Expression and function of CCAAT/enhancer binding proteinbeta (C/EBPbeta) LAP and LIP isoforms in mouse mammary gland, tumors and cultured mammary epithelial cells

CCAAT/Enhancer binding proteins (C/EBPs) play important roles in the regulation of cell growth and differentiation. This study investigated the expression and function of C/EBPbeta isoforms in the mouse mammary gland, mammary tumors, and a nontransformed mouse mammary epithelial cell line (HC11). C/EBPbeta mRNA levels are 2-5-fold higher in mouse mammary tumors derived from MMTV/c-neu transgenic mice compared with lactating and involuting mouse mammary gland. The "full-length" 38 kd C/EBPbeta LAP ("Liver-enriched Activator Protein") isoform is the predominant C/EBPbeta protein isoform in mammary tumor whole cell lysates, however, the truncated 20 kd C/EBPbeta LIP ("Liver-enriched Inhibitory Protein") isoform is also present at detectable levels (mean LAP:LIP ratio 5.3:1). The mammary tumor C/EBPbeta LAP:LIP ratio decreases 70% (from 5.3:1 to 1.6:1) when lysate preparation is switched from a rapid whole cell lysis protocol to a multistep nuclear/cytoplasmic fractionation protocol. In contrast to mammary tumors, only the C/EBPbeta LAP isoform is detectable in the mammary gland whole cell and nuclear lysates; the truncated "LIP" isoform is undetectable regardless of isolation protocol. Ectopic over expression of C/EBPbeta LIP or C/EBPbeta LAP did not alter HC11 growth rates. However, C/EBPbeta LIP over expressing HC11 cells (LAP:LIP ratio of approximately 1:1) exhibited a consistent 2-4 h delay in G(0)/S phase transition. C/EBPbeta LIP overexpressing HC11 cells did not express beta-casein mRNA (mammary epithelial cell differentiation marker) in response to lactogenic hormones. This defect in beta-casein expression was not corrected by carrying out the differentiation protocol in the presence of an artificial extracellular matrix. These results demonstrate that the "full-length" C/EBPbeta LAP isoform is the predominant C/EBPbeta protein isoform expressed in mouse mammary gland in vivo and mouse mammary epithelial cell cultures in vitro. C/EBPbeta LIP detected in mammary tumor lysates may result from in vivo production or ex vivo isolation-induced proteolysis of C/EBPbeta LAP. Ectopic overexpression of C/EBPbeta LIP (LAP:LIP ratio of approximately 1:1) inhibits mammary epithelial cell differentiation (beta-casein expression).     

CCAAT/enhancer-binding protein beta isoforms and the regulation of alpha-smooth muscle actin gene expression by IL-1 beta

The role of IL-1beta in inflammation is amply documented, but its ability to inhibit myofibroblast differentiation and, in particular, the suppression of alpha-smooth muscle actin (alpha-SMA) gene expression is less well understood. Because IL-1beta can induce C/EBPbeta expression, the role of C/EBPbeta isoforms in IL-1beta regulation of alpha-SMA gene expression was investigated in rat lung myofibroblasts. The results showed that IL-1beta inhibited alpha-SMA expression in a dose-dependent manner, which was associated with stimulation of the expression of both C/EBPbeta isoforms, liver-enriched activating protein (LAP) and liver-enriched inhibitory protein (LIP). However, a greater increase in LIP relative to LAP expression resulted in a reduced LAP/LIP ratio after IL-1beta treatment. Transfection with an LAP-expressing plasmid stimulated, whereas an LIP-expressing plasmid inhibited, alpha-SMA expression. Cells from C/EBPbeta-deficient mice had reduced levels of alpha-SMA expression and promoter activity, which failed to respond to IL-1beta treatment. Sequence analysis identified the presence of a C/EBPbeta consensus binding sequence in the alpha-SMA promoter, which, when mutated, resulted in diminished promoter activity and abolished its responsiveness to IL-1beta treatment. EMSA revealed binding of C/EBPbeta to this C/EBPbeta consensus binding sequence from the alpha-SMA promoter. Finally, IL-1beta enhanced the expression of eukaryotic initiation factor 4E, a stimulator of LIP expression, which may account for a mechanism by which IL-1beta could alter the LAP/LIP ratio. These data taken together suggest that C/EBPbeta isoforms regulate alpha-SMA gene expression, and that its inhibition by IL-1beta was due to preferential stimulation of LIP expression.     

