Hepatitis C Virus (HCV) cirrhotic patients showed coinduction of Collagen-I and cleaved OPN compared to healthy individuals. Likewise, inhibition of PI3K and NFκB blocked the OPN-mediated Collagen-I increase. Neutralization of integrin α(v) β(3) prevented the OPN-mediated activation of the PI3K/pAkt/NFκB-signaling cascade and Collagen-I up-regulation. OPN induction of Collagen-I occurred via integrin α(v)β(3) engagement and activation of the phosphoinositide 3-kinase/phosphorylated Akt/nuclear factor kappa B (PI3K/pAkt/NFκB)-signaling pathway, whereas cluster of differentiation 44 (CD44) binding and mammalian target of rapamycin/70-kDa ribosomal protein S6 kinase (mTOR/p70S6K) were not involved. HSCs isolated from wild-type (WT) mice were more profibrogenic than those from OPN knockout (Opn(-/-)) mice and infection of primary HSCs with an Ad-OPN increased Collagen-I, indicating correlation between both proteins. rOPN activated primary HSCs, confirmed by increased α-smooth muscle actin (αSMA) expression and enhanced their invasive and wound-healing potential.

Recombinant OPN (rOPN) up-regulated Collagen-I protein in primary HSCs in a transforming growth factor beta (TGFβ)-independent fashion, whereas it down-regulated matrix metalloprotease-13 (MMP13), thus favoring scarring. We hypothesized that osteopontin (OPN), an extracellular matrix (ECM) cytokine expressed in hepatic stellate cells (HSCs), could drive fibrogenesis by modulating the HSC pro-fibrogenic phenotype and Collagen-I expression.

Therefore, leucine exerts on hepatic stellate cells a prooxidant action through NADPH oxidase and mitochondrial Reactive oxygen species production and these effects mediate the activation of IR/IGF-IR and signaling pathways, finally leading to changes in translational regulation of collagen synthesis.Ī key feature in the pathogenesis of liver fibrosis is fibrillar Collagen-I deposition yet, mediators that could be key therapeutic targets remain elusive. Leucine also induced an increased phosphorylation of IR/IGF-R that was abolished by pretreatment with either rotenone or apocynin. The mitochondrial electron chain inhibitor rotenone and the NADPH oxidase inhibitor apocynin prevented reactive oxygen species production caused by leucine.

However, preincubation with glutathione prevented ERK, Akt and mTOR phosphorylation caused by treatment with leucine. Inhibitors for MEK-1 (PD98059), PI3K (wortmannin) or mTOR (rapamycin) did not affect leucine-induced reactive oxygen species production. Preincubation with glutathione also prevented 4E-BP1, eIF4E and Mnk-1 phosphorylation induced by leucine, as well as enhancement of procollagen alpha1(I) protein levels. Intracellular reactive oxygen species levels were increased in hepatic stellate cells incubated with leucine 5 mM at early time points, and this effect was abolished by pretreatment with the antioxidant glutathione. The aim of the present study was to evaluate the role played by reactive oxygen species on these effects. The amino acid leucine causes an increase of collagen alpha1(I) synthesis in hepatic stellate cells through the activation of translational regulatory mechanisms and PI3K/Akt/mTOR and ERK signaling pathways.

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