Hepatocyte growth factor stimulates root growth during the development of mouse molar teeth.

BACKGROUND AND OBJECTIVE: It is well known that tooth root formation is initiated by the development of Hertwig's epithelial root sheath (HERS). However, relatively little is known about the regulatory mechanisms involved in root development. As hepatocyte growth factor (HGF) is one of the mediators of epithelial-mesenchymal interactions in rodent tooth, the objective of this study was to examine the effects of HGF on the root development of mouse molars. MATERIAL AND METHODS: The HERS of mouse molars and HERS01a, a cell line originated from HERS, were used in this study. For detection of HGF receptors in vivo and in vitro, we used immunochemical procedures. Root development was assessed by implanting molar tooth germs along with HGF-soaked beads into kidney capsules, by counting cell numbers in HERS01a cell cultures and by performing a 5'-bromo-2'-deoxyuridine (BrdU) assay in an organ-culture system. RESULTS: HGF receptors were expressed in the enamel epithelium of molar germs as well as in HERS cells. HGF stimulated root development in the transplanted tooth germs, the proliferation of HERS01a cells in culture and HERS elongation in the organ-culture system. Examination using BrdU revealed that cell proliferation in HERS was increased by treatment with HGF, especially that in the outer layer of HERS. This effect was down-regulated when antibody against HGF receptor was present in the culture medium. CONCLUSION: Our results raise the possibility that HGF signaling controls root formation via the development of HERS. This study is the first to show that HGF is one of the stimulators of root development.

Origin-associated features of chondrocytes in mouse Meckel's cartilage and costal cartilage: an in vitro study.

Using a cell culture method, we histochemically and immunohistochemically investigated whether chondrocytes deriving from different origins, such as Meckel's or costal cartilages, express similar phenotypic characteristics. Chondrocytes isolated enzymatically from Meckel's and costal cartilages of 17-day embryonic mice both actively proliferated and formed cartilage nodules consisting of toluidine blue-positive proteoglycans and type II collagen. Both deposited calcified cartilaginous matrix as revealed by alkaline phosphatase (ALPase) activity and alizarin red staining throughout 3 weeks in culture. Immunostaining for osteopontin (OP), osteocalcin (OC), and osteonectin (ON) revealed that chondrocytes from both cartilages were positive for their proteins, but type I collagen was detected only in cells transforming from Meckel's chondrocytes late in the culture. Electron microscopy demonstrated that although costal and Meckel's chondrocytes had typical chondrocytic features during 2 weeks in culture, Meckel's chondrocytes transformed into osteocytic cells that produced thick, banded type I collagen fibrils. In contrast, costal chondrocytes maintained typical hypertrophic morphology throughout the final stage of culture. The present study suggests that Meckel's chondrocytes derived from neural crest-ectomesenchyme retain osteogenic potential, and differ from costal chondrocytes originating from mesoderm.

Formation of the sphenomandibular ligament by Meckel's cartilage in the mouse: possible involvement of epidermal growth factor as revealed by studies in vivo and in vitro.

In mammals, the midportion of the soft tissue of Meckel's cartilage at the degenerating stage forms a ligament known as the sphenomandibular ligament. To clarify the mechanism of formation of this ligament by Meckel's cartilage in mouse, we examined the effects of epidermal growth factor (EGF) on the chondrocytes in terms of the proliferation and differentiation of cells and calcification of the matrix in vivo and in vitro. The effects of EGF were examined by immunohistochemical staining, with EGF-soaked beads, by electron microscopy, and by general histochemical analysis of proteoglycans and calcification. Analysis of labeling with bromodeoxyuridine (BrdU) and the rate of cell growth revealed that EGF enhanced DNA synthesis and the proliferation of Meckel's chondrocytes. Histological findings in organ culture and in cell culture, with and without the application of EGF-soaked beads, revealed that EGF inhibited the differentiation of cells to chondrocytes and induced phenotypic changes in fibroblastic cells. The inhibition of alkaline phosphatase activity that resulted from exposure to EGF was accompanied by prolonged calcification of the matrix. Whole-mount staining revealed that subcutaneous injection of EGF enhanced the disappearance of Meckel's cartilage. Our results suggest a possible mechanism whereby the midportion of Meckel's cartilage remains uncalcified and is rapidly transformed into the sphenomandibular ligament.

