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U-159, Institut National de la Santé et de la Recherche Médicale, 75014 Paris, France; and the Division of Neurophysiology, National Institute for Medical Research (D.F.C., P.B., C.A.F.), London, United Kingdom NW7 1AA
Address all correspondence and requests for reprints to: Dr. M. T. Bluet-Pajot, INSERM U-159, 2ter rue d’Alésia, 75014 Paris, France. E-mail: bluetmt{at}broca.inserm.fr
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several years ago, Charlton et al. (5) described the spontaneous dwarf dw/dw rat. This dwarf strain bears an autosomal recessive mutation whose nature is still uncharacterized (6), but which gives rise to specific partial GH deficiency. The residual GH continues to be released in a sexually dimorphic secretory pattern and is responsive to GHRH and somatostatin (SRIH) (7), whereas replacement therapy with GH or insulin growth factor I (IGF-I) stimulates normal growth (8). More recently, transgenesis has been used to create new models of GH deficiency or excess. Mice with human GH (hGH) expressed in the central nervous system show reductions in GHRH that lead to secondary GH deficiency and dwarfism (9). We have recently developed such a rat model of dominant dwarfism by expressing hGH under control of the rat GHRH (rGHRH) transgene (10). These transgenic growth-retarded (Tgr) rats also continue to exhibit a sexually dimorphic reduced GH secretory pattern and remain responsive to GH secretagogues that stimulate their growth (11).
GH production and release from somatotrophs in the anterior pituitary are regulated by a complex interplay between GHRH and SRIH secreted, respectively, from hypothalamic arcuate and periventricular neurons that project to the median eminence (12). GH elicits a negative feedback on its own secretion (13) by modulating GHRH (14) and SRIH (15) messenger RNA (mRNA) expression. The short term regulation of GHRH or SRIH by GH feedback can be studied by injection or infusion of exogenous GH (16). Long term exposure to heterologous GH can be achieved by studying selected transgenic animals. Alternatively, high circulating levels of rat GH (rGH) can be maintained for several weeks in female rats by implantation of GC cells that form encapsulated rGH-secreting sc tumors (17). In this model, it is possible to study the effects of chronic GH exposure on GH peripheral action and hypothalamic GH feedback. We have now attempted to combine these approaches by comparing the effects of chronic rGH exposure from GC cell implants in two different strains of rat (dw/dw and Tgr) that show dwarfism due to either primary or secondary GH deficiency. We now describe the effects of chronic GH exposure in these dwarf rats and have compared their hypothalamic expression of GHRH, SRIH, and GH receptors (GH-R) both in the basal state and after chronic GH exposure.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The pituitary cell implant model (see below) was developed in Wistar-Furth (WF) rats obtained from Iffa Credo (L’Arbresle, France). In preliminary studies, GC cell implants were made in DW animals. However, no tumor formation was evident, and we suspected that the GC cells were being rejected by the immune system of these animals (which are of the AS strain). The implants were, therefore, repeated in a fresh group of DW rats treated with cyclosporin (10 mg/kg·day, ip, 2 days before and 2 weeks after GC cell implants). This treatment allowed the GC cell implants to survive and grow in the treated DW females, and the results presented here for DW rats were all obtained in cyclosporin-treated animals.
The Tgr rats are also on the AS background, but in this case the transgene is dominant, and dwarfism can, therefore, be induced in other rat strains by crossing with Tgr rats. For this study, the Tgr transgene was crossed onto a partial Wistar-Furth background as follows. At 12 weeks of age, six female WF rats were mated with two male hemizygote (Tgr/+) rats of the AS strain. All of the progeny were, therefore, an F1 strain of AS/WF, half of which should carry the Tgr transgene. The pups were genotyped for Tgr by Southern blotting for the rGHRH-hGH transgene on DNA from tail biopsies. When GC cells were implanted in these F1 animals, tumor formation occurred regardless of whether they carried the Tgr transgene, and no immunosuppressant therapy was required. All of the experiments with GC cells in Tgr animals were, therefore, carried out in these F1 crosses. However, for the sake of simplicity, the WF/AS animals that carry the rGHRH-hGH transgene are referred to as WF/Tgr animals, whereas the nontransgenic littermate controls are designated WF/-.
