0021-972X/90/7004-1725$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1990 by The Endocrine Society
Vol. 70, No. 6 Printed in U.S.A.
Visual Demonstration of Growth Hormone Receptors on Human Growth Plate Chondrocytes* G. A. WERTHER, K. M. HAYNES, R. BARNARD, AND M. J. WATERS Department of Endocrinology and Diabetes, Royal Children's Hospital (G.A.W., K.M.H.), Melbourne, Victoria; and The Department of Physiology, University of Queensland (R.B., M.J.W.), St. Lucia, Queensland, Australia
ABSTRACT. The sites of action of GH in the human infant remain unclear; recent evidence in animals suggests direct actions on growth plate and other tissues. We have used a monoclonal antibody recognizing the human GH receptor to visually identify and localize GH receptors in the human infant growth plate. Sternochondral cartilage was obtained at postmortem from infants dying of sudden infant death (n = 20), and either decalcified, fixed, and cut into longitudinal sections or digested with collagenase for monolayer culture of chondrocytes. Sections of cultured chondrocytes were stained immunocytochemically with a monoclonal antibody recognizing human GH receptor (MAb 263), using an avidin-biotin system. Sternochondral cartilage was also obtained at operation from adolescents undergoing sternochondroplasty. In infant tissue, GH receptor was identified in sections in chondrocytes of the proliferative and hypertrophic layers, in
perichondrium, in osteocytes in new bone, and in hemopoietic precursor cells in marrow. Cultured chondrocytes showed heterogeneous staining for GH receptor. With prolonged culture from 5-8 days, the pattern of staining changed from individual cells to groups of cells. [125I]Human (h)GH showed specific binding to chondrocyte monolayer (0.6 ± 0.3%), confirmed visually on emulsion autoradiography. In support of specificity of MAb263, it was able to displace [125I]hGH from monolayers by 35%. Adolescent cultured chondrocytes failed to demonstrate specific binding of [125I]hGH. We conclude that GH receptors are widely distributed in a range of mesenchyme cells in the human infant growth plate, including bone and hemopoietic precursors. The expression of these receptors appears to be maturation dependent in both intact tissue and culture, while they may no longer be expressed after the peak growth phase of puberty. (J Clin Endocrinol Metab 70:1725-1731, 1990)
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HE ORIGINAL somatomedin hypothesis suggested that the mitogenic actions of GH in man and animals were mediated by circulating somatomedins derived from the liver in response to pituitary-derived GH (1). However, recent evidence in animals and developing humans shows that somatomedins, or insulin-like growth factors (IGFs), are widely synthesized in supporting tissues (2, 3), and that they may be directly regulated by GH both in vivo (4, 5) and in vitro (6). Furthermore, such direct effects of GH have been shown to lead to unilateral rat epiphyseal growth when injected (7, 8) or infused into one leg (9) and, similarly, to promote chondrocyte proliferation in vitro (10, 11). To achieve a biological action, GH must first bind to its receptor; the gene for the human and rabbit forms has recently been cloned and sequenced (12). Little is known about the events leading to biological effects after the binding of
GH to its receptor. However, identification of specific receptors for GH points to likely target tissues for biological action. Using radiolabeled GH, specific receptors have been identified on rabbit chondrocytes (13), in keeping with its apparent direct mitogenic effects (10, 11). Furthermore, using one of a panel of monoclonal antibodies raised against rabbit liver GH receptors (14), we recently used immunohistochemical techniques to identify and localize GH receptors on developing rabbit tibial growth plate chondrocytes (15). This monoclonal antibody was found to recognize the human (h) GH receptor on IM-9 tumor cells (16), which are rich in GH receptors. Since GH receptors have never been identified in skeletal tissues of the developing human, we have used this monoclonal antibody recognizing GH receptor to identify and localize hGH receptors on human infant growth plate cells.
Received June 26,1989. Address all correspondence to: Dr. George Werther, Department of Endocrinology and Diabetes, Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia. * Presented in part at the Annual Meeting of The Endocrine Society, Indianapolis, IN June 1987. This work was supported by grants from the National Health and Medical Research Council of Australia.
Materials and Methods Subjects Sternochondral/costal cartilage was obtained at autopsy within 24 h of death from infants dying of sudden infant death syndrome (n = 20). The age range of infants was 3 days to 8
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months, and all were previously healthy, with no specific abnormalities found at postmortem. Sternochondral cartilage segments were also obtained at operation from adolescents, aged 14-16 yr, undergoing sternochondroplasty (n = 3). Preparation of growth plate sections Rib and costal cartilage were separated from the sternum and cut longitudinally and placed in buffered picric acid formaldehyde fixative for 24 h at 4 C. After washing with six changes of distilled H2O over 2 h, decalcification was performed in 0.38 M EDTA, pH 7.0, at 4 C for 72 h; the EDTA solution was changed several times, and the progress of decalcification was monitored by x-ray. After washing in phosphate-buffered saline (PBS) at 4° C, segments were paraffin embedded, and 6-/xm sections cut using a microtome for mounting on glass slides. Before immunohistochemical staining sections were deparaffinized and rehydrated. Monolayer culture of chondrocytes Sternum and costal cartilage was rinsed in 70% ethanol, followed by phosphate-buffered saline. Cartilage was dissected from bone and then scraped to remove all connective tissue and perichondrium. Cartilage was cut into small (~2 mm) slices with a scalpel blade and placed in 20 mL Ham's F-12 medium with 0.25% trypsin (Flow Laboratories, Sydney, Australia), mg/ mL collagenase (Sigma type XI, Sigma, St. Louis, MO), 20 mM HEPES, 0.29% glutamine, penicillin, and streptomycin. After incubation for 30 min at 37 C on a rotary mixer, incubation was stopped by the addition of 2 mL fetal calf serum (FCS) and spun at 300 X g for 10 min. Supernatant was removed, and the cartilage was resuspended in a similar solution without trypsin and with the addition of 5% FCS. Incubation was performed overnight at 37 C on a rotary mixer, usually leading to complete digestion by 16 h. The solution was filtered through coarse nylon mesh (500 nm) and then fine nylon mesh (60 jum) and spun three times for 10 min each, and the chondrocyte pellet was washed three times in Ham's F-12 medium with 10% FCS. Cells were counted, viability was checked, and then cells were plated in multiwell slide chambers (Lab-Tek Nunc, Naperville, IL) at a density of 0.5 x 106/cm2 for incubation at 37 C in 5% CO2. Cultures were fed after 3 days, and receptor studies were performed at least 3 days later without further feeding. Validation of chondrocyte phenotype in culture Chondrocyte phenotype was validated by the following methods. Morphology and staining. A typical cuboidal appearance of cells was noted, rather than a fibroblastoid shape. There was positive staining with alcian blue, which reacts with chondrocyte proteoglycans (17). Proteoglycan synthesis. Eighteen hours after the addition of [35S]proline, cells and medium were separately harvested and subjected to gel chromatography after digestion (kindly per-
