclin D1 promoter, and loss of GATA4-mediated cyclin D1 transcription diminished SMC proliferation. Stimulation with platelet-derived growth factor or serum activated FAK and redistributed FAK from the nucleus to cytoplasm, leading to concomitant increase in GATA4 protein and cyclin D1 expression. In a femoral artery wire injury model, increased neointimal hyperplasia was observed in parallel with elevated FAK activity, GATA4 and cyclin D1 expression following injury in control mice, but not in VS-4718-treated and SMC-specific FAK kinase-dead mice. Finally, lentiviral shGATA4 knockdown in the wire injury significantly reduced cyclin D1 expression, SMC proliferation, and neointimal hyperplasia compared with control mice. CONCLUSIONS Nuclear enrichment of FAK by inhibition of FAK catalytic activity during vessel injury blocks SMC proliferation and neointimal hyperplasia through regulation of GATA4-mediated cyclin D1 transcription.
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Affiliation(s)
- Kyuho Jeong
- From the Department of Biochemistry and Molecular Biology (K.J., J.M.M., H.P., S.-J.K., Y.A.R.R., W.T.G., J.-S.K., E.-Y.E.A., S.-T.S.L.), University of South Alabama, College of Medicine, Mobile
| | - Jung-Hyun Kim
- Mitchell Cancer Institute (J.-H.K., H.K., E.-Y.E.A), University of South Alabama, College of Medicine, Mobile
| | - James M Murphy
- From the Department of Biochemistry and Molecular Biology (K.J., J.M.M., H.P., S.-J.K., Y.A.R.R., W.T.G., J.-S.K., E.-Y.E.A., S.-T.S.L.), University of South Alabama, College of Medicine, Mobile
| | - Hyeonsoo Park
- From the Department of Biochemistry and Molecular Biology (K.J., J.M.M., H.P., S.-J.K., Y.A.R.R., W.T.G., J.-S.K., E.-Y.E.A., S.-T.S.L.), University of South Alabama, College of Medicine, Mobile
| | - Su-Jeong Kim
- From the Department of Biochemistry and Molecular Biology (K.J., J.M.M., H.P., S.-J.K., Y.A.R.R., W.T.G., J.-S.K., E.-Y.E.A., S.-T.S.L.), University of South Alabama, College of Medicine, Mobile
| | - Yelitza A R Rodriguez
- From the Department of Biochemistry and Molecular Biology (K.J., J.M.M., H.P., S.-J.K., Y.A.R.R., W.T.G., J.-S.K., E.-Y.E.A., S.-T.S.L.), University of South Alabama, College of Medicine, Mobile
| | - Hyunkyung Kong
- Mitchell Cancer Institute (J.-H.K., H.K., E.-Y.E.A), University of South Alabama, College of Medicine, Mobile
| | - Chungsik Choi
- Department of Physiology (C.C., T.M.L.), University of South Alabama, College of Medicine, Mobile
| | - Jun-Lin Guan
- Department of Cancer Biology, University of Cincinnati, College of Medicine, OH (J.-L.G.)
| | - Joan M Taylor
- Department of Pathology, University of North Carolina, School of Medicine, Chapel Hill (J.M.T.)
| | - Thomas M Lincoln
- Department of Physiology (C.C., T.M.L.), University of South Alabama, College of Medicine, Mobile
| | - William T Gerthoffer
- From the Department of Biochemistry and Molecular Biology (K.J., J.M.M., H.P., S.-J.K., Y.A.R.R., W.T.G., J.-S.K., E.-Y.E.A., S.-T.S.L.), University of South Alabama, College of Medicine, Mobile
| | - Jun-Sub Kim
- From the Department of Biochemistry and Molecular Biology (K.J., J.M.M., H.P., S.-J.K., Y.A.R.R., W.T.G., J.-S.K., E.-Y.E.A., S.-T.S.L.), University of South Alabama, College of Medicine, Mobile.,Department of Biotechnology, Korea National Transportation University, Chungbuk (J.-S.K.)
| | - Eun-Young Erin Ahn
- From the Department of Biochemistry and Molecular Biology (K.J., J.M.M., H.P., S.-J.K., Y.A.R.R., W.T.G., J.-S.K., E.-Y.E.A., S.-T.S.L.), University of South Alabama, College of Medicine, Mobile.,Mitchell Cancer Institute (J.-H.K., H.K., E.-Y.E.A), University of South Alabama, College of Medicine, Mobile
| | - David D Schlaepfer
- Department of Reproductive Medicine, Moores Cancer Center, University of California, San Diego, La Jolla (D.D.S.)
| | - Ssang-Taek Steve Lim
- From the Department of Biochemistry and Molecular Biology (K.J., J.M.M., H.P., S.-J.K., Y.A.R.R., W.T.G., J.-S.K., E.-Y.E.A., S.-T.S.L.), University of South Alabama, College of Medicine, Mobile
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8
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Huang L, Li L, Yang T, Li W, Song L, Meng X, Gu Q, Xiong C, He J. Transgelin as a potential target in the reversibility of pulmonary arterial hypertension secondary to congenital heart disease. J Cell Mol Med 2018; 22:6249-6261. [PMID: 30338626 PMCID: PMC6237561 DOI: 10.1111/jcmm.13912] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2018] [Revised: 08/13/2018] [Accepted: 08/20/2018] [Indexed: 12/21/2022] Open
Abstract
BACKGROUND The reversibility of pulmonary arterial hypertension (PAH) in congenital heart disease (CHD) is of great importance for the operability of CHD. Proteomics analysis found that transgelin was significantly up-regulated in the lung tissue of CHD-PAH patients, especially in the irreversible group. However, how exactly it participated in CHD-PAH development is unknown. METHODS Immunohistochemical staining and Western blot were performed for further qualitative and quantitative analysis of transgelin in the lung tissues of CHD-PAH patients. The mechanism of transgelin in CHD-PAH development was explored in vitro. Primary human pulmonary arterial smooth muscle cells (hPASMCs) were cultured and infected with TAGLN siRNA or TAGLN lentiviral vector. Cell morphologic change (Coomassie Brilliant Blue staining), proliferation (cell count and EdU assay), apoptosis (terminal deoxyribonucleotidyl transferase mediated dUTP nick end labeling assay and Annexin-V flow cytometry) and migration (transwell) were evaluated following the cell treatment. The mRNA and protein expression levels were detected in real-time PCR and Western blot. RESULTS In line with the proteomic findings, transgelin was obviously expressed in PASMC of the middle pulmonary arterioles, especially in the irreversible PAH group. Also, transgelin expression showed positive relation with pathological grading. Experiment in vitro demonstrated that transgelin overexpression promoted PASMC proliferation and migration, strengthened cytoskeleton and was accompanied by increased expression of synthetic phenotype markers (osteopontin, proliferating cell nuclear antigen) and anti-apoptotic protein (bcl-2). On the other hand, suppression of transgelin expression activated PASMC apoptosis, reducing cell proliferation and migration. CONCLUSIONS Transgelin may be a potential target in the development of irreversible CHD-PAH through inducing PASMC phenotype change, proliferation, migration and reducing cell apoptosis.
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Affiliation(s)
- Li Huang
- Center of Pulmonary Vascular DiseaseState Key Laboratory of Cardiovascular DiseaseFuwai HospitalNational Center for Cardiovascular DiseasesChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijingChina
| | - Li Li
- Department of PathologyState Key Laboratory of Cardiovascular DiseaseFuwai HospitalNational Center for Cardiovascular DiseasesChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijingChina
| | - Tao Yang
- Center of Pulmonary Vascular DiseaseState Key Laboratory of Cardiovascular DiseaseFuwai HospitalNational Center for Cardiovascular DiseasesChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijingChina
| | - Wen Li
- Center of Pulmonary Vascular DiseaseState Key Laboratory of Cardiovascular DiseaseFuwai HospitalNational Center for Cardiovascular DiseasesChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijingChina
| | - Li Song
- State Key Laboratory of Cardiovascular DiseaseFuwai HospitalNational Center for Cardiovascular DiseasesChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijingChina
| | - Xianmin Meng
- State Key Laboratory of Cardiovascular DiseaseFuwai HospitalNational Center for Cardiovascular DiseasesChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijingChina
| | - Qing Gu
- Center of Pulmonary Vascular DiseaseState Key Laboratory of Cardiovascular DiseaseFuwai HospitalNational Center for Cardiovascular DiseasesChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijingChina
| | - Changming Xiong
- Center of Pulmonary Vascular DiseaseState Key Laboratory of Cardiovascular DiseaseFuwai HospitalNational Center for Cardiovascular DiseasesChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijingChina
| | - Jianguo He
- Center of Pulmonary Vascular DiseaseState Key Laboratory of Cardiovascular DiseaseFuwai HospitalNational Center for Cardiovascular DiseasesChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijingChina
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9
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Zak TJ, Koshman YE, Samarel AM, Robia SL. Regulation of Focal Adhesion Kinase through a Direct Interaction with an Endogenous Inhibitor. Biochemistry 2017; 56:4722-4731. [PMID: 28782937 DOI: 10.1021/acs.biochem.7b00616] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
Focal adhesion kinase (FAK) plays a key role in integrin and growth factor signaling pathways. FAK-related non-kinase (FRNK) is an endogenous inhibitor of FAK that shares its primary structure with the C-terminal third of FAK. FAK S910 phosphorylation is known to regulate FAK protein-protein interactions, but the role of the equivalent site on FRNK (S217) is unknown. Here we determined that S217 is highly phosphorylated by ERK in cultured rat aortic smooth muscle cells. Blocking phosphorylation by mutation (S217A) greatly increased FRNK inhibitory potency, resulting in strong inhibition of FAK autophosphorylation at Y397 and induction of smooth muscle cell apoptosis. FRNK has been proposed to compete for FAK anchoring sites in focal adhesions, but we did not detect displacement of FAK by WT-FRNK or superinhibitory S217A-FRNK. Instead, we found FRNK interacted directly with FAK, and this interaction is markedly strengthened for the superinhibitory S217A-FRNK. The results suggest that FRNK limits growth and survival signaling pathways by binding directly to FAK in an inhibitory complex, and this inhibition is relieved by phosphorylation of FRNK at S217.
