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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.

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.

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We previously showed that the rate of hepatocyte proliferation in livers from newborn C/EBP␣ knockout mice was increased. An examination of cell cycle-related proteins showed that the cyclin-dependent kinase (CDK) inhibitor p21 level was reduced in the knockout animals compared to that in wild-type littermates. Here we show additional cell cycle-associated proteins that are affected by C/EBP␣. We have observed that C/EBP␣ controls the composition of E2F complexes through interaction with the retinoblastoma (Rb)-like protein, p107, during prenatal liver development. S-phase-specific E2F complexes containing E2F, DP, cdk2, cyclin A, and p107 are observed in the developing liver. In wild-type animals these complexes disappear by day 18 of gestation and are no longer present in the newborn animals. In the C/EBP␣ mutant, the S-phase-specific complexes do not diminish and persist to birth. The elevation of levels of the S-phase-specific E2F-p107 complexes in C/EBP␣ knockout mice correlates with the increased expression of several E2F-dependent genes such as those that encode cyclin A, proliferating cell nuclear antigen, and p107. The C/EBP␣-mediated regulation of E2F binding is specific, since the deletion of another C/EBP family member, C/EBP␤, does not change the pattern of E2F binding during prenatal liver development. The addition of bacterially expressed, purified His-C/EBP␣ to the E2F binding reaction resulted in the disruption of E2F complexes containing p107 in nuclear extracts from C/EBP␣ knockout mouse livers. Ectopic expression of C/EBP␣ in cultured cells also leads to a reduction of E2F complexes containing Rb family proteins. Coimmunoprecipitation analyses revealed an interaction of C/EBP␣ with p107 but none with cdk2, E2F1, or cyclin A. A region of C/EBP␣ that has sequence similarity to E2F is sufficient for the disruption of the E2F-p107 complexes. Despite its role as a DNA binding protein, C/EBP␣ brings about a change in E2F complex composition through a protein-protein interaction. The disruption of E2F-p107 complexes correlates with C/EBP␣-mediated growth arrest of hepatocytes in newborn animals.