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PubMed2000

Expression of Indian hedgehog, parathyroid hormone-related protein, and their receptors in the postnatal growth plate of the rat: evidence for a locally acting growth restraining feedback loop after birth.

A locally acting growth restraining feedback loop has been identified in the murine embryonic growth plate in which the level of parathyroid hormone-related peptide (PTHrP) expression regulates the pace of chondrocyte differentiation. To date, it is largely unknown whether this feedback loop also regulates the pace of chondrocyte differentiation in the growth plate after birth. We therefore characterized the spatio-temporal expression of Indian hedgehog (IHH), PTHrP, and their receptors in the postnatal growth plate from female and male rats of 1, 4, 7, and 12 weeks of age. These stages are representative for early life and puberty in rats. Using semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) on growth plate tissue, IHH and components of its receptor complex, patched (PTC) and smoothened (SMO), PTHrP and the type I PTH/PTHrP receptor messenger RNA (mRNA) were shown at all ages studied irrespective of gender. Using in situ hybridization, IHH, PTHrP, and PTH/PTHrP receptor mRNA were detected in prehypertrophic and hypertrophic chondrocytes in both sexes during development. In addition, especially in the younger age groups, faint expression of PTH/PTHrP receptor mRNA also was shown in stem cells and proliferative chondrocytes. Immunohistochemistry confirmed the observations made with in situ hybridization, by showing the presence of IHH, PTC, PTHrP, and PTH/PTHrP receptor protein in prehypertrophic and hypertrophic chondrocytes. In addition, staining for hedgehog, PTC, and PTHrP also was observed in growth plate stem cells. No differences in staining patterns were observed between the sexes. Furthermore, no mRNA or protein expression of the mentioned factors was detected in the perichondrium. Our data suggest that in contrast to the proposed feedback loop in the early embryonic growth plate, which requires the presence of the perichondrium, a feedback loop in the postnatal growth plate can be confined to the growth plate itself. In fact, two loops might exist: (1) a loop confined to the transition zone and early hypertrophic chondrocytes, which might in part be autocrine and (2) a loop involving the growth plate stem cells.

Animals↗
PubMed1998

Changes in the expression of Fas, osteonectin and osteocalcin with age in the rabbit growth plate.

Chondrocytes of the growth plate are generally assumed to undergo apoptosis, but the mechanisms which induce this cell death are not known. The Fas receptor is a mediator of the apoptotic signal in some systems. We studied its expression in situ in growth plates of rabbits aged from five to 20 weeks. In addition, we investigated the immunolocalisation in the growth plates of the bone proteins, osteonectin and osteocalcin, and the changes in their expression with age. The Fas-positive chondrocytes were found mostly in the hypertrophic zone, as were the osteonectin-positive and osteocalcin-positive cells. The percentage of Fas-positive cells increased with age whereas little change was found in the number of osteonectin-positive and osteocalcin-positive chondrocytes. Many of the Fas-positive chondrocytes were also TUNEL-positive. This strongly suggests that apoptosis in the growth plate is mediated through the Fas system. Double immunostaining for osteocalcin and Fas showed that not all hypertrophic chondrocytes were of the same cell type. Some chondrocytes stained for osteocalcin only, others for Fas only, while some were positive for both.

Aging↗
PubMed2000

Stability of alternatively spliced IGF-I mRNA in growth plate chondrocytes.

Insulin-like growth factor-I (IGF-I) enhances the proliferation and hypertrophy of growth plate chondrocytes leading to increased growth plate width thus promoting bone elongation. Differing quantities of the multiple IGF-I transcripts within the growth plate suggest differential regulation of IGF-I. To assess this, the relative stabilities of the 1Ea, 1Eb, and 2Ea IGF-I mRNA classes in 2-6 week old rat growth plates were evaluated. The mean estimated half-life of class 1Ea was 3.7+/-0.05 h, while classes 1Eb and 2Ea decayed gradually over the course of the experiment. Estimated half-lives for each IGF-I mRNA species were unchanged at all ages examined. Incubation with Act D enhanced the transcription of class 1Ea, 1Eb, and 2Ea mRNAs to varying degrees. This implies that the differential stability of alternative IGF-I mRNA classes may be an inherent regulatory component that is not influenced by an animal's developmental state, and the differential abundance of alternative IGF-I mRNA species in the growth plate throughout development is due to transcriptional differences.

