Esta Sterneck

MAPK3/1 (ERK1/2) in Ovarian Granulosa Cells Are Essential for Female Fertility

Regulating Oocyte Maturation Understanding exactly how ovarian follicles mature to generate fertile eggs is key to many aspects of fertility treatment. When the pituitary surge of luteinizing hormone (LH) binds to its receptor on granulosa cells of preovulatory follicles, a cascade of signaling events triggers granulosa cells to become luteal cells and the oocyte to resume meiosis. Fan et al. (p. 938; see the Perspective by Duggavathi and Murphy), using the mouse as a model system, targeted disruption of the kinases ERK1 and ERK2 selectively in granulosa cells. The kinases were essential in vivo mediators of LH induction of ovulation and luteinization. Targeted disruption of the kinases derails the molecular events that mediate induction of female reproductive development. 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/Enhancer-binding 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.

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.

CCAAT/enhancer binding protein-β is a mediator of keratinocyte survival and skin tumorigenesis involving oncogenic Ras signaling

The basic leucine zipper transcription factor CCAAT/enhancer binding protein-β (C/EBPβ) is expressed in many cell types, including keratinocytes. C/EBPβ activity can be increased by phosphorylation through pathways stimulated by oncogenic Ras, although the biological implications of Ras-C/EBPβ signaling are not currently understood. We report here that C/EBPβ-nullizygous mice are completely refractory to skin tumor development induced by a variety of carcinogens and carcinogenesis protocols, including 7,12-dimethylbenz[a]anthracene-initiation/12-O-tetradecanoylphorbol 13-acetate promotion, that produce tumors containing oncogenic Ras mutations. No significant differences in TPA-induced epidermal keratinocyte proliferation were observed in C/EBPβ-null versus wild-type mice. However, apoptosis was significantly elevated (17-fold) in the epidermal keratinocytes of 7,12-dimethylbenz[a]anthracene-treated C/EBPβ-null mice compared with wild-type mice. In v-Ha-ras transgenic mice, C/EBPβ deficiency also led to greatly reduced skin tumor multiplicity and size, providing additional evidence for a tumorigenesis pathway linking Ras and C/EBPβ. Oncogenic Ras potently stimulated C/EBPβ to activate a C/EBP-responsive promoter-reporter in keratinocytes and mutating an ERK1/2 phosphorylation site (T188) in C/EBPβ abolished this Ras effect. Finally, we observed that C/EBPβ participates in oncogenic Ras-induced transformation of NIH 3T3 cells. These findings indicate that C/EBPβ has a critical role in Ras-mediated tumorigenesis and cell survival and implicate C/EBPβ as a target for tumor inhibition.

Interleukin-6-Specific Activation of the C/EBPδ Gene in Hepatocytes Is Mediated by Stat3 and Sp1

ABSTRACT C/EBPδ (CCAAT/enhancer binding protein δ) has been implicated as a regulator of acute-phase response (APR) genes in hepatocytes. Its expression increases dramatically in liver during the APR and can be induced in hepatic cell lines by interleukin-6 (IL-6), an acute-phase mediator that activates transcription of many APR genes. Here we have investigated the mechanism by which C/EBPδ expression is regulated by IL-6 in hepatoma cells. C/EBPδ promoter sequences to −125 bp are sufficient for IL-6 inducibility of a reporter gene and include an APR element (APRE) that is essential for IL-6 responsiveness. DNA binding experiments and transactivation assays demonstrate that Stat3, but not Stat1, interacts with this APRE. Two Sp1 sites, one of which is adjacent to the APRE, are required for IL-6 induction and transactivation by Stat3. Thus, Stat3 and Sp1 function cooperatively to activate the C/EBPδ promoter. Replacement of the APRE with Stat binding elements (SBEs) from the ICAM-1 or C/EBPβ promoter, both of which recognize both Stat1 and Stat3, confers responsiveness to gamma interferon, a cytokine that selectively activates Stat1. Sequence comparisons suggest that the distinct Stat binding specificities of the C/EBPδ and C/EBPβ SBEs are determined primarily by a single base pair difference. Our findings indicate that the cytokine specificity of C/EBPδ gene expression is governed by the APRE sequence.

The ability of C/EBP beta but not C/EBP alpha to synergize with an Sp1 protein is specified by the leucine zipper and activation domain.

