Yuji Hiraki

Cellular Hypertrophy and Calcification of Embryonal Carcinoma‐Derived Chondrogenic Cell Line ATDC5 In Vitro

During the process of endochondral bone formation, proliferating chondrocytes give rise to hypertrophic cells, which then deposit a mineralized matrix to form calcified cartilage prior to replacement by bone. Previously, we reported that a clonal cell line, ATDC5, undergoes efficient chondrogenic differentiation through a cellular condensation stage. Here we report that the differentiated ATDC5 cells became hypertrophic at the center of cartilage nodules, when the cells ceased to grow. Formation of hypertrophic chondrocytes took place in association with type X collagen gene expression and a dramatic elevation of alkaline phosphate (ALPase) activity. After 5 weeks of culture, mineralization of the culture could be discerned as Alizarin red‐positive spots, which spread throughout the nodules even in the absence of β‐glycerophosphate. Electron microscopy and electron probe microanalysis revealed that calcification was first initiated at matrix vesicles in the territorial matrix and that it advanced progressively along the collagen fibers in a manner similar to that which occurs in vivo. The infrared spectrum of the mineralized nodules indicated two absorption doublets around 1030 cm−1 and 600 cm−1, which are characteristic of apatitic mineral. Calcifying cultures of ATDC5 cells retained responsiveness to parathyroid hormone (PTH): PTH markedly inhibited elevation of ALPase activity and calcification in the culture in a dose‐dependent manner. Thus, we demonstrated that ATDC5 cells keep track of the multistep differentiation process encompassing the stages from mesenchymal condensation to calcification in vitro. ATDC5 cells provide an excellent model to study the molecular mechanism underlying regulation of cartilage differentiation during endochondral bone formation.

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.

Functional Analysis of Diastrophic Dysplasia Sulfate Transporter ITS INVOLVEMENT IN GROWTH REGULATION OF CHONDROCYTES MEDIATED BY SULFATED PROTEOGLYCANS

Mutations in the diastrophic dysplasia sulfate transporter (DTDST) gene constitute a family of recessively inherited osteochondrodysplasias including achondrogenesis type 1B, atelosteogenesis type II, and diastrophic dysplasia. However, the functional properties of the gene product have yet to be elucidated. We cloned rat DTDST cDNA from rat UMR-106 osteoblastic cells. Northern blot analysis suggested that cartilage and intestine were the major expression sites for DTDST mRNA. Analysis of the genomic sequence revealed that the rat DTDST gene was composed of at least five exons. Two distinct transcripts were expressed in chondrocytes due to alternative utilization of the third exon, corresponding to an internal portion of the 5′-untranslated region of the cDNA. Injection of rat and human DTDST cRNA into Xenopus laevis oocytes induced Na+-independent sulfate transport. Transport activity of the expressed DTDST was markedly inhibited by extracellular chloride and bicarbonate. In contrast, canalicular Na+-independent sulfate transporter Sat-1 required the presence of extracellular chloride in the cRNA-injected oocytes. The activity profile of sulfate transport in growth plate chondrocytes was studied in the extracellular presence of various anions and found substantially identical to DTDST expressed in oocytes. Thus, sulfate transport of chondrocytes is dominantly dependent on the DTDST system. Finally, we demonstrate that undersulfation of proteoglycans by the chlorate treatment of chondrocytes significantly impaired growth response of the cells to fibroblast growth factor, suggesting a role for DTDST in endochondral bone formation.

Molecular cloning of a new class of cartilage-specific matrix, chondromodulin-I, which stimulates growth of cultured chondrocytes

Here we report the structure and bioactivity of 25 kDa glycoprotein (chondromodulin-I) as a tissue-specific functional matrix component identified and cloned for the first time. Chondromodulin-I purified from fetal bovine cartilage markedly stimulated DNA synthesis of cultured growth-plate chondrocytes in the presence of basic fibroblast growth factor (FGF). Bovine chondromodulin-I cDNA revealed that the mature protein consists of 121 amino acids with three possible glycosylation sites and is coded as the C-terminal part of a larger precursor. On northern blot analysis, expression of chondromodulin-I mRNA was observed only in cartilage.