Alternative Splicing↗
PubMed2003

Distinct expression of APO-1/Fas and caspase-8 in the human growth plate.

Chondrocytes in the growth plate undergo a characteristic sequel of differentiation processes, finally leading in their death by apoptosis. The mechanisms that mediate these remarkable biological phenomenon are still widely unknown. For this study, growth plates from five resected polydactylic infantile fingers were taken and immunostained for APO-1 and Caspase-8, two apoptosis-mediating proteins. The expression of both proteins was examined in the resting zone, proliferating zone, and hypertrophic zone, representing the maturation stages of the growth plate. Positive cells for APO-1 and Caspase-8 were found in all zones of the physis with a significant increase from the resting to the hypertrophic zone (from 17.4% to 33.067% for APO-1 and from 13.17% to 28.22% for Caspase-8). Our findings suggest that both APO-1 and Caspase-8 are involved in chondrocyte development in the growth plate, predominantly near to the closure area.

Apoptosis↗
PubMed2003

Short-term glucocorticoid treatment of prepubertal mice decreases growth and IGF-I expression in the growth plate.

The insulin-like growth factor (IGF) system is an important mediator of postnatal longitudinal growth, and the growth inhibiting effects of glucocorticoid (GC) treatment are suggested to be due to impaired action of the IGF system. However, the precise changes of the IGFs and the IGF-binding proteins (IGFBPs) in the growth plate, occurring upon short-term GC treatment have not been characterized. Prepubertal mice treated daily with dexamethasone (DXM) for 7 days, showed significant growth inhibition of total body length and weight and weight of the liver, thymus and spleen, whereas the weight of the kidneys was not affected. Analysis of the tibial growth plate showed that the total growth plate width significantly decreased to 84.5% of control values, caused by a significant decrease in the proliferative zone. The number of proliferating cell nuclear antigen (PCNA)-positive chondrocytes in the proliferative zone decreased significantly (to 40%) and TUNEL staining showed a significant 1.6-fold increase in apoptotic hypertrophic chondrocytes. In the growth plates, both IGF-I and IGF-II, as well as IGFBP-2 mRNAs were detected, mainly in the proliferative and prehypertrophic zones. DXM treatment significantly decreased the number of chondrocytes expressing IGF-I, whereas the number of chondrocytes expressing IGF-II and IGFBP-2 were not affected. The decrease in IGF-I expression in the growth plate indicates that GC treatment affects IGF-I at the local level of the growth plate, which could contribute to the GC-induced growth retardation.

Animals↗
PubMed1993

Regulation of growth-plate chondrocytes by insulin-like growth-factor I and basic fibroblast growth factor.

A study was performed in order to investigate the possible functional roles of insulin-like growth-factor I (IGFI) and basic fibroblast growth factor (bFGF) in the regulation of mitotic and metabolic activity of growth-plate chondrocytes. Chondrocytes from the distal radial growth plates of calves and the costal physeal cartilage of rats were exposed to these factors, individually and in combination, in primary monolayer culture, to assess their effects. The data showed that bFGF had both a greater potency and a greater efficacy as a mitogen for bovine growth-plate chondrocytes than did IGF-I. The maximum incorporation of 3H-thymidine by bFGF was 8.3 times that in serum-free (control) cultures; the maximum stimulation of incorporation by IGF-I was 2.5 times that in the control medium. In contrast, IGF-I stimulated a maximum incorporation of 35S-sulfate into glycosaminoglycans that was 2.6 times that in the IGF-I serum-free control cultures, while bFGF had no effect or was mildly inhibitory. When used together, these two factors acted synergistically. Incorporation of 3H-thymidine was more than two times greater than the sum of the effects of the growth factors when used alone and 20.5 times greater than that of the growth factor-free control cultures. Physeal chondrocytes from six-day-old rats were mitotically more responsive to bFGF than to IGF-I, but they were more responsive to IGF-I when they had been derived from twenty-eight-day-old rats. Interaction between bFGF and factors in the serum enhanced the mitotic activity of the rat chondrocytes, but bFGF did not interact with IGF-I under the same experimental conditions. In the presence of bFGF, there was a reduction in the stimulation by IGF-I of incorporation of 35S-sulfate and a decrease in the percentage of chondrocytes containing alkaline phosphatase. These growth factors also influenced cellular morphology in culture. In the presence of IGF-I or serum, the rat chondrocytes manifested the polygonal morphology typical of chondrocytes in culture, while bFGF promoted a more elongated spindle shape. Removal of bFGF and replacement by IGF-I restored the polygonal morphology, indicating that this transition is reversible.

