C/EBPalpha regulates formation of S-phase-specific E2F-p107 complexes in livers of newborn mice

We previously showed that the rate of hepatocyte proliferation in livers from newborn C/EBPalpha 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/EBPalpha. We have observed that C/EBPalpha 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/EBPalpha 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/EBPalpha 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/EBPalpha-mediated regulation of E2F binding is specific, since the deletion of another C/EBP family member, C/EBPbeta, does not change the pattern of E2F binding during prenatal liver development. The addition of bacterially expressed, purified His-C/EBPalpha to the E2F binding reaction resulted in the disruption of E2F complexes containing p107 in nuclear extracts from C/EBPalpha knockout mouse livers. Ectopic expression of C/EBPalpha in cultured cells also leads to a reduction of E2F complexes containing Rb family proteins. Coimmunoprecipitation analyses revealed an interaction of C/EBPalpha with p107 but none with cdk2, E2F1, or cyclin A. A region of C/EBPalpha 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/EBPalpha brings about a change in E2F complex composition through a protein-protein interaction. The disruption of E2F-p107 complexes correlates with C/EBPalpha-mediated growth arrest of hepatocytes in newborn animals.     

Inhibitor of cyclin-dependent kinase (CDK) interacting with cyclin A1 (INCA1) regulates proliferation and is repressed by oncogenic signaling

The cell cycle is driven by the kinase activity of cyclin·cyclin-dependent kinase (CDK) complexes, which is negatively regulated by CDK inhibitor proteins. Recently, we identified INCA1 as an interaction partner and a substrate of cyclin A1 in complex with CDK2. On a functional level, we identified a novel cyclin-binding site in the INCA1 protein. INCA1 inhibited CDK2 activity and cell proliferation. The inhibitory effects depended on the cyclin-interacting domain. Mitogenic and oncogenic signals suppressed INCA1 expression, whereas it was induced by cell cycle arrest. We established a deletional mouse model that showed increased CDK2 activity in spleen with altered spleen architecture in Inca1(-/-) mice. Inca1(-/-) embryonic fibroblasts showed an increase in the fraction of S-phase cells. Furthermore, blasts from acute lymphoid leukemia and acute myeloid leukemia patients expressed significantly reduced INCA1 levels highlighting its relevance for growth control in vivo. Taken together, this study identifies a novel CDK inhibitor with reduced expression in acute myeloid and lymphoid leukemia. The molecular events that control the cell cycle occur in a sequential process to ensure a tight regulation, which is important for the survival of a cell and includes the detection and repair of genetic damage and the prevention of uncontrolled cell division.     

HDAC1 promotes liver proliferation in young mice via interactions with C/EBPbeta

HDAC1 (histone deacetylase 1) regulates a number of biological processes in cells. Our previous studies revealed that HDAC1 inhibits proliferation of the livers in old mice. We have surprisingly observed that HDAC1 is also increased in young livers proliferating after partial hepatectomy (PH) and in human liver tumors. Increased levels of HDAC1 after PH lead to its interaction with a member of the C/EBP family, C/EBPbeta, which is also elevated after PH. At early time points after PH, the HDAC1-C/EBPbeta complex binds to the C/EBPalpha promoter and represses expression of C/EBPalpha. A detailed analysis of the role of HDAC1 and C/EBPbeta proteins in the regulation of C/EBPalpha promoter showed that, whereas C/EBPbeta alone activates the promoter, HDAC1 represses the promoter in a C/EBPbeta-dependent manner. The inhibition of HDAC1 in the livers of young mice inhibits liver proliferation after PH, which is associated with high levels of C/EBPalpha. Consistent with the positive role of HDAC1-C/EBPbeta complexes in liver proliferation, we have found that the CUGBP1-HDAC1-C/EBPbeta pathway is activated in human tumor liver samples, suggesting that HDAC1-C/EBPbeta complexes are involved in the development of liver tumors. The causal role of the CUGBP1-HDAC1 pathway in liver proliferation was examined in CUGBP1 transgenic mice, which display high levels of the CUGBP1-eIF2 complex. We have demonstrated that elevation of the HDAC1-C/EBPbeta complexes in CUGBP1 transgenic mice reduces expression of C/EBPalpha and increases the rate of liver proliferation. Thus, these studies have identified a new pathway that promotes liver proliferation in young mice and might contribute to the malignant transformations in the liver.     

Disruption of hepatic C/EBPalpha results in impaired glucose tolerance and age-dependent hepatosteatosis

C/EBPalpha is highly expressed in liver and regulates many genes that are preferentially expressed in liver. Because C/EBPalpha-null mice die soon after birth, it is impossible to analyze the function of C/EBPalpha in the adult with this model. To address the function of C/EBPalpha in adult hepatocytes, liver-specific C/EBPalpha-null mice were produced using a floxed C/EBPalpha allele and the albumin-Cre transgene. Unlike whole body C/EBPalpha-null mice, mice lacking hepatic C/EBPalpha expression did not exhibit hypoglycemia, nor did they show reduced hepatic glycogen in adult. Expression of liver glycogen synthase, phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase remained at normal levels. However, these mice exhibited impaired glucose tolerance due in part to reduced expression of hepatic glucokinase, and hyperammonemia from reduced expression of hepatic carbamoyl phosphate synthase-I. These mice also had reduced serum cholesterol and steatotic livers that was exacerbated with aging. This phenotype could be explained by increased expression of hepatic lipoprotein lipase and reduced expression of microsomal triglyceride transfer protein, apolipoproteins B100, and A-IV. These data demonstrate that hepatic C/EBPalpha is critical for ammonia detoxification and glucose and lipid homeostasis in adult mice.     

