OSTEOPONTIN, AN OXIDANT STRESS-SENSITIVE CYTOKINE, UP-REGULATES COLLAGEN-I VIA INTEGRIN α_Vβ₃ ENGAGEMENT AND PI3K-pAkt-NFκB SIGNALING

Abstract

Background & Aim

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 (HSC), could drive fibrogenesis by modulating the HSC profibrogenic phenotype and collagen-I expression.

Results

rOPN up-regulated collagen-I protein in primary HSC in a TGFβ-independent fashion whereas it down-regulated matrix metalloprotease-13 (MMP13) thus favoring scarring. rOPN activated primary HSC -confirmed by increased α-smooth muscle actin (α-SMA) expression- and enhanced their invasive and wound-healing potential. HSC isolated from wild type (WT) mice were more profibrogenic than those from Opn^-/- mice and infection of primary HSC with an Ad-OPN increased collagen-I, indicating correlation between both proteins. The OPN induction of collagen-I occurred via integrin α_vβ₃ engagement and activation of the PI3K-pAkt-NFκB signaling pathway, while CD44-binding and mTOR-p70S6K were not involved. Neutralization of integrin α_vβ₃ 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. HCV-cirrhotic patients showed co-induction of collagen-I and cleaved OPN compared to healthy individuals. Acute and chronic liver injury by carbon tetrachloride (CCl₄)-injection or thioacetamide (TAA)-treatment elevated OPN expression. Reactive oxygen species up-regulated OPN in vitro and in vivo and antioxidants prevented this effect. Opn^HEP Tg mice developed spontaneous liver fibrosis compared to WT mice. Lastly, chronic CCl₄-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 cytokine within the ECM protein network driving the increase in collagen-I protein contributing to scarring and liver fibrosis.

Fibrogenesis, or activation of the wound-healing response to persistent liver injury, is characterized by changes in the composition and quantity of the ECM deposits distorting the normal hepatic architecture by forming fibrotic scars. Failure to degrade accumulated ECM is a major reason why fibrosis progresses to cirrhosis. Emerging antifibrogenic therapies aim at inhibiting the activation of profibrogenic cells to prevent fibrillar collagen-I deposition, at degrading the excessive ECM to recover the normal liver architecture and at restoring functional liver mass.

Although different cell types contribute to the increase in fibrillar collagen-I during hepatic fibrogenesis, they all undergo a common process of differentiation and acquisition of a classical myofibroblast-like phenotype. HSC are considered a central ECM-producing cell within the injured liver (1), playing a significant role in collagen-I deposition when hepatocellular injury is concentrated within the liver lobule and sinusoids. In normal liver, they reside in the sinusoidal space of Disse; however, during chronic injury, they activate while acquiring motile, proinflammatory and profibrogenic properties (2). Activated HSC migrate and accumulate at the sites of tissue repair, secreting large amounts of ECM, mostly collagen-I, and regulating ECM remodeling. Up-regulation of fibrillar collagen-I is thus a key event leading to scarring, the pathophysiological hallmark of liver fibrosis.

While some current therapies have proven beneficial, dissecting key profibrogenic mechanisms, pathways and mediators of disease progression is vital. Several studies have identified OPN as significantly up-regulated during liver injury and in HSC (3-6). OPN is a soluble cytokine and a matrix-bound protein that can remain intracellular or is secreted; hence, allowing autocrine and paracrine signaling (7-8). OPN, as a matricellular phosphoglycoprotein, functions as adaptor and modulator of cell-matrix interactions (8). Among its many roles, it regulates cell migration, ECM-invasion and cell adhesion due to its ability to bind integrins -via its RGD motif- or CD44 -via a cryptic site (SVVYGLR)-exposed after cleavage by thrombin, plasminogen, plasmin, cathepsin-B and some MMPs (5, 9). OPN expression increases in tumorigenesis, angiogenesis and in response to inflammation, cellular stress and injury (10-14). OPN plays an important role in regulating tissue remodeling and cell survival as well as in chemoattracting inflammatory cells (15). Moreover, Opn^-/- mice show matrix disarrangement and alteration of collagen fibrillogenesis in cartilage compared to their WT littermates (16).

