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Department of Medicine, Section of Endocrinology and Metabolism, University of Illinois at Chicago, Chicago, Illinois 60612
Address all correspondence and requests for reprints to: Lawrence A. Frohman, M.D., Department of Medicine (M/C 787), University of Illinois at Chicago, 840 South Wood Street, Chicago, Illinois 60612.
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Introduction |
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Ligand-initiated receptor down-regulation has been theorized to include receptor/effector modification and uncoupling, internalization and degradation of existing receptors, and a decrease in receptor synthesis (6, 7, 8). With the recent cloning of various receptor complementary DNAs (cDNAs), it is now clear that transcriptional regulation of receptor synthesis can play an important role in homologous desensitization. Of the G protein-coupled receptors studied to date, messenger RNA (mRNA) levels of the receptors for TRH, TSH, GnRH, LH, CRH, and catecholamines are all decreased by in vitro exposure to their respective ligands (8, 9, 10, 11, 12, 13, 14, 15). The aim of the present study was to determine if transcriptional regulation of GHRH-receptor (GHRH-R) expression is also an important component of GHRH-induced somatotrope desensitization. To this end we have used quantitative reverse transcription (RT)-PCR to evaluate the effect of GHRH exposure on the level of GHRH-R mRNA in rat pituitary cells, in vitro.
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Materials and Methods |
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Unless otherwise stated, all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) and tissue culture reagents and molecular biology reagents were obtained from GIBCO-BRL (Grand Island, NY).
Primary rat pituitary cell cultures
Anterior pituitaries were enzymatically and mechanically
dissociated into single cells and cultured for 3 days before
experimental treatment. Specifically, pituitaries were rinsed in
sterile MEM containing 0.1% BSA, 100 µg/ml streptomycin, and 100
U/ml penicillin (S-MEM). Individual pituitaries were minced and tissue
fragments were suspended in 10 ml of S-MEM containing 0.15% trypsin
(DIFCO Laboratories, Detroit, MI). The enzymatic digestion was carried
out in 15 ml polypropylene tubes at 37 C with gentle and continuous
rotation, as previously described (16). After 45 min, tissue pieces
were mechanically disrupted using a siliconized, flame-polished Pasteur
pipette. Following an additional 15 min incubation (37 C with
agitation), cells were triturated, washed, and resuspended in MEM alpha
medium supplemented with 0.1% BSA and antibiotics (
MEM).
Approximately 3.25 x 106 cells were recovered from
each pituitary with viability consistently greater than 95%, as
assessed by trypan blue exclusion. Cells were plated onto 12-well
tissue culture plates at a density of 720,000 cells/500 µl
medium/well in -MEM and allowed to attach for 1 h at 37 C in a
humidified atmosphere containing 95% air:5% CO2.
Following cell attachment, 500 µl of fresh MEM supplemented with
20% horse serum was added, to achieve a final concentration of 10%
serum.
After a 3-day culture period, wells were rinsed twice with serum-free
MEM and incubated for 1 h at 37 C. Medium was then replaced
with fresh medium and treated with rat GHRH (1–44) NH2
(Peninsula Laboratories Inc., Belmont, CA), dexamethasone (American
Regent Laboratories Inc., Shirley, NY), forskolin, octreotide acetate
(Sandostatin; Sandoz, East Hanover, NJ) or the appropriate control
vehicle. Following incubation at 37 C with test substances, medium was
collected and stored at -20 C until analyzed for GH (17).
RNA extraction
Total cellular RNA was extracted according to the Tri Reagent
protocol (Molecular Research Center, Cincinnati, OH) with the exception
that the aqueous phase was further purified by extraction with
phenol:chloroform:isoamyl alcohol (25:24:1; pH 5.2; Fisher Scientific,
Pittsburgh, PA) to improve the efficiency of the reverse transcription
reaction. RNA was then precipitated with isopropanol, and the pellet
washed with 70% ethanol, air dried, and resuspended in TE buffer (10
mM Tris-HCl, pH 7.6, 1 mM EDTA). The
concentration of RNA was determined by spectrophotometric analysis at
OD 260/280 nm. This method yielded 5.48 ± 0.23 µg/well of total
RNA (i.e. 0.76 µg/100,000 cells).
