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Zhu YN, Pan F, Gan XW, Liu Y, Wang WS, Sun K. The Role of DNMT1 and C/EBPα in the Regulation of CYP11A1 Expression During Syncytialization of Human Placental Trophoblasts. Endocrinology 2023; 165:bqad195. [PMID: 38146648 DOI: 10.1210/endocr/bqad195] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/24/2023] [Revised: 12/19/2023] [Accepted: 12/22/2023] [Indexed: 12/27/2023]
Abstract
Progesterone synthesized in the placenta is essential for pregnancy maintenance. CYP11A1 is a key enzyme in progesterone synthesis, and its expression increases greatly during trophoblast syncytialization. However, the underlying mechanism remains elusive. Here, we demonstrated that passive demethylation of CYP11A1 promoter accounted for the upregulation of CYP11A1 expression during syncytialization with the participation of the transcription factor C/EBPα. We found that the methylation rate of a CpG locus in the CYP11A1 promoter was significantly reduced along with decreased DNA methyltransferase 1 (DNMT1) expression and its enrichment at the CYP11A1 promoter during syncytialization. DNMT1 overexpression not only increased the methylation of this CpG locus in the CYP11A1 promoter, but also decreased CYP11A1 expression and progesterone production. In silico analysis disclosed multiple C/EBPα binding sites in both CYP11A1 and DNMT1 promoters. C/EBPα expression and its enrichments at both the DNMT1 and CYP11A1 promoters were significantly increased during syncytialization. Knocking-down C/EBPα expression increased DNMT1 while it decreased CYP11A1 expression during syncytialization. Conclusively, C/EBPα plays a dual role in the regulation of CYP11A1 during syncytialization. C/EBPα not only drives CYP11A1 expression directly, but also indirectly through downregulation of DNMT1, which leads to decreased methylation in the CpG locus of the CYP11A1 promoter, resulting in increased progesterone production during syncytialization.
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Affiliation(s)
- Ya-Nan Zhu
- Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200135, P.R. China
- Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai 200135, P.R. China
- Center for Reproductive Medicine, Xiangyang Central Hospital, Hubei University of Arts and Science, Xiangyang, Hubei 441021, P.R. China
| | - Fan Pan
- Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200135, P.R. China
- Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai 200135, P.R. China
| | - Xiao-Wen Gan
- Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200135, P.R. China
- Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai 200135, P.R. China
| | - Yun Liu
- Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200135, P.R. China
- Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai 200135, P.R. China
| | - Wang-Sheng Wang
- Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200135, P.R. China
- Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai 200135, P.R. China
| | - Kang Sun
- Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200135, P.R. China
- Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai 200135, P.R. China
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Fuertes-Agudo M, Luque-Tévar M, Cucarella C, Martín-Sanz P, Casado M. Advances in Understanding the Role of NRF2 in Liver Pathophysiology and Its Relationship with Hepatic-Specific Cyclooxygenase-2 Expression. Antioxidants (Basel) 2023; 12:1491. [PMID: 37627486 PMCID: PMC10451723 DOI: 10.3390/antiox12081491] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2023] [Revised: 07/18/2023] [Accepted: 07/21/2023] [Indexed: 08/27/2023] Open
Abstract
Oxidative stress and inflammation play an important role in the pathophysiological changes of liver diseases. Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that positively regulates the basal and inducible expression of a large battery of cytoprotective genes, thus playing a key role in protecting against oxidative damage. Cyclooxygenase-2 (COX-2) is a key enzyme in prostaglandin biosynthesis. Its expression has always been associated with the induction of inflammation, but we have shown that, in addition to possessing other benefits, the constitutive expression of COX-2 in hepatocytes is beneficial in reducing inflammation and oxidative stress in multiple liver diseases. In this review, we summarized the role of NRF2 as a main agent in the resolution of oxidative stress, the crucial role of NRF2 signaling pathways during the development of chronic liver diseases, and, finally we related its action to that of COX-2, where it appears to operate as its partner in providing a hepatoprotective effect.
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Affiliation(s)
- Marina Fuertes-Agudo
- Instituto de Biomedicina de Valencia (IBV), CSIC, Jaume Roig 11, 46010 Valencia, Spain; (M.F.-A.); (M.L.-T.); (C.C.)
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Monforte de Lemos 3-5, 28029 Madrid, Spain
| | - María Luque-Tévar
- Instituto de Biomedicina de Valencia (IBV), CSIC, Jaume Roig 11, 46010 Valencia, Spain; (M.F.-A.); (M.L.-T.); (C.C.)
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Monforte de Lemos 3-5, 28029 Madrid, Spain
| | - Carme Cucarella
- Instituto de Biomedicina de Valencia (IBV), CSIC, Jaume Roig 11, 46010 Valencia, Spain; (M.F.-A.); (M.L.-T.); (C.C.)
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Monforte de Lemos 3-5, 28029 Madrid, Spain
| | - Paloma Martín-Sanz
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Monforte de Lemos 3-5, 28029 Madrid, Spain
- Instituto de Investigaciones Biomédicas (IIB) “Alberto Sols”, CSIC-UAM, Arturo Duperier 4, 28029 Madrid, Spain
| | - Marta Casado
- Instituto de Biomedicina de Valencia (IBV), CSIC, Jaume Roig 11, 46010 Valencia, Spain; (M.F.-A.); (M.L.-T.); (C.C.)
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Monforte de Lemos 3-5, 28029 Madrid, Spain
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3
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Yue Y, Zou L, Tao J, Yin L, Xie Z, Xia Y, Zhang Z, Wang K, Zhu M. Transcriptomics and metabolomics together reveal the underlying mechanism of heroin hepatotoxicity. Toxicology 2023; 483:153393. [PMID: 36502556 DOI: 10.1016/j.tox.2022.153393] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2022] [Revised: 12/03/2022] [Accepted: 12/07/2022] [Indexed: 12/14/2022]
Abstract
Researches on heroin are more about addiction and some infectious diseases it causes, but liver fibrosis caused by heroin abuse and the mechanism of heroin hepatotoxicity in addicts are ignored. To explore the mechanism of heroin hepatotoxicity, mice in heroin group were intraperitoneally injected by heroin (10 mg/kg) once a day for 14 consecutive days, while mice in heroin withdraw group underwent another 7 days without heroin administration after the same treatment as heroin group. The levels of alanine aminotransferase （ALT）and aspartate aminotransferase （AST） in serum, as biochemical indexes, were applied to evaluate liver damage. H & E staining and oil red O staining were used to observe the pathological changes of liver. Transcriptomics and metabolomics were applied to detect genes and metabolites in livers. The results of biochemical analysis and pathological examination showed that heroin induced liver damage and lipid loss in mice, and these mice did not return to normal completely after a short-term withdrawal. A total of 511 differential genes and 78 differential metabolites were identified by transcriptomics and metabolomics. These differential genes and metabolites were significantly enriched in pathways like lipid metabolism, arachidonic acid metabolism, glutathione metabolism, TCA cycle. And after undergoing 7-day withdrawal of heroin, most of the above differential genes and metabolites did not return to normal. Our study revealed the hepatotoxicity of heroin and that short-term withdrawal of heroin did not fully restore liver function. In addition, transcriptomics and metabolomics revealed that lipid metabolism and arachidonic acid metabolism may be potential therapeutic targets of heroin hepatotoxicity, providing a basis for the treatment of heroin addiction patients in the future.
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Affiliation(s)
- Yingbiao Yue
- National Health Commission Key Laboratory of Drug Addiction Medicine, Kunming Medical University, Kunming 650032, Yunnan, China
| | - Lei Zou
- Department of Hepatobiliary Surgery, The First People's Hospital of Yunnan Province, Kunming 650032, Yunnan, China
| | - Jie Tao
- Drug Rehabilitation Center of Kunming Public Security Bureau, Kunming 650032, Yunnan, China
| | - Lin Yin
- Drug Rehabilitation Center of Kunming Public Security Bureau, Kunming 650032, Yunnan, China
| | - Zhenrong Xie
- The Medical Biobank, the First Affiliated Hospital of Kunming Medical University, Kunming 650032, Yunnan, China
| | - Yu Xia
- Peking University Health Science Center, Beijing 100191, China
| | - Zunyue Zhang
- School of Medicine, Yunnan University, Kunming 650032, Yunnan, China.
| | - Kunhua Wang
- School of Medicine, Yunnan University, Kunming 650032, Yunnan, China.
| | - Mei Zhu
- First Affiliated Hospital of Kunming Medical University, Kunming 650032, Yunnan, China.
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4
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Fuertes-Agudo M, Luque-Tévar M, Cucarella C, Brea R, Boscá L, Quintana-Cabrera R, Martín-Sanz P, Casado M. COX-2 Expression in Hepatocytes Improves Mitochondrial Function after Hepatic Ischemia-Reperfusion Injury. Antioxidants (Basel) 2022; 11:1724. [PMID: 36139798 PMCID: PMC9495319 DOI: 10.3390/antiox11091724] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2022] [Revised: 08/23/2022] [Accepted: 08/27/2022] [Indexed: 12/15/2022] Open
Abstract
Cyclooxygenase 2 (COX-2) is a key enzyme in prostanoid biosynthesis. The constitutive hepatocyte expression of COX-2 has a protective role in hepatic ischemia-reperfusion (I/R) injury (IRI), decreasing necrosis, reducing reactive oxygen species (ROS) levels, and increasing autophagy and antioxidant and anti-inflammatory response. The physiopathology of IRI directly impacts mitochondrial activity, causing ATP depletion and being the main source of ROS. Using genetically modified mice expressing human COX-2 (h-COX-2 Tg) specifically in hepatocytes, and performing I/R surgery on the liver, we demonstrate that COX-2 expression has a beneficial effect at the mitochondrial level. Mitochondria derived from h-COX-2 Tg mice livers have an increased respiratory rate associated with complex I electron-feeding pathways compared to Wild-type (Wt) littermates, without affecting complex I expression or assembly. Furthermore, Wt-derived mitochondria show a loss of mitochondrial membrane potential (ΔΨm) that correlates to increased proteolysis of fusion-related OPA1 through OMA1 protease activity. All these effects are not observed in h-COX-2 Tg mitochondria, which behave similarly to the Sham condition. These results suggest that COX-2 attenuates IRI at a mitochondrial level, preserving the proteolytic processing of OPA1, in addition to the maintenance of mitochondrial respiration.
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Affiliation(s)
- Marina Fuertes-Agudo
- Instituto de Biomedicina de Valencia (IBV), CSIC, Jaume Roig 11, 46010 Valencia, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Monforte de Lemos 3-5, 28029 Madrid, Spain
| | - María Luque-Tévar
- Instituto de Biomedicina de Valencia (IBV), CSIC, Jaume Roig 11, 46010 Valencia, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Monforte de Lemos 3-5, 28029 Madrid, Spain
| | - Carme Cucarella
- Instituto de Biomedicina de Valencia (IBV), CSIC, Jaume Roig 11, 46010 Valencia, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Monforte de Lemos 3-5, 28029 Madrid, Spain
| | - Rocío Brea
- Instituto de Investigaciones Biomedicas Alberto Sols (IIBM), CSIC-UAM, Arturo Duperier 4, 28029 Madrid, Spain
| | - Lisardo Boscá
- Instituto de Investigaciones Biomedicas Alberto Sols (IIBM), CSIC-UAM, Arturo Duperier 4, 28029 Madrid, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERcv), Monforte de Lemos 3-5, 28029 Madrid, Spain
| | | | - Paloma Martín-Sanz
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Monforte de Lemos 3-5, 28029 Madrid, Spain
- Instituto de Investigaciones Biomedicas Alberto Sols (IIBM), CSIC-UAM, Arturo Duperier 4, 28029 Madrid, Spain
| | - Marta Casado
- Instituto de Biomedicina de Valencia (IBV), CSIC, Jaume Roig 11, 46010 Valencia, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Monforte de Lemos 3-5, 28029 Madrid, Spain
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5
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Alvarez MDL, Lorenzetti F. Role of eicosanoids in liver repair, regeneration and cancer. Biochem Pharmacol 2021; 192:114732. [PMID: 34411565 DOI: 10.1016/j.bcp.2021.114732] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2021] [Revised: 08/10/2021] [Accepted: 08/12/2021] [Indexed: 12/24/2022]
Abstract
Eicosanoids are lipid signaling molecules derived from the oxidation of ω-6 fatty acids, usually arachidonic acid. There are three major pathways, including the cyclooxygenase (COX), lipoxygenase (LOX), and P450 cytochrome epoxygenase (CYP) pathway. Prostanoids, which include prostaglandins (PG) and thromboxanes (Tx), are formed via the COX pathway, leukotrienes (LT) and lipoxins (LX) by the action of 5-LOX, and hydroxyeicosatetraenoic acids (HETEs) and epoxyeicosatrienoic acids (EETs) by CYP. Although eicosanoids are usually associated with pro-inflammatory responses, non-classic eicosanoids, as LX, have anti-inflammatory and pro-resolving properties. Eicosanoids like PGE2, LTB4 and EETs have been involved in promoting liver regeneration after partial hepatectomy. PGE2 and LTB4 have also been reported to participate in the regenerative phase after ischemia and reperfusion (I/R), while cysteinyl leukotrienes (Cys-LT) contribute to the inflammatory process associated with I/R and are also involved in liver fibrosis and cirrhosis. However, LX, another product of 5-LOX, have the opposite effect, acting as pro-resolving mediators in these pathologies. In liver cancer, most studies show that eicosanoids, with the exception of LX, promote the proliferation of hepatocellular carcinoma cells and favor metastasis. This review summarizes the synthesis of different eicosanoids in the liver and discusses key findings from basic research linking eicosanoids to liver repair, regeneration and cancer and the impact of targeting eicosanoid cascade. In addition, studies in patients are presented that explore the potential use of eicosanoids as biomarkers and show correlations between eicosanoid production and the course and prognosis of liver disease.
