C/EBP-ALPHA
The CCAAT/enhancer-binding protein bears sequence homology and functional similarities to liver activator protein
(LAP) According to the nomenclature proposed by
Cao et al. (1991) , the CCAAT/enhancer-binding protein is C/EBP-alpha and NF-IL6 (LAP) is C/EBP-beta, with the
corresponding genes being CEBPA and CEBPB. CEBPB was formerly symbolized TCF5.
The
transcription factor C/EBP beta (CCAAT/enhancer-binding protein beta) is expressed in ovaries and
testes, as well as many other tissues of adult mice.
This gene was found to be regulated by gonadotropins in DNA arrays.
NCBI Summary:
This intronless gene encodes a transcription factor that contains a basic leucine zipper (bZIP) domain. The encoded protein functions as a homodimer but can also form heterodimers with CCAAT/enhancer-binding proteins alpha, delta, and gamma. Activity of this protein is important in the regulation of genes involved in immune and inflammatory responses, among other processes. The use of alternative in-frame AUG start codons results in multiple protein isoforms, each with distinct biological functions. [provided by RefSeq, Oct 2013]
General function
Nucleic acid binding, DNA binding, Transcription factor
C/EBPβ regulates Vegf gene expression in granulosa cells undergoing luteinization during ovulation in female rats. Shinagawa M et al. (2019) The ovulatory LH-surge increases Vegf gene expression in granulosa cells (GCs) undergoing luteinization during ovulation. To understand the factors involved in this increase, we examined the roles of two transcription factors and epigenetic mechanisms in rat GCs. GCs were obtained from rats treated with eCG before, 4 h, 8 h, 12 h and 24 h after hCG injection. Vegf mRNA levels gradually increased after hCG injection and reached a peak at 12 h. To investigate the mechanism by which Vegf is up-regulated after hCG injection, we focused on C/EBPβ and HIF1α. Their protein expression levels were increased at 12 h. The binding activity of C/EBPβ to the Vegf promoter region increased after hCG injection whereas that of HIF1α did not at this time point. The C/EBPβ binding site had transcriptional activities whereas the HIF1α binding sites did not have transcriptional activities under cAMP stimulation. The levels of H3K9me3 and H3K27me3, which are transcriptional repression markers, decreased in the C/EBPβ binding region after hCG injection. The chromatin structure of this region becomes looser after hCG injection. These results show that C/EBPβ regulates Vegf gene expression with changes in histone modifications and chromatin structure of the promoter region in GCs undergoing luteinization during ovulation.//////////////////
Knockdown of CEBP by RNAi in porcine granulosa cells resulted in S phase cell cycle arrest and decreased progesterone and estradiol synthesis. Zhen YH 2014 et al.
Cultured ovarian granulosa cells (GCs) are essential models to study molecular mechanisms of gene regulation during folliculogenesis. CCAAT enhancer binding proteins (CEBP) has been identified in the ovary and is critical for follicular growth, ovulation and luteinization in mice. In the present study, hormonal treatment indicated that luteinizing hormone (LH) and exogenous human chorionic gonadotropins (hCG) significantly increased the expression of CEBP in porcine GCs. By RNAi-Ready pSIREN-RetroQ-ZsGreen Vector mediated recombinant pshRNA vectors, CEBP gene was successfully knocked down in porcine GCs, confirmed by mRNA and protein level analyzed by real time PCR and western blot, respectively. We further found that knockdown of CEBP significantly increased the expression of p-ERK1/2. Furthermore, CEBP knockdown arrested the GCs at S phase of cell cycle, but had no effects on cell apoptosis. More importantly, it markedly down regulated the concentration of estradiol (E2) and progesterone (P4) in the culture medium. To uncover the regulatory mechanism of CEBP knockdown on cell cycle and steroids synthesis, we found that the mRNA expression of bcl-2 (anti-apoptosis), StAR and Runx2 (steroid hormone synthesis) was up-regulated, while genes related to apoptosis (Caspase-3 and p53), hormonal synthesis (CYP11A1) and cell cycle (cyclinA1, cyclinB1, cyclinD1) were down-regulated, suggesting that knockdown of CEBP may inhibit apoptosis, regulate cell cycle and hormone secretions at the transcriptional level in porcine GCs. Furthermore, knockdown of CEBP significantly increased the expression of PTGS2 and decreased the expression of IGFBP4, Has2 and PTGFR which are important for folliculogenesis in porcine GCs. In conclusion, this study reveals that CEBP is a key regulator of porcine GCs through modulation of cell cycle, apoptosis, steroid synthesis, and other regulators of folliculogenesis.
