The STAT3 inhibitor Stattic acts independently of STAT3 to decrease histone acetylation and modulate gene expression.

Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor involved in many physiological functions including embryonic development and immune responses and is often activated under pathological conditions such as cancer. Strategies to inactivate STAT3 are being pursued as potential anticancer therapies and have led to the identification of Stattic (6-nitrobenzo[b]thiophene-1,1-dioxide) as a "specific" STAT3 inhibitor that is often used to interrogate STAT3-mediated gene expression in vitro and in vivo. Here, we show that Stattic exerts many STAT3-independent effects on cancer cells, calling for reassessment of results previously ascribed to STAT3 functions. Studies of the STAT3-deficient prostate cancer cell line PC-3 (PC3) along with STAT3-proficient breast cancer cell lines (MDA-MB-231, SUM149) revealed that Stattic attenuated histone acetylation and neutralized effects of the histone deacetylase (HDAC) inhibitor romidepsin. In PC3 cells, Stattic alone inhibited gene expression of CCL20 and CCL2, but activated expression of TNFA, CEBPD, SOX2, and MYC. In addition, we found that Stattic promoted autophagy and caused cell death. These data point to profound epigenetic effects of Stattic that are independent of its function as a STAT3 inhibitor. Our results demonstrate that Stattic directly or indirectly reduces histone acetylation and suggest reevaluation of Stattic and related compounds as polypharmacological agents through multipronged cytotoxic effects on cancer cells.

The tumour suppressor C/EBPδ inhibits FBXW7 expression and promotes mammary tumour metastasis.

Inflammation and hypoxia are known to promote the metastatic progression of tumours. The CCAAT/enhancer-binding protein-δ (C/EBPδ, CEBPD) is an inflammatory response gene and candidate tumour suppressor, but its physiological role in tumourigenesis in vivo is unknown. Here, we demonstrate a tumour suppressor function of C/EBPδ using transgenic mice overexpressing the Neu/Her2/ERBB2 proto-oncogene in the mammary gland. Unexpectedly, this study also revealed that C/EBPδ is necessary for efficient tumour metastasis. We show that C/EBPδ is induced by hypoxia in tumours in vivo and in breast tumour cells in vitro, and that C/EBPδ-deficient cells exhibit reduced glycolytic metabolism and cell viability under hypoxia. C/EBPδ supports CXCR4 expression. On the other hand, C/EBPδ directly inhibits expression of the tumour suppressor F-box and WD repeat-domain containing 7 gene (FBXW7, FBW7, AGO, Cdc4), encoding an F-box protein that promotes degradation of the mammalian target of rapamycin (mTOR). Consequently, C/EBPδ enhances mTOR/AKT/S6K1 signalling and augments translation and activity of hypoxia-inducible factor-1α (HIF-1α), which is necessary for hypoxia adaptation. This work provides new insight into the mechanisms by which metastasis-promoting signals are induced specifically under hypoxia.

Positional variations in mammary gland development and cancer.

Most mammals develop their mammary glands in pairs of which the two counterparts are symmetrically displaced away from the ventral midline. Based on this symmetry and the same functional outcome as a milk-producing organ, the mammary glands are easily presumed to be mere copies of one another. Based on our analysis of published data with inclusion of new results related to mammary development and pathology in mice, we argue that this presumption is incorrect: Between and within pairs, mammary glands differ from one another, and tumor incidence and biology depend on the position along the anterior-posterior and the left-right axis as well. This insight has implications for experimental designs with mouse models and for data extrapolation between mammary glands within and between species. We suggest that improved documentation of location-specific mammary gland features will lead to more insights into the molecular mechanisms of mammary gland development and cancer biology in both mice and humans.

The Janus kinase 1 is critical for pancreatic cancer initiation and progression.

