Search

Background/aims: To determine the effects of IFNalpha on MMP-13 gene expression in primary culture of hepatic stellate cells., Methods: We measured MMP-13 mRNA, MMP-13 protein, MMP-13 luciferase activity, binding of AP1 and PEA3 to DNA, and binding of PEA3 to Jak1 and Stat1., Results: IFNalpha increased MMP-13 mRNA, MMP-13 protein, and luciferase activity in cells transfected either with a luciferase plasmid driven by the MMP-13 promoter or with the same plasmid in which the AP1 binding site has been mutated. IFNalpha induced the binding of nuclear proteins to a radiolabeled PEA3 probe, but not to a AP1 probe. Supershift assays demonstrated that PEA3 and Stat1 are implicated in the formation of this complex. Immunoprecipitation assays showed that PEA3 interacts physically with Stat1 and that IFNalpha treatment increases this interaction. Downregulation of PEA3 or JAK1 with appropriated siRNAs or mutation of the PEA3 binding site in the MMP-13 promoter abrogated the effects of IFNalpha on MMP-13 gene expression. Finally, IFNalpha induced the binding of PEA3 to JAK1, as well as PEA3 tyrosine and serine phosphorylation., Conclusions: IFNalpha determines the binding of PEA3 to JAK1 and its tyrosine phosphorylation. Activated PEA3 binds to MMP-13 promoter and activates its expression.

Background/aims: Circulating neutrophils from cirrhotic patients have a reduced capacity to generate superoxide anion (O(2)(-)), which might contribute to frequent bacterial infections in these patients. We studied the signal transduction pathways involved in the generation of O(2)(-) in neutrophils from 98 cirrhotic patients and 46 healthy controls., Methods: We measured O(2)(-) production in neutrophils induced by fMLP, opsonized zymosan, TNF alpha, NaF, AlF(4)(-), A23187 and phorbol myristate acetate. Furthermore, we measured phospholipase C activity in neutrophils from healthy controls and end-stage cirrhotic patients., Results: O(2)(-) production was decreased in neutrophils from patients in response to fMLP, opsonized zymosan and TNF alpha. Likewise, response of these cells to G-protein stimulation by fluorides was also decreased. These reduced responses correlated significantly with the degree of liver dysfunction. On the contrary, neutrophils from patients responded normally to A23187 and phorbol esters stimulation indicating that Ca(2+)- and PKC-dependent pathways are intact in these cells. Finally, phospholipase C activity was markedly reduced in neutrophils from end-stage liver cirrhosis., Conclusions: These data confirm that O(2)(-) generation by neutrophils is decreased in patients with cirrhosis, particularly in those with more severe liver dysfunction, and suggest that this defect involves phosphatidylinositol specific phospholipase C activity.