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Sirois J et al reported the transcriptional regulation of the rat prostaglandin endoperoxide
synthase 2 gene in granulosa cells through a
cis-acting C/EBP beta promoter element.
Electrophoretic mobility shift assays revealed
that the DNA subregion -142/-120 inhibited protein/DNA binding observed between granulosa cell
nuclear extracts and the labeled PGS-2 fragment -192/-53. The subregion -142/-120 acting element
C/EBP beta, 5'-TTATGCAAT-3'. Point mutations within the C/EBP beta element abolished
protein/DNA binding and resulted in a 50% loss of forskolin/LH/FSH inducibility of reporter gene
activity.
Christenson LK et al reported that two putative CCAAT/enhancer-binding protein (C/EBP) response elements were identified in the
proximal promoter of the human steroidogenic acute regulatory protein (StAR) gene, which encodes a
key protein-regulating steroid hormone synthesis. Expression of C/EBPalpha and -beta increased StAR
promoter activity in COS-1 and HepG2 cells. Cotransfection of C/EBPalpha or -beta and steroidogenic
factor 1, a transcription factor required for cAMP regulation of StAR expression, into COS-1 augmented
8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP)-stimulated promoter activity. When the
putative C/EBP response elements were mutated, individually or together, a pronounced decline in basal
StAR promoter activity in human granulosa-lutein cells resulted, but the fold stimulation of promoter
activity by 8-Br-cAMP was unaffected. Recombinant C/EBPalpha and -beta bound to the two identified
sequences but not the mutated elements. Human granulosa-lutein cell nuclear extracts also bound these
elements but not the mutated sequences. An antibody to C/EBPbeta, but not C/EBPalpha, supershifted the
nuclear protein complex associated with the more distal element. Western blot analysis
revealed the presence of C/EBPalpha and C/EBPbeta in human granulosa-lutein cell nuclear extracts.
Concerted regulation of the porcine StAR gene promoter activity by FSH and IGF-I in granulosa cells involves GATA-4 and C/EBP{beta}
LaVoie HA, et al 2004 previously demonstrated that FSH alone or in combination with insulin-like growth factor I (IGF-I) activated the porcine steroidogenic acute regulatory protein (StAR) gene promoter in a concerted manner in primary cultures of granulosa cells. Studies were undertaken to further delineate cis- and trans-acting elements of the porcine promoter and mechanisms mediating FSH-stimulation and its augmentation by IGF-I. Mutation of several putative regulatory elements localized hormone-stimulated activity to the highly conserved GATA site and identified novel nucleotides downstream as a functional C/EBPbeta site. In granulosa cell nuclear extracts GATA-4 and C/EBPbeta formed a high molecular weight complex with an oligonucleotide spanning -76 to -32 bp of the porcine promoter. The intensity of this high molecular weight complex was increased in granulosa cell nuclear extracts by treatment with FSH alone and was enhanced with the combination of FSH and IGF-I at 2 to 3 h of treatment. GATA-4 and C/EBPbeta proteins were uniformly expressed with all treatments at time points associated with increased DNA binding. Treatment (2 h) with FSH alone or FSH + IGF-I increased phosphorylation of GATA-4 on a PKA-consensus site. The 38 kDa isoform of C/EBPbeta exhibited greater phosphorylation with FSH + IGF-I treatment. Porcine luteal cell nuclear extracts also demonstrated GATA-4 and C/EBPbeta binding to the -76 to -32 bp region of the promoter providing evidence for their cooperation in vivo. These data indicate that GATA-4 and C/EBPbeta are both required for FSH +/- IGF-I stimulation of the porcine StAR promoter in homologous granulosa cell cultures.
Expression regulated by
LH
Comment
Sterneck E et al showed that , C/EBP
beta mRNA is specifically induced by luteinizing hormone (LH/hCG) in the granulosa layer of
preovulatory antral follicles.
Sirois J et al reported that C/EBP beta mRNA and protein were induced rapidly in granulosa cells in vivo by an ovulatory dose of human chorionic gonadotropin (hCG). C/EPB beta
appears to play a key role in regulating induction of the PGS-2 gene in granulosa cells prior to ovulation.
Ovarian localization
Granulosa, Surface epithelium
Comment
Sundfeldt K et al reported that immunohistochemical experiments demonstrated that C/EBPalpha and C/EBPbeta were preferentially expressed in epithelial ovarian epithelial tumour cells irrespective of stage or grade of the tumour.
Follicle stages
Preovulatory, Corpus luteum
Comment
Repression of the Inhibin {alpha} Subunit Gene by the Transcription Factor CCAAT/Enhancer Binding Protein Beta (C/EBP{beta}) Burkart AD, et al .
Inhibin is a dimeric peptide hormone produced in ovarian granulosa cells that suppresses FSH synthesis and secretion in the pituitary. Expression of inhibin alpha and beta subunit genes in the rodent ovary is positively regulated by FSH and negatively regulated following the preovulatory LH surge. We have investigated the role of the transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) in repressing the inhibin alpha subunit gene. C/EBPbeta knockout mice fail to appropriately down-regulate inhibin alpha subunit mRNA levels following treatment with hCG, indicating that C/EBPbeta may function to repress inhibin gene expression. The expression and regulation of C/EBPbeta was examined in the rodent ovary, and these studies show that C/EBPbeta is expressed in the ovary and in granulosa cells and is induced in response to hCG. Transient cotransfections with an inhibin promoter-luciferase reporter in a mouse granulosa cell line, GRMO2 cells, show that C/EBPbeta is capable of repressing both basal and forskolin-stimulated inhibin gene promoter activity. An upstream binding site for C/EBPbeta in the inhibin alpha subunit promoter was identified by electrophoretic mobility shift assays, which, when mutated, results in elevated inhibin promoter activity. However, C/EBPbeta also represses shorter promoter constructs lacking this site, and this component of repression is dependent on the more proximal promoter cAMP response element (CRE). EMSA assays show that C/EBPbeta effectively competes with CREB for binding to this atypical CRE. Thus, there are two distinct mechanisms by which C/EBPbeta represses inhibin alpha subunit gene expression in ovarian granulosa cells.
Expression of CCAAT/Enhancer Binding Proteins Alpha and Beta in the Porcine Ovary and Regulation in Primary Cultures of Granulosa Cells Gillio-Meina C, et al .
The expression of C/EBPalpha and betatranscription factor mRNAs and proteins was evaluated in porcine ovarian structures during the estrous cycle. C/EBPbeta mRNA was present in antral follicles and significantly increased in healthy corpora lutea (CL), whereas C/EBPalpha mRNA was constitutively expressed in these structures. Both isoforms of C/EBPalpha (42 and 30 kDa) exhibited greater expression in preovulatory follicles and the 42 kDa isoform increased in CL whereas the 30 kDa isoform decreased. All major isoforms of C/EBPbeta (38, 34, and 20 kDa) were expressed, with the 34 and 20 kDa isoforms being more abundant in preovulatory follicles and further increased in CL. The effects of FSH and cyclic AMP analogue on the distribution of C/EBP isoforms were also evaluated in primary cultures of porcine granulosa cells. FSH and 8-Br-cAMP had little stimulatory effect on isoform distribution but cAMP treatment (24 h) tended to decrease the 30 kDa form of C/EBPalpha and the 34 kDa form of C/EBPbeta. The 34 kDa form of C/EBPbeta was decreased by the PKA inhibitor H89 at 4 h (with FSH treatment) and by both PKA and PI3-kinase inhibitors at 24-h treatment. In transfected granulosa cells, FSH and cAMP analogue stimulated a C/EBP consensus sequence-reporter construct that was blocked by H89. These data implicate PKA as the major regulator of C/EBPbeta isoform distribution and C/EBP-mediated transactivation in granulosa cells. The differential expression of specific C/EBPalpha/beta isoforms observed in maturing follicles and CL may contribute to changes in follicular cell differentiation and increasing steroidogenic capacity.
