LUTEINIZING HORMONE (LH) STIMULATES MTOR/S6K SIGNALING PATHWAY BY DEACTIVATING GSK3 AND AMPK IN CULTURED BOVINE CORPORA LUTEAL CELLS

Abstract

The corpus luteum (CL) is a transient reproductive gland that produces progesterone, a required product for the establishment and maintenance of pregnancy. Inadequate nutritional status or energy balance negatively impacts reproductive potential and reduces steroidogenesis. While it is appreciated that luteinizing hormone (LH) is a crucial factor involved in stimulating the secretion of progesterone by the CL, the cellular and molecular mechanisms that regulate the maintenance of the CL are poorly understood. Recent studies have implicated a variety of key protein kinases in cellular responses linked to cellular energy homeostasis. These include glycogen synthase kinase 3 (GSK3), AMP-activated protein kinase (AMPK), and the mammalian target of rapamycin (mTOR). While GSK3 and AMPK impair protein synthesis, mTOR stimulates protein translation via mechanisms involving the phosphorylation of the 70-kDa ribosomal S6 protein kinase (S6K) and the translation regulator eukaryotic initiation factor 4E-binding protein (4EBP1). A recent report suggests that GSK3 works in concert with AMPK to inhibit the mTOR pathway. Here, we studied the actions of LH on the regulation of mTOR/S6K signaling pathway in steroidogenic corpus luteum cells. Bovine luteal cells were derived from CL of early pregnancy. Primary cultures of luteal cells were treated and the phosphorylation status of S6K, GSK3, and AMPK was examined by western immuno-blot using phospho-specific antibodies. The activity of S6K was analyzed by an in vitro immunocomplex kinase assay using a GST-S6 peptide as substrate. Translational activation was monitored by 4EBP1 phosphorylation and the release of 4EBP1 binding in m7G cap binding assays. Treatment of luteal cells with LH (100 ng/ml), FSK (10 μM), a stimulator of adenylyl cyclase, or 8-BrcAMP (1 μM), a cAMP analogue significantly induced S6K phosphorylation and activation in a dose- and time-dependent manner. The phosphorylation of S6K1 in response to LH treatment was inhibited by a protein kinase A (PKA)-specific inhibitor Rp-8-Br-cAMP. Another cAMP analogue 8-CPT-2-Me-cAMP, which activates cAMP-guanine nucleotide exchange factors (EPAC) without activating PKA, had no effect on S6K phosphorylation. These findings suggest that LH and/or cAMP may regulate S6K phosphorylation via a PKA-dependent pathway. Treatment of luteal cells with LH also resulted in a time- and concentrationdependent inhibition of GSK3 activity by phosphorylating GSK3 at Ser9. The phosphorylation of GSK3 in response to LH was prevented by inhibitors of PKA. Deactivating GSK3 by treatment with GSK3 inhibitors, LiCl or GSKi, mimicked the stimulatory action of LH on S6K and S6 phosphorylation. Furthermore, LH treatment inhibited AMPK by phosphorylating AMPK on Thr172 residues. Pre-treatment of luteal cells with a cell-permeable AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) prevented S6K phosphorylation in response to LH. We also observed that LH activated mTOR/S6K down stream processes (i.e., 4EBP1 phosphorylation, release of 4EBP1 binding in m7G cap binding assay, and the phosphorylation of S6); and these events were inhibited by treatment with rapamycin or AICAR. In conclusion, LH and cAMP activate mTOR/S6K via a PKA-dependent signaling pathway that involves deactivating GSK3 and AMPK. We propose that mTOR/S6K integrates hormonal and energy signals to maintain CL function. Supported by VA, USDA, NIH and Olson Center for Women's Health. (platform)