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formed by Dr Chris Handley, Monash University) (18). Chromatographic profiles were examined for comparison with typical chondrocyte proteoglycan patterns. Collagen synthesis. [3H]Proline (Amersham, Bucks, United Kingdom) was added to culture medium for 48 h, after which cells were harvested, washed, and solubilized to run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of mol wt markers. Gels were exposed to x-ray film (Kodak XAR5, Eastman Kodak, Rochester, NY) for 24 h for autoradiography (19). Immunohistochemical staining Cultured cells in slide chambers were used at least 3-5 days after plating. Medium was aspirated, and cells were fixed for 30 min using 10% neutral buffered formalin. Sections or chondrocytes in slide chambers were incubated at room temperature in PBS for 20 min, followed by 20-min incubation in 1.5% normal horse serum, and then 1 h with monoclonal antibody recognizing hGH receptor (MAb263; 18 Mg/mL) (14). As well as nonimmune mouse ascites fluid, the following control antibodies were used: monoclonal antibodies to rabbit GH receptor of the same idiotype, not recognizing membrane-bound GH receptor (MAb5 and MAb7) (14), and a monoclonal antibody to brucella. Each of the antibodies was prepared in a similar fashion, and they were used as 33% NH4SO4 cuts (immunoglobulin fraction) from ascites fluid (14), as was the ascites fluid control. A further control was omission of the primary antibody. After three 10-min washes in PBS, incubation was carried out with biotinylated horse antimouse immunoglobulin G, followed by three further 10-min washes with PBS. Vectastain (Burlingame, CA) ABC reagent (peroxidase for cultured cells and glucose oxidase or peroxidase for sections) was then added for 30 min, followed by three further 10-min PBS washes. The final incubation of cultured cells (and some sections) was with 0.1% diaminobenzidine tetrachloride plus 0.02% hydrogen peroxidase in 0.1 M Tris buffer, pH 7.2. Sections were generally incubated in the dark with tetranitroblue tetrazolium in 50 mM Tris-HCl buffer, pH 8.2 (Vector Glucose Oxidase Substrate kit IITNBT, (Vector, Burlingame, CA). After washing cells or sections for 5 min with water, counterstaining was, respectively, with Harris hemotoxylin or light green. Radiolabeled hGH binding Cultured chondrocytes in multiwell slide chambers after 5 days culture were washed in PBS and incubated with [125I]hGH (SA, ~150 Ci/mol; kindly provided by Dr Adrian Herington, Melbourne, Australia) in Ham's F-12 medium, pH 7.4, without serum, but with 1% BSA. Incubation was carried our for 18 h at 22 C, and some wells had excess cold hGH added (10 ng/ mL) to determine nonspecific binding. After washing in PBS cells were solubilized with sodium hydroxide for counting in a 7-counter.
Results Distribution of hGH receptors on growth plate sections GH receptors were identified immunocytochemically on a variety of cell types on human infant growth plate
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GH RECEPTORS ON HUMAN CARTILAGE
longitudinal sections (n = 5; age, 1-5 months; Fig. 1). Chondrocytes were only occasionally stained in the reserve or stem cell zone, while staining was generally seen in the proliferative zone closest to the more mature hypertrophic zone, which consistently showed positive staining throughout. This staining generally extended into the degenerative zone, which leads to ossification (Fig. 1, a and c). Positive staining was also seen in perichondrial fibroblasts along the length of the growth plate (Fig. lc). In ossification centers, positive staining was seen on some, but not all, osteocytes in each section (Fig. Id). Occasional osteoclasts were also stained (not shown). Positive staining was also seen in a large number of hemopoietic precursor cells in marrow. These could not be precisely identified, but were mostly of erythroid origin (Fig. le). None of the control antibodies or ascites fluid gave any significant staining in sections (Fig. lb). Cultured human chondrocytes Validation of chondrocyte phenotype. Each of the means of validating chondrocyte phenotype was confirmatory. These included typical cuboidal morphology (Fig. 2), alcian blue staining (not shown) (16), typical proteoglycan profile (18), and type II, but not type I, collagen synthesis (19). Cultured human skin fibroblasts showed type I and III collagen production using similar methods (19). Immunocytochemical demonstration of hGH receptors. Positive staining for GH receptors was seen consistently on infant chondrocyte monolayers (n = 8; five males and three female; age, 3 days to 5 months; Fig. 2). Staining was seen over the cell cytoplasm in positive cells. Strikingly, only some cells in each monolayer showed positive staining (Fig. 2a), whereas uniform staining of most cells was seen in similarly stained human skin fibroblast monolayers (not shown). This patchy staining pattern of individual cells was seen in infant chondrocytes of all ages. With increasing time in culture (from 5-8 days) it was apparent that staining was concentrated in clumps of cells rather than individual cells, with many cells remaining unstained (Fig. 2, c and d). Controls showed no staining (Fig. 2b). Adolescent chondrocytes were not available for immunocytochemical examination. Autoradiographic demonstration and specificity of hGH receptors. Specific binding of [125I]hGH was demonstrated on monolayer chondrocyte cultures, representing 0.6 ± 0.3% (±SD) of the total counts per min added (n = 7; age, 1-8 months). A typical experiment is shown in Fig. 3. Nonspecific binding was 48% of the total binding (Fig. 3). This was demonstrated visually in emulsion
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autoradiographs, which show patchy distribution of silver grains and displacement of specifically bound labeled hormone in the presence of excess unlabeled hGH (nonspecific; data not shown). Specificity of the antibody for the hGH receptor was demonstrated by 35% displacement of [125I]hGH by MAb263 (Fig. 3) at a concentration similar to that used for immunocytochemistry. Control antibody did not displace the labeled hGH (Fig. 3). Adolescent chondrocytes (n = 3; two males and one female, aged 14-16 yr) consistently failed to show specific binding.
Discussion We have for the first time demonstrated sites of GH receptors on a variety of mesenchymally derived cells associated with the human infant growth plate. These include epiphyseal chondrocytes differentiating into bone, osteocytes in new bone, fibroblasts in perichondrium, as well as hemopoietic precursor cell in bone marrow. We have, furthermore, demonstrated that phenotypically preserved chondrocytes from human infant growth plate express GH receptors in primary culture, demonstrated by both immunocytochemical and classical radiolabeled hormone techniques. With the exception of liver (20), the demonstration of GH receptors on a variety of putative mesenchymal target tissues in both man and animals has been inconsistent, possibly reflecting low abundance of receptor and consequent failure to detect receptors by conventional radioligand techniques. Although radiolabeled GH has been reported to bind to human cells, including fibroblasts (21) and lymphocytes (22), a number of groups, including our own, have found these data difficult to reproduce. GH receptors have, nevertheless, been detected by such methods on rat chondrocytes in suspension (13) and more recently in monolayer culture (23), supported by immunocytochemical detection with the same antibody as that used in the current study. Our radioligand binding, although consistent, was very low and did not allow formal competition and kinetic studies. The immunocytochemical method is clearly more sensitive. The specificity of our immunohistochemical staining for the hGH receptor was supported by the lack of staining with a closely related monoclonal antibody to rabbit GH receptor of the same idiotype, which does not recognize hGH receptor (14). This was further supported by lack of staining with unrelated monoclonal antibody and immunoglobulin fractions of ascites fluid preparations. Highly purified hGH receptor was not available for classical immunodepletion studies. However, the specificity of the antibody was further established by our demonstration of competition for radiolabeled hGH
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FlG. 1. Immunohistochemical staining of GH-receptors on cells in and around the human infant rib growth plate. Positive staining is seen as purple-black, with counterstain (light green) seen as blue-green, a, Section (X120) of growth plate at epiphyseal junction, showing positive staining for GH receptor in chondrocytes of the proliferative (pr) and hypertrophic (hp) zones, extending into the degenerative (dg) zone, b, Section (X50) stained with control monoclonal antibody, showing no positive staining, c, Higher power (X300) section showing positive staining for GH receptor in proliferative zone (pr), hypertrophic zone (hp), and perichondrium (per), d, Section (X300) showing new bone formation near epiphysis. Positive staining for GH receptor is seen in several osteocytes (ob). e, Section (X120) of bone marrow showing positive staining on a range of hemopoietic precursor cells. The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 22:17 For personal use only. No other uses without permission. . All rights reserved.
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FIG. 2. Immunohistochemical staining of GH receptors on monolayer cultures of human infant growth plate chondrocytes, showing typical cuboidal morphology. Positive staining is seen as brown, with pink-purple counterstain. a, Positive staining of GH receptor, showing a heterogeneous distribution among cells after 5 days in culture (X300). b, Control slide showing no staining for GH receptor in the presence of control monoclonal antibody (X400). c, Heterogeneous staining of GH receptor seen in individual or groups of two or three cells after 5 days in culture (X300). d, Positive staining of larger groups or clones of cells after 8 days in culture (X300).
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WERTHER ET AL. cpm 600
500
400
300
200
100
TOTAL
1uM hGH
MAB 263
CONTROL AB
FIG. 3. [125I]hGH binding to cultured human infant growth plate chondrocyte monolayers. A typical experiment is shown (±SD of triplicates) indicating binding expressed as counts per min. The first two bars respectively represent total and nonspecific (in the presence of excess unlabeled hGH) binding. Specific binding (total minus nonspecific) was 240 cpm. The last two bars represent binding in the presence respectively of the monoclonal antibody to the GH receptor (MAb 263) and a control monoclonal antibody.
binding on cultured chondrocytes, whereas control antibody did not compete. This competition was in accord with our observation that MAb263 blocks binding of about 50% of [125I]hGH to IM-9 lymphocytes (16). Staining in sections was with a glucose oxidase system in order to avoid false positive staining due to endogenous peroxidase. Although it is not shown, the distribution of staining using peroxidase in sections was identical to that using glucose oxidase. In monolayer cultures, no false positive endogenous peroxidase staining was seen, and this staining system was, therefore, used. Our identification of hGH receptor on a variety of mesenchyme cell types involved in bone formation, including chondrocytes, perichondrial cells, and osteocytes, strongly supports a number of studies, mostly in animals, showing direct effects of GH on chondrocytes (10, 11) (24), osteoblasts (26, 27), as well as human erythroid precursor cells (28, 29). The demonstration of these appears to be dependent on culture conditions and may not be observed in monolayer cultures (30). Receptors for GH have similarly been identified on rat chondrocytes (13, 23) and human fibroblasts (21). Studies on human cells are few, although we recently demonstrated direct effects of hGH on DNA synthesis in human skin fibroblasts, involving local IGF-I synthesis (31). The demonstration of hGH receptor staining on
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a variety of hemopoietic precursor cells is consistent with the findings of Golde et al. (28) that hGH has direct effects on proliferation of human erythroid precursor cells in three-dimensional serum-substituted culture. Our findings in human growth plate chondrocytes, although not identical, have some similarities with our earlier findings in developing rabbit tibial growth plate in both receptor distribution and ontogeny (15). The distribution of receptors in the infant in mature proliferative and hypertrophic zones, but not significantly in the less mature reserve zone, is similar to the distribution in the newborn rabbit, where staining was limited to the hypertrophic zone (15). In contrast, the 20- and 50-day old rabbit (beyond infancy) showed staining of expressed GH receptor in the less mature reserve zone as well as the proliferative zone (15). We found occasional staining in the reserve zone of the human infant. However, further studies in children beyond early infancy will be required to determine whether a similar ontogeny of growth plate GH receptor expression is seen as in the rabbit. The distribution seen in human infants certainly suggests a maturational process of GH receptor expression, with receptors being expressed as chondrocytes line up in columns in the proliferative zone. Our monolayer culture findings further support this notion, in that the expression of receptors was heterogeneous, as similarly described recently in rat chondrocytes (23). Our cultured cell findings presumably reflect chondrocytes derived from different regions of the growth plate. Those chondrocytes expressing GH receptor were probably from the proliferative or hypertrophic zone, while those not expressing receptor were likely to be from the resting zone. Furthermore, the change over time in culture from scattered individually stained cells to groups of stained cells suggests the proliferation of clones of cells that are committed to hGH receptor expression. This may be similar to the process seen in the intact growth plate proliferative zone. As for the rabbit data, our human findings support the model for GH action proposed by Isakksson and colleagues (32) based on their studies of rat growth plate chondrocytes. According to this model, GH is primarily involved in the differentiation of prechondrocytes to mature chondrocytes, which then proliferate under the effect of locally produced IGF-I, which is also triggered by GH. The expression of hGH receptor, making the maturing chondrocyte sensitive to circulating GH, is of course an effective local regulator of GH action. In the developing rabbit, receptors were no longer seen by 180 days, when epiphyseal closure was nearly complete (15). Although we did not have growth plate sections from adolescents with near-completed growth, limited quantities of growth plate chondrocytes were available for culture. Whereas hGH receptors were consistently seen
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GH RECEPTORS ON HUMAN CARTILAGE
in monolayer cultures of infant human chondrocytes, adolescent chondrocytes failed to show radioligand binding for hGH receptors. It is likely, therefore, that, as in rabbits, hGH receptor is expressed in growth plate chondrocytes during the period of rapid growth in infancy and is no longer expressed as growth nears completion with epiphyseal closure. Such a process would enhance tissue sensitivitiy to circulating GH during the rapid growth period. Whether there is variation in growth plate hGH receptor expression during the rapid growth phase of puberty is unclear from our studies. However, our findings of apparent differences in abundance of hGH receptor in growth plate between infancy and adolescence is consistent with the model recently proposed by Daughaday and Rotwein (33). They argue that paracrine actions of IGF-I (possibly regulated directly by GH) predominate in infancy, while endocrine IGF-I actions (regulated by GH action on liver) predominate in adolescence (33). Our current findings provide clear evidence in human infant tissues for GH receptor expression on a variety of likely target cells associated with cartilage and bone growth and hemopoiesis.
