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Autoregulation of growth hormone receptor and growth hormone binding protein transcripts in brain and peripheral tissues of the rat
Growth Hormone and IGF Research, 1998, 8, 167-173
Autoregulation of growth hormone receptor and growth hormone binding protein transcripts in brain and peripheral tissues of the rat K.L. Hull* and S. Harvey Departmentof Physiology,Universityof Alberta,Edmonton,Alberta,Canada,T6G2H7 *Currentaddress:Departmentof Biology,Bishop'sUniversity, Lennoxville,Quebec,Canada,J1M 1Z7
Summary Growth hormone (GH) differs from other pituitary hormones in that it can affect a wide spectrum of cellular activities in many different tissues. These disparate actions are, however, mediated by a common receptor, suggesting tissue-specific differences in the post-receptor mechanisms and/or tissue sensitivities to GH stimulation may confer specificity. Tissue sensitivity depends upon the abundance of GH receptors (GHRs) and may be modulated by the amplitude and pulsatility of GH secretion. It may also be dependent upon the presence of non-signal transducing GHbinding proteins (GHBPs), which result from the alternate splicing of GHR gene transcripts. Tissue-specific autoregulation of GHRs and GHBPs could, therefore, contribute to differential tissue responsiveness to GH action. The autoregulation of GHR and GHBP gene transcription in novel central (hypothalamus, brainstem, and cortex/neocortex) and peripheral (spleen) tissues was therefore examined in adult, male Sprague-Dawley rats. For comparative purposes, GHR/GHBP gene expression was also examined in the liver, which has traditionally been considered the major GH-target site. Chronic hyposomatotropism, induced by hypophysectomy, exerted tissue-specific effects on the abundance of GHR gene products 10 days post-hypophysectomy. Both GHR and GHBP transcripts were reduced in the hypothalamus of hypophysectomized rats by 20% (P < 0.001), although neither transcript was affected in the liver, spleen, cortex/neocortex or brainstem. In contrast, 2 h after a single bolus GH injection that was designed to simulate a pulsatile increase in circulating GH concentrations, GHR and GHBP mRNA content was significantly increased by 25-30% (P < 0.001) in all brain regions and in the spleen of hypophysectomized or sham-hypophysectomized rats. Production of the two transcripts was differentially regulated, however, as GHBP, but not GHR, transcripts were increased in the liver (P < 0.001), whereas the GHR:GHBP ratio was decreased in the hypothalamus of GH-treated rats (P < 0.001). These results suggest that GHR gene transcription and splicing are acutely autoregulated in a tissue-specific way. 9 1998 Churchill Livingstone
Key words: growth hormone receptors, growth hormone, brain, immune system, regulation, autoregulation.
INTRODUCTION Growth hormone (GH) can directly influence cellular development and metabolism by binding to growth Correspondence to: StephenHarvey,Departmentof Physiology,University of Alberta,Edmonton,Alberta,Canada,T6G2H7.
1096-6374/98/020167+07$18.00/0
hormone receptors (GHRs) and activating intracellular signal transduction mechanisms. 1,2The magnitude of the resulting cellular change is dependent upon factors such as the amplitude and frequency of the GH pulse. 3,4Tissue responsiveness to GH can be further modulated by alterations in GHR gene transcription or translation. GHR regulation by GH itself may be a particularly important 9 1998 Churchill Livingstone
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K.L. Hull and S. Harvey
determinant of GH action, as it would enhance or reduce the effect of a particular GH dose. The relationship between GH and GHR abundance, however, is not clearly understood. 5 Indeed, hypophysectomy had been shown to increase 6,7 or have no effect8,9 on hepatic GHR mRNA and GH-binding. The relationship between GH and GHR abundance may therefore be complex and depend upon the sex, hormonal milieu, and age of the experimental animal. 5 The ability of GH to autoregulate its receptor may also be temporally related, especially as differential effects on hepatic GH binding and GHR mRNA have been observed in response to chronic and acute GH stimulation, z-1I The physiological relevance of changes induced by chronic GH stimulation are, however, uncertain, since GH secretion in almost all species is phasic rather than tonic. In addition to changes in GHR and GHR mRNA, the production of growth hormone binding proteins (GHBPs) may provide an additional locus for the regulation of somatotropic function. Tissue GHBPs may locally inhibit GH action by competing with GHRs for available GH, 12-14 thus, an increase in GHBP content could negate a corresponding increase in tissue GHR. Tissue GHBPs may also maintain high local GH concentrations by serving as a GH reservoir. ]5 The ratio between signal-transducing receptors and truncated binding proteins, rather than absolute receptor levels, may thus determine the magnitude of tissue responsiveness to GH stimulation. In rats, the GHBP is comprised of the extracellular domain of the GHR that is C-terminally extended by a short hydrophilic tail. This protein is synthesized from a splice variant of the GHR gene. 16,17Splice site usage and thus the GHR:GHBP ratio can, however, be regulated, since hepatic GHBP mRNA but not GHR m R N A is increased in hypophysectomized rats by long-term GH treatment.Is The liver is not, however, the only GH-target site, as GHRs and GH responsiveness have been demonstrated in numerous non-hepatic organs? These non-hepatic GHRs may also be dependent upon GH status, but may be regulated differently than the hepatic GHR. This possibility was first suggested by Frick et al., 7 who showed that GHR mRNA was increased in rat muscle and liver, but decreased in fat by hypophysectomy. Splice site usage is similarly tissue-specific, as the GHR mRNA: GHBP mRNA ratio differs between tissues? 