Liver-enriched Activator Protein Research Articles

P53 regulates CCAAT/Enhancer binding protein β gene expression

BackgroundThe transcription factor CCAAT/enhancer-binding protein β (C/EBPβ) is implicated in diverse processes and diseases. Its two isoforms, namely liver-enriched activator protein (LAP) and liver-enriched inhibitor protein (LIP) are translated from the same mRNA. They share the same C-terminal DNA binding domain except LAP has an extra N-terminal activation domain. Probably due to its higher affinity for its DNA cognate sequences, LIP can inhibit LAP transcriptional activity even at substoichiometric levels. However, the regulatory mechanism of C/EBPβ gene expression and the LAP: LIP ratio is unclear. MethodsIn this study, the C/EBPβ promoter sequence was scanned for conserved P53 response element (P53RE), and binding of P53 to the C/EBPβ promoter was tested by Electrophoretic Mobility Shift Assay (EMSA) and chromatin immunoprecipitation assay. P53 over-expression and dominant negative P53 expression plasmids were transfected into rat lung fibroblasts and tested for C/EBPβ gene transcription and expression. Western blot analysis was used to test the regulation of C/EBPβ LAP and LIP isoforms. Constructs containing the LAP 5′untranslated region (5′UTR) or the LIP 5′UTR region were used to test the importance of 5′UTR in the control of C/EBPβ LAP and LIP translation. ResultsThe C/EBPβ promoter sequence was found to contain a conserved P53 response element (P53RE), which binds P53 as demonstrated by Electrophoresis Mobility Shift Assay and chromatin immunoprecipitation assays. P53 over-expression suppressed while dominant negative P53 stimulated C/EBPβ gene transcription and expression. Western blot analysis showed that P53 differentially regulated the translation of the C/EBPβ LAP and LIP isoforms through the regulation of eIF4E and eIF4E-BP1. Further studies with constructs containing the LAP 5′untranslated region (5′UTR) or the LIP 5′UTR region showed that the 5′UTR is important in differential control of C/EBPβ LAP and LIP translation. ConclusionAnalysis of the effects of P53 on C/EBPβ expression revealed a novel mechanism by which P53 could antagonize the effects of C/EBPβ on its target gene expression. For the first time, P53 is shown to be a repressor of C/EBPβ gene expression at both transcriptional and translational levels, with a differential effect in the magnitude of the effect on LAP vs. LIP isoforms.

Tumor Environment Promotes Lnc57Rik-Mediated Suppressive Function of Myeloid-Derived Suppressor Cells.

Myeloid-derived suppressor cells (MDSCs) are pathologically activated neutrophils and monocytes with potent immunosuppressive activity that regulate immune responses in the tumor microenvironment. We identified a novel long noncoding RNA (lncRNA), named as lnc57Rik, in the MDSCs that controls their immunosuppressive functions. Lnc57Rik was induced in in vitro and in vivo inflammatory settings and upregulated the genes related to MDSC-mediated immunosuppression, including Arg-1, NOS2, NOX2, and COX2 Furthermore, Lnc57Rik can not only bind with the C/EBPβ isoform liver-enriched activator protein to activate C/EBPβ but also with the methyltransferase WD repeat-containing protein 5 that enables the enrichment of histone H3 trimethylated lysine 4 marks on the promoter regions of Arg-1, NOS2, NOX2, and COX2, eventually resulting in their transcriptional activation. Furthermore, the conserved human lnc57Rik has a similar function as murine lnc57Rik Taken together, upregulation of lnc57Rik in the tumor microenvironment promotes the immunosuppressive function of MDSCs.

C/EBPβ induces B‐cell acute lymphoblastic leukemia and cooperates with BLNK mutations