Interleukin-6 (IL-6)-class inflammatory cytokines signal through the Janus tyrosine kinase (JAK)/signal transducer and activator of transcription (STAT) pathway and promote the development of pancreatic ductal adenocarcinoma (PDAC); however, the functions of specific intracellular signaling mediators in this process are less well defined. Using a ligand-controlled and pancreas-specific knockout in adult mice, we demonstrate in this study that JAK1 deficiency prevents the formation of KRASG12D-induced pancreatic tumors, and we establish that JAK1 is essential for the constitutive activation of STAT3, whose activation is a prominent characteristic of PDAC. We identify CCAAT/enhancer binding protein δ (C/EBPδ) as a biologically relevant downstream target of JAK1 signaling, which is upregulated in human PDAC. Reinstating the expression of C/EBPδ was sufficient to restore the growth of JAK1-deficient cancer cells as tumorspheres and in xenografted mice. Collectively, the findings of this study suggest that JAK1 executes important functions of inflammatory cytokines through C/EBPδ and may serve as a molecular target for PDAC prevention and treatment.

Mismatch repair protein MLH1 suppresses replicative stress in BRCA2-deficient breast tumors.

Loss of BRCA2 (breast cancer 2) is lethal for normal cells. Yet it remains poorly understood how, in BRCA2 mutation carriers, cells undergoing loss of heterozygosity overcome the lethality and undergo tissue-specific neoplastic transformation. Here, we identified mismatch repair gene mutL homolog 1 (MLH1) as a genetic interactor of BRCA2 whose overexpression supports the viability of Brca2-null cells. Mechanistically, we showed that MLH1 interacts with Flap endonuclease 1 (FEN1) and competes to process the RNA flaps of Okazaki fragments. Together, they restrained the DNA2 nuclease activity on the reversed forks of lagging strands, leading to replication fork (RF) stability in BRCA2-deficient cells. In these cells, MLH1 also attenuated R-loops, allowing the progression of stable RFs, which suppressed genomic instability and supported cell viability. We demonstrated the significance of their genetic interaction by the lethality of Brca2-mutant mice and inhibition of Brca2-deficient tumor growth in mice by Mlh1 loss. Furthermore, we described estrogen as inducing MLH1 expression through estrogen receptor α (ERα), which might explain why the majority of BRCA2 mutation carriers develop ER-positive breast cancer. Taken together, our findings reveal a role of MLH1 in relieving replicative stress and show how it may contribute to the establishment of BRCA2-deficient breast tumors.

Transcription factor C/EBPbeta isoform ratio regulates osteoclastogenesis through MafB.

Disequilibrium between bone-forming osteoblasts and bone-resorbing osteoclasts is central to many bone diseases. Here, we show that dysregulated expression of translationally controlled isoforms of CCAAT/enhancer-binding protein beta (C/EBPbeta) differentially affect bone mass. Alternative translation initiation that is controlled by the mammalian target of rapamycin (mTOR) pathway generates long transactivating (LAP(*), LAP) and a short repressive (LIP) isoforms from a single C/EBPbeta transcript. Rapamycin, an inhibitor of mTOR signalling increases the ratio of LAP over LIP and inhibits osteoclastogenesis in wild type (WT) but not in C/EBPbeta null (c/ebpbeta(-/-)) or in LIP knock-in (L/L) osteoclast precursors. C/EBPbeta mutant mouse strains exhibit increased bone resorption and attenuated expression of MafB, a negative regulator of osteoclastogenesis. Ectopic expression of LAP and LIP in monocytes differentially affect the MafB promoter activity, MafB gene expression and dramatically affect osteoclastogenesis. These data show that mTOR regulates osteoclast formation by modulating the C/EBPbeta isoform ratio, which in turn affects osteoclastogenesis by regulating MafB expression.

CCAAT/enhancer binding protein delta (C/EBPdelta, CEBPD)-mediated nuclear import of FANCD2 by IPO4 augments cellular response to DNA damage.

Maintenance of genomic integrity is an essential cellular function. We previously reported that the transcription factor and tumor suppressor CCAAT/enhancer binding protein δ (C/EBPδ, CEBPD; also known as "NFIL-6ß") promotes genomic stability. However, the molecular mechanism was not known. Here, we show that C/EBPδ is a DNA damage-induced gene, which supports survival of mouse bone marrow cells, mouse embryo fibroblasts (MEF), human fibroblasts, and breast tumor cells in response to the DNA cross-linking agent mitomycin C (MMC). Using gene knockout, protein depletion, and overexpression studies, we found that C/EBPδ promotes monoubiquitination of the Fanconi anemia complementation group D2 protein (FANCD2), which is necessary for its function in replication-associated DNA repair. C/EBPδ interacts with FANCD2 and importin 4 (IPO4, also known as "Imp4" and "RanBP4") via separate domains, mediating FANCD2-IPO4 association and augmenting nuclear import of FANCD2, a prerequisite for its monoubiquitination. This study identifies a transcription-independent activity of C/EBPδ in the DNA damage response that may in part underlie its tumor suppressor function. Furthermore, we report a function of IPO4 and nuclear import in the Fanconi anemia pathway of DNA repair.