Tumor model
GC cells were grown in Ham’s F-10 medium supplemented with 15%
horse serum and 2.5% FCS. A suspension of 2 x 107 GC
cells in Hanks’ medium (0.3 ml vol) was injected sc into the flank of
7-week-old female rats. Controls were injected sc with saline. Animals
were weighed weekly. Tumors became palpable from 2–3 weeks after
implantation, and animals not developing palpable tumors were discarded
from the study. At 12 weeks, animals were killed, a blood sample taken,
and pituitary and brain were removed rapidly, frozen on dry ice, and
stored at -70 C. The encapsulated sc rGH cell tumors were dissected
and weighed.
rGHBP assay
Plasma rGHBP was measured as previously described (18) using a
polyclonal antibody against recombinant rGHBP and recombinant rGHBP for
iodination and as standard. The results are expressed as nanograms of
rGHBP per ml.
rGH RIAs
After decapitation, pituitaries were homogenized in 50
mM PBS, pH 7.4. rGH plasma levels and pituitary contents
were assayed using reagents obtained from the NIDDK, as previously
described (19).
SRIH, GHRH, and GH-R in situ hybridization
SRIH and GHRH.
In situ hybridization was carried
out as described previously (20). Briefly, a 45-base oligoprobe
[corresponding to amino acids 96–111 of the prepro-SRIH complementary
DNA (cDNA); Genofit, Geneva, Switzerland] was labeled with
[
-35S]deoxy-ATP (Amersham, Aylesbury, UK) using
terminal deoxynucleotidyl transferase (Boeh-ringer Mannheim,
Meylan, France) at a specific activity of 2000 Ci/mM.
Sections were fixed for 10 min at room temperature in potassium
phosphate buffer containing 4% paraformaldehyde. Then they were
prehybridized for 30 min in a solution containing 4 x SSC and
1 x Denhart’s solution (Sigma, Saint-Quentin Fallavier, France)
and for 10 min in 4 x SSC containing triethanolamine (1.33%) and
anhydrous acetic acid (0.25%; pH 8.0). Hybridization was performed for
18 h at 38 C in the hybridization solution [50% formamide,
4 x SSC, 1 x Denhart’s solution, 1% sarcosyl, 10
mM dithiothreitol, 100 mM potassium phosphate
(pH 7.4), 100 ng yeast transfer RNA, and 100 ng herring sperm DNA]
containing the labeled oligoprobe (2 nM). Sections were
rinsed at 36 C for 30 min in 4 x SSC, three times for 15 min each
time in 1 x SSC, and three times for 15 min each time in 0.1
x SSC; dried; and coated by dipping in RPN 40 LM1 emulsion (Amersham).
Exposure time was 7 days and 20 days at 4 C for the detection of SRIH
mRNA in the periventricular nucleus (PeV) and arcuate nucleus (ARC),
respectively. Autoradiograms were developed in Dektol (Eastman Kodak,
Rochester, NY), stained with toluidine blue, and coverslipped.
A similar method was used for GHRH mRNA hybridization in the ARC (20). Briefly, a 45-base oligoprobe (bases 31–75 of the rat GHRH cDNA) provided by Genset (Strasbourg, France), was 3'-labeled with a [33P]deoxy-ATP (Amersham). The hybridization was carried out at 36 C, and sections were rinsed at 34 C. The exposure time of the dipped slides was 6–8 weeks.
rGH-R.
A cDNA clone, pGO.9, containing the 900-bp
bglII fragment of the rGH-R cDNA cloned into the
BamHI site of the vector pT7T318U was provided by Prof. G.
Norstedt (Huddinge, Sweden). The antisense cDNA probe and the sense
cDNA probe were synthesized in vitro with T7 polymerase on a
plasmid linearized with XbaI and with T3 polymerase on
plasmid linearized with SpeI, respectively. The radiolabeled
complementary RNA was synthesized in vitro with
[-35S]UTP (Amersham) at a concentration of 5
mM. The template DNA was removed by deoxyribonuclease I
treatment, and the radiolabeled riboprobe was separated from unreacted
components by phenol-chloroform-isoamyl alcohol extraction. The
riboprobe was then hydrolyzed with sodium hydrogen carbonate (0.4
M NaHCO3) to obtain fragments approximately 200
bases in length.
Sections of the PeV and ARC region were dried for 10 min at room temperature and fixed in 4% paraformaldehyde. Sections were rinsed in PBS and treated in a triethanolamine (1.4%) and acetic anhydride (0.25%) solution. Slides were dehydrated in a series of alcohols, delipidated in chloroform, and dried. The riboprobe was dissolved in hybridization buffer [25 mM Tris (pH 7.4), 1 mM EDTA, 350 mM NaCl, 60% deionized formamide, 12% dextran sulfate, 50 x Denhardt’s solution, 5 mg yeast transfer RNA, 5 mg single stranded salmon sperm DNA, and 125 nM dithiothreitol].