BLNK (BASH/SLP‐65) encodes an adaptor protein that plays an important role in B‐cell receptor (BCR) signaling. Loss‐of‐function mutations in this gene are observed in human pre‐B acute lymphoblastic leukemia (ALL), and a subset of Blnk knock‐out (KO) mice develop pre‐B‐ALL. To understand the molecular mechanism of the Blnk mutation‐associated pre‐B‐ALL development, retroviral tagging was applied to KO mice using the Moloney murine leukemia virus (MoMLV). The Blnk mutation that significantly accelerated the onset of MoMLV‐induced leukemia and increased the incidence of pre‐B‐ALL Cebpb was identified as a frequent site of retroviral integration, suggesting that its upregulation cooperates with Blnk mutations. Transgenic expression of the liver‐enriched activator protein (LAP) isoform of Cebpb reduced the number of mature B‐lymphocytes in the bone marrow and inhibited differentiation at the pre‐BI stage. Furthermore, LAP expression significantly accelerated leukemogenesis in Blnk KO mice and alone acted as a B‐cell oncogene. Furthermore, an inverse relationship between BLNK and C/EBPβ expression was also noted in human pre‐B‐ALL cases, and the high level of CEBPB expression was associated with short survival periods in patients with BLNK‐downregulated pre‐B‐ALL. These results indicate the association between the C/EBPβ transcriptional network and BCR signaling in pre‐B‐ALL development and leukemogenesis. This study gives insight into ALL progression and suggests that the BCR/C/EBPβ pathway can be a therapeutic target.

C/EBPβ isoforms sequentially regulate regenerating mouse hematopoietic stem/progenitor cells

AbstractThe transcription factor CCAAT enhancer-binding protein β (C/EBPβ) is required for stress-induced granulopoiesis at the level of hematopoietic stem/progenitor cells (HSPCs); however, its role and mechanisms of action in HSPCs are unknown. In this study, we assessed the regulation and functions of C/EBPβ in HSPCs, especially under stress conditions. After 5-fluorouracil treatment or bone marrow transplantation, Cebpb−/− HSPCs exhibited impaired cell-cycle activation and myeloid differentiation at the early and late phases of regeneration, respectively, whereas at steady state, Cebpb deficiency did not affect HSPCs. C/EBPβ was upregulated in response to hematopoietic stress, especially in CD150high long term-hematopoietic stem cells (LT-HSCs). Intracellular flow cytometric analysis that detected distinct domains of C/EBPβ revealed that, among the 3 isoforms of C/EBPβ, liver-enriched inhibitory protein (LIP) was upregulated in LT-HSCs prior to liver-enriched activating protein (LAP)/LAP* during regeneration. Early upregulation of LIP promoted cell-cycle entry of LT-HSCs by positively regulating Myc and expanded the HSPCs pool. Subsequent myeloid differentiation of amplified HSPCs was mediated by LAP/LAP*, which were upregulated at a later phase of regeneration. Collectively, our findings show that stress-induced sequential upregulation of C/EBPβ isoforms is critical for fine-tuning the proliferation and differentiation of regenerating HSPCs.

Metformin Regulates the Expression of CD133 Through the AMPK-CEBPβ Pathway in Hepatocellular Carcinoma Cell Lines

CD133 is a cellular surface protein, which has been reported to be a cancer stem cell marker, and thus is considered a potential target for cancer treatment. Metformin, one of the biguanides used for the treatment of diabetes, is also known to reduce the risk of cancer development and cancer stem-like cells (CSCs), including the expression of CD133. However, the mechanism underlying the reduction of the expression of CD133 by metformin is not yet understood. This study shows that metformin suppressed CD133 expression mainly by affecting the CD133 P1 promoter via adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling but not the mammalian target of rapamycin (mTOR). AMPK inhibition rescued the reduction of CD133 by metformin. Further experiments demonstrated that CCAAT/enhancer-binding protein beta (CEBPβ) was upregulated by metformin and that two isoforms of CEBPβ reciprocally regulated the expression of CD133. Specifically, the liver-enriched activator protein (LAP) isoform increased the expression of CD133 by directly binding to the P1 promoter region, whereas the liver-enriched inhibitory protein (LIP) isoform suppressed the expression of CD133. Consistent with these findings, a three dimensional (3D) culture assay and drug sensitivity assay demonstrated that LAP-overexpressing cells formed large spheroids and were more resistant to 5-fluorouracil (5-FU) treatment, whereas LIP-overexpressing cells were more sensitive to 5-FU and showed combined effects with metformin. Our results indicated that metformin-AMPK-CEBPβ signaling plays a crucial role in regulating the gene expression of CD133. Additionally, regulating the ratio of LAP/LIP may be a novel strategy for targeting CSCs for the treatment of cancer.