9 Tissue difference in GHR and GHBP autoregulation could thus permit tissue-specific responses when stimulated by identical blood GH levels. Indeed, since GH secretion in male rats is highly pulsatile, acute (within 2-3 h) autoregulation of GHR/GHBP gene transcription may be a particularly important determinant of GH action. In recent years, the demonstration of GHRs and GHBPs in the central nervous system (CNS) 2~ and
immune system n,23 has suggested novel roles for GH in neural and neuroendocrine function24,25 and in cellular and humeral immunity,z3 The regulation of the GHR gene in these tissues is, however, poorly understood. The possibility that neural and immune GHR gene transcripts may be autoregulated has therefore been determined in this study and compared with the autoregulation of hepatic GHR gene transcription. MATERIALS AND METHODS Animals
The secretion of GH in male rats is pulsatile, with an episode of GH release every 3 h. 26 The possibility that a phasic rather than chronic increase in the circulating GH concentration might autoregulate GHR gene expression was therefore examined. Intact adult male Sprague-Dawley rats (Charles River, Wilmington, MA, USA) were intraperitoneally (i.p.) injected with ovine GH 27 (oGH)(NIH-oGH-; NIADKD, Bethesda, MD, USA) (150 ~tg/100 g body weight) or the 0.90/0 (w/v) NaC1 vehicle (100 ~d/100 g). Ovine GH has previously been shown to interact with GH binding sites in rat tissues. 2s For comparative purposes, oGH and the saline vehicle were similarly injected into hypophysectomized adult SpragueDawley rats (Charles River), approximately 10 days posthypophysectomy. The animals (n = 10 per group) were killed by decapitation 2 h after the injection, and central (brainstem, hypothalamus and neocortex/cortex) and peripheral (spleen, liver) tissues were rapidly dissected out and quick-frozen in liquid nitrogen. Northern analysis
Tissue GHR and GHBP mRNA were quantified by Northern analysis. Total RNA was extracted from the frozen (-80~ livers, spleens, hypothalami, brainstems and cortex/neocortex using commercial reagents, according to the manufacturer's instructions (RNA Now; Biocan Scientific, Mississauga, Ontario, Canada). The RNA was subjected to Northern analysis as previously described, 29 using a complementary DNA fragment (pRatl-20) (provided by Dr W Baumbach, American Cyanamid, Princeton, NJ, USA) encoding the majority of the extracellular domain of the rat GHR. GHR mRNA and GHBP mRNA content w e r e quantified by laser densitometry and corrected for loading error by comparison with the hybridization of an 18S cDNA probe (American Type Culture Collection, Baltimore, MA, USA). Autoradiograph exposure time was optimized for each tissue, although this prohibited comparisons between tissues. Treatmentinduced differences in transcript abundance in the liver, spleen, hypothalarnus, neocortex/cortex and brainstem
Autoregulation of GH receptors 169
were determined by one-way analysis of variance (ANOVA) and means were separated by the Least Significant Difference (LSD) test. RESULTS
As previously shown, 21 transcripts of 4.4 and 1.2 kb coding for the GHR and GHBP, respectively, were detected in central (hypothalamus, brainstem, and neocortex/ cortex) and immune (spleen) tissues that corresponded in size to transcripts found in hepatic extracts (data not shown). The abundance of hepatic GHR transcripts was not significantly (P > 0.05) affected by hypophysectomy or GH treatment (Figs 1 and 2). In contrast, hypophysectomy reduced (by 20%) the abundance of the GHR transcript in the hypothalarnus, although it had no effect on GHR mRNA in other brain regions or in the spleen (Fig. 2). In further contrast, the abundance of GHR mRNA was significantly (P < 0.00l) increased by GH treatment in the spleen, hypothalamus, cortex/neocortex and brainstem of the intact and hypophysectomized rats (Fig. 2). In response to GH, the relative increase in GHR mRNA abundance in these extrahepatic tissues was comparable (20-30%) and in most tissues the GH-induced increase in intact animals was also comparable to the increase in hypophysectomized rats. The GH-induced increase in GHR mRNA in the cortex/neocortex of intact rats was, however, greater (P < 0.05) than that in hypophysectomized rats. The abundance of hepatic GHBP transcripts was also unaffected by hypophysectomy, but was increased (P< 0.001) by oGH injection in both intact and hypophysectomized rats (Figs 1 and 3). Hypophysectomy also had no effect on the abundance of GHBP transcripts in the spleen, neocortex/cortex or brainstem, but reduced
