Corticotropin-releasing hormone receptor expression and functional signaling in murine gonadotrope-like cells

Abstract

Corticotropin-releasing hormone (CRH) is a key regulator of the mammalian stress response, mediating a wide variety of stress-associated behaviors including stress-induced inhibition of reproductive function. To investigate the potential direct action of CRH on pituitary gonadotrope function, we examined CRH receptor expression and second messenger signaling in αT3-1 cells, a murine gonadotrope-like cell line. Reverse transcriptase PCR (RT-PCR) studies demonstrated that αT3-1 cells express mRNA for the two CRH receptor subtypes, CRHR1 and CRHR2, with CRHR2α as the predominant CRHR2 isoform. Stimulation of the cells with CRH or urocortin (UCN) resulted in rapid, transient increases in the intracellular levels of cAMP that were completely blocked by the addition of α-helical CRH 9-41 or astressin, non-selective CRH receptor antagonists. Stimulation of the cells with CRHR2-specific ligands, urocortin 2 (UCN2) or urocortin 3 (UCN3), resulted in rapid increases in intracellular cAMP levels to 50–60% of the levels observed with UCN. Treatment with a selective CRHR2 antagonist, antisauvagine, completely blocked UCN3-mediated increases in cAMP and significantly reduced, but did not completely block UCN-mediated increases in cAMP, demonstrating that both CRHR1 and CRHR2 are functionally active in these gonadotrope-like cells. Finally, UCN treatment significantly increased the transcriptional activity of the glycoprotein hormone α-subunit promoter as assessed by α-luciferase transfection assays. Together, these results demonstrate the functional signaling of CRH receptors in αT3-1 cells, suggesting that CRH may also modulate pituitary gonadotrope function in vivo.