Effect of transforming growth factor β on cell proliferation and glycosaminoglycan synthesis by rabbit growth-plate chondrocytes in culture

The effects of the transforming growth factor beta (TGF-beta) on the growth and glycosaminoglycan synthesis of rabbit growth plate-chondrocytes in culture were studied. In serum-free medium, TGF-beta caused dose-dependent inhibition of DNA synthesis by chondrocytes, measured as [3H]thymidine incorporation (ED50 = 0.1-0.3 ng/ml). The inhibitory effect was maximal at a dose of 1 ng/ml, and extended for a duration of 16-42 h. In contrast, TGF-beta potentiated the synthesis of DNA stimulated by fetal calf serum (FCS). Addition of TGF-beta (1 ng/ml) to cultures containing 10% FCS increased [3H]thymidine incorporation to 1.6-times that in cultures with 10% FCS alone. Consistent with this finding, TGF-beta potentiated DNA synthesis stimulated by the purified growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and fibroblast growth factor (FGF). The maximal stimulation of DNA synthesis by FGF (0.4 ng/ml) was further potentiated dose dependently by TGF-beta (ED50 = 0.1 ng/ml, maximum at 1 ng/ml). When the cultures were treated with the optimal concentrations of TGF-beta (1 ng/ml) and FGF (0.4 ng/ml), [3H]thymidine incorporation was 3-times higher than that of cultures treated with FGF alone. This TGF-beta-induced potentiation of DNA synthesis was associated with replication of chondrocytes, as shown by a marked increase in the amount of DNA during treatment of sparse cultures of the cells with the growth factors for 5 days. In contrast, TGF-beta caused dose-dependent stimulation of glycosaminoglycan synthesis in confluent cultures of growth-plate chondrocytes (ED50 = 0.3 ng/ml, maximum at 1 ng/ml). This stimulatory effect of TGF-beta was greater than that of insulin-like growth factor I (IGF-I) or PDGF. Furthermore, TGF-beta stimulated glycosaminoglycan synthesis additively with IGF-I or PDGF. Recently, it has been suggested that bone and articular cartilage are rich sources of TGF-beta, whereas epiphyseal growth cartilage is not. Thus, the present data indicate that TGF-beta may be important in bone formation by modulating growth and phenotypic expression of chondrocytes in the growth plate, possibly via a paracrine mechanism.

Inhibition of DNA synthesis and tube morphogenesis of cultured vascular endothelial cells by chondromodulin-I

Cartilage is an avascular tissue, and exhibits anti‐angiogenic properties. Cartilage extracts have been shown to contain an inhibitor for DNA synthesis in vascular endothelial cells in vitro. Here we purified the inhibitory activity in the 10–50 kDa fraction of guanidine extracts from fetal bovine epiphyseal cartilage, and found that the inhibitor was identical with chondromodulin‐I (ChM‐I). Purified ChM‐I inhibited tube morphogenesis of cultured vascular endothelial cells, as well as DNA synthesis. These results indicate that cartilage‐specific glycoprotein ChM‐I may participate in the maintenance of avascularity and anti‐angiogenic properties of cartilage.

Stimulation of osteoblast proliferation by the cartilage-derived growth promoting factors chondromodulin-I and -II

We previously reported the isolation of the cartilage‐derived growth promoting factors chondromodulin‐I (ChM‐I) and chondromodulin‐II (ChM‐II) from fetal bovine epiphyseal cartilage. Both of these factors stimulate the growth and matrix formation of chondrocytes in vitro. In the present study, we found that ChM‐I and ChM‐II stimulated the proliferation of clonal mouse osteoblastic MC3T3‐E1 cells as well as primary mouse osteoblasts in culture. Unlike other known growth factors, these factors did not support the proliferation of fibroblasts. Concomitantly with growth stimulation of osteoblasts, there was a reduction of alkaline phosphatase (ALP) activity in the cells, the expression of the differentiated phenotype. These results suggest that epiphyseal cartilage may play a functional role in longitudinal bone growth by production of these unique growth‐promoting factors.

