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

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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/106 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.

Introduction

Growth hormone-releasing hormone (GHRH) is an important physiological stimulator of the anterior pituitary somatotroph cell type, causing increases in both growth hormone (GH) synthesis and secretion (Fukata et al., 1985, Theill and Karin, 1993, Torchia et al., 1998). As synthesis of GH is regulated at the transcriptional level, GHRH increases the GH transcription rate by 200–300% in primary pituitary cultures. This increase is evident in as little as 10 min, indicating that the initial events in stimulation occur rapidly (Barinaga et al., 1983, Barinaga et al., 1985). Following 24 h of GHRH treatment, steady-state levels of GH mRNA are increased by over 200%, and these increases are maintained for at least 4 days, suggesting that the effects of GHRH are not transient (Gick et al., 1984).

The effects of GHRH are mediated by a specific seven transmembrane G-protein coupled receptor which is expressed primarily in the anterior pituitary (Mayo, 1992). Ligand binding to the GHRH receptor leads to activation of adenylate cyclase (Bilezikjian and Vale, 1983). The resulting increase in intracellular cAMP levels activates protein kinase A (PKA) (Bilezikjian et al., 1987), which is required for cAMP-dependent stimulation of GH transcription (Shepard et al., 1994). Once PKA is activated, it translocates to the nucleus where it phosphorylates a number of targets (Hagiwara et al., 1993). However, the mechanism by which PKA stimulates GH transcription remains to be elucidated.

Delineating the mechanisms by which GHRH regulates GH mRNA expression has been hindered by the lack of GHRH responsive cell line models. Although primary pituitary cultures are derived from normal tissue and are GHRH responsive, they contain multiple hormone-secreting cell types (Tashjian, 1979, Billestrup et al., 1986, Hoeffler and Frawley, 1987, Hoeffler et al., 1987). The heterologous composition of these cultures makes molecular and biochemical techniques difficult to interpret. Several immortal cell lines derived from pituitary tumors produce GH at high levels (Bancroft, 1981). These cell lines typically also produce detectable prolactin (PRL) making them a model of the somatomammotroph cell, which is defined by the production of both GH and PRL. However, such cell lines do not typically express the GHRH receptor or respond to GHRH at the level of GH secretion, or synthesis of mRNA or protein (Zeytin et al., 1984).

The rat MtT/S cell line was cloned by limiting dilution from an estrogen induced pituitary tumor (Inoue et al., 1990). The cell line expresses GH at high levels without detectable PRL under normal conditions. GHRH treatment results in elevated MtT/S cell GH secretion and GH protein synthesis (Inoue et al., 1990). Further, MtT/S cells are unique in the production of GHRH-R mRNA (Miller and Mayo, 1997b). Thus, the MtT/S cell line may be a useful model to study control of GH transcription by GHRH, especially because transcription from the endogenous GH gene can be studied.

Section snippets

Preparation of media and hormone stocks

MtT/S cells were routinely cultured in serum-containing medium as previously described (Inoue et al., 1990). Complete medium (CM) was composed of DMEM/F12 (1:1) medium containing 3151 mg/l d-glucose, 365 mg/l l-glutamine, and 2.438 g/l sodium bicarbonate supplemented with 10% donor horse serum, 2% certified fetal bovine serum, 50 U/ml penicillin G sodium, 50 μg/ml streptomycin sulfate, and 100 μg/ml kanamycin sulfate (Life Technologies, Grand Island, NY). Serum-free medium (SFM) was CM without

Measurement of GH mRNA by chemiluminescent-ribonuclease protection assay (c-RPA)

The use of the c-RPA required testing for accuracy and linearity of measurement. Antisense GH probe was hybridized with yeast RNA followed by incubation without or with RNase. No protected fragments of the probe remained after RNase treatment, indicating that hybridization was specific and digestion was complete (Fig. 1, left lane). GH antisense probe was hybridized with MtT/S target RNA, then incubated with RNase. As expected, RNase-treated GH probe migrated farther than untreated probe

Discussion

MtT/S cells are a pure somatotrophic cell line expressing high levels of GH (Inoue et al., 1990). In order to investigate the potential use of MtT/S cells for the analysis of GH transcriptional regulation, GH mRNA levels in response to a variety of stimulatory conditions were examined. Under unstimulated growth conditions, MtT/S cells maintain a high level of GH mRNA transcripts. Results in serum-free conditions suggest that such high level GH mRNA expression requires some factor(s) present in

Acknowledgements

The authors sincerely appreciate the contribution of GHRH by Dr William A. Murphy, Peptide Research Laboratories, Tulane School of Medicine, as well as helpful comments and insight. We thank Dr Ron Evans, Salk Institute, for the rat GHRH cDNA. These studies were supported by PHS grant DK48071 to K.E.M., and NSF Career (Presidential Young Investigator) Award IBN-9600805 to D.L.H.

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