GHBP mRNA in the hypothalamus (Fig. 3). Two hours after the administration of oGH, the abundance of GHBP mRNA was consistently increased in the spleen, hypothalamus, cortex/neocortex and brainstem (Fig. 3). The relative increase in GHBP mRNA abundance in each tissue of the GH-treated intact rats was comparable to the increase induced in GH-treated hypophysectomized rats. In intact rats, the GHR:GHBP ratio in the liver, spleen, cortex/neocortex and brainstem was approximately 0.8:1 (Fig. 4), indicating a preferential expression of the GHBP transcript. The GHR:GHBP ratio in the liver, spleen and brainstem was unaffected by hypophysectomy or by oGH treatment. In contrast, the GHR:GHBP ratio in the cortex/neocortex was reduced (P < 0.001) by hypophysectomy and increased in intact rats by GH administration. The GHR:GHBP ratio in the hypothalamus was unaffected by hypophysectomy, but in intact and hypophysectomized rats it was much higher (P < 0.001) than in other tissues (by at least two-fold), as GHR transcripts predominated. In both groups, the GHR:GHBP ratio in the hypothalamus was reduced (P < 0.001) 2 h after GH injection. DISCUSSION
These data demonstrate, for the first time, that GHR/GHBP mRNA abundance in central and immune tissues of intact and hypophysectomized rats is rapidly autoregulated within 2 h of an episodic increase in the circulating GH concentration. The expression of the GHR gene has similarly been shown to be acutely increased in the rat ovary3~and adipose tissue 31 following a bolus GH injection. In contrast, it is well-established that a single GH injection or physiological pulse in the circulating GH concentration does not alter hepatic GHR
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E~ Fig. 1 Northern blot showing the abundance of growth hormone receptor (GHR) and growth hormone binding protein (GHBP) mRNA in hepatic extracts of representative groups of hypophysectomized (hypox) and sham-hypophysectomized (sham) rats 2 h after an injection of growth hormone (+ GH) or saline. The lower panel indicates transcript abundance, as determined by laser densitometry [(O), GHBP mRNA; (9 GHR mRNA] Mean densities for each group are indicated by the horizontal bars.
Fig. 2 The abundance of the 4.4 kb (GHR) transcript in the liver, spleen, hypothalamus (hypo.), neocortex/cortex (brain), and brainstem of sham-hypophysectomized (Intact) or hypophysectomized (Hypox) rats 2 h after an injection of saline or ovine growth hormone (+ GH). GHR mRNA abundance was quantified by laser densitometry and expressed relative to the abundance of 18SRNA in densitometric units (d.u.). *P < 0.001, as determined by ANOVA. (C3) Intact; (iFJ) intact + GH; (1~1)Hypox.; (11) Hypox. + GH.
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Fig 4. GHR mRNA:GHBP mRNA ratio in the liver, spleen, hypothalamus (hypo.), neocortex/cortex (brain), and brainstem of sham-hypophysectomized (Intact) or hypophysectomized (Hypox) rats 2 h after an injection of saline or ovine growth hormone (+ GH). The values represent the mean (+ SEM) of GHR:GHBP mRNA ratios obtained from individual rats. *P < 0.001, as determined by ANOVA. ([]) Control; ([]) Control + GH; (IN) Hypox.; (m) Hypox. + GH.
mRNA content in male rats, TM as confirmed in the present study. Chronic hyposomatotropism, as induced by hypophysectomy, similarly had n o effect on hepatic GHR/GHBP mRNA abundance. 9,18Indeed, only hypothalamic GHR transcripts were altered by hypophysectomy, thus, tissue GHR abundance may be differentially sensitive to acute and chronic changes in GH abundance. The autoregulation of the GHR gene is therefore clearly tissue specific and this could permit tissue-specific differences in responsiveness to episodic GH pulses and long-term changes in endogenous GH tone. Thus, while hepatic GH receptor status is widely used as a model of GH action, hepatic GHR gene status should not be tacitly assumed to reflect the GH-responsiveness of tissues other than the liver. Unlike hepatic GHR transcripts, GHR mRNA in the corterJneocortex, brainstem, hypothalamus and spleen was more abundant in GH-treated hypophysectomized or intact rats than in saline-treated rats. Central and immune GHR transcripts are thus more sensitive to GH autoregulation than the liver, suggesting important roles for these non-hepatic receptors. The abundance of GHR transcripts in the hypothalamus was particularly sensitive to somatotropic state, as GHR mRNA was reduced by 17 and 130/0, respectively, by hypophysectomy and increased by 20-40% by a single GH injection. Indeed, unlike other tissues, hypothalamic GHR transcripts were more abundant than GHBP transcripts. This predominance of GHR transcripts in the hypothalamus was also observed by Minami et al. 32 and postulated by Bennett eta/. 33 This abundance and GH- dependence of hypothalamic GHR mRNA may indicate that hypothalamic GHRs play a role in the regulation of GH secretion, especially as GHRs are present on somatostatin (SRIF) and growth hormone-releasing hormone (GHRH)-containing neurons. 34,35