The rGH-R complementary RNA probe in hybridization buffer was positioned on all sections, which were glass covered, sealed with rubber cement, and incubated overnight at 50 C in a humidified chamber. After hybridization, coverslips were gently lifted off in 2 x SSC at room temperature, and the slides were washed for 30 min in two changes of 2 x SSC-50% formamide at 50 C. Sections were then rinsed briefly in 2 x SSC at 37 C and incubated in 2 x SSC containing 20 µg/ml ribonuclease A for 30 min at 37 C. Sections were again rinsed in 2 x SSC, then washed three times for 15 min each time in 2 x SSC-50% formamide at 50 C, followed by two room temperature washes in 2 x SSC for 5 min each time. Slides were briefly dipped in water, then air-dried. The dried sections were dipped in RPN 40 LM1 emulsion (Amersham). Exposure time was 3 weeks.
Image analysis and quantification.
Eight sections per region
(PeV and ARC) in each rat were analyzed for SRIH, GHRH, and GH-R mRNA
in situ hybridization. Sections were visualized at x500
magnification (Ortho-plan, Leitz, Rockleigh, NJ) under fluorescent
epiillumination. Grain counting was performed with a Biocom 200 image
analyzer (Les Ulis, France) using the computer-based image analysis
system (RAG 200) that allows rapid estimation of grain numbers over
neuronal perikarya. An internal calibration curve was recorded for each
section, measuring the mean quantity of light reflected by a known
number of grains according to the procedure described by Bisconte
et al. (21). Labeled neurons were identified by toluidine
blue under brightfield illumination and delineated on the screen, and
the quantity of light reflected in the area was measured under
epiillumination.
Statistical analysis
Values are given as the mean ± SEM, and
statistical analysis was performed by ANOVA, using StatView 4.02
software (Abacus Concepts, Palo Alto, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As expected, crossing the rGHRH-hGH transgene onto the WF
background induced dominant dwarfism in the Tgr-positive progeny. The
mean body weight of adult hemizygote WF/Tgr animals was approximately
70–75% that of the WF/- littermates (144 ± 3 vs.
187 ± 2 g; P < 0.01). In DW, WF/-,
and WF/Tgr strains, body weight was markedly increased in
tumor-bearing animals (Fig. 1
). This was
accompanied by consistently high plasma GH levels measured after
decapitation. In line with their more active tumor growth, terminal
plasma GH levels were significantly (P < 0.01) higher
in both groups of WF+GC rats than in the DW+GC rats (3600 ± 1000
and 4210 ± 670 vs. 830 ± 140 ng/ml in WF/-+GC
and WF/Tgr+GC vs. DW+GC rats, respectively).
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). Pituitary GH contents were
reduced by GC cell implants in WF/- animals, but there was no change
after GC cell implants in DW or WF/Tgr rats. GHBP levels were markedly
increased by GC cell implants in all strains (Table 1).
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). In DW rats, GHRH
mRNA-containing cell number and GHRH mRNA levels per cell were
considerably higher compared with those in WF/- and WF/Tgr
(P < 0.001; Fig. 3). In
WF/Tgr animals, GHRH mRNA-containing cell numbers and GHRH mRNA levels
per cell were decreased in the ARC compared with those in WF/-
(P < 0.001 and P < 0.05,
respectively; Fig. 3), whereas SRIH mRNA-containing cell number and
SRIH mRNA levels were slightly increased in the PeV (P
< 0.01 and P < 0.05, respectively), but were not
altered in the ARC (Fig. 4).
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), whereas they
increased SRIH cell numbers and SRIH mRNA levels in the PeV
(P < 0.001), but not in the ARC (Fig. 4). Similar
effects were observed in WF/- animals (Figs. 3 and 4), although the
increase in SRIH mRNA-containing cell number in the PeV did not reach
significance. In contrast, GC cell implants in WF/Tgr animals had no
effect on GHRH mRNA-containing cell number and GHRH mRNA levels in the
ARC (Fig. 3) and caused only a slight increase in PeV SRIH cell numbers
(P < 0.05) and a decrease in ARC SRIH cell numbers and
mRNA levels (P < 0.05; Fig. 4).
