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Department of Medicine (X.-d.P., S.P., M.R.G., L.A.F., R.D.K.), University of Illinois at Chicago, Chicago, Illinois; and Edison Biotechnology Institute (K.T.C., J.J.K.), and Department of Biomedical Sciences, College of Osteopathic Medicine (J.J.K.), Ohio University, Athens, Ohio
Address all correspondence and requests for reprints to: Rhonda D. Kineman, Ph.D., Department of Medicine (M/C 640), University of Illinois at Chicago, 1819 West Polk, Chicago, Illinois 60612. E-mail: kineman{at}uic.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Further examination of the GH-axis of the SDR has revealed that GH-mediated changes in hypothalamic GHRH and SRIF expression are also associated with changes in the expression patterns of the pituitary receptors known to regulate GH synthesis and release. In the absence of GH, pituitary messenger RNA (mRNA) levels for the GHRH receptor (GHRH- R), the GH secretagogue receptor (GHS-R), and the SRIF receptor subtype, sst2, are increased; whereas mRNA levels for the SRIF receptor subtype, sst5, are suppressed (5, 7, 8). GH replacement restored SDR pituitary receptor expression levels to normal, indicating that GH regulates its own synthesis and release not only by altering the expression of hypothalamic neuropeptides but also by regulating pituitary receptor synthesis.
To determine whether the GH-mediated changes in hypothalamic neuropeptide and pituitary receptor expression observed in the SDR are unique to this model system or represent generalized mechanisms of GH negative feedback regulation, we have examined the hypothalamic-pituitary axis of two genetically engineered mouse strains with altered GH signaling and production: 1) the GH receptor/binding protein gene-disrupted mouse (GHR/BP-/-); and 2) the metallothionein promoter-driven human GHRH (MT-hGHRH) transgenic mouse. The GHR/BP-/- mouse was developed by targeted disruption of exon 4 (the GH binding domain) of the GH receptor gene (9). The lack of GH signaling leads to an increase in circulating GH levels, while IGF-I is reduced and growth is severely retarded. The MT-hGHRH mouse, originally developed by Hammer et al. (10), expresses the hGHRH transgene in most tissues of the body, including the pituitary and hypothalamus (11). Chronic GHRH stimulation leads to increased serum GH and IGF-I levels and a giant phenotype. In the present study, ribonuclease (RNase) protection assays (RPAs) were used to examine the effect of altered GH signaling and production on hypothalamic neuropeptide [GHRH, SRIF, and neuropeptide Y (NPY)] mRNA levels, while multiplex RT-PCR was used to examine pituitary receptor (GHRH-R, GHS-R, sst2, and sst5) expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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GH immunocytochemistry
To assess the proportion of somatotropes in anterior pituitaries
from male GHR/BP-/-, MT-hGHRH, and their respective normal
littermates, 2–3 anterior pituitaries were pooled and mechanically and
enzymatically dissociated into single cells (n = 2
experiments). Monodispersed cells were placed on
poly-L-lysine-coated microscope slides, fixed, and
immunostained for GH using a monkey antirat GH serum (1:100,000)
that exhibits complete cross-reactivity with mouse GH. Details of the
pituitary dispersion and immunocytochemistry procedures have been
previously reported (14).
RPA of hypothalamic GHRH, SRIF, and NPY mRNA
One microgram of mouse hypothalamic RNA was reverse transcribed
using Superscript II RT (Life Technologies, Inc., St.