These data also demonstrate, for the first time, that GHR/GHBP transcripts in non-hypothalamic regions of the brain and in the spleen are also regulated by GH, thus; GH may be a physiologically relevant regulator of neural and immune function. For instance, autoregulation of GHR mRNA in the neocortex/cortex and brainstem may modulate GH effects on sleep, neurotransmission, brain growth and development and locomotion. 24,z5 Conversely, GH-induced changes in macrophage proliferation and activity, the growth of immune organs, and the humeral response may be modulated by GHR mRNA autoregulation in the spleen and other immune organsY A recent study by Bennett et al.33 also examined the autoregulation of hypothalamic and central GHR transcripts, although their in situ analysis provided only minimal information regarding differential regulation of GHR and GHBP transcripts. As in our study, GHR/GHBP transcripts were reduced in the hypothalamus (arcuate nucleus) of hypophysectomized rats. They also observed that GH upregulated hypothalamic GHR/GHBP transcripts in GH-deficient animals, although they employed a chronic GH paradigm (continuous human GH infusion for 6 days). Unlike the present study, they did not observe any GH effect in intact animals or in extrahypothalamic brain regions (dentate gyrus and hippocampus). This discrepancy may indicate that GH-induced perturbations in hypothalamic GHR mRNA abundance are of short duration in intact animals but of longer duration in GH-deficient states. This discrepancy may also reflect the different exogenous GH preparation used by Bennett et al. 33 who employed lactogenic human GH rather than the purely somatogenic ovine GH employed in this study. As hGH binding sites in the rat median eminence are competitively inhibited by ovine prolactin, but not
Autoregulation of GH receptors 171
somatogenic bovine GH, 36 the resuks of Bennett e t al. may reflect GHR regulation by prolactin as well as, or instead of, GH. Although both GHR mRNA and GHBP mRNA were upregulated by oGH treatment in the spleen, brainstem, neocortexJcortex, and hypothalamus, only GHBP mRNA was upregulated in the liver. The abundance of the two transcripts can thus be co-ordinately or disparately regulated, in a tissue-specific way. It is thus possible that differentially increasing or decreasing the relative amount of tissue GHBP might provide a mechanism to modulate GH binding to tissue receptors and to buffer oscillations in extracellular GH concentrations. The changes in GHR/GHBP mRNA abundance induced by GH-treatment were highly significant (generally P < 0.001) and smaller in magnitude (15-350/0) than some studies on GHR autoregulation. 7,37 The magnitude of these changes in GHR/GHBP mRNA were, however, similar or greater (15-50%) than that observed in other studies on GHR mRNA and IGF-I mRNA regulation. 333s-40 These relatively small, yet significant, changes in GHR mRNA abundance in GH-treated rats may have important physiological functions, although a relationship between statistical and physiological significance has yet to be established. The changes in GHR/GHBP mRNA abundance in central tissues following peripheral GH administration would suggest that peripheral GH can rapidly access the central nervous system. Indeed, Mustafa e t al. 41 recently observed that GH (200 ng Ia25-GH or 20 ~tg unlabelled GH) can accumulate in the cortex, hippocampus, cerebellum, hypothalamus and brainstem within 5 min of intravenous administration. Although other studies have claimed that the blood-brain barrier is impermeable to GH, GH may access cortical and neocortical brain regions via circumventricular organs, in which the presence of GH-binding sites may reflect the presence of an active uptake mechanism.42,43 GH m a y also enter the hypothalamus via retrograde flow up the pituitary stalk or via the pituitary portal vessels. 44 Alternatively, peripheral GH may affect central GHRs indirectly, via IGF-I or other peripheral factors, although IGF-I cannot replace GH in stimulating hepatic GHR mRNA in dwarf rats. ]~ Curiously, Bennett e t al. 33 observed that chronic i.c.v. GH reduced hypothalamic GHR mRNA in intact, but not GHdeficient rats, whereas chronic peripheral GH increased hypothalamic GHR mRNA in GH-deficient, but not intact, rats. This finding may indicate that an additional factor mediates the central effects of peripheral GH, although Bennett e t al. 33 concluded, nonetheless, that peripheral GH was accessing the brain. Although GHR/GHBP mRNA was reduced in central and immune tissue(s) by a single GH injection, chronic GH-deprivation, induced by hypophysectomy, altered
GHR/GHBP mRNA abundance only in the hypothalamus. Previous studies have also observed unchanged GHR/GHBP mRNA levels in the liver 9.45 and kidney 46 of hypophysectomized male rats, although transcript abundance was altered in the hypothalamus, 3a muscle, 7 and fat z of hypophysectomized male rats and in the liver of hypophysectomized female rats. ]s,47 In addition, GHR/GHBP transcripts were generally acutely autoregulated to a similar extent in hypophysectomized and intact rats. Thus, at least in central tissues and in the spleen, short-term, rather than chronic, changes in GH tone may be more important determinants of GHR/GHBP transcript abundance. In conclusion, GHR and GHBP transcripts in central and immune tissue(s) are exquisitely sensitive to GH status, as they are upregulated by a single GH injection within 2 h. As hepatic GHBP, but not GHR, transcripts were similarly modulated, autoregulation of GHR gene products is both transcript- and tissue-specific.
ACKNOWLEDGEMENTS
The authors would like to thank Dr W. Baumbach (American Cyanamid, Princeton, NJ, USA) for kindly donating the rat GHR cDNA sequence used in Northern blotting and NIH for the donation of ovine GH. This work was supported by the Natural Sciences and Engineering Research Council of Canada and NATO.
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47. Baumbach WR, Bingham B. One class of growth hormone (GH) receptor and binding protein messenger ribonucleic acid in rat liver, GHR v is sexually dimorphic and regulated by GH. Endocrinology 1995; 136: 749-760.