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Affiliation(s)
- María de Luján Alvarez
- Instituto de Fisiología Experimental (IFISE), Facultad de Ciencias Bioquímicas y Farmacéuticas, CONICET, UNR, Suipacha 570 (S2002LRL), Rosario, Argentina; Área Morfología, Facultad de Ciencias Bioquímicas y Farmacéuticas, UNR, Suipacha 570 (S2002LRL), Rosario, Argentina; Centro de Altos Estudios en Ciencias Humanas y de la Salud (CAECIHS) Sede Regional Rosario, Universidad Abierta Interamericana, Av. Pellegrini 1618 (S2000BUG), Rosario, Argentina.
| | - Florencia Lorenzetti
- Instituto de Fisiología Experimental (IFISE), Facultad de Ciencias Bioquímicas y Farmacéuticas, CONICET, UNR, Suipacha 570 (S2002LRL), Rosario, Argentina
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6
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Lu J, Wang W, Zhou Q, Ling L, Ying H, Sun Y, Myatt L, Sun K. C/EBPδ drives key endocrine signals in the human amnion at parturition. Clin Transl Med 2021; 11:e416. [PMID: 34185432 PMCID: PMC8191398 DOI: 10.1002/ctm2.416] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2021] [Revised: 04/19/2021] [Accepted: 04/25/2021] [Indexed: 12/18/2022] Open
Abstract
Amnion-derived prostaglandin E2 (PGE2) and cortisol are key to labor onset. Identification of a common transcription factor driving the expression of both cyclooxygenase-2 (COX-2) and 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1), the key enzymes in their production, may hold the key to the treatment of pre-term labor. Here, we have found that the CCAAT enhancer binding protein δ (C/EBPδ) is such a transcription factor which underlies the feed-forward induction of COX-2 and 11β-HSD1 expression by their own products PGE2 and cortisol in human amnion fibroblasts so that their production would be ensured in the amnion for the onset of labor. Moreover, the abundance of C/EBPδ in the amnion increases along with COX-2 and 11β-HSD1 at term and further increases at parturition. Knockout of C/EBPδ in mice delays the onset of labor further supporting the concept. In conclusion, C/EBPδ pathway may be speculated to serve as a potential pharmaceutical target in the amnion for treatment of pre-term labor.
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Affiliation(s)
- Jiang‐Wen Lu
- Center for Reproductive Medicine, Ren Ji HospitalSchool of Medicine, Shanghai Jiao Tong UniversityShanghaiP.R. China
- Shanghai Key Laboratory for Assisted Reproduction and Reproductive GeneticsShanghaiP.R. China
| | - Wang‐Sheng Wang
- Center for Reproductive Medicine, Ren Ji HospitalSchool of Medicine, Shanghai Jiao Tong UniversityShanghaiP.R. China
- Shanghai Key Laboratory for Assisted Reproduction and Reproductive GeneticsShanghaiP.R. China
| | - Qiong Zhou
- Department of Obstetrics and GynecologyRen Ji Hospital, School of Medicine, Shanghai Jiao Tong UniversityShanghaiP.R. China
| | - Li‐Jun Ling
- Shanghai First Maternity and Infant HospitalTongji University School of MedicineShanghaiP.R. China
| | - Hao Ying
- Shanghai First Maternity and Infant HospitalTongji University School of MedicineShanghaiP.R. China
| | - Yun Sun
- Center for Reproductive Medicine, Ren Ji HospitalSchool of Medicine, Shanghai Jiao Tong UniversityShanghaiP.R. China
- Shanghai Key Laboratory for Assisted Reproduction and Reproductive GeneticsShanghaiP.R. China
| | - Leslie Myatt
- Department of Obstetrics and GynecologyOregon Health and Science UniversityPortlandOregonUSA
| | - Kang Sun
- Center for Reproductive Medicine, Ren Ji HospitalSchool of Medicine, Shanghai Jiao Tong UniversityShanghaiP.R. China
- Shanghai Key Laboratory for Assisted Reproduction and Reproductive GeneticsShanghaiP.R. China
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Wang EM, Hu TH, Huang CC, Chang YC, Yang SM, Huang ST, Wu JC, Ma YL, Chan HH, Liu LF, Lu WB, Kung ML, Wen ZH, Wang JC, Ko CY, Tsai WL, Chu TH, Tai MH. Hepatoma-derived growth factor participates in concanavalin A-induced hepatitis. FASEB J 2020; 34:16163-16178. [PMID: 33063394 DOI: 10.1096/fj.202000511rr] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2020] [Revised: 09/13/2020] [Accepted: 09/28/2020] [Indexed: 12/12/2022]
Abstract
Hepatitis is an important health problem worldwide. Novel molecular targets are in demand for detection and management of hepatitis. Hepatoma-derived growth factor (HDGF) has been delineated to participate in hepatic fibrosis and liver carcinogenesis. However, the relationship between hepatitis and HDGF remains unclear. This study aimed to elucidate the role of HDGF during hepatitis using concanavalin A (ConA)-induced hepatitis model. In cultured hepatocytes, ConA treatment-elicited HDGF upregulation at transcriptional level and promoted HDGF secretion while reducing intracellular HDGF protein level and cellular viability. Similarly, mice receiving ConA administration exhibited reduced hepatic HDGF expression and elevated circulating HDGF level, which was positively correlated with serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. By using HDGF knockout (KO) mice, it was found the ConA-evoked cell death was prominently alleviated in KO compared with control. Besides, it was delineated HDGF ablation conferred protection by suppressing the ConA-induced neutrophils recruitment in livers. Above all, the ConA-mediated activation of tumor necrosis factor-α (TNF-α)/interleukin-1β (IL-1β)/interleukin-6 (IL-6)/cyclooxygenase-2 (COX-2) inflammatory signaling was significantly abrogated in KO mice. Treatment with recombinant HDGF (rHDGF) dose-dependently stimulated the expression of TNF-α/IL-1β/IL-6/COX-2 in hepatocytes, further supporting the pro-inflammatory function of HDGF. Finally, application of HDGF antibody not only attenuated the ConA-mediated inflammatory cascade in hepatocytes, but also ameliorated the ConA-induced hepatic necrosis and AST elevation in mice. In summary, HDGF participates in ConA-induced hepatitis via neutrophils recruitment and may constitute a therapeutic target for acute hepatitis.
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Affiliation(s)
- E-Ming Wang
- Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan.,Division of Gastroenterology and Hepatology, Department of Internal Medicine, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan
| | - Tsung-Hui Hu
- Division of Hepato-Gastroenterology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan
| | - Chao-Cheng Huang
- Biobank and Tissue Bank, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan.,Department of Pathology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan
| | - Yi-Chen Chang
- Doctoral Degree Program in Marine Biotechnology, National Sun Yat-sen University and Academia Sinica, Kaohsiung, Taiwan
| | - Shih-Ming Yang
- Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan
| | - Shih-Tsung Huang
- Doctoral Degree Program in Marine Biotechnology, National Sun Yat-sen University and Academia Sinica, Kaohsiung, Taiwan.,LabTurbo Biotech Corporation, Taipei, Taiwan
| | - Jian-Ching Wu
- Biobank and Tissue Bank, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan.,Department of Pathology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan
| | - Yi-Ling Ma
- Division of Nephrology, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan
| | - Hoi-Hung Chan
- Division of Gastroenterology, Department of Medicine, Conde S. Januário Hospital, Macau, China
| | - Li-Feng Liu
- Department of Biological Science & Technology, I-Shou University, Kaohsiung, Taiwan
| | - Wen-Bin Lu
- Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan
| | - Mei-Lang Kung
- Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan
| | - Zhi-Hong Wen
- Department of Marine Biotechnology and Resources, Asia-Pacific Ocean Research Center, National Sun Yat-sen University, Kaohsiung, Taiwan
| | - Jui-Chu Wang
- Department of Pathology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan
| | - Chou-Yuan Ko
- Department of Gastroenterology, Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan
| | - Wei-Lun Tsai
- Division of Gastroenterology and Hepatology, Department of Internal Medicine, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan
| | - Tian-Huei Chu
- Biobank and Tissue Bank, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan.,Department of Medical Research, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan
| | - Ming-Hong Tai
- Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan.,Doctoral Degree Program in Marine Biotechnology, National Sun Yat-sen University and Academia Sinica, Kaohsiung, Taiwan.,Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan.,Center for Neuroscience, National Sun Yat-sen University, Kaohsiung, Taiwan.,Regenerative Medicine and Cell Therapy Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan
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8
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COX-2 in liver fibrosis. Clin Chim Acta 2020; 506:196-203. [PMID: 32184095 DOI: 10.1016/j.cca.2020.03.024] [Citation(s) in RCA: 37] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2020] [Revised: 03/13/2020] [Accepted: 03/13/2020] [Indexed: 02/07/2023]
Abstract
As a vital inducible sensor, cyclooxygenase-2 (COX-2) plays an important role in the progress of hepatic fibrogenesis. Activation of hepatic stellate cells (HSCs) in the liver can significantly accelerate the onset and development of liver fibrosis. COX-2 overexpression triggers inflammation that is an important inducer in hepatic fibrosis. Increasing evidence indicates that COX-2 is involved in the main pathogenesis of liver fibrosis, such as inflammation, apoptosis, and cell senescence. Moreover, COX-2 expression is altered in patients and animal models with non-alcoholic fatty liver disease or cirrhosis. These findings suggest that COX-2 has a broad and critical role in the development of liver fibrosis. In this review, we summarize the latest advances in the regulation and signal transduction of COX-2 and its impact on liver fibrosis.
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9
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Jaén RI, Prieto P, Casado M, Martín-Sanz P, Boscá L. Post-translational modifications of prostaglandin-endoperoxide synthase 2 in colorectal cancer: An update. World J Gastroenterol 2018; 24:5454-5461. [PMID: 30622375 PMCID: PMC6319129 DOI: 10.3748/wjg.v24.i48.5454] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/07/2018] [Revised: 11/13/2018] [Accepted: 11/13/2018] [Indexed: 02/06/2023] Open
Abstract
The biosynthesis of prostanoids is involved in both physiological and pathological processes. The expression of prostaglandin-endoperoxide synthase 2 (PTGS2; also known as COX-2) has been traditionally associated to the onset of several pathologies, from inflammation to cardiovascular, gastrointestinal and oncologic events. For this reason, the search of selective PTGS2 inhibitors has been a focus for therapeutic interventions. In addition to the classic non-steroidal anti-inflammatory drugs, selective and specific PTGS2 inhibitors, termed coxibs, have been generated and widely used. PTGS2 activity is less restrictive in terms of substrate specificity than the homeostatic counterpart PTGS1, and it accounts for the elevated prostanoid synthesis that accompanies several pathologies. The main regulation of PTGS2 occurs at the transcription level. In addition to this, the stability of the mRNA is finely regulated through the interaction with several cytoplasmic elements, ranging from specific microRNAs to proteins that control mRNA degradation. Moreover, the protein has been recognized to be the substrate for several post-translational modifications that affect both the enzyme activity and the targeting for degradation via proteasomal and non-proteasomal mechanisms. Among these modifications, phosphorylation, glycosylation and covalent modifications by reactive lipidic intermediates and by free radicals associated to the pro-inflammatory condition appear to be the main changes. Identification of these post-translational modifications is relevant to better understand the role of PTGS2 in several pathologies and to establish a correct analysis of the potential function of this protein in diseases progress. Finally, these modifications can be used as biomarkers to establish correlations with other parameters, including the immunomodulation dependent on molecular pathological epidemiology determinants, which may provide a better frame for potential therapeutic interventions.
