The authors have no conflict of interest.
Localization and Regulation of the Growth Hormone Receptor and Growth Hormone-Binding Protein in the Rat Growth Plate†
Article first published online: 1 AUG 2002
Copyright © 2002 ASBMR
Journal of Bone and Mineral Research
Volume 17, Issue 8, pages 1408–1419, August 2002
How to Cite
Gevers, E. F., Van Der Eerden, B. C. J., Karperien, M., Raap, A. K., Robinson, I. C. A. F. and Wit, J.-M. (2002), Localization and Regulation of the Growth Hormone Receptor and Growth Hormone-Binding Protein in the Rat Growth Plate. J Bone Miner Res, 17: 1408–1419. doi: 10.1359/jbmr.2002.17.8.1408
- Issue published online: 2 DEC 2009
- Article first published online: 1 AUG 2002
- Manuscript Accepted: 2 APR 2002
- Manuscript Revised: 6 MAR 2002
- Manuscript Received: 8 OCT 2001
- growth plate;
- growth hormone receptor;
- growth hormone binding protein;
- tyramide signal amplification
Growth hormone (GH) has direct effects on the growth plate to stimulate longitudinal growth, but it is not clear which chondrocyte populations GH acts on. The dual effector theory suggests that GH would act primarily on the “stem cells.” However, staining with a GH receptor (GHR) antibody is found in all layers of the growth plate in rabbits and humans. We now have investigated the localization and regulation of GHR and the related GH binding protein (GHBP) in the rat growth plate using a sensitive immunohistochemical method involving tyramide signal amplification (TSA) and antibodies specific for GHR or GHBP. Both GHR and GHBP were shown in the germinal and proliferative chondrocytes, but most clearly in early maturing chondrocytes at the interface between proliferative and hypertrophic cells. Staining for GHR and GHBP was located in both the cytoplasm and the nucleus. Expression of GHR mRNA and GHBP mRNA in the growth plate was confirmed by reverse-transcription polymerase chain reaction (RT-PCR). Immunohistochemical staining for GHR and GHBP decreased with age; in 12-week-old normal rats, only the early maturing chondrocytes were stained. In GH-deficient dwarf rats, staining seemed less than in normal rats, and in hypophysectomized (Hx) rats, staining for GHBP was clearly reduced. Treatment of Hx rats with thyroid hormones (T3 + T4), via subcutaneously (sc) implanted osmotic minipumps, induced little growth and induced a small layer of GHR-positive and GHBP-positive early maturing chondrocytes. Treatment with GH and thyroid hormones (TH) resulted in greater growth and a broader layer of GHR-positive and GHBP-positive cells, indistinguishable from normal rats. In contrast, dexamethasone treatment of normal rats inhibited their growth and reduced GHR and GHBP staining in the growth plate. These results show that GHR and GHBP in the growth plate are under hormonal control. The localization of GHR/GHBP suggests that in addition to actions on germinal and proliferative cells in young rats, GH also has effects on early maturing chondrocytes and may be involved in their differentiation to a fully hypertrophic chondrocyte.
THE GROWTH plate is divided into distinct layers of chondrocytes, resting (also referred to as “reserve,” “germinal,” or “stem”) cells, proliferating cells, and differentiating (maturing) hypertrophic chondrocytes.(1,2) The border between the zone of proliferating cells and differentiating/maturing cells consists of a few cell layers, often called the transition zone. The cells in this layer and the upper part of the hypertrophic cell layer are considered as “early maturing cells.” In addition to an indirect action via insulin-like growth factor (IGF)-I generated at the liver, there is strong evidence that growth hormone (GH) acts locally at the growth plate to stimulate longitudinal growth.(3,4) This hypothesis has been strengthened recently by experiments showing that growth of transgenic mice with a targeted disruption of the IGF-I gene in the liver is not different from normal mice.(5,6) There also is direct evidence that GH acts on the stem cells(7) resulting in differentiation to a proliferative cell type that produces IGF-I,(8) which then acts in an autocrine or paracrine fashion to induce further proliferation and clonal expansion of the chondrocytes.(9) Although this theory implicates the presence of GH receptors (GHRs), primarily in the stem cells in the rat, immunostaining has shown the presence of the GHR in all cell layers of the growth plate.(10) However, this study was performed in the rabbit and used a monoclonal antibody (MAb 263) directed against the extracellular domain of the GHR, which therefore does not discriminate between GHR and the GH binding protein (GHBP), which corresponds to the GHR extracellular domain.(11,12)
Although GHBP is produced mainly in the liver,(5,6) it also is produced in other tissues expressing GHR.(13) Its function is not clear because, on one hand, it prolongs the half-life of GH in vivo,(14) whereas, on the other hand, it competes with GHR for binding to GH in vitro and can reduce the effect of GH.(15,16) In rats, GHBP is produced by alternative splicing of the primary GHR RNA(17) and the GHR/GHBP mRNA ratio differs between tissues.(13,18,19) Furthermore, different GHR/GHBP transcripts exist with multiple 5′ untranslated exons and these might be responsible for tissue-specific regulation of GHR and GHBP expression.(20,21) Until now, regulation of GHR expression has been studied mostly in the liver and brain. However, the more relevant target for the regulation of skeletal growth by GH is the expression of GHR and GHBP in the growth plate itself.