Aerobic Glycolysis Controls Myeloid-Derived Suppressor Cells and Tumor Immunity via a Specific CEBPB Isoform in Triple-Negative Breast Cancer

Myeloid-derived suppressor cells (MDSCs) inhibit anti-tumor immunity. Aerobic glycolysis is a hallmark of cancer. However, the link between MDSCs and glycolysis is unknown in patients with triple-negative breast cancer (TNBC). Here, we detect abundant glycolytic activities in human TNBC. In two TNBC mouse models, 4T1 and Py8119, glycolysis restriction inhibits tumor granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulatingfactor (GM-CSF) expression and reduces MDSCs. These are accompanied with enhanced Tcell immunity, reduced tumor growth and metastasis, and prolonged mouse survival. Mechanistically, glycolysis restriction represses the expression of a specific CCAAT/enhancer-binding protein beta (CEBPB) isoform, liver-enriched activator protein (LAP), via the AMP-activated protein kinase (AMPK)-ULK1 and autophagy pathways, whereas LAP controls G-CSF and GM-CSF expression to support MDSC development. Glycolytic signatures that include lactate dehydrogenase A correlate with high MDSCs and low Tcells, and are associated with poor human TNBC outcome. Collectively, tumor glycolysis orchestrates a molecular network of the AMPK-ULK1, autophagy, and CEBPB pathways to affect MDSCs and maintain tumor immunosuppression.

Liver-enriched activator protein 1 as an isoform of CCAAT/enhancer-binding protein beta suppresses stem cell features of hepatocellular carcinoma

PurposeLiver cancer stem cells (CSCs) are known to be associated with the development, survival, proliferation, metastasis, and recurrence of liver tumors. The aim of this study was to investigate the association of liver-enriched activator protein 1 (LAP1) with hepatocellular carcinoma (HCC) and liver CSCs (LCSCs) and explore the impact of LAP1 on LCSCs.Materials and methodsDifferences in LAP1 expression in liver cancer tissues versus matched para-tumoral liver tissues and LCSCs versus non-CSCs were analyzed by Western blotting, real-time polymerase chain reaction, immunohistochemistry, and flow cytometry. The effect of LAP1 on liver cancer cells was evaluated by the expression of CSC markers, oncosphere formation, proliferation, migration, and invasion in vitro. Cell cycle distribution and the number of apoptotic cells were analyzed to assess cell cycle and cell apoptosis. Furthermore, a mouse subcutaneous tumor implant model was established to explore the role of LAP1 in the development of HCC in vivo. Finally, the expression of CSC markers in paraffin-embedded sections was evaluated by immunofluorescence.ResultsLAP1 was weakly expressed in HCC tumors and cell lines and even weaker in LCSCs. LAP1 inhibited the expression of stem cell–associated genes and reduced the abilities of oncosphere formation, proliferation, migration, and invasion in vitro. Cell cycle assay revealed that LAP1 induced G1/G0 arrest. Furthermore, LAP1 decreased subcutaneous tumor-formation ability and the expression of CSC markers and Ki67 in vivo.ConclusionLAP1 suppressed the stem cell features of HCC, indicating that it possessed an antitumor effect in liver cancer, both in vitro and in vivo; therefore, LAP1 may prove to be a potential target in liver CSC-targeted therapy.

Transcriptional and translational regulation of CCAAT/Enhancer Binding Protein β expression by P53

The transcription factor CCAAT/enhancer‐binding protein β (C/EBPβ) is highly expressed in various tissues during development, including liver, adipose tissue, blood cells, lung and the endocrine pancreas. Two isoforms of C/EBPβ namely liver‐enriched activator protein (LAP) and liver‐enriched inhibitor protein (LIP) translated from the same mRNA have been described. The LAP and LIP isoforms share the same C‐terminal DNA binding domain except LAP has an extra N‐terminal activation domain. Probably due to its higher affinity for its DNA cognate sequences, the LIP can counteract the effects of LAP isoforms even at substoichiometric levels. In vitro and in vivo experiments with cultured cells and knockout mice demonstrated that C/EBPβ play a critical role in the regulation of myofibroblast differentiation and bleomycin induced pulmonary fibrosis. However, the regulatory mechanism of C/EBPβ gene expression is unclear. Analysis of the C/EBPβ promoter sequence identified a conserved P53 response element (P53RE) in the C/EBPβ gene promoter. Gel shift mobility and chromatin immunoprecipitation assays confirmed that P53 binds to the P53RE. Promoter reporter assays and real time PCR analysis of samples from fibroblasts transiently transfected with P53 or dominant negative P53 expression plasmids indicated that P53 was a repressor of C/EBPβ gene transcription. Western blot analysis with protein extracts from similarly transfected fibroblasts or P53 deficient fibroblasts showed that P53 differentially regulated translation of the C/EBPβ LAP and LIP isoforms through the regulation of eIF4E and eIF4E‐BP1. Further studies with constructs containing the LAP 5′ untranslated region or the LIP 5′ untranslated region showed that the 5′ untranslated region is important in differential control of C/EBPβ LAP and LIP translation. Thus P53 could regulate C/EBPβ gene expression at both transcriptional and translational levels.Support or Funding InformationThis work was supported by grants HL052285 and HL112880 from the National Institute of Health.

