Cultured Growth Plate Research Articles

Autogenous cultured growth plate chondrocyte transplantation in the treatment of physeal injury in rabbits.

ObjectivesThe aim of this experimental study on New Zealand’s white rabbits was to investigate the transplantation of autogenous growth plate cells in order to treat the injured growth plate. They were assessed in terms of measurements of radiological tibial varus and histological characteristics. MethodsAn experimental model of plate growth medial partial resection of the tibia in 14 New Zealand white rabbits was created. During this surgical procedure the plate growth cells were collected and cultured. While the second surgery was being performed, the autologous cultured growth plate cells were grafted at the right tibia, whereas the left tibia was used as a control group. ResultsHistological examinations showed that the grafted right tibia presented the regular shape of the plate growth with hypertrophic maturation, chondrocyte columniation and endochondral calcification. Radiological study shows that the mean tibial deformity at the left angle was 20.29° (6.25 to 33) and 7.21° (5 to 10) in the right angle. ConclusionThis study has demonstrated that grafting of autogenous cultured growth plate cells into a defect of the medial aspect of the proximal tibial physis can prevent bone bridge formation, growth arrest and the development of varus deformity.Cite this article: Bone Joint Res 2014;3:310–16

Increased Expression of Fibroblast Growth Factor 21 (FGF21) during Chronic Undernutrition Causes Growth Hormone Insensitivity in Chondrocytes by Inducing Leptin Receptor Overlapping Transcript (LEPROT) and Leptin Receptor Overlapping Transcript-like 1 (LEPROTL1) Expression

During calorie restriction in mice, increased expression of FGF21 causes growth attenuation and growth hormone (GH) insensitivity. Previous evidence also indicates that fasting-associated increased expression of leptin receptor overlapping transcript (LEPROT) and LEPROT-like 1 (LEPROTL1) (two proteins that regulate intracellular protein trafficking) reduces GH receptor cell-surface expression in the liver. Thus, we hypothesized that FGF21 causes GH insensitivity through regulation of LEPROT and/or LEPROTL1 expression. After 4 weeks of food restriction, LEPROT and LEPROTL1 mRNA expression in the liver and in the tibial growth plate of wild-type (WT) mice was increased compared with WT mice fed ad libitum. In Fgf21 knock-out (KO) mice, LEPROT and LEPROTL1 mRNA expression in food-restricted and fed ad libitum was similar, with the exception of a subgroup of food-restricted Fgf21 KO mice treated with recombinant human (rh) FGF21 that experienced increased LEPROT and LEPROTL1 mRNA expression compared with untreated food-restricted Fgf21 KO mice. In cultured growth plate chondrocytes, FGF21 stimulated LEPROT and LEPROTL1 mRNA expression, with such effect being prevented in chondrocytes transfected with FGFR1 siRNA or ERK1 siRNA. In cells transfected with control siRNA, GH increased [(3)H]thymidine incorporation, collagen X, and IGF-1 mRNA expression, with all effects being prevented by rhFGF21. In addition, rhFGF21 decreased (125)I-GH binding. In LEPROT siRNA- and/or LEPROTL1 siRNA-transfected cells, rhFGF21 did not prevent the GH stimulatory effects on thymidine incorporation, collagen X, and IGF-1 expression; furthermore, rhFGF21 did not prevent (125)I-GH binding. Consistent with the effects of rhFGF21, LEPROT overexpression in chondrocytes resulted in the inhibition of GH action. Our findings indicate that the increased expression of FGF21 during chronic undernutrition inhibits GH action on chondrocytes by activating LEPROT and LEPROTL1.