Generation of the RNA standard (RPS-1)
Rat GHRH-R cDNA was generated by oligo (deoxythymidine)-primed
reverse transcription of rat pituitary total RNA, followed by PCR
amplification using the following specific primers:
5'-AGCTGAGAGACGATGAGCTT-3' (sense, nt 125–144) and
5'-CGACAGGAGATGACGAAGGA-3' (antisense, nt 1144–1163). The primers were
selected from the cDNA sequence reported by Mayo (18). The resulting
1038-bp fragment (GHRH-R; Fig. 1
) was subcloned into the
pGEM T vector (Promega, Madison, WI) according to the manufacturer’s
instructions. The nucleotide sequence of the inserted fragment was
confirmed by partial sequencing using the Sequenase Version 2.0 DNA
sequencing kit (USB, Cleveland, OH). A 235-bp fragment (nt 734–968)
was excised from GHRH-R using BlnI and EcoNI. The
ends of the resultant linearized plasmid were blunted by the Klenow
fragment of DNA polymerase I and the strand was recircularized by
T4 DNA ligase. This shortened cDNA is referred to as RPS-1
(Fig. 1).
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Quantitative RT-PCR
GHRH-R mRNA levels were assessed using quantitative RT-PCR. In
this protocol, RNA samples obtained from pituitary cell cultures were
co-reverse transcribed with the RNA standard, RPS-1, to correct for
RT-PCR efficiency. The reagents and protocol for the reverse
transcription were obtained from the Superscript Preamplification
System for First Strand cDNA Synthesis (GIBCO-BRL). Specifically,
1 x 106 copies of RPS-1 were added to 1 µg of total
pituitary RNA and subsequently reverse transcribed in a final volume of
20 µl containing 50 ng random hexamers, deoxy (d)-NTPs (0.5
mM each of dATP, dCTP, dGTP, and dTTP), PCR buffer [200
mM Tris-HCl (pH 8.4), 50 mM KCl], 2.5
mM MgCl2, 0.01 M DTT and 200 U of
Superscript II Rnase H- reverse transcriptase. One-tenth
(2 µl) of the RT reaction was used for PCR amplification with a
primer set that would amplify both cDNAs for GHRH-R and RPS-1. The
sense primer (5'-TGCTACTTTCATCCTCAAGG-3') and the antisense primer
(5'-GAATGTGTGTGCCCGAGTCA-3') spanned nt 534–553 and nt 978–997 of the
rat GHRH-R cDNA sequence, respectively (Fig. 1
). The PCR reaction was
performed in a 50 µl volume that included 1 x PCR buffer
(Perkin-Elmer, Norwalk, CT), 1.5 mM MgCl2, 200
µ M each of dATP, dCTP, dGTP, dTTP (Boehringer Mannheim,
Indianapolis, IN), 0.05 µM of each primer and 1.25 U
Amplitaq (Perkin-Elmer). PCR amplification was performed with the
following cycle profile: 94 C for 3 min, annealing at 60 C for 1 min,
and extension at 72 C for 2 min, followed by 25 cycles of 95 C for 40
sec, 60 C for 1 min, and 72 C for 2 min. After the last cycle, there
was a final extension for 15 min at 72 C.
A 30-µl aliquot of the PCR product was analyzed by gel
electrophoresis using a 1.5% agarose gel containing 0.5 µg/ml of
ethidium bromide, transferred to a nylon membrane (Schleicher and
Schuell, Inc., Keene, NH), and hybridized with a probe that recognized
a 89-bp sequence internal to the primers and common to both GHRH-R and
RPS-1 (nt 613–701; Fig. 1). The probe was labeled using the Pharmacia
Oligolabeling kit (Pharmacia). Hybridization was performed with a total
of 1.2 x 107 dpm [32P]dCTP-labeled
probe at 42 C for 18 h. Membranes were washed with 1 x
SSC/1% SDS at 25 C for 30 min, at 65 C for 30 min, and with 0.1
x SSC/0.1% SDS at 65 C for 30 min. RT-PCR products were visualized by
a Molecular Dynamics phosphorimager (Sunnyvale, CA). Two distinct
bands, 464 bp and 229 bp, were generated that corresponded to the
predicted size of the GHRH-R and RPS-1 fragments, respectively.