Alkaline Phosphatase↗
PubMed1989

Effect of aging on tensile mechanical properties of the rabbit distal femoral growth plate.

Tensile tests to failure were performed on the distal femoral growth plate of rabbits. The measured tensile forces, together with animal weight and dimensional measurements of the femur, were used to develop a noninvasive method of predicting growth failure loads and to examine the effects of age on the tensile strength of the growth plate. Growth plate failure load correlated with rabbit weight, (r = 0.93), femoral length, (r = 0.91), and contoured growth plate surface area, (r = 0.74). Regression analysis for these relationships allowed for failure load prediction if animal weight, femoral length, or growth plate surface area was known. Nominal stress (failure load/surface area) was calculated and correlated with animal weight (age) for each distal femoral growth plate utilizing two surface area estimating techniques: growth plate surface contour and growth plate cross-sectional enlargement. A strong association was found with either technique. The growth and maturation of growth plate cartilage is of major importance in pediatric orthopaedics. This study demonstrate that the age-dependent increases in tensile mechanical properties of the growth plate do not result merely from growth plate enlrgement or conformational changes. The changing tensile mechanical properties of the growth plate may explain why transphyseal distraction used clinically has produced varied and somewhat unpredictable results. Our study suggests that dose-response investigations are needed to define safe and optimal transphyseal distraction tensions before further clinical application is pursued.

Aging↗
PubMed2004

Action of estradiol on epiphyseal growth plate chondrocytes.

Estrogen plays an important role in the human growth plate by accelerating growth and promoting epiphyseal fusion in both sexes. Nevertheless, the precise mechanisms responsible for these effects are poorly understood. In the present study, we examined the role of 17beta-estradiol (E2) on cell proliferation and viability, type X collagen synthesis, alkaline phosphatase activity, and matrix calcification in primary cultures of resting, proliferating, and prehypertrophic chondrocytes derived from explants of the bovine fetal epiphyseal growth plate. Growth plate chondrocytes were isolated and separated into maturationally distinct subpopulations, which were cultured for 7-21 days to high density in either (1) serum-free medium, (2) 1 nM thyroid hormone (T3), (3) E2 concentrations ranging from 10(-13) M to 10(-7) M, or (4) a combination of T3 and E2. To compare E2 effects in both sexes, chondrocytes were harvested from 8 fetuses of both sexes. After hormone treatment, cell cultures were analyzed for cell number and viability, collagen type X, alkaline phosphatase (ALP), and matrix calcification. Neither DNA content nor cell viability were affected by the duration or type of hormone treatment. By itself, E2 stimulated maturation of all subpopulations only in pharmacologic doses (10(-7) M). Physiologic E2 concentrations were no different than negative controls treated with ITS (insulin, transferrin, and selenite). Regardless of E2 concentrations, the addition of E2 to 1 nM T3 did not appreciably affect the response to T3 alone, which stimulates maturation of the phenotype. All effects were comparable in both male and female chondrocytes, in all cell subpopulations (maturation stages) and fetuses of varying gestational age. These findings indicate that at physiologic concentrations, the effects of E2 on fetal bovine growth plate chondrocyte appear to be indirect and independent of T3, suggesting that, in vivo, E2 acts in concert with other factors or hormones to induce fusion of the growth plate.

