Nongenomic glucocorticoid receptor action regulates gap junction intercellular communication and neural progenitor cell proliferation

Abstract

Glucocorticoids (GCs) are used to treat pregnant women at risk for preterm delivery; however, prenatal exposure to GCs may trigger adverse neurological side effects due to reduced neural progenitor cell (NPC) proliferation. Whereas many established cell-cycle regulators impact NPC proliferation, other signaling molecules, such as the gap junction protein connexin-43 (Cx43), also influence proliferation. Gap junction intercellular communication (GJIC) is influenced by GCs in some cells, but such hormone effects have not been examined in coupled stem cells. We found that both continuous and transient exposure of embryonic day 14.5 mouse neurosphere cultures to dexamethasone (DEX) limits proliferation of coupled NPCs, which is manifested by both a reduction in Sphase progression and enhanced cell-cycle exit. A short (i.e., 1-h) DEX treatment also reduced GJIC as measured by live-cell fluorescence recovery after photobleaching, and altered the synchrony of spontaneous calcium transients in coupled NPCs. GC effects on GJIC in NPCs are transcription-independent and mediated through plasma membrane glucocorticoid receptors (GRs). This nongenomic pathway operates through lipid raft-associated GRs via a site-specific, MAPK-dependent phosphorylation of Cx43, which is linked to GR via caveolin-1 (Cav-1) and c-src. Cav-1 is essential for this nongenomic action of GR, as DEX effects on GJIC, Cx43 phosphorylation, and MAPK activation are not observed in Cav-1 knockout NPCs. As transient pharmacologic inhibition of GJIC triggers reduced S-phase progression but not enhanced cell-cycle exit, the nongenomic GR signaling pathway may operate via distinct downstream effectors to alter the proliferative capacity of NPCs.

Caveolin-1 Regulates Genomic Action of the Glucocorticoid Receptor in Neural Stem Cells

“…We previously established that inhibition of GJIC by the rapid action of GR was associated with an inhibition of S-phase progression in cultured NPSCs (17). Our observation that transient pharmacologic inhibition of GJIC is also sufficient to reduce NPSC proliferation (17) was recently confirmed in cultured embryonic stem cell-derived neural progenitors and in embryonic neural progenitors isolated from the cerebral cortex (34). Recent studies in human hippocampal neural progenitor cells indicate that the antiproliferative properties of GCs are dependent upon at least one genomic GR target gene, Sgk-1 (19).…”

“…NPSCs were derived from embryonic (E14.5) cerebral cortex of wild-type C57BL/6 (C57) or Cav-1 knockout (KO) mice and grown as three-dimensional neurosphere cultures (17). Cells were passaged every 7 days, and experiments were performed at passage 3 unless indicated otherwise.…”

“…Caveolae can act as signaling organizers, and previous work from our laboratory established a role for Cav-1 in a rapid GC signaling pathway that triggers MAPK activation in embryonic mouse NPSC cultures (17). One of the consequences of Cav-1-dependent activation of MAPK by GCs is an inhibition of gap junction intercellular communication (GJIC) between coupled NPSCs (17). Studies with other steroid hormone receptors, such as the androgen and estrogen receptor, have revealed mechanisms of cross talk between genomic and rapid actions of the receptors (reviewed in reference 18).…”

“…These kinases can then influence GR transcriptional response through direct modification of the receptor or its associated transcriptional coregulators (16). Although both rapid and genomic GR signaling operates in NPSCs (17), the degree of interaction between the two pathways and how they intersect to influence GC effects on NPSC biology are unknown.…”

“…Given the role of Cav-1 in mediating the rapid response of GR (16,17), we set out to examine whether Cav-1 could also participate in classical genomic GR signaling and impact the antiproliferative response of NPSCs to Dex. Here we show that loss of the Dex-mediated antiproliferative response in NPSCs derived from Cav-1 knockout (KO) mice is associated with the alterations in Dex-induced expression of an established negative regulator of the cell cycle in NPSCs, the serum-and GC-induced kinase 1 gene, Sgk-1 (19).…”