C/EBPβ Isoforms Distinctively and Collaboratively Regulate the Behavior of Hematopoietic Stem and Progenitor Cells in Regenerative Conditions

CCAAT Enhancer Binding Protein b (C/EBPb) is a leucine zipper type transcription factor. While C/EBPa plays a critical role in maintaining steady-state granulopoiesis, C/EBPb is required for stress-induced granulopoiesis (Hirai et al., 2006). We have been focusing on the functions of C/EBPb in the regulation of hematopoietic stem and progenitor cells (HSPCs) especially under stressed conditions. Last year in this meeting, we have shown that 1) C/EBPb was upregulated at protein level in HSPCs after hematopoietic stresses, 2) C/EBPb was required for initial expansion of HSPCs after transplantation, and 3) C/EBPb promoted exhaustion of HSPCs under repetitive hematopoietic stresses (56th ASH, abstract #67850). Here, we further investigated the significance of C/EBPb in cell cycle regulation of HSPCs and the distinct roles of C/EBPb isoforms in HSPCs during regenerative conditions.To clarify the involvement of C/EBPb in cell cycle regulation of HSPCs, we compared the cell cycle status of wild-type (WT) and Cebpb knockout (KO) HSPCs by intracellular Ki67 staining and short-term BrdU incorporation assay in combination with multi-color flow cytometric analysis. In order to exclude the difference in the bone marrow microenvironment, CD45.2+ WT or Cebpb KO bone marrow (BM) cells were transplanted into lethally irradiated CD45.1+ WT mice. At steady state (12 weeks after the BM transplantation), the cell cycle status of Cebpb KO HSPCs was identical to that of WT HSPCs. Then cell cycle status of HSPCs was assessed at various time points during regeneration after intraperitoneal administration of 5-fluorouracil (5-FU, 150mg/kg). We found that significantly more Cebpb KO HSPCs remained in the G0 phase than WT HSPCs (in LT-HSCs on days 3-10; in MPPs on days 6-12). Significantly less Cebpb KO HSPCs were BrdU+ and were in the S/G2/M phase on day 7. These findings suggest that C/EBPb, in a cell-intrinsic manner, facilitates cell cycle entry, progression and consequent earlier expansion of HSPCs in response to hematopoietic stresses.Next, we investigated the distinct roles of C/EBPb isoforms in regulation of HSPCs. C/EBPb is a unique single exon gene and utilization of three different initiating codons result in three distinct isoforms. Liver-enriched activating protein* (LAP*) and LAP are the longer isoforms containing transactivating domains, DNA binding and dimerization domains, and liver-enriched inhibitory protein (LIP) is the shortest isoform which lacks the transactivating domains. In order to examine the expression pattern of C/EBPb isoforms in vivo in scarce populations of regenerating HSPCs, we developed a novel flow cytometric method to distinguish the cells predominantly expressing shorter isoform (LIP) from the cells expressing both LIP and the longer isoforms (LAP* and LAP) by intracellular double staining. Using this method, we found that predominantly LIP-expressing cells transiently emerged within MPP fraction in the regenerating bone marrow (on days 5-6 after administration of 5-FU, Figure below), while overall C/EBPb expression levels were significantly upregulated in most cells.To examine the roles of respective C/EBPb isoforms in regulation of HSPCs, EML cells, a murine hematopoietic stem cell line, were retrovirally transduced with one of the C/EBPb isoforms and the transduced cells were subjected to further analysis (vectors are kind gifts from Dr Watanabe-Okouchi N and Dr Kurokawa M, Tokyo Univ). LIP-expressing EML cells were more proliferative and actively cycling than EML cells transduced with a control vector, whereas the proliferation of LAP*- or LAP-expressing cells were markedly suppressed. LIP-expressing cells remained undifferentiated status (c-kithigh CD11b-) for more than 2 weeks, while LAP*- or LAP-expressing cells rapidly differentiated into c-kitlow CD11b+ myeloid cells and eventually exhausted within a week. These results indicate LIP plays quite distinct roles from LAP* and LAP in regulation of HSPCs.Collectively, our data suggest that C/EBPb isoforms distinctively and collaboratively regulate HSPCs in regenerative conditions: early transient elevation of LIP contributes to cell cycle activation and rapid expansion of HSPC population, which is in turn converted into supply of mature myeloid cells by more abundant upregulation of LAP* and LAP. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Suppression of the pregnane X receptor during endoplasmic reticulum stress is achieved by down-regulating hepatocyte nuclear factor-4α and up-regulating liver-enriched inhibitory protein.

