THYROTROPIN-RELEASING-HORMONE GENE-EXPRESSION IN THE ANTERIOR-PITUITARY .2. STIMULATION BY GLUCOCORTICOIDS

The present studies were undertaken to determine whether glucocorticoids (GC) regulate TRH gene expression in cultured anterior pituitary (AP) cells. AP cells derived from 15-day-old male rats were cultured for up to 18 days in Dulbecco's Modified Eagle's Medium-L-15 medium supplemented with 1) fetal calf serum (FCS), 2) charcoal-treated FCS, 3) normal rat serum, or 4) serum from rats that were adrenalectomized, rendered hypothyroid, and gonadectomized (ATG rat serum). Dexamethasone (Dex) or corticosterone (Cort) was added to the culture medium at various concentrations with exposure times ranging from 4-18 days. TRH and prepro-TRH-(25-50) in cellular extracts and release media were measured by RIA, and pro-TRH mRNA was determined by Northern blot analysis and in situ hybridization. Dex substantially stimulated cellular TRH and prepro-TRH-(25-50) accumulation under all culture conditions investigated, i.e. in medium supplemented with any of the four sera. TRH gene expression did not occur in medium supplemented with charcoal-treated FCS or ATG rat serum. Pretreatment with 10(-8) M Dex caused a significant increase in basal as well as cAMP- or phorbol ester-stimulated release of the peptide. Steady state pro-TRH mRNA levels rose 6.8- and 4.2-fold (both P<0.01) after treatment with 10(-8) M Dex for 4 and 12 days, respectively. In situ. hybridization experiments revealed that this rise in pro-TRH mRNA levels was probably the result of an increase in the number of AP cells expressing pro-TRH. Both Dex and Cort caused a dose-dependent increase in TRH accumulation, but Cort was approximately 40 times less potent than Dex. These results indicate that GC stimulate TRH gene expression in cultured AP cells. The presence of GC in culture medium is a prerequisite for the occurrence of TRH gene expression in the AP. As GC have been reported to reduce pro-TRH mRNA levels in the hypothalamus in vivo, our results may provide an example of the tissue-specific effects of GC on TRH gene expression.