There is limited information on the contribution of OPN to the HSC profibrogenic behavior and the molecular mechanisms and signaling pathways involved in governing collagen-I protein expression during the fibrogenic response to liver injury (3-6, 17). Since OPN is expressed in HSC (3-6), we hypothesized that OPN could trigger signals capable of up-regulating collagen-I per se; hence, acting as a feed-forward mechanism promoting scarring. Therefore, the major goal of this work was to determine how OPN could become a profibrogenic ‘switch’ and to characterize the underlying cellular mechanism for this effect. In the present study, we identified a role for intracellular OPN in increasing collagen-I, the HSC membrane proteins engaged by extracellular OPN, the proximal signaling molecules/stress-sensitive kinases activated upon binding that trigger the profibrogenic cascade, the ability of OPN to respond to oxidant stress and the effect of Opn ablation or overexpression on collagen-I deposition in vivo.

MATERIAL AND METHODS

Please see Supplementary Experimental Procedures

RESULTS

rOPN slightly increases HSC proliferation rates and promotes HSC migration

rOPN did not alter HSC viability but slightly induced proliferation rates both in rat and in human HSC (Supplementary Figure 1); however, rOPN caused a 2-fold increase in the invasive potential or chemotaxis (Supplementary Figure 2A-2B) and enhanced the wound closure ability of rat HSC (Supplementary Figure 2C), important functions gained by HSC during their activation that contribute to their profibrogenic ability. Neutralizing Abs to α_vβ₃ integrin and OPN blocked the effects on HSC invasion (not shown) and on closure ability (Supplementary Figure 2C).

rOPN induces profibrogenic effects in primary HSC

Upon stimulation with rOPN, rat HSC up-regulated intra- and extracellular collagen-I in a time-dependent fashion (Figure 1A, left). Denatured rOPN did not elevate collagen-I; thus, confirming the specificity of the rOPN effect on collagen-I in HSC (not shown). rOPN lowered extracellular MMP13 protein by 50%, contributing to extracellular collagen-I accumulation. Reciprocal modulation of MMP13 and collagen-I has been previously described in rat HSC (18). Extracellular pro-, intermediate and active MMP2 and MMP9 remained unchanged (Figure 1A, left). Likewise, tissue inhibitor of MMP1 was comparable (not shown). rOPN induced rat HSC activation as shown by the increase in collagen-I and α-SMA proteins (Figure 1A, right). Analogous results were observed in human HSC (Figure 1B).

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.

Because of the ability of HSC to secrete TGFβ (19) along with its well-known profibrogenic effect (20), rat HSC were treated with anti-TGFβ Ab. Neutralization of TGFβ did not alter the rOPN-mediated induction of collagen-I; thus, implying a mechanism independent of TGFβ production by HSC (Supplementary Figure 3).

To dissect whether intracellular OPN could play an autocrine role in modulating collagen-I expression, HSC were isolated from WT and Opn^-/- mice. WT HSC appeared more profibrogenic than Opn^-/- HSC since intracellular collagen-I expression at 7 days of culture was higher in WT HSC than in Opn^-/- HSC (Figure 1C). Infection of rat HSC with an Ad-OPN increased intracellular collagen-I and intra- and extracellular OPN compared to HSC infected with Ad-LacZ (Figure 1D). Therefore, a novel autocrine role for intracellular OPN in modulating collagen-I deposition was identified.

Anti-α_vβ₃ integrin Ab blocks the rOPN-driven collagen-I increase

Since OPN is also a soluble cytokine and a matrix-bound protein, next we evaluated the role of extracellular OPN-mediated signaling (paracrine role) on collagen-I induction in HSC. OPN signals via integrins - mostly integrin α_vβ₃ highly expressed in HSC- (21-23) and via CD44 -also expressed in HSC- (24). Incubation with anti-α_vβ₃ integrin blocked the rOPN-driven total collagen-I (intra-plus extracellular) increase in rat HSC, while no major effect was observed by anti-CD44 (Figure 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 HSC (not shown).

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.

rOPN increases collagen-I protein via activation of the PI3K-pAkt-NFκB signaling pathway

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 pp38 (25-26), pERK1/2 (27), pJNK (28-29), PI3K and pAkt (26, 30). Only PI3K and the ratio pAkt ⁴⁷³Ser/Akt were elevated time-dependently by rOPN up to 3 hours in rat HSC (Figure 2B) and up to 1 hour in human HSC (Supplementary Figure 4A).