Further evidence for secretion of matrix metalloproteinase-1 by Meckel's chondrocytes during degradation of the extracellular matrix.

We examined the possibility that chondrocytes in Meckel's cartilage might secrete matrix metalloproteinase-1 (MMP-1) during degradation of the extracellular matrix. Evidence for the secretion of MMP-1 was obtained by immunohistochemical staining and immunoelectron microscopy, in addition to general histochemical staining for proteoglycans. Not only staining with toluidine blue and alcian blue but also immunostaining for chondroitin sulfate proteoglycan (CSPG) revealed that levels of glycoproteins are rapidly reduced at the late stage of degradation. MMP-1 was detected continuously in cells from chondrocytes at the early stage to hypertrophic chondrocytes at the late stage. Immunoelectron microscopy revealed that the deposition of colloidal golds shifted from an intracellular localization in chondrocytes at the early stage to pericellular spaces at the late stage. The localization of tissue inhibitor of the metalloproteinase-1 (TIMP-1) at the early stage was similar to that of MMP-1, but the level of TIMP-1 decreased significantly in hypertrophic cartilage. These findings suggest that MMP-1 is present continuously in Meckel's chondrocytes but that the active form, which degrades the extracellular matrix, is the MMP-1 that accumulates in the pericellular spaces around hypertrophic chondrocytes.

Histochemical and immunohistochemical analysis of the mechanism of calcification of Meckel's cartilage during mandible development in rodents.

It is widely accepted that Meckel's cartilage in mammals is uncalcified hyaline cartilage that is resorbed and is not involved in bone formation of the mandible. We examined the spatial and temporal characteristics of matrix calcification in Meckel's cartilage, using histochemical and immunocytochemical methods, electron microscopy and an electron probe microanalyser. The intramandibular portion of Meckel's cartilage could be divided schematically into anterior and posterior portions with respect to the site of initiation of ossification beneath the mental foramen. Calcification of the matrix occurred in areas in which alkaline phosphatase activity could be detected by light and electron microscopy and by immunohistochemical staining. The expression of type X collagen was restricted to the hypertrophic cells of intramandibular Meckel's cartilage, and staining with alizarin red and von Kossa stain revealed that calcification progressed in both posterior and anterior directions from the primary centre of ossification. After the active cellular resorption of calcified cartilage matrix, new osseous islands were formed by trabecular bone that intruded from the perichondrial bone collar. Evidence of such formation of bone was supported by results of double immunofluorescence staining specific for type I and type II collagens, in addition to results of immunostaining for osteopontin. Calcification of the posterior portion resembled that in the anterior portion of intramandibular Meckel's cartilage, and our findings indicate that the posterior portion also contributes to the bone formation of the mandible by an endochondral-type mechanism of calcification.

Expression of osteopontin in Meckel's cartilage cells during phenotypic transdifferentiation in vitro, as detected by in situ hybridization and immunocytochemical analysis.

The localization of osteopontin (OP) was examined in Meckel's cartilage cells that bipotentially expressed cartilage and bone phenotypes during cellular transformation in vitro. Cultured cells were analyzed by in situ hybridization, immunostaining followed by light and electron microscopy, electron microscopy, and electron probe microanalysis. The combination of ultrastructural analysis and immunoperoxidase staining indicated that OP-synthesizing cells were cells that were autonomously undergoing a change from chondrocytes to bone-forming cells at the top of nodules. Double immunofluorescence staining of 2-week-old cultures revealed that OP was first synthesized by chondrocytic cells at the top of nodules. After further time in culture, the distribution of OP expanded from the central toward the peripheral regions of the nodules. Electron probe microanalysis revealed that the localization of OP was associated with matrices of calcified cartilage and osteoid nodules that contained calcium and phosphorus. Immunoperoxidase electron microscopy revealed that, in addition to the intracellular immunoreactivity in chondrocytes and small round cells that were undergoing transformation, matrix foci of calcospherites and matrix vesicles, in particular, included growing crystals that were immunopositive for OP. An intense signal due to mRNA for OP in 3-week-old cultures was detected in nodule-forming round cells, while fibroblastic cells, spreading in a monolayer over the periphery of nodules, were only weakly labeled. These findings indicate that OP might be expressed sequentially by chondrocytes and by cells that are transdifferentiating further and exhibit an osteocytic phenotype, and moreover, that expression of OP is closely associated with calcifying foci in the extracellular matrix.

