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.

Abstract

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.