The rat CYP2D5 P-450 gene is activated in the liver during postnatal development. We previously showed that liver-specific transcription of the CYP2D5 gene is dictated by a proximal promoter element, termed 2D5, that is composed of a binding site for Sp1 or a related factor, and an adjacent cryptic C/EBP (CCAAT/enhancer-binding protein) site. Despite the fact that both C/EBP alpha and C/EBP beta are expressed abundantly in liver, only C/EBP beta is capable of stimulating the 2D5 promoter in HepG2 hepatocarcinoma cells. In addition, activation of the 2D5 promoter by C/EBP beta is completely dependent on the presence of the Sp1 site. Domain switch experiments reveal that C/EBP beta proteins containing either the leucine zipper or the activation domain of C/EBP alpha are unable to stimulate the 2D5 promoter yet are fully capable of transactivating an artificial promoter bearing a high-affinity C/EBP site. Thus, the leucine zipper and the activation domain of C/EBP beta are absolutely required to support transactivation of the 2D5 promoter. Using Drosophila cells that lack endogenous Sp1 activity, we show that the serine/threonine- and glutamine-rich activation domains A and B of Sp1 are required for efficient cooperatively with C/EBP beta. Furthermore, analysis of c/ebp beta-deficient mice shows that mutant animals are defective in expression of a murine CYP2D5 homolog in hepatic cells, confirming the selective ability of C/EBP beta to activate this liver-specific P-450 gene in vivo. Our findings illustrate that two members of a transcription factor family can achieve distinct target gene specificities through differential interactions with a cooperating Sp1 protein.

C/EBPbeta modulates the early events of keratinocyte differentiation involving growth arrest and keratin 1 and keratin 10 expression.

ABSTRACT The epidermis is a stratified squamous epithelium composed primarily of keratinocytes that become postmitotic and undergo sequential changes in gene expression during terminal differentiation. The expression of the transcription factor CCAAT/enhancer binding protein β (C/EBPβ) within mouse epidermis and primary keratinocytes has recently been described; however, the function of C/EBPβ within the epidermal keratinocyte is unknown. We report here that transient transfection of mouse primary keratinocytes with a C/EBP-responsive promoter-reporter construct resulted in a sevenfold increase in luciferase activity when keratinocytes were switched to culture conditions that induce growth arrest and differentiation. Forced expression of C/EBPβ in BALB/MK2 keratinocytes inhibited growth, induced morphological changes consistent with a more differentiated phenotype, and upregulated two early markers of differentiation, keratin 1 (K1) and keratin 10 (K10) but had a minimal effect on the expression of late-stage markers, loricrin and involucrin. Analysis of the epidermis of C/EBPβ-deficient mice revealed a mild epidermal hyperplasia and decreased expression of K1 and K10 but not of involucrin and loricrin. C/EBPβ-deficient primary keratinocytes were partially resistant to calcium-induced growth arrest. Analysis of terminally differentiated spontaneously detached keratinocytes or those induced to differentiate by suspension culture revealed that C/EBPβ-deficient keratinocytes displayed striking decreases in K1 and K10, while expression of later-stage markers was only minimally altered. Our results demonstrate that C/EBPβ plays an important role in the early events of stratified squamous differentiation in keratinocytes involving growth arrest and K1 and K10 expression.

CCAAT/Enhancer Binding Protein β Is a Neuronal Transcriptional Regulator Activated by Nerve Growth Factor Receptor Signaling

Abstract: CCAAT/enhancer binding protein β (C/EBPβ) is a transcriptional regulator of the basic leucine zipper family. By in situ hybridization analysis, we found that C/EBPβ is widely expressed in the CNS of adult mice, including cells of the hippocampus and dentate gyrus and cerebellar Purkinje and granule cells. Expression of C/EBPβ had also been reported in the PC12 cell line, which undergoes differentiation to neuron‐like cells in response to nerve growth factor (NGF). We show that C/EBPβ mRNA expression increases while protein levels decrease during differentiation of PC12 cells. In transactivation assays, C/EBPβ activity was stimulated by NGF receptor signaling. Mutations of a phosphorylation site for mitogen‐activated protein kinase in C/EBPβ affected its capacity to transactivate in a promoter‐specific manner. Our data identify the C/EBPβ protein and gene as direct downstream targets of the NGF receptor and suggest a role for C/EBPβ in neurotrophin signaling in the brain.

C/EBPδ is a crucial regulator of pro-apoptotic gene expression during mammary gland involution

The STAT3 transcription factor is an important initiator of mammary gland involution in the mouse. This work shows that the STAT3 target gene CCAAT/enhancer binding protein delta (C/EBPδ) is a crucial mediator of pro-apoptotic gene expression events in mammary epithelial cells. In the absence of C/EBPδ, involution is delayed, the pro-apoptotic genes encoding p53, BAK, IGFBP5 and SGP2/clusterin are not activated, while the anti-apoptotic genes coding for BFL1 and Cyclin D1 are not repressed. Consequently, p53 targets such as survivin, BRCA1, BRCA2 and BAX are not regulated appropriately and protease activation is delayed. Furthermore, expression of MMP3 and C/EBPδ during the second phase of involution is perturbed in the absence of C/EBPδ. In HC11 cells, C/EBPδ alone is sufficient to induce IGFBP5 and SGP2. It also suppresses Cyclin D1 expression and cooperates with p53 to elicit apoptosis. This study places C/EBPδ between STAT3 and several pro- and anti-apoptotic genes promoting the physiological cell death response in epithelial cells at the onset of mammary gland involution.

Interleukin‐6 Induces Expression of Peripherin and Cooperates with Trk Receptor Signaling to Promote Neuronal Differentiation in PC12 Cells

Abstract: In contrast to the intensively studied nerve growth factor (NGF)‐related family of cytokines, relatively little is known about the mechanisms of neurotrophic activity elicited by the cytokine interleukin‐6 (IL‐6). We have examined the mechanisms of IL‐6‐induced neuronal differentiation of the pheochromocytoma cell line PC12. IL‐6 independently induced the expression of peripherin, identifying this gene as the first neuronal‐specific target of IL‐6. However, IL‐6 alone failed to elicit neurite outgrowth in PC12 cells and instead required low levels of Trk/NGF receptor tyrosine kinase activity to induce neuronal differentiation. The cooperating Trk signal could be provided by either overexpression of Trk or exposure to low concentrations of NGF. IL‐6 also functioned cooperatively with basic fibroblast growth factor to promote PC12 differentiation. IL‐6 and Trk/NGF synergized in enhancing tyrosine phosphorylation of the Erk‐1 mitogen‐activated protein kinase and in activating expression of certain NGF target genes. NGF also induced expression of the gp80 subunit of the IL‐6 receptor, providing another potential mechanism of cooperativity between NGF and IL‐6 signaling. We propose that IL‐6 functions as an enhancer of NGF signaling rather than as an autonomous neuronal differentiation signal. Moreover, our results demonstrate that a Trk receptor‐specific cellular response can be achieved in the absence of NGF through amplification of its basal signaling activity by the IL‐6 receptor system.

Feedback Inhibition of the Retinaldehyde Dehydrogenase GeneALDH1 by Retinoic Acid through Retinoic Acid Receptor α and CCAAT/Enhancer-binding Protein β

Aldehyde dehydrogenase 1 (ALDH1) plays a major role in the biosynthesis of retinoic acid (RA), a hormone required for several essential life processes. Recent evidence, using the aryl hydrocarbon receptor-null mouse, suggests that elevated hepatic RA down-regulates ALDH1 in a unique feedback pathway to control RA biosynthesis. To determine the mechanism of suppression of theALDH1 gene by RA, transactivation studies were carried out in Hepa-1 mouse hepatoma cells. RA decreased expression of an ALDH1-CAT construct containing −2536 base pairs of DNA upstream of the transcription start site. Retinoic acid receptor α (RARα) transactivates the ALDH1 gene promoter through a complex with an RA response-like element (RARE) located at −91/−75 bp, which bound to the RARα/retinoid X receptor β heterodimer. CCAAT/enhancer-binding protein (C/EBPβ) also transactivates theALDH1 gene promoter through a CCAAT box located 3′ and directly adjacent to the RARE, and the ALDH1 gene is down-regulated in C/EBPβ-null mouse liver. Exposure of Hepa-1 cells to RA results in a decrease in C/EBPβ mRNA levels; however, there was no difference in mRNA and protein levels between wild-type and AHR-null mouse liver. These data support a model in which the RARα and C/EBPβ activate the ALDH1 gene promoter through the RARE and C/EBP response elements, and in Hepa-1 cells, high levels of RA inhibit this activation by decreasing cellular levels of C/EBPβ.