References 1. Salmon WH, Daughaday WH. A hormonally controlled serum factor which stimulates sulfate incorporation in cartilage in vitro. J Lab Clin Med. 1957;49:825. 2. Hoyt EC, Van Wyk JJ, Lund PK. Tissue and development specific regulation of a complex family of rat insulin-like growth factor I messenger ribonucleic acids. Mol Endocrinol 1988;2:1077-86. 3. Underwood LE, D'Ercole JA, Clemmons DR, Van Wyk JJ. Paracrine functions of somatomedins. Clin Endocrinol Metab. 1986;15:59-77. 4. Nilsson A, Isgaard J, Lindahl A, Dahlstrom A, Skottner A, Isaksson 0. Regulation by growth hormone of number of chondrocytes containing IGF-I in rat growth plate. Science. 1986;233:571-4. 5. Isgaard J, Moller C, Isaksson OG, Nilsson A, Mathews LS, Norstedt G. Regulation of insulin-like growth factor messenger ribonucleic acid in rat growth plate by growth hormone. Endocrinology. 1988;122:1515-20. 6. Stracke H, Schulz A. Moeller D, Rossol S, Schatz H. Effect of growth hormone on osteoblasts and demonstration of somatomedin-C/IGF-I in bone organ culture. Acta Endocrinol (Copenh). 1984;107:16-24. 7. Isaksson OGP, Jansson J-0, Gause IAM. Growth hormone stimulates longitudinal bone growth directly. Science 1982;216:1237. 8. Russell SM, Spencer EM. Local injections of human or rat growth hormone or of purified human somatomedin-C stimulate unilateral tibial epiphyseal growth in hypophysectomized rats. Endocrinology. 1985;116:2563. 9. Schlechter NL, Russell SM, Spencer EM, Nicoll CS. Evidence suggesting that the direct growth-promoting effect of growth hormone on cartilage in vivo is mediated by local production of somatomedin. Proc Natl Acad Sci USA. 1986;83:7932. 10. Madsen K, Friberg U, Roos P, Eden S, Isaksson 0. Growth hormone stimulates the proliferation of cultured chondrocytes from rabbit ear and rat rib growth cartilage. Nature. 1983;304:545. 11. Lindahl A, Isgaard J, Nilsson A, Isaksson OGP. Growth hormone potentiates colony formation of epiphyseal chondrocytes in suspension culture. Endocrinology. 1986;118:1843-8. 12. Leung DW, Spencer SA, Cachiane G, Hammonds RG, Collins C, Henzel WJ, Barnard R, Waters MJ, Wood WI. Growth hormone
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receptor and serum binding protein: purification, cloning and expression. Nature. 1987;330:537-43. 13. Eden S, Isaksson OGP, Madsen K, Friberg U. Specific binding of growth hormone to isolated chondrocytes from rabbit ear and epiphyseal plate. Endocrinology. 1983;112:1127. 14. Barnard R, Bundesen PG, Rylatt DB, Waters MJ. Evidence from the use of monoclonal antibody probes for structural hetrogeneity of the GH receptor. Biochem J. 1985;231:459-68. 15. Barnard R, Haynes KM, Werther GA, Waters MJ. The ontogeny of growth hormone receptors in the rabbit tibia. Endocrinology. 1988;122:2562-9. 16. Asakawa K, Hedo JA, MacElduff A, Rouiller DG, Waters MJ, Gorden P. The human growth hormone receptor of cultured human lymphocytes: structural characteristics and glycosylation properties. Biochem J. 1986;238:379-86. 17. Kiernan JA. Histological and histochemical methods: theory and practice. Toronto: Pergamon Press; 1981;162. 18. McQuillan DJ, Handley CJ, Campbell MA, Bolis S, Milway VE, Herington AC. Stimulation of proteoglycan biosynthesis by serum and insulin-like growth factor-I in cultured bovine articular cartilage. Biochem J. 1986;240:423-30. 19. Bateman JF, Chan D, Mascara T, Rogers JG, Cole WG. Collagen defects in lethal perinatal osteogenesis imperfecta. Biochem J. 1986;240:699-708. 20. Herington AC. Ontogenesis of hepatic growth hormone receptors in the rat. Horm Metab Res. 1982;14:422-4. 21. Murphy LJ, Vrhosek E, Lazarus L. Identification and characterization of specific growth hormone receptors in cultured human fibroblasts. J Clin Endocrinol Metab. 1983;57:1117-1124. 22. Kiess W, Butenandt 0. Specific growth hormone receptors on human peripheral mononuclear cells: reexpression, identification, and characterization. J Clin Endocrinol Metab. 1985;60:740-6. 23. Nilsson A, Lindahl A, Eden S, Isaksson OGP. Demonstration of growth hormone receptors in cultured rat epihyseal chondrocytes by specific binding of growth hormone and immunocytochemistry. J Endocrinol. In Press. 24. Lindahl A, Nilsson A, Isaksson OGP. Effects of growth hormone and insulin-like growth factor-I on colony formation of rabbit epiphyseal chondrocytes at different stages of maturation. J Endocrinol. 1987;115:263-71. 25. Corvol M, Dumontier MF, Prevot C, Bonaventure J, de la Tour B, Willeput J. Effect of growth hormone on the differentiation of chondrocytes from prepuberal rabbits in serum-free culture and on the radioimmunologic activity of Sm-C/IGFl measured in the culture medium. Reprod Nutr Dev. 1988;28(2A):233-40. 26. Ernst M, Froesch ER. Growth hormone dependent stimulation of osteoblast-like cells in serum-free cultures via local synthesis of insulin-like growth factor I. Biochem Biophys Res Commun. 1988;26:142-7. 27. Ernst M, Froesch ER. Growth hormone dependent stimulation of osteoblast-like cells in serum-free cultures via local synthesis of insulin-like growth factor I. Biochem Biophys Res Commun. 1988;151:142-7. 28. Golde DW. Bersch N, Li CH. Growth hormone species-specific stimulation of erythropoiesis in vitro. Science 1976;196:1112-3. 29. Merchav S, Tatarsky I, Hochberg Z. Enhancement of erythropoiesis in vitro by human growth hormone is mediated by insulin-like growth factor-I. Br J Haematol. 1988;70:267-271. 30. Trippel SB, Corvol MT, Dumontier MF, Rappaport R, Hung HH, Mankin HJ. Effect of somatomedin-C/insulin-like growth factor I and growth hormone on cultured growth plate and articular chondrocytes. Pediatr Res. 1989;25:76-82. 31. Cook JJ, Haynes KM, Werther GA. Mitogenic effects of growth hormone in cultured human fibroblasts. Evidence for action via local insulin-like growth factor I production. J Clin Invest. 81:20612. 32. Isaksson OG, Lindahl A, Nilsson A, Isgaard J. Mechanism of the stimulatory effect of growth hormone on longitudinal bone growth. Endocr Rev. 1987;8:426-38. 33. Daughaday WH, Rotwein P. Insulin-like growth factors I and II. Peptide, messenger ribonucleic acid and gene structures, serum, and tissue concentrations. Endocr Rev. 1989;10:68-91.