Epidermal growth factor receptor signalling mediates growth hormone-induced growth of chondrocytes from sex hormone-inhibited adolescent rats

1. Growth hormone (GH) has been demonstrated to overcome the inappropriate deceleration of growth rate in children with central precocious puberty treated with gonadotropin-releasing hormone analogue (GnRHa). However, the underlying mechanisms remain largely unclear. In the present study, we investigated the potential involvement of the epidermal growth factor receptor (EGFR) pathway in the growth promotion by GH using in vitro cultured growth plate chondrocytes isolated from adolescent rats treated with GnRHa. 2. Chondrocytes were stimulated with GH in the presence or absence of the Janus tyrosine kinase (JAK) 2 inhibitor AG490 (1, 10 and 100 nmol/L), the EGFR kinase inhibitor AG1478 (0.1, 1 and 10 nmol/L), U0126 (an inhibitor of extracellular signal-regulated kinase (Erk) activation; 10 μmol/L) or a neutralizing antibody against epidermal growth factor (EGF Ab; 0.1, 1 and 10 μg/mL). The proliferation of chondrocytes was assessed by the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay and immunostaining for proliferating cell nuclear antigen (PCNA). Phosphorylation of Erk1/2 and EGFR was detected by western-blotting. Intracellular mRNA and extracellular protein levels of EGF were detected using reverse transcription-polymerase chain reaction and ELISA, respectively. 3. Growth hormone promoted the proliferation of chondrocytes, which was correlated with increased phosphorylation of Erk1/2 and EGFR and enhanced expression of EGF. Pretreatment with AG490, AG1478, U0126 or EGF Ab completely or partially inhibited the proliferation of chondrocytes and activation of Erk1/2 and EGFR. Pretreatment with AG490, AG1478, or U0126 partially inhibited the expression of EGF. 4. The findings indicate that GH promotes chondrocyte proliferation by activating EGFR signalling.

Effects of stanozolol on collagen synthesization of cultured growth plate chondrocytes from pubertal female rat treated with GnRHa in vitro

Objective To observe the effects of stanozolol on collagen I1 and X synthesization of cul- tured growth plate ehondrocytes from pubertal female rat treated with GnRHa in vitro. Methods Primary Chon-drocytes from 6 female rats were divided into two groups ( time-effect groups and concentration-effect groups). immunohistochemical staining of collagen Ⅱ, X were conducted. Results the results of type Ⅱ collagen synthe- sization demonstrated stanozolol enhanced the proliferation of the chondrocytes, time-course studies had shown that the proliferation were maximally stimulated by stanozolol after 3 days of incubation and decreased again after longer periods of incubation. Stanozolol did not stimulated type X collagen synthesization from 10-11 M-10-8 M, but experiments showed that type X collagen was already stimulated after incubation in stanozolol (10-7-10-5 M). Time-course studies had shown those type X collagen synthesizations were stimulated by stanozolol after 4-5 days of incubation. Conclusion Stanozoiol enhances the proliferation of ehondrocytes of pubertal female rat treated with GnRHa in vitro( time-course-dependent and concentration-dependent). Key words: Chondrocyte; Epiphyseal growth plate; Stanozolol; Collagen

Nuclear Factor-κB p65 Facilitates Longitudinal Bone Growth by Inducing Growth Plate Chondrocyte Proliferation and Differentiation and by Preventing Apoptosis

NF-kappaB is a group of transcription factors involved in cell proliferation, differentiation, and apoptosis. Mice deficient in the NF-kappaB subunits p50 and p52 have retarded growth, suggesting that NF-kappaB is involved in bone growth. Yet, it is not clear whether the reduced bone growth of these mice depends on the lack of NF-kappaB activity in growth plate chondrocytes. Using cultured rat metatarsal bones and isolated growth plate chondrocytes, we studied the effects of two NF-kappaB inhibitors (pyrrolidine dithiocarbamate (PDTC) or BAY11-7082 (BAY)), p65 short interference RNA (siRNA), and of the overexpression of p65 on chondrocyte proliferation, differentiation, and apoptosis. To further define the underlying mechanisms, we studied the functional interaction between NF-kappaB p65 and BMP-2 in chondrocytes. PDTC and BAY suppressed metatarsal linear growth. Such growth inhibition resulted from decreased chondrocyte proliferation and differentiation and from increased chondrocyte apoptosis. In cultured chondrocytes, the inhibition of NF-kappaB p65 activation (by PDTC and BAY) and expression (by p65 siRNA) led to the same findings observed in cultured metatarsal bones. In contrast, overexpression of p65 in cultured chondrocytes induced chondrocyte proliferation and differentiation and prevented apoptosis. Although PDTC, BAY, and p65 siRNA reduced the expression of BMP-2 in cultured growth plate chondrocytes, the overexpression of p65 increased it. The addition of Noggin, a BMP-2 antagonist, neutralized the stimulatory effects of p65 on chondrocyte proliferation and differentiation, as well as its anti-apoptotic effect. In conclusion, our findings indicate that NF-kappaB p65 expressed in growth plate chondrocytes facilitates growth plate chondrogenesis and longitudinal bone growth by inducing BMP-2 expression and activity.

Cyclooxygenases and prostaglandin E2 receptors in growth plate chondrocytes in vitro and in situ – prostaglandin E2 dependent proliferation of growth plate chondrocytes

Prostaglandin E2 (PGE2) plays an important role in bone development and metabolism. To interfere therapeutically in the PGE2 pathway, however, knowledge about the involved enzymes (cyclooxygenases) and receptors (PGE2 receptors) is essential. We therefore examined the production of PGE2 in cultured growth plate chondrocytes in vitro and the effects of exogenously added PGE2 on cell proliferation. Furthermore, we analysed the expression and spatial distribution of cyclooxygenase (COX)-1 and COX-2 and PGE2 receptor types EP1, EP2, EP3 and EP4 in the growth plate in situ and in vitro. PGE2 synthesis was determined by mass spectrometry, cell proliferation by DNA [3H]-thymidine incorporation, mRNA expression of cyclooxygenases and EP receptors by RT-PCR on cultured cells and in homogenized growth plates. To determine cellular expression, frozen sections of rat tibial growth plate and primary chondrocyte cultures were stained using immunohistochemistry with polyclonal antibodies directed towards COX-1, COX-2, EP1, EP2, EP3, and EP4. Cultured growth plate chondrocytes transiently secreted PGE2 into the culture medium. Although both enzymes were expressed in chondrocytes in vitro and in vivo, it appears that mainly COX-2 contributed to PGE2-dependent proliferation. Exogenously added PGE2 stimulated DNA synthesis in a dose-dependent fashion and gave a bell-shaped curve with a maximum at 10-8 M. The EP1/EP3 specific agonist sulprostone and the EP1-selective agonist ONO-D1-004 increased DNA synthesis. The effect of PGE2 was suppressed by ONO-8711. The expression of EP1, EP2, EP3, and EP4 receptors in situ and in vitro was observed; EP2 was homogenously expressed in all zones of the growth plate in situ, whereas EP1 expression was inhomogenous, with spared cells in the reserve zone. In cultured cells these four receptors were expressed in a subset of cells only. The most intense staining for the EP1 receptor was found in polygonal cells surrounded by matrix. Expression of receptor protein for EP3 and EP4 was observed also in rat growth plates. In cultured chrondrocytes, however, only weak expression of EP3 and EP4 receptor was detected. We suggest that in growth plate chondrocytes, COX-2 is responsible for PGE2 release, which stimulates cell proliferation via the EP1 receptor.

Insulin-Like Growth Factor (IGF)-I Stimulates Cell Proliferation and Induces IGF Binding Protein (IGFBP)-3 and IGFBP-5 Gene Expression in Cultured Growth Plate Chondrocytes via Distinct Signaling Pathways

The bioactivity of IGF-I in the cellular microenvironment is modulated by both inhibitory and stimulatory IGF binding proteins (IGFBPs), whose production is partially under control of IGF-I. However, little is known on the IGF-mediated regulation of these IGFBPs in the growth plate. We therefore studied the effect of IGF-I on IGFBP synthesis and the involved intracellular signaling pathways in two cell culture models of rat growth plate chondrocytes. In growth plate chondrocytes in primary culture, incubation with IGF-I increased the concentrations of IGFBP-3 and IGFBP-5 in conditioned cell culture medium in a dose- and time-dependent manner. Coincubation of IGF-I with specific inhibitors of the p42/44 MAPK pathway (PD098059 or U0126) completely abolished the stimulatory effect of IGF-I on IGFBP-3 mRNA expression but did not affect increased IGFBP-5 mRNA levels. In contrast, inhibition of the phosphatidylinositol-3 kinase signaling pathway by LY294002 abrogated both IGF-I-stimulated IGFBP-3 and -5 mRNA expression. Comparable results regarding IGFBP-5 were obtained in the mesenchymal chondrogenic cell line RCJ3.1C5.18, which does not express IGFBP-3. The IGF-I-induced IGFBP-5 gene expression required de novo mRNA transcription and de novo protein synthesis. These data suggest that IGF-I modulates its activity in cultured rat growth plate chondrocytes by the synthesis of both inhibitory (IGFBP-3) and stimulatory (IGFBP-5) binding proteins. The finding that IGF-I uses different and only partially overlapping intracellular signaling pathways for the regulation of two IGFBPs with opposing biological functions might be important for the modulation of IGF bioactivity in the cellular microenvironment.

Hyaluronidase expression in cultured growth plate chondrocytes during differentiation

Hyaluronan (HA) is a major component of the extracellular matrix of cartilage, contributes to its structural and functional integrity, and has various important roles in the differentiation of chondrocytes. HA metabolism is regulated by both anabolic and catabolic processes; however, the details have not yet been clarified. The purpose of this study was to clarify the expression patterns of hyaluronidase (HAase) mRNAs (from the relevant HAase genes: the HYALs) and HAase activity during chondrocyte differentiation. Cartilage tissue and growth plate chondrocytes were isolated from the ribs of 4-week-old male Japanese rabbits. The expression of HYAL mRNAs in cartilage was analyzed by in situ hybridization. The expression levels of HYAL mRNAs in the culture were analyzed for each of the chondrocyte differentiation stages by means of quantitative real-time polymerase chain reaction analysis. Enzymatic activity in the conditioned medium from the cultures was examined by using HA zymography and an enzyme-linked immunosorbent-like assay. The expression levels of HYAL1 and HYAL2 mRNAs were enhanced about 2.8-fold and 3.2-fold at the maximum during the early matrix forming stage, respectively, and by about 3.2-fold and 2.0-fold at the maximum in the hypertrophic stage, respectively. HYAL3 mRNA was not detected throughout the experimental period. HAase activity was enhanced at the early matrix forming and hypertrophic stages. These results suggest that selective expression of HYALs is essential for extracellular HA metabolism during chondrocyte differentiation.

Rat growth plate chondrocytes express low levels of 25-hydroxy-1α-hydroxylase

Long standing disturbances of Vitamin D-metabolism as well as null-mutant animals for 25-hydroxy-1α-hydroxylase results in disorganised growth plates. Cultured chondrocytes were shown to be target for the hydroxylated Vitamin D-metabolites 1α,25(OH) 2D 3 and 24,25(OH) 2D 3. Because studies on production of these metabolites were inconclusive in in vitro systems, the expression of the Vitamin D-system was examined in rat growth plate chondrocytes in vitro as well as ex vivo. Gene expression for 25-hydroxy-1α-hydroxylase, 25-hydroxy-24-hydroxylase as well as Vitamin D-receptor and collagen II and X were analysed on mRNA level by RT-PCR and quantitative real-time PCR, on protein level by western blotting and by immunohistochemistry in isolated growth plate chondrocytes or intact growth plates. Compared to UMR or CaCo 2 cells and renal homogenates cultured growth plate chondrocytes expressed low levels of 25-hydroxy-1α-hydroxylase mRNA and 25-hydroxy-24-hydroxylase mRNA. The expression of both was modulated by 25(OH)D 3, but 1α,25(OH) 2D 3 affected only 25-hydroxy-24-hydroxylase. These data were confirmed by Western blotting. Immunohistochemistry demonstrated predominant staining for 25-hydroxy-1α-hydroxylase in chondrocyte nodules and cells embedded in matrix in vitro. Ex vivo, 25-hydroxy-1α-hydroxylase was detected predominantly in late proliferative and hypertrophic zone of the growth plate. In conclusion, growth plate chondrocytes express the key components for a paracrine/autocrine Vitamin D-system.

