Abraham Amsterdam

The ERK signaling cascade inhibits gonadotropin-stimulated steroidogenesis.

The response of granulosa cells to luteinizing hormone (LH) and follicle-stimulating hormone (FSH) is mediated mainly by cAMP/protein kinase A (PKA) signaling. Notably, the activity of the extracellular signal-regulated kinase (ERK) signaling cascade is elevated in response to these stimuli as well. We studied the involvement of the ERK cascade in LH- and FSH-induced steroidogenesis in two granulosa-derived cell lines, rLHR-4 and rFSHR-17, respectively. We found that stimulation of these cells with the appropriate gonadotropin induced ERK activation as well as progesterone production downstream of PKA. Inhibition of ERK activity enhanced gonadotropin-stimulated progesterone production, which was correlated with increased expression of the steroidogenic acute regulatory protein (StAR), a key regulator of progesterone synthesis. Therefore, it is likely that gonadotropin-stimulated progesterone formation is regulated by a pathway that includes PKA and StAR, and this process is down-regulated by ERK, due to attenuation of StAR expression. Our results suggest that activation of PKA signaling by gonadotropins not only induces steroidogenesis but also activates down-regulation machinery involving the ERK cascade. The activation of ERK by gonadotropins as well as by other agents may be a key mechanism for the modulation of gonadotropin-induced steroidogenesis.

Distribution of an α-bungarotoxin-binding cholinergic nicotinic receptor in rat brain

Cholinergic nicotinic receptors in rat brain were demonstrated by the use of the potent nicotinic antagonist [125I]alpha-bungarotoxin [125I]alpha-Btx). Biochemical studies on binding of [125I]alpha-Btx to rat hippocampal homogenates revealed saturable binding sites which are protected by nicotine, D-tuborcurarine and acetylcholine but not by atropine or oxotremorine. The hippocampus and hypothalamus displayed relatively high [125I]alpha-Btx specific binding whereas the cerebellum was devoid of specific binding. Other regions displayed intermediate binding levels. Analysis of the regional distribution of [125I]alpha-Btx binding by autoradiography of frontal brain sections revealed high labeling in the hippocampus, hypothalamic supraoptic, suprachiasmatic and periventricular nuclei, ventral lateral geniculate and the mesencephalic dorsal tegmental nucleus. It is suggested that the limbic forebrain and midbrain structures as well as sensory nuclei are the main nicotinic cholinoceptive structures in the brain.

Cell-specific regulation of apoptosis by glucocorticoids: implication to their anti-inflammatory action.

Glucocorticoids play a major role in attenuation of the inflammatory response. These steroid hormones are able to induce apoptosis in cells of the hematopoietic system such as monocytes, macrophages, and T lymphocytes that are involved in the inflammation reaction. In contrast, it was discovered recently that in glandular cells such as the mammary gland epithelia, hepatocytes, ovarian follicular cells, and in fibroblasts glucocorticoids protect against apoptotic signals evoked by cytokines, cAMP, tumor suppressors, and death genes. The anti-apoptotic effect of glucocorticoids is exerted by modulation of several survival genes such as Bcl-2, Bcl-x(L), and NFkB, in a cell-specific manner. Moreover, upregulation or downregulation of the same gene product can occur in a cell-dependent manner following stimulation by glucocorticoids. This phenomenon is probably due to composite regulatory cross-talk among multiple nuclear coactivators or corepressors, which mediate the transcription regulation of the genes, by their interaction with the glucocorticoid receptor. These observations suggest that the anti-inflammatory action of glucocorticoids is exerted by two complementary mechanisms: on one hand, they induce death of the cells that provoke the inflammation, and on the other hand they protect the resident cells of the inflamed tissue by arresting apoptotic signals. Moreover, the complementary action of glucocorticoids provides a new insight to the therapeutic potential of these hormones.