PCR amplification of rat GAPDH cDNA
Rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was
amplified from one-tenth (2 µl) of the RT reaction using sense
(5'-AGGGCTGCCTTCTCTTGTGGAC -3') and antisense
(5'-CAGCATCAAAGGTGGAGGAAT-3') primers previously reported by Siegfried
et al. (19), to correct for variations in mRNA used in the
RT reaction. The PCR protocol was identical to that described for
GHRH-R/RPS-1 with the exception that the annealing temperature was 64
C. As with GHRH-R/RPS-1, the GAPDH PCR products were electrophoresed on
1.5% agarose gels, transferred to nylon membranes and hybridized with
a radiolabeled probe that corresponded to sequences internal to the
primers (nt 196–357).
RT-PCR data analysis
Triplicate wells were used for each treatment group within each
experiment. The intensity of each band (GHRH-R, RPS-1 and GAPDH) was
evaluated using the image analysis software package, ImageQuant
(Molecular Dynamics), where band intensity is expressed in pixels. The
relative level of GHRH-R mRNA/well was determined by the equation:
GHRH-R x 1/RPS-1 x 1/GAPDH. Thus, GHRH-R values were
normalized by RPS-1 to account for variability in RT-PCR efficiency and
further adjusted by GAPDH to correct for variations in starting mRNA
concentrations. Where appropriate, data was logarithmically transformed
and then subjected to ANOVA. Differences among treatment means were
determined by Bonferroni’s test. A P value less than 0.05
was considered significant.
GHRH-induced intracellular cAMP generation
To determine if GHRH and GH pretreatment altered somatotrope
sensitivity to subsequent GHRH exposure, pituitary cells were plated in
24-well tissue culture plates at 50,000 cells/well as described above.
After a 3-day culture period, wells were rinsed with serum-free medium
and preincubated for 4 h in the presence or absence of 10
nM GHRH or 8 µg/ml rat GH (National Hormone and Pituitary
Program, NIH, Bethesda, MD). Medium was then removed and replaced
with 1 ml of medium to which was added GHRH to achieve a final
concentration of 1 or 10 nM. Cultures were incubated for 15
min and cells were extracted with 0.1 M HCl in 95% EtOH
for assay of intracellular cAMP (20). Four wells were assigned to a
given treatment group and differences between groups were determined by
Student’s t test. A value of P < 0.05 was
interpreted as a significant difference.
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Results |
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, increasing
the number of RPS-1 copies from 1 x 104 to 3 x
106 did not affect the amplification efficiency of GHRH-R
(slope = -0.0052). The regression lines generated from the
amplification curves for RPS-1 and GHRH-R intersect at a point which
corresponds to 7 x 105 copies of GHRH-R mRNA/µg of
total RNA. By extrapolation, using the amount of RNA/pituitary cell
(7.6 pg) and the proportion of somatotropes/pituitary [40%; (21)],
we can estimate that each somatotrope contains approximately 13 copies
of GHRH-R mRNA. This experiment was repeated using a different RNA
preparation and gave similar results (1 x 106 copies
of GHRH-R mRNA/µg of total RNA; data not shown). In all subsequent
experiments, 1 x 106 copies of RPS-1 were added to
each RNA sample analyzed.
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. Although total RNA appeared to compete with
RPS-1 at high concentrations (3 and 5 µg) the ratio of GHRH-R/RPS-1
remained proportional to the amount of RNA added over a 10-fold
concentration range. In addition, this experiment confirmed our
previous observation that 1 µg of pituitary total RNA contains
approximately 1 x 106 copies of GHRH-R transcript
(i.e. a 1:1 relationship was observed between GHRH-R and
RPS-1 when 1 µg of total RNA was used).
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. The linear portions of the two
curves (which extends from 20–30 cycles) have similar slopes
indicating GHRH-R and RPS-1 have comparable amplification efficiencies.
Transcripts were undetectable at 20 cycles while amplification reached
a plateau at 30 cycles. Therefore, to accurately assess both increases
and decreases in the endogenous transcript (GHRH-R), 26 cycles of PCR
amplification, representing the midpoint of the linear portion of the
curve, were used for all subsequent experiments.
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, 24-h treatment of
pituitary cell cultures with 10 nM dexamethasone, in the
presence of 2% serum, resulted in a 4-fold increase in the GHRH-R mRNA
level. As previously reported (23, 24) dexamethasone treatment had
little effect on basal GH release. However, 24-h treatment with
octreotide (a somatostatin analogue) reduced the GH level to 22% of
the control value while having no significant effect on the GHRH-R mRNA
concentration.
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).
Pituitary cells cultured for 3 days in the presence of 10% horse serum
(HS) had no significant decrease in GHRH-R mRNA levels when compared
with values obtained from fresh whole pituitaries. However, GHRH-R mRNA
levels decreased to 25% of control levels (in 10% HS) following
4 h of culture in serum-free medium and were further reduced to
6% after 8 h in the absence of serum. Likewise, after 24 h
of culture in 2% serum, GHRH-R mRNA levels were significantly reduced
from those observed in the presence of 10% HS. In light of these
results, the effect of GHRH on GHRH-R mRNA was examined in subsequent
experiments at 4 h under serum-free conditions, thus providing an
environment where GHRH-R mRNA is within a measurable range and GHRH is
not subjected to degradation by serum.
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. GHRH inhibited
GHRH-R gene expression in a dose-related manner (Fig. 6A), with 0.1
nM GHRH effecting a 50% reduction in GHRH-R mRNA levels
compared with untreated controls. However, a higher dose of GHRH (1
nM) was required to elicit maximal GH release (Fig. 6B). To
determine if the high concentration of medium GH elicited by GHRH
stimulation was responsible for the down-regulation of GHRH mRNA, cells
were pretreated with GH and GHRH-R function was assessed by a
subsequent 15 min challenge with GHRH (Fig. 7). GH
pretreatment did not significantly alter the ability of GHRH to
stimulate a rise in cAMP while GHRH pretreatment resulted in the
predicted decrease in GHRH-induced intracellular cAMP accumulation.
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).
Forskolin treatment resulted in a 40% reduction of GHRH-R mRNA which
was comparable to that observed with 0.1 nM GHRH (Fig. 6A).
Forskolin, like GHRH, potently stimulated GH release, resulting in a
3-fold increase in medium GH concentrations. The ability of
dexamethasone to stimulate GHRH-R expression was also observed during a
short term exposure (4 h) and in the absence of serum.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These results lead to the question of whether GHRH acts to down-regulate its own receptors in vivo. Recently, Miki et al. (26) presented compelling evidence that this indeed does occur. They found that systemic administration of GHRH antibodies or a catecholamine synthesis inhibitor (diethyl-dithiocarbamate; DDC), both of which reduce GHRH concentrations, up-regulated GHRH-R mRNA, as assessed by a ribonuclease protection assay. Restoration of GHRH concentrations by simultaneous GHRH administration prevented the inhibitory effect of DDC. These in vivo results coupled with our in vitro findings indicate that GHRH can tonically inhibit the expression of its receptor by a direct interaction at the pituitary level.
Our results also demonstrate the inhibitory effect of GHRH is mediated by binding to its receptor and activation of the adenylate cyclase second messenger system cascade since forskolin, which bypasses membrane receptors and directly activates adenylate cyclase, also inhibited GHRH-R mRNA accumulation. Somatostatin has been shown to inhibit basal adenylate cyclase activity in rat pituitary preparations (27, 28, 29). However, treatment of pituitary cell cultures with the somatostatin analogue, octreotide, failed to alter GHRH-R mRNA levels. This suggests that cAMP does not exhibit a tonic inhibitory effect on GHRH-R mRNA accumulation but rather mediates ligand (GHRH)-induced receptor desensitization.
The ability of ligands to modulate the production of their own receptors by a receptor mediated mechanism is not unique to the GHRH system. For example, a recent report by Pozzoli et al. (14) demonstrated that CRH rapidly (3 h) inhibits CRH-R mRNA levels in a dose-dependent manner. Stimulation of adenylate cyclase, a known component of the CRH-activated second messenger system pathway (30), also reduced CRH-R mRNA concentrations. Similarly, both TSH and TRH have been reported to reduce their own receptor mRNA levels by activation of receptor-dependent cAMP pathways (10, 31, 32). Therefore, the same second messenger system cascade which acts to stimulate hormone release and synthesis can also serve as a mechanism to attenuate the ligand-initiated response.