Effects of Leptin to Cultured Growth Plate Chondrocytes

Objective: This study aimed to evaluate whether leptin has any effect on growth plate chondrocytes. Methods: We studied the effects of exogenous leptin on cultured rabbit growth plate chondrocytes. This involved assessing [³H]thymidine incorporation, alkaline phosphatase (ALP) activity, proteoglycan production, leptin receptor (Ob-R) activity, and detection of Ob-R using Western blot analysis. Results: The existence of Ob-R in growth plate chondrocytes was revealed by Western blot and Ob-R activity. Prior to semiconfluence, leptin increased [³H]thymidine incorporation while at the semiconfluent and early confluent stages, leptin promoted ALP activity and tended to promote proteoglycan production. Conclusion: Growth plate chondrocytes possess Ob-Rs, and leptin enhance chondrocyte proliferation and subsequent cell differentiation.

Differential effects of insulin-like growth factor binding proteins-1, -2, -3, and -6 on cultured growth plate chondrocytes

In children with chronic renal failure (CRF), impairment of longitudinal growth is in part due to excess amounts of circulating high-affinity insulin-like growth factor binding proteins (IGFBPs) that might decrease or prevent insulin-like growth factor (IGF) binding to its signaling receptor. However, it appears from the clinical studies that various IGFBPs may have contrasting effects on longitudinal growth. Because of the potential importance of the IGFBPs as modulators of longitudinal growth in pediatric CRF, the aim of the present study was to investigate the biological effects of IGFBP-1, -2, -3, and -6 on cultured growth plate chondrocytes that express the type 1 IGF receptor. The effects of exogenous IGFBPs on IGF-independent and IGF-dependent proliferation of rat growth plate chondrocytes in primary culture were investigated. Proliferation was assessed by colony formation of agarose-stabilized long-term suspension cultures and by the [3H]thymidine assay. The effects of IGFBPs on IGF-I binding and the binding of IGFBPs to chondrocytes were assessed by binding studies with radiolabeled proteins in monolayer culture. Intact IGFBP-1, IGFBP-2 and IGFBP-6 inhibited in equimolar concentration the IGF-I- and IGF-II-stimulated DNA synthesis and cell proliferation, whereas the biological activity of IGFBP-3 was complex. It had an IGF-independent antiproliferative effect and also inhibited IGF-dependent chondrocyte proliferation under coincubation conditions, whereas under preincubation conditions IGFBP-3 enhanced IGF-I-responsiveness. Studies on the mechanism by which IGFBP-3 potentiated IGF activity demonstrated that under preincubation conditions IGFBP-3 is capable to associate with the cell membrane and to facilitate IGF-I cell surface binding. Intact IGFBP-1, IGFBP-2 and IGFBP-6 act exclusively as growth inhibitors on IGF-dependent proliferation of growth plate chondrocytes. IGFBP-3, however, can either inhibit IGF-independent and IGF-dependent cell proliferation, or enhance IGF responsiveness of chondrocytes dependent on the temporal relationship to the IGF exposure.

A vitamin makes minerals

Growth plate chondrocytes help turn cartilage into bone by releasing specialized matrix vesicles, which contain the crystaline seeds for calcification. On page 1061, Wang and Kirsch show how retinoic acid (vitamin A) controls this process of mineralization. Figure Vitamin A induces bone mineralization. Previous work showed that retinoic acid stimulates mineralization and induces the formation of matrix vesicles, but its molecular targets remained unknown. In the new study, the authors found that, in cultured growth plate chondrocytes, retinoic acid acts by increasing the transcription of three annexin family proteins and causing them to form calcium channels in the plasma membrane. Retinoic acid is known to regulate transcription after binding to the retinoic acid receptor complex, but the new results imply that it can also control calcium homeostasis, perhaps through receptors yet to be discovered. After retinoic acid induces an initial calcium influx, the formation of induced annexins into channels in the plasma membrane further boosts cytosolic calcium, increasing annexin expression again and causing the release of matrix vesicles. These vesicles contain annexin channels that allow the entry of crystal-forming calcium. Excess mineralization has been implicated in several disease processes, including osteoarthritis and the calcification of cardiovascular tissues. Recently, the authors have determined that annexin expression is induced in osteoarthritic chondrocytes. If annexins are as central to pathogenic mineralization as they appear to be in healthy tissue, they could be promising targets for a variety of therapies. ▪