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Affiliation(s)
- Rafael I Jaén
- Department of Metabolism and Physiopathology of Inflammatory Diseases, Instituto de Investigaciones Biomédicas Alberto Sols (CSIC-UAM), Madrid 28029, Spain
| | - Patricia Prieto
- Department of Metabolism and Physiopathology of Inflammatory Diseases, Instituto de Investigaciones Biomédicas Alberto Sols (CSIC-UAM), Madrid 28029, Spain
| | - Marta Casado
- Department of Biomedicine, Instituto de Biomedicina de Valencia (CSIC), Valencia 46010, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares, y Hepáticas y Digestivas, ISCIII, Madrid 28029, Spain
| | - Paloma Martín-Sanz
- Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares, y Hepáticas y Digestivas, ISCIII, Madrid 28029, Spain
- Unidad Asociada IIBM-ULPGC, Universidad de las Palmas de Gran Canaria (ULPGC), Las Palmas de Gran Canaria 35001, Spain
| | - Lisardo Boscá
- Department of Metabolism and Physiopathology of Inflammatory Diseases, Instituto de Investigaciones Biomédicas Alberto Sols (CSIC-UAM), Madrid 28029, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares, y Hepáticas y Digestivas, ISCIII, Madrid 28029, Spain
- Unidad Asociada IIBM-ULPGC, Universidad de las Palmas de Gran Canaria (ULPGC), Las Palmas de Gran Canaria 35001, Spain
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10
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Martín-Sanz P, Casado M, Boscá L. Cyclooxygenase 2 in liver dysfunction and carcinogenesis: Facts and perspectives. World J Gastroenterol 2017; 23:3572-3580. [PMID: 28611510 PMCID: PMC5449414 DOI: 10.3748/wjg.v23.i20.3572] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/25/2017] [Revised: 03/03/2017] [Accepted: 04/12/2017] [Indexed: 02/06/2023] Open
Abstract
The biosynthesis of prostaglandins and thromboxanes has been a focus of interest in the management of many liver diseases. Cyclooxygenases are the enzymes involved in the first step of the biosynthesis of these lipid mediators and selective inhibitors for these isoenzymes as well as pharmacological analogues of prostaglandins have been developed and are currently applied therapeutically. Here we discuss the implications of these enzymes in the onset of metabolic and lipid disorders in the liver and their potential role in the progression of the diseases towards fibrosis and hepatocellular carcinogenesis.
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11
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García-París M, López-Estrada EK. First records of Eupompha imperialis (Wellman, 1912) (Coleoptera: Meloidae) in Mexico. GRAELLSIA 2015. [DOI: 10.3989/graellsia.2015.v71.131] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
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12
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Francés DE, Motiño O, Agrá N, González-Rodríguez Á, Fernández-Álvarez A, Cucarella C, Mayoral R, Castro-Sánchez L, García-Casarrubios E, Boscá L, Carnovale CE, Casado M, Valverde ÁM, Martín-Sanz P. Hepatic cyclooxygenase-2 expression protects against diet-induced steatosis, obesity, and insulin resistance. Diabetes 2015; 64:1522-1531. [PMID: 25422106 DOI: 10.2337/db14-0979] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/25/2014] [Accepted: 11/18/2014] [Indexed: 02/07/2023]
Abstract
Accumulation evidence links obesity-induced inflammation as an important contributor to the development of insulin resistance, which plays a key role in the pathophysiology of obesity-related diseases such as type 2 diabetes and nonalcoholic fatty liver disease. Cyclooxygenase (COX)-1 and -2 catalyze the first step in prostanoid biosynthesis. Because adult hepatocytes fail to induce COX-2 expression regardless of the proinflammatory stimuli used, we have evaluated whether this lack of expression under mild proinflammatory conditions might constitute a permissive condition for the onset of insulin resistance. Our results show that constitutive expression of human COX-2 (hCOX-2) in hepatocytes protects against adiposity, inflammation, and, hence, insulin resistance induced by a high-fat diet, as demonstrated by decreased hepatic steatosis, adiposity, plasmatic and hepatic triglycerides and free fatty acids, increased adiponectin-to-leptin ratio, and decreased levels of proinflammatory cytokines, together with an enhancement of insulin sensitivity and glucose tolerance. Furthermore, hCOX-2 transgenic mice exhibited increased whole-body energy expenditure due in part by induction of thermogenesis and fatty acid oxidation. The analysis of hepatic insulin signaling revealed an increase in insulin receptor-mediated Akt phosphorylation in hCOX-2 transgenic mice. In conclusion, our results point to COX-2 as a potential therapeutic target against obesity-associated metabolic dysfunction.
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Affiliation(s)
- Daniel E Francés
- Institute of Experimental Physiology (Instituto de Fisiología Experimental), Consejo Nacional de Investigaciones Científicas y Técnicas, Rosario, Argentina
| | - Omar Motiño
- Institute of Biomedical Research Alberto Sols, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain
| | - Noelia Agrá
- Institute of Biomedical Research Alberto Sols, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain
| | - Águeda González-Rodríguez
- Institute of Biomedical Research Alberto Sols, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain CIBERDEM, Instituto de Salud Carlos III, Madrid, Spain
| | - Ana Fernández-Álvarez
- Institute of Experimental Physiology (Instituto de Fisiología Experimental), Consejo Nacional de Investigaciones Científicas y Técnicas, Rosario, Argentina
| | - Carme Cucarella
- Biomedical Institute of Valencia, Instituto de Biomedicina de Valencia-Consejo Superior de Investigaciones Científicas, Valencia, Spain
| | - Rafael Mayoral
- CIBERehd, Instituto de Salud Carlos III, Madrid, Spain Department of Medicine, University of California, San Diego, La Jolla, CA
| | - Luis Castro-Sánchez
- Institute of Biomedical Research Alberto Sols, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain
| | - Ester García-Casarrubios
- Institute of Biomedical Research Alberto Sols, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain
| | - Lisardo Boscá
- Institute of Biomedical Research Alberto Sols, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain CIBERehd, Instituto de Salud Carlos III, Madrid, Spain
| | - Cristina E Carnovale
- Institute of Experimental Physiology (Instituto de Fisiología Experimental), Consejo Nacional de Investigaciones Científicas y Técnicas, Rosario, Argentina
| | - Marta Casado
- Biomedical Institute of Valencia, Instituto de Biomedicina de Valencia-Consejo Superior de Investigaciones Científicas, Valencia, Spain CIBERehd, Instituto de Salud Carlos III, Madrid, Spain
| | - Ángela M Valverde
- Institute of Biomedical Research Alberto Sols, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain CIBERDEM, Instituto de Salud Carlos III, Madrid, Spain
| | - Paloma Martín-Sanz
- Institute of Biomedical Research Alberto Sols, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain CIBERehd, Instituto de Salud Carlos III, Madrid, Spain
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13
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Llorente-Izquierdo C, Mayoral R, Cucarella C, Grau C, Alvarez MS, Flores JM, García-Palencia P, Agra N, Castro-Sánchez L, Boscá L, Martín-Sanz P, Casado M. Progression of liver oncogenesis in the double transgenic mice c-myc/TGF α is not enhanced by cyclooxygenase-2 expression. Prostaglandins Other Lipid Mediat 2013; 106:106-115. [PMID: 23579063 DOI: 10.1016/j.prostaglandins.2013.03.006] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2013] [Revised: 03/19/2013] [Accepted: 03/28/2013] [Indexed: 02/07/2023]
Abstract
Cyclooxygenase-2 (COX-2) has been associated with cell growth regulation, tissue remodeling and carcinogenesis. Overexpression of COX-2 in hepatocytes constitutes an ideal condition to evaluate the role of prostaglandins (PGs) in liver pathogenesis. The effect of COX-2-dependent PGs in genetic hepatocarcinogenesis has been investigated in triple c-myc/transforming growth factor α (TGF-α) transgenic mice that express human COX-2 in hepatocytes on a B6CBAxCD1xB6DBA2 background. Analysis of the contribution of COX-2-dependent PGs to the development of hepatocarcinogenesis, evaluated in this model, suggested a minor role of COX-2-dependent prostaglandins to liver oncogenesis as indicated by liver histopathology, morphometric analysis and specific markers of tumor progression. This allows concluding that COX-2 is insufficient for modifying the hepatocarcinogenesis course mediated by c-myc/TGF-α.
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Affiliation(s)
- Cristina Llorente-Izquierdo
- Instituto de Investigaciones Biomédicas Alberto Sols, CSIC-UAM Madrid, Arturo Duperier, 4, 28029 Madrid, Spain
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14
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Francés DEA, Ingaramo PI, Mayoral R, Través P, Casado M, Valverde ÁM, Martín-Sanz P, Carnovale CE. Cyclooxygenase-2 over-expression inhibits liver apoptosis induced by hyperglycemia. J Cell Biochem 2013; 114:669-80. [PMID: 23059845 DOI: 10.1002/jcb.24409] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2012] [Accepted: 09/24/2012] [Indexed: 12/22/2022]
Abstract
Increased expression of COX-2 has been linked to inflammation and carcinogenesis. Constitutive expression of COX-2 protects hepatocytes from several pro-apoptotic stimuli. Increased hepatic apoptosis has been observed in experimental models of diabetes. Our present aim was to analyze the role of COX-2 as a regulator of apoptosis in diabetic mouse liver. Mice of C57BL/6 strain wild type (Wt) and transgenic in COX-2 (hCOX-2 Tg) were separated into Control (vehicle) and SID (streptozotocin induced diabetes, 200 mg/kg body weight, i.p.). Seven days post-injection, Wt diabetic animals showed a decrease in PI3K activity and P-Akt levels, an increase of P-JNK, P-p38, pro-apoptotic Bad and Bax, release of cytochrome c and activities of caspases-3 and -9, leading to an increased apoptotic index. This situation was improved in diabetic COX-2 Tg. In addition, SID COX-2 Tg showed increased expression of anti-apoptotic Mcl-1 and XIAP. Pro-apoptotic state in the liver of diabetic animals was improved by over-expression of COX-2. We also analyzed the roles of high glucose-induced apoptosis and hCOX-2 in vitro. Non-transfected and hCOX-2-transfected cells were cultured at 5 and 25 mM of glucose by 72 h. At 25 mM there was an increase in apoptosis in non-transfected cells versus those exposed to 5 mM. This increase was partly prevented in transfected cells at 25 mM. Moreover, the protective effect observed in hCOX-2-transfected cells was suppressed by addition of DFU (COX-2 selective inhibitor), and mimicked by addition of PGE(2) in non-transfected cells. Taken together, these results demonstrate that hyperglycemia-induced hepatic apoptosis is protected by hCOX-2 expression.
