cAMP-mediated signals as determinants for apoptosis in primary granulosa cells.
Journal - Experimental cell research (UNITED STATES )
Differentiation and luteinization of granulosa cells are induced by gonadotrophic hormones and other substances elevating intracellular levels of cyclic AMP (cAMP). We have investigated the correlation between the potency of these substances to enhance steroidogenesis and to induce apoptosis in primary granulosa cell cultures obtained from rat preovulatory follicles. The cAMP analog, 8-Br cAMP, induced apoptosis in more than 90% of the cell population within 15 h of incubation at 37 degrees C in serum-free medium. The physiological stimulants of these cells, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which caused a moderate cAMP response in these cells, followed by a desensitization period, increased progesterone production by fourfold with no apparent effect on cell death. In contrast, forskolin, a potent activator of adenylate cyclase, stimulated both the cAMP and steroidogenic response by an order of magnitude greater than the gonadotropin stimulation, concomitantly with a pronounced increase in cell death (25%). Moreover, blocking of the cellular phosphodiesterase activity in forskolin-stimulated cells by isobutylmethylxanthine (IBMX), which maintains high levels of intracellular cAMP, led to further enhancement of cell death following 40 h of incubation (50%). Basic fibroblast growth factor (bFGF) and gonadotropin-releasing hormone (GnRH), which stimulated steroidogenesis in these cells in a cAMP-independent manner, did not promote cell death. Moreover, costimulation of the cells with forskolin and bFGF led to a substantial decrease in the incidence of apoptosis relative to forskolin alone. In order to examine whether the expression of tumor suppressor genes is involved in granulosa cell differentiation and apoptosis induced by cAMP, we examined the effect of cAMP in SV40 transformed granulosa cells, in which T-antigen expression is expected to block the activity of p53 as well as of the retinoblastoma gene product (pRB) and its related proteins. Cultures of three different cell lines established by SV40 transformation demonstrated resistance to 8-Br-cAMP- or forskolin plus IBMX-induced apoptosis, in contrast to the severe apoptotic response in primary cells. We suggest that stimulation of primary granulosa cells by high levels of cAMP catalyzes programmed cell death, while stimulation of the cells by gonadotropic hormones, which result in a moderate cAMP response, followed by desensitization to further stimulation, can prolong the lifespan of the luteinized granulosa cells. Moreover, one or more tumor suppressor proteins may mediate the cAMP generated signal leading to cell death.
|ISSN : ||0014-4827|
|Mesh Heading : ||1-Methyl-3-isobutylxanthine 8-Bromo Cyclic Adenosine Monophosphate Animals Apoptosis Cells, Cultured Chorionic Gonadotropin Culture Media, Serum-Free Cyclic AMP DNA Female Fibroblast Growth Factor 2 Follicle Stimulating Hormone Forskolin Gonadotropin-Releasing Hormone Granulosa Cells Kinetics Microscopy, Electron Microvilli Mitochondria Progesterone Rats Time Factors pharmacology drug effects pharmacology analysis pharmacology pharmacology pharmacology pharmacology cytology drug effects drug effects ultrastructure drug effects ultrastructure biosynthesis|
|Mesh Heading Relevant : ||Signal Transduction pharmacology physiology metabolism physiology|
Cross-talk between adenylate cyclase activation and tyrosine phosphorylation leads to modulation of the actin cytoskeleton and to acute progesterone secretion in ovarian granulosa cells.
Journal - Endocrinology (UNITED STATES )
Steroidogenesis in granulosa cells can be stimulated by gonadotropic hormones and substances elevating cAMP. This cAMP-dependent metabolic event can be enhanced by peptide growth factors such as insulin, insulin-like growth factor, and epidermal growth factor, but the mechanism of cooperation between these two different signaling pathways is not yet clear. We have tested whether enhancement of tyrosine phosphorylation by vanadate, which blocks tyrosine phosphatases, is able to mimic the effect of growth factors on cAMP-induced steroidogenesis and investigated the cellular components involved in such modulation. Ortho- and metavanadate at 0.1-1.0 mM, when added to primary granulosa cell cultures, stimulated by gonadotropic hormones or forskolin, enhanced progesterone production by 1.5- to 9.0-fold within 120 min. Pervanadate showed a similar effect on steroidogenesis at a concentration one order of magnitude lower than ortho- or meta-vanadate. Phenylarsine-oxide, another blocker of tyrosine phosphatase, stimulated forskolin-induced steroidogenesis by 2.5-fold at 30 microM. In contrast, okadaic acid and calyculin A, which block specifically serine and threonine phosphatase, had no effect on steroidogenesis, when used at concentrations of 1 microM and 10 nM, respectively. The stimulation by vanadate was associated with a pronounced change in cell shape and total collapse of the actin network, which retracts to form a few large actin aggregates of 1-7 microns in diameter in the perinuclear region as revealed by visualization of actin by rhodamine-phalloidin staining under the fluorescent microscope. Steroidogenesis is not affected in cells treated with vanadate alone; the effect of vanadate on the actin cytoskeleton is much less pronounced. Electron microscopy of ultra-thin sections showed massive breakdown of thin filament cables in cells stimulated with vanadate together with gonadotropic hormone or forskolin. Massive clustering of lipid droplets and mitochondria as well as sharp increase in the electron-density of mitochondrial matrix was also observed in the stimulated cells. The action of vanadate in cAMP-stimulated cells leads to massive tyrosine phosphorylation of intracellular proteins in the range of 22-200 kilodaltons. It is suggested that the cross-talk between the cAMP pathway and tyrosine phosphorylation, which leads to enhanced steroidogenesis may be mediated by phosphorylation of cytoskeleton or associated proteins. The marked changes in lipid droplet-mitochondria interaction suggests that this enhanced steroidogenesis is due in part to mobilization of cholesterol into mitochondria in cells costimulated with vanadate and gonadotropins.
|ISSN : ||0013-7227|
|Mesh Heading : ||Actins Adenylate Cyclase Animals Cells, Cultured Chorionic Gonadotropin Cytoskeleton Enzyme Activation Female Follicle Stimulating Hormone Forskolin Granulosa Cells Hydrogen Peroxide Microscopy, Electron Phosphorylation Phosphotyrosine Progesterone Rats Tyrosine Vanadates pharmacology pharmacology pharmacology drug effects ultrastructure pharmacology biosynthesis metabolism pharmacology|
|Mesh Heading Relevant : ||metabolism metabolism metabolism physiology secretion analogs & derivatives|