Intact IGF-Binding Protein-4 and -5 and Their Respective Fragments Isolated from Chronic Renal Failure Serum Differentially Modulate IGF-I Actions in Cultured Growth Plate Chondrocytes

Impairment of longitudinal growth among children with chronic renal failure (CRF) may be partly attributable to the inhibition of insulin-like growth factor (IGF) activity by an excess amount of high-affinity IGF-binding proteins (IGFBP). Elevated levels of immunoreactive IGFBP-4 in CRF serum are inversely correlated with the standardized heights of these children, whereas levels of IGFBP-5, which circulates mainly as proteolyzed fragments, are positively correlated with growth parameters. To delineate the respective effects of these IGFBP on growth cartilage, the biologic effects of intact and fragmented forms of IGFBP-4 and IGFBP-5 on rat growth plate chondrocytes in primary cultures were characterized. Intact IGFBP-4 and IGFBP-5 and the amino-terminal fragment IGFBP-5(1-169) were recombinant proteins; the carboxy-terminal fragments IGFBP-5(144-252) and IGFBP-4(136-237) and the amino-terminal fragment IGFBP-4(1-122) were purified to homogeneity from CRF hemofiltrates. Intact IGFBP-4 and, to a lesser extent, IGFBP-4(1-122) inhibited IGF-I-induced cell proliferation. In contrast, intact IGFBP-5 was stimulatory in the absence or presence of exogenous IGF-I, whereas the amino-terminal fragment IGFBP-5(1-169) was inhibitory. Studies on the mechanism by which IGFBP-4 and IGFBP-5 exert opposite effects on chondrocyte proliferation demonstrated that intact IGFBP-4 prevented the binding of (125)I-IGF-I to chondrocytes, whereas intact IGFBP-5 enhanced ligand binding and was able to bind specifically to the cell membrane. These data suggest that intact IGFBP-4 and, to a lesser extent, IGFBP-4(1-122) act exclusively as growth-inhibitory binding proteins in the growth cartilage. IGFBP-5, however, can either stimulate (if it remains intact) or inhibit (if amino-terminal forms predominate) IGF-I-stimulated chondrocyte proliferation.

TGFbeta1 regulates 25-hydroxyvitamin D3 1alpha- and 24-hydroxylase activity in cultured growth plate chondrocytes in a maturation-dependent manner.

