Mode of action of prostaglandin F 2α in human luteinized granulosa cells: role of protein kinase C

Abstract

It is well documented that prostaglandin F 2α (PGF 2α) inhibits progesterone production in luteal cells, but its mode of action is uncertain. It has recently been suggested that PGF 2α acts by activating the calcium and phospholipid-dependent protein kinase, protein kinase C (PKC). This hypothesis has been tested by comparing the site and mode of action of PGF 2α, a PGF 2α analogue (cloprostenol) and the PKC activator phorbol myristate acetate (4βPMA) in human granulosa-lutein cells. PGF 2α and cloprostenol exerted similar concentration-dependent inhibitory actions on gonadotrophin-stimulated cyclic AMP (cAMP) accumulation and progesterone production by human granulosa-lutein cells. The similarity in the actions of PGF 2α and cloprostenol in human granulosa-lutein cells suggests that they can be used interchangeably to study the role of PGF 2α in the regulation of steroidogenesis in the human ovary. Gonadotrophin-stimulated cAMP accumulation and progesterone production was also concentration-dependently inhibited by 4β PMA. In addition, cloprostenol and 4β PMA also inhibited dibutyryl cAMP-stimulated progesterone production, suggesting that these compounds inhibit LH action at sites before and after the generation of cAMP. The pre-cAMP site of action can be localised to the stimulatory G-protein (G s) as both compounds inhibited cholera toxin-stimulated cAMP accumulation without affecting forskolin-stimulated cAMP accumulation. The post cAMP site of action can be localised to actions on cholesterol side chain cleavage enzyme, as both cloprostenol and 4β PMA inhibited 22R hydroxycholesterol-supported progesterone production without affecting pregnenolone-supported progesterone production. The finding that cloprostenol and 4βPMA interact with the steroidogenic cascade in a similar manner is indicative of a shared common mediator of their actions in human granulosa-lutein cells, i.e. PKC. The inhibitory actions of PGF 2α and 4βPMA on hLH-stimulated progesterone production were abolished in the presence of the PKC inhibitor, staurosporine. In addition, in PKC-depleted cells (achieved by exposure to 4β PMA for 20 h) the inhibitory actions of PGF 2α and 4βPMA were abolished. These results support the hypothesis that the inhibitory actions of PGF 2α are mediated by PKC in human granulosa-lutein cells.