Autoregulation of growth hormone receptor and growth hormone binding protein transcripts in brain and peripheral tissues of the rat K.L. Hull* and S. Harvey Departmentof Physiology,Universityof Alberta,Edmonton,Alberta,Canada,T6G2H7 *Currentaddress:Departmentof Biology,Bishop'sUniversity, Lennoxville,Quebec,Canada,J1M 1Z7
Summary Growth hormone (GH) differs from other pituitary hormones in that it can affect a wide spectrum of cellular activities in many different tissues. These disparate actions are, however, mediated by a common receptor, suggesting tissue-specific differences in the post-receptor mechanisms and/or tissue sensitivities to GH stimulation may confer specificity. Tissue sensitivity depends upon the abundance of GH receptors (GHRs) and may be modulated by the amplitude and pulsatility of GH secretion. It may also be dependent upon the presence of non-signal transducing GHbinding proteins (GHBPs), which result from the alternate splicing of GHR gene transcripts. Tissue-specific autoregulation of GHRs and GHBPs could, therefore, contribute to differential tissue responsiveness to GH action. The autoregulation of GHR and GHBP gene transcription in novel central (hypothalamus, brainstem, and cortex/neocortex) and peripheral (spleen) tissues was therefore examined in adult, male Sprague-Dawley rats. For comparative purposes, GHR/GHBP gene expression was also examined in the liver, which has traditionally been considered the major GH-target site. Chronic hyposomatotropism, induced by hypophysectomy, exerted tissue-specific effects on the abundance of GHR gene products 10 days post-hypophysectomy. Both GHR and GHBP transcripts were reduced in the hypothalamus of hypophysectomized rats by 20% (P < 0.001), although neither transcript was affected in the liver, spleen, cortex/neocortex or brainstem. In contrast, 2 h after a single bolus GH injection that was designed to simulate a pulsatile increase in circulating GH concentrations, GHR and GHBP mRNA content was significantly increased by 25-30% (P < 0.001) in all brain regions and in the spleen of hypophysectomized or sham-hypophysectomized rats. Production of the two transcripts was differentially regulated, however, as GHBP, but not GHR, transcripts were increased in the liver (P < 0.001), whereas the GHR:GHBP ratio was decreased in the hypothalamus of GH-treated rats (P < 0.001). These results suggest that GHR gene transcription and splicing are acutely autoregulated in a tissue-specific way. 9 1998 Churchill Livingstone
Key words: growth hormone receptors, growth hormone, brain, immune system, regulation, autoregulation.
INTRODUCTION Growth hormone (GH) can directly influence cellular development and metabolism by binding to growth Correspondence to: StephenHarvey,Departmentof Physiology,University of Alberta,Edmonton,Alberta,Canada,T6G2H7.
1096-6374/98/020167+07$18.00/0
hormone receptors (GHRs) and activating intracellular signal transduction mechanisms. 1,2The magnitude of the resulting cellular change is dependent upon factors such as the amplitude and frequency of the GH pulse. 3,4Tissue responsiveness to GH can be further modulated by alterations in GHR gene transcription or translation. GHR regulation by GH itself may be a particularly important 9 1998 Churchill Livingstone
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K.L. Hull and S. Harvey
determinant of GH action, as it would enhance or reduce the effect of a particular GH dose. The relationship between GH and GHR abundance, however, is not clearly understood. 5 Indeed, hypophysectomy had been shown to increase 6,7 or have no effect8,9 on hepatic GHR mRNA and GH-binding. The relationship between GH and GHR abundance may therefore be complex and depend upon the sex, hormonal milieu, and age of the experimental animal. 5 The ability of GH to autoregulate its receptor may also be temporally related, especially as differential effects on hepatic GH binding and GHR mRNA have been observed in response to chronic and acute GH stimulation, z-1I The physiological relevance of changes induced by chronic GH stimulation are, however, uncertain, since GH secretion in almost all species is phasic rather than tonic. In addition to changes in GHR and GHR mRNA, the production of growth hormone binding proteins (GHBPs) may provide an additional locus for the regulation of somatotropic function. Tissue GHBPs may locally inhibit GH action by competing with GHRs for available GH, 12-14 thus, an increase in GHBP content could negate a corresponding increase in tissue GHR. Tissue GHBPs may also maintain high local GH concentrations by serving as a GH reservoir. ]5 The ratio between signal-transducing receptors and truncated binding proteins, rather than absolute receptor levels, may thus determine the magnitude of tissue responsiveness to GH stimulation. In rats, the GHBP is comprised of the extracellular domain of the GHR that is C-terminally extended by a short hydrophilic tail. This protein is synthesized from a splice variant of the GHR gene. 16,17Splice site usage and thus the GHR:GHBP ratio can, however, be regulated, since hepatic GHBP mRNA but not GHR m R N A is increased in hypophysectomized rats by long-term GH treatment.Is The liver is not, however, the only GH-target site, as GHRs and GH responsiveness have been demonstrated in numerous non-hepatic organs? These non-hepatic GHRs may also be dependent upon GH status, but may be regulated differently than the hepatic GHR. This possibility was first suggested by Frick et al., 7 who showed that GHR mRNA was increased in rat muscle and liver, but decreased in fat by hypophysectomy. Splice site usage is similarly tissue-specific, as the GHR mRNA: GHBP mRNA ratio differs between tissues? 9 Tissue difference in GHR and GHBP autoregulation could thus permit tissue-specific responses when stimulated by identical blood GH levels. Indeed, since GH secretion in male rats is highly pulsatile, acute (within 2-3 h) autoregulation of GHR/GHBP gene transcription may be a particularly important determinant of GH action. In recent years, the demonstration of GHRs and GHBPs in the central nervous system (CNS) 2~ and
