3). To dissect whether intracellular OPN could play an autocrine role in modulating Collagen-I expression, HSCs were isolated from WT and Opn−/− mice. WT HSCs appeared more profibrogenic than Opn−/− HSCs, because intracellular Collagen-I expression at 7 days of culture was higher in WT HSCs than in Opn−/− HSCs (Fig. 1C). Infection of rat HSCs with an Ad-OPN increased intracellular Collagen-I and intra- and extracellular OPN, compared to HSCs infected with Ad-LacZ (Fig. 1D). Therefore, a novel autocrine role for intracellular OPN in modulating Collagen-I deposition was identified. Because OPN is also a soluble cytokine and a matrix-bound protein, we next evaluated
the role of extracellular OPN-mediated signaling (i.e., paracrine role) on Collagen-I induction in HSCs.
OPN signals via integrins—mostly integrin Quizartinib chemical structure αvβ3 highly expressed in HSCs21-23—and via CD44, also expressed in HSCs.24 Incubation with anti-αvβ3 integrin blocked the rOPN-driven DAPT total Collagen-I (intra- plus extracellular) increase in rat HSCs, whereas no major effect was observed by anti-CD44 (Fig. 2A). Neutralization of other integrins (i.e., β1, β5 and β6) failed to prevent the increase in Collagen-I by rOPN (not shown). Similar results were observed in human HSCs (not shown). Given that Collagen-I protein is highly responsive to oxidant stress-sensitive kinases, we analyzed the expression of protein kinases involved in regulating Collagen-I expression, such as phosphorylated p38 (pp38),25, 26 phosphorylated extracellular signal-related kinase (pERK1/2),27 pJNK,28, 29 phosphoinositide 3-kinase (PI3K), and phosphorylated Akt (pAkt).26, 30 Only PI3K and the ratio of pAkt 473Ser/Akt were elevated time dependently by rOPN up to 3 hours in rat HSCs (Fig.
2B) and up to 1 hour in human HSCs (Supporting Fig. 4A). Because PI3K/pAkt are upstream of I kappa B kinase (IKK) and the IKK complex is central for the activation of nuclear factor kappa B (NFκB) to regulate Collagen-I,26, 31 we focused on analyzing this signaling Non-specific serine/threonine protein kinase pathway. There was up-regulation of the ratios pIKKα,β 176/180Ser/IKKα,β, pIκBα 32Ser/IκBα as well as of nuclear/cytosolic p65 in OPN-treated rat HSCs (Fig. 2C). However, involvement of the target of rapamycin/70-kDa ribosomal protein S6 kinase (mTOR-p70S6K) cascade, a translational regulatory mechanism downstream of PI3K and pAkt 473Ser for regulating Collagen-I,32 was precluded, because rOPN neither altered mTOR and p706SK expression (Fig. 2D) nor induced mTOR phosphorylation at 2448Ser or 2481Ser in rat HSCs (undetectable). To further define the molecular mechanism for Collagen-I induction under rOPN challenge, we evaluated the potential role of the activation of these two stress-sensitive kinases (i.e., PI3K and pAkt) and of the NFκB-signaling pathway.