Evidence for transformation of chondrocytes and site-specific resorption during the degradation of Meckel's cartilage.

It is unknown whether cells in the midportion of Meckel's cartilage undergo transformation into other kinds of cell or whether resorption of cells occurs during development. Therefore, the midportion of Meckel's cartilage from the mouse and the rat was subdivided into anterior and posterior portions. The ultimate fates of these tissues were analyzed with a focus on resorption -related cells, death of chondrocytes by apoptosis, and transformation of the chondrocytes themselves. Cellular and extracellular features of mouse Meckel's cartilage were observed after von Kossa's staining and staining for acid phosphatase (APase) activity, as well as by light and electron microscopy. To identify resorbing cells, immunostaining specific for macrophages and staining for tartrate-resistant acid phosphatase (TRAP) were performed. The DNA nick end-labeling (TUNEL) method was used for the detection of death of chondrocytes by apoptosis. The replacement of the extracellular matrix of rat Meckel's cartilage was examined with double immunofluorescence staining for type I and type II collagens. When the anterior midportion from embryonic mice on day 18 was examined after von Kossa's staining, it was clear that the extracellular matrix had already calcified and vascularization had been initiated that reflected the calcified matrix. TRAP staining and immunostaining for macrophages revealed two types of osteoclast and macrophages that were involved in resorption of the matrix. In the posterior midportion, no vascular invasion was evident, and chondrocytes were transformed directly into fibroblastic cells by phenotypic conversion. In such cells we found reaction products specific for APase activity, suggestive of the intracellular degradation of fine collagenous fibrils. Double immunofluorescence staining showed that cartilage-specific type II collagen was replaced by type I collagen with the phenotypic transformation to fibroblastic cells. There were no significant changes in the number of TUNEL-positive apoptotic cells from day 17 of gestation to day 6 after parturition. Death of chondrocytes by apoptosis was not, therefore, involved directly in the disappearance of Meckel's cartilage. These results in the posterior midportion served as an instance of phenotypic switches in differentiated cells from chondrocytes to fibroblast-like cells. The present study indicates that there is a difference between the ultimate fate of cells in the posterior part and that of cells in the anterior part in the midportion of Meckel's cartilage in the mouse and rat.

Inhibition of endogenous expression of connective tissue growth factor by its antisense oligonucleotide and antisense RNA suppresses proliferation and migration of vascular endothelial cells.

Previously, we cloned an mRNA predominantly expressed in hypertrophic chondrocytes by differential display-PCR from a human chondrosarcoma-derived chondrocytic cell line (HCS-2/8) that is identical to that of connective tissue growth factor (CTGF). In the present study, we investigated the roles of CTGF in the proliferation and migration of vascular endothelial cells using its antisense oligonucleotide and antisense RNA, because angiogenesis into the hypertrophic zone of cartilage occurs at the final step of endochondral ossification. Immunohistochemical and immunofluorescence techniques revealed that not only hypertrophic chondrocytes but also endothelial cells in the cost-chondral junctions of mouse ribs were stained with an anti-CTGF antibody in vivo. Northern blot analysis revealed that CTGF was strongly expressed in chondrocytic cells as well as bovine aorta endothelial (BAE) cells in culture, but not in other types of cells such as osteoblastic cells. Its expression in BAE cells was greater in the growing phase than in the confluent phase. When one-half of a monolayer of a confluent culture of BAE cells had been peeled off, only the cells proliferating and extending into the vacant area were stained with the anti-CTGF antibody. The addition of an antisense oligonucleotide inhibited the proliferation and extension of the BAE cells into the vacant area. The antisense oligonucleotide also inhibited the proliferation of BAE cells in the rapidly proliferating phase. In a Boyden chamber assay, pretreatment with the antisense oligonucleotide markedly inhibited the migration of BAE cells. Furthermore, the abilities to proliferate and migrate of BAE cells, which were stably transfected with expression vectors that generate the antisense RNA of CTGF cDNA, were markedly lower than those of the control. These findings suggest that endogenous CTGF expression is involved in the proliferation and migration of BAE cells.

Immunocytochemical expression of type I and type II collagens by rat Meckel's chondrocytes in culture during phenotypic transformation.

In culture, chondrocytes of Meckel's cartilage can differentiate further to become bone-type collagen-synthesizing cells. Here, the replacement of type II collagen by type I collagen, accompanying expression of the osteocytic phenotype, was analysed by double immunofluorescence staining, histochemistry and electron microscopy. After 1 week in culture, formation of a toluidine blue-positive matrix, demonstrating the synthesis of cartilaginous proteoglycans, and the expression of type II collagen were detected. After 2 weeks, immunoreactivity specific for type II collagen was detected along the cartilaginous areas of the nodules, and type I collagen appeared in association with the immunopositive extracellular matrix around spindle-shaped cells. Electron microscopy revealed that the extracellular matrix at this stage was composed of homogeneous fine fibrils of type II collagen and thick cross-banded bundles of type I collagen: there was also continuity between the type I and II collagens. Double immunofluorescence staining of 3 week-old cultures revealed that type II collagen had been replaced by type I which was synthesized by small round cells that appeared at the top of the nodules. With further passage of time in culture, the distribution of type I collagen expanded further towards the peripheral areas from the central areas of the nodules. The present combination of ultrastructural analysis and double immunofluorescence staining shows that the transition from synthesis of cartilage-specific type II collagen to expression of type I collagen occurred sequentially in spindle-shaped cells located at the top of nodules in conjunction with the further differentiation of Meckel's cartilage cells.

Ultrastructural and histochemical changes and apoptosis of inner enamel epithelium in rat enamel-free area.

The formation of an enamel-free area (EFA), a region of the dentin without an enamel cap at the cusp tip of rodent molar, is thought to depend on the specific differentiation and function of inner enamel epithelium of EFAs (EFA cells). The authors attempted to clarify both the ultrastructure and alkaline phosphatase (ALPase) activity of EFA cells up until tooth eruption by using rat mandibular first molars. Apoptosis was also examined. The EFA cells differentiated into secretory cells resembling differentiating ameloblasts but without Tomes' processes (postnatal day 1-3). No reactivity for ALPase was observed in the EFA cells. Enamel-like crystals were detected in close vicinity to dentin crystals at this stage. Thereafter, EFA cells became maturative ameloblast-like with ruffled border-like structures (postnatal day 5-8) and exhibited a strong reactivity for ALPase. These findings suggest that EFA cells change from secretory to absorptive cells within a short period and become reduced enamel epithelium at the early stage of tooth development. Apoptosis occurs in EFA cells, as it does in ameloblasts, but its significance seems to differ between the two cell types.

Split-course accelerated hyperfractionation radiotherapy for advanced head and neck cancer: influence of split time and overall treatment time on local control.

We analyzed 52 patients with stage III and IV head and neck cancer who were given split-course accelerated hyperfractionated radiotherapy with curative intent, focusing particularly on the influence of split-time on local control. An initial complete response was achieved in 16 patients (31%), and the rate of persistent local control at 3 years was 23%. The cause specific survival rate at 3 years was 29%. Univariate analysis of local control according to the split-time duration and overall treatment time showed that shorter duration (< or = 14 days or < or = 45 days, respectively) had a significantly positive impact on local control (P < 0.05). Multivariate analysis using local control as an endpoint also demonstrated that gender (women showing a better outcome than men) and split-time (< or = 14 days was better than > 14 days) were statistically significant factors for local control. These results suggest that shortening the split-time during radiotherapy might improve local control in accelerated hyperfractionation.

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 beta-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.

Sequential synthesis of cartilage and bone marker proteins during transdifferentiation of mouse Meckel's cartilage chondrocytes in vitro.

Meckel's cartilage cells cultured in vitro undergo phenotypic transformation toward osteogenic cells. We examined whether these cells synthesize type X collagen and bone morphogenetic protein-2 (BMP-2). We also examined the results of Alcian blue staining and the expression of type I and type II collagen, osteocalcin and chondroitin sulfate proteoglycan (CSPG) during this transdifferentiation. Meckel's chondrocytes, isolated from day-17 mouse embryos, were inoculated at 1 x 10(4)/penicylinder and cultured in alpha-MEM for periods up to 4 weeks. Alcian blue staining and immunostaining of type II collagen and CSPG confirmed that, after cell culture for 2 weeks, the cartilaginous phenotype was expressed most intensely. Later in culture, chondrocytes underwent modification through the synthesis of bone-type proteins; nodule-forming small round cells showed ALPase activity and were immunoreactive for type I collagen and osteocalcin. Immunoreactivity for type X collagen was detected in the small round cells at the top of the nodules prior to calcification of the matrix, as well as in large hypertrophic cells. BMP-2 was also expressed first in similar small round cells after 3 weeks in culture, and it subsequently extended along the extracellular matrix in the calcified nodules. These results indicate that small round cells that are differentiating toward osteocyte-like cells from Meckel's chondrocytes express type X collagen and BMP-2 sequentially.

Characterization of the inner enamel epithelium in the enamel-free area based on the ability to secrete enamel protein demonstrated by in situ hybridization and immunohistochemistry.

Both the expression of amelogenin mRNA and secretion of amelogenin were investigated in rat molars by in situ hybridization and immunohistochemistry. Probes were designed by multiple-labeling of oligonucleotide probes for in situ hybridization. Amelogenin mRNA first appeared in differentiating ameloblasts of the distal region and some inner enamel epithelial cells of enamel-free area (EFA cells) of the second cusp at postnatal day 0. At the same time, amelogenin protein was detected in the extracellular matrix between dentin and differentiating ameloblasts and in some EFA cells of the second cusp. At postnatal day 1-3, amelogenin was expressed in the secretory ameloblasts, and in the matrix beneath these cells. Both amelogenin mRNA and amelogenin were detected in the EFA cells and their extracellular matrix. After postnatal day 5, amelogenin mRNA and amelogenin were detected in the secretory ameoloblasts and extracellular matrix in the enamel-forming region, respectively. At this time, amelogenin mRNA was not detected in the EFA cells, but a small amount of amelogenin was found in the matrix beneath the EFA cells. These findings suggest that EFA cells differentiate into amelogenin-secreting cells, i.e. ameloblasts, but that the secretion lasts for only a short period at the early stage of tooth development.

A case of pulmonary ossified adenocarcinoma with marked osteoplastic bone metastasis.

We report the case of a 53-year-old man suffering from a pulmonary adenocarcinoma with ossification and diffuse metastatic osteoplastic lesions throughout the skeletal system. This is a rare condition in lung carcinomas. Radiographs of the chest and bones demonstrated mineral densities in the primary tumor and multiple expansive osseous lesions with a diffuse sclerotic pattern resembling multiple bone metastases from prostatic carcinoma.

Clinical experience of radiation therapy for Graves' ophthalmopathy.

The effect of radiation therapy for Graves' ophthalmopathy was evaluated. Ten patients with Graves' ophthalmopathy were treated with radiation therapy between 1992 and 1993 in Gunma University Hospital. All patients had a past history of hyperthyroidism and received 2,000 cGy to the retrobulbar tissues in 20 fractions. Nine of ten patients were treated with radiation therapy after the failure of corticosteroids. Six patients (60%) showed good or excellent responses. The exophthalmos type was more responsive to radiation therapy than the double vision type in this series. Two of five patients with the exophthalmos type demonstrated excellent responses, and their symptoms disappeared almost completely. The improvement of symptoms appeared within 3-6 months, and obvious clinical effects were demonstrated after 6 months of radiotherapy. Radiation therapy was well tolerated, and we have not observed any side effects of radiation therapy. In conclusion, radiation therapy is effective treatment for Graves' ophthalmopathy.