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Affiliation(s)
- Daniel E A Francés
- Instituto de Fisiología Experimental (IFISE-CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina
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15
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Siegmund SV, Wojtalla A, Schlosser M, Zimmer A, Singer MV. Fatty acid amide hydrolase but not monoacyl glycerol lipase controls cell death induced by the endocannabinoid 2-arachidonoyl glycerol in hepatic cell populations. Biochem Biophys Res Commun 2013; 437:48-54. [PMID: 23806692 DOI: 10.1016/j.bbrc.2013.06.033] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2013] [Accepted: 06/11/2013] [Indexed: 12/21/2022]
Abstract
The endogenous cannabinoids anandamide (N-arachidonoylethanolamide, AEA) and 2-arachidonoyl glycerol (2-AG) are upregulated during liver fibrogenesis and selectively induce cell death in hepatic stellate cells (HSCs), the major fibrogenic cells in the liver, but not in hepatocytes. In contrast to HSCs, hepatocytes highly express the AEA-degrading enzyme fatty acid amide hydrolase (FAAH) that protects them from AEA-induced injury. However, the role of the major 2-AG-degrading enzyme monoacylglycerol lipase (MGL) in 2-AG-induced hepatic cell death has not been investigated. In contrast to FAAH, MGL protein expression did not significantly differ in primary mouse hepatocytes and HSCs. Hepatocytes pretreated with selective MGL inhibitors were not sensitized towards 2-AG-mediated death, indicating a minor role for MGL in the cellular resistance against 2-AG. Moreover, while adenoviral MGL overexpression failed to render HSCs resistant towards 2-AG, FAAH overexpression prevented 2-AG-induced death in HSCs. Accordingly, 2-AG caused cell death in hepatocytes pretreated with the FAAH inhibitor URB597, FAAH(-/-) hepatocytes, or hepatocytes depleted of the antioxidant glutathione (GSH). Moreover, 2-AG increased reactive oxygen species production in hepatocytes after FAAH inhibition, indicating that hepatocytes are more resistant to 2-AG treatment due to high GSH levels and FAAH expression. However, 2-AG was not significantly elevated in FAAH(-/-) mouse livers in contrast to AEA. Thus, FAAH exerts important protective actions against 2-AG-induced cellular damage, even though it is not the major 2-AG degradation enzyme in vivo. In conclusion, FAAH-mediated resistance of hepatocytes against endocannabinoid-induced cell death may provide a new physiological concept allowing the specific targeting of HSCs in liver fibrosis.
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16
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Fernández-Alvarez A, Llorente-Izquierdo C, Mayoral R, Agra N, Boscá L, Casado M, Martín-Sanz P. Evaluation of epigenetic modulation of cyclooxygenase-2 as a prognostic marker for hepatocellular carcinoma. Oncogenesis 2012; 1:e23. [PMID: 23552739 PMCID: PMC3412654 DOI: 10.1038/oncsis.2012.23] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2012] [Revised: 06/11/2012] [Accepted: 06/14/2012] [Indexed: 02/07/2023] Open
Abstract
Cyclooxygenases (COX-1 and 2) catalyze the first step in prostanoid biosynthesis. They are implicated in homeostatic processes with an important role in inflammation and carcinogenesis. In the liver, COX-2 expression is restricted to proliferation or dedifferentiation situations. The COX-2 promoter contains numerous CpG islands that, when hypermethylated, result in transcriptionally silencing thus regulating the growth of carcinoma cells. In this work, we investigated whether a correlation exists between COX-2 expression and methylation signatures at the 5'region of the gene in hepatoma cell lines and human hepatocellular carcinoma (HCC). We also examined the acetylation status of the COX-2 promoter and the effects of histone deacetylase (HDAC) inhibitors on COX-2 expression. Our results suggest a significant association between reduced COX-2 expression and promoter hypermethylation of COX-2 and histone deacetylation in some hepatoma cell lines and in HCC. Treatment with demethylating agents or HDAC inhibitors restored the expression of COX-2. Moreover, in an HCC cohort, a statistically significant inverse association was observed between COX-2 mRNA levels and promoter methylation. In agreement with these data, a reduction of overall survival of the patients was observed after decreased COX-2 expression by promoter hypermethylation and histone H3 hypoacetylation.
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Affiliation(s)
| | | | - R Mayoral
- Instituto de Investigaciones Biomédicas Alberto Sols, CSIC-UAM, Madrid, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain
| | - N Agra
- Instituto de Investigaciones Biomédicas Alberto Sols, CSIC-UAM, Madrid, Spain
| | - L Boscá
- Instituto de Investigaciones Biomédicas Alberto Sols, CSIC-UAM, Madrid, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain
| | - M Casado
- Instituto de Biomedicina de Valencia, IBV-CSIC, Valencia, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain
| | - P Martín-Sanz
- Instituto de Investigaciones Biomédicas Alberto Sols, CSIC-UAM, Madrid, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain
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17
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Rivero S, Ruiz-García A, Díaz-Guerra MJM, Laborda J, García-Ramírez JJ. Characterization of a proximal Sp1 response element in the mouse Dlk2 gene promoter. BMC Mol Biol 2011; 12:52. [PMID: 22185379 PMCID: PMC3296630 DOI: 10.1186/1471-2199-12-52] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2011] [Accepted: 12/20/2011] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND DLK2 is an EGF-like membrane protein, closely related to DLK1, which is involved in adipogenesis. Both proteins interact with the NOTCH1 receptor and are able to modulate its activation. The expression of the gene Dlk2 is coordinated with that of Dlk1 in several tissues and cell lines. Unlike Dlk1, the mouse Dlk2 gene and its locus at chromosome 17 are not fully characterized. RESULTS The goal of this work was the characterization of Dlk2 mRNA, as well as the analysis of the mechanisms that control its basal transcription. First, we analyzed the Dlk2 transcripts expressed by several mouse cells lines and tissues, and mapped the transcription start site by 5' Rapid Amplification of cDNA Ends. In silico analysis revealed that Dlk2 possesses a TATA-less promoter containing minimal promoter elements associated with a CpG island, and sequences for Inr and DPE elements. Besides, it possesses six GC-boxes, considered as consensus sites for the transcription factor Sp1. Indeed, we report that Sp1 directly binds to the Dlk2 promoter, activates its transcription, and regulates its level of expression. CONCLUSIONS Our results provide the first characterization of Dlk2 transcripts, map the location of the Dlk2 core promoter, and show the role of Sp1 as a key regulator of Dlk2 transcription, providing new insights into the molecular mechanisms that contribute to the expression of the Dlk2 gene.
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Affiliation(s)
- Samuel Rivero
- Facultad de Medicina/Centro Regional de Investigaciones Biomédicas, Universidad de Castilla-La Mancha, Calle Almansa 14, 02006 Albacete, Spain
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18
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Au AY, Hasenwinkel JM, Frondoza CG. Silybin inhibits interleukin-1β-induced production of pro-inflammatory mediators in canine hepatocyte cultures. J Vet Pharmacol Ther 2011; 34:120-9. [PMID: 21395602 DOI: 10.1111/j.1365-2885.2010.01200.x] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Hepatocytes are highly susceptible to cytokine stimulation and are fundamental to liver function. We established primary canine hepatocyte cultures to study effects of anti-inflammatory agents with hepatoprotective properties. Hepatocyte cultures were incubated with control media alone, silybin (SB), or the more bioavailable silybin-phosphatidylcholine complex (SPC), followed by activation with interleukin-1 beta (IL-1β; 10 ng/mL). Inflammatory response was measured by prostaglandin E2 (PGE(2) ), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1) production and also nuclear factor-kappa B (NF-κB) translocation. Hepatocyte cultures continued production of the phenotypic marker albumin for more than 7 days in culture. IL-1β exposure increased PGE(2) , IL-8, and MCP-1 production, which was paralleled by NF-κB translocation from the cytoplasm to the nucleus. Pretreatment with SB and SPC significantly inhibited IL-1β-induced production of pro-inflammatory markers and attenuated NF-κB nuclear translocation. We demonstrate for the first time that primary canine hepatocyte cultures can be maintained in culture without phenotypic loss. The observation that hepatocyte cultures respond to pro-inflammatory IL-1β activation indicates hepatocytes as primary cellular targets of extrinsic IL-1β. The ability of SB and SPC to inhibit hepatocyte culture activation by IL-1β reinforces the notion of their hepatoprotective effects. Our primary canine hepatocyte culture model facilitates identification of hepatoprotective agents and their mechanism of action.
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Affiliation(s)
- A Y Au
- Research and Development, Nutramax Laboratories, Inc., Edgewood, MD 21040, USA
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19
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Llorente Izquierdo C, Mayoral R, Flores JM, García-Palencia P, Cucarella C, Boscá L, Casado M, Martín-Sanz P. Transgenic mice expressing cyclooxygenase-2 in hepatocytes reveal a minor contribution of this enzyme to chemical hepatocarcinogenesis. THE AMERICAN JOURNAL OF PATHOLOGY 2011; 178:1361-1373. [PMID: 21356386 PMCID: PMC3069875 DOI: 10.1016/j.ajpath.2010.11.074] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/06/2010] [Revised: 10/21/2010] [Accepted: 11/23/2010] [Indexed: 02/07/2023]
Abstract
Cyclooxygenase-2 (COX-2) has been associated with cell growth regulation, tissue remodeling, and carcinogenesis. Ectopic expression of COX-2 in hepatocytes constitutes a nonphysiological condition ideal for evaluating the role of prostaglandins (PGs) in liver pathogenesis. The effect of COX-2-dependent PGs in chronic liver disease, hepatitis, fibrosis, and chemical hepatocarcinogenesis, has been investigated in transgenic (Tg) mice that express human COX-2 in hepatocytes and in Tg hepatic human cell lines. We have used three different complementary approaches: i) diethylnitrosamine (DEN)-induced chemical hepatocarcinogenesis in COX-2 Tg mice, ii) DEN/phenobarbital treatment of human COX-2 Tg hepatocyte-like cells, and iii) COX-2 Tg hepatocyte-like cells implants in nude mice. The data suggest that PGs produced by COX-2 in hepatocytes promoted mild hepatitis in 60-week-old mice, as assessed by histological examination, but failed to contribute to the development of liver fibrogenesis after methionine- and choline-deficient diet treatment. Moreover, liver injury, collagen content, and hepatic stellate cell activation were equally severe in wild-type and COX-2 Tg mice. The contribution of COX-2-dependent PGs to the development of DEN-induced hepatocarcinogenesis was evaluated in Tg mice, Tg hepatocyte-like cells, and nude mice and the analysis revealed that COX-2 expression favors the development of preneoplastic foci without affecting malignant transformation. Endogenous COX-2 expression in wild-type mice is a late event in the development of hepatocellular carcinoma.
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Affiliation(s)
| | - Rafael Mayoral
- Institute of Biomedical Research Alberto Sols (CSIC-UAM), Madrid, Spain
- Biomedical Research Centre Network of Hepatic and Digestive Diseases (CIBERehd), Barcelona, Spain
| | - Juana María Flores
- Department of Medicine and Animal Surgery, Veterinary Faculty, Complutense University, Madrid, Spain
| | - Pilar García-Palencia
- Department of Medicine and Animal Surgery, Veterinary Faculty, Complutense University, Madrid, Spain
| | - Carme Cucarella
- Institute of Biomedicine of Valencia (IBV-CSIC), Valencia, Spain
| | - Lisardo Boscá
- Institute of Biomedical Research Alberto Sols (CSIC-UAM), Madrid, Spain
- Biomedical Research Centre Network of Hepatic and Digestive Diseases (CIBERehd), Barcelona, Spain
| | - Marta Casado
- Biomedical Research Centre Network of Hepatic and Digestive Diseases (CIBERehd), Barcelona, Spain
- Institute of Biomedicine of Valencia (IBV-CSIC), Valencia, Spain
| | - Paloma Martín-Sanz
- Institute of Biomedical Research Alberto Sols (CSIC-UAM), Madrid, Spain
- Biomedical Research Centre Network of Hepatic and Digestive Diseases (CIBERehd), Barcelona, Spain
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20
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Ejarque-Ortiz A, Gresa-Arribas N, Straccia M, Mancera P, Solà C, Tusell JM, Serratosa J, Saura J. CCAAT/enhancer binding protein delta in microglial activation. J Neurosci Res 2010; 88:1113-23. [PMID: 19908286 DOI: 10.1002/jnr.22272] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
The transcription factor CCAAT/enhancer binding protein delta (C/EBP delta) regulates transcription of genes that play important roles in glial activation. Previous studies have shown the astroglial expression of C/EBP delta but the microglial expression of C/EBP delta remains virtually unexplored, with the exception of two microarray studies. In this report, using murine primary cultures and BV2 cells we clearly demonstrate that C/EBP delta is expressed by microglia and it is upregulated in microglial activation. Lipopolysaccharide upregulates C/EBP delta both in microglia and in astrocytes. This effect is time-dependent, with a maximum effect at 3 hr at mRNA level and at 4-8 hr at protein level, and concentration-dependent, with a maximum effect at 100 ng/mL. The lipopolysaccharide-induced C/EBP delta upregulation in BV2 microglia is mimicked by agonists of the toll-like receptors 2, 3 and 9 and can be prevented by an inhibitor of extracellular signal-regulated kinase activation. C/EBP delta from activated BV2 microglia binds to the cyclooxygenase-2 promoter and forms complexes with C/EBP beta isoforms. These results point to C/EBP delta as a putative key regulator of proinflammatory gene expression in microglial activation.