Chondrocytes metabolize 25-(OH)D3 to the two active dihydroxylated forms of the secosteroid, 1,25-(OH)2D3 and 24,25-(OH)2D3. The aim of the present study was to examine the activity of the enzymes responsible for this metabolism, 1alpha-hydroxylase and 24R-hydroxylase, and their regulation by TGFbeta1. Basal 1alpha- and 24R-hydroxylase activities were measured in homogenates of confluent, fourth passage rat costochondral resting zone and growth zone chondrocytes and mouse cortico-tubular cells (MCT) were used as a positive control. The cells were harvested and homogenized in buffer optimized to maintain the activity and stability of the hydroxylases. Homogenates were incubated for 90 minutes and 1alpha- and 24R-hydroxylase activities determined by measuring the conversion of [3H]-25-(OH)D3 to [3H]-1,25-(OH)2D3 and [3H]-24,25-(OH)2D3 using an HPLC with an inline radioisotope detector. Resting zone cells were also treated with various concentrations of recombinant human TGFbeta1 for 24 hours, and enzyme activity in total cell homogenates as well as 24-hydroxylase mRNA levels were determined. In addition, [3H]-1,25-(OH)2D3 and [3H]-24,25-(OH)2D3 released into the conditioned media by resting zone chondrocyte cultures in response to TGFbeta1 were measured. In culture, all three cell types were found to contain 1alpha- and 24R-hydroxylase activities. Basal 1alpha-hydroxylase specific activity was significantly higher than 24R-hydroxylase specific activity in all cells. RT-PCR confirmed that resting zone and growth zone cells expressed mRNA for 24R-hydroxylase. Treatment of resting zone cells with TGFbeta1 increased 24R-hydroxylase mRNA levels in a dose-dependent manner. TGFbeta1 also increased 24R-hydroxylase activity 2- to 5-fold and decreased 1alpha-hydroxylase activity by 20-30%. Similar changes were observed with MCT cells, but not growth zone cells. Production of [3H]-24,25-(OH)2D3 by resting zone cells increased with TGFbeta1 treatment, while [3H]-1,25-(OH)2D3 production decreased. The effect was time- and dose-dependent, correlating with hydroxylase activity and 24-hydroxylase gene expression. These results demonstrate that growth plate chondrocytes contain the necessary enzymes to produce 1, 25-(OH)2D3 and 24,25-(OH)2D3 from 25-(OH)D3. In addition, the activity of these enzymes in resting zone cells, but not growth zone cells, is regulated by TGFbeta1 by increasing gene transcription, indicating that cell maturation-dependent autocrine/paracrine pathways exist for regulating vitamin D metabolite production.

Identification of Chondromodulin I as a Novel Endothelial Cell Growth Inhibitor: PURIFICATION AND ITS LOCALIZATION IN THE AVASCULAR ZONE OF EPIPHYSEAL CARTILAGE

Cartilage is unique among tissues of mesenchymal origin in that it is resistant to vascular invasion due to an intrinsic angiogenic inhibitor. During endochondral bone formation, however, calcified cartilage formed in the center of the cartilaginous bone rudiment allows vascular invasion, which initiates the replacement of cartilage by bone. The transition of cartilage from the angioresistant to the angiogenic status thus plays a key role in bone formation. However, the molecular basis of this phenotypic transition of cartilage has been obscure. We report here purification of an endothelial cell growth inhibitor from a guanidine extract of bovine epiphyseal cartilage. The N-terminal amino acid sequence indicated that the inhibitor was identical to chondromodulin I (ChM-I), a cartilage-specific growth-modulating factor. Purified ChM-I inhibited DNA synthesis and proliferation of vascular endothelial cells as well as tube morphogenesis in vitro. Expression of ChM-I cDNA in COS7 cells indicated that mature ChM-I molecules were secreted from the cells after post-translational modifications and cleavage from the transmembrane precursor at the predicted processing signal. Recombinant ChM-I stimulated DNA synthesis and proteoglycan synthesis of cultured growth plate chondrocytes, but inhibited tube morphogenesis of endothelial cells. In situ hybridization and immunohistochemical studies indicated that ChM-I is specifically expressed in the avascular zone of cartilage in developing bone, but not present in calcifying cartilage. These results suggest a regulatory role of ChM-I in vascular invasion during endochondral bone formation.

A Novel Growth-Promoting Factor Derived from Fetal Bovine Cartilage, Chondromodulin II: PURIFICATION AND AMINO ACID SEQUENCE

