A Role for Mitogen-Activated Protein Kinase in Mediating Activation of the Glycoprotein Hormone α-Subunit Promoter by Gonadotropin-Releasing Hormone

Abstract

Gonadotropin-releasing hormone (GnRH) interacts with a G protein-coupled receptor and increases the transcription of the glycoprotein hormone alpha-subunit gene. We have explored the possibility that mitogen-activated protein kinase (MAPK) plays a role in mediating GnRH effects on transcription. Activation of the MAPK cascade by an expression vector for a constitutively active form of the Raf-1 kinase led to stimulation of the alpha-subunit promoter in a concentration-dependent manner. GnRH treatment was found to increase the phosphorylation of tyrosine residues of MAPK and to increase MAPK activity, as determined by an immune complex kinase assay. A reporter gene assay using the MAPK-responsive, carboxy-terminal domain of the Elk1 transcription factor was also consistent with GnRH-induced activation of MAPK. Interference with the MAPK pathway by expression vectors for kinase-defective MAPKs or vectors encoding MAPK phosphatases reduced the transcription-stimulating effects of GnRH. The DNA sequences which are required for responses to GnRH include an Ets factor-binding site. An expression vector for a dominant negative form of Ets-2 was able to reduce GnRH effects on expression of the alpha-subunit gene. These findings provide evidence that GnRH treatment leads to activation of the MAPK cascade in gonadotropes and that activation of MAPK contributes to stimulation of the alpha-subunit promoter. It is likely that an Ets factor serves as a downstream transcriptional effector of MAPK in this system.

Calcium Influx through L-type Channels Is Required for Selective Activation of Extracellular Signal-regulated Kinase by Gonadotropin-releasing Hormone

“…Plasmids and Transfection Experiments-The expression vector for Gal4 DNA binding domain-Elk 1 transactivation domain and the luciferase reporter containing five Gal4 DNA binding sites upstream of the E1B TATA box and luciferase coding sequences have been previously described (9). All plasmids used in transfection studies were prepared by centrifugation through cesium chloride using standard methods.…”

“…Prior to all studies, cells were split to fresh media and cultured to approximately 60 -70% confluence. All transient transfection studies were conducted as described previously (9). Briefly, for transient transfection studies, cells were transfected by electroporation using a single electrical pulse at 220 V and 950 microfarads.…”

“…Inhibitors were added again approximately 16 h following electroporation. Cells were collected by scraping 6 h following the final administration of inhibitors and lysed by three freeze thaw cycles, and luciferase activity was determined as described (9).…”

“…Stimulation of ␣T3-1 cells with the GnRH-R agonist buserelin results in activation of three members of the MAPK family (9 -14), increased mRNA levels of the IEGs c-fos and c-jun (15), transcriptional activation of the gene for the common ␣-subunit of luteinizing hormone and follicle-stimulating hormone (16), and activation of the transcription factor Elk 1 (9). Previous studies have shown that activation of a MAPK family member, ERK, is absolutely required for buserelin-induced transcription of the ␣-subunit and the GnRH receptor gene (9,17). Following exposure of ␣T3-1 cells to GnRH, a biphasic calcium response consisting of an initial IP 3 -dependent spike phase followed by a sustained extracellular calciumrequiring plateau phase is observed (2).…”

Hypoxia-associated Induction of Early Growth Response-1 Gene Expression

“…Expression of tissue factor was monitored using the tissue factor promoter-luciferase construct spanning the hypoxia responsive Egr-1 elements (pTF[Ϫ111/ϩ14]Luc; Fig. 7E) (39). Although hypoxia caused increased luciferase activity/expression with the tissue factor promoter-luciferase construct alone (pTF[Ϫ111/ϩ14]Luc), cotransfection with the DN-PKC␤II suppressed luciferase activity (Fig.…”