Alkaline Phosphatase↗
PubMed2000

Thyroid hormone acts directly on growth plate chondrocytes to promote hypertrophic differentiation and inhibit clonal expansion and cell proliferation.

T3 is an important regulator of endochondral bone formation in epiphyseal growth plates. Growth arrest in juvenile hypothyroidism results from disorganization of growth plate chondrocytes and their failure to undergo hypertrophic differentiation, but it is unclear how T3 acts directly on chondrocytes or whether its actions involve other pathways. To address this issue, we investigated whether thyroid hormone receptors (TR) were localized to discrete regions of the unfused epiphysis by immunohistochemistry performed in tibial growth plates from 21-day-old rats and examined the effects of T3 on growth plate chondrocytes in agarose suspension cultures in vitro. TRalpha1, -alpha2, and -beta1 were expressed in reserve and proliferating zone chondrocytes, but not in hypertrophic cells, suggesting that progenitor cells and immature chondrocytes are the major T3 target cells in the growth plate. Chondrocytes in suspension culture expressed TRalpha1, -alpha2, and -beta1 messenger RNAs and matured by an ordered process of clonal expansion, colony formation, and terminal hypertrophic differentiation. Clonal expansion and proliferation of chondrocytes were inhibited by T3, which also induced alkaline phosphatase activity, expression of collagen X messenger RNA, and secretion of an alcian blue-positive matrix as early as 7 days after hormone stimulation. Thus, T3 inhibited chondrocyte clonal expansion and cell proliferation while simultaneously promoting hypertrophic chondrocyte differentiation. These data indicate that thyroid hormones concurrently and reciprocally regulate chondrocyte cell growth and differentiation in the endochondral growth plate.

Animals↗
PubMed1994

Autocrine, paracrine, and hormonal signals involved in growth plate chondrocyte differentiation.

Longitudinal bone growth depends upon intricate steps in the metabolism and differentiation of epiphyseal growth plate chondrocytes. These steps involve cell proliferation followed by the synthesis of gene products that are characteristic of the subsequent stages of differentiation. Until recently, the major control of growth plate metabolism was thought to reside with systemic hormones, principally growth hormone. However, it is now apparent that locally produced peptide growth factors play important autocrine and paracrine roles in normal growth plate physiology. Investigations with cultured chondrocytes have demonstrated the importance of the peptide growth factors insulin-like growth factor-I, transforming growth factor-beta, and basic fibroblast growth factor for cell proliferation, extracellular matrix biosynthesis, and changes in morphology associated with the progressive stages of chondrocyte differentiation. All three of these factors have been shown to be present in specific zones of the epiphyseal growth plate. A preliminary map of the epiphyseal growth plate is proposed, which delineates the site and mode of action of systemic and locally produced factors involved in longitudinal bone growth.

Animals↗
PubMed1997

Interaction of epidermal growth factor and insulin-like growth factor-I in the regulation of growth plate chondrocytes.

The action of growth factors on the cells of the epiphyseal growth plate is an important mechanism in the regulation of skeletal growth. Insulin-like growth factor-I (IGF-I) is known to play a central role in the regulation of bone growth. In contrast, the role, if any, of epidermal growth factor (EGF) is not yet clear. In these studies, we tested the hypothesis that EGF interacts with IGF-I in the regulation of growth plate chondrocyte mitotic and metabolic activities. Chondrocytes isolated from bovine radioulnar growth plates and incubated in suspension culture were analyzed for their responsiveness to EGF with respect to synthesis of DNA, proteins, and proteoglycans, responsiveness to IGF-I, and ability to specifically bind [125I]IGF-I. Treatment of growth plate chondrocytes with maximally effective concentrations (10-100 ng/ml) of EGF produced a 16-27% increase in specific binding of [125I]IGF-I. Scatchard analysis indicated that this increase in specific binding was due to an increase in the number of receptors/cell with no change in receptor affinity. EGF stimulated protein synthesis by 30-35%. Pretreatment with EGF increased the responsiveness of chondrocytes to IGF-I, resulting in 90 and 60% augmentation of IGF-I-stimulated mitotic activity and proteoglycan synthesis, respectively. Given the prominent role of IGF-I in skeletal development and the presence of EGF in the growth plate, this study suggests an important role for interactions between these growth factors in the regulation of skeletal growth.

