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TGF-beta down regulates factor VII gene promoter by inhibiting the binding of HNF-4
Category 3: Molecular and cell biology (gene expression, signalling, fibrosis) an anti-CD81 neutralizing rnAb. The same effects were detected in human CD81 NIH3T3 transfected cells with identical time-course features. This time-dependency suggests that upregulation of MMP-2 expression occurs through transcriptional mechanisms. Indeed CD81, by acting as a molecular facilitator, could theoretically associate with membrane proteins involved in intracellular signaling. Accordingly, E2-CD81 binding on HSC was associated to a type II phosphatidylinositol 4-kinase (PI 4-K) activity. Therefore, human HSC could represent a novel potential cellular target for HCV within the liver: E2 engagement of CD81 on HSC surface leads to an increased expression of MMP-2, likely mediated by PI 4-K activation and phosphatidylinoside-intracellular signaling.
-~ ROLE OF TRANSFORMING GROWTH FACTOR B (TGF-B) IN HEPATIC FIBROSIS OF NON-ALCOHOLIC STEATOHEPATITIS (NASH) PATIENTS Javier Crespo, Amalia Cayon, Marta Mayorga, Agustin Dominguez-Diez, Manuel Hernandez-Guerra, Pedro Fernandez-Gil, Fernando Pons-Romero. Institute of Digestive Diseases, University
Hospital Valdecilla, School of Medicine, Santander, Spain Previous findings suggest that the TNF-alpha system may be involved in the pathogenesis of NASH. However, TNF-alpha has not been directly implicated in hepatic fibrosis. The aim of this work was to analyze the pathogenic role of TGF-beta in the development of hepatic fibrosis in NASH patients. Methods: Fifty subjects diagnosed of obesity were studied (NASH = 35 patients; non-NASH obese group = 15). The following aspects were investigated: TNF-alpha, p55, p75 and TGF-beta mRNA expression by quantitative RT-PCR in liver tissue. TGF-beta expression was related to the grade of hepatic fibrosis and the expression of TNF-alpha. Results: A remarkable increase of TGF-beta mRNA expression in patients with NASH was noticed in liver tissue (1.6 + 1.3 vs 0.4 + 0.4 in control group; p < 0.01). Furthermore, we have noticed a significant increase in mRNA levels of TNF receptor p55 (2.42 4- 1.81; vs 1.56 4- 1.17; p < 0.05). Those patients with significant fibrosis presented an increase in the expression of TGF-beta compared to those with a slight or non-existent fibrosis (p < 0.001). There was a close correlation between TGF-beta and TNF-alpha expression in liver tissue (p < 0.006). Conclusions: There is an overexpression of TGF-beta in NASH patients. This overexpression is more elevated in patients with more advanced fibrosis stages. There is a close correlation between TGF-beta and TNF-aipha expression. These findings suggest that the TNF-alpha system may be involved in the pathogenesis of NASH and that the expression of this cytokine may be upregulated by TGB-beta, one of the major modulator in hepatic fibrosis.