C/EBP{delta} targets cyclin D1 for proteasome-mediated degradation via induction of CDC27/APC3 expression.

The transcription factor CCAAT/enhancer binding protein delta (C/EBPdelta, CEBPD, NFIL-6beta) has tumor suppressor function; however, the molecular mechanism(s) by which C/EBPdelta exerts its effect are largely unknown. Here, we report that C/EBPdelta induces expression of the Cdc27 (APC3) subunit of the anaphase promoting complex/cyclosome (APC/C), which results in the polyubiquitination and degradation of the prooncogenic cell cycle regulator cyclin D1, and also down-regulates cyclin B1, Skp2, and Plk-1. In C/EBPdelta knockout mouse embryo fibroblasts (MEF) Cdc27 levels were reduced, whereas cyclin D1 levels were increased even in the presence of activated GSK-3beta. Silencing of C/EBPdelta, Cdc27, or the APC/C coactivator Cdh1 (FZR1) in MCF-10A breast epithelial cells increased cyclin D1 protein expression. Like C/EBPdelta, and in contrast to cyclin D1, Cdc27 was down-regulated in several breast cancer cell lines, suggesting that Cdc27 itself may be a tumor suppressor. Cyclin D1 is a known substrate of polyubiquitination complex SKP1/CUL1/F-box (SCF), and our studies show that Cdc27 directs cyclin D1 to alternative degradation by APC/C. These findings shed light on the role and regulation of APC/C, which is critical for most cellular processes.

Stabilization of E-cadherin adhesions by COX-2/GSK3ß signaling is a targetable pathway in metastatic breast cancer.

Metastatic progression of epithelial cancers can be associated with epithelial-mesenchymal transition (EMT) including transcriptional inhibition of E-cadherin (CDH1) expression. Recently, EM plasticity (EMP) and E-cadherin-mediated, cluster-based metastasis and treatment resistance have become more appreciated. However, the mechanisms that maintain E-cadherin expression in this context are less understood. Through studies of inflammatory breast cancer (IBC) and a 3D tumor cell "emboli" culture paradigm, we discovered that cyclooxygenase 2 (COX-2; PTGS2), a target gene of C/EBPδ (CEBPD), or its metabolite prostaglandin E2 (PGE2) promotes protein stability of E-cadherin, ß-catenin, and p120 catenin through inhibition of GSK3ß. The COX-2 inhibitor celecoxib downregulated E-cadherin complex proteins and caused cell death. Coexpression of E-cadherin and COX-2 was seen in breast cancer tissues from patients with poor outcome and, along with inhibitory GSK3ß phosphorylation, in patient-derived xenografts (PDX) including triple negative breast cancer (TNBC).Celecoxib alone decreased E-cadherin protein expression within xenograft tumors, though CDH1 mRNA levels increased, and reduced circulating tumor cell (CTC) clusters. In combination with paclitaxel, celecoxib attenuated or regressed lung metastases. This study has uncovered a mechanism by which metastatic breast cancer cells can maintain E-cadherin-mediated cell-to-cell adhesions and cell survival, suggesting that some patients with COX-2+/E-cadherin+ breast cancer may benefit from targeting of the PGE2 signaling pathway.

PERK signaling through C/EBPδ contributes to ER stress-induced expression of immunomodulatory and tumor promoting chemokines by cancer cells.

Cancer cells experience endoplasmic reticulum (ER) stress due to activated oncogenes and conditions of nutrient deprivation and hypoxia. The ensuing unfolded protein response (UPR) is executed by ATF6, IRE1 and PERK pathways. Adaptation to mild ER stress promotes tumor cell survival and aggressiveness. Unmitigated ER stress, however, will result in cell death and is a potential avenue for cancer therapies. Because of this yin-yang nature of ER stress, it is imperative that we fully understand the mechanisms and dynamics of the UPR and its contribution to the complexity of tumor biology. The PERK pathway inhibits global protein synthesis while allowing translation of specific mRNAs, such as the ATF4 transcription factor. Using thapsigargin and tunicamycin to induce acute ER stress, we identified the transcription factor C/EBPδ (CEBPD) as a mediator of PERK signaling to secretion of tumor promoting chemokines. In melanoma and breast cancer cell lines, PERK mediated early induction of C/EBPδ through ATF4-independent pathways that involved at least in part Janus kinases and the STAT3 transcription factor. Transcriptional profiling revealed that C/EBPδ contributed to 20% of thapsigargin response genes including chaperones, components of ER-associated degradation, and apoptosis inhibitors. In addition, C/EBPδ supported the expression of the chemokines CXCL8 (IL-8) and CCL20, which are known for their tumor promoting and immunosuppressive properties. With a paradigm of short-term exposure to thapsigargin, which was sufficient to trigger prolonged activation of the UPR in cancer cells, we found that conditioned media from such cells induced cytokine expression in myeloid cells. In addition, activation of the CXCL8 receptor CXCR1 during thapsigargin exposure supported subsequent sphere formation by cancer cells. Taken together, these investigations elucidated a novel mechanism of ER stress-induced transmissible signals in tumor cells that may be particularly relevant in the context of pharmacological interventions.

Slug and E-Cadherin: Stealth Accomplices?

During physiological epithelial-mesenchymal transition (EMT), which is important for embryogenesis and wound healing, epithelial cells activate a program to remodel their structure and achieve a mesenchymal fate. In cancer cells, EMT confers increased invasiveness and tumor-initiating capacity, which contribute to metastasis and resistance to therapeutics. However, cellular plasticity that navigates between epithelial and mesenchymal states and maintenance of a hybrid or partial E/M phenotype appears to be even more important for cancer progression. Besides other core EMT transcription factors, the well-characterized Snail-family proteins Snail (SNAI1) and Slug (SNAI2) play important roles in both physiological and pathological EMT. Often mentioned in unison, they do, however, differ in their functions in many scenarios. Indeed, Slug expression does not always correlate with complete EMT or loss of E-cadherin (CDH1). For example, Slug plays important roles in mammary epithelial cell progenitor cell lineage commitment and differentiation, DNA damage responses, hematopoietic stem cell self-renewal, and in pathologies such as pulmonary fibrosis and atherosclerosis. In this Perspective, we highlight Slug functions in mammary epithelial cells and breast cancer as a "non-EMT factor" in basal epithelial cells and stem cells with focus reports that demonstrate co-expression of Slug and E-cadherin. We speculate that Slug and E-cadherin may cooperate in normal mammary gland and breast cancer/stem cells and advocate for functional assessment of such Slug+/E-cadherinlow/+ (SNAI2+/CDH1low/+) "basal-like epithelial" cells. Thus, Slug may be regarded as less of an EMT factor than driver of the basal epithelial cell phenotype.

C/EBPδ protects from radiation-induced intestinal injury and sepsis by suppression of inflammatory and nitrosative stress.

Ionizing radiation (IR)-induced intestinal damage is characterized by a loss of intestinal crypt cells, intestinal barrier disruption and translocation of intestinal microflora resulting in sepsis-mediated lethality. We have shown that mice lacking C/EBPδ display IR-induced intestinal and hematopoietic injury and lethality. The purpose of this study was to investigate whether increased IR-induced inflammatory, oxidative and nitrosative stress promote intestinal injury and sepsis-mediated lethality in Cebpd-/- mice. We found that irradiated Cebpd-/- mice show decreased villous height, crypt depth, crypt to villi ratio and expression of the proliferation marker, proliferating cell nuclear antigen, indicative of intestinal injury. Cebpd-/- mice show increased expression of the pro-inflammatory cytokines (Il-6, Tnf-α) and chemokines (Cxcl1, Mcp-1, Mif-1α) and Nos2 in the intestinal tissues compared to Cebpd+/+ mice after exposure to TBI. Cebpd-/- mice show decreased GSH/GSSG ratio, increased S-nitrosoglutathione and 3-nitrotyrosine in the intestine indicative of basal oxidative and nitrosative stress, which was exacerbated by IR. Irradiated Cebpd-deficient mice showed upregulation of Claudin-2 that correlated with increased intestinal permeability, presence of plasma endotoxin and bacterial translocation to the liver. Overall these results uncover a novel role for C/EBPδ in protection against IR-induced intestinal injury by suppressing inflammation and nitrosative stress and underlying sepsis-induced lethality.