In this study, we present data on the expression of GHR and GHBP and mRNA in the growth plate at different ages. By using antibodies specific for GHR, GHBP, or both, we discriminated between GHR and GHBP and compared their localization in the rat growth plate to identify target cells for GH action.
A sensitive immunohistochemical method, using tyramide signal amplification (TSA) with biotin-labeled tyramides, readily detected GHR and GHBP. In the rat, GHBP is produced by alternative splicing of exon 8, and this generates a GHBP product with a unique 17 residue hydrophilic tail.(17) Thus, an antibody specific for this peptide tail could be used to localize GHBP specifically(22) while an antibody against the intracellular domain of the GHR could be used to identify GHR specifically. Reverse-transcription polymerase chain reaction (RT-PCR) was used to confirm the presence of GHR and GHBP mRNA transcripts in the growth plate. To investigate hormonal regulation of GHR and GHBP in the growth plate, immunohistochemistry was performed on sections of growth plates from normal rats, dwarf rats with a specific GH deficiency,(23) and hypophysectomized (Hx) rats with and without thyroid hormone (TH) and/or GH treatment. We also studied the effect of treatment with dexamethasone in normal-growing rats to investigate whether growth inhibition by dexamethasone is accompanied by changes of GHR expression or localization in the growth plate.
MATERIALS AND METHODS
Unless stated otherwise, rats were kept in a light- and temperature-controlled room (12 h of light and 23-25°C) with food and water available ad libitum. Experiments were approved by the local ethical committee for animal experiments.
Male and female normal rats (Lewis strain; Harlan, Zeist, The Netherlands) and GH-deficient dwarf rats(23) (Lewis strain; Harlan), 1, 4, 7, and 12 weeks of age (n = 5-6 per group), were killed by decapitation. The proximal part of the tibia was isolated, cut in half longitudinally, fixed overnight in buffered picric acid/formaldehyde, and then decalcified for 3 weeks in 20% EDTA (pH 8.0) as described by Barnard and colleagues.(10)
Male rats were Hx at the age of 21-28 days (n = 6 per group; Iffa Credo, Brussels, Belgium). One week later osmotic minipumps (Alzet; Broekman Institute, Someren, The Netherlands), delivering either 100 μg/day of recombinant human GH (hGH; Pharmacia-Upjohn, Stockholm, Sweden) or 1.0 μg/day of T4 plus 0.125 μg/day of T3 (T4/T3) (Sigma, St. Louis, USA) or both GH and T4/T3, were implanted subcutaneously (sc) under halothane/O2/N2O anesthesia. Control Hx or normal rats were implanted with teflon rods. Five percent glucose and 0.9% NaCl were added to the drinking water. Treatment was for 2 weeks after which tibias were isolated as described previously. Body weight was recorded weekly.
Four-week-old normal Lewis female rats (n = 5 per group) received dexamethasone in the drinking water in a dose of 0.3 mg/liter or 2 mg/liter of tap water with 0.02% alcohol. Two control groups received tap water with 0.02% alcohol. One of these groups was pair fed to match the food intake of the group that received the highest dose of dexamethasone. Treatment was for 1 week after which tibias were isolated as described previously. Body weight and body length were recorded.
Results of growth are shown as mean ± SEM. Data were subjected to ANOVA, followed by the Student-Newman-Keuls test. Statistical significant difference was accepted below p = 0.05.
Preparation of tibial growth plates and the immunohistochemical procedures were modifications of the method used by Barnard and colleagues(10) to visualize GHR/BP in rabbit growth plate. Decalcified tibias were stored frozen. One day before immunohistochemical analysis, 10 μm of cryostat sections were mounted on gelatin-KCr(SO4)-glutaraldehyde-coated slides. Endogenous peroxidase activity was blocked in 1% H2O2 in PBS/40% methanol. Sections then were subjected to pepsin degradation (0.05% pepsin [Sigma] in HCl [pH = 2]) for 7 minutes at 37°C and subsequently to 1% hydroxyl ammonium chloride for 15 minutes at room temperature. Next, sections were incubated in 0.5% Boehringer milk powder (in 0.1M TrisHCl/0.15M NaCl/0.05% Tween 20 (Sigma; 60 minutes at 37°C) to block nonspecific binding, followed by incubation with the following antibodies: MAb 263 (1:10; Sanver Tech, Boechout, Belgium), raised to the extracellular part of the rat GHR(11); MAb 4.3, raised to a peptide corresponding to the 17 amino acids tail of GHBP (1:10)(22); or a polyclonal antibody raised to the intracellular part of GHR (PAb iGHR; 1:20). The latter two antibodies were kindly provided by Dr. W. Baumbach (Cyanamid, Princeton, NJ, USA). A nonrelevant MAb (mouse antibromodeoxyuridine; Dako A/S, Glostrup, Denmark) was used as a control. Incubations with the primary antibodies was for 60 minutes at 37°C. Sections then were incubated with peroxidase-labeled rabbit anti-mouse antibody or swine anti-rabbit antibody (1:100 for 60 minutes at 37°C; Dako). Next, sections were incubated with biotin-labeled tyramides (a generous gift of Perkin Elmer, Boston, MA, USA; 1:300 in 0.1 M of Tris/0.1 M of NaCl/10% dextran sulfate/10 mM of imidazol/1:1000 H2O2, for 30 minutes at room temperature), followed by a peroxidase-labeled antibiotin antibody (1:500 for 45 minutes at 37°C; Dako). The last antibody was visualized with diaminobenzidine (DAB; Sigma; 0.05% DAB in 50 mM of TrisHCl/1 mM of imidazol/0.085% H2O2, for 14 minutes at room temperature). Finally, sections were washed in tap water and dried and mounted in Fluoromount (BDH, Poole, UK). Only in some sections hematoxylin counterstain was used because we found that counterstaining reduced the visibility of the immunostaining. In control sections, the first antibody was omitted. Also, in other sections, immunohistochemistry was performed without TSA to show the level of enhancement by tyramides, using only a biotin-labeled second antibody followed by a peroxidase-labeled antibiotin antibody. When comparing staining between different groups, immunohistochemistry of the sections of all animals in those groups was performed at the same time. Representative sections are shown in the figures.