The Proteasome Is Involved in the First Metaphase-to-Anaphase Transition of Meiosis in Rat Oocytes

Abstract The proteasome engages in protein degradation as a regulatory process in biological transactions. Among other cellular processes, the proteasome participates in degradation of ubiquinated cyclins in mitosis. However, its role in meiosis has not been established. Resumption of meiosis in the oocyte involves the activation of maturation promoting factor (MPF), a complex of p34cdc2 and cyclin B. Inactivation of this factor, occurring between the two meiotic divisions, is associated with degradation of cyclin B. In this study, we examined the possible involvement of the proteasome in regulation of the exit from metaphase I in spontaneously maturing rat oocytes. We found that upon resumption of meiosis, proteasomes translocate to the spindle apparatus. We further demonstrated that specific inhibitors of proteasome catalytic activity, MG132 and lactacystin, blocked polar body extrusion. Chromosome and microtubule fluorescent staining verified that MG132-treated oocytes were arrested at metaphase I. Intervention of proteasomal action with this inhibitor also resulted in accumulation of cyclin B and elevated activity of MPF. These data demonstrate that proteasomal catalytic activity is absolutely essential for the decrease in MPF activity and completion of the first meiotic division. Its translocation to the spindle apparatus may facilitate the timely degradation of cyclin B.

Induction of Mdm2 and enhancement of cell survival by bFGF

Basic fibroblast growth factor (bFGF) can exert mitogenic and viability-promoting effects in a wide range of biological systems. The biochemical activities mediating the cell survival function of bFGF are largely unknown. We report here that exposure of fibroblasts to bFGF, which confers upon them increased survival, also causes at the same time an increase in cellular levels of the Mdm2 oncoprotein. Cells constitutively exposed to a bFGF autocrine loop are more refractory to killing by cisplatin. This increased chemoresistance coincides with elevated Mdm2 and reduced activation of the endogenous p53, resulting in inefficient transcriptional activation of the bax gene promoter. Importantly, unlike Mdm2 accumulation in fibroblasts exposed to DNA damage, induction of Mdm2 by bFGF does not occur through a p53-mediated pathway. The role of p53 in DNA damage-induced apoptosis and the ability of Mdm2 to block p53-mediated cell death are well established. These findings therefore suggest that induction of Mdm2 and the subsequent inhibition of p53 function may contribute, at least partially, to the anti-apoptotic effects of bFGF and possibly some other survival factors.

The anti-inflammatory action of glucocorticoids is mediated by cell type specific regulation of apoptosis

Glucocorticoids play a major role in attenuation of the inflammatory response. These steroid hormones are able to induce apoptosis in cells of the hematopoietic system such as monocytes, macrophages and T-lymphocytes that are involved in the inflammation reaction. In contrast, it was discovered recently that in glandular cells such as the mammary gland epithelia, hepatocytes, ovarian follicular cells and in fibroblasts glucocorticoids protect against apoptotic signals evoked by cytokines, cAMP, tumor suppressors and death genes. The anti-apoptotic effect of glucocorticoids is exerted by modulation of several survival genes such as Bcl-2, Bcl-x(L) and NFkappaB, in a cell type-specific manner. Moreover, up regulation or down regulation of the same gene product can occur in a cell type-dependent manner following stimulation by glucocorticoids. This phenomenon is probably due to composite regulatory cross-talk among multiple nuclear coactivators or corepressors, which mediate the transcriptional regulation of the genes, by their interaction with the glucocorticoid receptor (GR). These observations suggest that the anti-inflammatory action of glucocorticoids is exerted by two complementary mechanisms: on the one hand, they induce death of the cells that provoke the inflammation, and on the other hand, they protect the resident cells of the inflamed tissue by arresting apoptotic signals.

