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Stimulation of matrix metalloproteinase-9 in activated hepatic stellate cells is mediated by TNF through TNF receptor1

AuthorsTarrats, Núria ; Moles, Anna ; Morales, Albert ; Fernández-Checa, José C. ; Marí, Montserrat
Issue Date6-Sep-2010
CitationThe Liver Meeting (2010)
AbstractHepatic stellate cells (HSC) undergo a phenotypic transformation during their activation, and play a key role in liver pathologies and fibrosis. Matrix metalloproteinases (MMPs) participate in liver fibrogenesis and it is known that TNF-α can regulate their expression, but the signaling mechanism remains still unclear. The aim of this study was to analyse the role of TNFR1 or TNFR2 on the activation and fibrogenic potential of HSC using knockout mice for TNFR1, TNFR2 or both (TNFRDKO). Methods: Primary mouse HSC from wild type and KO mice were isolated and cultured for up to 14 days. Cell proliferation was determined by H3-thymidine incorporation into DNA. AKT, p-AKT, MMP-9 and PDGFβR were detected by western blot. TNFR1 and TNFR2 antagonism in the human HSC cell line LX2 was achieved using commercially available siRNAs and neutralizing antibodies. mRNA levels were assessed by quantitative real time RT-PCR. Gelatinolytic MMP-9 activity was detected by zymography assay. Results: While the absence of TNFR1 and TNFR2, as in TNFRDKO HSC, did not affect the expression of α-SMA or TGF-β during the activaction process, the levels of collagen type I mRNA and the global proliferation rate decreased in TNFRDKO as compared to wild type. In addition, activated TNFRDKO HSCs were unable to phosphorylate AKT in response to PDGF despite of similar PDGFβR expression compared to wild type HSC, indicating potential cross-talk between PDGFβR signaling and TNF receptors. Moreover, the role that MMP-9 plays in matrix replacement during the fibrogenic process is well established. Using HSC from knockout mice (TNFR1KO, TNFR2KO and TNFRDKO) we observed that while MMP-9 expression and gelatinolytic activity in WT and TNFR2KO HSC increased upon TNF-α stimualtion, this response was blunted in TNFRDKO and TNFR1KO HSC after TNF stimulation. These results where further confirmed in the human HSC cell line LX2 by using neutralizing antibodies against TNFR1, while TNFR2 antagonism did not prevent MMP-6 activation following TNF exposure. Conclusion: All together, these results point to TNFR1 as the key receptor for the profibrogenic action of TNF through MMP-9 activation, and suggest that TNF acts as a profibrogenic mediator in HSCs.
DescriptionPóster presentado en el 61th Annual Meeting of the American Association for the Study of Liver Diseases (2010), celebrado del 29 de octubre al 2 de noviembre de 2010 en Boston (Estados Unidos)
Appears in Collections:(IIBB) Comunicaciones congresos
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