Growth plates of 1, 4, 7, and 12-week-old male and female rats and livers of 8-week-old female rats were dissected carefully. The edges of the growth plate were cut off to avoid contamination with other cell types. Total RNA was extracted following the method of Chomczynski and Sacchi.(24) Two hundred fifty nanograms of total RNA was transcribed into cDNA with 50 U of Moloney murine leukemia virus (MMLV) reverse transcriptase, 7.5 pmol of deoxynucleoside triphosphate (dNTP), 50 ng of random primer, 0.1 μmol of dithiothreitol (DTT), and 0.5 μl of RNasin in a total volume of 20 μl of buffer. The reaction was carried out at 37°C for 1 h and then heated to 70°C for 10 minutes. Fifty units of MMLV reverse transcriptase and 0.5 μl of RNasin were added again and the mixture was incubated for 30 minutes at 37°C and heated to 70°C for 5 minutes. Finally, 180 μl of 10 mM Tris (pH = 8.0) was added.
PCR was performed in a total volume of 50 μl, containing buffer, 2.5 mM of MgCl2, 0.3 mM of dNTP, 10 pmol of forward and reverse primer, and 0.25 U of Taq polymerase (Eurogentec, Seraing, Belgium), using 30 cycles (Hybaid Omnigene System [Biozyme, Landgraaf, The Netherlands]). Each cycle consisted of 30 s of denaturation at 96°C, 30 s annealing at 56°C, and 1 minute extension at 72°C, preceded by an initial denaturation at 96°C for 3 minutes and followed by a final step at 72°C for 10 minutes. In each PCR experiment, negative controls included tubes with distilled water instead of cDNA or with 25 ng of RNA instead of cDNA for each tissue. PCR products were analyzed by gel electrophoresis.
Oligonucleotide primers were purchased from Isogen Bioscience BV (Maarssen, The Netherlands). For the amplification of GHR cDNA, a primer set was chosen from the intracellular sequence of the GHR: the sense and antisense primers were 5′-GAGGAGGTGAACACCATCTTGGGC-3′ and 5′-ACCACCTGCCTGGTGTAATGTC-3′, respectively. Sense and antisense primers for the amplification of GHBP cDNA were 5′-TGGTGATTTGTTGGACGAAA-3′and 5′-GCTAGGGATGGCAGATCCTC-3′, respectively.
Enhancement of signal by biotin-labeled tyramides
Figure 1 shows immunohistochemistry with PAb iGHR in the growth plate of a 12-week-old female rat, performed with and without the use of TSA. Without the use of TSA, the staining was very weak (Fig. 1A). When TSA was used, specific staining was enhanced whereas background staining in the growth plate was still very low (Fig. 1B). Staining mainly was in early maturing chondrocytes (transition zone/upper hypertrophic zone) but with the use of TSA staining also could be visualized, using higher magnification, in germinal cells. There was no staining when the nonrelevant MAb was used (antibromodeoxyuridine).
Comparison of MAb 263, PAb iGHR, and MAb 4.3
Figure 2 compares the staining obtained with the three different antibodies recognizing either the common extracellular part of the GHR/GHBP (MAb 263), the internal part of the GHR (PAb iGHR), or the tail of GHBP (MAb 4.3) in the growth plate of a 12-week-old female rat. Specificity of the antibodies and the tyramides was shown by the disappearance of staining when the first antibody was omitted (Fig. 2A). As can be seen, background staining in bone tissue (but not in the growth plate) was seen. This probably was caused by cross-reaction with rat antigen of the rabbit anti-mouse second antibody used to detect the monoclonal primary antibodies, because this background was not observed when a swine anti-rabbit second antibody was used (not shown). The specificity of these antibodies for their respective antigens has been shown in other experiments by competition with excess antigen.(22,25)
All three antibodies showed staining mainly in the transition zone/upper hypertrophic chondrocyte layer (Figs. 2B, 2C, and 2D showing staining with MAb 263, PAb iGHR, and MAb 4.3). Immunohistochemistry with MAb 263 and PAb iGHR also revealed staining in proliferative and germinal chondrocytes (Figs. 2E and 2F). Although not the primary object of this study, we noted that immunoreactivity also was intense in bone and bone marrow proximal and distal to the growth plate (data not shown).
Detection of GHR- and GHBP mRNA in the growth plate
RT-PCR confirmed the presence of GHR mRNA and GHBP mRNA in the growth plate (Fig. 3). GHR and GHBP RNA were clearly present in growth plate and liver and no band was observed in the appropriate controls. GHR and GHBP RNA were shown in the growth plate of both male and female rats at all ages studied.
Expression of GHR and GHBP in growth plates at different ages (experiment 1)
In 1, 4, and 7-week-old rats, staining with all three antibodies was seen in stem cells, proliferative chondrocytes, and early hypertrophic chondrocytes with faint staining in some more mature chondrocytes. In 12-week-old rats, staining in stem cells and proliferative chondrocytes was reduced greatly and staining was seen mostly in the transition zone and the early hypertrophic chondrocytes. This is illustrated in Fig. 4, showing the antibody PAb iGHR, recognizing GHR only (Figs. 4A–4D), and MAb 4.3, recognizing GHBP only (Figs. 4E–4H). At all ages, cells in the transition zone showed the most intense staining, although cells in the stem cell zone also stained positive for both GHR and GHBP, shown at a higher magnification with PAb iGHR and MAb 4.3 in Fig. 5. In 1, 4, and 12-week-old rats there were no differences between gender but in 7-week-old rats, staining with MAb 4.3 in stem cells and proliferative chondrocytes consistently appeared more abundant in male rats than in female rats (Figs. 6A and 6B). In dwarf rats, localization of GHR/GHBP staining was similar to normal rats, but intensity seemed less when using MAb 4.3 (Figs. 4I–4L). This difference was less clear when using the other two antibodies.
Staining with all three antibodies was present in both the cytoplasm, and in many chondrocytes, the nucleus (Figs. 7A and 7B). Nuclear staining was most prominent in the early hypertrophic chondrocytes. Interestingly, GHBP staining in growth plates of dwarf rats was almost exclusively cytoplasmic (Fig. 7C).
Regulation of GHR and GHBP by GH and thyroid hormones (experiment 2)
To gain insight in the regulation of GHR and GHBP in the growth plate, Hx rats were treated with GH, T3/T4, or both, and normal rats were treated with dexamethasone, after which immunohistochemistry was performed on their growth plates. Hx rats showed virtually no growth when treated with saline for 2 weeks. T3/T4 induced a small weight gain of 5.2 ± 0.2 g/week, and GH was more effective and the weight gain was not increased further by additional T3/T4 treatment (Table 1).
Figure 8 shows staining for GHBP in the growth plate of rats from these different groups. Compared with normal rats (Fig. 8E), staining with MAb 4.3 was greatly reduced in Hx rats (Fig. 8A). T3/T4 treatment alone induced a small row of positive cells in the transition zone between the proliferative and hypertrophic zone (Fig. 8B). GH treatment resulted in a wider growth plate and a broader band of GHBP-positive cells (Fig. 8C). Staining was indistinguishable in tibias from GH-treated, GH + T3/T4-treated, and normal rats (Figs. 8C–8E).
Similar results were obtained for MAb 263, but differences between groups were less clear when PAb iGHR was used, suggesting that GHBP and GHR are regulated separately in the growth plate.
Regulation of GHR and GHBP by dexamethasone (experiment 3)
Table 2 shows the effect of low-dose (0.3 mg/liter drinking water) and high-dose (2 mg/liter drinking water) dexamethasone treatment on gain in weight and length, compared with pair-fed and normal-fed rats. Dexamethasone-treated rats gained less weight and length (p < 0.01) than both the ad libitum fed group and the pair-fed group (paired to the high-dose group only). Figure 9 shows that GHBP staining was clearly reduced in proliferative chondrocytes of the dexamethasone-treated rats and was hardly detectable in the rats treated with the highest dose, compared with both ad libitum and pair-fed rats. Staining in the early hypertrophic cell layer also was clearly diminished in the dexamethasone-treated rats. Similar results were obtained when PAb iGHR and MAb 263 were used (not shown).
There is an increasing amount of evidence that, in addition to the hepatic effects on IGF-I generation, GH affects the growth plate directly to stimulate longitudinal growth in the rat.(3–6,26) However, its exact cellular target in the growth plate is not clear. The dual effector theory(9) implicates GH action on the resting cells, but GHR is not exclusively limited to this zone in the growth plate, at least in the rabbit, where GHR has been shown not only in resting cells but also in proliferative chondrocytes and in hypertrophic chondrocytes.(10) One problem with most earlier studies is that the antibody used does not distinguish between GHR and GHBP, the soluble extracellular part of the GHR, which also might be present in chondrocytes. To readdress these questions in the rat, we have used a sensitive immunohistochemical method to localize GHR and GHBP specifically in the rat growth plate, using antibodies directed to the internal part of GHR and to the hydrophilic tail of GHBP, which enabled us to recognize GHR and GHBP separately in this species.
To enhance the sensitivity of immunostaining, we used biotin-labeled tyramides for amplification.(27,28) Tyramides precipitate on interaction with peroxidase and hydrogen peroxide. We used a peroxidase-labeled second antibody and biotin-labeled tyramides so that a large deposit of biotins is formed, which we detected with a peroxidase-labeled antibiotin antibody. Specificity of this TSA was shown by a complete disappearance of staining when either primary or secondary antibody was omitted or when a nonrelevant first antibody was used. The increased sensitivity achieved may account for the fact that we could detect staining with all three antibodies in rat growth plates at all ages, up to 12 weeks, whereas Barnard and colleagues did not detect GHR/BP in rabbits older than 50 days.(10)
Both GHR and GHBP protein were located in the rat growth plate. GHBP is produced mainly in the liver, but many extrahepatic tissues also synthesize GHBP.(13) It is likely that GHBP is produced also locally in the chondrocytes, but the immunostaining done could not exclude the possibility that the GHBP in chondrocytes was obtained from the circulation and then internalized by chondrocytes. However, RT-PCR of growth plate cDNA confirmed the presence of GHR mRNA and GHBP mRNA transcripts in the growth plate and thus suggests local production of GHR and GHBP in the growth plate.
The localization of both GHR and GHBP in the same cell is not surprising because both proteins are transcribed from the same gene(17) but the functional implications of the local GH-GHR-GHBP interactions in the growth plate are intriguing. GHBP could be produced locally to capture free GH to enable it to bind to the receptor or, alternatively, protect cells continuously exposed to GH by preventing it to bind to the receptor. It could also participate in the dimerization process and affect signaling.(29)
In young rats, immunoreactive GHR and GHBP were present in cells of the resting zone, as has been found before in rat embryos,(30) young rats,(31) and young rabbits.(10) Staining was especially clear in the groove of Ranvier, where cells migrate from the resting zone.(32) However, as also discussed by Lupu et al.,(33) it is difficult to demarcate the resting zone from epiphyseal chondrocytes involved in the growth of the secondary ossification center and it is unclear whether cells in the resting zone are stem cells/precursors of proliferative chondrocytes. The localization of CCAAT/enhancer-binding protein (C/EBP), an antimitogenic factor involved in differentiation of other mesenchymal cells like preadipocytes,(34) in these cells(31) could suggest that these cells are chondrocyte precursors differentiating into chondrocytes. Thus, the localization of GHR in these cells supports a direct action of GH on resting cells. However, it does not exclude a role for IGF-I, either produced in the liver or produced locally in these cells, in the differentiation of germinal chondrocytes to proliferative chondrocytes.(35,36)
More faint immunostaining was seen in proliferating chondrocytes of rats up to the age of 7 weeks but not in the 12-week-old rats. This is in agreement with the original study of GHR/BP in rabbits, in which a similar localization was found in 20- and 50-day-old rabbits but not in older rabbits,(10) and with a recent report of GHR/BP in uremic rats(37) and with GHR mRNA in situ hybridization data.(33) In other studies in rat tibial growth plates, immunostaining with MAb 263 was not found in proliferative chondrocytes,(31) but this might reflect the lower sensitivity of standard immunochemistry. The localization of GHR in proliferative chondrocytes suggests that GH also might act directly on these cells in young animals to stimulate mitogenesis. In culture, GH stimulates growth of large chondrocyte colonies thought to represent rapidly proliferating cells.(38,39)
However, the most prominently stained layer was seen in a band of early maturing chondrocytes at the transition between proliferative and hypertrophic zones. Staining in the most distal layer of fully mature hypertrophic chondrocytes was absent or faint, similar to findings in rat fetal growth plates(30) and localization of GHR mRNA.(33) This is the first time, maybe because of the ultrasensitive immunostaining technique, that the localization in this transition zone is so apparent. In older rats, GHR and GHBP localization in this zone remained visible whereas GHR and GHBP staining disappeared from proliferative chondrocytes. The difference in staining between the early maturing and the fully mature chondrocyte might indicate that GH receptor signaling is involved in the maturation process of chondrocytes.
Interestingly, we previously showed that GH treatment in dwarf rats results in a decrease in alkaline phosphatase activity in those chondrocytes(40); thus, this could be a direct effect of GH. In addition, in the fetal rat, these chondrocytes express GHR/BP mRNA but not IGF-I mRNA,(41) also suggesting that GH can act directly on these transitional chondrocytes without IGF-I as mediator, at least in the fetus.
Although not the primary aim of this study, we noted that all three antibodies stained GHR or GHBP in nuclei as well as cytoplasm, as has been shown before for GHR(42) and GHBP(43) and also for GH.(44) The significance of GHR and GHBP in the nucleus is not clear. They might provide an additional signal transduction pathway or act as transcription factors,(42–44) although this is still controversial. Translocation of the GHR to the nucleus requires the intracellular part of the GHR but also GH binding.(42) We observed a reduction of nuclear staining in GH-deficient dwarf rats most clearly with MAb 4.3, which may suggest that GHBP requires GH binding for translocation to the nucleus. Nuclear staining was not abolished completely but this might be because of the residual GH in these dwarf rats,(23) because in Hx rats staining completely disappeared.
Interestingly, nuclear staining was seen mainly in chondrocytes in the transition zone between proliferation and hypertrophy and rarely in stem cells or proliferating chondrocytes. This raises the possibility that proliferative chondrocytes use a different intracellular signal transduction pathway than hypertrophic chondrocytes (e.g., plasma membrane-associated protein phosphorylation vs. nuclear translocation).
Having established the localization of GHR and GHBP in the growth plate, we then wished to study its regulation. In the rat, hepatic GHR and plasma GHBP are developmentally regulated with a gender difference occurring at the age of puberty.(45–47) However, plasma GHBP mainly reflects hepatic GHBP production and little is known about GHR/GHBP regulation in other target tissues. In the growth plate, GHR and GHBP showed clear developmental regulation. Throughout sexual maturation, staining was very strong and was reduced as animals became older (and grew more slowly), by which time staining was only detected in the early maturing chondrocytes. A mild sex difference was detected only at the end of puberty (7 weeks of age) with staining less intense in female animals than in male animals, but this suppression is difficult to quantitate. At 7 weeks of age, growth velocity is less in female animals than in male animals; therefore, although we cannot quantify it from these data, it is tempting to speculate that there may be a relation between growth velocity and GHR/BP expression in the growth plate in this period of sexually dimorphic growth velocity.
A logical candidate for the in vivo regulation of growth plate GHR expression is GH itself, because in vitro studies have shown regulation of chondrocyte GHR by GH(8) and GH is a main regulator of hepatic GHR and plasma GHBP in vivo.(48) Like plasma GHBP,(47) GHBP staining appeared less intense in dwarf rats compared with normal rats and this difference in staining disappeared in the older animals when normals and dwarves were growing at a more similar rate. However, the polyclonal antibody specific for GHR showed less difference between normal and dwarf growth plates. The relative expression of GHR and GHBP in the growth plate may vary, but this will require confirmation with more quantitative techniques.
In the multiple hormone-deficiency state of hypophysectomy, in which growth has completely ceased, GHR and GHBP were virtually undetectable in the growth plate. GHR and GHBP expression in chondrocytes was induced again by T3/T4 and GH treatment, especially in the transition zone. Lewinson et al.(49) concluded from experiments in hypothyroid rats that condylar chondrocytes were compromised in their response to GH although they could not show differences in GHR staining using MAb 263.
The early maturing chondrocytes on the transition from the proliferative zone to the hypertrophic zone may be a pivotal zone in chondrocyte differentiation. For example, Indian hedgehog (Ihh), parathyroid hormone-related protein (PTHrP), PTH/PTHrP receptors, and basic FGF (bFGF), which are involved in chondrocyte maturation, are localized to the same chondrocytes.(28,50–54) We now show here that it is again these cells in which GHR/GHBP expression is restored after GH/T3/T4 replacement therapy.
We found that dexamethasone acts as a negative regulator of GHR/GHBP in the growth plate. Staining for GHR and GHBP had almost disappeared not only from resting zone cells and proliferative chondrocytes but also from early maturing chondrocytes. This agrees with earlier results on dexamethasone suppression of GHR expression(55,56) but seems in contrast to the results of Heinrichs et al., who showed that GHR mRNA in the rabbit growth plate was up-regulated by dexamethasone.(57) Of course, posttranscriptional regulation and species difference could be involved in this discrepancy. In the rabbit, GHR and GHBP are derived from the same mRNA, whereas in rats GHR and GHBP are derived from two separate mRNAs so they could be regulated independently. In line with our results, it has been shown in vitro that dexamethasone decreases GHR mRNA in rat chondrocytes.(58) Thus, apart from acting via corticosteroid receptors in the growth plate,(59) the growth retardation seen after corticosteroid treatment could be indirect due to a down-regulation of GHR and GHBP, resulting in a relative insensitivity of chondrocytes to direct effects of GH.
The localization of GHR in the growth plate suggests a direct effect of GH not only on germinal cells, but also on proliferation of chondrocytes and maturation into hypertrophic chondrocytes. It is still unclear whether the effect of GH can be replaced fully by IGF-I; mice transgenic for GH have increased skeletal growth,(60) and mice transgenic for IGF-I do not.(61) On the other hand, GH-deficient mice transgenic for IGF-I have normal growth,(62) although mice with a deletion of hepatic IGF-I also grow normally.(5) Double knockouts for GHR and IGF-I are dwarfed more severely than the single knockout mice, and growth plates of IGF-I knockout mice, with high circulating GH levels, have a smaller resting zone, a higher proliferation rate, and a greater height of individual hypertrophic chondrocytes than the double GHR/IGF-I knockout mice.(33) This is in line with the localization of GHR that we found.
Both GH and IGF-I are capable of shortening cell cycle time of resting cells and duration of the hypertrophic phase of chondrocytes in Hx rats.(35) However, IGF-I treatment of GHR knockout mice does not restore completely femoral length(63) and IGF-I treatment of GHR-deficient children does not have sustaining effects on growth.(64)
The localization of GHR in the growth plate suggests that the zone of chondrocytes in transition from a proliferative to a hypertrophic phenotype has to be considered as a major site of direct action for GH. It remains to be studied whether the effect of GH on these cells can be replaced by IGF-I.
We thank Dr. W. Baumbach (American Cyanamid Co.) for the antibodies against GHBP and the internal part of GHR. We also thank Dr. R. Dirks and F. van der Rijke (Laboratory of Cytochemistry and Cytology, Leiden University Medical Center) for their help in setting up the immunohistochemistry. We are grateful to Pharmacia-Upjohn for their continuing supply of recombinant hGH, and we thank Dr. C.W.G.M. Löwik (Department of Endocrinology, Leiden University Medical Center) for helpful comments.