Before concluding that the effect of GHRH on its receptor expression was direct, it was first necessary to consider the possibility that GH, rather than GHRH, is responsible for the reduction in GHRH-R mRNA observed because 1) GHRH induced increases in GH concentrations reached 5–8 µg/ml in our static culture system; and 2) GH receptors are present on pituitary cells (33). Therefore, the possibility exists that GH could act through GH receptors to inhibit GHRH-R expression. To test this hypothesis, pituitary cell cultures were pretreated with GH at concentrations comparable to those induced by GHRH and then stimulated by GHRH to elicit a rise in intracellular cAMP concentrations as a functional assay of GHRH-R levels. Consistent with previous reports (3, 4), GHRH pretreatment decreased subsequent GHRH-induced cAMP accumulation. However, GH pretreatment did not alter GHRH-R function. These results strongly suggest that the effect of GHRH on GHRH-R mRNA levels is independent of its effects on GH secretion.
The reduction or removal of serum from the culture media dramatically and rapidly reduced basal levels of GHRH-R mRNA. These findings imply that serum contains factors necessary for GHRH-R maintenance in vitro. Two likely candidates are glucocorticoids and T4, both of which have been previously shown to positively regulate GHRH-R levels. Glucocorticoids increase somatotrope sensitivity to GHRH (24) by increasing GHRH receptor numbers (22). This increase in GHRH-binding sites has been recently correlated with an increase in GHRH-R mRNA. Both Tamaki et al. (23) and our current report demonstrated that in vitro exposure of pituitary cell cultures to dexamethasone, for as little as 4 h, increased GHRH-R levels above untreated controls. Similarly, T4 has been shown to enhance somatotrope sensitivity to GHRH in vitro (34). Results from in vivo studies suggest that T4 exerts its positive effect on GHRH-R function by an increase in receptor synthesis. Miki et al. (35) reported that thyroidectomy decreased pituitary GHRH-R mRNA levels by 60%, and this decrease could be partially reversed by T4 replacement therapy. Comparable results were reported by Tam et al. (36) using rats rendered hypothyroid by propylthiouracil and methimazole treatment. Clearly, glucocorticoids and T4 augment somatotrope sensitivity to GHRH. However, it should be noted that neither hormone can prevent the homologous desensitization observed in vitro (3).
The results of the present study demonstrate that GHRH inhibits GHRH-R mRNA accumulation. However, the methodology used (RT-PCR) does not reveal if this decrease is due to a reduction in GHRH-R gene expression, a reduction in GHRH-R mRNA stability, or both. The ß-adrenergic receptor (ßAD-R) is often used as a prototype for G protein-coupled receptors and, like GHRH-R, ßAD-R mRNA levels are down-regulated by homologous stimulation. In this model, the mechanism that reduces receptor mRNA levels involves a cAMP-dependent destabilization of the receptor mRNA (8, 15). Ligand-initiated degradation of TRH receptor mRNA has also been reported (9, 32). In contrast, TSH suppresses the synthesis of its receptor by decreasing receptor gene transcription (31). However, LH both promotes its receptor degradation and decreases gene expression (12, 37). It remains to be determined if the mechanism by which GHRH-induces GHRH-R mRNA down-regulation includes transcriptional and/or posttranscriptional regulation.
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Acknowledgments |
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Footnotes |
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Received October 1, 1996.
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T3–1 cells.
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 S. Petersenn, A. C. Rasch, M. Heyens, and H. M. Schulte Structure and Regulation of the Human Growth Hormone-Releasing Hormone Receptor Gene Mol. Endocrinol., February 1, 1998; 12(2): 233 - 247. [Abstract] [Full Text]
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M. Kovacs, R. D. Kineman, A. V. Schally, M. Zarandi, K. Groot, and L. A. Frohman Effects of Antagonists of Growth Hormone-Releasing Hormone (GHRH) on GH and Insulin-Like Growth Factor I Levels in Transgenic Mice Overexpressing the Human GHRH Gene, an Animal Model of Acromegaly Endocrinology, November 1, 1997; 138(11): 4536 - 4542. [Abstract] [Full Text] [PDF]
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