PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a tumor suppressor that antagonizes signaling through the phosphatidylinositol-3-kinase-Akt pathway. We have demonstrated that subtle decreases in PTEN abundance can have critical consequences for tumorigenesis. Here, we used a computational approach to identify miR-22, miR-25, and miR-302 as three PTEN-targeting microRNA (miRNA) families found within nine genomic loci. We showed that miR-22 and the miR-106b∼25 cluster are aberrantly overexpressed in human prostate cancer, correlate with abundance of the miRNA processing enzyme DICER, and potentiate cellular transformation both in vitro and in vivo. We demonstrated that the intronic miR-106b∼25 cluster cooperates with its host gene MCM7 in cellular transformation both in vitro and in vivo, so that the concomitant overexpression of MCM7 and the miRNA cluster triggers prostatic intraepithelial neoplasia in transgenic mice. Therefore, the MCM7 gene locus delivers two simultaneous oncogenic insults when amplified or overexpressed in human cancer. Thus, we have uncovered a protooncogenic miRNA-dependent network for PTEN regulation and defined the MCM7 locus as a critical factor in initiating prostate tumorigenesis.

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The farnesoid X receptor (FXR) is expressed by and regulates hepatic stellate cells (HSCs). In the present study, we investigated whether 6-ethyl chenodeoxycholic acid (6-ECDCA or INT-747), a semisynthetic derivative of chenodeoxycholic acid (CDCA), modulates tissue metalloproteinase inhibitor (TIMP)-1 and matrix metalloprotease (MMP)-2 expression/activity in HSCs and in the liver of rats rendered cirrhotic by 4-week administration of CCl 4 . Exposure of HSCs to FXR ligands increases small heterodimer partner (SHP) mRNA by 3-fold and reduces basal and thrombin-stimulated expression of ␣1(I)collagen, ␣-smooth muscle actin (␣-SMA), TIMP-1, and TIMP-2 by Ϸ60 to 70%, whereas it increased matrix metalloprotease (MMP)-2 activity by 2-fold. In coimmunoprecipitation, electromobility shift, and transactivation experiments, FXR activation/ overexpression caused a SHP-dependent inhibition of JunD binding to its consensus element in the TIMP-1 promoter.Inhibition of TIMP-1 expression by SHP overexpression enhanced the sensitivity of HSCs to proapoptogenic stimuli. Administration of 3 mg/kg 6-ECDCA, but not 15 mg/kg ursodeoxycholic acid, resulted in early (3-5-day) induction of SHP and prevention of early up-regulation of TIMP-1 mRNA induced by CCl 4 . In the prevention protocol, 4-week administration of 6-ECDCA reduced ␣1(I)collagen, ␣-SMA, and TIMP-1 mRNA by 60 to 80%, whereas it increased MMP-2 activity by 5-fold. In the resolution protocol, administration of 3 mg/kg 6-ECDCA promoted liver fibrosis resolution and increased the apoptosis of nonparenchyma liver cells. By demonstrating that a FXR-SHP regulatory cascade promotes the development of a quiescent phenotype and increases apoptosis of HSCs, this study establishes that FXR ligands may be beneficial in treatment of liver fibrosis.Hepatic fibrosis is a scarring process of the liver that includes components of both increased and altered deposition of extracellular matrix (ECM) and reduced breakdown of ECM components (Mann and Smart, 2002;Friedman, 2003).In chronic liver disease, hepatic stellate cells (HSCs), the major source of ECM in the liver, undergo a process of transdifferentiation from a resting, fat-storing phenotype to a myofibroblast-like phenotype characterized by expression of fibroblastic cell markers such as ␣-smooth muscle actin (␣-SMA) (Mann and Smart, 2002;Friedman, 2003). In addition, there is now considerable evidence that HSCs are a source of both matrix-degrading metalloproteinases (MMPs), including those that degrade type I collagen and the tissue inhibiThis study was partially supported by a research grant from Intercept Pharmaceuticals.Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.105.084905.ABBREVIATIONS: ECM, extracellular matrix; HSC, hepatic stellate cell; ␣-SMA, ␣-smooth muscle actin; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of matrix metalloproteinase; AP-1, activator protein-1; FXR, farnesoid X receptor; RXR, retinoid X receptor; CDCA, chenodeoxycho...

The nuclear receptors farnesoid X receptor (FXR) and peroxisome proliferator-activated receptor (PPAR)␥ exert counterregulatory effects on hepatic stellate cells (HSCs) and protect against liver fibrosis development in rodents. Here, we investigated whether FXR ligands regulate PPAR␥ expression in HSCs and models of liver fibrosis induced in rats by porcine serum and carbon tetrachloride administration and bile duct ligation. Our results demonstrate that HSCs trans-differentiation associated with suppression of PPAR␥ mRNA expression, whereas FXR mRNA was unchanged. Exposure of cells to natural and synthetic ligands of FXR, including 6-ethyl chenodeoxycholic acid (6-ECDCA), a synthetic derivative of chenodeoxycholic acid, reversed this effect and increased PPAR␥ mRNA by Ϸ40-fold. Submaximally effective concentrations of FXR and PPAR␥ ligands were additive in inhibiting ␣1(I) collagen mRNA accumulation induced by transforming growth factor (TGF)␤ 1 . Administration of 6-ECDCA in rats rendered cirrhotic by porcine serum and carbon tetrachloride administration or bile duct ligation reverted down-regulation of PPAR␥ mRNA expression in HSCs. Cotreatment with 6-ECDCA potentiates the antifibrotic activity of rosiglitazone, a PPAR␥ ligand, in the porcine serum model as measured by morphometric analysis of liver collagen content, hydroxyproline, and liver expression of ␣1(I) collagen mRNA, ␣-smooth muscle actin, fibronectin, TGF␤ 1 , and tissue inhibitor of metalloprotease 1 and 2, whereas it enhanced the expression of PPAR␥ and uncoupling protein 2, a PPAR␥-regulated gene, by 2-fold. In conclusion, by using an in vitro and in vivo approach, we demonstrated that FXR ligands up-regulate PPAR␥ mRNA in HSCs and in rodent models of liver fibrosis. A FXR-PPAR␥ cascade exerts counterregulatory effects in HSCs activation.Hepatic fibrosis is a scarring process of the liver that includes both increased and altered deposition of extracellular matrix components (Friedman, 2003). In chronic liver disease, hepatic stellate cells (HSCs) undergo a process of trans-differentiation (Friedman, 2003) from a resting, fatstoring, phenotype toward a myofibroblast-like phenotype characterized by expression of fibroblastic cell markers such as ␣1(I) collagen and ␣-smooth muscle actin (␣-SMA). Although the mediators involved in this process are not completely understood, a growing body of evidence suggests that members of the nuclear receptor (NR) superfamily (Fiorucci et al., 2004b) exert counter-regulatory effects acting as braking signals to prevent HSCs trans-differentiation.The farnesoid X receptor (FXR) is a ligand-activated transcription factor that regulates cholesterol and fatty acid metabolism and functions as an endogenous sensor for bile acids (Forman et al., 1995;Makishima et al., 1999;Parks et al., This study was partially supported by a research grant from Intercept Pharmaceuticals (New York, NY).Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.doi:10.1124/jpet.105.085597. AB...