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Affiliation(s)
- Taylor J Zak
- Department of Cell and Molecular Physiology and ‡Department of Medicine, Cardiovascular Research Institute, Stritch School of Medicine, Loyola University Chicago , Maywood, Illinois 60153, United States
| | - Yevgenia E Koshman
- Department of Cell and Molecular Physiology and ‡Department of Medicine, Cardiovascular Research Institute, Stritch School of Medicine, Loyola University Chicago , Maywood, Illinois 60153, United States
| | - Allen M Samarel
- Department of Cell and Molecular Physiology and ‡Department of Medicine, Cardiovascular Research Institute, Stritch School of Medicine, Loyola University Chicago , Maywood, Illinois 60153, United States
| | - Seth L Robia
- Department of Cell and Molecular Physiology and ‡Department of Medicine, Cardiovascular Research Institute, Stritch School of Medicine, Loyola University Chicago , Maywood, Illinois 60153, United States
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11
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Weise-Cross L, Taylor JM, Mack CP. Inhibition of Diaphanous Formin Signaling In Vivo Impairs Cardiovascular Development and Alters Smooth Muscle Cell Phenotype. Arterioscler Thromb Vasc Biol 2015; 35:2374-83. [PMID: 26381868 DOI: 10.1161/atvbaha.115.305879] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2015] [Accepted: 09/03/2015] [Indexed: 01/13/2023]
Abstract
OBJECTIVE We and others have previously shown that RhoA-dependent stimulation of myocardin-related transcription factor nuclear localization promotes smooth muscle cell (SMC) marker gene expression. The goal of this study was to provide direct in vivo evidence that actin polymerization by the diaphanous-related formins contributes to the regulation of SMC differentiation and phenotype. APPROACH AND RESULTS Conditional Cre-based genetic approaches were used to overexpress a well-characterized dominant-negative variant of mDia1 (DNmDia) in SMC. DNmDia expression in SM22-expressing cells resulted in embryonic and perinatal lethality in ≈20% of mice because of defects in myocardial development and SMC investment of peripheral vessels. Although most DNmDia(+)/SM22Cre(+) mice exhibited no overt phenotype, the re-expression of SMC differentiation marker gene expression that occurs after carotid artery ligation was delayed, and this effect was accompanied by a significant decrease in myocardin-related transcription factor-A nuclear localization. Interestingly, neointima growth was inhibited by expression of DNmDia in SMC and this was likely because of a defect in directional SMC migration and not to defects in SMC proliferation or survival. Finally, by using the tamoxifen-inducible SM MHC-CreER(T2) line, we showed that SMC-specific induction of DNmDia in adult mice decreased SMC marker gene expression. CONCLUSIONS Our demonstration that diaphanous-related formin signaling plays a role in heart and vascular development and the maintenance of SMC phenotype provides important new evidence that Rho/actin/myocardin-related transcription factor signaling plays a critical role in cardiovascular function.
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Affiliation(s)
- Laura Weise-Cross
- From the Department of Pathology, University of North Carolina, Chapel Hill
| | - Joan M Taylor
- From the Department of Pathology, University of North Carolina, Chapel Hill
| | - Christopher P Mack
- From the Department of Pathology, University of North Carolina, Chapel Hill.
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12
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Kang I, Barth JL, Sproul EP, Yoon DW, Workman GA, Braun KR, Argraves WS, Wight TN. Expression of V3 Versican by Rat Arterial Smooth Muscle Cells Promotes Differentiated and Anti-inflammatory Phenotypes. J Biol Chem 2015; 290:21629-41. [PMID: 26152723 DOI: 10.1074/jbc.m115.657486] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2015] [Indexed: 01/03/2023] Open
Abstract
Arterial smooth muscle cells (ASMCs) undergo phenotypic changes during development and pathological processes in vivo and during cell culture in vitro. Our previous studies demonstrated that retrovirally mediated expression of the versican V3 splice variant (V3) by ASMCs retards cell proliferation and migration in vitro and reduces neointimal thickening and macrophage and lipid accumulation in animal models of vascular injury and atherosclerosis. However, the molecular pathways induced by V3 expression that are responsible for these changes are not yet clear. In this study, we employed a microarray approach to examine how expression of V3 induced changes in gene expression and the molecular pathways in rat ASMCs. We found that forced expression of V3 by ASMCs affected expression of 521 genes by more than 1.5-fold. Gene ontology analysis showed that components of the extracellular matrix were the most significantly affected by V3 expression. In addition, genes regulating the formation of the cytoskeleton, which also serve as markers of contractile smooth muscle cells (SMCs), were significantly up-regulated. In contrast, components of the complement system, chemokines, chemokine receptors, and transcription factors crucial for regulating inflammatory processes were among the genes most down-regulated. Consistently, we found that the level of myocardin, a key transcription factor promoting contractile SMC phenotype, was greatly increased, and the proinflammatory transcription factors NFκB1 and CCAAT/enhancer-binding protein β were significantly attenuated in V3-expressing SMCs. Overall, these findings demonstrate that V3 expression reprograms ASMCs promoting differentiated and anti-inflammatory phenotypes.