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Affiliation(s)
- Aroa Ejarque-Ortiz
- Department of Cerebral Ischaemia and Neurodegeneration, IIBB, CSIC, IDIBAPS, Barcelona, Spain
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21
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Martín-Sanz P, Mayoral R, Casado M, Boscá L. COX-2 in liver, from regeneration to hepatocarcinogenesis: what we have learned from animal models? World J Gastroenterol 2010; 16:1430-1435. [PMID: 20333781 PMCID: PMC2846246 DOI: 10.3748/wjg.v16.i12.1430] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/17/2009] [Revised: 01/15/2010] [Accepted: 01/22/2010] [Indexed: 02/06/2023] Open
Abstract
The use of animals lacking genes or expressing genes under the control of cell-specific promoters has significantly increased our knowledge of the genetic and molecular basis of physiopathology, allowing testing of functional hypotheses and validation of biochemical and pharmacologic approaches in order to understand cell function. However, with unexpected frequency, gene knockout animals and, more commonly, animal models of transgenesis give experimental support to even opposite conclusions on gene function. Here we summarize what we learned on the role of cyclooxygenase 2 (COX-2) in liver and revise the results obtained in 3 independent models of mice expressing a COX-2 transgene specifically in the hepatocyte. Upon challenge with pro-inflammatory stimuli, the animals behave very differently, some transgenic models having a protective effect but others enhancing the injury. In addition, one transgene exerts differential effects on normal liver physiology depending on the transgenic animal model used.
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22
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López-Fontal R, Zeini M, Través PG, Gómez-Ferrería M, Aranda A, Sáez GT, Cerdá C, Martín-Sanz P, Hortelano S, Boscá L. Mice lacking thyroid hormone receptor Beta show enhanced apoptosis and delayed liver commitment for proliferation after partial hepatectomy. PLoS One 2010; 5:e8710. [PMID: 20090848 PMCID: PMC2806828 DOI: 10.1371/journal.pone.0008710] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2009] [Accepted: 12/22/2009] [Indexed: 02/07/2023] Open
Abstract
BACKGROUND The role of thyroid hormones and their receptors (TR) during liver regeneration after partial hepatectomy (PH) was studied using genetic and pharmacologic approaches. Roles in liver regeneration have been suggested for T3, but there is no clear evidence distinguishing the contribution of increased amounts of T3 from the modulation by unoccupied TRs. METHODOLOGY/PRINCIPAL FINDINGS Mice lacking TRalpha1/TRbeta or TRbeta alone fully regenerated liver mass after PH, but showed delayed commitment to the initial round of hepatocyte proliferation and transient but intense apoptosis at 48h post-PH, affecting approximately 30% of the remaining hepatocytes. Pharmacologically induced hypothyroidism yielded similar results. Loss of TR activity was associated with enhanced nitrosative stress in the liver remnant, due to an increase in the activity of the nitric oxide synthase (NOS) 2 and 3, caused by a transient decrease in the concentration of asymmetric dimethylarginine (ADMA), a potent NOS inhibitor. This decrease in the ADMA levels was due to the presence of a higher activity of dimethylarginineaminohydrolase-1 (DDAH-1) in the regenerating liver of animals lacking TRalpha1/TRbeta or TRbeta. DDAH-1 expression and activity was paralleled by the activity of FXR, a transcription factor involved in liver regeneration and up-regulated in the absence of TR. CONCLUSIONS/SIGNIFICANCE We report that TRs are not required for liver regeneration; however, hypothyroid mice and TRbeta- or TRalpha1/TRbeta-deficient mice exhibit a delay in the restoration of liver mass, suggesting a specific role for TRbeta in liver regeneration. Altered regenerative responses are related with a delay in the expression of cyclins D1 and E, and the occurrence of liver apoptosis in the absence of activated TRbeta that can be prevented by administration of NOS inhibitors. Taken together, these results indicate that TRbeta contributes significantly to the rapid initial round of hepatocyte proliferation following PH, and improves the survival of the regenerating liver at later times.
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Affiliation(s)
| | - Miriam Zeini
- Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain
| | - Paqui G. Través
- Instituto de Investigaciones Biomédicas ‘Alberto Sols’ (CSIC-UAM), Madrid, Spain
| | | | - Ana Aranda
- Instituto de Investigaciones Biomédicas ‘Alberto Sols’ (CSIC-UAM), Madrid, Spain
| | - Guillermo T. Sáez
- Departamento de Bioquímica y Biología Molecular-Servicio de Análisis Clínicos, Hospital General Universitario, Valencia, Spain
| | - Concha Cerdá
- Departamento de Bioquímica y Biología Molecular-Servicio de Análisis Clínicos, Hospital General Universitario, Valencia, Spain
| | - Paloma Martín-Sanz
- Instituto de Investigaciones Biomédicas ‘Alberto Sols’ (CSIC-UAM), Madrid, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (Ciberehd), Barcelona, Spain
| | - Sonsoles Hortelano
- Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain
| | - Lisardo Boscá
- Instituto de Investigaciones Biomédicas ‘Alberto Sols’ (CSIC-UAM), Madrid, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (Ciberehd), Barcelona, Spain
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23
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Du ZY, Ma T, Winterthun S, Kristiansen K, Frøyland L, Madsen L. beta-oxidation modulates metabolic competition between eicosapentaenoic acid and arachidonic acid regulating prostaglandin E(2) synthesis in rat hepatocytes-Kupffer cells. Biochim Biophys Acta Mol Cell Biol Lipids 2010; 1801:526-36. [PMID: 20079880 DOI: 10.1016/j.bbalip.2010.01.002] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2009] [Revised: 12/29/2009] [Accepted: 01/06/2010] [Indexed: 01/27/2023]
Abstract
The ability of n-3 PUFA to competitively inhibit the use of arachidonic acid (AA) for membrane phospholipid synthesis and prostaglandin E(2) (PGE(2)) production has been well demonstrated in single cell models. In the present study, we investigated the metabolic competition between AA and eicosapentaenoic acid (EPA) for PGE(2) synthesis in a rat hepatocyte-Kupffer cell (HPC/KC) co-culture system when the cellular oxidation capacity was enhanced by exogenous l-carnitine. We demonstrate that in the absence of l-carnitine, 1) beta-oxidation rates of EPA and AA were comparable in HPCs and in KCs; 2) AA and not EPA was preferentially incorporated into glycerolipids; and 3) addition of EPA significantly decreased AA-dependent PGE(2) synthesis in HPCs and cyclooxygenase-2 (COX-2) expression in co-cultured HPCs/KCs. However, enhancing the cellular oxidation capacity by the addition of l-carnitine 1) significantly increased beta-oxidation of EPA in HPCs, but only marginally elevated the oxidation of AA in HPCs and the oxidation of both fatty acids in KCs; 2) decreased the esterification, but did not alter the preferential incorporation of AA into glycerolipids; and 3) alleviated the significant competitive inhibition of AA-dependent PGE(2) synthesis and COX-2 expression by EPA. Taken together, the results strongly suggest that l-carnitine affects competition between AA and EPA in PG synthesis in liver cells by enhancing oxidation of EPA in HPCs. This implies that the beneficial effects of n-3 PUFA, especially EPA, are affected by the cellular oxidation capacity.
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Affiliation(s)
- Zhen-Yu Du
- National Institute of Nutrition and Seafood Research (NIFES), P.O. Box 2029 Nordnes, N-5817 Bergen, Norway.
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24
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Han C, Li G, Lim K, DeFrances MC, Gandhi CR, Wu T. Transgenic expression of cyclooxygenase-2 in hepatocytes accelerates endotoxin-induced acute liver failure. THE JOURNAL OF IMMUNOLOGY 2008; 181:8027-35. [PMID: 19017995 DOI: 10.4049/jimmunol.181.11.8027] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Bacterial LPS (endotoxin) is implicated in the pathogenesis of acute liver failure and several chronic inflammatory liver diseases. To evaluate the effect of hepatocyte cyclooxygenase (COX)-2 in LPS-induced liver injury, we generated transgenic mice with targeted expression of COX-2 in the liver by using the albumin promoter-enhancer driven vector and the animals produced were subjected to a standard experimental protocol of LPS-induced acute fulminant hepatic failure (i.p. injection of low dose of LPS in combination with d-galactosamine (d-GalN)). The COX-2 transgenic mice exhibited earlier mortality, higher serum aspartate aminotransferase and alanine aminotransferase levels and more prominent liver tissue damage (parenchymal hemorrhage, neutrophilic inflammation, hepatocyte apoptosis, and necrosis) than wild-type mice. Western blot analysis of the liver tissues showed that LPS/d-GalN treatment for 4 h induced much higher cleavage of poly(ADP-ribose) polymerase, caspase-3, and caspase-9 in COX-2 transgenic mice than in wild-type mice. Increased hepatic expression of JNK-2 in COX-2 transgenic mice suggest that up-regulation of JNK-2 may represent a potential mechanism for COX-2-mediated exacerbation of liver injury. Blocking the prostaglandin receptor, EP(1), prevented LPS/d-GalN-induced liver injury and hepatocyte apoptosis in COX-2 transgenic mice. Accordingly, the mice with genetic ablation of EP(1) showed less LPS/d-GalN-induced liver damage and less hepatocyte apoptosis with prolonged survival when compared with the wild-type mice. These findings demonstrate that COX-2 and its downstream prostaglandin receptor EP(1) signaling pathway accelerates LPS-induced liver injury. Therefore, blocking COX-2-EP(1) pathway may represent a potential approach for amelioration of LPS-induced liver injury.
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Affiliation(s)
- Chang Han
- Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.
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25
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Mayoral R, Mollá B, Flores JM, Boscá L, Casado M, Martín-Sanz P. Constitutive expression of cyclo-oxygenase 2 transgene in hepatocytes protects against liver injury. Biochem J 2008; 416:337-346. [PMID: 18671671 DOI: 10.1042/bj20081224] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The effect of COX (cyclo-oxygenase)-2-dependent PGs (prostaglandins) in acute liver injury has been investigated in transgenic mice that express human COX-2 in hepatocytes. We have used three well-established models of liver injury: in LPS (lipopolysaccharide) injury in D-GalN (D-galactosamine)-preconditioned mice; in the hepatitis induced by ConA (concanavalin A); and in the proliferation of hepatocytes in regenerating liver after PH (partial hepatectomy). The results from the present study demonstrate that PG synthesis in hepatocytes decreases the susceptibility to LPS/D-GalN or ConA-induced liver injury as deduced by significantly lower levels of the pro-inflammatory profile and plasmatic aminotransferases in transgenic mice, an effect suppressed by COX-2-selective inhibitors. These Tg (transgenic) animals express higher levels of anti-apoptotic proteins and exhibit activation of proteins implicated in cell survival, such as Akt and AMP kinase after injury. The resistance to LPS/D-GalN-induced liver apoptosis involves an impairment of procaspase 3 and 8 activation. Protection against ConA-induced injury implies a significant reduction in necrosis. Moreover, hepatocyte commitment to start replication is anticipated in Tg mice after PH, due to the expression of PCNA (proliferating cell nuclear antigen), cyclin D1 and E. These results show, in a genetic model, that tissue-specific COX-2-dependent PGs exert an efficient protection against acute liver injury by an antiapoptotic/antinecrotic effect and by accelerated early hepatocyte proliferation.