Endoplasmic reticulum (ER) stress is recognized as a common theme in the development of metabolic syndrome and other diseases. Chronic liver diseases develop ER stress and also show decreased capacity of drug metabolism. The pregnane X receptor (PXR) is a master regulator of genes involved in drug elimination. This study was performed to determine whether ER stress condition decreases the expression of PXR and whether the decrease alters the induction of cytochrome P450 3A4 (CYP3A4). Human primary hepatocytes and HepG2 cell line (human hepatocellular carcinoma) were treated with brefeldin A and thapsigargin, 2 well-established ER stressors. Without exceptions, both stressors significantly decreased the expression of PXR. The decrease led to reduced induction of CYP3A4. Reporter dissection study, electrophoretic mobility shift assay, and chromatin immunoprecipitation located in the PXR promoter region 2 adjacent elements recognized by hepatocyte nuclear factor-4α (HNF-4α) and cytidine-cytidine-adenosine-adenosine-thymidine enhanced binding proteins (C/EBPs), respectively. Additional studies demonstrated that HNF-4α was down-regulated during ER stress but the expression of C/EBPβ varied depending on a particular form of C/EBPβ. Liver-enriched activator protein (LAP) was down-regulated but liver-enriched inhibitory protein (LIP) was highly induced. Nevertheless, over-expression of HNF-4α or LAP restored the expression of PXR. Interestingly, the very same sequence also responded to interleukin-6 (IL-6), and primary hepatocytes treated with thapsigargin significantly increased the level of IL-6 mRNA. These findings establish a functional interconnection between ER stress and signaling of proinflammatory cytokines in the regulation of PXR expression.

CCAAT/enhancer binding protein beta (C/EBPβ) isoform balance as a regulator of epithelial-mesenchymal transition in mouse mammary epithelial cells

Activation of the epithelial-mesenchymal transition (EMT) program promotes cell invasion and metastasis, and is reversed through mesenchymal-epithelial transition (MET) after formation of distant metastases. Here, we show that an imbalance of gene products encoded by the transcriptional factor C/EBPβ, LAP (liver-enriched activating protein) and LIP (liver-enriched inhibitory protein), can regulate both EMT- and MET-like phenotypic changes in mouse mammary epithelial cells. By using tetracycline repressive LIP expression constructs, we found that SCp2 cells, a clonal epithelial line of COMMA1-D cells, expressed EMT markers, lost the ability to undergo alveolar-like morphogenesis in 3D Matrigel, and acquired properties of benign adenoma cells. Conversely, we found that inducible expression of LAP in SCg6 cells, a clonal fibroblastic line of COMMA1-D cells, began to express epithelial keratins with suppression of proliferation. The overexpression of the C/EBPβ gene products in these COMMA1-D derivatives was suppressed by long-term cultivation on tissue culture plastic, but gene expression was maintained in cells grown on Matrigel or exposed to proteasome inhibitors. Thus, imbalances of C/EBPβ gene products in mouse mammary epithelial cells, which are affected by contact with basement membrane, are defined as a potential regulator of metastatic potential.