Animals↗
PubMed1993

Quantification and immunolocalisation of porcine articular and growth plate cartilage collagens.

The collagens of growth plate and articular cartilage from 5-6 month old commercial pigs were characterised. Growth plate cartilage was found to contain less total collagen than articular cartilage as a proportion of the dry weight. Collagen types I, II, VI, IX and XI are present in both growth plate and articular cartilage whereas type X is found exclusively in growth plate cartilage. Types III and V collagen could not be detected in either cartilage. Type I collagen makes up at least 10% of the collagenous component of both cartilages. There are significant differences in the ratios of the quantifiable collagen types between growth plate and articular cartilage. Collagen types I, II, and XI were less readily extracted from growth plate than from articular cartilage following pepsin treatment, although growth plate cartilage contains less of the mature collagen cross-links, hydroxylysyl-pyridinoline and lysyl-pyridinoline. Both cartilages contain significant amounts of the divalent reducible collagen cross-links, hydroxylysyl-ketonorleucine and dehydro-hydroxylysinonorleucine. Immunofluorescent localisation indicated that type I collagen is located predominantly at the surface of articular cartilage but is distributed throughout the matrix in growth plate. Types II and XI are located in the matrix of both cartilages whereas type IX is predominantly pericellular in the calcifying region of articular cartilage and the hypertrophic region of the growth plate. Collagen type VI is located primarily as a diffuse area at the articular surface.

Animals↗
PubMed1995

Transforming growth factor-beta 1 and fibroblast growth factors in rat growth plate.

Chondrocytes in the growth plate progress in an orderly fashion from resting through proliferating to hypertrophic cells. In the region of hypertrophic chondrocytes, the cartilage is invaded by capillary loops and endochondral ossification is initiated. It is currently believed that growth factors may regulate the proliferation and maturation of chondrocytes and the synthesis of extracellular matrix in the growth plate. The ordered sequence of proliferation and differentiation observed in the growth plate provides a unique opportunity to study the role of acidic fibroblast growth factor, basic fibroblast growth factor, and transforming growth factor-beta 1 in the regulation of these processes. In this study, expression of the mRNA of these growth factors was examined using total RNA extracted from the physis and epiphysis of rat tibias. Transforming growth factor-beta 1 mRNA was detected by Northern hybridization. Expression of the genes encoding acidic and basic fibroblast growth factors was demonstrated by polymerase chain reaction amplification. In addition, using polyclonal antibodies against these growth factors, we localized them by immunohistochemical analysis. Strong intracellular staining with a predominantly nuclear pattern was observed in chondrocytes from the proliferating and upper hypertrophic zones. In contrast, chondrocytes in the resting zone stained only faintly for the presence of these growth factors. Some chondrocytes in the resting zone adjacent to the proliferating zone stained with these antibodies, and the antibodies also stained cells in the zone of Ranvier, which regulates latitudinal bone growth. Lastly, the location of transforming growth factor-beta 1 was examined further with use of a polyclonal antipeptide antibody specific for its extracellular epitope.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Vascular invasion of the epiphyseal growth plate: analysis of metaphyseal capillary ultrastructure and growth dynamics.