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TNF-alpha AND TNF-alpha RECEPTORS SERUM LEVELS DO NOT CORRELATE WITH GENE EXPRESSION OF TNF-alpha AND THEIR RECEPTORS IN THE NON-ALCOHOLIC STEATOHEPATITIS (NASH) PATIENTS
Amalia Cayon, Javier Crespo, Mafia Teresa Garcia-Unzueta, Juan Carlos Fernandez-Escalante, Marta Mayorga, Pedro Fernandez-Gil, Manuel Hernandez-Guerra, Fernando Pons-Romero. Inst. of Digestive
Diseases, Univ. Hospital Valdecilla, School of Medicine, Santander, Spain It has been reported that NASH patients have an overexpression of TNFalpha mRNA and TNF-alpha receptor p55; these findings suggest that the TNF-alpha system may be involved in the pathogenesis of NASH. The aim of this work was to analyze TNF-alpha, p55 and p75 levels in patients with NASH, and to correlate these findings with the gene expression of these molecules. Material and Methods: Sixty-seven subjects diagnosed of obesity were studied (NASH = 43; non-NASH = 24). The following aspects were investigated: 1) Gene expression of TNF-alpha, p55 and p75 by quantitative RT-PCR in liver tissue, 2) Serum levels of TNF-alpha, p55 and p75, 3) The
69
correlation between gene expression and TNF-alpha, p55 and p75 serum levels. Results: TNF-alpha levels were higher in the obese patients than in control subjects. Furthermore, the levels of TNF-alpha of patients with NASH compared with obese patients without NASH was similar (3.1 + 2.1 vs 3.2 + 1.4 pg/ml; p = NS). We found no differences between levels of p55 receptor (2.2 + 0.9 vs 2.5 + 1.5 ng/ml; p = NS) and p75 receptor (3.5 + 1.0 vs 3.7 + 0.8 ng/ml; p = NS) in the NAH patients compared with obese patients without NASH. These serum levels were compared to the gene expression of TNF-alpha and their receptors (data previously published) and we did not found any correlation. Conclusions: We reaffirmed the existence of an overexpression of TNFalpha and p55 in NASH patients; although we did not found any correlation between gene expression and serum levels.
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TGF-beta DOWNREGULATES FACTOR Vll GENE PROMOTER BY INHIBITING THE BINDING OF HNF-4
Susana De Lucas, Javier Bartolome, Cristina Navacerrada, Vicente Carreno. Fundacion Estudio Hepatitis Virales and Institute of
Hepatology, Hospital Pardo de Aravaca, Madrid, Spain Coagulation Factor VII (FVII) levels decrease during progression of chronic liver disease. We have shown that this decrease is not due to a loss of liver parenchyma cells but to a decay in the percentage of hepatocytes expressing FVII mRNA. It has been demonstrated that TGF-beta downregulates FVII mRNA levels in hepatoma cell lines. However it is not established if TGFobeta inhibits FVII gene expression at the transcriptional level. Furthermore, the molecular mechanism implicated in this inhibition is unknown. To study these issues a 616 pb fragment of the FVII gene promoter was amplified by PCR and cloned upstream of the CAT gene to generate the pVII-CAT plasmid. This fragment contains the binding sites for the SP1 and HNF-4 transcription factors (essentials for the FVII gene expression) and is able to drive the expression of a heterologous gene in a hepatocyte specific manner. HepG2 cells were transfected with the pVIICAT plasmid and treated with different concentrations of TGF-beta (from 5 to 80 ng/ml). CAT analysis demonstrated that TGF-beta downregulates FVII gene promoter in a dose-dependentmanner. Electrophoretic mobility shift assay using nuclear extracts from TGF-beta treated cells showed a decrease in HNF-4 binding capacity while SPI binding was not affected. In conclusion, our results suggest that TGF-beta downregulates FVII gene expression by inhibiting the binding of HNF-4 to its sequence in the promoter.
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DISTINCT PATTERN OF BIOLOGICAL EFFECTS ON IFN SIGNALING AND CELL TRANSFORMATION BETWEEN HCC TUMOUR AND NON-TUMOUR CELL-DERIVED CORE PROTEIN
Nadira Delhem, Abdelmajid Sabile, Carlo Giannini, Magali Moreau, Christian Brechot. Department of Liver Cancer and Molecular Virology,
lnserm U370, CHU Necker, Paris, France HCV core protein shows pleiotropic biological effects and has a potential role in HCV-related liver carcinogenesis. To overcome the difficulties in interpretating data obtained from ectopic expression of isolated core protein, our approach is based on a comparative analysis of HCV core natural mutants expressed from tumour and non-tumour-derived tissus from patients with HCC; we have recently validated this approach by reporting activation of PKR by tumour-derived, and not non-tumour derived core sequences (Delhem, Net al; oncogene, 2001). Methods: 1) Transient efficient (40-50%) expression in a human hepatocytic (HUH7) and Rat embryonic fibroblasts (REF) cells. Stable expression in a human hepatocytic (HUH7) and a rat fibroblastic (Ratl) cell lines. 2) Tests performed with three full lenght core sequences isolated from tumour (1) and non-tumour (2) liver section of a genotype lb-infected patient with HCC and a reference lb sequence (3).
