Osteopontin: A missing link between hedgehog signaling and fibrosis in nonalcoholic steatohepatitis
Authors: Mariana Verdelho Machado, Helena Cortez-Pinto
Journal: Hepatology (2010)

EMT, epithelial-to-mesenchymal transition; Gli, glioma-associated oncogene; Hh, hedgehog; HSC, hepatic stellate cell; MCD, methionine choline–deficient; NASH, nonalcoholic steatohepatitis; PDGF, platelet-derived growth factor; PI3K, phosphatidyl inositol-3 kinase; Ptc, patched; Smo, smoothened; TGF-β, transforming growth factor β. The progression of nonalcoholic steatohepatitis (NASH) involves hepatocyte cell death, which elicits both regenerative and fibrogenic responses. First, steatosis renders hepatocytes more susceptible to apoptosis,1 which correlates with fibrosis. Second, hepatocyte replication is inhibited in patients with nonalcoholic fatty liver disease.2 In an adaptive reaction, dying hepatocytes release signals that induce oval cells to proliferate and differentiate in hepatocytes; this allows repair and regeneration.3 Oval cells are progenitor cells that can differentiate into hepatocytes or cholangiocytes. Upon stimulation, they assume the intermediate phenotype of a ductular hepatocyte; they appear in a ductular reaction before they differentiate into hepatocytes. Previously, Diehl's group4 suggested that the hedgehog (Hh) pathway is the link between dying hepatocytes and progenitor cells. Hh signaling regulates cell proliferation and differentiation during embryogenesis and in postembryonic tissues to maintain stem cells. However, in contrast to oval cells, hepatocytes are unresponsive to Hh. In fact, Hh signaling maintains resident hepatic progenitors throughout life, and the progenitor population expands or shrinks according to the availability of the Hh ligand.5 The Hh ligand binds to its receptor, patched (Ptc), and deactivates it. Thus, in the absence of Hh, Ptc suppresses the activity of smoothened (Smo), a transmembrane protein. Yet, upon Hh binding, Smo is derepressed, activates transcription factors such as glioma-associated oncogene (Gli) proteins, and up-regulates several target genes such as fibroblast growth factor 4, phosphatidyl inositol-3 kinase (PI3K), and vascular endothelial growth factor. Recently, osteopontin (OPN) has been suggested as a target gene of Gli-1.6 Simultaneously with a proliferative response, a fibrogenic response occurs. Immature ductular cells and fibroblastic cells proliferate in parallel with bridging fibrosis as nonalcoholic fatty liver disease progresses to cirrhosis.3 Hh signaling can induce epithelial-to-mesenchymal transition (EMT) responses in ductular-type progenitors that assume a myofibroblast phenotype.7 An EMT response occurs after exposure to transforming growth factor β (TGF-β), an inducer of Hh signaling.8 Hepatic stellate cells (HSC) are also responsive to Hh, which induces the activation of quiescent HSCs into myofibroblasts and maintains viability while inhibiting the apoptosis of HSCs and promoting proliferation.9 Leptin, a powerful profibrogenic cytokine, activates HSCs through the Hh ligand; this mechanism is dependent on PI3K/protein kinase B induction.10 OPN, a pleomorphic glycoprotein, mediates inflammation and carcinogenesis. Its expression is increased in the obese11 and correlates with insulin resistance and steatosis.12 OPN triggers fibrogenesis; this has been demonstrated in vitro, in in vivo animal models, and in human liver diseases. HSC activation is associated with OPN up-regulation; additionally, HSC incubation with OPN induces proliferative and migratory effects as well as collagen production and TGF-β receptor up-regulation.13 In viral hepatitis, OPN correlates with fibrosis and the risk and severity of hepatic cirrhosis.14 Also, in NASH, OPN seems crucial to fibrogenesis. Rats fed a high-fat diet presented OPN up-regulation correlating with α-smooth muscle actin and fibrosis in steatotic livers.15 In the methionine choline–deficient (MCD) mice model, steatosis and fibrosis were correlated with OPN up-regulation.16 In OPN knockout mice, an MCD diet induced less hepatic inflammation and fibrosis. OPN has been linked to oval cell induction17 and hepatic carcinogenesis and is associated with decreased survival in patients with hepatocellular carcinoma.18 In the January 2011 issue of HEPATOLOGY, Syn et al.19 report that OPN is a missing link between Hh signaling and fibrosis in NASH. In the first stage, they fed an MCD diet to wild-type mice and two sets of knockout mice: Ptc+/− mice partially deficient in Ptc with overly inducible Hh signaling and OPN−/− mice deficient in OPN. Ptc+/− mice developed more severe fibrosis that was associated with greater increases in OPN in comparison with wild-type mice. In contrast, OPN−/− mice developed significantly less fibrosis, despite similar Hh induction, according to Gli-2 staining. In the second stage, they cultured HSCs with S-antigen (an Hh agonist) and cyclopamine (an Hh antagonist). In HSCs, OPN production was increased by Hh agonists and decreased by antagonists, and this demonstrated that OPN production was dependent on Hh signaling. Also, incubating HSCs with different concentrations of recombinant OPN promoted fibrogenesis and increased the expression of α-smooth muscle actin and collagen, which were attenuated when OPN was neutralized with RNA aptamers. Finally, in NASH human livers, OPN levels were correlated with the Hh activity (Gli-2 immunohistochemistry) and fibrosis. In those samples, ductular cells expressed both OPN and Gli-2 and were localized in fibrous septa. OPN was also increased in other forms of liver cirrhosis, such as alcoholic and autoimmune cirrhosis, and this suggests that it might be a monotonous response to liver injury. Use of the MCD diet model, which does not reflect the usual phenotype of human NASH with insulin resistance, is subject to criticism; in fact, Hh signaling regulates insulin receptor substrate 1 expression, which can act as a compensatory mechanism decreasing insulin resistance in NASH patients.20 In short, in patients with NASH, injured hepatocytes are committed to apoptosis, and because their regenerative capacity is blunted, they produce the Hh ligand, which acts in a paracrine way, in oval and stellate cells: Oval cell proliferation is the basis of regeneration and the activation of HSCs into myofibroblasts is the basis of fibrogenesis; hence, regeneration and fibrogenesis progress side by side. OPN, a downstream effector of Hh, has already been associated with fibrosis and advanced cirrhosis as well as hepatic carcinogenesis, and this may indicate a deregulated repair and regenerative response. PI3K activation is probably an intermediate in Hh-induced OPN up-regulation and cytokine-induced Hh production. Several profibrogenic cytokines [i.e., leptin, platelet-derived growth factor (PDGF), and TGF-β] may share this pathway (Fig. 1). Knowledge of these mediators creates potential targets for treatments designed to reduce fibrosis progression and hepatocellular carcinoma development in patients with NASH and other liver diseases because these pathways are highly conserved responses to chronic liver injury. Dying hepatocytes, committed to apoptosis, produce the Hh ligand, which stimulates both oval and stellate cells. Oval cells proliferate and differentiate in hepatocytes (replacing the dying ones) and in myofibroblasts; this process is known as EMT. Several cytokines elicit Hh production, probably through PI3K. OPN is one of the Hh target genes and is believed to be directly involved in the regenerative and fibrogenic responses.