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/EBPβ (CCAAT/Enhancerbinding protein-β) is a critical downstream mediator of ERK1/2 activation. Thus, ERK1/2 and C/ EBPβ constitute an in vivo LH-regulated signaling pathway that controls ovulation-and luteinization-related events.In the mammalian ovary, the female germ cells (oocytes) reside within the ovarian follicles and are surrounded by somatic cell-derived granulosa cells (GCs) and cumulus cells that have endocrine functions and control oocyte maturation. Female reproductive success depends on the growth of ovarian follicles and differentiation of GCs as well as oocyte maturation and ovulation (1,2). Although LH plays a critical role in the initiation of ovulation and in the terminal differentiation of GCs to luteal cells that compose the corpora lutea (CLs) and produce progesterone, the precise molecular targets in these processes remain ill-defined. Cyclic adenosine 3´,5´-monophosphate (cAMP) is a well-known mediator of LH action, but LH also induces expression of the epidermal growth factor

A large number of intercellular signaling molecules have been identified that orchestrate female reproductive physiology. However, with the exception of steroid hormone receptors, little information exists about the transcriptional regulators that mediate cellular responses to these signals. The transcription factor C/EBP␤ (CCAAT/enhancer-binding protein ␤) is expressed in ovaries and testes, as well as many other tissues of adult mice. Here we show that mice carrying a targeted deletion of the C/EBP␤ 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. In normal ovaries, C/EBP␤ mRNA is specifically induced by luteinizing hormone (LH/hCG) in the granulosa layer of preovulatory antral follicles. C/EBP␤-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␤ is essential for periovulatory granulosa cell differentiation in response to LH. C/EBP␤ is thus established as a critical downstream target of G-protein-coupled LH receptor signaling and one of the first transcription factors, other than steroid hormone receptors, known to be required for ovarian follicle development in vivo.

Studies of C/EBP␤-deficient mice have demonstrated a pivotal role for this transcription factor in hematopoiesis, adipogenesis, and ovarian function. Here we show that C/EBP␤ is also essential for normal development and function of the mammary gland. Ductal morphogenesis in virgin C/EBP␤-deficient mice was disrupted, with ducts displaying reduced growth and branching. To distinguish whether the effect of C/EBP␤ deficiency on mammary epithelium is indirect or cell autonomous, we performed ovarian and mammary gland transplants. Transplants of wild-type ovaries into mutant females partially restored ductal morphogenesis during puberty but failed to support mammopoiesis during pregnancy. At term, mutant mice harboring wild-type ovaries exhibited reduced alveolar proliferation and impaired epithelial cell differentiation, including a complete absence of milk protein expression. Mammary gland transplant experiments demonstrated that development of C/EBP␤-deficient epithelium was defective within a wild-type stroma and host background. Cell proliferation during pregnancy was reduced and differentiation, as measured by the activity of milk protein genes, was inhibited. However, wild-type epithelium developed in a C/EBP␤-deficient stroma. Thus, C/EBP␤ plays an essential, cell autonomous role in the proliferation and differentiation of mammary secretory epithelial cells and is required for the activation of milk protein genes.

The basic leucine zipper transcription factor CCAAT͞enhancer binding protein-␤ (C͞EBP␤) is expressed in many cell types, including keratinocytes. C͞EBP␤ activity can be increased by phosphorylation through pathways stimulated by oncogenic Ras, although the biological implications of Ras-C͞EBP␤ signaling are not currently understood. We report here that C͞EBP␤-nullizygous mice are completely refractory to skin tumor development induced by a variety of carcinogens and carcinogenesis protocols, including 7,12-dimethylbenz-[a]anthracene-initiation͞12-O-tetradecanoylphorbol 13-acetate promotion, that produce tumors containing oncogenic Ras mutations. No significant differences in TPA-induced epidermal keratinocyte proliferation were observed in C͞EBP␤-null versus wild-type mice. However, apoptosis was significantly elevated (17-fold) in the epidermal keratinocytes of 7,12-dimethylbenz[a]anthracene-treated C͞EBP␤-null mice compared with wild-type mice. In v-Ha-ras transgenic mice, C͞EBP␤ deficiency also led to greatly reduced skin tumor multiplicity and size, providing additional evidence for a tumorigenesis pathway linking Ras and C͞EBP␤. Oncogenic Ras potently stimulated C͞EBP␤ to activate a C͞EBP-responsive promoter-reporter in keratinocytes and mutating an ERK1͞2 phosphorylation site (T188) in C͞EBP␤ abolished this Ras effect. Finally, we observed that C͞EBP␤ participates in oncogenic Ras-induced transformation of NIH 3T3 cells. These findings indicate that C͞EBP␤ has a critical role in Ras-mediated tumorigenesis and cell survival and implicate C͞EBP␤ as a target for tumor inhibition.T he Ras family of GTP binding proteins function as intracellular mediators of extracellular signals to regulate cell proliferation, apoptosis, survival, senescence, and differentiation (1-5). Ras protooncogenes are frequently mutated in tumors, and Ϸ25% of human cancers contain transforming mutations in ras. Therefore, understanding oncogenic Ras-signaling pathways is critical for elucidating the mechanisms that underlie cellular transformation and for designing effective therapeutic strategies to prevent the development or block the growth of many classes of tumors. Ras has numerous effectors, and its pathways are multifaceted (3, 6, 7). Ras activation by growth factors or oncogenic mutations elicits activation of several transcription factors, which in turn regulate the expression of genes that control the cellular responses to Ras signaling, including oncogenesis. The transcription factors Ets, c-jun, c-myc, and NF-B are known to have roles in oncogenic ras-induced cellular transformation (8-11).The basic leucine zipper (bZIP) transcription factor CCAAT͞ enhancer binding protein-␤ (C͞EBP␤, also known as NF-IL6, IL-6DBP, LAP, CRP2, and NF-M) is expressed in a variety of cell types (12, 13) including keratinocytes (14,15), where it plays a role in squamous differentiation (16). C͞EBP␤ is also involved in regulating differentiation of specific mesenchymal, epithelial, and hematopoietic cell types (17-21). C͞EBP␤ activity can be activ...

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.

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Inflammation and hypoxia are known to promote the metastatic progression of tumours. The CCAAT/enhancerbinding protein-d (C/EBPd, 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/EBPd using transgenic mice overexpressing the Neu/Her2/ERBB2 proto-oncogene in the mammary gland. Unexpectedly, this study also revealed that C/EBPd is necessary for efficient tumour metastasis. We show that C/EBPd is induced by hypoxia in tumours in vivo and in breast tumour cells in vitro, and that C/EBPd-deficient cells exhibit reduced glycolytic metabolism and cell viability under hypoxia. C/EBPd supports CXCR4 expression. On the other hand, C/EBPd 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/EBPd enhances mTOR/AKT/S6K1 signalling and augments translation and activity of hypoxia-inducible factor-1a (HIF-1a), which is necessary for hypoxia adaptation. This work provides new insight into the mechanisms by which metastasis-promoting signals are induced specifically under hypoxia.

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/enhancerbinding protein b (C/EBPb) 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/EBPb 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/EBPb null (c/ebpb À/À ) or in LIP knock-in (L/L) osteoclast precursors. C/EBPb 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/EBPb isoform ratio, which in turn affects osteoclastogenesis by regulating MafB expression.