-~ ROLE OF TRANSFORMING GROWTH FACTOR B (TGF-B) IN HEPATIC FIBROSIS OF NON-ALCOHOLIC STEATOHEPATITIS (NASH) PATIENTS Javier Crespo, Amalia Cayon, Marta Mayorga, Agustin Dominguez-Diez, Manuel Hernandez-Guerra, Pedro Fernandez-Gil, Fernando Pons-Romero. Institute of Digestive Diseases, University
Hospital Valdecilla, School of Medicine, Santander, Spain Previous findings suggest that the TNF-alpha system may be involved in the pathogenesis of NASH. However, TNF-alpha has not been directly implicated in hepatic fibrosis. The aim of this work was to analyze the pathogenic role of TGF-beta in the development of hepatic fibrosis in NASH patients. Methods: Fifty subjects diagnosed of obesity were studied (NASH = 35 patients; non-NASH obese group = 15). The following aspects were investigated: TNF-alpha, p55, p75 and TGF-beta mRNA expression by quantitative RT-PCR in liver tissue. TGF-beta expression was related to the grade of hepatic fibrosis and the expression of TNF-alpha. Results: A remarkable increase of TGF-beta mRNA expression in patients with NASH was noticed in liver tissue (1.6 + 1.3 vs 0.4 + 0.4 in control group; p < 0.01). Furthermore, we have noticed a significant increase in mRNA levels of TNF receptor p55 (2.42 4- 1.81; vs 1.56 4- 1.17; p < 0.05). Those patients with significant fibrosis presented an increase in the expression of TGF-beta compared to those with a slight or non-existent fibrosis (p < 0.001). There was a close correlation between TGF-beta and TNF-alpha expression in liver tissue (p < 0.006). Conclusions: There is an overexpression of TGF-beta in NASH patients. This overexpression is more elevated in patients with more advanced fibrosis stages. There is a close correlation between TGF-beta and TNF-aipha expression. These findings suggest that the TNF-alpha system may be involved in the pathogenesis of NASH and that the expression of this cytokine may be upregulated by TGB-beta, one of the major modulator in hepatic fibrosis.
~-]
TNF-alpha AND TNF-alpha RECEPTORS SERUM LEVELS DO NOT CORRELATE WITH GENE EXPRESSION OF TNF-alpha AND THEIR RECEPTORS IN THE NON-ALCOHOLIC STEATOHEPATITIS (NASH) PATIENTS
Amalia Cayon, Javier Crespo, Mafia Teresa Garcia-Unzueta, Juan Carlos Fernandez-Escalante, Marta Mayorga, Pedro Fernandez-Gil, Manuel Hernandez-Guerra, Fernando Pons-Romero. Inst. of Digestive
Diseases, Univ. Hospital Valdecilla, School of Medicine, Santander, Spain It has been reported that NASH patients have an overexpression of TNFalpha mRNA and TNF-alpha receptor p55; these findings suggest that the TNF-alpha system may be involved in the pathogenesis of NASH. The aim of this work was to analyze TNF-alpha, p55 and p75 levels in patients with NASH, and to correlate these findings with the gene expression of these molecules. Material and Methods: Sixty-seven subjects diagnosed of obesity were studied (NASH = 43; non-NASH = 24). The following aspects were investigated: 1) Gene expression of TNF-alpha, p55 and p75 by quantitative RT-PCR in liver tissue, 2) Serum levels of TNF-alpha, p55 and p75, 3) The
69
correlation between gene expression and TNF-alpha, p55 and p75 serum levels. Results: TNF-alpha levels were higher in the obese patients than in control subjects. Furthermore, the levels of TNF-alpha of patients with NASH compared with obese patients without NASH was similar (3.1 + 2.1 vs 3.2 + 1.4 pg/ml; p = NS). We found no differences between levels of p55 receptor (2.2 + 0.9 vs 2.5 + 1.5 ng/ml; p = NS) and p75 receptor (3.5 + 1.0 vs 3.7 + 0.8 ng/ml; p = NS) in the NAH patients compared with obese patients without NASH. These serum levels were compared to the gene expression of TNF-alpha and their receptors (data previously published) and we did not found any correlation. Conclusions: We reaffirmed the existence of an overexpression of TNFalpha and p55 in NASH patients; although we did not found any correlation between gene expression and serum levels.