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Vol. 70, No. 6 Printed in U.S.A.
Visual Demonstration of Growth Hormone Receptors on Human Growth Plate Chondrocytes* G. A. WERTHER, K. M. HAYNES, R. BARNARD, AND M. J. WATERS Department of Endocrinology and Diabetes, Royal Children's Hospital (G.A.W., K.M.H.), Melbourne, Victoria; and The Department of Physiology, University of Queensland (R.B., M.J.W.), St. Lucia, Queensland, Australia
ABSTRACT. The sites of action of GH in the human infant remain unclear; recent evidence in animals suggests direct actions on growth plate and other tissues. We have used a monoclonal antibody recognizing the human GH receptor to visually identify and localize GH receptors in the human infant growth plate. Sternochondral cartilage was obtained at postmortem from infants dying of sudden infant death (n = 20), and either decalcified, fixed, and cut into longitudinal sections or digested with collagenase for monolayer culture of chondrocytes. Sections of cultured chondrocytes were stained immunocytochemically with a monoclonal antibody recognizing human GH receptor (MAb 263), using an avidin-biotin system. Sternochondral cartilage was also obtained at operation from adolescents undergoing sternochondroplasty. In infant tissue, GH receptor was identified in sections in chondrocytes of the proliferative and hypertrophic layers, in
perichondrium, in osteocytes in new bone, and in hemopoietic precursor cells in marrow. Cultured chondrocytes showed heterogeneous staining for GH receptor. With prolonged culture from 5-8 days, the pattern of staining changed from individual cells to groups of cells. [125I]Human (h)GH showed specific binding to chondrocyte monolayer (0.6 ± 0.3%), confirmed visually on emulsion autoradiography. In support of specificity of MAb263, it was able to displace [125I]hGH from monolayers by 35%. Adolescent cultured chondrocytes failed to demonstrate specific binding of [125I]hGH. We conclude that GH receptors are widely distributed in a range of mesenchyme cells in the human infant growth plate, including bone and hemopoietic precursors. The expression of these receptors appears to be maturation dependent in both intact tissue and culture, while they may no longer be expressed after the peak growth phase of puberty. (J Clin Endocrinol Metab 70:1725-1731, 1990)
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HE ORIGINAL somatomedin hypothesis suggested that the mitogenic actions of GH in man and animals were mediated by circulating somatomedins derived from the liver in response to pituitary-derived GH (1). However, recent evidence in animals and developing humans shows that somatomedins, or insulin-like growth factors (IGFs), are widely synthesized in supporting tissues (2, 3), and that they may be directly regulated by GH both in vivo (4, 5) and in vitro (6). Furthermore, such direct effects of GH have been shown to lead to unilateral rat epiphyseal growth when injected (7, 8) or infused into one leg (9) and, similarly, to promote chondrocyte proliferation in vitro (10, 11). To achieve a biological action, GH must first bind to its receptor; the gene for the human and rabbit forms has recently been cloned and sequenced (12). Little is known about the events leading to biological effects after the binding of
GH to its receptor. However, identification of specific receptors for GH points to likely target tissues for biological action. Using radiolabeled GH, specific receptors have been identified on rabbit chondrocytes (13), in keeping with its apparent direct mitogenic effects (10, 11). Furthermore, using one of a panel of monoclonal antibodies raised against rabbit liver GH receptors (14), we recently used immunohistochemical techniques to identify and localize GH receptors on developing rabbit tibial growth plate chondrocytes (15). This monoclonal antibody was found to recognize the human (h) GH receptor on IM-9 tumor cells (16), which are rich in GH receptors. Since GH receptors have never been identified in skeletal tissues of the developing human, we have used this monoclonal antibody recognizing GH receptor to identify and localize hGH receptors on human infant growth plate cells.
Received June 26,1989. Address all correspondence to: Dr. George Werther, Department of Endocrinology and Diabetes, Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia. * Presented in part at the Annual Meeting of The Endocrine Society, Indianapolis, IN June 1987. This work was supported by grants from the National Health and Medical Research Council of Australia.
