Osteopontin, an oxidant stress sensitive cytokine, up-regulates collagen-I via integrin α(V)β(3) engagement and PI3K/pAkt/NFκB signaling

Abstract

A key feature in the pathogenesis of liver fibrosis is fibrillar Collagen-I deposition; yet, mediators that could be key therapeutic targets remain elusive. 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. 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. rOPN activated primary HSCs, confirmed by increased α-smooth muscle actin (αSMA) expression and enhanced their invasive and wound-healing potential. 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. 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. Neutralization of integrin α(v) β(3) prevented the OPN-mediated activation of the PI3K/pAkt/NFκB-signaling cascade and Collagen-I up-regulation. Likewise, inhibition of PI3K and NFκB blocked the OPN-mediated Collagen-I increase. Hepatitis C Virus (HCV) cirrhotic patients showed coinduction of Collagen-I and cleaved OPN compared to healthy individuals. Acute and chronic liver injury by CCl(4) injection or thioacetamide (TAA) treatment elevated OPN expression. Reactive oxygen species up-regulated OPN in vitro and in vivo and antioxidants prevented this effect. Transgenic mice overexpressing OPN in hepatocytes (Opn(HEP) Tg) mice developed spontaneous liver fibrosis compared to WT mice. Last, chronic CCl(4) injection and TAA treatment caused more liver fibrosis to WT than to Opn(-/-) mice and the reverse occurred in Opn(HEP) Tg mice.

Conclusion: OPN emerges as a key cytokine within the ECM protein network driving the increase in Collagen-I protein contributing to scarring and liver fibrosis.

Figure 1. Profibrogenic effects of rOPN in primary HSC

Primary rat HSC cultured for 7 days were treated with 0-50 nM rOPN for 6 and 24 hours. Western blot analysis of intra- and extracellular collagen-I, extracellular MMP13 and β-tubulin. Gelatine zymography showing extracellular pro-, intermediate and active MMP2 and MMP9 (A, left). Western blot analysis of intracellular collagen-I, α-SMA and actin in rat HSC treated with 0-50 nM rOPN for 24 hours (A, right). Human HSC cultured for 7 days were treated with 0-100 nM rOPN for 1 and 24 hours. Western blot analysis of intra- and extracellular collagen-I, extracellular MMP13 and MMP1 and β-tubulin. Pro-MMP2 activity was measured by gelatine zymography. Extracellular MMP9 activity was undetectable (not shown) (B). Western blot analysis of intracellular collagen-I and actin in HSC from WT and Opn^-/- mice. Extracellular collagen-I was undetectable (C). Western blot analysis of intracellular collagen-I and OPN as well as extracellular OPN in rat HSC infected with Ad-LacZ or with Ad-OPN for 48 hours. Extracellular collagen-I was undetectable (D). Results are expressed as average values. Experiments were performed in triplicates four times. **p<0.01 and ***p<0.001 for rOPN, Opn^-/- and Ad-OPN vs control, WT and Ad-LacZ, respectively.

Figure 2. Role of α_vβ₃ integrin and the PI3K-pAkt-NFκB signaling pathway in the rOPN-mediated effects on collagen-I

Primary rat HSC cultured for 7 days were incubated with 0-50 nM rOPN plus 5 μg/ml of non-immune IgG, anti-α_vβ₃ or anti-CD44 for 6 hours. Western blot analysis of intra- and extracellular collagen-I and actin (A). Western blot analysis of PI3K, pAkt ⁴⁷³Ser, Akt and β-tubulin up to 3 hours of 0-50 nM rOPN treatment in rat HSC (B). Western blot analysis of pIKKα,β ^176/180Ser, IKKα,β, pIκBα ³²Ser, IκBα, nuclear and cytosolic p65 and actin up to 30 min of 0-50 nM rOPN treatment in rat HSC (C). Western blot analysis of mTOR, p70S6K and actin up to 1 hour of 0-50 nM rOPN treatment in rat HSC (D). Results are expressed as average values. Experiments were performed in triplicates four times. *p<0.05, **p<0.01 and ***p<0.001 for rOPN-treated vs control at any given time-point. •p<0.05, ••p<0.01 and •••p<0.001 for co-treated vs Ab-treated.