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Affiliation(s)
- Rafael Mayoral
- Instituto de Investigaciones Biomédicas Alberto Sols Consejo Superior de Investigaciones Científicas, CSIC-UAM, Arturo Duperier 4, 28029 Madrid, Spain
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26
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Gong R, Latif S, Morris DJ, Brem AS. Co-localization of glucocorticoid metabolizing and prostaglandin synthesizing enzymes in rat kidney and liver. Life Sci 2008; 83:725-31. [DOI: 10.1016/j.lfs.2008.09.015] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2008] [Revised: 07/10/2008] [Accepted: 09/15/2008] [Indexed: 11/28/2022]
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Fernandez P, de Beer PM, van der Merwe L, Heyns CF. COX-2 promoter polymorphisms and the association with prostate cancer risk in South African men. Carcinogenesis 2008; 29:2347-50. [PMID: 18974063 DOI: 10.1093/carcin/bgn245] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Cyclooxygenase-2 (COX-2) converts arachidonic acid to prostaglandins, which are important mediators of cell proliferation and inflammation. Evidence indicates that COX-2 plays a role in carcinogenesis and that it is over-expressed in prostate tumours. We investigated the role of COX-2 variants in prostate cancer in a case-control study of South African Coloured men, consisting of 151 cases and 134 controls. The genotype frequencies of four single-nucleotide polymorphisms (SNPs) in the COX-2 promoter were initially determined in 50 control subjects. One SNP, rs20417 (-899G>C), was monomorphic and excluded from further investigation. Three SNPs, rs3918304 (-1285A>G), rs20415 (-1265C>T) and rs5270 (-297C>G), were genotyped in all the case patients and control subjects. The -1285 G-allele and -1265 T-allele were associated with increased risk of prostate cancer [odds ratio (OR) = 3.53; confidence interval (CI) = 2.14-5.90; P < 0.0001 and OR = 3.01; CI = 1.82-5.02; P < 0.0001] after adjusting for age. Haplotype GTC conferred increased risk of prostate cancer in South African Coloured men (OR = 3.54 versus ACC; CI = 2.12-5.92; P < 0.0001). These findings in conjunction with findings in other populations of African descent might suggest a common causal variant for prostate cancer in COX-2, or a variant in a nearby gene.
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Affiliation(s)
- Pedro Fernandez
- Department of Urology, Stellenbosch University, Tygerberg, South Africa.
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28
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Wu CC, Lin JC, Yang SC, Lin CW, Chen JJW, Shih JY, Hong TM, Yang PC. Modulation of the expression of the invasion-suppressor CRMP-1 by cyclooxygenase-2 inhibition via reciprocal regulation of Sp1 and C/EBPalpha. Mol Cancer Ther 2008; 7:1365-75. [PMID: 18524846 DOI: 10.1158/1535-7163.mct-08-0091] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Collapsin response mediator protein-1 (CRMP-1) controls neural development and axonal growth but also acts as a cancer invasion suppressor. In this study, we investigated the transcriptional regulation of CRMP-1 expression. Using a serial deletion strategy, we identified a basal promoter region between nucleotides -100 and -180 in the 5' flanking region of CRMP-1 (nucleotides -1,920 to +50) that contains multiple putative Sp1 and C/EBPalpha sites. Site-directed mutagenesis and deletion analysis revealed that the two C/EBPalpha sites, from nucleotides -122 to -133 and from nucleotides -101 to -113, are the most important regulatory elements. Gel-shift and antibody supershift assays showed that Sp1 protein was also present at this C/EBPalpha site, which overlaps with a Sp1 site. Overexpression of Sp1 decreased CRMP-1 promoter activity and protein expression, whereas overexpression of C/EBPalpha produced the opposite effect. Chromatin immunoprecipitation assays confirmed that Sp1 and C/EBPalpha compete for binding at the overlapping C/EBPalpha and Sp1 sites and reciprocally regulate CRMP-1 expression. Overexpression of cyclooxygenase-2 (COX-2) decreased CRMP-1 mRNA and protein expression. Conversely, the COX-2 inhibitor, celecoxib, induced a dose-dependent increase in CRMP-1 expression. COX-2 inhibition also decreased Sp1-DNA complex formation and inhibited cell invasion. We conclude that transcription of the invasion suppressor, CRMP-1, is reciprocally regulated at the promoter region by C/EBPalpha and Sp1. COX-2 inhibitors increase CRMP-1 expression by inhibiting Sp1-DNA complex formation and enhancing DNA binding of C/EBPalpha at the promoter.
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Affiliation(s)
- Cheng-Chung Wu
- Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
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Ejarque-Ortiz A, Tusell JM, Serratosa J, Saura J. CCAAT/enhancer binding protein-alpha is down-regulated by toll-like receptor agonists in microglial cells. J Neurosci Res 2007; 85:985-93. [PMID: 17253645 DOI: 10.1002/jnr.21195] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
The transcription factor CCAAT/enhancer binding protein-alpha (C/EBPalpha) can regulate the expression of important genes in the inflammatory response, but little is known about its role in glial activation. By using primary cortical murine glial cultures, we show that C/EBPalpha is expressed by microglial cells in vitro. Lipopolysaccharide (LPS) down-regulates C/EBPalpha mRNA at 2 hr and all C/EBPalpha protein isoforms at 4 hr. This effect is elicited by LPS concentrations >/=100 pg/ml. LPS-induced C/EBPalpha down-regulation occurs in microglial cells both in mixed glial and in microglial-enriched cultures. As seen with LPS, other toll-like receptor agonists (polyinosinic-polycytidylic acid, peptidoglycan from Staphylococcus aureus, and the oligonucleotide CpG1668) also down-regulate C/EBPalpha whereas cytokines such as interleukin-1beta, interleukin-6, macrophage-colony stimulating factor, and interferon-gamma do not. These findings suggest that C/EBPalpha down-regulation in activated microglia could play an important role in the increased expression of genes that are potentially pathogenic in a variety of neurological disorders.
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Affiliation(s)
- Aroa Ejarque-Ortiz
- Department of Pharmacology and Toxicology, IIBB-CSIC, IDIBAPS, Barcelona, Spain
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30
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Casado M, Mollá B, Roy R, Fernández-Martínez A, Cucarella C, Mayoral R, Boscá L, Martín-Sanz P. Protection against Fas-induced liver apoptosis in transgenic mice expressing cyclooxygenase 2 in hepatocytes. Hepatology 2007; 45:631-638. [PMID: 17326157 DOI: 10.1002/hep.21556] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
UNLABELLED Cyclooxygenase-2 (COX-2) is upregulated in many cancers, and the prostanoids synthesized increase proliferation, improve angiogenesis, and inhibit apoptosis in several tissues. To explore the function of COX-2 in liver, transgenic (Tg) mice were generated containing a fusion gene (LIVhCOX-2) consisting of human COX-2 cDNA under the control of the human ApoE promoter. Six lines were developed; all of them expressed the LIVhCOX-2 transgene selectively in hepatocytes. The Tg mice exhibited a normal phenotype, and the increased levels of PGE2 found were due to the constitutively expressed COX-2. Histological analysis of different tissues and macroscopic examination of the liver showed no differences between wild-type (Wt) and Tg animals. However, Tg animals were resistant to Fas-mediated liver injury, as demonstrated by low levels of plasmatic aminotransferases, a lesser caspase-3 activation, and Bax levels and an increase in Bcl-2, Mcl-1, and xIAP proteins, when compared with the Wt animals. Moreover, the resistance to Fas-mediated apoptosis is suppressed in the presence of COX-2-selective inhibitors, which prevented prostaglandin accumulation in the liver of Tg mice. CONCLUSION These results demonstrate that expression of COX-2-dependent prostaglandins exerted a protection against liver apoptosis.
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Affiliation(s)
- Marta Casado
- Instituto de Biomedicina de Valencia, IBV-CSIC, Valencia, Spain.
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31
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Pérez-Rodríguez R, Fuentes MP, Oliván AM, Martínez-Palacián A, Roncero C, González MP, Oset-Gasque MJ. Mechanisms of nitric oxide-induced apoptosis in bovine chromaffin cells: Role of mitochondria and apoptotic proteins. J Neurosci Res 2007; 85:2224-38. [PMID: 17523167 DOI: 10.1002/jnr.21342] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
The aim of this work was to establish the possible involvement of mitochondria in the apoptotic event triggered by nitric oxide (NO) in chromaffin cells. Using bovine chromaffin cells in primary culture and several NO donors (SNP, SNAP, and GSNO) at apoptotic concentrations (50 microM-1 mM), we have shown that NO induces a time-dependent decrease in the mitochondrial transmembrane potential (DeltaPsi(m)), which correlates with the appearance of hypodiploid cells. Disruption in DeltaPsi(m) is followed by cytochrome c release to the cytosol, which in turn precedes caspase 3 activation. In this mechanism participates the Bcl-2 protein family, because NO donors downregulate the expression of anti-apoptotic members of the family such as Bcl-2 and Bcl-XL, and increase the expression of pro-apoptotic members, Bax and Bcl-Xs, inductors of cytochrome c release to cytosol. Different cell signaling pathways seem to regulate Bax induction and Bcl-2 inhibition because decreased Bcl-2 levels are detected later than enhanced Bax expression. The tumour suppressor protein p53 is also upregulated in a very early phase (30 min) of the NO-induced apoptosis and may be responsible for the further induction of Bax expression. Finally, the translocation of NF-kappaB to the nucleus seems to be another early event in NO-induced apoptosis and it may be involved in the regulation of p53 expression. These results support strongly the participation of mitochondrial mechanisms in NO-induced apoptosis in chromaffin cells and suggest that these cells may be good models for the investigation of molecular basis of neurodegeneration and neuroprotection.
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Affiliation(s)
- Rocío Pérez-Rodríguez
- Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, Complutense University of Madrid, Madrid, Spain
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Abstract
Many epidemiological studies demonstrate that treatment with non-steroidal anti-inflammatory drugs (NSAIDs) reduce the incidence and mortality of certain malignancies, especially gastrointestinal cancer. The cyclooxygenase (COX) enzymes are well-known targets of NSAIDs. However, conventional NSAIDs non-selectively inhibit both the constitutive form COX-1, and the inducible form COX-2. Recent evidence indicates that COX-2 is an important molecular target for anticancer therapies. Its expression is undetectable in most normal tissues, and is highly induced by pro-inflammatory cytokines, mitogens, tumor promoters and growth factors. It is now well-established that COX-2 is chronically overexpressed in many premalignant, malignant, and metastastic cancers, including hepatocellular carcinoma (HCC). Overexpression of COX-2 in patients with HCC is generally higher in well-differentiated HCCs compared with less-differentiated HCCs or histologically normal liver, suggesting that COX-2 may be involved in the early stages of hepatocarcinogenesis, and increased expression of COX-2 in noncancerous liver tissue has been significantly associated with shorter disease-free survival in patients with HCC.
In tumors, overexpression of COX-2 leads to an increase in prostaglandin (PG) levels, which affect many mechanisms involved in carcinogenesis, such as angiogenesis, inhibition of apoptosis, stimulation of cell growth as well as the invasiveness and metastatic potential of tumor cells.
The availability of novel agents that selectively inhibit COX-2 (COXIB), has contributed to shedding light on the role of this molecule. Experimental studies on animal models of liver cancer have shown that NSAIDs, including both selective and non-selective COX-2 inhibitors, exert chemopreventive as well as therapeutic effects. However, the key mechanism by which COX-2 inhibitors affect HCC cell growth is as yet not fully understood.
Increasing evidence suggests the involvement of molecular targets other than COX-2 in the anti-proliferative effects of COX-2 selective inhibitors. Therefore, COX-inhibitors may use both COX-2-dependent and COX-2-independent mechanisms to mediate their antitumor properties, although their relative contributions toward the in vivo effects remain less clear.
Here we review the features of COX enzymes, the role of the expression of COX isoforms in hepatocarcinogenesis and the mechanisms by which they may contribute to HCC growth, the pharmacological properties of COX-2 selective inhibitors, the antitumor effects of COX inhibitors, and the rationale and feasibility of COX-2 inhibitors for the treatment of HCC.
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Abstract
Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer related mortality worldwide. The incidence of HCC is rising worldwide, especially in the United States. The overall survival of patients with HCC is grim and currently no efficient secondary prevention or systemic treatments are available. Recent evidence suggests that COX-2 signaling is implicated in hepatocarcinogenesis and COX-2 inhibitors prevent HCC cell growth in vitro and in animal models. However, given the recently reported side effect associated with some of the COX-2 inhibitors, it is imperative to develop chemotherapeutic strategy that simultaneously targets COX-2 and other related key molecules in hepatocarcinogenesis or to utilize agents inhibiting COX-2 signaling in conjunction with other standard chemotherapy or radiation therapy. Such combinational therapeutic approaches are expected to provide synergistic anti-tumor effect with lesser side effect. In this regard, the recently delineated interplay between COX-2-derived PG signaling and other growth-regulatory pathways such as EGFR, Met, iNOS, VEGF and n-3 polyunsaturated fatty acids is expected to provide important therapeutic implications. This review summarizes the recent advances in understanding the mechanisms for COX-2-derived PG signaling in hepatocarcinogenesis and focuses on the newly unveiled interactions between PG cascade and other key signaling pathways that coordinately regulate HCC growth. Understanding these mechanisms and interplays will facilitate the development of more effective chemopreventive and therapeutic strategies.