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Affiliation(s)
- Inkyung Kang
- From the Matrix Biology Program, Benaroya Research Institute at Virginia Mason, Seattle, Washington 98101 and
| | - Jeremy L Barth
- the Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, South Carolina 29425
| | - Erin P Sproul
- the Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, South Carolina 29425
| | - Dong Won Yoon
- From the Matrix Biology Program, Benaroya Research Institute at Virginia Mason, Seattle, Washington 98101 and
| | - Gail A Workman
- From the Matrix Biology Program, Benaroya Research Institute at Virginia Mason, Seattle, Washington 98101 and
| | - Kathleen R Braun
- From the Matrix Biology Program, Benaroya Research Institute at Virginia Mason, Seattle, Washington 98101 and
| | - W Scott Argraves
- the Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, South Carolina 29425
| | - Thomas N Wight
- From the Matrix Biology Program, Benaroya Research Institute at Virginia Mason, Seattle, Washington 98101 and
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13
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Pelargonidin attenuates PDGF-BB-induced aortic smooth muscle cell proliferation and migration by direct inhibition of focal adhesion kinase. Biochem Pharmacol 2014; 89:236-45. [PMID: 24582770 DOI: 10.1016/j.bcp.2014.02.015] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2013] [Revised: 02/07/2014] [Accepted: 02/19/2014] [Indexed: 12/28/2022]
Abstract
Pelargonidin is a natural red pigment found in fruits and vegetables, and has been reported to exhibit various effects potentially beneficial for human health. However, the possible preventive effects of pelargonidin toward atherosclerosis and mechanisms involved have not been investigated to date. Here, we compared the effects of pelargonidin and its glucoside-conjugated form, pelargonidin-3-glucoside (P3G), on proliferation and migration induced by platelet-derived growth factor (PDGF)-BB in human aortic smooth muscle cells (HASMCs). Pelargonidin, but not P3G, exhibited strong inhibitory effects against PDGF-BB-induced HASMC proliferation and migration, while suppressing PDGF-BB-induced ex vivo rat aortic ring sprouting. Immunoblot analysis revealed that pelargonidin inhibited PDGF-BB-induced phosphorylation of focal adhesion kinase (FAK) as well as F-actin reduction, whereas Src, mitogen-activated protein kinases (MAPKs) and Akt phosphorylation status were not altered. We also observed that the anti-proliferative and migratory effects of both pelargonidin and P3G corresponded with the extent of FAK inhibition. Both in vitro and ex vivo pull-down assays revealed that pelargonidin binds directly with FAK in an adenosine triphosphate-competitive manner, suggesting that FAK could be a molecular target of pelargonidin. Interestingly, pelargonidin did not exhibit inhibitory effects on the proliferation, migration or FAK phosphorylation of human umbilical vein endothelial cells (HUVECs). Taken together, our results suggest that pelargonidin exhibits potential preventive effects toward atherosclerosis through the attenuation of HASMC proliferation and migration, as well as aortic sprouting via the direct inhibition of FAK activity.
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Son JE, Lee E, Jung SK, Kim JE, Oak MH, Lee KW, Lee HJ. Anthocyanidins, novel FAK inhibitors, attenuate PDGF-BB-induced aortic smooth muscle cell migration and neointima formation. Cardiovasc Res 2013; 101:503-12. [DOI: 10.1093/cvr/cvt337] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
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15
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Li M, Schwerbrock NMJ, Lenhart PM, Fritz-Six KL, Kadmiel M, Christine KS, Kraus DM, Espenschied ST, Willcockson HH, Mack CP, Caron KM. Fetal-derived adrenomedullin mediates the innate immune milieu of the placenta. J Clin Invest 2013; 123:2408-20. [PMID: 23635772 DOI: 10.1172/jci67039] [Citation(s) in RCA: 48] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2012] [Accepted: 02/22/2013] [Indexed: 12/11/2022] Open
Abstract
The remodeling of maternal uterine spiral arteries (SAs) is an essential process for ensuring low-resistance, high-capacitance blood flow to the growing fetus. Failure of SAs to remodel is causally associated with preeclampsia, a common and life-threatening complication of pregnancy that is harmful to both mother and fetus. Here, using both loss-of-function and gain-of-function genetic mouse models, we show that expression of the pregnancy-related peptide adrenomedullin (AM) by fetal trophoblast cells is necessary and sufficient to promote appropriate recruitment and activation of maternal uterine NK (uNK) cells to the placenta and ultimately facilitate remodeling of maternal SAs. Placentas that lacked either AM or its receptor exhibited reduced fetal vessel branching in the labyrinth, failed SA remodeling and reendothelialization, and markedly reduced numbers of maternal uNK cells. In contrast, overexpression of AM caused a reversal of these phenotypes with a concomitant increase in uNK cell content in vivo. Moreover, AM dose-dependently stimulated the secretion of numerous chemokines, cytokines, and MMPs from uNK cells, which in turn induced VSMC apoptosis. These data identify an essential function for fetal-derived factors in the maternal vascular adaptation to pregnancy and underscore the importance of exploring AM as a biomarker and therapeutic agent for preeclampsia.
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Affiliation(s)
- Manyu Li
- Department of Cell Biology and Physiology, The University of North Carolina, Chapel Hill, North Carolina 27599, USA
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FAK-related nonkinase is a multifunctional negative regulator of pulmonary fibrosis. THE AMERICAN JOURNAL OF PATHOLOGY 2013; 182:1572-84. [PMID: 23499373 DOI: 10.1016/j.ajpath.2013.01.026] [Citation(s) in RCA: 45] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/26/2012] [Revised: 12/07/2012] [Accepted: 01/14/2013] [Indexed: 12/18/2022]
Abstract
Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease whose underlying molecular mechanisms are largely unknown. Herein, we show that focal adhesion kinase-related nonkinase (FRNK) plays a key role in limiting the development of lung fibrosis. Loss of FRNK function in vivo leads to increased lung fibrosis in an experimental mouse model. The increase in lung fibrosis is confirmed at the histological, biochemical, and physiological levels. Concordantly, loss of FRNK function results in increased fibroblast migration and myofibroblast differentiation and activation of signaling proteins that drive these phenotypes. FRNK-deficient murine lung fibroblasts also have an increased capacity to produce and contract matrix proteins. Restoration of FRNK expression in vivo and in vitro reverses these profibrotic phenotypes. These data demonstrate the multiple antifibrotic actions of FRNK. More important, FRNK expression is down-regulated in human IPF, and down-regulation of FRNK in normal human lung fibroblasts recapitulates the profibrotic phenotype seen in FRNK-deficient cells. The effect of loss and gain of FRNK in the experimental model, when taken together with its down-regulation in human IPF, suggests that FRNK acts as an endogenous negative regulator of lung fibrosis by repressing multiple profibrotic responses.