Differential effect of components of the extracellular matrix on differentiation and apoptosis

BACKGROUND Epithelial cells are closely associated with a basement membrane, but the intimate relationships that affect growth, differentiation and survival remain enigmatic. We have previously reported that granulosa cells adjacent to the basement membrane of the ovarian follicle have a higher degree of differentiation compared with cells located distal to the basement membrane. By contrast, granulosa cells distal to the basement membrane are the first to undergo apoptosis during follicular atresia. Moreover, growth of granulosa cells in vitro on a naturally produced basement-membrane-like extracellular matrix (ECM) enhances progesterone production and the cellular response to gonadotropic hormones by an undefined mechanism. RESULTS To investigate the effect of the ECM on granulosa cell differentiation and death, primary granulosa cells were cultured on ECMs that lacked or contained bFGF (basic fibroblast growth factor). These otherwise identical ECMs were deposited by HR9 mouse endodermal cells, which do not synthesize bFGF, or by HR9 cells transfected with the bFGF gene. Both ECMs provided protection against apoptosis in serum-free medium, but only the bFGF-containing ECM maintained expression of the steroidogenic P450scc enzyme system and the production of progesterone. Moreover, culturing the cells on this ECM enhanced the expression of the 30 kDa steroid acute regulatory protein which plays a key role in steroid hormone biosynthesis. Laminin, but not fibronectin, was able to replace the ECM in protecting the cells from apoptosis; but not in maintaining steroidogenesis, whereas bFGF was able to enhance steroidogenesis without protecting the cells against apoptosis. Cells cultured on both ECMs or laminin had a well-developed actin cytoskeleton compared with cells cultured on non-coated dishes, which underwent apoptosis. CONCLUSIONS Cellular responses to ECM are mediated by the combined action of macromolecular constituents and regulatory molecules, such as bFGF, that are sequestered and stored in the ECM. ECM or laminin protects against cell death by interacting with specific integrin receptors and maintaining a well-developed actin cytoskeleton. ECM-bound bFGF provides differentiation signals for granulosa cells, which are in intimate contact with the ECM. Thus, a clear distinction can be made between the survival activity and the differentiation stimulus exerted by the ECM on epithelial cells.

Gap junction communication and connexin 43 gene expression in a rat granulosa cell line : Regulation by follicle-stimulating hormone

Abstract Follicle-stimulating hormone is the major regulator of growth and development of antral follicles in the ovary. Granulosa cells (GCs) in these follicles are coupled via gap junctions (GJs) consisting of connexin 43 (Cx 43). Because we and others have found that Cx 43 and GJs, respectively, are more abundant in large antral follicles compared with small antral and preantral follicles, we hypothesized that FSH may control Cx 43 gene expression, GJ formation, and intercellular communication. To directly address these points, we chose a rat GC line (GFSHR-17) expressing the FSH receptor and the Cx 43 gene. The functionality of FSH receptors was shown by the effects of porcine FSH, namely cell rounding, reduced cellular proliferation, and stimulation of progesterone production of GFSHR-17 cells, which are effects that were detectable within hours. Treatment with FSH also statistically significantly increased Cx 43 mRNA levels, as shown after 6 to 9 h in Northern blots. These effects were antedated by altered GJ communication, which was observed within seconds. Using a single-cell/whole-cell patch clamp technique, we showed that FSH rapidly and reversibly enhanced electrical cell coupling of GFSHR-17 cells. Increased GJ communication was associated with statistically significantly decreased phosphorylation of Cx 43, which was observed within 10 min after FSH addition, during immunoprecipitation experiments. Our results demonstrate, to our knowledge for the first time, that the gonadotropin FSH acutely and directly stimulates intercellular communication of GFSHR-17 cells through existing GJs. Moreover, FSH also increases levels of Cx 43 mRNA. These changes are associated with reduced proliferation and enhanced differentiation of GFSHR-17 cells. In vivo factors in addition to FSH may be involved in the regulation of GJ/GJ communication between GCs in the follicle, but our results suggest that improved cell-to-cell coupling, enhanced Cx 43 gene expression, and possibly, formation of new GJs are direct consequences of FSH receptor activation and may antedate and/or initiate the pivotal effects of FSH on GCs.