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Affiliation(s)
- Tong Wu
- Department of Pathology, University of Pittsburgh School of Medicine, MUH E-740, 200 Lothrop Street, Pittsburgh, PA 15213, USA.
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Halmos B, Bassères DS, Monti S, D'Aló F, Dayaram T, Ferenczi K, Wouters BJ, Huettner CS, Golub TR, Tenen DG. A Transcriptional Profiling Study of CCAAT/Enhancer Binding Protein Targets Identifies Hepatocyte Nuclear Factor 3β as a Novel Tumor Suppressor in Lung Cancer. Cancer Res 2004; 64:4137-47. [PMID: 15205324 DOI: 10.1158/0008-5472.can-03-4052] [Citation(s) in RCA: 60] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
We showed previously that CCAAT/enhancer binding protein alpha (C/EBP alpha), a tissue-specific transcription factor, is a candidate tumor suppressor in lung cancer. In the present study, we have performed a transcriptional profiling study of C/EBP alpha target genes using an inducible cell line system. This study led to the identification of hepatocyte nuclear factor 3beta (HNF3 beta), a transcription factor known to play a role in airway differentiation, as a downstream target of C/EBP alpha. We found down-regulation of HNF3 beta expression in a large proportion of lung cancer cell lines examined and identified two novel mutants of HNF3 beta, as well as hypermethylation of the HNF3 beta promoter. We also developed a tetracycline-inducible cell line model to study the cellular consequences of HNF3 beta expression. Conditional expression of HNF3 beta led to significant growth reduction, proliferation arrest, apoptosis, and loss of clonogenic ability, suggesting additionally that HNF3 beta is a novel tumor suppressor in lung cancer. This is the first study to show genetic abnormalities of lung-specific differentiation pathways in the development of lung cancer.
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Affiliation(s)
- Balazs Halmos
- Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Boston, USA
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35
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Núñez Martínez O, Clemente Ricote G, García Monzón C. [Role of cyclooxygenase-2 in the pathogenesis of chronic liver diseases]. Med Clin (Barc) 2004; 121:743-8. [PMID: 14678698 DOI: 10.1016/s0025-7753(03)74082-2] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
Abstract
Cyclooxygenase (COX) is a crucial enzyme in the biosynthesis of prostaglandins. There are two COX isoforms: COX-1 is constitutively expressed in a number of cell types and is involved in the homeostatic functions of prostaglandins, whereas COX-2 is inducible by a variety of proinflammatory stimuli, such as cytokines and lipopolysaccharide. In the liver, COX-2 and prostaglandins production has been implicated in hepatic regeneration, liver matrix remodeling and portal hypertension. In animal models of alcoholic-induced liver disease has been demonstrated its relation with necro-inflammatory activity. In viral hepatitis, hepatocellular COX-2 expression was observed and associated with fibrosis progression. More interestingly it has been the demonstration of COX-2 role in the development of hepatocellular carcinoma and cholangiocarcinoma, such in experimental models as in human samples. It has also been demonstrated that COX-2 was implicated in carcinogenesis through apoptosis inhibition and increased proliferation of human tumor cells. Experimental evidences show that selective pharmacologic inhibition of COX-2 could be useful in chemoprevention of primary liver tumors.
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Affiliation(s)
- Oscar Núñez Martínez
- Instituto de Hepatología Clínica-Experimental y Trasplante Hepático, Unidad Funcional Interhospitalaria Gregorio Marañón-Santa Cristina, Madrid, España
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Callejas NA, Fernández-Martínez A, Castrillo A, Boscá L, Martín-Sanz P. Selective inhibitors of cyclooxygenase-2 delay the activation of nuclear factor kappa B and attenuate the expression of inflammatory genes in murine macrophages treated with lipopolysaccharide. Mol Pharmacol 2003; 63:671-677. [PMID: 12606776 DOI: 10.1124/mol.63.3.671] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
The effect of rofecoxib, a selective cyclooxygenase-2 inhibitor, on inflammatory signaling has been investigated in elicited murine peritoneal macrophages. Macrophages treated with 10 microM rofecoxib exhibited an important inhibition in the early activation of nuclear factor kappa B (NF-kappa B) and the mitogen-activated protein kinase p38, the extracellular-regulated kinase p44, and the c-Jun N-terminal kinase. Moreover, this drug decreased the protein levels of nitric-oxide synthase-2 and cyclooxygenase-2 in lipopolysaccharide (LPS)-treated macrophages. Rofecoxib delayed and attenuated NF-kappa B activation, which impaired significantly the expression of kappa B-dependent genes. This drug and related coxibs did not affect cell viability and protected against LPS-induced apoptosis through the impairment of the inflammatory response. These data show an additional anti-inflammatory mechanism of selective cyclooxygenase-2 inhibitors through the attenuation of macrophage activation.
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Affiliation(s)
- Nuria A Callejas
- Instituto de Bioquímica, Centro Mixto Consejo Superior de Investigaciones Cientificas-Universidad Complutense de Madrid, Facultad de Farmacia, Universidad Complutense, Madrid, Spain
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Joseph SB, Castrillo A, Laffitte BA, Mangelsdorf DJ, Tontonoz P. Reciprocal regulation of inflammation and lipid metabolism by liver X receptors. Nat Med 2003; 9:213-9. [PMID: 12524534 DOI: 10.1038/nm820] [Citation(s) in RCA: 990] [Impact Index Per Article: 45.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2002] [Accepted: 12/20/2002] [Indexed: 02/08/2023]
Abstract
Macrophages have important roles in both lipid metabolism and inflammation and are central to the pathogenesis of atherosclerosis. The liver X receptors (LXRs) are established mediators of lipid-inducible gene expression, but their role in inflammation and immunity is unknown. We demonstrate here that LXRs and their ligands are negative regulators of macrophage inflammatory gene expression. Transcriptional profiling of lipopolysaccharide (LPS)-induced macrophages reveals reciprocal LXR-dependent regulation of genes involved in lipid metabolism and the innate immune response. In vitro, LXR ligands inhibit the expression of inflammatory mediators such as inducible nitric oxide synthase, cyclooxygenase (COX)-2 and interleukin-6 (IL-6) in response to bacterial infection or LPS stimulation. In vivo, LXR agonists reduce inflammation in a model of contact dermatitis and inhibit inflammatory gene expression in the aortas of atherosclerotic mice. These findings identify LXRs as lipid-dependent regulators of inflammatory gene expression that may serve to link lipid metabolism and immune functions in macrophages.
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Affiliation(s)
- Sean B Joseph
- Howard Hughes Medical Institute, Department of Pathology and Laboratory Medicine, University of California, Los Angeles, California, USA
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Ivanov AI, Pero RS, Scheck AC, Romanovsky AA. Prostaglandin E(2)-synthesizing enzymes in fever: differential transcriptional regulation. Am J Physiol Regul Integr Comp Physiol 2002; 283:R1104-17. [PMID: 12376404 DOI: 10.1152/ajpregu.00347.2002] [Citation(s) in RCA: 108] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The febrile response to lipopolysaccharide (LPS) consists of three phases (phases I-III), all requiring de novo synthesis of prostaglandin (PG) E(2). The major mechanism for activation of PGE(2)-synthesizing enzymes is transcriptional upregulation. The triphasic febrile response of Wistar-Kyoto rats to intravenous LPS (50 microg/kg) was studied. Using real-time RT-PCR, the expression of seven PGE(2)-synthesizing enzymes in the LPS-processing organs (liver and lungs) and the brain "febrigenic center" (hypothalamus) was quantified. Phase I involved transcriptional upregulation of the functionally coupled cyclooxygenase (COX)-2 and microsomal (m) PGE synthase (PGES) in the liver and lungs. Phase II entailed robust upregulation of all enzymes of the major inflammatory pathway, i.e., secretory (s) phospholipase (PL) A(2)-IIA --> COX-2 --> mPGES, in both the periphery and brain. Phase III was accompanied by the induction of cytosolic (c) PLA(2)-alpha in the hypothalamus, further upregulation of sPLA(2)-IIA and mPGES in the hypothalamus and liver, and a decrease in the expression of COX-1 and COX-2 in all tissues studied. Neither sPLA(2)-V nor cPGES was induced by LPS. The high magnitude of upregulation of mPGES and sPLA(2)-IIA (1,257-fold and 133-fold, respectively) makes these enzymes attractive targets for anti-inflammatory therapy.
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Affiliation(s)
- Andrei I Ivanov
- Trauma Research, Barrow Neurological Institute, St. Joseph's Hospital and Medical Center, Phoenix, Arizona 85013, USA
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Johnston DE. Hepatocyte prostaglandin synthesis and Mdr1 expression. Hepatology 2002; 36:510-1; author reply 511-2. [PMID: 12143066 DOI: 10.1053/jhep.2002.34289] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/07/2022]
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Tanabe T, Tohnai N. Cyclooxygenase isozymes and their gene structures and expression. Prostaglandins Other Lipid Mediat 2002; 68-69:95-114. [PMID: 12432912 DOI: 10.1016/s0090-6980(02)00024-2] [Citation(s) in RCA: 288] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
The cyclooxygenase (COX, prostaglandin endoperoxide synthase) is a key enzyme in prostaglandin biosynthesis. Two isoforms of COX, COX-1 and COX-2, have been identified by molecular biological methods. The amino acid sequence homology between COX-1 and COX-2 is about 60% for the human enzymes. COX-1 is constitutively expressed in most tissues and cells in animal species. The COX-1 promoter region lacks a canonical TATA or CAAT box and is GC-rich. These features are consistent with those of a housekeeping gene. On the other hand, COX-2 is an inducible enzyme and is induced by various cytokines and mitogenic factors. The induction of COX-2 is suppressed by dexamethasone and PGJ2. There are many consensus cis-elements in the 5'-flanking region to regulate the expression of COX-2. Among them, a CRE, an NF-kappaB site, a NF-IL6 motif and an E-box, regulate transcription independently or synergistically. Most of the transcriptional signaling pathways require activation of the mitogen-activated protein kinase (MAPK) cascade. Moreover, MAPK signaling pathways are involved in regulating COX-2 gene expression at the post-transcriptional level.
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Affiliation(s)
- Tadashi Tanabe
- Department of Pharmacology, National Cardiovascular Center Research Institute, Suita, Osaka, Japan.
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Tian G, Yu JP, Luo HS, Yu BP, Yue H, Li JY, Mei Q. Effect of Nimesulide on proliferation and apoptosis of human hepatoma SMMC-7721 cells. World J Gastroenterol 2002; 8:483-7. [PMID: 12046075 PMCID: PMC4656426 DOI: 10.3748/wjg.v8.i3.483] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: Cyclooxygenase-2 (COX-2) has been suggested to be associated with carcinogenesis. We sought to investigate the effect of the selective COX-2 inhibitor, Nimesulide on proliferation and apoptosis of SMMC-7721 human hepatoma cells.
METHODS: This study was carried out on the culture of hepatic carcinoma SMMC-7721 cell line. Various concentrations of Nimesulide (0, 200 μmol/L, 300 μmol/L, 400 μmol/L) were added and incubated. Cell proliferation was detected with MTT colorimetric assay, cell apoptosis by electron microscopy, flow cytometry and TUNEL.
RESULTS: Nimesulide could significantly inhibit SMMC-7721 cells proliferation dose-dependent and in a dependent manner compared with that of the control group. The duration lowest inhibition rate produced by Nimesulide in SMMC-7721 cells was 19.06%, the highest inhibition rate was 58.49%. After incubation with Nimesulide for 72 h, the most highest apoptosis rate and apoptosis index of SMMC-7721 cells comparing with those of the control were 21.20% ± 1.62% vs 2.24% ± 0.26% and 21.23 ± 1.78 vs 2.01 ± 0.23 (P < 0.05).
CONCLUSION: The selective COX-2 inhibitor, Nimesulide can inhibit the proliferation of SMMC-7721 cells and increase apoptosis rate and apoptosis index of SMMC-7721 cells. The apoptosis rate and the apoptosis index are dose-dependent. Under electron microscope SMMC-7721 cells incubated with 300 μmol and 400 μmol Nimesulide show apoptotic characteristics. With the clarification of the mechanism of selective COX-2 inhibitors, These COX-2 selective inhibitors can become the choice of prevention and treatment of cancers.