C/EBPbeta, when expressed from the C/ebpalpha gene locus, can functionally replace C/EBPalpha in liver but not in adipose tissue

Knockout of C/EBPalpha causes a severe loss of liver function and, subsequently, neonatal lethality in mice. By using a gene replacement approach, we generated a new C/EBPalpha-null mouse strain in which C/EBPbeta, in addition to its own expression, substituted for C/EBPalpha expression in tissues. The homozygous mutant mice C/ebpalpha(beta/beta) are viable and fertile and show none of the overt liver abnormalities found in the previous C/EBPalpha-null mouse line. Levels of hepatic PEPCK mRNA are not different between C/ebpalpha(beta/beta) and wild-type mice. However, despite their normal growth rate, C/ebpalpha(beta/beta) mice have markedly reduced fat storage in their white adipose tissue (WAT). Expression of two adipocyte-specific factors, adipsin and leptin, is significantly reduced in the WAT of C/ebpalpha(beta/beta) mice. In addition, expression of the non-adipocyte-specific genes for transferrin and cysteine dioxygenase is reduced in WAT but not in liver. Our study demonstrates that when expressed from the C/ebpalpha gene locus, C/EBPbeta can act for C/EBPalpha to maintain liver functions during development. Moreover, our studies with the C/ebpalpha(beta/beta) mice provide new insights into the nonredundant functions of C/EBPalpha and C/EBPbeta on gene regulation in WAT.     

Dephosphorylated C/EBPalpha accelerates cell proliferation through sequestering retinoblastoma protein

CCAAT/enhancer-binding protein alpha (C/EBPalpha) has been previously considered a strong inhibitor of cell proliferation which uses multiple pathways to cause growth arrest. In this paper, we describe a new function of C/EBPalpha, which is an acceleration of cell proliferation. This new function of C/EBPalpha is created in proliferating livers by protein phosphatase 2A-mediated dephosphorylation of C/EBPalpha at Ser193. The Ser193-dephosphorylated C/EBPalpha interacts with retinoblastoma protein (Rb) independently on E2Fs and sequesters Rb, leading to a reduction of E2F-Rb repressors and to acceleration of proliferation. This new function of C/EBPalpha requires Rb, since the dephosphorylated C/EBPalpha does not promote proliferation in Rb-negative cells. We also show that a balance of Rb and Ser193-dephosphorylated C/EBPalpha determines if the cells are growth arrested or have an increased rate of proliferation. Consistently with these findings, a significant portion of Rb is sequestered into Rb-C/EBPalpha complexes in proliferating livers, and E2F-Rb complexes are not detectable in these livers. Our data demonstrate a new pathway by which the phosphorylation-dependent switch of biological functions of C/EBPalpha promotes liver proliferation.     

Impaired hepatocyte DNA synthetic response posthepatectomy in insulin-like growth factor binding protein 1-deficient mice with defects in C/EBP beta and mitogen-activated protein kinase/extracellular signal-regulated kinase regulation

After a two-thirds hepatectomy, normally quiescent liver cells are stimulated to reenter the cell cycle and proliferate to restore the original liver mass. One of the most rapidly and highly induced genes and proteins in regenerating liver is insulin-like growth factor binding protein 1 (IGFBP-1), a secreted protein that may modulate the activities of insulin-like growth factors (IGFs) or signal via IGF-independent mechanisms. To assess the functional role of IGFBP-1 in liver regeneration, mice with a targeted disruption of the IGFBP-1 gene were generated. Although IGFBP-1(-/-) mice demonstrated normal development, they had abnormal liver regeneration after partial hepatectomy, characterized by liver necrosis and reduced and delayed hepatocyte DNA synthesis. The abnormal regenerative response was associated with blunted activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and a reduced induction of C/EBP beta protein expression posthepatectomy. Like cell cycle abnormalities observed in hepatectomized C/EBP beta(-/-) mice, cyclin A and cyclin B1 expression was delayed and reduced in IGFBP-1(-/-) livers, whereas cyclin D1 expression was normal. Treatment of IGFBP-1(-/-) mice with a preoperative dose of IGFBP-1 induced MAPK/ERK activation and C/EBP beta expression, suggesting that IGFBP-1 may support liver regeneration at least in part via its effect on MAPK/ERK and C/EBP beta activities. These findings are the first demonstration of the involvement of IGFBP-1 in the regulation of in vivo mitogenic signaling pathways.     

C/EBPalpha triggers proteasome-dependent degradation of cdk4 during growth arrest

CCAAT/enhancer binding protein alpha (C/EBPalpha) causes growth arrest via direct interaction with the cyclin-dependent kinases cdk2 and cdk4. In this paper, we present evidence showing that C/EBPalpha enhances a proteasome-dependent degradation of cdk4 during growth arrest in liver of newborn mice and in cultured cells. Overexpression of C/EBPalpha in several biological systems leads to a reduction of cdk4 protein levels, but not mRNA levels. Experiments with several tissue culture models reveal that C/EBPalpha enhances the formation of cdk4-ubiquitin conjugates and induces degradation of cdk4 through a proteasome-dependent pathway. As a result, the half-life of cdk4 is shorter and protein levels of cdk4 are reduced in cells expressing C/EBPalpha. Gel filtration analysis of cdk4 complexes shows that a chaperone complex cdk4-cdc37-Hsp90, which protects cdk4 from degradation, is abundant in proliferating livers that lack C/EBPalpha, but this complex is weak or undetectable in livers expressing C/EBPalpha. Our studies show that C/EBPalpha disrupts the cdk4-cdc37-Hsp90 complex via direct interaction with cdk4 and reduces protein levels of cdk4 by increasing proteasome-dependent degradation of cdk4.