Phenotypes
Mutations
3 mutations
Species: mouse
Mutation name: None
type: null mutation fertility: infertile - ovarian defect Comment:Sterneck E et al showed that mice carrying a targeted deletion of the C/EBP beta gene exhibit reproductive defects. Although these animals develop normally and males
are fertile, adult females are sterile. Transplantation of normal ovaries into mutant females restored
fertility, thus localizing the primary reproductive defect to the ovary proper. C/EBP beta-deficient ovaries lack corpora lutea and fail to down-regulate expression of the prostaglandin endoperoxidase synthase 2 and P450 aromatase genes in response to gonadotropins. These findings demonstrate that C/EBP beta is essential for periovulatory granulosa cell differentiation in response to LH. C/EBP beta is thus established as a critical downstream target of G-protein-coupled LH receptor signaling and one of the first transcription factors known to be required for ovarian follicle development in vivo.
Species: mouse
Mutation name: None
type: null mutation fertility: subfertile Comment: MAPK3/1 (ERK1/2) in ovarian granulosa cells are essential for female fertility. Fan HY et al. A surge of luteinizing hormone (LH) from the pituitary gland triggers ovulation, oocyte maturation, and luteinization for successful reproduction in mammals. Because the signaling molecules RAS and ERK1/2 (extracellular signal-regulated kinases 1 and 2) are activated by an LH surge in granulosa cells of preovulatory follicles, we disrupted Erk1/2 in mouse granulosa cells and provide in vivo evidence that these kinases are necessary for LH-induced oocyte resumption of meiosis, ovulation, and luteinization. In addition, biochemical analyses and selected disruption of the Cebpb gene in granulosa cells demonstrate that C/EBPbeta (CCAAT/Enhancer-binding protein-beta) is a critical downstream mediator of ERK1/2 activation. Thus, ERK1/2 and C/EBPbeta constitute an in vivo LH-regulated signaling pathway that controls ovulation- and luteinization-related events.
Species: mouse
Mutation name: None
type: null mutation fertility: infertile - ovarian defect Comment: CCAAT/Enhancer-Binding Proteins (C/EBP)-{alpha} and -{beta} Are Essential for Ovulation, Luteinization, and the Expression of Key Target Genes. Fan HY et al. LH activation of the epidermal growth factor receptor/RAS/ERK1/2 pathway is essential for ovulation and luteinization because granulosa cell (GC) depletion of Erk1/2 (Erk1/2(gc-/-) mice) renders mice infertile. As mediators of ERK1/2-dependent GC differentiation, the CCAAT/enhancer-binding proteins, (C/EBP)a and C/EBP? were also disrupted. Female Cebpb(gc-/-) mutant mice, but not Cebpa(gc-/-) mice, were subfertile whereas Cebpa/b(gc-/-) double-mutant females were sterile. Follicles failed to ovulate, ovaries were devoid of corpora lutea, luteal cell marker genes (Lhcgr, Prlr, Ptgfr, Cyp11a1, and Star) were absent, and serum progesterone levels were low. Microarray analyses identified numerous C/EBPa/?target genes in equine chorionic gonadotropin (eCG)-human (h)CG-treated mice. At 4 h post-hCG, a subset (19%) of genes altered in the Cebpa/b-depleted cells was also altered in Erk1/2-depleted cells; hence they are common effectors of ERK1/2. Additional genes down-regulated in the Cebpa/b-depleted cells at 8 and 24 h post-hCG include known (Akr1b7, Runx2, Star, Saa3) and novel (Abcb1b, Apln, Igfbp4, Prlr, Ptgfr Timp4) C/EBP targets and effectors of luteal and vascular cell development. Bhmt, a gene controlling methionine metabolism and thought to be expressed exclusively in liver and kidney, was high in wild-type luteal cells but totally absent in Cebpa/b mutant cells. Because numerous genes potentially associated with vascular development were suppressed in the mutant cells, C/EBPa/?appear to dictate the luteinization process by also controlling genes that regulate the formation of the extensive vascular network required to sustain luteal cells. Thus, C/EBPa/?mediate the terminal differentiation of GCs during the complex process of luteinization.