GH-R expression in PeV and ARC in the WF/Tgr rats remained unchanged
compared with that in their WF/- littermates (Fig. 5). In WF/- animals given GC cell
implants, there was an increase in GH-R-positive cells in ARC
(P < 0.001), but not in the PeV and no change in
number of grains per cell in either area. In contrast, GC cell implants
in the WF/Tgr animals reduced GH-R cell numbers and expression
significantly in both PeV (P < 0.05) and ARC
(P < 0.001 and P < 0.01,
respectively) compared with those in the untreated WF/Tgr group (Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As the tumor cells derive from the WF rats and fail to survive in AS rats, we thought it possible that cell survival might be possible if the host immune system were suppressed. Cyclosporin treatment did indeed make it possible to grow GC cell tumors in severely GH-deficient DW rats, and the GH produced from these tumors clearly stimulated growth. However, this approach was not completely successful, as the tumors grew more slowly, the GH levels achieved were not as high as those in WF rats with the same implants, and the increase in body weight was not as great. Furthermore, interpretation of these results is complicated by the possible side-effects of cyclosporine. This treatment did not modify the body weight increase, but induced a slight decrease in pituitary GH content in normal rats (our unpublished findings). The mechanism is unknown, but could be direct at the pituitary, as cyclosporin modifies the PRL and GH responses to dopamine and TRH in vitro (23).
Dwarfism induced in rat by a dominant transgene, provides a completely different model with the advantage that it could be induced in the WF strain by simple cross-breading with the AS Tgr line, and the progeny (WF/Tgr) may retain sufficient WF background to allow tumor formation by implanted GC cells. This proved to be the case and provided additional confirmation that the dwarf phenotype can be transferred to an unrelated rat strain in a single hemizygote cross. When the progeny were inoculated with GC cells, tumors formed in a high proportion of animals and subsequently grew as efficiently and produced as much circulating GH as those in their nontransgenic WF littermates. This completely overcame the dwarfism induced by the transgene and showed that peripheral GH-R in these animals are functional and capable of mediating a rapid growth response to GH excess indistinguishable from that in nontransgenic animals.
Chronic GH release from GC cells is continuous (24) rather than pulsatile, which is the hallmark of endogenous GH secretion (25). The GH-binding protein derived from alternative splicing of the GHR mRNA in the rat is particularly sensitive to continuous GH, although these observations have been limited to relatively short term exposure to heterologous GHs (26). Our data confirm that extended exposure to continuous rGH elevates plasma GHBP levels in three strains. We have previously shown that GHBP levels in DW females are lower than those in normal females (18). This study also provides the first data on plasma GHBP levels in female Tgr rats, which are lower than those in normal females, but not as low as those in DW rats, in line with their relative GH deficiencies. Thus, in the three conditions, GHBP is always regulated in parallel with GH levels and can regulate GH metabolic clearance and action on target cell receptors (27)
The second purpose of this study was to compare the responses to GC cell implants in strains that represent different types of dwarfism. DW rats have a primary pituitary defect, and hypothalamic GHRH is increased, but without subsequent effect, as in this model it has no trophic effect on the pituitary somatotrophs, nor does GHRH treatment stimulate their growth (28). In contrast, dwarfism in the Tgr animal is due to hypothalamic expression of the hGH transgene and reduction in GHRH expression, which results in secondary pituitary GH deficiency. In these animals, GHRH treatment readily stimulates growth (11). Thus, the two models of GH deficiency exhibit diametrically opposite changes in hypothalamic GHRH, and we attempted to determine how these would be affected by the transition from dwarfism to the acromegalic state by peripheral exposure to GH excess.
Normal animals exposed to chronic GH excess show a decrease in pituitary GH content, whether the GH arises from peripheral tumors (20) or from ectopically expressing GH transgenes (9, 29). Although trace amounts of GH-R mRNA can be identified in pituitary tissue (30, 31), there is no functional evidence for a direct feedback effect of GH on its own release (32). However, chronic overexposure to GH also increases circulating IGF-I levels, and this can inhibit both the synthesis and secretion of GH at the pituitary level (33, 34). Instead, it seems that the main site of direct GH feedback is hypothalamic, via GH-R in both PeV and ARC, and is expressed mainly on SRIH- and neuropeptide Y-containing cells, respectively, and possibly on a few GHRH containing-ARC cells (35, 36, 37). In a variety of GH deficiency models, PeV SRIH expression is reduced, whereas GHRH expression is increased; these changes are reduced by GH treatment and completely reversed by GH excess (38). Other evidence for a role of GH feedback in GH control is that GH pulses can be synchronized by serial GH injections (39). More recent evidence in favor of a direct effect of GH via GH-R is that antisense RNA for the GH-R enhances GH pulsatility and decreases hypothalamic SRIH expression without marked modification of GHRH expression (40).