~-~
TGF-beta DOWNREGULATES FACTOR Vll GENE PROMOTER BY INHIBITING THE BINDING OF HNF-4
Susana De Lucas, Javier Bartolome, Cristina Navacerrada, Vicente Carreno. Fundacion Estudio Hepatitis Virales and Institute of
Hepatology, Hospital Pardo de Aravaca, Madrid, Spain Coagulation Factor VII (FVII) levels decrease during progression of chronic liver disease. We have shown that this decrease is not due to a loss of liver parenchyma cells but to a decay in the percentage of hepatocytes expressing FVII mRNA. It has been demonstrated that TGF-beta downregulates FVII mRNA levels in hepatoma cell lines. However it is not established if TGFobeta inhibits FVII gene expression at the transcriptional level. Furthermore, the molecular mechanism implicated in this inhibition is unknown. To study these issues a 616 pb fragment of the FVII gene promoter was amplified by PCR and cloned upstream of the CAT gene to generate the pVII-CAT plasmid. This fragment contains the binding sites for the SP1 and HNF-4 transcription factors (essentials for the FVII gene expression) and is able to drive the expression of a heterologous gene in a hepatocyte specific manner. HepG2 cells were transfected with the pVIICAT plasmid and treated with different concentrations of TGF-beta (from 5 to 80 ng/ml). CAT analysis demonstrated that TGF-beta downregulates FVII gene promoter in a dose-dependentmanner. Electrophoretic mobility shift assay using nuclear extracts from TGF-beta treated cells showed a decrease in HNF-4 binding capacity while SPI binding was not affected. In conclusion, our results suggest that TGF-beta downregulates FVII gene expression by inhibiting the binding of HNF-4 to its sequence in the promoter.
~-~
DISTINCT PATTERN OF BIOLOGICAL EFFECTS ON IFN SIGNALING AND CELL TRANSFORMATION BETWEEN HCC TUMOUR AND NON-TUMOUR CELL-DERIVED CORE PROTEIN
Nadira Delhem, Abdelmajid Sabile, Carlo Giannini, Magali Moreau, Christian Brechot. Department of Liver Cancer and Molecular Virology,
lnserm U370, CHU Necker, Paris, France HCV core protein shows pleiotropic biological effects and has a potential role in HCV-related liver carcinogenesis. To overcome the difficulties in interpretating data obtained from ectopic expression of isolated core protein, our approach is based on a comparative analysis of HCV core natural mutants expressed from tumour and non-tumour-derived tissus from patients with HCC; we have recently validated this approach by reporting activation of PKR by tumour-derived, and not non-tumour derived core sequences (Delhem, Net al; oncogene, 2001). Methods: 1) Transient efficient (40-50%) expression in a human hepatocytic (HUH7) and Rat embryonic fibroblasts (REF) cells. Stable expression in a human hepatocytic (HUH7) and a rat fibroblastic (Ratl) cell lines. 2) Tests performed with three full lenght core sequences isolated from tumour (1) and non-tumour (2) liver section of a genotype lb-infected patient with HCC and a reference lb sequence (3).