C/EBPδ links IL-6 and HIF-1 signaling to promote breast cancer stem cell-associated phenotypes.

To improve cancer patient outcome significantly, we must understand the mechanisms regulating stem-like cancer cells, which have been implicated as a cause of metastasis and treatment resistance. The transcription factor C/EBPδ can exhibit pro- and anti-tumorigenic activities, but the mechanisms underlying the complexity of its functions are poorly understood. Here we identify a role for breast cancer cell intrinsic C/EBPδ in promoting phenotypes that have been associated with cancer stem cells (CSCs). While C/EBPδ expression is not abundant in most metastatic breast cancers, our data support a pro-tumorigenic role of C/EBPδ when expressed in subsets of tumor cells and/or through transient activation by the tumor microenvironment or loss of substrate adhesion. Using genetic mouse models and human breast cancer cell lines, we show that deletion or depletion of C/EBPδ reduced expression of stem cell factors and stemnness markers, sphere formation and self-renewal, along with growth of tumors and established experimental metastases in vivo. C/EBPδ is also known as a mediator of the innate immune response, which is enhanced by hypoxia and interleukin-6 (IL-6) signaling, two conditions that also play important roles in cancer progression. Our mechanistic data reveal C/EBPδ as a link that engages two positive feedback loops, in part by directly targeting the IL-6 receptor (IL6RA) gene, and, thus, amplifying IL-6 and HIF-1 signaling. This study provides a molecular mechanism for the synergism of tumor microenvironmental conditions in cancer progression with potential implications for the targeting of CSCs.

An essential role for a MEK-C/EBP pathway during growth factor-regulated cortical neurogenesis.

Mammalian neurogenesis is determined by an interplay between intrinsic genetic mechanisms and extrinsic cues such as growth factors. Here we have defined a signaling cascade, a MEK-C/EBP pathway, that is essential for cortical progenitor cells to become postmitotic neurons. Inhibition of MEK or of the C/EBP family of transcription factors inhibits neurogenesis while expression of a C/EBPbeta mutant that is a phosphorylation-mimic at a MEK-Rsk site enhances neurogenesis. C/EBP mediates this positive effect by direct transcriptional activation of neuron-specific genes such as Talpha1 alpha-tubulin. Conversely, inhibition of C/EBP-dependent transcription enhances CNTF-mediated generation of astrocytes from the same progenitor cells. Thus, activation of a MEK-C/EBP pathway enhances neurogenesis and inhibits gliogenesis, thereby providing a mechanism whereby growth factors can selectively bias progenitors to become neurons during development.

SLC5A8 triggers tumor cell apoptosis through pyruvate-dependent inhibition of histone deacetylases.

Tumor cells up-regulate glycolysis but convert pyruvate into lactate instead of oxidizing it. Here, we show that pyruvate, but not lactate, is an inhibitor of histone deacetylases (HDAC) and an inducer of apoptosis in tumor cells and that SLC5A8, a Na(+)/monocarboxylate cotransporter, is obligatory for this process. We found that SLC5A8 is expressed in nontransformed breast epithelial cell lines but silenced by DNA methylation in tumor cell lines. The down-regulation of the gene is also evident in primary breast tumors. When MCF7 breast tumor cells are transfected with SLC5A8 cDNA, the cells undergo pyruvate-dependent apoptosis. Butyrate and propionate also induce apoptosis in SLC5A8-expressing cells, whereas lactate does not. The differential ability of these monocarboxylates to cause apoptosis in SLC5A8-expressing MCF7 cells correlates with their ability to inhibit HDACs. Apoptosis induced by SLC5A8/pyruvate in MCF7 cells is associated with up-regulation of p53, Bax, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), TRAIL receptor (TRAILR) 1, and TRAILR2 and down-regulation of Bcl2 and survivin. Lactate dehydrogenase isozymes are differentially expressed in nontransformed cells and tumor cells such that the latter convert pyruvate into lactate. Silencing of SLC5A8 coupled with conversion of pyruvate into lactate in tumor cells correlates with increased HDAC activity in these cells compared with nontransformed cells. Our studies thus identify pyruvate as a HDAC inhibitor and indicate that the Na(+)-coupled pyruvate transport underlies the tumor-suppressive role of SLC5A8. We propose that tumor cells silence SLC5A8 and convert pyruvate into lactate as complementary mechanisms to avoid pyruvate-induced cell death.