Louis, MO) with an oligo d(T) primer. To generate riboprobes,
complementary DNA (cDNA) was amplified by PCR using primers for mouse
GHRH, SRIF, NPY, or 
-actin. The antisense primers were modified to
contain a 17-base T7 RNA polymerase recognition sequence
(5'-TAATACGACTCACTATA-3'), a 6-base transcription initiation sequence,
and 15 or 20 bases of nonspecific (not hybridizable) sequence appended
at the 5' end. The nonspecific sequence was added to the primer to
allow for the differentiation of protected and unprotected probe after
RNase digestion. The amplified PCR products were used as templates for
in vitro transcription performed using the MAXIscript kit
(Ambion, Inc., Austin, TX) in the presence of
[
-32P]CTP. Radiolabeled riboprobes
were gel-purified before use. The primer sequences and the expected
sizes of full-length riboprobes and protected fragments are summarized
in Table 1
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-actin (4 x
103 cpm; specific activity, 8 x
107 cpm/µg) were incubated for 20 min at 68 C
in 10 µl HybSpeed Hybridization Buffer containing 50% of the total
RNA isolated from a single hypothalamus, or 50 µg yeast RNA (negative
control). Unhybridized probes were digested by treating the reactions
with RNase A/T1 mix (1.0 µg/20 U) for 1 h at 37 C. Protected
fragments were separated by electrophoresis through a 5%
polyacrylamide/8M urea gel. Gels were dried on chromatography
paper and exposed to a phosphorimage screen. Band intensity was
evaluated by ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA). It should be noted that the GHRH riboprobe
was specific for the mouse GHRH transcript and did not hybridize to the
transcript produced by the hGHRH transgene.
Multiplex RT-PCR of pituitary GHRH-R, GHS-R, sst2, and sst5
mRNA
The relative levels of pituitary GHRH-R, GHS-R, sst2, and sst5
mRNA were measured by multiplex RT-PCR using glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) as an internal control. One microgram of total
RNA was reverse transcribed using the Superscript Preamplification
System for First Strand Synthesis (Life Technologies, Inc.) with random hexamer priming in a 20-µl vol. The
resultant cDNA was used in three separate PCR reaction mixtures
containing specific primers for either GHRH-R and GAPDHa (reaction no.
1); GHS-R and GAPDHb (reaction no. 2); or sst2, sst5, and GAPDHa
(reaction no. 3). Primer sequences used in each PCR reaction (see Table 2) were selected on the basis of: 1)
comparable annealing temperatures; 2) transcript specificity (as
determined by a GenBank search); 3) exclusion of primers that showed
secondary structure or primer/primer interactions; and 4) product size.
Primer concentrations were empirically determined to achieve a final
signal that was comparable for all PCR products within each reaction
and that would provide noncompetitive and specific amplification for
each PCR product. Therefore, this technique can only be used to compare
the expression level of a single receptor type between experimental
groups and not the relative expression levels between the various
receptor types. Each PCR reaction was performed in a 50-µl vol
containing 1.5 µl RT (reaction nos. 1 and 3) or 2.0 µl RT (reaction
no. 2) and 1x PCR buffer, 1.5 mM
MgCl2, 0.2 mM deoxynucleotide
triphosphates, 2U Taq Gold polymerase (Perkin-Elmer Corp., Branchburg, NJ), and 5 µCi
[-32P]dCTP (deoxycytidine triphosphate)
(specific activity, 800 Ci/mmol). All PCR reactions consisted of a
predenaturing step at 95 C for 10 min, 26–29 cycles of 1-min
denaturation (95 C), 1-min annealing (62–69 C), and 1-min extension
(72 C), followed by a final extension at 72 C for 10 min (see Table 2
for specific reaction details). PCR products from reactions no. 1 and
no. 3 were separated on 5% polyacrylamide/8M urea gels while
transcripts from reaction no. 2 were separated on 7% polyacrylamide/8M
urea gels. Gels were dried on chromatography paper and exposed to a
phosphorimage screen. Band intensity was evaluated by image analysis
software (Molecular Dynamics, Inc.). There was no
significant difference in GAPDH mRNA levels between experimental
groups. Therefore, signal intensity for each of the pituitary receptor
subtypes was adjusted by that of GAPDH to control for variability in
the amount of total RNA used in the RT reaction and the efficiency of
conversion of RNA to cDNA.
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); 25 and 29 cycles for GHS-R and
GAPDHb (Fig. 1B); and 24 and 32 cycles for sst2, sst5, and GAPDHa (Fig. 1C). Similar results were obtained in at least two independent
experiments indicating that: 1) there is no competition between primer
sets; 2) reagents are not limiting; and 3) amplification efficiencies
are similar between all transcripts within each reaction. Therefore,
all subsequent amplifications were performed using 26, 29, and 28
cycles, respectively.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Although the pituitaries
were reduced in size, the proportion of GH immunopositive cells
represented 64% of all pituitary cells, in contrast to the 51%
observed in normal mouse pituitaries. Hypothalamic GHRH mRNA levels in
GHR/BP-/- mice were 232 ± 20% (P < 0.01) of
GHR/BP+/+ littermates, whereas SRIF and NPY mRNA levels were reduced to
86 ± 2% (P < 0.05) and 46 ± 3%
(P < 0.01), respectively (Fig. 2A). Pituitary GHRH-R and GHS-R mRNA
levels of GHR/BP-/- mice were elevated to 275 ± 55% and
319 ± 68% of GHR/BP+/+ values (P < 0.05),
whereas the sst2 and sst5 mRNA levels did not differ from controls
(Fig. 2B).
