Sp1 and Sp3 transcription factors mediate leptin-induced collagen α1(I) gene expression in primary culture of male rat hepatic stellate cells.

Mechanisms by which leptin stimulates collagen α(1)(I) [Col1a(I)] gene expression are unclear. The purposes of this study were to identify the trans-acting factors and cis-acting elements in Col1a(I) promoter involved in this effect as well as the pathways that are implicated. In primary cultures of rat hepatic stellate cells (HSCs), we measured the effects of leptin on Col1a(I) gene and protein expression and on the binding of nuclear proteins to the Col1a(I) promoter. We found that leptin increased Col1a(I) gene and protein expression in activated HSCs. Transient transfections showed that leptin exerted its effects through elements located between -220 and -112 bp of the Col1a(I) promoter. Gel retardation assays demonstrated that leptin induced the binding of transcription factors specific protein (Sp)-1 and Sp3 to two elements located between -161 and -110 bp of the Col1a(I) promoter. Leptin-induced Sp1/Sp3 phosphorylation, but this effect was suppressed by inhibiting or silencing Janus kinase-2, phosphatidylinositol-3-kinase, nonphagocytic adenine dinucleotide phosphate (NADPH) oxidase, or ERK1/2, by the use of antioxidants or catalase, or by preventing protein-aldehyde adduct formation. Leptin provoked oxidative stress, aldehyde-protein adduct formation, and increased gene expression of some components of the NADPH oxidase complex. In conclusion, in HSCs, leptin up-regulates Col1a(I) gene expression after activating NADPH oxidase, inducing oxidative stress, aldehyde-protein adduct formation, and ERK1/2 phosphorylation, which in turn activates Sp1/Sp3 and provokes the binding of these two factors to regulatory elements located between -161 and -110 bp of the Col1a(I) promoter. These findings may contribute to a better understanding of mechanisms involved in the leptin-induced liver fibrosis.

Protein-tyrosine phosphatases are involved in interferon resistance associated with insulin resistance in HepG2 cells and obese mice.

Insulin resistance is a risk factor for non-response to interferon/ribavirin therapy in patients with chronic hepatitis C. The aim of this study was to determine the role played by protein-tyrosine phosphatases (PTPs) in the absence of interferon-α (IFNα) response associated with insulin resistance. We induced insulin resistance by silencing IRS-2 or by treating HepG2 cells with tumor necrosis factor-α (TNFα) and analyzed insulin response by evaluating Akt phosphorylation and IFNα response by measuring Stat-1 tyrosine phosphorylation and 2',5'-oligoadenylate synthase and myxovirus resistance gene expression. The response to IFNα was also measured in insulin-resistant obese mice (high fat diet and ob/ob mice) untreated and treated with metformin. Silencing IRS-2 mRNA induces insulin resistance and inhibits IFNα response. Likewise, TNFα suppresses insulin and IFNα response. Treatment of cells with pervanadate and knocking down PTP-1B restores insulin and IFNα response. Both silencing IRS-2 and TNFα treatment increase PTP and PTP-1B activity. Metformin inhibits PTP and improves IFNα response in insulin-resistant cells. Insulin-resistant ob/ob mice have increased PTP-1B gene expression and activity in the liver and do not respond to IFNα administration. Treatment with metformin improves this response. In HepG2 cells, insulin resistance provokes IFNα resistance, which is associated with an increased PTP-1B activity in the liver. Inhibition of PTP-1B activity with pervanadate and metformin or knocking down PTP-1B reestablishes IFNα response. Likewise, metformin decreases PTP-1B activity and improves response to IFNα in insulin-resistant obese mice. The use of PTP-1B inhibitors may improve the response to IFNα/ribavirin therapy.

Melatonin improves mitochondrial respiratory chain activity and liver morphology in ob/ob mice.

In previous studies, we have shown that mitochondrial respiratory chain (MRC) activity is decreased in patients with nonalcoholic steatohepatitis and in ob/ob mice and that peroxynitrite plays a pathogenic role. The present study examined whether melatonin, a peroxynitrite scavenger, prevents: (i) the in vitro effects of peroxynitrite on normal mitochondrial proteins and (ii) the development of nonalcoholic liver disease, MRC dysfunction and proteomic changes found in the mitochondrial complexes from ob/ob mice. We studied MRC activity, assembly of mitochondrial complexes and its subunits in normal mitochondrial proteins exposed to peroxynitrite in the absence and presence of melatonin. The same studies were done in mitochondrial proteins from ob/ob mice untreated and treated with melatonin. Preincubation of mitochondrial proteins from wild-type mice with melatonin prevented 3-tyrosine nitration of these proteins, eliminated the reduction in the MRC activity, the defect in the assembly of mitochondrial complexes and degradation of their subunits induced by peroxynitrite in vitro. Moreover, treatment of ob/ob mice with 10 mg/kg/day melatonin for 12 wk reduced oxidative and nitrosative stress, prevented the loss of MRC activity, protected their complexes and subunits from degradation, and favored assembling of mitochondrial complexes. In addition, this treatment improved fatty liver, decreased hepatic triglyceride concentration and increased apolipoprotein B100 in liver tissue. In conclusion, melatonin prevents the effects of peroxynitrite on mitochondrial proteins in vitro and administration of melatonin to ob/ob mice normalizes liver morphology, mitochondrial dysfunction and assembly of MRC complexes.

Fibronectin increases survival of rat hepatic stellate cells--a novel profibrogenic mechanism of fibronectin.

UNLABELLED: The aims of this study were to determine whether fibronectin increases survival of hepatic stellate cells (HSCs) in starving conditions, and to identify the signal transduction pathways involved in this effect. METHODS: Primary culture of rat HSCs were plated on fibronectin-uncoated or coated culture wells, and grown in the presence of 0.2% or 20% fetal calf serum. Cell apoptosis was measured by an ELISA procedure. Signal transduction pathways were analyzed by inhibiting major intracellular transduction pathways with appropriated inhibitors and by detecting phosphorylated proteins. RESULTS: Fibronectin increased survival of serum deprived HSCs. This effect was abrogated by the presence of the RGD peptide, by silencing FAK expression, and by inhibiting PI3K with LY294002 or wortmannin. Growth of HSCs on fibronectin induced integrin alpha5beta1 expression, tyr397, ser473, and ser136 phosphorylation of FAK, Akt, and Bad, respectively, and the binding of phosphorylated Bad to 14-3-3 proteins. Likewise, fibronectin increased Bcl2/Bax ratio and reduced release of mitochondrial cytochrome c into the cytoplasm, formation of apoptosome, and caspase 9 and 3 activity. These effects were avoided by treatment of cells with PI3K inhibitors. CONCLUSION: Fibronectin increases survival of HSCs via a pathway involving integrin alpha5beta1 receptors, FAK, PI3K, Akt and proteins of Bcl2 family.