Figure 3. Blocking α_vβ₃ integrin, PI3K-pAkt activation and the NFκB signaling pathway prevents the rOPN-mediated effects on collagen-I

Primary rat HSC cultured for 7 days were treated with 0-50 nM rOPN or co-treated with 0-10 μM wortmannin, 0-10 μM LY294002, 0-10 μM PDTC, 0-5 μM CAY10512 or with 5 μg/ml of non-immune IgG or a neutralizing Ab to integrin α_vβ₃. Western blot analysis showing that the rOPN-mediated induction of collagen-I in HSC was blunted by 0.1, 1 and 10 μM wortmannin (A, top), LY294002 (A, bottom), PDTC (B, top) and CAY10512 (B, middle). Infection of HSC with Ad-NFκB-Luc for 48 hours and treatment with 50 nM rOPN for 24 hours increased luciferase activity over that of non-treated Ad-NFκB-Luc-infected cells (B, bottom). Both, an integrin α_vβ₃ Ab and wortmannin blunted the rOPN-mediated induction of the ratios pIKKα,β ^176/180Ser/IKKα,β, pIκBα ³²Ser/IκBα and nuclear/cytosolic p65 (C). A neutralizing Ab to integrin α_vβ₃ prevented the induction of PI3K, the ratio pAkt ⁴⁷³Ser/Akt and collagen-I by rOPN in HSC (D). Results are expressed as average values. Experiments were performed in triplicates four times. **p<0.01 and ***p<0.001 for rOPN-treated vs control. •p<0.05, ••p<0.01 and •••p<0.001 for inhibitor or Ab-cotreated vs rOPN-treated or control.

Figure 4. OPN expression is induced during liver injury and under oxidant stress conditions

Western blot analysis of cleaved OPN, total collagen-I and actin in livers from control and from patients with stage-3 HCV-induced cirrhosis (A). Western blot analysis of cleaved OPN and actin in livers of mice injected with CCl₄ for 24 hours (acute liver injury), with CCl₄ for 1 month or with TAA for 4 months (chronic liver injury) (B). In (A) and (B) fully modified (glycosylated and phosphorylated) monomeric OPN was not detected. Immunocytochemistry for OPN in primary HSC isolated from WT mice and cultured for 6 days (C, left). IHC depicting significant OPN expression in HSC, biliary epithelial cells and hepatocytes at 1 month of CCl₄-injection (C, middle) and in HSC, biliary epithelial cells, oval cells and hepatocytes after 4 months of TAA-treatment (C, right). The insets show OPN⁺ HSC in both models (). Immunofluorescence showing co-localization of OPN⁺ with α-SMA⁺ (a HSC activation marker) after 4 months of TAA-treatment (D). Western blot analysis of intracellular OPN and β-tubulin in HSC in the presence of two prooxidants (H₂O₂ and BSO) and an antioxidant (GSH-EE: glutathione-ethyl ester) (E). Results are expressed as average values. Experiments were performed in triplicates four times. ***p<0.001 for HCV, CCl₄, TAA or prooxidant treated vs control, mineral oil (MO) or water, respectively. •••p<0.001 for BSO + GSH-EE co-treated vs BSO-treated.

Figure 5. SAM protects WT mice from CCl₄-induced chronic liver injury

C57BL/6J WT mice were injected MO, SAM plus MO, CCl₄ or CCl₄ plus SAM for 1 month. Co-treated mice showed decreased OPN expression (A), which was quantified by morphometry analysis (B). Likewise, fibrosis was less apparent in co-treated mice than in CCl₄-injected mice as shown by Sirius red/fast green staining (C) and morphometry analysis (D). Results are expressed as mean values ± SEM. n=8/group; ***p<0.001 for CCl₄ or CCl₄ + SAM vs MO or SAM; ••p<0.01 for CCl₄ + SAM vs CCl₄.

