The role of hepatocyte nuclear factor 4alpha1 (HNF4alpha1) in cell cycle regulation Free

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Hepatocellular carcinoma (HCC), primarily caused by hepatitis virus infection and aflatoxin exposure, is one of the most widespread cancers. Hepatocellular carcinogenesis is a multi-step process caused by the accumulation of genetic changes that alter the hepatocellular phenotype and lead to HCC. Hepatocyte nuclear factor (HNF) 4alpha is an abundant and constitutively active transcription factor, and a member of the nuclear receptor superfamily. It is known as a key regulator of both liver development and maintenance of a differentiated liver phenotype in adults. Previous research has shown that loss of HNF4alpha expression may be a determinant in HCC progression: Ectopic expression of HNF4alpha1 can cause tumors to revert to a less invasive, more differentiated, slow-growing phenotype. Recently, one study reported that HNF4alpha over-expression could induce the expression of CDK inhibitor p21 and arrest cell cycle progression in F9 murine embryonal carcinoma cells. However, the mechanism of HNF4alpha1 regulation of p21 was not investigated. Furthermore, microarray analysis from our laboratory has indicated that HNF4alpha1 may also regulate other cell cycle proteins. Thus, up-regulation of p21 by HNF4alpha1 via direct and/or indirect mechanisms may be one of the factors that prevent progression of HCC.To elucidate the mechanism by which expression of HNF4alpha1 regulates cell cycle progression and activates the p21 promoter, we used a tetracycline-regulated adenovirus system containing HNF4alpha1 cDNA to over express HNF4alpha1 in mammalian cells. Fluorescence activated cell sorter (FACS) analysis showed that expression of HNF4alpha1 caused cell cycle arrest in both HCT116 and COS-7 cells at G1 phase. This cell cycle arrest was correlated with activation of p21 expression, independent of the p53 pathway. Importantly, expression of HNF4alpha1 in HCT116 p21 knockout cells showed that p21 is critical for the HNF4alpha1-mediated cell cycle arrest. We also performed chromatin immunoprecipitation (ChIP) assays which showed that HNF4alpha1 was associated with the p21 promoter, and transient reporter assays showed that HNF4alpha1 activated p21 transcription. Interestingly, transactivation of the p21 promoter occurred even when a minimal p21 promoter construct (which consisted only of 6 Sp1 sites) was used. We also observed that expression of HNF4alpha1 induced the expression of Ninjurin-1 (NINJ-1), a novel adhesion molecule that has been associated with increased p21 expression and induction of cellular senescence in human hepatoma cells.Our results suggest that HNF4alpha1 may activate p21 expression via at least two potential mechanisms: Sp1-mediated recruitment to the p21 promoter and NINJ1-mediated activation of p21 expression. One or more of these mechanisms may be involved in HNF4alpha-mediated inhibition of HCC progression.