C/EBP-β Regulates Endoplasmic Reticulum Stress–Triggered Cell Death in Mouse and Human Models

Endoplasmic reticulum (ER) stress elicits the unfolded protein response (UPR), initially aimed at coping with the stress, but triggering cell death upon further stress. ER stress induces the C/EBP-® variant Liver-enriched Activating Protein (LAP), followed by the dominant-negative variant, Liver Inhibitory Protein (LIP). However, the distinct role of LAP and LIP in ER stress is unknown. We found that the kinetics of the ER stress-induced expression of LIP overlapped with that of the cell death in mouse B16 melanoma cells. Furthermore, inducible over-expression of LIP augmented ER stress-triggered cell death whereas over-expression of LAP attenuated cell death. Similar results were obtained in human 293T cells. Limited vasculature in tumors triggers hypoxia, nutrient shortage and accumulation of toxic metabolites, all of which eliciting continuous ER stress. We found that LAP promoted and LIP inhibited B16 melanoma tumor progression without affecting angiogenesis or accelerating the cell cycle. Rather, LAP attenuated, whereas LIP augmented tumor ER stress. We therefore suggest that C/EBP-® regulates the transition from the protective to the death–promoting phase of the UPR. We further suggest that the over-expression of LAP observed in many solid tumors promotes tumor progression by attenuating ER stress–triggered tumor cell death.     

Role of CREB in transcriptional regulation of CCAAT/enhancer-binding protein beta gene during adipogenesis

The proximal promoter of the C/EBPbeta gene possesses dual cis regulatory elements (TGA1 and TGA2), both of which contain core CREB binding sites. Comparison of the activities of C/EBPbeta promoter-reporter genes with 5'-truncations or site-directed mutations in the TGA elements showed that both are required for maximal promoter function. Electrophoretic mobility shift and chromatin immunoprecipitation (ChIP) analyses with antibodies specific to CREB and ATF1 showed that these CREB family members associate with the proximal promoter both in vitro and ex vivo. Immunoblotting and ChIP analysis revealed that other CREB family members, CREM and ATF1, are up-regulated and associate with the proximal C/EBPbeta promoter in mouse embryonic fibroblasts (MEFs) from CREB(-/-) mice. ChIP analysis of wild-type MEFs and 3T3-L1 preadipocytes revealed that interaction of phospho-CREB, the active form of CREB, with the C/EBPbeta gene promoter occurs only after induction of differentiation of 3T3-L1 preadipocytes and MEFs. Consistent with the interaction of CREB and ATF1 at the TGA regulatory elements, expression of constitutively active CREB strongly activated C/EBPbeta promoter-reporter genes, induced expression of endogenous C/EBPbeta, and caused adipogenesis in the absence of the hormonal inducers normally required. Conversely, expression of a dominant-negative CREB blocked promoter-reporter activity, expression of C/EBPbeta, and adipogenesis. When subjected to the standard adipocyte differentiation protocol, wild-type MEFs differentiate into adipocytes at high frequency, whereas CREB(-/-) MEFs exhibit greatly reduced expression of C/EBPbeta and differentiation. The low level of expression of C/EBPbeta and differentiation in CREB(-/-) MEFs appears to be due to up-regulation of other CREB protein family members, i.e. ATF1 and CREM.     

Degradation of polyomavirus JC T-antigen by stress involves the LIP isoform of C/EBP

Endoplasmic reticulum (ER) stress is caused by the accumulation of misfolded or unfolded proteins in the lumen of the endoplasmic reticulum. CCAAT/enhancer binding proteins are one of the cellular proteins whose expression is upregulated during ER stress. Previously, we have identified C/EBPbeta isoforms, especially LIP, as a negative regulator of polyomavirus JC (JCV), the causative agent of the demyelinating disease progressive multifocal leukoencephalopathy (PML). Here, we show that the induction of ER stress by thapsigargin increase the expression of endogenous LIP and the degradation of JCV T-antigen in a JCV-transgenic mouse tumor cell line. Our results also revealed that overexpression of LIP significantly reduced the level of T-Ag and this effect is reversed upon siRNA-mediated silencing of LIP. Immunoprecipitation/Western blot experiments indicated that LIP interacts with T-antigen directly. Treatment of cells that overexpress LIP with MG115, a proteasome inhibitor, partially rescued LIP-mediated degradation of T-antigen. Our observations point to a role of LIP in ER stress regulation of T-antigen stability and may open a new avenue to study host-virus interaction during ER stress.