We confirmed that normal WF rats showed reductions in hypothalamic GHRH expression and pituitary GH when implanted with GC cells (20). However, the WF/Tgr animals showed no further reduction in GHRH or pituitary GH after GC cell implants despite the marked stimulation of growth. This shows that the hypothalamic changes induced by peripheral GH excess in normal WF animals are not simply due to growth stimulation or increases in IGF-I, but most likely reflect a direct effect of GH. These are not seen in Tgr animals, which already show reduced GHRH expression and pituitary GH levels caused by the transgene. The original study in these rats only documented reduced GHRH expression in hypothalamic extracts (10). The present study extends these observations to show that both GHRH cell number and cell expression are reduced by the transgene-derived hGH, and that this effect is probably maximal, as no additional effect was seen with GC cell implants.
This contrasts completely with DW rats, whose pituitary GH deficiency leads to an increased arcuate GHRH cell number and expression (38, 41), which was reversed by GC cell implants. This treatment did not cause any further reduction in the already low DW pituitary GH content. Although this could be due to the comparatively smaller GC cell tumors leading to lower (but still elevated) circulating GH levels, the simplest explanation is that feedback effects of GH on GHRH are not reflected in GH production in these animals. Although DW rats are weakly responsive to the releasing effects of GHRH (7), they do not show increased cAMP (6), somatotroph proliferation, or body growth (42), suggesting a defect in GHRH transduction pathways that may be causally linked to the GH deficiency (6). As their high endogenous GHRH levels are unable to restore normal GH synthesis, suppression of these high GHRH levels by GC cell implants would thus be unlikely to reduce GH synthesis further. Indeed, pituitary GH contents might even rise, if the stimulus to release GH is lessened.
Somatostatin expression is also sensitive to GH feedback. The primary hypophysiotropic SRIH projections arise in the PeV, and their expression is up-regulated by GH excess (20) and reduced in GH deficiency (43). There is also a significant population of SRIH-containing cells in the ARC (44) whose function is unclear but which may have a role within the ARC to regulate GHRH neuronal activity (45). This is the first study of SRIH expression in Tgr rats and showed that the number of cells expressing SRIH is increased in PeV, but not in ARC. These results indicate that the direct feedback inhibition of hypothalamic hGH exerted on SRIH neurons still occurred despite the marked deficiency in peripheral GH, low IGF-I, and dwarfism exhibited by these rats (11). Other mouse models with central GH excess (9, 29) or central administration of GH to DW rats (38) also led to increased PeV SRIH, suggesting that the hypothalamic hGH produced by the transgene can gain access to receptors that regulate PeV SRIH expression. GC cell implants increased SRIH cell numbers and SRIH mRNA levels in the PeV in both WF/- and DW animals, but not in WF/Tgr animals, again suggesting that this effect was exerted locally by the hGH transgene.
GC cell implants in WF Tgr animals did cause a decrease in ARC SRIH cell numbers and mRNA levels, and the reason for this is unclear if the hGH feedback on GHRH in this site is already maximal. One obvious difference is that chronic GH excess will in addition raise IGF-I levels, and it is possible that the changes in ARC SRIH reflect an additional hypothalamic IGF-I feedback. However, similar IGF-I changes would have been expected in DW or WF/- rats, but no alterations in their ARC SRIH expression were seen.
One factor that must be taken into account is that the hypothalamic GH-R themselves are subject to complex feedback regulation by GH, and therefore, the strength of the feedback signal from peripheral GH may be altered by effects on GH-R expression. For example, we have previously reported that DW rats show a reduction in GH-R expression in the ARC that can be restored by peripheral treatment with GH, whereas large doses of GH given centrally to normal rats reduce their ARC GH-R expression (16). In the present study, GC cell treatment reduced ARC GH-R expression in the WF/Tgr dwarves, whereas GH-R expression was slightly increased in the normal animals receiving GC cell implants. The reason for these differences is not clear and may relate to the high local concentrations of hGH in the ARC of transgenic animals, but not in wild-type rats, or may be secondary to the effects on GHRH/SRIH expression provoked by the high circulating GH levels via down-regulation or desensitization of GH-R.
In conclusion, we have shown that chronic GH excess has differential feedback effects in different models of dwarfism with primary or secondary GH deficiency, and that the GC cell model can be successfully adapted to a transgenic model of dominant dwarfism. The combination of these approaches now makes it possible to study the effects of a wide range of pathophysiological concentrations of GH, ranging from dwarfism to acromegaly.
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Acknowledgments |
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This article is dedicated to the memory of our colleague Françoise Mounier.
Received March 17, 1997.
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