GH mRNA levels are elevated by forskolin but not GH releasing hormone in GHRH receptor-expressing MtT/S somatotroph cell line

Abstract

The MtT/S somatotroph cell line should be a growth hormone-releasing hormone (GHRH)-responsive model system for the study of physiological control of growth hormone (GH) transcription because GH secretion from these cells is stimulated by GHRH. To examine the GH transcriptional activity of these cells, endogenous GH mRNA levels were measured using a ribonuclease protection assay following treatment under a variety of hormonal conditions. While omission of serum led to reduction of GH mRNA to 22% of control levels by 2 days and to 8% by 5 days ( P<0.05 for both), GH mRNA levels were maintained at control values in serum-free medium containing 5 nM dexamethasone and 30 pM triiodothyronine (TDM). However, the addition of 10 nM GHRH under any treatment condition did not significantly alter GH mRNA levels. Characterization of the MtT/S cells showed that GHRH-receptor (GHRH-R) mRNA was detectable by reverse transcription-polymerase chain reaction (RT-PCR) amplification. Measurement of extracellular cAMP showed that the MtT/S cells have basal levels of ≥20 nmol/10 6 cells per h in both serum-containing and serum-free media, and that GHRH had no effect on cAMP levels, suggesting constitutive activation. To rule out the possibility of autocrine stimulation by GHRH produced endogenously, GHRH mRNA was not detectable in MtT/S cells using RT-PCR amplification. The stimulatory G-protein α subunit, mutations of which are known to activate adenylate cyclase constitutively in acromegaly, was sequenced but found not to differ from normal pituitary in the regions most commonly mutated. Finally, treatment with 10 μM forskolin, to directly activate adenylate cyclase, increased GH mRNA to 140% of controls in TDM, and to 163% in serum-free medium after 2 days, and to 166% in TDM-treated cells and 174% in serum-free culture after 5 days (all P<0.05). Taken together, these data indicate that although MtT/S cells express the GHRH-R, GHRH cannot stimulate adenylate cyclase to increase GH transcription due to constitutive elevation of cAMP levels, by a means that may be similar to that in cases of acromegaly not caused by oncogenic gsp mutations.