The involvement of phosphatidylinositol 3-kinase in gonadotropin-releasing hormone-induced gonadotropin alpha- and FSHbeta-subunit genes expression in clonal gonadotroph LbetaT2 cells.
Akt/protein kinase B (Akt/PKB), which is activated by phosphatidylinositol-3 kinase (PI3-kinase), plays an important role in cell survival and cell proliferation. Using the well differentiated, clonal gonadotroph cell line, LbetaT2, we examined (1) whether Akt/PKB was activated by gonadotropin-releasing hormone (GnRH); (2) the contribution of PI3-kinase-Akt/PKB pathway in each of gonadotropin subunit gene expression; (3) crosstalk between extracellular signal-regulated kinase (ERK) and Akt/PKB pathways. Insulin-like growth factor-1 (IGF-1) was used as Akt/PKBs classic activator. Western blot analyses using antibodies specific for the phosphorylated forms of ERK and Akt/PKB demonstrated that both were rapidly phosphorylated following treatment with GnRH and IGF-1. Akt/PKB activation by GnRH and IGF-1 was completely eliminated in the presence of the PI3-kinase inhibitor, LY 294002, but not in the presence of an Akt/PKB inhibitor. Interestingly, the total amount of Akt/PKB protein was dramatically increased in the presence of LY 294002. Phosphorylation of ERK was significantly increased in the presence of LY 294002 alone, and was further increased when GnRH was used in combination with LY 294002. In experiments using a luciferase reporter construct containing the serum response element (SRE), a known target of the ERK pathway, LY 294002 but not the Akt/PKB inhibitor increased SRE-luciferase activity. GnRH-induced SRE-luciferase activity was significantly increased by LY 294002. GnRH stimulation resulted in gonadotropin LHbeta, FSHbeta, and alpha-subunit promoter activation, while IGF-1 failed to stimulate any of them. GnRH-induced gonadotropin promoter activities were not modulated in the presence of an Akt/PKB inhibitor, but treatment with LY 294002 or Wortmannin resulted in a significant increase in alpha- and FSHbeta-subunit promoter activation, both with and without GnRH. LY 294002, but not the Akt/PKB inhibitor, significantly inhibited cell proliferation. These results suggest that GnRH-induced gonadotropin gene expression is not regulated through the Akt/PKB pathway; however, PI3-kinase may be involved in the negative regulation of alpha- and FSHbeta-subunit gene expression as well as cell proliferation.
Mutiara
Sandra
S
Department of Obstetrics and Gynecology, Shimane University, School of Medicine, 89-1 Enya Cho, Izumo City 693-8501, Shimane Prefecture, Japan.
Kanasaki
Haruhiko
H
Harada
Takashi
T
Oride
Aki
A
Miyazaki
Kohji
K
D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D002999:Clone Cells / Q000166:cytology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D037201:Follicle Stimulating Hormone, beta Subunit / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D015292:Glycoprotein Hormones, alpha Subunit / Q000235:genetics* / Q000378:metabolism; D052681:Gonadotrophs / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology*; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D008954:Models, Biological; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000235:genetics; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors; D051381:Rats; D013997:Time Factors
D004791:Enzyme Inhibitors; D048049:Extracellular Signal-Regulated MAP Kinases; D037201:Follicle Stimulating Hormone, beta Subunit; D015292:Glycoprotein Hormones, alpha Subunit; D007987:Gonadotropin-Releasing Hormone; D007334:Insulin-Like Growth Factor I; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt