1,25-Dihydroxyvitamin D3-induced upregulation of the thyrotropin-releasing hormone receptor in clonal rat pituitary GH3 cells.
1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) is active in primary dispersed and clonal pituitary cells where it stimulates pituitary hormone production and agonist-induced hormone release. We have studied the effect of 1,25-(OH)2D3 on thyrotropin-releasing hormone (TRH) binding in clonal rat pituitary tumour (GH3) cells. Compared with vehicle-treated cells, 1,25-(OH)2D3 (10 nmol/l) increased specific [3H]MeTRH binding by 26% at 8 h, 38% at 16 h, 35% at 24 h and reached a maximum at 48 h (90%). In dose-response experiments, specific [3H]MeTRH binding increased with 1,25-(OH)2D3 concentration and reached a maximum at 10 nmol/l. Half-maximal binding occurred at 0.5 nmol 1,25-(OH)2D3/l. The vitamin D metabolite, 25-OH D3, increased [3H]MeTRH binding but was 1000-fold less potent than 1,25-(OH)2D3. In equilibrium binding assays, treatment with 10 nmol 1,25-(OH)2D3/l for 48 h increased the maximum binding from 67.4 +/- 8.8 fmol/mg protein in vehicle-treated cells to 96.7 +/- 12.4 fmol/mg protein in treated cells. There was no difference in apparent Kd (1.08 +/- 0.10 nmol/l for 1,25-(OH)2D3-treated and 0.97 +/- 0.11 nmol/l for vehicle-treated cells). Molecular investigations revealed that 10 nmol 1,25-(OH)2D3/l for 24 h caused an 8-fold increase in TRH receptor-specific mRNA. Actinomycin D (2 micrograms/ml, 6 h) abrogated the 1,25-(OH)2D3-induced increase in [3H]MeTRH binding. Cortisol also increased [3H]MeTRH binding but showed no additivity or synergism with 1,25-(OH)2D3. TRH-stimulated prolactin release was not enhanced by 1,25-(OH)2D3. We conclude that the active vitamin D metabolite, 1,25-(OH)2D3, caused a time- and dose-dependent increase in [3H]MeTRH binding. The effect was vitamin D metabolite-specific and resulted from an upregulation of the TRH receptor. Further studies are needed to determine the functional significance of this novel finding.
University of Melbourne, Department of Medicine, Royal Melbourne Hospital, Parkville, Victoria, Australia.
L M
LM
Atley
N
N
Lefroy
J D
JD
Wark
D000818:Animals; D015152:Blotting, Northern; D002117:Calcitriol / Q000494:pharmacology*; D002460:Cell Line; D002478:Cells, Cultured; D002999:Clone Cells; D004305:Dose-Response Relationship, Drug; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000378:metabolism; D010911:Pituitary Neoplasms / Q000378:metabolism* / Q000473:pathology; D011388:Prolactin / Q000032:analysis / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D012333:RNA, Messenger / Q000032:analysis; D011863:Radioimmunoassay; D051381:Rats; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D015854:Up-Regulation
D012333:RNA, Messenger; D018025:Receptors, Thyrotropin-Releasing Hormone; D011388:Prolactin; D002117:Calcitriol