Materials and Methods Subjects Sternochondral/costal cartilage was obtained at autopsy within 24 h of death from infants dying of sudden infant death syndrome (n = 20). The age range of infants was 3 days to 8
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months, and all were previously healthy, with no specific abnormalities found at postmortem. Sternochondral cartilage segments were also obtained at operation from adolescents, aged 14-16 yr, undergoing sternochondroplasty (n = 3). Preparation of growth plate sections Rib and costal cartilage were separated from the sternum and cut longitudinally and placed in buffered picric acid formaldehyde fixative for 24 h at 4 C. After washing with six changes of distilled H2O over 2 h, decalcification was performed in 0.38 M EDTA, pH 7.0, at 4 C for 72 h; the EDTA solution was changed several times, and the progress of decalcification was monitored by x-ray. After washing in phosphate-buffered saline (PBS) at 4° C, segments were paraffin embedded, and 6-/xm sections cut using a microtome for mounting on glass slides. Before immunohistochemical staining sections were deparaffinized and rehydrated. Monolayer culture of chondrocytes Sternum and costal cartilage was rinsed in 70% ethanol, followed by phosphate-buffered saline. Cartilage was dissected from bone and then scraped to remove all connective tissue and perichondrium. Cartilage was cut into small (~2 mm) slices with a scalpel blade and placed in 20 mL Ham's F-12 medium with 0.25% trypsin (Flow Laboratories, Sydney, Australia), mg/ mL collagenase (Sigma type XI, Sigma, St. Louis, MO), 20 mM HEPES, 0.29% glutamine, penicillin, and streptomycin. After incubation for 30 min at 37 C on a rotary mixer, incubation was stopped by the addition of 2 mL fetal calf serum (FCS) and spun at 300 X g for 10 min. Supernatant was removed, and the cartilage was resuspended in a similar solution without trypsin and with the addition of 5% FCS. Incubation was performed overnight at 37 C on a rotary mixer, usually leading to complete digestion by 16 h. The solution was filtered through coarse nylon mesh (500 nm) and then fine nylon mesh (60 jum) and spun three times for 10 min each, and the chondrocyte pellet was washed three times in Ham's F-12 medium with 10% FCS. Cells were counted, viability was checked, and then cells were plated in multiwell slide chambers (Lab-Tek Nunc, Naperville, IL) at a density of 0.5 x 106/cm2 for incubation at 37 C in 5% CO2. Cultures were fed after 3 days, and receptor studies were performed at least 3 days later without further feeding. Validation of chondrocyte phenotype in culture Chondrocyte phenotype was validated by the following methods. Morphology and staining. A typical cuboidal appearance of cells was noted, rather than a fibroblastoid shape. There was positive staining with alcian blue, which reacts with chondrocyte proteoglycans (17). Proteoglycan synthesis. Eighteen hours after the addition of [35S]proline, cells and medium were separately harvested and subjected to gel chromatography after digestion (kindly per-
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formed by Dr Chris Handley, Monash University) (18). Chromatographic profiles were examined for comparison with typical chondrocyte proteoglycan patterns. Collagen synthesis. [3H]Proline (Amersham, Bucks, United Kingdom) was added to culture medium for 48 h, after which cells were harvested, washed, and solubilized to run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of mol wt markers. Gels were exposed to x-ray film (Kodak XAR5, Eastman Kodak, Rochester, NY) for 24 h for autoradiography (19). Immunohistochemical staining Cultured cells in slide chambers were used at least 3-5 days after plating. Medium was aspirated, and cells were fixed for 30 min using 10% neutral buffered formalin. Sections or chondrocytes in slide chambers were incubated at room temperature in PBS for 20 min, followed by 20-min incubation in 1.5% normal horse serum, and then 1 h with monoclonal antibody recognizing hGH receptor (MAb263; 18 Mg/mL) (14). As well as nonimmune mouse ascites fluid, the following control antibodies were used: monoclonal antibodies to rabbit GH receptor of the same idiotype, not recognizing membrane-bound GH receptor (MAb5 and MAb7) (14), and a monoclonal antibody to brucella. Each of the antibodies was prepared in a similar fashion, and they were used as 33% NH4SO4 cuts (immunoglobulin fraction) from ascites fluid (14), as was the ascites fluid control. A further control was omission of the primary antibody. After three 10-min washes in PBS, incubation was carried out with biotinylated horse antimouse immunoglobulin G, followed by three further 10-min washes with PBS. Vectastain (Burlingame, CA) ABC reagent (peroxidase for cultured cells and glucose oxidase or peroxidase for sections) was then added for 30 min, followed by three further 10-min PBS washes. The final incubation of cultured cells (and some sections) was with 0.1% diaminobenzidine tetrachloride plus 0.02% hydrogen peroxidase in 0.1 M Tris buffer, pH 7.2. Sections were generally incubated in the dark with tetranitroblue tetrazolium in 50 mM Tris-HCl buffer, pH 8.2 (Vector Glucose Oxidase Substrate kit IITNBT, (Vector, Burlingame, CA). After washing cells or sections for 5 min with water, counterstaining was, respectively, with Harris hemotoxylin or light green. Radiolabeled hGH binding Cultured chondrocytes in multiwell slide chambers after 5 days culture were washed in PBS and incubated with [125I]hGH (SA, ~150 Ci/mol; kindly provided by Dr Adrian Herington, Melbourne, Australia) in Ham's F-12 medium, pH 7.4, without serum, but with 1% BSA. Incubation was carried our for 18 h at 22 C, and some wells had excess cold hGH added (10 ng/ mL) to determine nonspecific binding. After washing in PBS cells were solubilized with sodium hydroxide for counting in a 7-counter.
Results Distribution of hGH receptors on growth plate sections GH receptors were identified immunocytochemically on a variety of cell types on human infant growth plate
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longitudinal sections (n = 5; age, 1-5 months; Fig. 1). Chondrocytes were only occasionally stained in the reserve or stem cell zone, while staining was generally seen in the proliferative zone closest to the more mature hypertrophic zone, which consistently showed positive staining throughout. This staining generally extended into the degenerative zone, which leads to ossification (Fig. 1, a and c). Positive staining was also seen in perichondrial fibroblasts along the length of the growth plate (Fig. lc). In ossification centers, positive staining was seen on some, but not all, osteocytes in each section (Fig. Id). Occasional osteoclasts were also stained (not shown). Positive staining was also seen in a large number of hemopoietic precursor cells in marrow. These could not be precisely identified, but were mostly of erythroid origin (Fig. le). None of the control antibodies or ascites fluid gave any significant staining in sections (Fig. lb). Cultured human chondrocytes Validation of chondrocyte phenotype. Each of the means of validating chondrocyte phenotype was confirmatory. These included typical cuboidal morphology (Fig. 2), alcian blue staining (not shown) (16), typical proteoglycan profile (18), and type II, but not type I, collagen synthesis (19). Cultured human skin fibroblasts showed type I and III collagen production using similar methods (19). Immunocytochemical demonstration of hGH receptors. Positive staining for GH receptors was seen consistently on infant chondrocyte monolayers (n = 8; five males and three female; age, 3 days to 5 months; Fig. 2). Staining was seen over the cell cytoplasm in positive cells. Strikingly, only some cells in each monolayer showed positive staining (Fig. 2a), whereas uniform staining of most cells was seen in similarly stained human skin fibroblast monolayers (not shown). This patchy staining pattern of individual cells was seen in infant chondrocytes of all ages. With increasing time in culture (from 5-8 days) it was apparent that staining was concentrated in clumps of cells rather than individual cells, with many cells remaining unstained (Fig. 2, c and d). Controls showed no staining (Fig. 2b). Adolescent chondrocytes were not available for immunocytochemical examination. Autoradiographic demonstration and specificity of hGH receptors. Specific binding of [125I]hGH was demonstrated on monolayer chondrocyte cultures, representing 0.6 ± 0.3% (±SD) of the total counts per min added (n = 7; age, 1-8 months). A typical experiment is shown in Fig. 3. Nonspecific binding was 48% of the total binding (Fig. 3). This was demonstrated visually in emulsion
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autoradiographs, which show patchy distribution of silver grains and displacement of specifically bound labeled hormone in the presence of excess unlabeled hGH (nonspecific; data not shown). Specificity of the antibody for the hGH receptor was demonstrated by 35% displacement of [125I]hGH by MAb263 (Fig. 3) at a concentration similar to that used for immunocytochemistry. Control antibody did not displace the labeled hGH (Fig. 3). Adolescent chondrocytes (n = 3; two males and one female, aged 14-16 yr) consistently failed to show specific binding.