Metaphyseal blood vessels which invade the calcifying epiphyseal growth plate were examined by a variety of techniques to determine their morphology, cell division, and growth patterns as they relate to endochondral ossification. Four regions of these vessels were characterized: 1) sprout tips--the terminal ends of the capillary sprouts which impinge upon the hypertrophic chondrocytes of the growth plate; 2) region of extended calcified cartilage--those deeper vessels within the metaphysis which are surrounded by an extracellular matrix predominantly composed of extended septa of calcified cartilage; 3) region of bone deposition--further still from the epiphysis these microvessels are contained within a network of active bone deposition laid upon a scaffold of calcified cartilage; 4) region of primary vessels--at a distance of 350-500 microns from the epiphysis are dilated vessels with one or two layers of smooth muscle in their walls, which supply and drain the metaphyseal capillary plexus. The sprout tips are continuous blind-ended vessels lined with an attenuated endothelium with no underlying basement membrane. Dividing endothelial cells are most frequently found in the region of bone deposition 175-200 microns behind the apices of the growing sprout tips. A time-coursed, autoradiographic examination of cytokinesis revealed radio-labelled endothelial cells to appear at the epiphysis after a 24 hr period. The metaphyseal capillary sprouts represent a continuous, unidirectional angiogenic vascular network which grows by elongation from the region of bone deposition; this region remains a fixed distance behind the sprout tips. These findings are discussed in light of the growth dynamics between this vascular plexus and the epiphyseal growth plate.

Animals↗
PubMed2001

Testosterone stimulates growth of tibial epiphyseal growth plate and insulin-like growth factor-1 receptor abundance in hypophysectomized and castrated rats.

Puberty is associated with an increase in the plasma concentration of sex steroids, growth hormone (GH), and insulin-like growth factor-1 (IGF-1). Gonadal steroid hormones are important for the normal pubertal growth spurt and skeletal growth. The mechanism by which gonadal steroids induces skeletal growth is still not fully understood. To study the GH-independent effect of testosterone on growth, we investigated the effect of testosterone injections on the tibial epiphyseal growth plate (EGP) in an in vivo model of hypophysectomized and castrated male rats. Four groups (six animals each) of 28-d-old male rats were studied. Groups A, B, and C were hypophysectomized and castrated and received 500 microg/(kg x d) of hydrocortisone and 15 microg/(kg x d) of levothyroxine sodium. Groups A and B were also treated with daily sc injections of 10 microg of testosterone/100 g of body wt and 100 microg of testosterone/100 g of body wt, respectively, for 7 d. Group C was injected with vehicle alone. Group D were intact animals injected with saline (controls). Animals were sacrificed on 8 d. As expected, serum GH levels were found to be very low (1.13+/-0.1 ng/mL) in the hypophysectomized animals (group C, hypopit), and testosterone treatment did not change them significantly. Serum IGF-1 decreased from 502.9+/-13 ng/mL in group D to 167+/-41.4 ng/mL in group C (p < 0.001). Testosterone therapy had no stimulatory effect on serum IGF-1 levels in the hypopit + low-dose group (A) (220+/-94.8 ng/mL) and had an inhibitory effect in the hypopit + high-dose group (B) (39.3+/-17.5). Histomorphometric determinations demonstrated an EGP width of 472.3+/-39 microm in the intact animals but only 336.9+/-1.6 microm in the hypopit group (C) (p < 0.01). High-dose testosterone treatment (group B) significantly increased the EGP width (to 438.8+/-27.8), (p < 0.001), whereas low-dose testosterone (group A) did not. Immunohistochemistry studies revealed that the levels of IGF-1 in the EGP of the control animals were almost negligible and that testosterone did not change them. However, testosterone increased in a dose-dependent manner the abundance of IGF-1 receptor EGP. We conclude that testosterone has a direct, local, GH-independent effect on the EGP growth and IGF-1 receptor abundance.

Animals↗
PubMed1994

Dexamethasone increases growth hormone receptor messenger ribonucleic acid levels in liver and growth plate.