Figure 6. WT mice show more CCl₄-induced chronic liver injury than Opn^-/- mice

C57BL/6J WT and Opn^-/- mice were injected CCl₄ or MO for 1 month. H&E staining revealed more centrilobular necrosis (), centrilobular inflammation () and hepatocyte ballooning degeneration () in CCl₄-injected WT than in Opn^-/- mice (A). ALT activity (B), centrilobular and parenchymal inflammation scores (C), hepatocyte ballooning degeneration score (D) and centrilobular and parenchymal necrosis scores (E). A Western blot analysis showing similar CYP2E1 expression in WT and Opn^-/- mice (F). Results are expressed as mean values ± SEM. n=8/group; ***p<0.001 for CCl₄ vs MO; •p<0.05, ••p<0.01 and •••p<0.001 for Opn^-/- + CCl₄ vs WT + CCl₄.

Figure 7. WT mice show more CCl₄-induced liver fibrosis than Opn^-/- mice

C57BL/6J WT and Opn^-/- mice were injected CCl₄ or MO for 1 month. Sirius red/fast green staining indicated fibrosis stage ~3 in CCl₄-injected WT and ~1-2 in CCl -injected Opn^-/- 4 mice (portal and bridging fibrosis) as well as greater scar thickness in WT compared to Opn^-/- mice () (A). Collagen-I IHC confirmed the extent of portal fibrosis (), bridging fibrosis () and scar thickness () in CCl₄-injected mice (B). Brunt fibrosis score (C), Sirius red morphometry (D) and collagen-I morphometry analysis (E). Results are expressed as mean values ± SEM. n=8/group; **p<0.01 and ***p<0.001 for CCl₄ vs MO; ••p<0.01 and •••p<0.001 for Opn^-/- + CCl₄ vs WT + CCl₄.

Figure 8. Opn^HEP Tg mice in C57BL/6J genetic background show more CCl₄-induced fibrosis than WT mice

WT and Opn^HEP Tg mice were injected MO or CCl₄ for 1 month. H&E staining revealed similar centrilobular necrosis () and inflammation () in CCl₄-injected Opn^HEP Tg and in WT mice (A). ALT activity (B). Necrosis and inflammation scores (C). Sirius red/fast green staining and IHC for collagen-I demonstrated more portal fibrosis (), bridging fibrosis () and sinusoidal fibrosis () in CCl -injected Opn^HEP 4 Tg than in WT mice (D-E). Brunt fibrosis score, collagen-I and Sirius red/fast green morphometry (F). Results are expressed as mean values ± SEM. n=8/group; **p<0.01 and ***p<0.001 for CCl₄ vs MO; ••p<0.01 for Opn^HEP Tg + CCl₄ vs WT + CCl₄.

Figure 8. Opn^HEP Tg mice in C57BL/6J genetic background show more CCl₄-induced fibrosis than WT mice

WT and Opn^HEP Tg mice were injected MO or CCl₄ for 1 month. H&E staining revealed similar centrilobular necrosis () and inflammation () in CCl₄-injected Opn^HEP Tg and in WT mice (A). ALT activity (B). Necrosis and inflammation scores (C). Sirius red/fast green staining and IHC for collagen-I demonstrated more portal fibrosis (), bridging fibrosis () and sinusoidal fibrosis () in CCl -injected Opn^HEP 4 Tg than in WT mice (D-E). Brunt fibrosis score, collagen-I and Sirius red/fast green morphometry (F). Results are expressed as mean values ± SEM. n=8/group; **p<0.01 and ***p<0.001 for CCl₄ vs MO; ••p<0.01 for Opn^HEP Tg + CCl₄ vs WT + CCl₄.