Luteinizing Hormone Receptor-Stimulated Progesterone Production by Preovulatory Granulosa Cells Requires Protein Kinase A-Dependent Activation/Dephosphorylation of the Actin Dynamizing Protein Cofilin

Abstract

Activation of the LH receptor (LHR) on preovulatory granulosa cells stimulates the cAMP/protein kinase A (PKA) pathway to regulate expression of genes required for ovulation and luteinization. LHR signaling also initiates rearrangement of the actin cytoskeleton. Because disruption of the actin cytoskeleton has been causally linked to steroidogenesis in various cell models, we sought to identify the cellular mechanisms that may modulate reorganization of the actin cytoskeleton and to determine whether cytoskeletal reorganization is required for steroidogenesis. Herein we report that LHR signaling in preovulatory granulosa cells promotes rapid dephosphorylation of the actin-depolymerizing factor cofilin at Ser3 that is dependent on PKA. The LHR-stimulated dephosphorylation of cofilin(Ser3) switches on cofilin activity to bind actin filaments and enhance their dynamics. Basal phosphorylation of cofilin(Ser3) is mediated by active/GTP-bound Rho and downstream protein kinases; LHR signaling promotes a decrease in active/GTP-bound Rho by a PKA-dependent mechanism. LHR-dependent Rho inactivation and subsequent activation of cofilin does not involve ERK, epidermal growth factor receptor, or phosphatidylinositol 3-kinase pathways downstream of PKA. To understand the biological significance of cofilin activation, preovulatory granulosa cells were transduced with a mutant cofilin adenoviral vector in which Ser3 was mutated to Glu (S-E cofilin). Inactive S-E cofilin abolished LHR-mediated reorganization of the actin cytoskeleton and caused a 70% decrease in LHR-stimulated progesterone that is obligatory for ovulation. Taken together, these results show that LHR signaling via PKA activates a cofilin-regulated rearrangement of the actin cytoskeleton and that active cofilin is required to initiate progesterone secretion by preovulatory granulosa cells.

Dephosphorylation of MAP2D enhances its binding to vimentin in preovulatory ovarian granulosa cells

“…F-actin is reorganized from long fibers into a nondescript punctate distribution within both the cell body and extensions of granulosa cells (Karlsson et al, 2010). Consistent with our previous results (Karlsson et al, 2010), dual staining of cells with antibodies to vimentin and MAP2D showed that hCG promoted the contraction of granulosa cells and appearance of long cellular extensions (Fig. 1D).…”

“…Untreated fixed granulosa cells appear flattened and fibroblastic with long bundles of phalloidin-stained F-actin (Karlsson et al, 2010). Staining with antibodies against β-tubulin, vimentin [the predominant intermediate protein in granulosa cells (Albertini and Kravit, 1981)], actin and MAP2D proteins also showed that each of these proteins reached into the periphery of the cells (Fig.…”

“…Treatment with 1 IU hCG/ml for 30 min results in the rounding of granulosa cells and the appearance of dendritic-like cellular extensions (Karlsson et al, 2010). F-actin is reorganized from long fibers into a nondescript punctate distribution within both the cell body and extensions of granulosa cells (Karlsson et al, 2010).…”

“…Preovulatory granulosa cells in primary culture on fibronectin substratum appear fibroblastic, with long bundles of F-actin (Karlsson et al, 2010). hCG promotes the PKA-dependent dephosphorylation of the actin-depolymerizing factor cofilin on Ser3, resulting in cell rounding and the appearance of spindly processes that appear neuronal-like (Karlsson et al, 2010).…”

“…hCG promotes the PKA-dependent dephosphorylation of the actin-depolymerizing factor cofilin on Ser3, resulting in cell rounding and the appearance of spindly processes that appear neuronal-like (Karlsson et al, 2010). These events are required for the progesterone synthetic response (Karlsson et al, 2010) necessary for ovulation (Lydon et al, 1995;Robker et al, 2000).…”