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Bai X, Lenhart KC, Bird KE, Suen AA, Rojas M, Kakoki M, Li F, Smithies O, Mack CP, Taylor JM. The smooth muscle-selective RhoGAP GRAF3 is a critical regulator of vascular tone and hypertension. Nat Commun 2013; 4:2910. [PMID: 24335996 PMCID: PMC4237314 DOI: 10.1038/ncomms3910] [Citation(s) in RCA: 51] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2013] [Accepted: 11/11/2013] [Indexed: 12/13/2022] Open
Abstract
Although hypertension is a worldwide health issue, an incomplete understanding of its aetiology has hindered our ability to treat this complex disease. Here we identify arhgap42 (also known as GRAF3) as a Rho-specific GAP expressed specifically in smooth muscle cells (SMCs) in mice and humans. We show that GRAF3-deficient mice exhibit significant hypertension and increased pressor responses to angiotensin II and endothelin-1; these effects are prevented by treatment with the Rho-kinase inhibitor, Y27632. RhoA activity and myosin light chain phosphorylation are elevated in GRAF3-depleted SMCs in vitro and in vivo, and isolated vessel segments from GRAF3-deficient mice show increased contractility. Taken together, our data indicate that GRAF3-mediated inhibition of RhoA activity in vascular SMCs is necessary for maintaining normal blood pressure homoeostasis. Moreover, these findings provide a potential mechanism for a hypertensive locus recently identified within arhgap42 and provide a foundation for the future development of innovative hypertension therapies.
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Affiliation(s)
- Xue Bai
- Department of Pathology, University of North Carolina, Chapel Hill, NC 27599, USA
| | - Kaitlin C. Lenhart
- Department of Pathology, University of North Carolina, Chapel Hill, NC 27599, USA
| | - Kim E. Bird
- Department of Pathology, University of North Carolina, Chapel Hill, NC 27599, USA
| | - Alisa A. Suen
- Department of Pathology, University of North Carolina, Chapel Hill, NC 27599, USA
| | - Mauricio Rojas
- McAllister Heart Institute, University of North Carolina, Chapel Hill, NC 27599, USA
| | - Masao Kakoki
- Department of Pathology, University of North Carolina, Chapel Hill, NC 27599, USA
| | - Feng Li
- Department of Pathology, University of North Carolina, Chapel Hill, NC 27599, USA
| | - Oliver Smithies
- Department of Pathology, University of North Carolina, Chapel Hill, NC 27599, USA
- McAllister Heart Institute, University of North Carolina, Chapel Hill, NC 27599, USA
| | - Christopher P. Mack
- Department of Pathology, University of North Carolina, Chapel Hill, NC 27599, USA
- McAllister Heart Institute, University of North Carolina, Chapel Hill, NC 27599, USA
| | - Joan M. Taylor
- Department of Pathology, University of North Carolina, Chapel Hill, NC 27599, USA
- McAllister Heart Institute, University of North Carolina, Chapel Hill, NC 27599, USA
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18
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Cheng Z, DiMichele LA, Hakim ZS, Rojas M, Mack CP, Taylor JM. Targeted focal adhesion kinase activation in cardiomyocytes protects the heart from ischemia/reperfusion injury. Arterioscler Thromb Vasc Biol 2012; 32:924-33. [PMID: 22383703 DOI: 10.1161/atvbaha.112.245134] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
OBJECTIVE We previously reported that cardiac-restricted deletion of focal adhesion kinase (FAK) exacerbated myocyte death following ischemia/reperfusion (I/R). Here, we interrogated whether targeted elevation of myocardial FAK activity could protect the heart from I/R injury. METHODS AND RESULTS Transgenic mice were generated with myocyte-specific expression of a FAK variant (termed SuperFAK) that conferred elevated allosteric activation. FAK activity in unstressed transgenic hearts was modestly elevated, but this had no discernable effect on anabolic heart growth or cardiac function. Importantly, SuperFAK hearts exhibited a dramatic increase in FAK activity and a reduction in myocyte apoptosis and infarct size 24 to 72 hours following I/R. Moreover, serial echocardiography revealed that the transgenic mice were protected from cardiac decompensation for up to 8 weeks following surgery. Mechanistic studies revealed that elevated FAK activity protected cardiomyocytes from I/R-induced apoptosis by enhancing nuclear factor-κB (NF-κB)-dependent survival signaling during the early period of reperfusion (30 and 60 minutes). Moreover, adenoviral-mediated expression of SuperFAK in cultured cardiomyocytes attenuated H(2)O(2) or hypoxia/reoxygenation-induced apoptosis, whereas blockade of the NF-κB pathway using a pharmacological inhibitor or small interfering RNAs completely abolished the beneficial effect of SuperFAK. CONCLUSIONS Enhancing cardiac FAK activity attenuates I/R-induced myocyte apoptosis through activation of the prosurvival NF-κB pathway and may represent a novel therapeutic strategy for ischemic heart diseases.