Discussion We have for the first time demonstrated sites of GH receptors on a variety of mesenchymally derived cells associated with the human infant growth plate. These include epiphyseal chondrocytes differentiating into bone, osteocytes in new bone, fibroblasts in perichondrium, as well as hemopoietic precursor cell in bone marrow. We have, furthermore, demonstrated that phenotypically preserved chondrocytes from human infant growth plate express GH receptors in primary culture, demonstrated by both immunocytochemical and classical radiolabeled hormone techniques. With the exception of liver (20), the demonstration of GH receptors on a variety of putative mesenchymal target tissues in both man and animals has been inconsistent, possibly reflecting low abundance of receptor and consequent failure to detect receptors by conventional radioligand techniques. Although radiolabeled GH has been reported to bind to human cells, including fibroblasts (21) and lymphocytes (22), a number of groups, including our own, have found these data difficult to reproduce. GH receptors have, nevertheless, been detected by such methods on rat chondrocytes in suspension (13) and more recently in monolayer culture (23), supported by immunocytochemical detection with the same antibody as that used in the current study. Our radioligand binding, although consistent, was very low and did not allow formal competition and kinetic studies. The immunocytochemical method is clearly more sensitive. The specificity of our immunohistochemical staining for the hGH receptor was supported by the lack of staining with a closely related monoclonal antibody to rabbit GH receptor of the same idiotype, which does not recognize hGH receptor (14). This was further supported by lack of staining with unrelated monoclonal antibody and immunoglobulin fractions of ascites fluid preparations. Highly purified hGH receptor was not available for classical immunodepletion studies. However, the specificity of the antibody was further established by our demonstration of competition for radiolabeled hGH
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FlG. 1. Immunohistochemical staining of GH-receptors on cells in and around the human infant rib growth plate. Positive staining is seen as purple-black, with counterstain (light green) seen as blue-green, a, Section (X120) of growth plate at epiphyseal junction, showing positive staining for GH receptor in chondrocytes of the proliferative (pr) and hypertrophic (hp) zones, extending into the degenerative (dg) zone, b, Section (X50) stained with control monoclonal antibody, showing no positive staining, c, Higher power (X300) section showing positive staining for GH receptor in proliferative zone (pr), hypertrophic zone (hp), and perichondrium (per), d, Section (X300) showing new bone formation near epiphysis. Positive staining for GH receptor is seen in several osteocytes (ob). e, Section (X120) of bone marrow showing positive staining on a range of hemopoietic precursor cells. The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 22:17 For personal use only. No other uses without permission. . All rights reserved.
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FIG. 2. Immunohistochemical staining of GH receptors on monolayer cultures of human infant growth plate chondrocytes, showing typical cuboidal morphology. Positive staining is seen as brown, with pink-purple counterstain. a, Positive staining of GH receptor, showing a heterogeneous distribution among cells after 5 days in culture (X300). b, Control slide showing no staining for GH receptor in the presence of control monoclonal antibody (X400). c, Heterogeneous staining of GH receptor seen in individual or groups of two or three cells after 5 days in culture (X300). d, Positive staining of larger groups or clones of cells after 8 days in culture (X300).
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WERTHER ET AL. cpm 600
500
400
300
200
100
TOTAL
1uM hGH
MAB 263
CONTROL AB
FIG. 3. [125I]hGH binding to cultured human infant growth plate chondrocyte monolayers. A typical experiment is shown (±SD of triplicates) indicating binding expressed as counts per min. The first two bars respectively represent total and nonspecific (in the presence of excess unlabeled hGH) binding. Specific binding (total minus nonspecific) was 240 cpm. The last two bars represent binding in the presence respectively of the monoclonal antibody to the GH receptor (MAb 263) and a control monoclonal antibody.
binding on cultured chondrocytes, whereas control antibody did not compete. This competition was in accord with our observation that MAb263 blocks binding of about 50% of [125I]hGH to IM-9 lymphocytes (16). Staining in sections was with a glucose oxidase system in order to avoid false positive staining due to endogenous peroxidase. Although it is not shown, the distribution of staining using peroxidase in sections was identical to that using glucose oxidase. In monolayer cultures, no false positive endogenous peroxidase staining was seen, and this staining system was, therefore, used. Our identification of hGH receptor on a variety of mesenchyme cell types involved in bone formation, including chondrocytes, perichondrial cells, and osteocytes, strongly supports a number of studies, mostly in animals, showing direct effects of GH on chondrocytes (10, 11) (24), osteoblasts (26, 27), as well as human erythroid precursor cells (28, 29). The demonstration of these appears to be dependent on culture conditions and may not be observed in monolayer cultures (30). Receptors for GH have similarly been identified on rat chondrocytes (13, 23) and human fibroblasts (21). Studies on human cells are few, although we recently demonstrated direct effects of hGH on DNA synthesis in human skin fibroblasts, involving local IGF-I synthesis (31). The demonstration of hGH receptor staining on
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a variety of hemopoietic precursor cells is consistent with the findings of Golde et al. (28) that hGH has direct effects on proliferation of human erythroid precursor cells in three-dimensional serum-substituted culture. Our findings in human growth plate chondrocytes, although not identical, have some similarities with our earlier findings in developing rabbit tibial growth plate in both receptor distribution and ontogeny (15). The distribution of receptors in the infant in mature proliferative and hypertrophic zones, but not significantly in the less mature reserve zone, is similar to the distribution in the newborn rabbit, where staining was limited to the hypertrophic zone (15). In contrast, the 20- and 50-day old rabbit (beyond infancy) showed staining of expressed GH receptor in the less mature reserve zone as well as the proliferative zone (15). We found occasional staining in the reserve zone of the human infant. However, further studies in children beyond early infancy will be required to determine whether a similar ontogeny of growth plate GH receptor expression is seen as in the rabbit. The distribution seen in human infants certainly