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Keywords:

  • growth plate;
  • chondrocyte;
  • growth hormone receptor;
  • growth hormone binding protein;
  • tyramide signal amplification

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

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.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

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

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Animal experiments

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.

Experiment 1

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)

Experiment 2

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.

Experiment 3

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.

Immunohistochemistry

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.

RT-PCR

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.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

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).

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Figure FIG. 1. Immunohistochemistry with PAb iGHR in the growth plate of a 12-week-old female rat performed (A) without and (B) with the use of TSA. The line indicates 100 μm. Immunohistochemistry was performed with DAB as chromogen (hematoxylin background staining).

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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)

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Figure FIG. 2. Immunohistochemical staining in the growth plate of a 12-week-old female rat with three different antibodies: (A) omission of first antibody; (B) MAb 263, recognizing specifically the extracellular part of GHR/GHBP; (C and E) PAb iGHR, recognizing the iGHR; and (D and F) MAb 4.3, recognizing the 17 amino acids tail specific for GHBP. Staining is mainly in the zone of early hypertrophic chondrocytes (E and F). At higher magnification, staining in cells in the resting zone (arrows) and some proliferative cells is revealed. The line indicates 100 μm. Immunohistochemistry was performed with DAB as chromogen (hematoxylin background staining).

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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.

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Figure FIG. 3. RT-PCR showing presence of GHR mRNA and GHBP mRNA in growth plate tissue and liver extracts of male and female rats at 1, 4, 7 and 12 weeks of age. Lane 1-8: growth plate tissue extracts in the following order: lane 1: 1-week-old female, lane 2: 4-week-old female, lane 3: 7-week-old female, lane 4: 12-week-old female and lane 5: 1-week-old male, lane 6: 4-week-old male, lane 7: 7-week-old male, lane 8: 12-week-old male. Lane 9: control using water instead of DNA. Lane 10: liver tissue extract of 8-week-old female. Lane 11-20: controls: PCR reactions in same growth plate tissue of lane 1-10 but without reverse transcription.

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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.

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Figure FIG. 4. (A-D) Immunohistochemistry for GHR, using PAb iGHR in normal male rat growth plates at (A) 1week, (B) 4 weeks, (C) 7 weeks, and (D) 12 weeks of age. Staining in 1-, 4-, and 7-week-old rats is in cells of the resting zone, proliferative cells, and early hypertrophic chondrocytes. In 12-week-old rats, staining mainly is in early hypertrophic chondrocytes in the transition zone between the proliferative and hypertrophic cell layer. The line indicates 100 μm. (E-H) Immunohistochemistry for GHBP, using a MAb recognizing the 17 amino acids tail specific for GHBP (MAb 4.3) in growth plates of the same rats as used in panels A-D. Staining for GHBP is similar as for GHR. (I-L) Immunohistochemistry for GHBP, using MAb 4.3, in growth plates of GH-deficient dwarf rats of (I) 1 week, (J) 4 weeks, (K) 7 weeks, and (L) 12 weeks of age. GHBP staining seems less in dwarf rats than in normal rats (cf. with panels E-H). The line indicates 100 μm. Immunohistochemistry was performed with DAB as chromogen (no background staining).

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Figure FIG. 5. Presence of GHR and GHBP immunostaining in cells of the resting zone (arrows) in 4-week-old male rats. (A) PAb iGHR, recognizing GHR; (B) MAb 4.3, recognizing GHBP. The line indicates 25 μm. Immunohistochemistry was performed with DAB as chromogen (no background staining).

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Figure FIG. 6. GHBP immunostaining using MAb 4.3 in a 7-week-old (A) male and (B) female rat. The growth plate appears smaller and the staining less in the female rat. The line indicates 100 μm. Immunohistochemistry was performed with DAB as chromogen (no background staining).

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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).

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Figure FIG. 7. Nuclear localization of (A) GHR immunostaining (PAb iGHR) and (B) GHBP immunostaining (MAb 4.3) in hypertrophic chondrocytes in a 4-week-old normal rat; (C) GHBP immunostaining (MAb 4.3) in hypertrophic chondrocytes in a 4-week-old GH-deficient dwarf rat. Note the decrease in nuclear localization of GHBP in the dwarf rat. The line indicates (A) 25 μm or (B and C) 100 μm. Immunohistochemistry was performed with DAB as chromogen (no background staining).

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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).

Table Table 1.. Weight Gain in Normal and Hormone-Treated 4- to 5-Week-Old Rats
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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).

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Figure FIG. 8. Induction of GHBP immunostaining in the growth plate after T3/T4 and GH treatment for 2 weeks in 4- to 5-week-old Hx rats. (A) Saline-treated Hx rat, (B) T3/T4-treated, (C) GH-treated, (D) GH + T3/T4-treated, and (E) normal intact rat. See text for more details. The line indicates 100 μm. Immunohistochemistry was performed with DAB as chromogen (no background staining).

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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).

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Figure FIG. 9. Reduction of GHBP immunostaining after 1 week of dexamethasone treatment in two different doses in 4-week-old female rats compared with both ad libitum-fed and pair-fed rats (pair fed with rats receiving the highest dexamethasone dose). (A) Controls, ad libitum-fed (B) low-dose dexamethasone treatment (0.3 mg/liter of drinking water), (C) high-dose dexamethasone treatment (2 mg/liter of drinking water), and (D) controls, pair-fed to rats receiving 2 mg/liter of dexamethasone. The line indicates 100 μm. Immunohistochemistry was performed with DAB as chromogen (no background staining).

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Table Table 2.. Weight Gain and Body Length Gain During Dexamethasone Treatment for 1 Week in 4-Week-Old Female Rats
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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

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.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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