immune system n,23 has suggested novel roles for GH in neural and neuroendocrine function24,25 and in cellular and humeral immunity,z3 The regulation of the GHR gene in these tissues is, however, poorly understood. The possibility that neural and immune GHR gene transcripts may be autoregulated has therefore been determined in this study and compared with the autoregulation of hepatic GHR gene transcription. MATERIALS AND METHODS Animals
The secretion of GH in male rats is pulsatile, with an episode of GH release every 3 h. 26 The possibility that a phasic rather than chronic increase in the circulating GH concentration might autoregulate GHR gene expression was therefore examined. Intact adult male Sprague-Dawley rats (Charles River, Wilmington, MA, USA) were intraperitoneally (i.p.) injected with ovine GH 27 (oGH)(NIH-oGH-; NIADKD, Bethesda, MD, USA) (150 ~tg/100 g body weight) or the 0.90/0 (w/v) NaC1 vehicle (100 ~d/100 g). Ovine GH has previously been shown to interact with GH binding sites in rat tissues. 2s For comparative purposes, oGH and the saline vehicle were similarly injected into hypophysectomized adult SpragueDawley rats (Charles River), approximately 10 days posthypophysectomy. The animals (n = 10 per group) were killed by decapitation 2 h after the injection, and central (brainstem, hypothalamus and neocortex/cortex) and peripheral (spleen, liver) tissues were rapidly dissected out and quick-frozen in liquid nitrogen. Northern analysis
Tissue GHR and GHBP mRNA were quantified by Northern analysis. Total RNA was extracted from the frozen (-80~ livers, spleens, hypothalami, brainstems and cortex/neocortex using commercial reagents, according to the manufacturer's instructions (RNA Now; Biocan Scientific, Mississauga, Ontario, Canada). The RNA was subjected to Northern analysis as previously described, 29 using a complementary DNA fragment (pRatl-20) (provided by Dr W Baumbach, American Cyanamid, Princeton, NJ, USA) encoding the majority of the extracellular domain of the rat GHR. GHR mRNA and GHBP mRNA content w e r e quantified by laser densitometry and corrected for loading error by comparison with the hybridization of an 18S cDNA probe (American Type Culture Collection, Baltimore, MA, USA). Autoradiograph exposure time was optimized for each tissue, although this prohibited comparisons between tissues. Treatmentinduced differences in transcript abundance in the liver, spleen, hypothalarnus, neocortex/cortex and brainstem
Autoregulation of GH receptors 169
were determined by one-way analysis of variance (ANOVA) and means were separated by the Least Significant Difference (LSD) test. RESULTS
As previously shown, 21 transcripts of 4.4 and 1.2 kb coding for the GHR and GHBP, respectively, were detected in central (hypothalamus, brainstem, and neocortex/ cortex) and immune (spleen) tissues that corresponded in size to transcripts found in hepatic extracts (data not shown). The abundance of hepatic GHR transcripts was not significantly (P > 0.05) affected by hypophysectomy or GH treatment (Figs 1 and 2). In contrast, hypophysectomy reduced (by 20%) the abundance of the GHR transcript in the hypothalarnus, although it had no effect on GHR mRNA in other brain regions or in the spleen (Fig. 2). In further contrast, the abundance of GHR mRNA was significantly (P < 0.00l) increased by GH treatment in the spleen, hypothalamus, cortex/neocortex and brainstem of the intact and hypophysectomized rats (Fig. 2). In response to GH, the relative increase in GHR mRNA abundance in these extrahepatic tissues was comparable (20-30%) and in most tissues the GH-induced increase in intact animals was also comparable to the increase in hypophysectomized rats. The GH-induced increase in GHR mRNA in the cortex/neocortex of intact rats was, however, greater (P < 0.05) than that in hypophysectomized rats. The abundance of hepatic GHBP transcripts was also unaffected by hypophysectomy, but was increased (P< 0.001) by oGH injection in both intact and hypophysectomized rats (Figs 1 and 3). Hypophysectomy also had no effect on the abundance of GHBP transcripts in the spleen, neocortex/cortex or brainstem, but reduced