suggests a maturational process of GH receptor expression, with receptors being expressed as chondrocytes line up in columns in the proliferative zone. Our monolayer culture findings further support this notion, in that the expression of receptors was heterogeneous, as similarly described recently in rat chondrocytes (23). Our cultured cell findings presumably reflect chondrocytes derived from different regions of the growth plate. Those chondrocytes expressing GH receptor were probably from the proliferative or hypertrophic zone, while those not expressing receptor were likely to be from the resting zone. Furthermore, the change over time in culture from scattered individually stained cells to groups of stained cells suggests the proliferation of clones of cells that are committed to hGH receptor expression. This may be similar to the process seen in the intact growth plate proliferative zone. As for the rabbit data, our human findings support the model for GH action proposed by Isakksson and colleagues (32) based on their studies of rat growth plate chondrocytes. According to this model, GH is primarily involved in the differentiation of prechondrocytes to mature chondrocytes, which then proliferate under the effect of locally produced IGF-I, which is also triggered by GH. The expression of hGH receptor, making the maturing chondrocyte sensitive to circulating GH, is of course an effective local regulator of GH action. In the developing rabbit, receptors were no longer seen by 180 days, when epiphyseal closure was nearly complete (15). Although we did not have growth plate sections from adolescents with near-completed growth, limited quantities of growth plate chondrocytes were available for culture. Whereas hGH receptors were consistently seen
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in monolayer cultures of infant human chondrocytes, adolescent chondrocytes failed to show radioligand binding for hGH receptors. It is likely, therefore, that, as in rabbits, hGH receptor is expressed in growth plate chondrocytes during the period of rapid growth in infancy and is no longer expressed as growth nears completion with epiphyseal closure. Such a process would enhance tissue sensitivitiy to circulating GH during the rapid growth period. Whether there is variation in growth plate hGH receptor expression during the rapid growth phase of puberty is unclear from our studies. However, our findings of apparent differences in abundance of hGH receptor in growth plate between infancy and adolescence is consistent with the model recently proposed by Daughaday and Rotwein (33). They argue that paracrine actions of IGF-I (possibly regulated directly by GH) predominate in infancy, while endocrine IGF-I actions (regulated by GH action on liver) predominate in adolescence (33). Our current findings provide clear evidence in human infant tissues for GH receptor expression on a variety of likely target cells associated with cartilage and bone growth and hemopoiesis.
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receptor and serum binding protein: purification, cloning and expression. Nature. 1987;330:537-43. 13. Eden S, Isaksson OGP, Madsen K, Friberg U. Specific binding of growth hormone to isolated chondrocytes from rabbit ear and epiphyseal plate. Endocrinology. 1983;112:1127. 14. Barnard R, Bundesen PG, Rylatt DB, Waters MJ. Evidence from the use of monoclonal antibody probes for structural hetrogeneity of the GH receptor. Biochem J. 1985;231:459-68. 15. Barnard R, Haynes KM, Werther GA, Waters MJ. The ontogeny of growth hormone receptors in the rabbit tibia. Endocrinology. 1988;122:2562-9. 16. Asakawa K, Hedo JA, MacElduff A, Rouiller DG, Waters MJ, Gorden P. The human growth hormone receptor of cultured human lymphocytes: structural characteristics and glycosylation properties. Biochem J. 1986;238:379-86. 17. Kiernan JA. Histological and histochemical methods: theory and practice. Toronto: Pergamon Press; 1981;162. 18. McQuillan DJ, Handley CJ, Campbell MA, Bolis S, Milway VE, Herington AC. Stimulation of proteoglycan biosynthesis by serum and insulin-like growth factor-I in cultured bovine articular cartilage. Biochem J. 1986;240:423-30. 19. Bateman JF, Chan D, Mascara T, Rogers JG, Cole WG. Collagen defects in lethal perinatal osteogenesis imperfecta. Biochem J. 1986;240:699-708. 20. Herington AC. Ontogenesis of hepatic growth hormone receptors in the rat. Horm Metab Res. 1982;14:422-4. 21. Murphy LJ, Vrhosek E, Lazarus L. Identification and characterization of specific growth hormone receptors in cultured human fibroblasts. J Clin Endocrinol Metab. 1983;57:1117-1124. 22. Kiess W, Butenandt 0. Specific growth hormone receptors on human peripheral mononuclear cells: reexpression, identification, and characterization. J Clin Endocrinol Metab. 1985;60:740-6. 23. Nilsson A, Lindahl A, Eden S, Isaksson OGP. Demonstration of growth hormone receptors in cultured rat epihyseal chondrocytes by specific binding of growth hormone and immunocytochemistry. J Endocrinol. In Press. 24. Lindahl A, Nilsson A, Isaksson OGP. Effects of growth hormone and insulin-like growth factor-I on colony formation of rabbit epiphyseal chondrocytes at different stages of maturation. J Endocrinol. 1987;115:263-71. 25. Corvol M, Dumontier MF, Prevot C, Bonaventure J, de la Tour B, Willeput J. Effect of growth hormone on the differentiation of chondrocytes from prepuberal rabbits in serum-free culture and on the radioimmunologic activity of Sm-C/IGFl measured in the culture medium. Reprod Nutr Dev. 1988;28(2A):233-40. 26. Ernst M, Froesch ER. Growth hormone dependent stimulation of osteoblast-like cells in serum-free cultures via local synthesis of insulin-like growth factor I. Biochem Biophys Res Commun. 1988;26:142-7. 27. Ernst M, Froesch ER. Growth hormone dependent stimulation of osteoblast-like cells in serum-free cultures via local synthesis of insulin-like growth factor I. Biochem Biophys Res Commun. 1988;151:142-7. 28. Golde DW. Bersch N, Li CH. Growth hormone species-specific stimulation of erythropoiesis in vitro. Science 1976;196:1112-3. 29. Merchav S, Tatarsky I, Hochberg Z. Enhancement of erythropoiesis in vitro by human growth hormone is mediated by insulin-like growth factor-I. Br J Haematol. 1988;70:267-271. 30. Trippel SB, Corvol MT, Dumontier MF, Rappaport R, Hung HH, Mankin HJ. Effect of somatomedin-C/insulin-like growth factor I and growth hormone on cultured growth plate and articular chondrocytes. Pediatr Res. 1989;25:76-82. 31. Cook JJ, Haynes KM, Werther GA. Mitogenic effects of growth hormone in cultured human fibroblasts. Evidence for action via local insulin-like growth factor I production. J Clin Invest. 81:20612. 32. Isaksson OG, Lindahl A, Nilsson A, Isgaard J. Mechanism of the stimulatory effect of growth hormone on longitudinal bone growth. Endocr Rev. 1987;8:426-38. 33. Daughaday WH, Rotwein P. Insulin-like growth factors I and II. Peptide, messenger ribonucleic acid and gene structures, serum, and tissue concentrations. Endocr Rev. 1989;10:68-91.
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