Pediatr Nephrol(1991) 5:451-453 9 IPNA1991
Growth Review article
Regulation of cartilage growth by growth hormone and insulin-like growth factor I Olle G. P. Isaksson, Claes Ohlsson, Anders Nilsson, J6rgen Isgaard, and Anders Lindahl Departmentof Physiology,Universityof Gothenburg,S-40033 Gothenburg,Sweden ReceivedOctober 18 1990
Abstract. A number of studies have shown that growth hormone (GH) and insulin-like growth factor-I (IGF-I) have important regulatory roles for skeletal growth. However, it has been a matter of controversy whether GH acts directly on cells in the growth plate or if the growth-promoting effects of GH are mediated by liver-derived (endocrine-acting) IGF-I. With the recognition that GH regulates the production of IGF-I in multiple extra-hepatic tissues, autocrine and paracrine functions of IGF-I have been suggested as important components of GH action. This review focuses on recent developments in our understanding of the cellular mechanisms by which GH promotes longitudinal bone growth and the inter-relationship between GH and IGF-I in the growth plate. Key words: Growth hormone - Insulin-like growth factor-I - Epiphyseal growth plate - Chondrocytes
Growth hormone effects on cartilage growth Longitudinal bone growth is the result of recruitment of new cells from the stem cell layer and the subsequent proliferation of the differentiating cells in the growth plate of the long bones. In growing individuals the rate of multiplication of cartilage cells is in balance with the rate of calcification of cells at the diaphyseal end of the plate. Therefore the width of the growth plate is approximately the same although the accumulated length of the bone increases. As new cells start their programme of differentiation, and undergo a limited clonal expansion, the cells in the growth plate are continuously renewed until epiphyseal closure at the time of sexual maturation. Of the endocrine factors regulating skeletal growth, growth hormone (GH) is the only recognized hormone that stimulates longitudinal bone growth in a dose-dependent manner over a wide range of doses. This fact forms the
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basis for the in vivo assay known as the tibia test in which the width of the tibial growth plate is determined by a histomorphometric technique. Difficulties in demonstrating in vitro effects of GH on various growth-related parameters, such as thymidine incorporation and sulphate uptake in costal cartilage of hypophysectomized rats in combination with the finding that addition of serum from normal but not hypophysectomized rats stimulated these parameters, formed the basis for the somatomedin hypothesis of GH action . According to this theory, GH stimulated growth indirectly by stimulating the production of serum factors which mediated the growth-promoting effects of GH. The "somatomedin" hypothesis was further strengthened by the identification of insulin-like growth factor-I (IGF-I) as the principal serum somatomedin and the observation that GH stimulated the production and release of this peptide from the liver. Several recent investigations have shown that local administration of GH at the site of the epiphyseal growth plate of hypophysectomized rats stimulates unilateral bone growth. It has also been demonstrated that infnsion of GH into one of the femoral arteries of hypophysectomized rats stimulates longitudial bone growth of the treated leg, giving strong evidence for the hypothesis that GH stimulates the differentiation/proliferation of some - as yet unidentified - epiphyseal chondrocytes . Schlechter et al.  made the important observation that the stimulatory effect of locally administered GH on longitudinal bone growth was completely abolished if antiserum to IGF-I was co-infused with the hormone, suggesting that the stimulatory effects of GH on longitudinal bone growth was dependent on the presence of "endocrine" or locally produced (antocrine/paracrine action) IGF-I. Immunohistochemistry using monoclonal antibodies against the GH receptor have revealed receptors in the germinative, proliferative and hypertrophic zones of sagittal sections of the proximal rabbit tibia growth plate, providing a structural basis for a direct interaction between GH and epiphyseal chondrocytes at various maturational stages . GH receptors have also been visualized in cultured rabbit and rat epiphyseal chondrocytes [5, 6]. Recently it has been demonstrated that epiphyseal chondro-
Fig. 1 A - C. Photomicrographs of in situ hybridizations of insulin-like growth factor 1 (IGF-I) mRNA in sagittal sections from epiphyseal growth plate using 35S-labelled antisense RNA. A A 35-day-old agematched normal rat; B Hypophysectomized rat; C growth hormone
(GH)-treated (200 gg human GH administered subcutaneously every 4 h for 24 h) hypophysectomized rat (Magnification • 105). Reproduced from [ 11] with permission.
