Calcium and glycoprotein hormone alpha-subunit gene expression and secretion in alpha T3-1 gonadotropes.
GnRH stimulates both transcription and secretion of the alpha-subunit in pituitary cells, but the precise role of the calcium- signaling mechanisms mediating these actions are unclear. We have examined the role of calcium using alpha T3-1 gonadotropes transfected with alpha-promoter constructs linked to a luciferase reporter gene and concomitant measurement of alpha-subunit secretion. The calcium channel agonist, BayK8644 (1 microM) significantly stimulated alpha-subunit transcription (4.9-fold, P < 0.05) but to a lesser extent than either GnRH (100 nM, 20.7-fold, P < 0.001) or phorbol-12-myristate-13-acetate (TPA, 100 nM, 8.7-fold, P < 0.05). The transcriptional response to a combination of BayK8644 and TPA was approximately additive. Despite stimulating alpha-subunit gene expression, BayK8644 had no effect on alpha-subunit secretion at 24 h, and co-addition of BayK8644 and TPA did not produce any further stimulation of alpha-subunit secretion (3.0-fold, P < 0.001) compared with TPA alone (3.2-fold, P < 0.001). Pretreatment of alpha T3-1 cells with the calcium channel blocker, nifedipine (1 microM for 5 min), essentially blocked GnRH-stimulated alpha-promoter activity without affecting GnRH-stimulated alpha-subunit release. In contrast, thapsigargin pretreatment (1 microM for 5 min), which depletes intracellular calcium stores, significantly reduced basal and GnRH-stimulated secretion without affecting the ability of GnRH to increase alpha-promoter activity. Incubation of alpha T3-1 cells in calcium-depleted media showed that the transcriptional response was dependent on extracellular calcium concentration, with maximum stimulation by GnRH seen at a calcium concentration of 1.7 mM. Deletion analysis indicated that sequences between -420 and -244 bp were involved in mediating the response to BayK8644. Constructs containing only upstream alpha-promoter sequences from -517 to -98 bp, fused to the heterologous thymidine kinase promoter, exhibited loss of responsiveness to BayK8644 below -298 bp. These upstream elements were also found to be important for basal expression of the alpha-promoter and for mediating the response to TPA but were distinct from GnRH responsiveness of the human promoter in alpha T3-1 cells. These studies suggest differential regulation of GnRH-stimulated alpha-subunit gene transcription and secretion by extracellular calcium influx and intracellular calcium mobilization. The transcriptional response to extracellular calcium influx is mediated through two or more elements between -420 and -244 bp, which are also involved in basal and TPA-stimulated expression of the alpha-subunit promoter.
Holdstock
J G
JG
Department of Clinical Biochemistry, London Hospital Medical College, United Kingdom.
Aylwin
S J
SJ
Burrin
J M
JM
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002120:Calcium Channel Agonists / Q000494:pharmacology; D002460:Cell Line; D005786:Gene Expression Regulation; D015292:Glycoprotein Hormones, alpha Subunit / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology; D006801:Humans; D051379:Mice; D010902:Pituitary Gland / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects
D002120:Calcium Channel Agonists; D015292:Glycoprotein Hormones, alpha Subunit; D011994:Recombinant Proteins; D007987:Gonadotropin-Releasing Hormone; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D013937:Thymidine Kinase; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium