RNA CUG-binding protein 1 increases translation of 20-kDa isoform of CCAAT/enhancer-binding protein beta by interacting with the alpha and beta subunits of eukaryotic initiation translation factor 2

Expression of a dominant negative 20-kDa isoform of CCAAT/enhancer-binding protein (C/EBPbeta), LIP, is increased in proliferating livers and in tumor cells. Two RNA-binding proteins, CUGBP1 and calreticulin, have been implicated in the translational regulation of C/EBPbeta. In this paper, we present evidence showing several critical steps by which liver increases translation of LIP after partial hepatectomy. At early stages after partial hepatectomy, liver activates CUGBP1 by a hyperphosphorylation. The activated CUGBP1 binds to the 5' region of C/EBPbeta mRNA and replaces calreticulin, which partially represses translation of C/EBPbeta in quiescent livers. The hyperphosphorylated CUGBP1 also interacts with the alpha and beta subunits of initiation factor eIF2. Our data demonstrate that the interaction of CUGBP1 with the eIF2alpha enhances the association of CUGBP1 with ribosomes and correlates with increased translation of LIP in the liver after partial hepatectomy. Our data support the hypothesis that CUGBP1 increases translation of LIP by the interaction with the eIF2alpha subunit. This facilitates subsequent recruitment of larger numbers of ribosomes to initiate translation of LIP.     

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.     

CCAAT/enhancer binding protein beta mediates the activation of the murine alpha1(I) collagen promoter by acetaldehyde

Acetaldehyde was previously shown to activate the alpha1(I) and alpha2(I) collagen promoters and to increase collagen production in activated stellate cells. Also, CCAAT/enhancer binding protein beta (C/EBPbeta) binds and activates the mouse alpha1(I) collagen promoter. This study investigates the role of C/EBPbeta in mediating the activation of the alpha1(I) collagen promoter by acetaldehyde. Nuclear extracts isolated from cultured activated rat hepatic stellate cells formed four protein-DNA complexes on electrophoretic mobility shift assay with an oligonucleotide including the C/EBP binding site between -365 and -335 in the alpha1(I) collagen promoter. The four complexes were identified to represent C/EBPbeta binding to the oligonucleotide by supershift with C/EBPbeta antibody. The principal C/EBP isoform found in the nuclear extracts from stellate cells was C/EBPbeta, with very low amounts of C/EBPalpha detected. Acetaldehyde (200 microM) increased C/EBPbeta protein in stellate nuclear extracts, increased its binding to the promoter, and activated the alpha1(I) collagen promoter in transfected stellate cells. Mutation of the C/EBPbeta binding site markedly decreased nuclear protein binding. A transfected promoter, mutated at the C/EBP binding site, had decreased basal activity, was not activated by acetaldehyde, and was not activated when cotransfected with a C/EBPbeta expression vector. This study shows that C/EBPbeta is the predominant C/EBP isoform found in activated stellate cells and that increased C/EBPbeta protein and C/EBPbeta binding to a proximal C/EBP binding site in the promoter mediates the activating effect of acetaldehyde.     

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.