Mary Hunzicker-Dunn

Follicle-stimulating Hormone Activates Extracellular Signal-regulated Kinase but Not Extracellular Signal-regulated Kinase Kinase through a 100-kDa Phosphotyrosine Phosphatase

In this report we sought to elucidate the mechanism by which the follicle-stimulating hormone (FSH) receptor signals to promote activation of the p42/p44 extracellular signal-regulated protein kinases (ERKs) in granulosa cells. Results show that the ERK kinase MEK and upstream intermediates Raf-1, Ras, Src, and L-type Ca2+ channels are already partially activated in vehicle-treated cells and that FSH does not further activate them. This tonic stimulatory pathway appears to be restrained at the level of ERK by a 100-kDa phosphotyrosine phosphatase that associates with ERK in vehicle-treated cells and promotes dephosphorylation of its regulatory Tyr residue, resulting in ERK inactivation. FSH promotes the phosphorylation of this phosphotyrosine phosphatase and its dissociation from ERK, relieving ERK from inhibition and resulting in its activation by the tonic stimulatory pathway and consequent translocation to the nucleus. Consistent with this premise, FSH-stimulated ERK activation is inhibited by the cell-permeable protein kinase A-specific inhibitor peptide Myr-PKI as well as by inhibitors of MEK, Src, a Ca2+ channel blocker, and chelation of extracellular Ca2+. These results suggest that FSH stimulates ERK activity in immature granulosa cells by relieving an inhibition imposed by a 100-kDa phosphotyrosine phosphatase.

Follicle-stimulating hormone/cAMP regulation of aromatase gene expression requires β-catenin

Estrogens profoundly influence the physiology and pathology of reproductive and other tissues. Consequently, emphasis has been placed on delineating the mechanisms underlying regulation of estrogen levels. Circulating levels of estradiol in women are controlled by follicle-stimulating hormone (FSH), which regulates transcription of the aromatase gene (CYP19A1) in ovarian granulosa cells. Previous studies have focused on two downstream effectors of the FSH signal, cAMP and the orphan nuclear receptor steroidogenic factor-1 (NR5A1). In this report, we present evidence for β-catenin (CTNNB1) as an essential transcriptional regulator of CYP19A1. FSH induction of select steroidogenic enzyme mRNAs, including Cyp19a1, is enhanced by β-catenin. Additionally, β-catenin is present in transcription complexes assembled on the endogenous gonad-specific CYP19A1 promoter, as evidenced by chromatin immunoprecipitation assays. Transient expression and RNAi studies demonstrate that FSH- and cAMP-dependent regulation of this promoter is sensitive to alterations in the level of β-catenin. The stimulatory effect of β-catenin is mediated through functional interactions with steroidogenic factor-1 that involve four acidic residues within its ligand-binding domain, mutation of which attenuates FSH/cAMP-induced Cyp19a1 mRNA accumulation. Together, these data demonstrate that β-catenin is essential for FSH/cAMP-regulated gene expression in the ovary, identifying a central and previously unappreciated role for β-catenin in estrogen biosynthesis, and a potential broader role in other aspects of follicular maturation.

Role of the phosphatidylinositol-3-kinase and extracellular regulated kinase pathways in the induction of hypoxia-inducible factor (HIF)-1 activity and the HIF-1 target vascular endothelial growth factor in ovarian granulosa cells in response to follicle-stimulating hormone.

FSH stimulation of granulosa cells (GCs) results in increased hypoxia-inducible factor (HIF)-1alpha protein levels and HIF-1 activity that is necessary for up-regulation of certain FSH target genes including vascular endothelial growth factor. We report that the role of the phosphatidylinositol (PI)-3-kinase/AKT pathway in increasing HIF-1alpha protein in FSH-stimulated GCs extends beyond an increase in mammalian target of rapamycin-stimulated translation. FSH increases phosphorylation of the AKT target mouse double-minute 2 (MDM2); a phosphomimetic mutation of MDM2 is sufficient to induce HIF-1 activity. The PI3-kinase/AKT target forkhead box-containing protein O subfamily 1 (FOXO1) also effects the accumulation of HIF-1alpha as evidenced by the ability of a constitutively active FOXO1 mutant to inhibit the induction by FSH of HIF-1alpha protein and HIF-1 activity. Activation of the PI3-kinase/AKT pathway in GCs by IGF-I is sufficient to induce HIF-1alpha protein but surprisingly not HIF-1 activity. HIF-1 activity also appears to require a PD98059-sensitive protein (kinase) activity stimulated by FSH that is both distinct from mitogen-activated ERK kinase1/2 or 5 and independent of the PI3-kinase/AKT pathway. These results indicate that FSH-stimulated HIF-1 activation leading to up-regulation of targets such as vascular endothelial growth factor requires not only PI3-kinase/AKT-mediated activation of mammalian target of rapamycin as well as phosphorylation of FOXO1 and possibly MDM2 but also a protein (kinase) activity that is inhibited by the classic ERK kinase inhibitor PD98059 but not ERK1/2 or 5. Thus, regulation of HIF-1 activity in GCs by FSH under normoxic conditions is complex and requires input from multiple signaling pathways.