Since PI3K/pAkt are upstream of IKK and the IKK complex is central for the activation of NFκB to regulate collagen-I (26, 31), we focused on analyzing this signaling pathway. There was up-regulation of the ratios pIKKα,β 176/180Ser/IKKα,β and pIκBα 32Ser/IκBα as well as of nuclear/cytosolic p65 in OPN-treated rat HSC (Figure 2C). However, involvement of the mTOR-p70S6K cascade, a translational regulatory mechanism downstream of PI3K and pAkt ⁴⁷³Ser for regulating collagen-I (32), was precluded since rOPN neither altered mTOR and p706SK expression (Figure 2D) nor induced mTOR phosphorylation at 2448Ser or ²⁴⁸¹Ser in rat HSC (undetectable).

To define further the molecular mechanism for the 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. Wortmannin, a PI3K inhibitor, neither altered rat HSC viability (100% by the MTT assay), morphology nor proliferation rates (Supplementary Figure 4B and not shown); however, three different doses of wortmannin down-regulated total collagen-I expression in rat HSC co-treated with rOPN (Figure 3A, top). Similar effects were observed by co-incubation with LY294002, a second PI3K inhibitor (Figure 3A, bottom); thus, linking OPN, PI3K-pAkt activation and collagen-I up-regulation in rat HSC. Comparable results were observed in human HSC (Supplementary Figure 4C). Lastly, inhibitors of pp38, pERK1/2 and pJNK signaling did not prevent the increase in collagen-I by rOPN (not shown).

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.

Addition of pyrrolidine dithiocarbamate (PDTC) to block NFκB signaling prevented the rOPN-driven increase in collagen-I in rat HSC (Figure 3B, top). Analogous effects were observed by co-incubation with CAY10512 -a second inhibitor of NFκB signaling- (Figure 3B, middle). Moreover, HSC infected with Ad-NFκB-Luc and treated with rOPN for 24 hours showed a 2-fold increase in luciferase activity compared to non-rOPN-treated Ad-NFκB-Luc-infected cells (Figure 3B, bottom). Both, wortmannin and an α_vβ₃ integrin neutralizing Ab, blunted the rOPN-mediated effect on the ratios pIKKα,β 176/180Ser/IKKα,β and pIκBα 32Ser/IκBα as well as on nuclear/cytosolic p65 (Figure 3C), suggesting engagement of OPN with integrin α_vβ₃, PI3K-pAktactivation and NFκB signaling to up-regulate collagen-I expression in rat HSC. Lastly, blocking α_vβ₃ integrin prevented the increase in PI3K, the ratio pAkt ⁴⁷³Ser/Akt and collagen-I by rOPN in rat HSC (Figure 3D). In summary, these results established a connection among rOPN, α_vβ₃ integrin, PI3K-pAkt activation and the NFκB signaling pathway to drive collagen-I up-regulation in rat HSC in a paracrine manner.

OPN expression is up-regulated in liver fibrosis

Samples from stage-3 HCV-cirrhotic patients displayed correlation between elevated collagen-I and cleaved OPN protein (~55, ~42 and ~25 kDa isoforms) compared to healthy individuals. Fully modified (glycosylated and phosphorylated) monomeric OPN, typically running at ~75 kDa, was not detectable (Figure 4A).

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.

To determine whether OPN also increased during liver injury in mice, we used well-established in vivo models to induce liver fibrosis such as CCl₄-injection and TAA-treatment (33). These drugs undergo cytochrome P450 metabolism leading to significant oxidant stress, inflammation and hepatocyte necrosis within hours. The ~25 kDa OPN form was markedly induced in acute and chronic models of liver injury, while the ~55 kDa OPN form was elevated only under chronic CCl₄-injection and TAA-treatment (Figure 4B). Hence, there was association between OPN induction, OPN proteolytic processing and the extent of liver fibrosis both in humans and in mice.

Next, we evaluated the specific localization of the OPN induction in the liver. Non-treated livers showed OPN⁺ biliary epithelial cells (not shown). Primary HSC isolated from WT mice and cultured for 6 days were OPN⁺ (Figure 4C, left). Immunohistochemical (IHC) analysis revealed OPN expression in HSC (30), biliary epithelial cells (4, 6, 34), oval cells and mostly in damaged hepatocytes in WT mice injected CCl₄ for 1 month (Figure 4C, middle). Similar results were observed under TAA-treatment, although hepatocytes showed punctated staining (Figure 4C, right). The insets show OPN⁺ HSC in both models. In the early stages of CCl₄- and TAA-mediated liver injury, Kupffer cells were also OPN⁺ (not shown); however, the staining faded with disease progression. Of note, granular OPN⁺ staining -typical of secreted proteins-appeared in focal-septal hepatocytes (Figure 4C, middle). There was co-localization of OPN⁺ staining with α-SMA⁺ (a HSC activation marker) under TAA-treatment (Figure 4D) and by CCl₄-injection (not shown).

