No abstract

The transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha) is a strong inhibitor of cell proliferation. We found that C/EBPalpha directly interacts with cdk2 and cdk4 and arrests cell proliferation by inhibiting these kinases. We mapped a short growth inhibitory region of C/EBPalpha between amino acids 175 and 187. This portion of C/EBPalpha is responsible for direct inhibition of cyclin-dependent kinases and causes growth arrest in cultured cells. C/EBPalpha inhibits cdk2 activity by blocking the association of cdk2 with cyclins. Importantly, the activities of cdk4 and cdk2 are increased in C/EBPalpha knockout livers, leading to increased proliferation. Our data demonstrate that the liver-specific transcription factor C/EBPalpha brings about growth arrest through direct inhibition of cdk2 and cdk4.

An RNA CUG triplet repeat binding protein, CUGBP1, regulates splicing and translation of various RNAs. Expansion of RNA CUG repeats in the 3-untranslated repeat of the mutant myotonin protein kinase (DMPK) mRNA in myotonic dystrophy (DM) is associated with alterations in binding activity of CUGBP1. To investigate whether CUGBP1 is directly affected by expansion of CUG repeats in DM tissues, we examined the intracellular status of CUGBP1 in DM patients as well as in cultured cells over expressing RNA CUG repeats. The analysis of RNA⅐protein complexes showed that, in control tissues, the majority of CUGBP1 is free of RNA, whereas in DM patients the majority of CUGBP1 is associated with RNA containing CUG repeats. Similarly to DM patients, overexpression of RNA CUG repeats in cultured cells results in the re-allocation of CUGBP1 from a free state to the RNA⅐protein complexes containing CUG repeats. CUG repeat-dependent translocation of CUGBP1 into RNA⅐protein complexes is associated with increased levels of CUGBP1 protein and its binding activity. Experiments with cyclohexamide-dependent block of protein synthesis showed that the half-life of CUGBP1 is increased in cells expressing CUG repeats. Alteration of CUGBP1 in DM is accompanied by alteration in translation of a transcription factor CCAAT/ enhancer-binding protein ␤ (C/EBP␤), which has been previously described to be a target of CUGBP1. Analysis of C/EBP␤ isoforms in DM patients with altered levels of CUGBP1 showed that translation of a dominant negative isoform, LIP, is induced by CUGBP1. Results of this paper demonstrate that the expansion of CUG repeats in DM affects RNA-binding proteins and leads to alteration in RNA processing.

CCAAT/enhancer binding protein ␣ (C/EBP␣) is expressed at high levels in quiescent hepatocytes and in differentiated adipocytes. In cultured cells, C/EBP␣ inhibits cell proliferation in part via stabilization of the p21 protein. The role of C/EBP␣ in regulating hepatocyte proliferation in vivo is presented herein. In C/EBP␣ knockout newborn mice, p21 protein levels are reduced in the liver, and the fraction of hepatocytes synthesizing DNA is increased. Greater than 30% of the hepatocytes in C/EBP␣ knockout animals continue to proliferate at day 17 of postnatal life when cell division in wild-type littermates is low (3%). p21 protein levels are relatively high in wild-type neonates but undetectable in C/EBP␣ knockout mice. The reduction of p21 protein in the highly proliferating livers that lack C/EBP␣ suggests that p21 is responsible for C/EBP␣-mediated control of liver proliferation in newborn mice. During rat liver regeneration, the amounts of both C/EBP␣ and p21 proteins are decreased before DNA synthesis (6 to 12 h) and then return to presurgery levels at 48 h. Although C/EBP␣ controls p21 protein levels, p21 mRNA is not influenced by C/EBP␣ in liver. Using coimmunoprecipitation and a mammalian two-hybrid assay system, we have shown the interaction of C/EBP␣ and p21 proteins. Study of p21 stability in liver nuclear extracts showed that C/EBP␣ blocks proteolytic degradation of p21. Our data demonstrate that C/EBP␣ regulates hepatocyte proliferation in newborn mice and that in liver, the level of p21 protein is under posttranscriptional control, consistent with the hypothesis that protein-protein interaction with C/EBP␣ determines p21 levels.

The liver is capable of completely regenerating itself in response to injury and after partial hepatectomy. In liver of old animals, the proliferative response is dramatically reduced, the mechanism for which is unknown. The liver specific protein, C/EBPalpha, normally arrests proliferation of hepatocytes through inhibiting cyclin dependent kinases (cdks). We present evidence that aging switches the liver-specific pathway of C/EBPalpha growth arrest to repression of E2F transcription. We identified an age-specific C/EBPalpha-Rb-E2F4 complex that binds to E2F-dependent promoters and represses these genes. The C/EBPalpha-Rb-E2F4 complex occupies the c-myc promoter and blocks induction of c-myc in livers of old animals after partial hepatectomy. Our results show that the age-dependent switch from cdk inhibition to repression of E2F transcription causes a loss of proliferative response in the liver because of an inability to induce E2F target genes after partial hepatectomy providing a possible mechanism for the age-dependent loss of liver regenerative capacity.