Protein kinase C and meiotic regulation in isolated mouse oocytes.

In this study, the possible role of protein kinase C (PKC) in mediating both positive and negative actions on meiotic maturation in isolated mouse oocytes has been examined. When cumulus cell–enclosed oocytes (CEO) were cultured for 17–18 hr in a medium containing 4 mM hypoxanthine (HX) to maintain meiotic arrest, each of the five different activators and five different antagonists of PKC stimulated germinal vesicle breakdown (GVB) in a dose‐dependent fashion. One of the activators, phorbol‐12‐myristate 13‐acetate (PMA), also triggered GVB in CEO arrested with isobutylmethylxanthine or guanosine, but not in those arrested with dibutyryl cyclic AMP. When denuded oocytes (DO) were cultured for 3 hr in inhibitor‐free medium, all PKC activators suppressed maturation (< 10% GVB compared to 94% in controls), while the effect of PKC antagonists was negligible. Four of the five antagonists reversed the meiosis‐arresting action of HX in DO. PMA transiently arrested the spontaneous maturation of both CEO and DO, with greater potency in DO. The stimulatory action of PMA in HX‐arrested oocytes was dependent on cumulus cells, because meiotic induction occurred in CEO but not DO. PKC activators also preferentially stimulated cumulus expansion when compared to antagonists. A cell–cell coupling assay determined that the action of PMA on oocyte maturation was not due to a loss of metabolic coupling between the oocyte and cumulus oophorus. Finally, Western analysis demonstrated the presence of PKCs α, β1, δ, and ζ in both cumulus cells and oocytes, but only PKC ϵ was detected in the cumulus cells. It is concluded that direct activation of PKC in the oocyte suppresses maturation, while stimulation within cumulus cells generates a positive trigger that leads to meiotic resumption. Mol. Reprod. Dev. 58:101–115, 2001.

Signal Transduction Pathways Activated by Chorionic Gonadotropin in the Primate Endometrial Epithelial Cells

Abstract Successful implantation requires synergism between the developing embryo and the receptive endometrium. In the baboon, infusion of chorionic gonadotropin (CG) modulates both morphology and physiology of the epithelial and stromal cells of the receptive endometrium. This study explored the signal transduction pathways activated by CG in endometrial epithelial cells from baboon (BE) and human (HES). Incubations of BE and HES cells with CG did not significantly alter adenylyl cyclase activity or increase intracellular cAMP when compared with Chinese hamster ovarian cells stably transfected with the full-length human CG/luteinizing hormone (LH) receptor (CHO-LH cells). However, in BE and HES cells, CG induced the phosphorylation of several proteins, among them, extracellular signal-regulated protein kinases 1 and 2 (ERK 1/2). Phosphorylation of ERK 1/2 in uterine epithelial cells was protein kinase A (PKA) independent. This novel signaling pathway is functional because, in response to CG stimulation, prostaglandin E2 (PGE2) was released into the media and increased significantly 2 h following CG stimulation. CG-stimulated PGE2 synthesis in epithelial cells was inhibited by a specific mitogen-activated protein kinase (MEK 1/2) inhibitor, PD 98059. In conclusion, immediate signal transduction pathways induced by CG in endometrial epithelial cells are cAMP independent and stimulate phosphorylation of ERK 1/2 via a MEK 1/2 pathway, leading to an increase in PGE2 release as the possible result of cyclooxygenase-2 activation.

Conditional Deletion of Beta-Catenin Mediated by Amhr2cre in Mice Causes Female Infertility

Abstract Follicle-stimulating hormone (FSH) regulation of aromatase gene expression in vitro requires the transcriptional coactivator beta-catenin. To ascertain the physiological significance of beta-catenin in granulosa cells during folliculogenesis, mice homozygous for floxed alleles of beta-catenin were intercrossed with Amhr2cre mice. Conditional deletion of beta-catenin in 8-wk-old females occurred in derivatives of the Müllerian duct, granulosa cells and, surprisingly, in brain, pituitary, heart, liver, and tail. Female mice deficient for beta-catenin were infertile, despite reaching puberty and ovulating at the expected age, indications of apparently normal ovarian function. In contrast, their oviducts were grossly distended, with fewer but healthy oocytes. In addition, their uteri lacked implantation sites. Together, these two phenotypes could explain the complete loss of fertility. Nevertheless, although the ovary appeared normal, with serum estradiol concentrations in the normal range, there was marked animal-to-animal variation of mRNAs encoding beta-catenin and aromatase. Similarly, inhibin-alpha and luteinizing hormone receptor mRNAs varied considerably in whole ovaries, whereas pituitary Fshb mRNA was significantly reduced. Collectively, these features suggested cyclization recombination (CRE)-mediated recombination of beta-catenin may be unstable in proliferating granulosa cells, and therefore may mask the suspected steroidogenic requirement for beta-catenin. We tested this possibility by transducing primary cultures of granulosa cells from mice homozygous for floxed alleles of beta-catenin with a CRE-expressing adenovirus. Reduction of beta-catenin significantly compromised FSH stimulation of aromatase mRNA and subsequent production of estradiol. Collectively, these data suggest that FSH regulation of steroidogenesis requires beta-catenin, a role that remains hidden when tested through Amhr2cre-mediated recombination in vivo.

Association of PKC δ and active Src in PMA-treated MCF-7 human breast cancer cells

Phorbol ester treatment of MCF-7 cells led to the tyrosine phosphorylation and activation of PKC δ. However, through Western blot analysis and in vitro immunecomplex kinase assays, we detected a differential localization of tyrosine-phosphorylated PKC δ and catalytically active PKC δ. Catalytically active PKC δ was concentrated in Triton X-100 solubilized-membrane fractions while tyrosine-phosphorylated PKC δ was localized to the cytosol fraction. Phorbol ester treatment of MCF-7 cells stimulated both the time-dependent in vivo association of Src with PKC δ, evidenced in Src immunoprecipitates by the co-immunoprecipitation of PKC δ, and activation of Src, evidenced in Src immunoprecipitates as an increase in reactivity with a Src antibody (clone 28) reactive only with active Src (de-phosphorylated on residue 530) and in Src and PKC δ immunoprecipitates by an increase in Src kinase activity. While our data are consistent with reports in the literature showing the activator/stimulus-dependent tyrosine phosphorylation of PKC δ, our data show that the tyrosine phosphorylation of PKC δ is not essential for kinase activity. These results are the first to demonstrate an in vivo association between PKC δ and active Src in the absence of over-expression of either PKC δ or Src, and support the association of Src and PKC δ towards a physiological function.

ARF6: a newly appreciated player in G protein-coupled receptor desensitization

The luteinizing hormone/choriogonadotropin hormone receptor (LH/CG R) signals to regulate ovulation, corpus luteum formation, and fetal survival during pregnancy. Agonist binding to the LH/CG R is poorly reversible, emphasizing the importance of a cellular mechanism to temper signaling by a potentially persistently active receptor. Like other G protein‐coupled receptors (GPCRs), signaling by this receptor is modulated by its binding of an arrestin. We have identified ADP ribosylation factor 6 (ARF6) as a protein whose activation state is regulated by the LH/CG R and which functions to regulate the availability of plasma membrane‐docked arrestin 2 to this receptor. We hypothesize that ARF6 might also serve GPCRs other than the LH/CG R to regulate the availability of arrestin 2 for receptor desensitization.

Regulation of protein kinase C δ by estrogen in the MCF-7 human breast cancer cell line☆

Abstract We have previously shown that estrogen up-regulates expression of protein kinase C (PKC) δ in the rat and rabbit corpus luteum as well as in luteinized rat granulosa primary cell cultures. To determine whether a similar regulation of the PKC δ isoform by estrogen occurred in another estrogen responsive system, we investigated the estrogen receptor positive MCF-7 human breast cancer cells. In a characterization of PKC isoforms in MCF-7 cells we determined that PKC δ was the predominant PKC isoform. However in contrast to the effect of estrogen on PKC δ expression in ovarian cells, estrogen treatment of MCF-7 cells resulted in a significant decrease in PKC δ protein and mRNA expression in a time and dose dependent manner. Treatment of MCF-7 cells with 10 −10 –10 −8 M estrogen for 7 days down-regulated specifically PKC δ mRNA and protein while expression of other PKC isoforms was unchanged. The opposite regulation of PKC δ expression in ovarian and breast cancer cells prompted us to evaluate the type of estrogen receptor present in both cell types. Results showed that luteinized rat granulosa cells expressed predominantly estrogen receptor β while the MCF-7 cells expressed predominantly estrogen receptor α and barely detectable levels of estrogen receptor β. These results suggest that the differential ability of estrogen to regulate PKC δ expression could potentially be a result of differential signaling through the two estrogen receptor subtypes.

Luteinizing hormone/choriogonadotropin receptor-mediated activation of heterotrimeric guanine nucleotide binding proteins in ovarian follicular membranes

The LH/CG receptor signals to adenylyl cyclase via the stimulatory heterotrimeric GTP binding regulatory protein, Gs, and to phospholipase C and potentially to other effectors, such as ion channels, via a G protein or proteins that have not been identified in gonadal cells. To identify G proteins activated in a physiological membrane environment upon LH/CG receptor activation, we used the ability of activated G proteins to bind GTP and incubated ovarian follicular membranes with the photoaffinity GTP analog, P3-(4-azidoanilido)-P1-5′-GTP ([32P]AAGTP). Results showed that human CG (hCG) stimulated the binding of [32P]AAGTP to a 45-kDa protein(s) in follicular membranes that comigrated with immunoreactive Gαs, Gαq/11, and Gα13. When Gα proteins were immunoprecipitated from Triton X-100 solubilized membrane extracts after photoaffinity labeling with [32P]AAGTP, a time-dependent increase in hCG-dependent [32P]AAGTP binding to Gαs, Gαq/11, and Gαi was detected. hCG-dependent [32P]AAGTP binding to Gα13 was also...