The Luteinizing Hormone-releasing Hormone Inhibits the Anti-apoptotic Activity of Insulin-like Growth Factor-1 in Pituitary αT3 Cells by Protein Kinase Cα-mediated Negative Regulation of Akt

Abstract

The luteinizing hormone-releasing hormone (LHRH) receptor is a G protein-coupled receptor involved in the synthesis and release of pituitary gonadotropins and in the proliferation and apoptosis of pituitary cells. Insulin-like growth factor-1 receptor (IGF-1R) is a tyrosine kinase receptor that has a mitogenic effect on pituitary cells. In this study, we used the ␣T3 gonadotrope cell line as a model to characterize the IGF-1R signaling pathways and to investigate whether this receptor interacts with the LHRH cascade. We found that IGF-1 activated the IGF-1R, insulin receptor substrate (IRS)-1, phosphatidylinositol 3-kinase, and Akt in a timedependent manner in ␣T3 cells. The MAPK (ERK1/2, p38, and JNK) pathways were only weakly activated by IGF-1. In contrast, LHRH strongly stimulated the MAPK pathways but had no effect on Akt activation. Cotreatment with IGF-1 and LHRH had various effects on these signaling pathways. 1) It strongly increased IGF-1-induced tyrosine phosphorylation of IRS-1 and IRS-1-associated phosphatidylinositol 3-kinase through activation of the epidermal growth factor receptor. 2) It had an additive effect on ERK1/2 activation without modifying the phosphorylation of p38 and JNK1/2. 3) It strongly reduced IGF-1 activation of Akt. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and cell cycle analysis revealed that, in addition to having an additive effect on ERK1/2 activation, cotreatment with IGF-1 and LHRH also had an additive effect on cell proliferation. The LHRH-induced inhibition of Akt stimulated by IGF-1 was completely blocked by Safingol, a protein kinase C (PKC) ␣-specific inhibitor, and by a dominant negative form of PKC␣. Finally, we showed that the inhibitory effect of LHRH on IGF-1-induced PKC␣-mediated Akt activation was associated with a marked reduction in Bad phosphorylation and a substantial decrease in the ability of IGF-1 to rescue ␣T3 cells from apoptosis induced by serum starvation. Our results demonstrate for the first time that several interactions take place between IGF-1 and LHRH receptors in gonadotrope cells. Luteinizing hormone-releasing hormone (LHRH)1 is a hypothalamic decapeptide, which plays a crucial role in normal reproductive function. In the pituitary, LHRH-I (mammalian LHRH) stimulates the synthesis and the release of the gonadotropins, LH and FSH, and promotes cell proliferation and apoptosis in pituitary cells (1, 2). The effects of LHRH-I are mediated by a cell surface receptor (LHRH-R) belonging to the G protein-coupled receptor superfamily (3, 4). LHRH-I binding to its cognate receptor leads to interaction of the receptor with heterotrimeric G proteins, including G q/11 . These G proteins in turn activate phospholipase C, leading to the production of diacylglycerol and the subsequent activation of protein kinase C (PKC) (5-9). Activation of PKC then leads to activation of downstream protein kinases belonging to the MAPK family (9 -15). Activation of LHRH-R also triggers calcium influx, resulting in an increase in intracellular c...

FOXO1 Transcription Factor Inhibits Luteinizing Hormone β Gene Expression in Pituitary Gonadotrope Cells

“…Our results showed that insulin treatment of L␤T2 cells increased FOXO1 serine 256 phosphorylation and cytoplasmic localization. These data are in agreement with a previous study that showed IGF-1 signaling increased FOXO1 phosphorylation in ␣T3-1 cells (26). We also used the PI3K inhibitor LY294002 to demonstrate that regulation of FOXO1 phosphorylation and cellular localization by insulin signaling in L␤T2 cells was dependent on PI3K, which is consistent with the insulin effect on FOXO1 in other tissues (33).…”

“…After demonstrating that FOXO1 is expressed in gonadotrope cells in vivo, we also showed that the FOXO1 protein can be detected in immortalized gonadotrope-derived cell lines, in agreement with a previous study (26). We then investigated whether FOXO1 is a target of insulin signaling in gonadotropes, as previously reported for IRS1/2 and AKT (24,27).…”

Gq Protein-induced Apoptosis Is Mediated by AKT Kinase Inhibition That Leads to Protein Kinase C-induced c-Jun N-terminal Kinase Activation

“…This effect might be due to the relatively high basal activity of AKT in these nonstimulated cells. Therefore, it is possible that the inhibition of AKT activity by G q -dependent stimulations in mitogenes (insulin, insulin-like growth factor-1, or EGF)-stimulated cells (23,24) occurs via a mechanism similar to the one identified here.…”

GnRH-II Antagonists Induce Apoptosis in Human Endometrial, Ovarian, and Breast Cancer Cells via Activation of Stress-Induced MAPKs p38 and JNK and Proapoptotic Protein Bax

“…Apoptosis also could be induced in the other cell lines used in which basal activity of PKB/Akt is not increased. In aT3-1 pituitary cells, it could be shown that the GnRH-I agonist-induced reduction of insulin-like growth factor-induced activation of PKB/Akt could be restored by GnRH-I antagonists (31). PKB/Akt does not play a relevant role in induction of apoptosis by GnRH-II antagonists.…”