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(10)]. The increase in pituitary size has been attributed
to hyperplasia of the somatotrope population (16), which
is reflected in this study by the increase in the proportion of GH
immunopositive cells, representing 70% of all pituitary cells (Table 3). Within the hypothalamus of the MT-hGHRH mouse, endogenous (mouse)
GHRH mRNA levels were reduced to 69 ± 6% (P <
0.01) of nontransgenic controls, whereas SRIF mRNA levels were
increased to 128 ± 6% (P < 0.01; Fig. 3A). There was no significant difference
in NPY mRNA levels between MT-hGHRH mice and their nontransgenic
littermates (100% ± 7% vs. 112 ± 8%,
P > 0.05). Despite changes in hypothalamic GHRH and
SRIF mRNA levels, MT-hGHRH pituitary GHRH-R and GHS-R mRNA levels did
not differ from controls (100 ± 8% vs. 112 ±
8%, and 100 ± 16% vs. 104 ± 15%,
respectively; P > 0.05; Fig. 3B). In contrast, sst2
and sst5 mRNA levels were increased to 147 ± 18% and 143 ±16%
of normal values, respectively (P < 0.05; Fig. 3B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Of interest is what drives these GH-mediated changes in hypothalamic
GHRH and SRIF expression. A schematic, representing the putative
interactions of the various components of the GH-axis, is shown in Fig. 4, to serve as a reference for the
discussion that follows. Evidence from functional as well as anatomical
studies demonstrate that NPY neurons within the arcuate nucleus express
GHRs. These studies also suggest that NPY may be required for
GH-mediated changes in GHRH and SRIF expression (reviewed in
20, 21). It is hypothesized that, in the presence
of elevated circulating GH, NPY neurons are activated, thus decreasing
GHRH production within the arcuate nucleus and increasing SRIF
production within the periventricular nucleus. This hypothesis is
supported by the fact that genetic disorders with decreased GH
production [SDR; (5)] or GH signaling (GHR/BP-/-,
current report) are characterized by reduced hypothalamic NPY
expression. If GH-stimulated NPY expression leads to a decrease in GHRH
and an increase in SRIF, we might have expected NPY mRNA levels to be
elevated in the hypothalamus of the MT-hGHRH mouse; however, this was
not the case. One possibility that would account for this discrepancy
may be that maximal GH effects on NPY expression are achieved at
physiologic concentrations; therefore, GH hypersecretion, as observed
in the MT-hGHRH mouse, would not further stimulate NPY gene expression.
This hypothesis is consistent with the fact that GH-mediated changes in
hypothalamic NPY mRNA levels have been reported only after GH
replacement in GH-deficient states (5, 20, 21).
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Because GHS-R expression parallels GHRH-R mRNA levels in both the GHR/BP-/- mouse and the SDR, it is tempting to speculate that the genes for the GHRH-R and GHS-R share common regulators. And indeed, increases in circulating GH levels were associated with a reduction in pituitary GHS-R mRNA levels in SDRs (7) and in rats implanted with GH-producing tumors (28). Direct evidence supporting a role for SRIF and IGF-I in these GH-induced changes awaits further experimentation. However, a positive role for GHRH in GHS-R regulation has been suggested. Hypophysectomized rats with pituitaries transplanted under the kidney capsule maintain responsiveness to GHRP-6 by prior GHRH priming (29). Also, acute GHRH exposure (4 h; iv) of normal rats or SDRs results in a 2-fold increase in GHS-R mRNA levels (26). However, the acute stimulatory effect of GHRH on GHS-R mRNA levels in vivo could not be reproduced in vitro (26), suggesting that GHRH modulates additional factors important in pituitary GHS-R gene regulation.