GHBP mRNA in the hypothalamus (Fig. 3). Two hours after the administration of oGH, the abundance of GHBP mRNA was consistently increased in the spleen, hypothalamus, cortex/neocortex and brainstem (Fig. 3). The relative increase in GHBP mRNA abundance in each tissue of the GH-treated intact rats was comparable to the increase induced in GH-treated hypophysectomized rats. In intact rats, the GHR:GHBP ratio in the liver, spleen, cortex/neocortex and brainstem was approximately 0.8:1 (Fig. 4), indicating a preferential expression of the GHBP transcript. The GHR:GHBP ratio in the liver, spleen and brainstem was unaffected by hypophysectomy or by oGH treatment. In contrast, the GHR:GHBP ratio in the cortex/neocortex was reduced (P < 0.001) by hypophysectomy and increased in intact rats by GH administration. The GHR:GHBP ratio in the hypothalamus was unaffected by hypophysectomy, but in intact and hypophysectomized rats it was much higher (P < 0.001) than in other tissues (by at least two-fold), as GHR transcripts predominated. In both groups, the GHR:GHBP ratio in the hypothalamus was reduced (P < 0.001) 2 h after GH injection. DISCUSSION
These data demonstrate, for the first time, that GHR/GHBP mRNA abundance in central and immune tissues of intact and hypophysectomized rats is rapidly autoregulated within 2 h of an episodic increase in the circulating GH concentration. The expression of the GHR gene has similarly been shown to be acutely increased in the rat ovary3~and adipose tissue 31 following a bolus GH injection. In contrast, it is well-established that a single GH injection or physiological pulse in the circulating GH concentration does not alter hepatic GHR
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Fig. 2 The abundance of the 4.4 kb (GHR) transcript in the liver, spleen, hypothalamus (hypo.), neocortex/cortex (brain), and brainstem of sham-hypophysectomized (Intact) or hypophysectomized (Hypox) rats 2 h after an injection of saline or ovine growth hormone (+ GH). GHR mRNA abundance was quantified by laser densitometry and expressed relative to the abundance of 18SRNA in densitometric units (d.u.). *P < 0.001, as determined by ANOVA. (C3) Intact; (iFJ) intact + GH; (1~1)Hypox.; (11) Hypox. + GH.
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Fig. 3 The abundance of the 1.2 kb (GHBP) transcript in the liver, spleen, hypothalamus (hypo.), neocortex/cortex (brain), and brainstem of sham-hypophysectomized (Intact) or hypophysectomized (Hypox) rats 2 h after an injection of saline or ovine growth hormone (+ GH). GHBP mRNA abundance was quantified by laser densitometry and expressed relative to the abundance of RNA in densitometric units (d.u.). *P < 0.001, as determined by ANOVA. (C~)Intact; ([]) intact + GH; ([]) Hypox.; (m) Hypox. + GH.
Fig 4. GHR mRNA:GHBP mRNA ratio in the liver, spleen, hypothalamus (hypo.), neocortex/cortex (brain), and brainstem of sham-hypophysectomized (Intact) or hypophysectomized (Hypox) rats 2 h after an injection of saline or ovine growth hormone (+ GH). The values represent the mean (+ SEM) of GHR:GHBP mRNA ratios obtained from individual rats. *P < 0.001, as determined by ANOVA. ([]) Control; ([]) Control + GH; (IN) Hypox.; (m) Hypox. + GH.
mRNA content in male rats, TM as confirmed in the present study. Chronic hyposomatotropism, as induced by hypophysectomy, similarly had n o effect on hepatic GHR/GHBP mRNA abundance. 9,18Indeed, only hypothalamic GHR transcripts were altered by hypophysectomy, thus, tissue GHR abundance may be differentially sensitive to acute and chronic changes in GH abundance. The autoregulation of the GHR gene is therefore clearly tissue specific and this could permit tissue-specific differences in responsiveness to episodic GH pulses and long-term changes in endogenous GH tone. Thus, while hepatic GH receptor status is widely used as a model of GH action, hepatic GHR gene status should not be tacitly assumed to reflect the GH-responsiveness of tissues other than the liver. Unlike hepatic GHR transcripts, GHR mRNA in the corterJneocortex, brainstem, hypothalamus and spleen was more abundant in GH-treated hypophysectomized or intact rats than in saline-treated rats. Central and immune GHR transcripts are thus more sensitive to GH autoregulation than the liver, suggesting important roles for these non-hepatic receptors. The abundance of GHR transcripts in the hypothalamus was particularly sensitive to somatotropic state, as GHR mRNA was reduced by 17 and 130/0, respectively, by hypophysectomy and increased by 20-40% by a single GH injection. Indeed, unlike other tissues, hypothalamic GHR transcripts were more abundant than GHBP transcripts. This predominance of GHR transcripts in the hypothalamus was also observed by Minami et al. 32 and postulated by Bennett eta/. 33 This abundance and GH- dependence of hypothalamic GHR mRNA may indicate that hypothalamic GHRs play a role in the regulation of GH secretion, especially as GHRs are present on somatostatin (SRIF) and growth hormone-releasing hormone (GHRH)-containing neurons. 34,35