Differential effects of parathyroid hormone and somatomedin-like growth factors on the sizes of proteoglycan monomers and their synthesis in rabbit costal chondrocytes in culture.

In the proteoglycans extracted from rabbit costal chondrocytes in culture, two populations of proteoglycans were distinguished by density gradient centrifugation under dissociative conditions. The major component was the faster sedimenting population (proteoglycan I), the putative cartilage-specific proteoglycans, and the minor component was the slower sedimenting population (proteoglycan II). The monomeric size of proteoglycan I was closely related to the differentiation-state of chondrocytes and was a good marker of the differentiated chondrocytes. Treatment of the cultures with parathyroid hormone (PTH) induced an increase in the monomeric size of proteoglycan I. This increase was ascribed to an increase in the molecular size of the glycosaminoglycan chain in proteoglycan I. On the other hand, somatomedin-like growth factors, such as multiplication-stimulating activity (MSA) and cartilage-derived factor (CDF), did not affect the size of proteoglycan I, while they markedly stimulated the synthesis of proteoglycan I. In contrast, treatment with nonsomatomedin growth factors, such as fibroblast growth factor (FGF) and epidermal growth factor (EGF), resulted in not only a decrease in glycosaminoglycan synthesis but also a slight decrease in size of proteoglycan I. However, synthesis and size of proteoglycan II were little affected by these agents. Thus, the present study clearly shows that PTH and somatomedin-like growth factors have differential functions in bringing about the expression of the differentiated phenotype of chondrocytes: PTH influences chain elongation and termination of glycosaminoglycans in proteoglycan I, while somatomedin-like growth factors affect primarily the synthesis and secretion of proteoglycan I.

Bone morphogenetic proteins (BMP‐2 and BMP‐3) induce the late phase expression of the proto‐oncogene c‐fos in murine osteoblastic MC3T3‐E1 cells

Here we report that bone morphogenetic proteins 2 and 3 (BMP‐2 and BMP‐3) induced marked expression of c‐fos mRNA in a biphasic manner, i.e. the late phase (48 to 60 h) as well as the immediate‐early phase (0.5 h), in murine osteoblastic MC3T3‐El cells in vitro. The BMP‐induced late phase c‐fos gene expression was temporally associated with the onset of marked expression of the genes for osteocalcin and alkaline phosphatase, differentiation markers of mature osteoblasts. In contrast, none of TGF‐β1, 10% FBS, IGF‐I and IGF‐II, which induced only the immediate‐early c‐fos mRNA expression, stimulated the expression of osteocalcin and alkaline phosphatase genes. These data suggest that in osteoblasts BMP‐2 and BMP‐3 induce the late phase expression of c‐fos, which may play a role in transcriptional activation of the genes involved in differentiation of osteoblasts.

Fracture healing induces expression of the proto-oncogene c-fos in vivo. Possible involvement of the Fos protein in osteoblastic differentiation.

Here we report marked in vivo expression of the c‐fos gene in the external soft callus (ESC) and periosteal hard callus (PHC) at the fracture site of adult rat tibia. Northern‐blot analysis showed that the ESC expressed a high level of c‐fos mRNA from post‐fracture day 10 to day 28, the time when endochondral ossification progressed, and that the ossifying PHC also expressed c‐fos mRNA. This c‐fos expression was followed by sequential expression of the genes for alkaline phosphatase, osteopontin and osteocalcin, which are osteoblastic markers. Immunohistochemical analysis showed that the c‐Fos protein was predominantly located in osteoblasts in the ossifying calluses.