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Affiliation(s)
- Zhaokang Cheng
- Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, 27599, USA
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19
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Koshman YE, Chu M, Engman SJ, Kim T, Iyengar R, Robia SL, Samarel AM. Focal adhesion kinase-related nonkinase inhibits vascular smooth muscle cell invasion by focal adhesion targeting, tyrosine 168 phosphorylation, and competition for p130(Cas) binding. Arterioscler Thromb Vasc Biol 2012; 31:2432-40. [PMID: 21852560 DOI: 10.1161/atvbaha.111.235549] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
OBJECTIVE Focal adhesion kinase-related nonkinase (FRNK), the C-terminal domain of focal adhesion kinase (FAK), is a tyrosine-phosphorylated, vascular smooth muscle cell (VSMC)-specific inhibitor of cell migration. FRNK inhibits both FAK and proline-rich tyrosine kinase 2 (PYK2) in cultured VSMCs, and both kinases may be involved in VSMC invasion during vascular remodeling. METHODS AND RESULTS Adenovirally mediated gene transfer of green fluorescent protein-tagged, wild-type (wt) FRNK into balloon-injured rat carotid arteries confirmed that FRNK overexpression inhibited both FAK and PYK2 phosphorylation and downstream signaling in vivo. To identify which kinase was involved in regulating VSMC invasion, adenovirally mediated expression of specific short hairpin RNAs was used to knock down FAK versus PYK2 in cultured VSMCs, but only FAK short hairpin RNA was effective in reducing VSMC invasion. The role of FRNK tyrosine phosphorylation was then examined using adenoviruses expressing nonphosphorylatable (Tyr168Phe-, Tyr232Phe-, and Tyr168,232Phe-) green fluorescent protein-FRNK mutants. wtFRNK and all FRNK mutants localized to FAs, but only Tyr168 phosphorylation was required for FRNK to inhibit invasion. Preventing Tyr168 phosphorylation also increased FRNK-paxillin interaction, as determined by coimmunoprecipitation, total internal reflection fluorescence microscopy, and fluorescence recovery after photobleaching. Furthermore, wtFRNK competed with FAK for binding to p130(Cas) (a critically important regulator of cell migration) and prevented its phosphorylation. However, Tyr168Phe-FRNK was unable to bind p130(Cas). CONCLUSION We propose a 3-stage mechanism for FRNK inhibition: focal adhesion targeting, Tyr168 phosphorylation, and competition with FAK for p130 binding and phosphorylation, which are all required for FRNK to inhibit VSMC invasion.
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Affiliation(s)
- Yevgeniya E Koshman
- Cardiovascular Institute, Loyola University Chicago Stritch School of Medicine, Maywood, IL, USA
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20
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Mack CP. Signaling mechanisms that regulate smooth muscle cell differentiation. Arterioscler Thromb Vasc Biol 2011; 31:1495-505. [PMID: 21677292 PMCID: PMC3141215 DOI: 10.1161/atvbaha.110.221135] [Citation(s) in RCA: 194] [Impact Index Per Article: 13.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2011] [Accepted: 04/25/2011] [Indexed: 01/05/2023]
Abstract
Extensive studies over the last 30 years have demonstrated that vascular smooth muscle cell (SMC) differentiation and phenotypic modulation is controlled by a dynamic array of environmental cues. The identification of the signaling mechanisms by which these environmental cues regulate SMC phenotype has been more difficult because of our incomplete knowledge of the transcription mechanisms that regulate SMC-specific gene expression. However, recent advances in this area have provided significant insight, and the goal of this review is to summarize the signaling mechanisms by which extrinsic cues control SMC differentiation.
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Affiliation(s)
- Christopher P Mack
- Department of Pathology, University of North Carolina, Chapel Hill, NC 27599-7525, USA.
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Zhang DH, Zhou JP. Role of focal adhesion kinase in the pathogenesis of colorectal carcinoma. Shijie Huaren Xiaohua Zazhi 2011; 19:1279-1284. [DOI: 10.11569/wcjd.v19.i12.1279] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Focal adhesion kinase (FAK) was initially identified as a nonreceptor protein tyrosine kinase localized to the focal contact protein clusters. This enzyme has been shown to facilitate generation of integrin-stimulated signals to downstream targets. It has been reported that activation of FAK leads to a number of processes, including cell attachment, migration, proliferation, and survival. The expression of FAK in colon carcinoma is significantly higher than that in matched cancer-adjacent normal tissue, suggesting that FAK may be an important target for the therapy of colon carcinoma. The inhibition of FAK activation can interrupt many signal pathways involved in colon carcinogenesis and may represent a new therapy strategy for colon carcinoma.
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Koshman YE, Kim T, Chu M, Engman SJ, Iyengar R, Robia SL, Samarel AM. FRNK inhibition of focal adhesion kinase-dependent signaling and migration in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 2010; 30:2226-33. [PMID: 20705914 PMCID: PMC4058887 DOI: 10.1161/atvbaha.110.212761] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
OBJECTIVE To examine whether interference with FRNK targeting to focal adhesions (FAs) affects its inhibitory activity and tyrosine phosphorylation. METHODS AND RESULTS Focal adhesion kinase and its autonomously expressed C-terminal inhibitor, focal adhesion kinase-related nonkinase (FRNK), regulate vascular smooth muscle cell (VSMC) signaling and migration. FRNK-paxillin binding was reduced by a point mutation in its FA targeting domain (L341S-FRNK). Green fluorescent protein-tagged wild type and L341S-FRNK were then adenovirally expressed in VSMCs. L341S-FRNK targeted to VSMC FAs, despite previous studies in other cell types. L341S-FRNK affected FA binding kinetics (assessed by total internal reflection fluorescnece [TIRF] microscopy and fluorescence recovery after photobleaching [FRAP]) and reduced its steady-state paxillin interaction (determined by coimmunoprecipitation). Both wt-FRNK and L341S-FRNK lowered basal and angiotensin II-stimulated focal adhesion kinase, paxillin, and extracellular signal-regulated kinase 1/2 phosphorylation. However, the degree of inhibition was significantly reduced by L341S-FRNK. L341S-FRNK also demonstrated significantly greater migratory activity compared with wt-FRNK-expressing VSMCs. Angiotensin II-induced Y168 phosphorylation was Src dependent, as evident by a significant reduction in Y168 phosphorylation by the Src family kinase inhibitor PP2 is 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Surprisingly, Y168 phosphorylation was unaffected by its targeting. Furthermore, Y232 phosphorylation increased approximately 3-fold in L341S-FRNK, which was less sensitive to PP2. CONCLUSIONS FRNK inhibition of VSMC migration requires both FA targeting and Y168 phosphorylation by Src family kinases. FRNK-Y232 phosphorylation occurs outside of FAs, probably by a PP2-insensitive kinase.
