Similar results were observed under TAA treatment, although hepatocytes showed punctated staining (Fig. 4C, right). Insets show OPN+ HSCs 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 (Fig. 4C, middle). There was colocalization of OPN+ staining with αSMA+ RXDX-106 (an HSCs activation marker) under TAA treatment (Fig. 4D) and by CCl4 injection (not shown). Because 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 (GSH). Both treatments increased OPN expression in HSCs, whereas cotreatment with glutathione ethyl ester (GSH-EE) to restore GSH levels, blunted this effect (Fig. 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 GSH levels. Coinjection with SAM lowered OPN protein (Fig. 5A, 5B) and the extent of liver fibrosis (Fig. 5C, 5D) by 50% when compared to mice injected anti-EGFR antibody inhibitor with CCl4 alone. In summary, these data proved the ability of OPN to respond to drug-induced liver injury and to oxidant stress. Fibrosis typically develops as a result of 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 (Fig. 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 the MCE公司 result of different CCl4 metabolism (Fig. 6F). 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 (Fig. 7A-7E). The above-described results were validated in WT and Opn−/− 129sv mice (Supporting Figs. 5 and 6). Transgenic mice overexpressing OPN in hepatocytes (OpnHEP Tg) injected with 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 (Fig. 8).