CCAAT/Enhancer-Binding Protein δ Is a Critical Mediator of Lipopolysaccharide-Induced Acute Lung Injury

Although inflammation plays a central role in the pathogenesis of acute lung injury, the molecular mechanisms underlying inflammatory responses in acute lung injury are poorly understood, and therapeutic options remain limited. CCAAT/enhancer-binding proteins, C/EBPβ and C/EBPδ, are expressed in the lung and have been implicated in the regulation of inflammatory mediators. However, their functions in lung pathobiological characteristics are not well characterized. Herein, we show that C/EBPβ and C/EBPδ are activated in mouse lung after intrapulmonary deposition of lipopolysaccharide (LPS). Mice carrying a targeted deletion of the C/EBPδ gene displayed significant attenuation of the lung permeability index (lung vascular leak of albumin), lung neutrophil accumulation (myeloperoxidase activity), and neutrophils in bronchial alveolar lavage fluids compared with wild-type mice. These phenotypes were consistent with morphological evaluation of lung, which showed reduced inflammatory cell influx and minimal intra-alveolar hemorrhage. Moreover, mutant mice expressed considerably less tumor necrosis factor-α, IL-6, and macrophage inflammatory protein-2 in bronchial alveolar lavage fluids in LPS-injured lung compared with wild-type mice. In contrast, C/EBPβ deficiency had no effect on LPS-induced lung injury. By using small-interfering RNA-mediated knockdown for C/EBPδ, we demonstrate, for the first time to our knowledge, that C/EBPδ plays a critical role for the tumor necrosis factor-α, IL-6, and macrophage inflammatory protein-2 production in LPS-stimulated alveolar macrophages. These findings demonstrate that C/EBPδ, but not C/EBPβ, plays an important role in LPS-induced lung inflammatory responses and injury.

Transcriptional Repression of ATF4 Gene by CCAAT/Enhancer-binding Protein β (C/EBPβ) Differentially Regulates Integrated Stress Response

Different environmental stresses induce the phosphorylation of eIF2 (eIF2∼P), repressing global protein synthesis coincident with preferential translation of ATF4. ATF4 is a transcriptional activator of genes involved in metabolism and nutrient uptake, antioxidation, and regulation of apoptosis. Because ATF4 is a common downstream target that integrates signaling from different eIF2 kinases and their respective stress signals, the eIF2∼P/ATF4 pathway is collectively referred to as the integrated stress response. Although eIF2∼P elicits translational control in response to many different stresses, there are selected stresses, such as exposure to UV irradiation, that do not increase ATF4 expression despite robust eIF2∼P. The rationale for this discordant induction of ATF4 expression and eIF2∼P in response to UV irradiation is that transcription of ATF4 is repressed, and therefore ATF4 mRNA is not available for preferential translation. In this study, we show that C/EBPβ is a transcriptional repressor of ATF4 during UV stress. C/EBPβ binds to critical elements in the ATF4 promoter, resulting in its transcriptional repression. Expression of C/EBPβ increases in response to UV stress, and the liver-enriched inhibitory protein (LIP) isoform of C/EBPβ, but not the liver-enriched activating protein (LAP) version, represses ATF4 transcription. Loss of the liver-enriched inhibitory protein isoform results in increased ATF4 mRNA levels in response to UV irradiation and subsequent recovery of ATF4 translation, leading to enhanced expression of its target genes. Together these results illustrate how eIF2∼P and translational control combined with transcription factors regulated by alternative signaling pathways can direct programs of gene expression that are specifically tailored to each environmental stress.

Regulation of Adipocyte 11β-Hydroxysteroid Dehydrogenase Type 1 (11β-HSD1) by CCAAT/Enhancer-Binding Protein (C/EBP) β Isoforms, LIP and LAP