Since liver fibrosis is associated with significant oxidant stress, to dissect whether OPN was responsive to reactive oxygen species, HSC were challenged with H₂O₂ -a prooxidant typically generated during CCl₄ metabolism- or with L-buthionine sulfoximine (BSO) -which depletes glutathione. Both treatments increased OPN expression in HSC, whereas co-treatment with glutathione-ethyl ester to restore glutathione levels, blunted this effect (Figure 4E). To validate the induction of OPN by oxidant stress in vivo, WT mice were CCl₄-injected for 1 month in the presence or absence of S-adenosylmethionine (SAM), an antioxidant known to restore glutathione levels. Co-injection with SAM lowered OPN protein (Figure 5A-5B) and the extent of liver fibrosis (Figure 5C-5D) by 50% when compared to mice injected CCl₄ alone. In summary, these data proved the ability of OPN to respond to drug-induced liver injury and to oxidant stress.

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.

WT show more CCl₄-induced chronic liver injury and fibrosis than Opn^-/- mice

Fibrosis typically develops due to chronic liver injury. To decipher the role of OPN in the progression of liver disease, we tested whether chronic CCl₄-injection could lead to differences in the extent of liver fibrosis. CCl₄-injected C57BL/6J WT showed greater alanine aminotransferase (ALT) activity and more inflammation, hepatocyte ballooning degeneration and necrosis than Opn^-/- mice (Figure 6A-6E). Cytochrome P450 2E1 (CYP2E1) expression was similar in WT and Opn^-/- mice, indicating that the extent of liver injury in these mice was not due to different CCl₄metabolism (Figure 6F).

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.

In addition, CCl₄-injected WT mice presented elevated collagenous proteins, portal fibrosis, bridging fibrosis, scar thickness, Brunt fibrosis score and Sirius red and collagen-I morphometry compared to Opn^-/- mice (Figure 7A-7E). The above results were validated in WT and Opn^-/- 129sv mice (Supplementary Figures 5-6). Transgenic mice overexpressing OPN in hepatocytes (Opn^HEP Tg) and injected CCl₄ for 1 month showed similar ALT activity, necrosis and inflammation but significant periportal, bridging and sinusoidal fibrosis along with increased collagen-I scar thickness compared to WT mice (Figure 8). Moreover, Opn^HEP Tg mice developed spontaneous perivenular, perisinusoidal and portal fibrosis over time (1 yr) in the absence of any profibrogenic treatment (Supplementary Figure 7). In aggregate, the data suggest that OPN plays a major role in chronic CCl₄-induced hepatic fibrosis by regulating scar formation.

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.

WT mice show significant liver fibrosis under chronic TAA-treatment compared to Opn^-/- mice

To confirm the results obtained under chronic CCl₄-injection, we used the TAA-treatment as a second model of chronic drug-induced liver fibrosis. Sirius red/fast green staining and collagen-I IHC showed stage >3 fibrosis in TAA-treated WT and ~1-2 in Opn^-/- mice with clear induction of collagen-I deposition in TAA-treated WT compared to Opn^-/- mice, extensive portal fibrosis, bridging fibrosis and a ~3-fold increase in scar thickness (Supplementary Figure 8A-8B). Thus, fibrosis was more distinct in TAA-treated WT than in Opn^-/- mice as quantified by the Brunt fibrosis score and by the Sirius red and collagen-I morphometry (Supplementary Figure 8C-8E).

Collectively, these results suggest that increased OPN expression per se or following chronic liver injury and oxidant stress can stimulate collagen-I deposition in vivo. In addition, the in vitrostudies demonstrate that intracellular OPN plays an autocrine role in regulating collagen-I expression in HSC. Moreover, treatment with rOPN to resemble the paracrine actions of secreted OPN, increases HSC invasion, chemotaxis and wound healing potential and up-regulates collagen-I via integrin α_vβ₃ engagement, activation of PI3K-pAkt and NFκB signaling (Supplementary Figure 9).