In contrast to the GHR/BP-/- mouse, mRNA levels for GHRH-R and GHS-R were unchanged in the MT-hGHRH mouse. These divergent observations may be explained by the fact that, in the MT-hGHRH mouse, unlike the GHR/BP-/- mouse, functional GHRs are present; and therefore, GH feedback regulation is intact. Hyperstimulation of the anterior pituitary by hGHRH leads to an increase in circulating GH that stimulates the production of peripheral IGF-I, in addition to increasing hypothalamic expression of SRIF. Elevated IGF-I and SRIF would, in turn, counteract the stimulatory actions of GHRH on its own receptor expression. It remains to be determined whether IGF-I and SRIF also suppress GHS-R synthesis.
Results obtained from this and previous studies demonstrate that GH feedback regulation includes regulation of the GH-stimulatory pituitary receptors. However, the association between GH and its inhibitory receptors (i.e. SRIF receptors) is not as clear. Seven SRIF receptor subtypes, encoded by five separate genes (sst1–5), have been cloned and characterized. Although all SRIF receptor subtypes have been detected in the anterior pituitary, in situ hybridization and immunocytochemistry studies indicate that sst2 and sst5 are the most dominant isoforms within the somatotrope population (reviewed in 30, 31). In addition, sst2 and sst5 selective agonists suppress GH release from primary pituitary cell cultures at concentrations 1000-fold less than sst1, 3, and 4 agonists (32), suggesting that sst2 and sst5 are primary mediators of pituitary GH release. Therefore, in the present report, we chose to examine the impact of genetic disruption of the GH-axis on pituitary sst2 and sst5 mRNA levels. In the GHR/BP-/- mouse, sst2 and sst5 expression levels did not differ from GHR/BP intact controls; whereas, in the MT-hGHRH mouse, both sst2 and sst5 mRNA levels were elevated to 140% of wild-type littermates. These observations were unexpected, because we had previously reported that in the SDR, the lack of GH was associated with an increase in sst2 and a decrease in sst5 mRNA levels, which could both be normalized with GH replacement (8). Taken together, these data suggest that GH-mediated regulation of pituitary SRIF receptor subtype expression may be species-specific.
It should be noted that comparisons between GH-regulatory pituitary receptor expression in wild-type and genetic mutant strains were made on a per-microgram-of-total-RNA basis. However, the proportion of somatotropes was significantly elevated in both GHR/BP-/- mice (64%) and MT-hGHRH mice (70%), compared with their respective controls (50%). These observations are consistent with previous histological evaluations demonstrating somatotrope hyperplasia in GHR/BP-/- (33) and MT-hGHRH (16) mice. Therefore, it can be argued that changes in pituitary receptor expression may be related to changes in the number of cells expressing the receptor and not to changes in the level of receptor expression per cell. Of all the pituitary receptor types studied, only the GHRH-R has been shown to be exclusively expressed in the somatotrope (34, 35). Therefore, a portion (but not all) of the more than 2-fold increase in GHRH-R mRNA levels observed in the GHR/BP-/- mice may be attributable to the relative increase in somatotrope number. Applying this same rationale to the other pituitary receptors (GHS-R, sst2, and sst5) is more difficult, in that the expression of these receptors is not limited to the somatotrope population. Lactotropes, thyrotropes, and corticotropes, as well as somatotropes, have been shown to express the GHS-R (36, 37, 38), whereas all pituitary cell types express sst2 and sst5 (30, 31). Therefore, it remains to be determined whether changes in the relative proportion of somatotropes contributed to the changes in GHS-R, sst2, and sst5 expression observed in this study.
In summary, examination of the GH axis of GHR/BP-/- and MT-hGHRH mice demonstrates that changes in GH signaling or production alters both hypothalamic neuropeptide and pituitary receptor expression. In the absence of GH negative feedback, as observed in the GHR/BP-/- mouse, both hypothalamic and pituitary expression is altered to favor stimulation of GH synthesis and release; whereas, in the presence of GH negative feedback, as in the MT-hGHRH mouse, both hypothalamic and pituitary expression is altered to favor suppression of GH synthesis and release.
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Footnotes |
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2 Visiting Scientist from the Department of Pharmacology, Kyunghee
University School of Medicine, Seoul 130–701, Korea.
Received August 22, 2000.
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