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) catalyses intracellular regeneration of active glucocorticoids, notably in liver and adipose tissue. 11β-HSD1 is increased selectively in adipose tissue in human obesity, a change implicated in the pathogenesis of metabolic syndrome. With high fat (HF)-feeding, adipose tissue 11β-HSD1 is down-regulated in mice, plausibly to counteract metabolic disease. Transcription of 11β-HSD1 is directly regulated by members of the CCAAT/enhancer binding protein (C/EBP) family. Here we show that while total C/EBPβ in adipose tissue is unaltered by HF diet, the ratio of the C/EBPβ isoforms liver-enriched inhibitor protein (LIP) and liver-enriched activator protein (LAP) (C/EBPβ-LIP:LAP) is increased in subcutaneous adipose. This may cause changes in 11β-HSD1 expression since genetically modified C/EBPβ(+/L) mice, with increased C/EBPβ-LIP:LAP ratio, have decreased subcutaneous adipose 11β-HSD1 mRNA levels, whereas C/EBPβΔuORF mice, with decreased C/EBPβ-LIP:LAP ratio, show increased subcutaneous adipose 11β-HSD1. C/EBPβ-LIP:LAP ratio is regulated by endoplasmic reticulum (ER) stress and mTOR signalling, both of which are altered in obesity. In 3T3-L1 adipocytes, 11β-HSD1 mRNA levels were down-regulated following induction of ER stress by tunicamycin but were up-regulated following inhibition of mTOR by rapamycin. These data point to a central role for C/EBPβ and its processing to LIP and LAP in transcriptional regulation of 11β-HSD1 in adipose tissue. Down-regulation of 11β-HSD1 by increased C/EBPβ-LIP:LAP in adipocytes may be part of a nutrient-sensing mechanism counteracting nutritional stress generated by HF diet.

CCAAT/Enhancer-binding Protein β Inhibits Proliferation in Monocytic Cells by Affecting the Retinoblastoma Protein/E2F/Cyclin E Pathway but Is Not Directly Required for Macrophage Morphology

Monocytic differentiation is orchestrated by complex networks that are not fully understood. This study further elucidates the involvement of transcription factor CCAAT/enhancer-binding protein β (C/EBPβ). Initially, we demonstrated a marked increase in nuclear C/EBPβ-liver-enriched activating protein* (LAP*)/liver-enriched activating protein (LAP) levels and LAP/liver-enriched inhibiting protein (LIP) ratios in phorbol 12-myristate 13-acetate (PMA)-treated differentiating THP-1 premonocytic cells accompanied by reduced proliferation. To directly study C/EBPβ effects on monocytic cells, we generated novel THP-1-derived (low endogenous C/EBPβ) cell lines stably overexpressing C/EBPβ isoforms. Most importantly, cells predominantly overexpressing LAP* (C/EBPβ-long), but not those overexpressing LIP (C/EBPβ-short), exhibited a reduced proliferation, with no effect on morphology. PMA-induced inhibition of proliferation was attenuated in C/EBPβ-short cells. In C/EBPβWT macrophage-like cells (high endogenous C/EBPβ), we measured a reduced proliferation/cycling index compared with C/EBPβKO. The typical macrophage morphology was only observed in C/EBPβWT, whereas C/EBPβKO stayed round. C/EBPα did not compensate for C/EBPβ effects on proliferation/morphology. Serum reduction, an independent approach known to inhibit proliferation, induced macrophage morphology in C/EBPβKO macrophage-like cells but not THP-1. In PMA-treated THP-1 and C/EBPβ-long cells, a reduced phosphorylation of cell cycle repressor retinoblastoma was found. In addition, C/EBPβ-long cells showed reduced c-Myc expression accompanied by increased CDK inhibitor p27 and reduced cyclin D1 levels. Finally, C/EBPβ-long and C/EBPβWT cells exhibited low E2F1 and cyclin E levels, and C/EBPβ overexpression was found to inhibit cyclin E1 promoter-dependent transcription. Our results suggest that C/EBPβ reduces monocytic proliferation by affecting the retinoblastoma/E2F/cyclin E pathway and that it may contribute to, but is not directly required for, macrophage morphology. Inhibition of proliferation by C/EBPβ may be important for coordinated monocytic differentiation.

Liver-enriched Inhibitory Protein (LIP) Actively Inhibits Preadipocyte Differentiation through Histone Deacetylase 1 (HDAC1)