- 11960 Cell divisions in endochondral ossification. A study of cell proliferation in rat bones by the method of tritiated thymidine autoradiography. J Bone Joint Surg Br 42:824–839.
- 21993 Review paper: Endocrine regulation of longitudinal bone growth. Acta Paed Suppl 391:33–40., , , , ,
- 31982 Growth hormone stimulates longitudinal bone growth directly. Science 216:1237–1239.
- 41991 Passive immunization against insulin-like growth factor-I does not inhibit growth hormone-stimulated growth of dwarf rats. Endocrinology 128:2103–2109., ,
- 51999 Liver-derived insulin-like growth factor I (IGF-I) is the principal source of IGF-I in blood but is not required for postnatal body growth in mice. Proc Natl Acad Sci USA 96:7088–7092., , , , , , , , , ,
- 61999 Normal growth and development in the absence of hepatic insulin-like growth factor I. Proc Natl Acad Sci USA 96:7324–7329., , , , , ,
- 71992 Growth hormone induces multiplication of the slowly cycling germinal cells of the rat tibial growth plate. Proc Natl Acad Sci USA 89:9826–9830., , ,
- 81986 Regulation by growth hormone of number of chondrocytes containing IGF-I in rat growth plate. Science 233:571–574., , , , ,
- 91987 Review paper: Mechanism of the stimulatory effect of growth hormone on longitudinal bone growth. Endocr Rev 8:426–438., , ,
- 101988 The ontogeny of growth hormone-receptors in the rabbit tibia. Endocrinology 122:2562–2569., , ,
- 111984 Monoclonal antibodies to the rabbit liver growth hormone receptor; production and characterization. Endocrinology 115:1805–1813., , ,
- 121987 Growth hormone receptor and serum binding protein: Purification, cloning and expression. Nature 330:537–543., , , , , , , ,
- 131990 Expression of the growth hormone-binding protein messenger-RNA in the liver and extrahepatic tissues in the rat—coexpression with the growth-hormone receptor. Mol Cell Endocrinol 73:R1–R6., , ,
- 141992 Growth hormone (GH) binding protein and GH interaction in vivo in the guinea pig. Endocrinology 131:1963–1969., ,
- 151990 Regulation of growth hormone (GH) bioactivity by a recombinant human GH-binding protein. Endocrinology 127:1287–1291., , ,
- 161994 Inhibition of growth hormone bioactivity by recombinant human growth hormone-binding protein in the eluted stain assay system. J Endocrinol 140:445–453., , , ,
- 171989 The growth hormone-binding protein in rat serum is an alternatively spliced form of the rat growth hormone receptor. Genes Dev 3:1199–1205., ,
- 181991 Tissue distribution, characterization, and regulation of messenger-ribonucleic-acid for growth hormone receptor and serum binding-protein in the rat. Endocrinology 129:1628–1634.,
- 191992 Tissue-specific developmental regulation of the messenger ribonucleic-acids encoding the growth hormone receptor and the growth-hormone binding-protein in rat fetal and postnatal tissues. Pediaetr Res 31:335–339., , ,
- 201995 One class of growth hormone (GH) receptor and binding protein messenger ribonucleic acid in rat liver, GHR1, is sexually dimorphic and regulated by GH. Endocrinology 136:749–760.,
- 211995 Rat growth hormone receptor/growth hormone-binding protein mRNAs with divergent 5′-untranslated regions are expressed in a tissue-specific manner. DNA Cell Biol 14:195–204., , , ,
- 221990 Identification of the origin of the growth hormone-binding protein in rat serum. Mol Endocrinol 4:1799–1805., , , ,
- 231988 Growth hormone-deficient dwarfism in the rat: A new mutation. J Endocrinol 119:51–58., , , , , ,
- 241987 Single step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159.,
- 251994 Expression of growth hormone-binding protein with a hydrophilic carboxyl terminus by the mouse placenta: Studies in vivo and in vitro. J Endocrinol 140:125–135., , , , , , ,
- 261983 Growth hormone stimulates the proliferation of cultured chondrocytes from rabbit ear and rat rib growth cartilage. Nature 304:545–547., , , ,
- 271995 Ultra-sensitive FISH using peroxidase-mediated deposition of biotin- or fluorochrome tyramides. Hum Mol Genet 4:529–534., , , , ,
- 282000 Expression of Indian hedgehog, parathyroid hormone-related protein, and their receptors in the postnatal growth plate of the rat: Evidence for a locally acting growth restraining feedback loop after birth. J Bone Miner Res 15:1045–1055., , , ,
- 291999 Truncated growth hormone receptor isoforms. Acta Paediatr Suppl 88:164–166.
- 301992 Prenatal expression of the growth hormone (GH) receptor/binding protein in the rat: A role for GH in embryonic and fetal development? Development 114:869–876., , , , ,
- 311997 The CCAAT/enhancer-binding protein-alpha is expressed in the germinal layer of the growth plate: Colocalisation with the growth hormone receptor. J Endocrinol 155:433–441., , , ,
- 321993 Vital staining indicating cell migration towards the periphery in the growth plate. Studies of fibular heads in rabbits. Acta Orthop Scand 64:683–687., ,
- 332001 Roles of growth hormone and insulin-like growth factor 1 in mouse postnatal growth. Dev Biol 229:141–162., , , ,
- 341994 CCAAT/enhancer binding protein alpha is sufficient to initiate the 3T3-L1 adipocyte differentiation program. Proc Natl Acad Sci USA 91:8757–8761.,
- 351994 Differential effects of insulin-like growth factor I and growth hormone on developmental stages of rat growth plate chondrocytes in vivo. J Clin Invest 93:1078–1086., ,
- 362000 Effect of growth hormone and insulin-like growth factor I (IGF-I) on the expression of IGF-I messenger ribonucleic acid and peptide in rat tibial growth plate and articular chondrocytes in vivo. Endocrinology 141:2847–2853., , , ,
- 372000 Growth hormone receptor abundance in tibial growth plates of uremic rats: GH/IGF-I treatment. Kidney Int 58:62–70., , , , ,
- 381987 Effects of growth hormone and insulin-like growth factor-I on colony formation of rabbit epiphyseal chondrocytes at different stages of maturation. J Endocrinol 115:263–271., ,
- 391987 Differential effects of growth hormone and insulin-like growth factor I on colony formation of epiphyseal chondrocytes in suspension culture in rats of different ages. Endocrinology 121:1061–1069., , ,
- 401996 Single cell enzyme activity and proliferation in the growth plate: Effects of growth hormone. J Bone Miner Res 11:1103–1111., , ,
- 411995 Localization of growth hormone receptor/binding protein messenger ribonucleic acid (mRNA) during rat fetal development: Relationship to insulin-like growth factor-I mRNA. Endocrinology 136:4602–4609., , , , ,
- 421994 Nuclear translocation and anchorage of the growth hormone receptor. J Biol Chem 269:31735–31746., , , ,
- 431992 Cellular localization of the GH-binding protein in the rat. Endocrinology 130:3057–3065., , , ,
- 441994 Receptor-mediated nuclear translocation of growth hormone. J Biol Chem 269:21330–21339., , , , ,
- 451983 Ontogeny of liver somatotropic and lactogenic binding sites in male and female rats. Endocrinology 113:1325–1332., , ,
- 461990 Initial characterization and sexual dimorphism of serum growth hormone-releasing binding protein in adult rats. Endocrinology 126:1976–1980., , ,
- 471992 Growth hormone (GH)-binding protein in normal and GH-deficient dwarf rats. J Endocrinol 135:447–457., , , ,
- 481993 Growth hormone (GH) receptors, GH binding protein and GH: An autoregulatory system? Int J Paed Suppl 82:22–28., ,
- 491994 Differential effects of hypothyroidism on the cartilage and the osteogenic process in the mandibular condyle: Recovery by growth hormone and thyroxine. Endocrinology 135:1504–1510., ,
- 501995 Transforming growth factor-beta1 and fibroblast growth factors in rat growth plate. J Orthop Res 13:761–768., , , ,
- 511996 Regulation of rate of cartilage differentiation by Indian Hedgehog and PTH-related protein. Science 273:613–621., , , , ,
- 521996 PTH/PTHrP receptor in early development and indian hedgehog-regulated bone growth. Science 273:663–666., , , , , , , , , , , , ,
- 531990 Fibroblast growth factor is an inhibitor of chondrocyte terminal differentiation. J Biol Chem 265:5903–5909.,
- 541995 Inhibitory effects of basic fibroblast growth factor on chondrocyte differentiation. J Bone Miner Res 10:735–742.,
- 551995 Steroid regulation of growth hormone (GH) receptor and GH binding protein messenger ribonucleic acids in the rat. Endocrinology 136:209–217., , ,
- 561995 Dexamethasone-induced antagonism of growth hormone (GH) action by down-regulation of GH binding in 3T3-F442A fibroblasts. Endocrinology 136:4796–803.,
- 571994 Dexamethasone increases growth hormone receptor messenger ribonucleic acid levels in liver and growth plate. Endocrinology 135:1113–1118., , , , , , ,
- 581998 Dexamethasone impairs growth hormone (GH)-stimulated growth by suppression of local insulin-like growth factor (IGF)-I production and expression of GH- and IGF-I-receptor in cultured rat chondrocytes. Endocrinology 139:3296–3305., , , , , ,
- 592000 The localization of the functional glucocorticoid receptor alpha in human bone. J Clin Endocrinol Metab 85:883–889., , , ,
- 601999 Transgenic models of growth hormone action. Annu Rev Nutr 19:437–461., ,
- 611988 Growth enhancement of transgenic mice expressing human insulin-like growth factor I. Endocrinology 123:2827–2833., , , , , ,
- 621990 Expression of insulin-like growth factor I stimulates normal somatic growth in growth hormone-deficient transgenic mice. Endocrinology 127:1033–1040., , , , ,
- 632000 Bone homeostasis in growth hormone receptor-null mice is restored by IGF-I but independent of Stat5. J Clin Invest 106:1095–1103., , , , , , , , , , ,
- 642001 Therapy for 6.5–7.5 years with recombinant insulin-like growth factor I in children with growth hormone insensitivity syndrome: A clinical research center study. J Clin Endocrinol Metab 86:1504–1510.,