Scientific Summary from the Morgan Welch MD Anderson Cancer Center Inflammatory Breast Cancer (IBC) Program 10th Anniversary Conference.

In 2006, a remarkable collaboration between University of Texas MD Anderson Cancer Center clinicians and Texas and New Mexico State legislators led to the formation of a dedicated IBC Research Program and Clinic at MD Anderson. This initiative provided funding and infrastructure to foster coordination of an IBC World Consortium of national and international experts, and launch the first ever IBC international conference in 2008, which brought together experts from around the world to facilitate collaborations and accelerate progress. Indeed great progress has been made since then. National and international experts in IBC convened at the 10th Anniversary Conference of the MD Anderson IBC Clinic and Research Program and presented the most extensive sequencing analysis to date comparing IBC to non-IBC, gene- and protein-based immunoprofiling of IBC versus non-IBC patients, and converging lines of evidence on the specific role of the microenvironment in IBC. Novel models, unique metabolic mechanisms, and prominent survival pathways have been identified and were presented. Multiple clinical trials based on the work of the last decade are in progress or in development. The important challenges ahead were discussed. This progress and a coordinated summary of these works are presented herein.

Comparison of mammary gland involution between 129S1 and C57BL/6 inbred mouse strains: differential regulation of Bcl2a1, Trp53, Cebpb, and Cebpd expression.

Genetic engineering has made the mouse an invaluable tool to address the function of individual genes in a targeted manner. Over the last decade it has become apparent that the genetic mouse strain background can significantly influence the phenotype of an engineered mouse. Therefore, it is essential to characterize the biology of the different wild-type background strains. In this study, we have compared mouse mammary gland involution in the 129S1 and C57BL/6 inbred strains and report significant differences at the molecular level with differential expression of Bcl2a1 (Bfl1), Trp53 (p53), Cebpb (C/EBP beta), and Cebpd (C/EBP delta). The C57BL/6 strain exhibits dynamic responses with induction of Trp53 and Cebpd and concomitant downregulation of Bcl2a1 during the first phase of involution. In contrast, expression of these genes does not change significantly in 129S1 mice. During the second phase, C57BL/6 glands contain more Cebpb than 129S1 glands. Nevertheless, involution proceeds morphologically with similar kinetics in both strains. The data demonstrate that the genetic response of mammary tissue varies significantly between 129S1 and C57BL/6. These results may provide a basis for the interpretation of strain-specific phenotypes in engineered mice and underline the importance of pure strains for large-scale expression studies with mutant mice.

Loss of CCAAT/enhancer binding protein delta promotes chromosomal instability.

The transcription factor CCAAT/enhancer binding protein delta (Cebpd, also known as C/EBPdelta, CRP3, CELF, NF-IL6beta) is implicated in diverse cellular functions such as the acute phase response, adipocyte differentiation, learning and memory, and mammary epithelial cell growth control. Here, we report that lack of Cebpd causes genomic instability and centrosome amplifications in primary embryonic fibroblasts derived from 129S1 mice. Upon spontaneous immortalization, Cebpd-deficient fibroblasts acquire transformed features such as impaired contact inhibition and reduced serum dependence. These data identify a novel role for Cebpd in the maintenance of chromosomal stability and suggest a potential tumor suppressor function in vivo.

Repression of the inhibin alpha-subunit gene by the transcription factor CCAAT/enhancer-binding protein-beta.

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 after 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 after treatment with human chorionic gonadotropin, indicating that C/EBPbeta may function to repress inhibin gene expression. The expression and regulation of C/EBPbeta were examined in rodent ovary, and these studies show that C/EBPbeta is expressed in ovary and granulosa cells and is induced in response to human chorionic gonadotropin. 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 activities. 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). Electrophoretic mobility shift assays show that C/EBPbeta effectively competes with CRE-binding protein 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.