Activation of the Luteinizing Hormone β Promoter by Gonadotropin-releasing Hormone Requires c-Jun NH2-terminal Protein Kinase

Abstract

Regulation of the mitogen-activated protein kinase (MAPK) family by gonadotropin-releasing hormone (GnRH) in the gonadotrope cell line L␤T2 was investigated. Treatment with gonadotropin-releasing hormone agonist (GnRHa) activates extracellular signal-regulated kinase (ERK) and c-Jun NH 2 -terminal kinase (JNK). Activation of ERK by GnRHa occurred within 5 min, and declined thereafter, whereas activation of JNK by GnRHa occurred with a different time frame, i.e. it was detectable at 5 min, reached a plateau at 30 min, and declined thereafter. GnRHa-induced ERK activation was dependent on protein kinase C or extracellular and intracellular Ca 2؉ , whereas GnRHa-induced JNK activation was not dependent on protein kinase C or on extracellular or intracellular Ca 2؉ . To determine whether a mitogen-activated protein kinase family cascade regulates rat luteinizing hormone ␤ (LH␤) promoter activity, we transfected the rat LH␤ (؊156 to ؉7)-luciferase construct into L␤T2 cells. GnRH activated the rat LH␤ promoter activity in a time-dependent manner. Neither treatment with a mitogen-activated protein kinase/ERK kinase (MEK) inhibitor, PD98059, nor cotransfection with a catalytically inactive form of a mitogenactivated protein kinase construct inhibited the induction of the rat LH␤ promoter by GnRH. Furthermore, cotransfection with a dominant negative Ets had no effect on the response of the rat LH␤ promoter to GnRH. On the other hand, cotransfection with either dominant negative JNK or dominant negative c-Jun significantly inhibited the induction of the rat LH␤ promoter by GnRH. In addition, GnRH did not induce either the rat LH␤ promoter activity in L␤T2 cells transfected stably with dominant negative c-Jun. These results suggest that GnRHa differentially activates ERK and JNK, and a JNK cascade is necessary to elicit the rat LH␤ promoter activity in a c-Jun-dependent mechanism in L␤T2 cells. GnRH,1 a hypothalamic decapeptide, serves as a key regulator of the reproductive system. GnRH acts on anterior pituitary gonadotropes to stimulate the synthesis and release of the pituitary gonadotropins LH and FSH. The gonadotropins are subunit hormones, each containing noncovalently linked ␣-and ␤-subunits (1, 2). Within a species, the ␣-subunits are identical, while the ␤-subunits differ and confer the physiological specificity of the heterodimeric hormone. Each ␤-subunit as well as the common ␣-subunit is encoded by different genes on separate chromosomes. When GnRH binds to its seven-transmembrane receptor (3), it induces interaction of the receptor with the heterotrimeric G q protein, which leads to activation of phospholipase C and formation of inositol 1,4,5-triphosphate and diacylglycerol, leading to elevation of intracellular Ca 2ϩ

Estrogen Induces the Akt-dependent Activation of Endothelial Nitric-oxide Synthase in Vascular Endothelial Cells

“…Assay of Akt Activity Using a Transient Expression System-CHO cells cultured in 100-mm dishes were transfected with 1 g of pSG5, 1 g of ER␣ expression vector (pSG5-HEGO), or 1 g of ER␤ expression vector (pSG5-mER␤) using LipofectAMINE plus as described previously (52,53). Seventy-two hours after transfection, serum-deprived cells were incubated with 10 Ϫ7 M 17␤-E2 for 15 min, and the Akt activity was measured as described above.…”

“…eNOS activity was determined as the conversion of radiolabeled L-arginine to L-citrulline by a method described previously (50, 51) with a minor modification. Briefly, 10 l of a sample was incubated for 10 min at 37°C in a solution consisting of 50 mM HEPES, 1 mM dithiothreitol, 1 mM CaCl 2 , 0.1 mM tetrahydro-L-biopterin, 1 mM NADPH, 10 g/ml calmodulin, 10 M FAD, and 1.55 M L-[guanidino- Assay of eNOS Activity Using a Transient Expression SystemTRLECs cultured in 100-mm dishes were transfected with 1 g of CMV-6, 1 g of CMV-6 containing the gene for HA-AktK179M, 1 g of pSG5, 1 g of ER␣ expression vector (pSG5-HEGO), or 1 g of ER␤ expression vector (pSG5-mER␤) using LipofectAMINE plus (Life Technologies, Inc.) as described previously (52,53). Seventy-two hours after transfection, serum-deprived cells were incubated with 10 Ϫ7 M 17␤-E2 for 15 min, and the eNOS activity was measured as described above.…”

“…Cell lysates were subjected to immunoprecipitation with anti-Akt antibody. For assay using a transient expression system, TRLECs cultured in 100-mm dishes were transfected with 1 g of HA-Akt, 1 g of HA-AktK179M, or 1 g of HA-m⌬4 -129Akt using LipofectAMINE plus (Life Technologies, Inc.) as described previously (52,53). Seventy-two hours after transfection, serum-deprived cells were incubated with 10 Ϫ7 M 17␤-E2 for 15 min, and lysates were immunoprecipitated with anti-HA antibody.…”

“…6B). Treatment with 50 M 1,2-bis(o-amino-phenoxy)ethane-N,N,NЈ-tetraacetic-acetoxymethyl ester (BAPTA-AM) for 20 min to eliminate intracellular Ca 2ϩ (52,53) completely blocked the 17␤-E2-induced Akt (Fig. 6B, upper panel) and eNOS (Fig.…”

“…6B, lower panel) activation. Moreover, elimination of both extracellular and intracellular Ca 2ϩ by treatment with 3 mM EGTA for 15 min (52,53,60) abolished the 17␤-E2-induced Akt (Fig. 6B, upper panel) and eNOS (Fig.…”