Novel genes modulated by FSH in normal and immortalized FSH-responsive cells: new insights into the mechanism of FSH action

Follicle‐stimulating hormone (FSH) controls the development of follicle‐enclosed oocytes in the mammalian ovary by interacting with specific receptors located exclusively on granulosa cells. Its biological activity involves stimulation of intercellular communication, intracellular signaling, and up‐regulation of steroidogenesis; the entire spectrum of genes regulated by FSH is not yet fully characterized. We have established monoclonal rat FSH‐responsive granulosa cell lines that express FSH receptors at 20‐fold higher rates than with primary cells, and thus increased the probability of yielding a distinct spectrum of genes modulated by FSH. Using Affymetrix DNA microarrays, we discovered 11 genes not reported earlier to be up‐regulated by FSH and 9 genes not reported earlier to be down‐regulated by FSH. Modulation of signal transduction associated with G‐protein signaling, phosphorylation of proteins, and intracellular‐extracellular ion balance was suggested by up‐regulation of decay accelerating factor GPI‐form precursor (DAF), membrane interacting protein RGS16, protein tyrosine phosphatase (PTPase), oxidative stress‐inducible protein tyrosine phosphatase (OSIPTPase), and down‐regulation of rat prostatic acid phosphatase (rPAP), Na+, K+‐ATPase, and protein phosphatase 1β. Elevation in granzyme‐like proteins 1 and 3, and natural killer (NK) cell protease 1 (NKP‐1) along with reduction in carboxypeptidase E indicates possible FSH‐mediated preparation of the cells for apoptosis. Up‐regulation of vascular endothelial growth factors indicates the ability of FSH to produce angiogenic factors upon their maturation; whereas, reduction in insulin‐like growth factor binding protein (IGFBP3) indicates its increased potential to promote p53‐induced apoptosis. Striking similarities in FSH modulation of gene expression were found in primary cultures of human granulosa cells obtained from IVF patients although these cells expressed only 1% of FSH receptor compared with immortalized rat cells, as indicated by microarray technique, which probably is in the normal range of expression of this receptor in nontransformed cells. These findings should increase our understanding of the mechanism of FSH action in stimulating development of the ovarian follicular cells, of intracellular and intercellular communication, and of increasing the potential of ovarian follicular cells to undergo apoptosis during the process of selection of the dominant follicle.—OSasson, R., Dantes, A., Tajima, K., Amsterdam, A. Novel genes modulated by FSH in normal and immortalized FSH‐responsive cells: new insights into the mechanism of FSH action. FASEB J. 17, 1256–1266 (2003)

Growth Differentiation Factor-9 Stimulates Rat Theca-Interstitial Cell Androgen Biosynthesis

Abstract Growth differentiation factor-9 (GDF-9) was shown recently to be essential for early follicular development, including the appearance of the theca layer. Theca cells provide the androgen substrate for aromatization and estrogen production by granulosa cells. Using biologically active recombinant GDF-9 (rGDF-9) and an androgen-producing immortalized theca-interstitial cell (TIC) line or primary TIC, we have examined the action of this paracrine hormone on theca cell steroidogenesis. The effect of GDF-9 on TIC progesterone synthesis was marginal and inconsistent in the primary cultures. In immortalized theca cells, GDF-9 attenuated the forskolin-stimulated progesterone accumulation. More significantly, this oocyte-derived growth factor enhanced both basal and stimulated androstenedione accumulation in the primary and transformed TIC cultures. The effects of GDF-9 on steroidogenesis by preovulatory follicles were relatively modest. Likewise, it did not affect the maturation of follicle-enclosed oocytes. The effect of GDF-9, an oocyte product, on TIC androgen production suggests a regulatory role of the oocyte on theca cell function and hence on follicle development and differentiation. This direct effect of GDF-9 on thecal steroidogenesis is consistent with its recently demonstrated actions on thecal cell recruitment and differentiation.