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Affiliation(s)
- Yevgeniya E Koshman
- Cardiovascular Institute, Loyola University Medical Center, 2160 S First Ave, Maywood, IL 60153, USA
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Li G, Jin R, Norris RA, Zhang L, Yu S, Wu F, Markwald RR, Nanda A, Conway SJ, Smyth SS, Granger DN. Periostin mediates vascular smooth muscle cell migration through the integrins alphavbeta3 and alphavbeta5 and focal adhesion kinase (FAK) pathway. Atherosclerosis 2010; 208:358-65. [PMID: 19695571 PMCID: PMC2841688 DOI: 10.1016/j.atherosclerosis.2009.07.046] [Citation(s) in RCA: 126] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/02/2009] [Revised: 07/23/2009] [Accepted: 07/23/2009] [Indexed: 11/30/2022]
Abstract
Smooth muscle cell (SMC) migration involves interactions of integrin receptors with extracellular matrix (ECM) and is an important process of neointimal formation in atherosclerosis and restenosis after vascular interventions. Previous studies have shown that periostin (PN), a novel ECM protein, is upregulated in rat carotid artery after balloon injury, and growth factor-stimulated expression of PN promotes SMC migration in vitro. Here, we address the mechanism by which PN-integrin interaction mediates SMC migration in vitro. Aortic SMCs isolated from PN null mice exhibited a significantly reduced ability to migrate and proliferate in vitro. Endogenous PN protein was absent and very low in the culture medium from the primary cultures of PN-/- and wildtype SMCs, respectively. In both types of SMCs, adenovirus-mediated overexpression of HA-tagged PN to a similar extent, which induced a robust cell migration concomitantly with an increase in beta3-integrin expression and phosphorylation of FAK (Tyr397). Furthermore, in cultured human SMCs, specific integrin blocking antibodies showed that interactions of PN-alphanubeta3 and PN-alphanubeta5, but not PN-beta1 integrins, are required for SMC migration. Inhibition of FAK signaling by overexpression of an endogenous FAK inhibitor termed FRNK (FAK-related nonkinase) significantly attenuated FAK (Tyr397) phosphorylation and the SMC migration induced by PN. These results reveal a mechanism whereby PN mediates vascular SMC migration through an interaction with alphaV-integrins (mainly alphanubeta3) and subsequent activation of FAK pathway.
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Affiliation(s)
- Guohong Li
- Department of Neurosurgery, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA, United States.
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Chen CH, Wu ML, Lee YC, Layne MD, Yet SF. Intronic CArG box regulates cysteine-rich protein 2 expression in the adult but not in developing vasculature. Arterioscler Thromb Vasc Biol 2010; 30:835-42. [PMID: 20075421 DOI: 10.1161/atvbaha.109.197251] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
OBJECTIVE An absence of cysteine-rich protein 2 (CRP2) enhances vascular smooth muscle cell (VSMC) migration and increases neointima formation after arterial injury; therefore, CRP2 plays an important role in the response to vascular injury. The goal of the present study was to elucidate the molecular mechanisms that preserve CRP2 expression in the adult vasculature and thus might serve to inhibit the response to injury. METHODS AND RESULTS We generated a series of transgenic mice harboring potential Csrp2 regulatory regions with a lacZ reporter. We determined that the 12-kb first intron was necessary for transgene activity in adult but not in developing vasculature. Within the intron we identified a 6.3-kb region that contains 2 CArG boxes. Serum response factor preferentially bound to CArG2 box in gel mobility shift and chromatin immunoprecipitation assays; additionally, serum response factor coactivator myocardin factors activated CRP2 expression via the CArG2 box. Mutational analysis revealed that CArG2 box was important in directing lacZ expression in VSMC of adult vessels. CONCLUSIONS Although CRP2 expression during development is independent of CArG box regulatory sites, CRP2 expression in adult VSMC requires CArG2 element within the first intron. Our results suggest that distinct mechanisms regulate CRP2 expression in VSMC that are controlled by separate embryonic and adult regulatory modules.
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Affiliation(s)
- Chung-Huang Chen
- Institute of Cellular and System Medicine, National Health Research Institutes, 35 Keyan Road, Zhunan, Miaoli County 35053, Taiwan
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Lilly B, Clark KA, Yoshigi M, Pronovost S, Wu ML, Periasamy M, Chi M, Paul RJ, Yet SF, Beckerle MC. Loss of the serum response factor cofactor, cysteine-rich protein 1, attenuates neointima formation in the mouse. Arterioscler Thromb Vasc Biol 2010; 30:694-701. [PMID: 20056913 DOI: 10.1161/atvbaha.109.200741] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
OBJECTIVE Cysteine-rich protein (CRP) 1 and 2 are cytoskeletal lin-11 isl-1 mec-3 (LIM)-domain proteins thought to be critical for smooth muscle differentiation. Loss of murine CRP2 does not overtly affect smooth muscle differentiation or vascular function but does exacerbate neointima formation in response to vascular injury. Because CRPs 1 and 2 are coexpressed in the vasculature, we hypothesize that CRPs 1 and 2 act redundantly in smooth muscle differentiation. METHODS AND RESULTS We generated Csrp1 (gene name for CRP1) null mice by genetic ablation of the Csrp1 gene and found that mice lacking CRP1 are viable and fertile. Smooth muscle-containing tissues from Csrp1-null mice are morphologically indistinguishable from wild-type mice and have normal contractile properties. Mice lacking CRPs 1 and 2 are viable and fertile, ruling out functional redundancy between these 2 highly related proteins as a cause for the lack of an overt phenotype in the Csrp1-null mice. Csrp1-null mice challenged by wire-induced arterial injury display reduced neointima formation, opposite to that seen in Csrp2-null mice, whereas Csrp1/Csrp2 double-null mice produce a wild-type response. CONCLUSIONS Smooth muscle CRPs are not essential for normal smooth muscle differentiation during development, but may act antagonistically to modulate the smooth muscle response to pathophysiological stress.