During endochondral bone formation, cartilage cells show increased matrix synthesis and rapid proliferation. We found that cartilage matrix contains at least two types of heparin binding growth-promoting components. One, with a higher affinity to heparin, was identified as chondromodulin I (Hiraki, Y., Tanaka, H., Inoue, H. , Kondo, J., Kamizono, A., and Suzuki, F. (1991) Biochem. Biophys. Res. Commun. 175, 871-977). In this study, we isolated a novel growth-promoting component, chondromodulin II, which has a lower heparin affinity, from the dissociative extracts of fetal bovine epiphyseal cartilage. Chondromodulin II stimulated the proteoglycan synthesis in rabbit cultured growth plate chondrocytes, an expression of the differentiated phenotype of chondrocytes. It also stimulated DNA synthesis in chondrocytes in both the absence and the presence of fibroblast growth factor-2. The apparent molecular mass of chondromodulin II on SDS-polyacrylamide gel electrophoresis was 16 kDa. Its complete amino acid sequence was determined by overlapping sequences of the peptides released by endopeptidase digestion and CNBr cleavage. Chondromodulin II consists of 133 amino acids (calculated Mr = 14,548). The sequence was unique but homologous to the repeats 1 and 2 of the deduced amino acid sequence of the chicken mim-1 gene, which is specifically transactivated by the v-Myb oncogene product in promyelocytes. We also found a minor component with a higher heparin affinity, chondromodulin III, in cartilage extracts. Chondromodulin III stimulated DNA synthesis in chondrocytes in vitro, and its N-terminal sequence was identical with ribosomal protein L31 lacking the N-terminal three amino acids. These findings suggest that the growth and differentiation of chondrocytes are regulated by multiple components in the cartilage matrix.

Changes in the irradiation sensitivity of cultured growth plate chondrocytes during cytodifferentiation

The effects of X-ray irradiation on the proliferation and differentiation of growth plate chondrocytes were examined. Chondrocytes were isolated from the ribs of 4-week-old New Zealand white rabbits and cultured as a packed mass in centrifuge tubes. The cells in the centrifuge tubes proliferated, matured and hypertrophied likein vivo chondrocytes. Irradiation at a dose as low as 3 Gy to proliferating chondrocytes significantly decreased the DNA content, alkaline phosphatase (ALPase) activity, and matrix calcification, without decreasing the proteoglycan content. Irradiation at 10Gy to mature chondrocytes caused significant decrease in ALPase activity and matrix calcification, although it did not have marked effects on DNA and proteoglycan contents. On the other hand, irradiation at 10Gy to hypertrophic chondrocytes did not cause any change in ALPase activity or cartilage-matrix calcification. These findings suggest that the irradiation sensitivity of chondrocytes changes during cytodifferentiation. Furthermore, the inhibition of ALPase expression and matrix calcification by irradiation proved to be independent of the suppression of proliferation.

Analysis of type II and type X collagen synthesis in cultured growth plate chondrocytes by in situ hybridization: Rapid induction of type X collagen in culture

Type X collagen is produced by hypertrophic chondrocytes and serves as a highly specific marker for chondrocyte maturation. This study was designed to compare the expression of type II and type X collagen in growth plate sections and in distinct populations of chondrocytes in culture by in situ hybridization. Growth plate sections were treated with type II and type X collagen cDNA probes. Type II collagen mRNA was present throughout the growth plate but greatest in the lower proliferating and upper hypertrophic regions. In contrast, type X collagen was expressed only in the hypertrophic region. Northern analysis confirmed the specificity of the probe for type X collagen mRNA. Chick growth plate chondrocytes were separated by countercurrent centrifugal elutriation into five distinct populations and plated in serum-containing medium. These cultures were examined at varying times after plating for the expression of type II and type X collagen mRNA. At 3 h, type II collagen was present in the majority of the cells in all fractions, and approximately 15-20% of the cells expressed type X collagen mRNA. The cells expressing type X were from the hypertrophic region. At 24 h, however, nearly all cells in culture expressed type X mRNA, and there was a decrease in expression of type II collagen mRNA. Similar results were obtained in cultures in the absence of serum, and SDS-PAGE analysis of collagen synthesis confirmed the expression of type X collagen in all populations of fractionated cells at 24 h at the protein level. Type X collagen is an important marker through which cellular matruation can be evaluated in culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Parathormone and calcitonin alter alkaline phosphatase activity and matrix calcification in cultured growth plate chondrocytes

Parathormone and calcitonin alter alkaline phosphatase activity and matrix calcification in cultured growth plate chondrocytes

Effects of 1,25(OH) 2D 3-pulses on cultured growth plate chondrocytes of the rat

Effects of 1,25(OH) 2D 3-pulses on cultured growth plate chondrocytes of the rat