The CCAAT/enhancer-binding protein β (C/EBPβ) is expressed as three isoforms (LAP*, liver-enriched activating protein (LAP), and liver-enriched inhibitory protein (LIP)) that differentially regulate gene expression. The interplay between LAP*, LAP, and LIP in regulating cellular processes is largely unknown, and LIP has been largely regarded to repress transcription through a passive heterodimerization-dependent mechanism. Recently, we have shown that p300/GCN5 and mSin3A/HDAC1 differentially regulate the ability of C/EBPβ to stimulate preadipocyte differentiation through activation of C/ebpα transcription. Here, we have mapped requirements for binding of mSin3A/HDAC1 to LAP/LAP* and LIP to a 4-amino acid motif in the central region of LAP/LAP* (residues 153-156) and the N terminus of LIP. Reducing mSin3A/HDAC1 binding to LAP/LAP* and LIP through deletion of this motif reduced the recruitment of HDAC1 to the C/ebpα promoter and increased preadipocyte differentiation stimulated by insulin and 1-methyl-3-isobutylxanthine. Additional studies showed that the interaction of HDAC1 with LIP provides for active repression of C/ebpα transcription and is largely responsible for the ability of LIP and HDAC1 to repress preadipocyte differentiation. Thus, although mSin3A/HDAC1 interacted readily with LAP/LAP* in addition to LIP and that expression of LAP/LAP* was sufficient to recruit HDAC1 to the C/ebpα promoter, mutations in C/ebpβ that abrogated HDAC1 association to LAP/LAP* in the absence of LIP provided no additional stimulation of differentiation or transcription beyond the deletion of LIP alone. The implication of these results for the interaction between p300/GCN5 and mSin3A/HDAC1 in regulating C/EBPα transcription and preadipocyte differentiation are discussed.

Characterization of the transcriptional signature of C/EBPbeta isoforms (LAP/LIP) in Hep3B cells: Implication of LIP in pro-survival functions

C/EBPbeta is an important mediator of several cellular processes, such as differentiation, proliferation, and survival of hepatic cells. However, a complete catalog of the targets of C/EBPbeta or the mechanism by which this transcription factor regulates certain liver-dependent pathways has not been clearly determined. Two major natural isoforms of this transcription factor exist: the liver-enriched activating protein (LAP) and the liver-enriched inhibitory protein (LIP), a functional LAP antagonist. In this study, we used the opposing transcriptional effects driven by LAP and LIP to determine the genuine C/EBPbeta molecular signature in the Hep3B human hepatoma cell line. We subsequently investigated the role of each of the LAP and LIP isoforms in drug-induced Hep3B cell death. We engineered Hep3B cells with regulated LAP or LIP expression using the Tet-off expression system. The genes that showed inverse regulation by LAP and LIP were identified by cDNA array analysis. The cohort of direct-C/EBPbeta-targets was distinguished from indirect-targets by ChIP-on-chip analysis. We characterized 676 genes by this approach. Among these genes, 39 are novel direct targets of C/EBPbeta. Eleven of these new direct targets are involved in cell survival, suggesting critical roles for LAP/LIP isoforms in this cellular process. Therefore, we examined the effects of LAP and LIP over-expression on cell survival. We show that LIP promotes survival in staurosporine- or taxol-induced Hep3B cell death. Our study provides new molecular and cellular insights into the role of C/EBPbeta in cells of hepatic origin.

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

There is an error with the beta symbols in the abstract. The abstract should read: 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-β 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-β 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.

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.

Lactogenic Hormonal Induction of Long Distance Interactions between β-Casein Gene Regulatory Elements

Lactogenic hormone regulation of beta-casein gene expression in mammary epithelial cells provides an excellent model in which to study the mechanisms by which steroid and peptide hormone signaling control gene expression. Prolactin- and glucocorticoid-mediated induction of beta-casein gene expression involves two principal regulatory regions, a proximal promoter and a distal enhancer located in the mouse approximately -6 kb upstream of the transcription start site. Using a chromosome conformation capture assay and quantitative real time PCR, we demonstrate that a chromatin loop is created in conjunction with the recruitment of specific transcription factors and p300 in HC11 mammary epithelial cells. Stimulation with both prolactin and hydrocortisone is required for the induction of these long range interactions between the promoter and enhancer, and no DNA looping was observed in nontreated cells or cells treated with each of the hormones separately. The lactogenic hormone-induced interaction between the proximal promoter and distal enhancer was confirmed in hormone-treated primary three-dimensional mammary acini cultures. In addition, the developmental regulation of DNA looping between the beta-casein regulatory regions was observed in lactating but not in virgin mouse mammary glands. Furthermore, beta-casein mRNA induction and long range interactions between these regulatory regions were inhibited in a progestin-dependent manner following stimulation with prolactin and hydrocortisone in HC11 cells expressing human PR-B. Collectively, these data suggest that the communication between these regulatory regions with intervening DNA looping is a crucial step required to both create and maintain active chromatin domains and regulate transcription.