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Affiliation(s)
- Geng Tian
- Gastroenterology department. Renmin hospital of Wuhan university, 238 Jie-fang Road,Wuhan 430060,Hubei Province,China
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Wu YL, Wiltbank MC. Transcriptional regulation of the cyclooxygenase-2 gene changes from protein kinase (PK) A- to PKC-dependence after luteinization of granulosa cells. Biol Reprod 2002; 66:1505-14. [PMID: 11967217 DOI: 10.1095/biolreprod66.5.1505] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/01/2022] Open
Abstract
This study was designed to elucidate the molecular mechanism(s) mediating cyclooxygenase-2 (Cox-2) regulation during differentiation of the granulosa cell. The 5' flanking sequence of the Cox-2 gene was linked to a vector with a luciferase reporter gene, and this vector was transfected into freshly isolated bovine granulosa cells or granulosa cells after culture with or without forskolin to induce luteinization in vitro. The Cox-2 promoter was inducible by 8-bromo cAMP but not by phorbol esters in fresh granulosa cells, and maximal expression by cAMP was delayed until 48 h after treatment. In contrast, after luteinization of granulosa cells by 8-day treatment with forskolin, the Cox-2 promoter was immediately inducible by phorbol esters but not by cAMP. In granulosa cells cultured for 8 days without forskolin, the Cox-2 promoter continued to be inducible only by cAMP and not by phorbol esters. Unexpectedly, no delay was observed in the induction of Cox-2 by cAMP in granulosa cells that were cultured without forskolin, compared with an approximately 1 day delay in Cox-2 induction by cAMP in fresh granulosa cells. Myristoylated protein kinase (PK) A and PKC inhibitory peptides were utilized to further confirm the PKA- or PKC-dependence of Cox-2 induction. Time-course experiments showed that only 2 days of forskolin treatment could induce PKC-responsiveness of the Cox-2 promoter, although maximal responsiveness was not observed until 10 days of luteinization. Promoter activity was also analyzed in a series of deletion mutants as well as site-directed mutants of C/EBP, CRE, and E-box. A 282-base pair sequence in the Cox-2 5' flanking region maintained full inducibility by PKA in granulosa cells and by PKC in luteinized granulosa cells. The E-box element was found to be the critical regulatory element for Cox-2 induction by either PKA in granulosa cells or by PKC in luteinized granulosa cells. Electrophoretic mobility shift assays were performed on nuclear extracts from fresh or luteinized granulosa cells. Upstream stimulatory factor (USF)-1 and USF-2 bound to the E-box of the Cox-2 gene, and binding was similar for nuclear extracts from fresh, cultured, or luteinized granulosa cells. Thus, although luteinization changes transcriptional regulation of Cox-2 from PKA- to PKC-dependence, the crucial role of the E-box element in this transcriptional activation is conserved.
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Affiliation(s)
- Yuh-Lin Wu
- Endocrinology-Reproductive Physiology Program, University of Wisconsin-Madison, 1675 Observatory Drive, Madison, WI 53706, USA
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Ziemann C, Schäfer D, Rüdell G, Kahl GF, Hirsch-Ernst KI. The cyclooxygenase system participates in functional mdr1b overexpression in primary rat hepatocyte cultures. Hepatology 2002; 35:579-88. [PMID: 11870370 DOI: 10.1053/jhep.2002.31778] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
Overexpression of mdr1-type P-glycoproteins (P-gps) is thought to contribute to primary chemotherapy resistance of untreated hepatocellular carcinoma. However, mechanisms of endogenous multidrug resistance 1 (mdr1) gene activation still remain unclear. Because recent studies have demonstrated overexpression of cyclooxygenase-2 (COX-2) in hepatocytes during early stages of hepatocarcinogenesis, we investigated whether the COX system, which catalyzes the rate-limiting step in prostaglandin synthesis, participates in mdr1 gene regulation. In the present study, primary rat hepatocyte cultures, exhibiting time-dependent mdr1b overexpression, demonstrated basal COX-2 and COX-1 mRNA expression and liberation of prostaglandin E(2) (PGE(2)), indicative of an active COX-dependent arachidonic acid metabolism. PGE(2) accumulation in culture supernatants was further enhanced by arachidonic acid (1mumol/L) and epidermal growth factor (EGF) (16 nmol/L). PGE(2) and prostaglandin F(2alpha) (PGF(2)alpha) (3-6mug/mL), added directly to the culture medium, significantly up-regulated intrinsic mdr1b mRNA overexpression and mdr1-dependent transport activity. Up-regulation was maximal after 3 days of culture. Like prostaglandins, the COX substrate, arachidonic acid, also induced mdr1b gene expression. Apart from this, structurally different COX inhibitors (indomethacin, meloxicam, NS-398) mediated significant inhibition of time-dependent and EGF-induced mdr1b mRNA overexpression, resulting in enhanced intracellular accumulation of the mdr1 substrate, rhodamine 123 (Rho123). Thus, the present data support the conclusion that the release of prostaglandins through activation of the COX system participates in endogenous mdr1b gene regulation. COX-2 inhibition might constitute a new strategy to counteract primary mdr1-dependent chemotherapy resistance.
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Affiliation(s)
- Christina Ziemann
- Department of Toxicology, Institute of Pharmacology and Toxicology, University of Göttingen, Robert-Koch-Strasse 40, D-37075 Göttingen, Germany.
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Callejas NA, Casado M, Boscá L, Martín-Sanz P. Absence of nuclear factor kappaB inhibition by NSAIDs in hepatocytes. Hepatology 2002; 35:341-348. [PMID: 11826407 DOI: 10.1053/jhep.2002.31163] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Stimulation of fetal hepatocytes with proinflammatory cytokines and lipopolysaccharide promotes the expression of cyclooxygenase-2 (COX-2) and nitric oxide synthase-2 (NOS-2), whereas the hepatoma cell line HepG2 exhibits a behavior similar to that described for adult hepatocytes and only expresses NOS-2. The effect of nonsteroidal anti-inflammatory drugs (NSAIDs) on the inflammatory onset was analyzed in these cells since in addition to the inhibition of cyclooxygenase activity, these drugs interfere with other signaling pathways related with the inflammatory response. Inhibition of nuclear factor kappaB (NF-kappaB) activation by aspirin and salicylate has been described in many cells. However, incubation of hepatic cells with salicylate, aspirin, indomethacin, ibuprofen, or 5,5-dimethyl-3(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5H)-furanone (DFU), a fluorinated derivative of rofecoxib, failed to impair IkappaB kinase activity, the processing of NF-kappaB, and the expression of NF-kappaB-dependent genes, such as NOS-2. Moreover, selective COX-2 inhibitors did not promote apoptosis in hepatocytes under inflammatory conditions, suggesting that prostaglandins are not required to maintain cell viability. In conclusion, these data indicate that hepatocytes are not sensitive to NF-kappaB inhibition by NSAIDs and that these drugs, especially the COX-2 selective inhibitors, do not alter cell viability.
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Affiliation(s)
- Nuria A Callejas
- Instituto de Bioquímica, Centro Mixto CSIC-UCM, Facultad de Farmacia, Universidad Complutense, Madrid, Spain
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45
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Wu YL, Wiltbank MC. Transcriptional regulation of cyclooxygenase-2 gene in ovine large luteal cells. Biol Reprod 2001; 65:1565-72. [PMID: 11673276 DOI: 10.1095/biolreprod65.5.1565] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023] Open
Abstract
There is positive feedback pathway in the ovine large luteal cell, such that prostaglandin (PG) F(2 alpha) stimulation induces intraluteal PGF(2 alpha) production as the result of induction of one of the rate-limiting enzymes in PG production, cyclooxygenase-2 (Cox-2). The objective of the present study was to evaluate the intracellular effector systems and important DNA transcriptional element(s) involved in regulating the Cox-2 gene in ovine large luteal cells. In transient transfection assays, Cox-2 promoter was rapidly induced (4 h) by phorbol didecanoate (a protein kinase [PK] C activator), ionomycin, and cloprostenol (PGF(2 alpha) analogue), with a peak induction at 12 h. Cloprostenol-mediated promoter activation was not blocked by inhibition of various second messenger systems, including PKA, calcium calmodulin kinase II, or mitogen-activated protein kinases. However, myristoylated PKC pseudosubstrate peptide inhibited cloprostenol stimulation of Cox-2 promoter, indicating the critical role of PKC in this stimulation. The Cox-2 promoter could be reduced to 282 base pairs (bp) of the 5' flanking sequence with retention of full inducibility by cloprostenol. Mutation of three critical cis-responsive elements within this 282-bp region (C/EBP, cAMP responsive element [CRE], and E-box) indicated that E-box was critical in both basal and cloprostenol-induced promoter activity. However, there was also significant but less dramatic inhibition of cloprostenol stimulation by mutation of C/EBP and CRE in the Cox-2 promoter, and mutation of all three elements eliminated cloprostenol induction of this promoter. Electrophoretic mobility shift assays of nuclear extracts from large luteal cells revealed that upstream stimulatory factor (USF)-1 and USF-2 bound to the E-box in Cox-2. Thus, PKC directly regulates transcription of the Cox-2 gene in large luteal cells by acting through DNA elements close to the putative transcriptional start point, particularly an E-box region at -50 bp.
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Affiliation(s)
- Y L Wu
- Endocrinology-Reproductive Physiology Program and. Department of Dairy Science, University of Wisconsin, Madison, Wisconsin 53706, USA
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Casado M, Callejas NA, Rodrigo J, Zhao X, Dey SK, Boscá L, Martín-Sanz P. Contribution of cyclooxygenase 2 to liver regeneration after partial hepatectomy. FASEB J 2001; 15:2016-2018. [PMID: 11511527 DOI: 10.1096/fj.01-0158fje] [Citation(s) in RCA: 76] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
Partial hepatectomy (PH) triggers a rapid regenerative response in the remaining tissue to reinstate the organ function and the cell numbers. Among the molecules that change in the course of regeneration is an accumulation of prostaglandin E2 in the sera of rats with PH. Analysis of the cyclooxygenase (COX) isoenzymes in the remnant liver showed the preferential expression of COX-2 in hepatocytes. Cultured regenerating hepatocytes expressed significant levels of COX-2, a process that was not observed in the sham counterparts. Maximal expression of COX-2 was detected 16 h after PH with increased levels present even at 96 h. Pharmacological inhibition of COX-2 activity with NS398 shunted the up-regulation of cell proliferation after PH, which suggests a positive interaction of prostaglandins with the progression of the cell cycle. Similar results were obtained after PH of mice lacking the COX-2 gene. The expression of COX-2 in regenerating liver was concomitant with a decrease in CCAAT-enhancer binding protein (C/EBP-a) level and an increase in the expression of C/EBP-b and C/EBP-d. These results suggest a contribution of the enhanced synthesis of prostaglandins to liver regeneration observed after PH.
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Affiliation(s)
- M Casado
- Instituto de Bioquímica, Centro Mixto CSIC-UCM, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain
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Souto EO, Miyoshi H, Dubois RN, Gores GJ. Kupffer cell-derived cyclooxygenase-2 regulates hepatocyte Bcl-2 expression in choledocho-venous fistula rats. Am J Physiol Gastrointest Liver Physiol 2001; 280:G805-11. [PMID: 11292587 DOI: 10.1152/ajpgi.2001.280.5.g805] [Citation(s) in RCA: 22] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
We have previously demonstrated that after bile duct ligation hepatocytes express Bcl-2, although the mechanisms regulating Bcl-2 expression were not identified. Our aim was to determine if biliary constituents induce hepatocellular expression of Bcl-2 by a cyclooxygenase-2 (COX-2)-dependent mechanism. We used the choledocho-venous fistula (CVF) rat model for these studies and inhibited COX-2 by feeding the animals nimesulide, a selective inhibitor of COX-2 activity. Serum bile acids were 70-fold greater in CVF animals compared with controls, although liver histology and serum alanine aminotransferase values remained normal for the duration of the study. Neither Bcl-2 nor COX-2 was detected in sham-operated animals. However, Bcl-2 was expressed in hepatocytes but not in other liver cells in the CVF animals. In contrast, COX-2 protein was identified in Kupffer cells but not in hepatocytes of CVF animals. Hepatic Bcl-2 protein expression was fourfold lower in the livers from nimesulide-treated CVF rats. In conclusion, high circulating concentrations of biliary constituents are associated with stimulation of de novo hepatocyte expression of Bcl-2 and Kupffer cell expression of COX-2. These data suggest Kupffer cell-derived prostanoids may regulate Bcl-2 expression in the hepatocyte.
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Affiliation(s)
- E O Souto
- Division of Gastroenterology and Hepatology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA
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