DISCUSSION

It is becoming clearer that OPN is significantly induced during liver injury both in humans and in rodents (4-6, 17). In the past few years, work from several groups (4-6, 17, 34) studied the potential role of OPN in liver fibrosis albeit with inconclusive results. Studies by Lee et al (5) demonstrated an OPN increase in the culture medium from culture-activated HSC and under oral CCl₄-administration; however, no mechanistic studies were performed to dissect how OPN regulates collagen-I protein deposition. Lorena et al (6) suggested increased susceptibility to CCl₄-injection in Opn^-/- mice. Although the authors claimed that the protection observed in WT mice was due to enhancement of hepatocyte survival and reduction in NOS2 expression; yet, they neither provided IHC for cell survival markers nor measured the concentration of NO· or ONOO^- to support their conclusions, and no studies on collagen-I regulation were performed. Lastly, a recent publication from Syn et al (4) proposes a role for the Hedgehog signaling pathway in activating OPN and promoting fibrosis progression in non-alcoholic steatohepatitis; however, it is not clear which OPN isoform the authors are referring to and it is Gli1, and not Gli2, expression that it is widely considered the most reliable readout for cells undergoing active Hedgehog signaling.

Thus, there is a well timed need for dissecting the molecular mechanism on how this matricellular protein could regulate the fibrogenic response to liver injury, and specifically collagen-I protein expression by HSC. Currently, there are many unresolved questions on how OPN could act as a feed-forward mechanism to promote scarring, among these are: Does extracellular OPN have the ability to increase the HSC profibrogenic potential and HSC-derived collagen-I protein? Which HSC receptors are involved in the profibrogenic cascade triggered by OPN? What intracellular signals are activated upon OPN-receptor binding that drive the collagen-I increase in HSC? Is autocrine OPN signaling involved in regulating collagen-I in HSC? Is OPN sensitive to oxidant stress? Which OPN isoforms appear during the course of liver fibrosis? Does OPN induce sinusoidal fibrosis?

Thus, the overall goal of this work was to address these questions, identify the mechanism for the OPN-driven collagen-I up-regulation in HSC and determine the functional role of OPN in the pathogenesis of liver fibrosis. The idea that OPN mediates liver fibrosis is relevant for several reasons. First, because the observation that OPN is up-regulated in HSC during hepatic injury provides an excellent conceptual advance in our understanding of liver fibrogenesis, as it appears that OPN up-regulates HSC collagen-I protein in an autocrine and paracrine fashion. Second, it supports the clinicopathological finding that injury occurring in the central region is accompanied by fibrosis. Third, it opens the possibility of linking a soluble cytokine/matricellular protein with fibrogenesis. Lastly, the identification of the mechanism and mediators involved in the profibrogenic actions of OPN could help in devising strategies for therapeutic targeting.

Our in vitro experiments validated the hypothesis of the profibrogenic and proinvasive actions of OPN in HSC. Mechanistic studies identified the HSC membrane proteins engaged by OPN and the proximal signaling molecules/oxidant stress-sensitive kinases activated upon OPN binding that trigger the fibrogenic cascade. The experimental data identified integrin α_vβ₃ as an efficient conveyor of the OPN-mediated profibrogenic actions in HSC and pointed at the PI3K-pAkt activation and the NFκB signaling pathway as highly involved in this process.

Since OPN signals via integrins and CD44, it is feasible that following liver injury, a ligand for α_vβ₃ integrin such as OPN accumulates in the space of Disse and acts in a α_vβ₃ integrin-dependent manner to maintain collagen-I induction, HSC activation, invasion and migration. Because OPN binds ECM proteins (35-36), this binding ability may enhance HSC activation,migration and invasion, key HSC features for the development of fibrosis. The finding that blocking CD44 did not prevent the effect of rOPN on collagen-I may be related to the ability of hyaluronic acid -a glycosaminglycan synthesized during HSC activation (24, 37)- to bind CD44; thus, competitive inhibition between hyaluronic acid and rOPN for CD44 binding could occur in HSC, although this possibility needs further investigation.