Glucocorticoid inhibits linear growth and renders target tissues, particularly liver and growth plate, insensitive to GH. We hypothesized that glucocorticoid-induced GH insensitivity is due to decreased gene expression of the GH receptor at the messenger RNA (mRNA) level. To test this hypothesis, we treated 4.5-wk-old male rabbits (n = 6-9 per group) with ip dexamethasone or vehicle and measured GH receptor mRNA levels (by RNase protection assay) and serum GH-binding protein levels (by radioimmunoprecipitation assay). Contrary to our hypothesis, dexamethasone administered in growth-suppressing doses did not decrease GH receptor mRNA levels in liver or growth plate. Instead a tissue-specific stimulation of GH receptor mRNA levels was observed. The dose-response relationship of this effect was biphasic, since the lower growth-suppressing dose of dexamethasone (0.1 mg/kg.day) caused the greater increase in GH receptor mRNA levels, whereas the higher growth-suppressing dose (4 mg/kg.day) had less effect. The dexamethasone-induced increase in GH receptor mRNA was observed in growth plate and liver, target tissues important for linear growth, but not in kidney. Serum GH-binding protein levels also showed a stimulatory response to dexamethasone treatment, with a biphasic dose-response relationship. These data suggest that glucocorticoid-induced GH insensitivity cannot be explained by decreased GH receptor mRNA levels. To the contrary, dexamethasone causes a tissue-specific stimulation in GH receptor mRNA levels with a biphasic dose-response relationship.

Animals↗
PubMed2002

Glucocorticoids inhibit vascular endothelial growth factor expression in growth plate chondrocytes.

Vascular endothelial growth factor (VEGF) plays an essential role in angiogenesis in the growth plate and ultimately in regulating endochondral ossification. Since longitudinal bone growth is often disturbed in children who are treated with glucocorticoids, we investigated the effects of dexamethasone on VEGF expression by epiphyseal chondrocytes. Cells were cultured from tibial growth plates of neonatal piglets. Using Northern blotting and RT-PCR techniques, the chondrocyte-specific markers aggrecan, collagen II and CD-RAP were detected. Also the glucocorticoid receptor (GR) was expressed. VEGF protein secreted from these cells was examined by ELISA and Western immunoblotting. The VEGF(121) and VEGF(165) isoforms were detected in the supernatant. As determined by RT-PCR, all three major mRNA splice variants were produced, including the species encoding VEGF(189). Dexamethasone (100 nM) inhibited both protein and mRNA expression by approximately 45%. Hydrocortisone (cortisol) and prednisolone also inhibited VEGF secretion, but they were less active than dexamethasone. The inhibitory actions of dexamethasone were almost completely blocked by the GR antagonist Org34116, indicating that the GR mediates these actions. Degradation of the VEGF mRNA was not accelerated by dexamethasone. Therefore, a transcriptional mechanism seems likely. Downregulation of this important growth factor could lead to disruption of the normal invasion of blood vessels in the growth plate, which could contribute to disturbed endochondral ossification and growth.

Animals↗
PubMed1996

Hepatocyte growth factor and its actions in growth plate chondrocytes.

Hepatocyte growth factor (HGF) has been implicated as a paracrine regulator of organogenesis and repair in many tissues. Here we have studied the expression and actions of HGF in intact rachitic rat growth plate and derived cultures of proliferative zone chondrocytes. In vivo and in vitro chondrocytes express HGF mRNA; 1,25(OH)2 has a three-fold maximal stimulatory effect, which can be blocked by H-7, an inhibitor of protein kinase C. Although HGF elaboration and action generally follow a paracrine model, chondrocytes appear capable of both expressing and responding to HGF. mRNA encoding the HGF receptor (c-met) was detected in both growth cartilage and derived chondrocyte cultures. HGF addition to chondrocyte cultures increased collagen II mRNA and alkaline phosphatase enzymatic activity to degrees comparable to that observed for active vitamin D metabolites. Combining HGF and 1,25-D evoked a synergistic response (ninefold) of alkaline phosphatase activity. To assess whether a similar stimulatory effect might be seen with bioactive peptides and HGF, we investigated the effect of HGF pretreatment on acute responses of chondrocytes to synthetic human calcitonin, an anabolic chondrocyte regulator whose skeletal action are mediated principally by cAMP elevation and subsequent protein kinase A activation. CT's maximal activation of protein kinase A was increased by prior HGF treatment from 56% to 78%. In concert, our findings indicate that in addition to HGF's classical paracrine role during skeletal growth, this growth factor may modulate hormonal sensitivity of the chondrocyte during proliferation, differentiation, and/or apoptosis.

Alkaline Phosphatase↗