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Affiliation(s)
- Brenda Lilly
- Huntsman Cancer Institute, Department of Biology, University of Utah, 2000 Circle of Hope, Salt Lake City, UT 84112, USA
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Koshman YE, Engman SJ, Kim T, Iyengar R, Henderson KK, Samarel AM. Role of FRNK tyrosine phosphorylation in vascular smooth muscle spreading and migration. Cardiovasc Res 2009; 85:571-81. [PMID: 19793767 DOI: 10.1093/cvr/cvp322] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
AIMS Focal adhesion kinase (FAK) and its autonomously expressed, C-terminal inhibitor FAK-related non-kinase (FRNK), are important regulators of vascular smooth muscle cell (VSMC) spreading and migration. However, the mechanisms of FRNK-mediated inhibition of FAK-dependent signalling are not fully defined. The aim of this study was to determine the potential role of FRNK tyrosine phosphorylation in regulating these processes. METHODS AND RESULTS Rat carotid arteries were balloon-injured and FAK and FRNK expression and phosphorylation were examined by immunocytochemistry, immunoprecipitation, and western blotting with total and phosphospecific antibodies. FAK and FRNK expression increased four- and nine-fold, respectively, in alpha-smooth muscle actin-positive VSMCs of injured arteries when compared with contralateral control arteries, and the upregulated FRNK was phosphorylated at residues Y168 and Y232. In A7r5 cells (an embryonic rat VSMC line), endogenously expressed FRNK was also phosphorylated at Y168 and Y232 under basal conditions, and Y168/Y232 phosphorylation increased in response to angiotensin II treatment. When overexpressed in A7r5 cells and adult rat aortic smooth muscle cells (RASM), wild-type (wt) GFP-tagged FRNK was also phosphorylated at residues Y168 and Y232, and GFP-wtFRNK inhibited cell spreading and migration. Mutation of GFP-FRNK at Y168 (GFP-Y168F-FRNK) abrogated FRNK-mediated inhibition of cell spreading and migration, but did not affect its localization in VSMC focal adhesions or its ability to inhibit FAK tyrosine phosphorylation. CONCLUSION Phosphorylation of Y168 on FRNK may represent a novel mechanism by which FRNK inhibits cell spreading and migration in VSMCs.
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Affiliation(s)
- Yevgeniya E Koshman
- The Cardiovascular Institute, Loyola University Chicago Stritch School of Medicine, 2160 South First Avenue, Maywood, IL 60153, USA
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DiMichele LA, Hakim ZS, Sayers RL, Rojas M, Schwartz RJ, Mack CP, Taylor JM. Transient expression of FRNK reveals stage-specific requirement for focal adhesion kinase activity in cardiac growth. Circ Res 2009; 104:1201-8. [PMID: 19372463 DOI: 10.1161/circresaha.109.195941] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
Focal adhesion kinase (FAK) is strongly activated by integrins and growth factors and is essential for embryonic development. We previously showed that the C terminus of FAK is expressed as a separate protein termed FAK-related nonkinase (FRNK) in a smooth muscle cell-selective fashion and that FRNK functions to buffer FAK-dependent signals. We now show that FRNK is also transiently expressed in the neonatal myocardium, with peak levels occurring 5 to 7 days postnatal, just before cell cycle withdrawal. Using novel mouse models, we demonstrate that cardiac-selective expression of FRNK (leading to inhibition of FAK) starting at embryonic day 10.5 leads to a severe ventricular noncompaction defect associated with reduced cardiomyocyte proliferation. Remarkably, postnatal expression of nearly identical levels of FRNK is well tolerated and does not affect viability or anabolic cardiac growth. Nonetheless, FRNK expression in the adult heart does attenuate pathological cardiac hypertrophy following aortic banding, confirming and extending our previous data that this compensatory response is blunted in FAK null hearts. Our mechanistic studies in cultured neonatal cardiomyocytes reveal that FRNK expression induces p38/p27(kip)-dependent cell cycle withdrawal and attenuates extracellular signal-regulated kinase-dependent hypertrophic growth. These findings indicate that dynamic expression of FRNK in the neonatal heart may function to promote cardiomyocyte quiescence in an environment that is particularly rich in growth factors and growth promoting extracellular matrices.
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Affiliation(s)
- Laura A DiMichele
- Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599, USA
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Lemmon JA, Wamhoff BR. "FRNKly, smooth muscle, I don't give a CArG!": a novel mechanism for smooth muscle cell differentiation. Arterioscler Thromb Vasc Biol 2008; 28:2091-3. [PMID: 19020312 PMCID: PMC2749302 DOI: 10.1161/atvbaha.108.176875] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
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