Several observations support the role for the PI3K-pAkt activation and the NFκB signaling pathway in the effects mediated by rOPN on collagen-I. First, rOPN rapidly increased PI3K, the ratios pAkt 473Ser/Akt, pIKKα,β 176/180Ser/IKKα,β and pIκBα 32Ser/IκBα as well as nuclear translocation of p65. Second, inhibitors of PI3K activation and NFκB signaling blunted the rOPN-mediated increase in intra- and extracellular collagen-I protein. Third, blockade of α_vβ₃ integrin signaling with a neutralizing Ab and incubation with wortmannin or LY294002 prevented the induction of PI3K, the increase in the ratios pAkt ⁴⁷³Ser/Akt, pIKKα,β ^176/180Ser/IKKα,β and pIκBα 32Ser/IκBα, nuclear translocation of p65 and the up-regulation of collagen-I protein by rOPN. Involvement of the mTOR cascade was ruled out since rOPN altered neither mTOR-p706SK expression nor mTOR phosphorylation. Therefore, this study linked extracellular and/or secreted OPN (i.e. paracrine effect) with α_vβ₃ integrin binding, PI3K-pAkt activation, NFκB signaling and scarring.

Work from several laboratories (3-6), including our own, suggests that HSC are an important source of OPN during liver injury. To date, OPN was believed to exert its effects by binding the RGD motif in integrins and the cell-surface receptor CD44; however, an intracellular function of OPN in liver fibrosis was largely unknown. Because HSC isolated from Opn^-/- mice were less profibrogenic than those from WT mice and infection of HSC with Ad-OPN increased intracellular collagen-I, these results suggested a novel autocrine mechanism whereby intracellular OPN could modulate collagen-I deposition in HSC. Alternatively, extracellular OPNeither from HSC or from neighboring cells may activate HSC through its receptor (α_vβ₃ integrin), as suggested above; thus, creating a positive feedback loop.

To further validate our hypothesis, we then assessed whether OPN contributed to the fibrogenic response in vivo using two mouse models of drug-induced liver injury. The data from human samples and from the mouse models showed that most of the OPN found in liver injury appears to be cleaved at least at the end-point of the experiments. The role of each cleaved isoform in regulating the fibrogenic response to liver injury as well as the identification of the proteases that cleave hepatic OPN is currently under active investigation in our laboratory since additional integrin binding sites, other than α_vβ₃ integrin, are likely to be uncovered by proteolytic processing of the protein.

Upon the onset of liver injury in mice, the increase in OPN likely results from oxidant stress because CCl₄ and TAA metabolism via cytochrome P450s generates a considerable amount of free radicals (33), and the in vitro data demonstrated the OPN-responsiveness to oxidant stress, which was blocked by antioxidant treatment. Furthermore, co-treatment with SAM, known to elevate glutathione levels, prevented the increase in OPN and the fibrogenic response in WT mice injected CCl₄ for 1 month.

Although it is possible that the chronic effects of Opn ablation could be secondary to its effects on liver injury itself (i.e. inflammation and ductular reaction, unpublished observations), the data clearly reveal a direct action of OPN on collagen-I protein expression, a key event in liver fibrosis. Hence, OPN appears to induce scarring per se. This is indeed also supported by the finding that while ALT activity and the necrosis and inflammation scores were similar, there was increased portal, bridging and sinusoidal fibrosis along with enhanced width of the collagenous septa in CCl₄-injected Opn^HEP Tg mice compared to their WT littermates. Notably, Opn^HEP Tg mice developed spontaneous fibrosis over time while WT mice did not. Lastly, in line with the results using Opn^HEP Tg mice and the in vitro data, fibrilar collagen-I content and scar thickness was significantly lowered by OPN ablation in vivo. It is likely that secreted OPN allows paracrine signaling to HSC, while endogenous OPN expression in HSC signals in an autocrine fashion amplifying the fibrogenic response. The cell- and matrix-binding ability of OPN may also facilitate a proper stromal and fibrillar collagen network organization. Overall, it is reasonable to propose that OPN may drive the fibrogenic response, among others, by directly regulating collagen-I deposition. Thus, OPN emerges as a key soluble cytokine and ECM-bound molecule promoting liver fibrosis.

List of Abbreviations

ALT

alanine aminotransferase

BSO

L-buthionine sulfoximine

CCl₄

carbon tetrachloride

CYP2E1

cytochrome P450 2E1

ECM

extracellular matrix

IHC

immunohistochemistry

IOD

integrated optical density

HSC

hepatic stellate cells

MMP

matrix metalloprotease

MO

mineral oil

OPN

osteopontin

Opn^-/-

osteopontin knockout mice

Opn^HEP Tg

osteopontin transgenic mice in hepatocytes

PDTC

pyrrolidine dithiocarbamate

rOPN

recombinant OPN

SAM

S-adenosylmethionine

α-SMA

α-smooth muscle actin

TAA

thioacetamide

WT

wild-type