@H Germinal cell
Proliferative cell layer
G) ;13 0 -q I
Hypertrophic cell layer
I. Development of IGF-I responsiveness 2. Expression of IGF-I gene I. Local production of IGF-I 2. Stimulation of clona[ expansion by autocrine/ /paracrine mechanisms
cytes in monolayer culture express GH receptor mRNA and that this expression is in part regulated by GH  providing evidence for a direct interaction between GH and epiphyseal condrocytes.
Local production of IGF-I in the growth plate and its regulation by GH Some years ago, D'Ercole et al.  demonstrated IGF-I-tike peptides in a number of different tissues, and that administration of GH to hypophysectomized rats increased the level of IGF-I in several tissues. A GH-dependent IGF-I immunoreactivity in the growth plate of hypophysectomized
Fig. 2. Hypothetical model for the stimulatory effect of GH on longitudinal bone growth. Reproduced with permission from 
rats has been demonstrated suggesting that GH stimulates the local production of IGF-I-like peptides in the epiphyseal growth plate . This hypothesis has received further support from studies showing that GH increases steady-state levels of IGF-I mRNA in the growth plate, suggesting that GH stimulates the transcription of the gene coding for IGF-I . Subsequent in situ~hybridization studies have demonstrated expression of IGF-I mRNA in proliferative and hypertrophic condrocytes of rat tibial growth plates (Fig. 1). Hypophysectomy reduced the hybridization signal and the number of cells containing significant amounts of signal activity. By GH replacement therapy the relative hybridization signal and the hybridization pattern were restored ,
453 confirming earlier results f r o m our laboratory using a solution hybridization assay [ 10]. There are several studies showing that a pulsatile pattern o f plasma G H promotes longitudinal bone growth more effectively than a constant plasma level. In recent studies it was found that a pulsatile pattern o f plasma G H was more effective than a constant level o f G H in increasing I G F - I m R N A in the growth plate o f h y p o p h y s e c tomized male rats. In contrast, levels o f IGF-I m R N A in the liver did not differ significantly by the two different modes o f G H administration, suggesting differential tissue effects on the transcription of the IGF-I gene in response to the plasma pattern of G H .
In vitro effects of GH and IGF-I on cartilage and isolated ehondroeytes M a o r et al.  have recently shown that G H stimulates chondrogenesis as well as osteogenesis in an organ culture system o f n e w b o r n murine mandibular condyles. These investigators observed that GH-treated explants were about 4 times larger in cell mass than controls after 6 days o f culture, providing strong evidence for an in vitro effect on cartilage growth. By optimizing the culture conditions it has recently been possible to demonstrate consistent stimulatory effects of G H on sulphate and thymidine incorporation o f epiphyseal chondrocytes in monolayer. It has been found that seeding the cells at a low density and maintaining the cells in culture for approximately 2 weeks in order to obtain confluent cells resulted in an increased number o f G H receptors . Concomitant to the development of G H receptors the cells b e c a m e responsive to the stimulatory effect o f G H in terms o f sulphate and thymidine incorporation, suggesting that the expression of G H receptors partially determines G H responsiveness [ 18]. The results of earlier studies using epiphyseal chondrocytes in suspension culture showed that both G H and IGF-I stimulated the formation o f chondrocyte colonies. By counting the number o f colonies and determining the size distribution o f colonies 2 - 3 weeks after start o f culture, consistent differencies between cultures containing G H or IGF-I became apparent. Thus, G H selectively promoted the formation o f large chondrocyte clusters, whereas I G F - I preferentially stimulated the formation of middle-sized and small colonies, suggesting that G H and I G F - I interact with different progenitor ceils. F r o m these studies it appears that G H interacts with pre-chondrocytes or y o u n g differentiating cells that have a high inherent capacity to divide, but IGF-I stimulates older differentiated cells that have a restricted capacity to divide [14, 15].
Unifying hypothesis - dual regulation of cartilage growth by GH and IGF-I Although the precise cellular mechanism(s) for the interaction between G H and IGF-I in epiphyseal cartilage remains to be elucidated, the results of a number o f studies, as mentioned above are in line with the proposed hypothesis o f G H action on tissue growth [16, 17]. According to this hypothesis, longitudinal bone growth is the result of an increased rate o f recruitment of chondrocyte stem cells directly stimulated by G H - which is followed by a limited
clonal expansion o f differentiating chondrocytes, promoted by the local production o f IGF-I (Fig. 2).
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