These data also demonstrate, for the first time, that GHR/GHBP transcripts in non-hypothalamic regions of the brain and in the spleen are also regulated by GH, thus; GH may be a physiologically relevant regulator of neural and immune function. For instance, autoregulation of GHR mRNA in the neocortex/cortex and brainstem may modulate GH effects on sleep, neurotransmission, brain growth and development and locomotion. 24,z5 Conversely, GH-induced changes in macrophage proliferation and activity, the growth of immune organs, and the humeral response may be modulated by GHR mRNA autoregulation in the spleen and other immune organsY A recent study by Bennett et al.33 also examined the autoregulation of hypothalamic and central GHR transcripts, although their in situ analysis provided only minimal information regarding differential regulation of GHR and GHBP transcripts. As in our study, GHR/GHBP transcripts were reduced in the hypothalamus (arcuate nucleus) of hypophysectomized rats. They also observed that GH upregulated hypothalamic GHR/GHBP transcripts in GH-deficient animals, although they employed a chronic GH paradigm (continuous human GH infusion for 6 days). Unlike the present study, they did not observe any GH effect in intact animals or in extrahypothalamic brain regions (dentate gyrus and hippocampus). This discrepancy may indicate that GH-induced perturbations in hypothalamic GHR mRNA abundance are of short duration in intact animals but of longer duration in GH-deficient states. This discrepancy may also reflect the different exogenous GH preparation used by Bennett et al. 33 who employed lactogenic human GH rather than the purely somatogenic ovine GH employed in this study. As hGH binding sites in the rat median eminence are competitively inhibited by ovine prolactin, but not
Autoregulation of GH receptors 171
somatogenic bovine GH, 36 the resuks of Bennett e t al. may reflect GHR regulation by prolactin as well as, or instead of, GH. Although both GHR mRNA and GHBP mRNA were upregulated by oGH treatment in the spleen, brainstem, neocortexJcortex, and hypothalamus, only GHBP mRNA was upregulated in the liver. The abundance of the two transcripts can thus be co-ordinately or disparately regulated, in a tissue-specific way. It is thus possible that differentially increasing or decreasing the relative amount of tissue GHBP might provide a mechanism to modulate GH binding to tissue receptors and to buffer oscillations in extracellular GH concentrations. The changes in GHR/GHBP mRNA abundance induced by GH-treatment were highly significant (generally P < 0.001) and smaller in magnitude (15-350/0) than some studies on GHR autoregulation. 7,37 The magnitude of these changes in GHR/GHBP mRNA were, however, similar or greater (15-50%) than that observed in other studies on GHR mRNA and IGF-I mRNA regulation. 333s-40 These relatively small, yet significant, changes in GHR mRNA abundance in GH-treated rats may have important physiological functions, although a relationship between statistical and physiological significance has yet to be established. The changes in GHR/GHBP mRNA abundance in central tissues following peripheral GH administration would suggest that peripheral GH can rapidly access the central nervous system. Indeed, Mustafa e t al. 41 recently observed that GH (200 ng Ia25-GH or 20 ~tg unlabelled GH) can accumulate in the cortex, hippocampus, cerebellum, hypothalamus and brainstem within 5 min of intravenous administration. Although other studies have claimed that the blood-brain barrier is impermeable to GH, GH may access cortical and neocortical brain regions via circumventricular organs, in which the presence of GH-binding sites may reflect the presence of an active uptake mechanism.42,43 GH m a y also enter the hypothalamus via retrograde flow up the pituitary stalk or via the pituitary portal vessels. 44 Alternatively, peripheral GH may affect central GHRs indirectly, via IGF-I or other peripheral factors, although IGF-I cannot replace GH in stimulating hepatic GHR mRNA in dwarf rats. ]~ Curiously, Bennett e t al. 33 observed that chronic i.c.v. GH reduced hypothalamic GHR mRNA in intact, but not GHdeficient rats, whereas chronic peripheral GH increased hypothalamic GHR mRNA in GH-deficient, but not intact, rats. This finding may indicate that an additional factor mediates the central effects of peripheral GH, although Bennett e t al. 33 concluded, nonetheless, that peripheral GH was accessing the brain. Although GHR/GHBP mRNA was reduced in central and immune tissue(s) by a single GH injection, chronic GH-deprivation, induced by hypophysectomy, altered
GHR/GHBP mRNA abundance only in the hypothalamus. Previous studies have also observed unchanged GHR/GHBP mRNA levels in the liver 9.45 and kidney 46 of hypophysectomized male rats, although transcript abundance was altered in the hypothalamus, 3a muscle, 7 and fat z of hypophysectomized male rats and in the liver of hypophysectomized female rats. ]s,47 In addition, GHR/GHBP transcripts were generally acutely autoregulated to a similar extent in hypophysectomized and intact rats. Thus, at least in central tissues and in the spleen, short-term, rather than chronic, changes in GH tone may be more important determinants of GHR/GHBP transcript abundance. In conclusion, GHR and GHBP transcripts in central and immune tissue(s) are exquisitely sensitive to GH status, as they are upregulated by a single GH injection within 2 h. As hepatic GHBP, but not GHR, transcripts were similarly modulated, autoregulation of GHR gene products is both transcript- and tissue-specific.
ACKNOWLEDGEMENTS
The authors would like to thank Dr W. Baumbach (American Cyanamid, Princeton, NJ, USA) for kindly donating the rat GHR cDNA sequence used in Northern blotting and NIH for the donation of ovine GH. This work was supported by the Natural Sciences and Engineering Research Council of Canada and NATO.
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47. Baumbach WR, Bingham B. One class of growth hormone (GH) receptor and binding protein messenger ribonucleic acid in rat liver, GHR v is sexually dimorphic and regulated by GH. Endocrinology 1995; 136: 749-760.