In vitro characterization of enamel epithelium and pulp cells in mouse tooth germs.

Mixed cell populations consisting of enamel epithelium and pulp cells obtained from 18-day embryonic mouse tooth germs were cultured in vitro. Epithelial cells and pulp cells were also cultured individually and examined. Isolated cells were cultured in alpha-MEM supplemented with 10% fetal bovine serum for up to four weeks and examined morphologically using histological procedures including immunostaining, light and electron microscopy, and electron probe microanalysis. The pulp cells proliferated and differentiated in the absence of epithelium, but the number of epithelial cells showed a strong dependence on the pulp cells. Pulp cells showing fibroblastic morphology in the mixed culture gradually became elliptical, and eventually transformed into spherical cells surrounded by a calcified extracellular matrix. Alkaline phosphatase (ALPase) activity was expressed on the pulp cells prior to calcification of the extracellular matrix, as shown by von Kossa's and alizarin-red staining. Calcification deposition, which is closely associated with thick banded type-I collagen fibrils, was shown to be composed of calcium and phosphorous using electron probe microanalysis. Type-I collagen immunoreactivity was detected on the extracellular matrix after two weeks of culturing. The present results show that the proliferation and differentiation of pulp cells, and subsequent calcification of the extracellular matrix occur in the presence or absence of epithelial elements, but that the proliferation of epithelial cells depends on the presence of pulp cells.

Mouse Meckel's cartilage chondrocytes evoke bone-like matrix and further transform into osteocyte-like cells in culture.

BACKGROUND: We reported that when Meckel's cartilage was transplanted ectopically, chondrocytes transformed into osteocyte-like cells accompanying the extracellular calcified matrix. However, we could not determine whether the osteocyte-like cells were derived from host tissues or from Meckel's cartilage itself. Therefore, we examined whether the Meckel's cartilage chondrocytes, which have a retrogressive ultimate fate, are capable of inducing the observed calcification and further transform into osteocyte-like cells in culture. METHODS: Meckelian chondrocytes isolated enzymatically were plated at a low density and grown in alpha-MEM containing 10% FBS at 37 degrees C under 5% CO2 in air for up to 4 weeks. RESULTS: Chondrocytes were fibroblast-like cells early in culture, but gradually transformed from polygonal cells into typical chondrocytes showing metachromasia with toluidine blue staining. After an additional week of culture, the chondrocytes transformed from large to small round cells accompanying nodule formations. Small round cells multiple-layered actively, and showed more intense alkaline phosphatase (ALPase) activity. Immunostaining identified type II collagen in the extracellular matrix at 2 weeks of culture, and type I collagen and osteocalcin were later synthesized by round cells. von Kossa's reaction showed extensive precipitation of calcification throughout the flocculent materials. Ultrastructural analysis showed that the cells surrounded by calcified matrix strongly resembled osteocytes. CONCLUSIONS: The present study suggested that the Meckel's cartilage chondrocytes can express the osteocyte-like phenotype in vitro during synthesis of bone-type marker proteins such as osteocalcin or type I collagen.

Chondrogenic differentiation of clonal mouse embryonic cell line ATDC5 in vitro: differentiation-dependent gene expression of parathyroid hormone (PTH)/PTH-related peptide receptor.

The regulatory role of parathyroid hormone (PTH)/PTH-related peptide (PTHrP) signaling has been implicated in embryonic skeletal development. Here, we studied chondrogenic differentiation of the mouse embryonal carcinoma-derived clonal cell line ATDC5 as a model of chondrogenesis in the early stages of endochondral bone development. ATDC5 cells retain the properties of chondroprogenitor cells, and rapidly proliferate in the presence of 5% FBS. Insulin (10 micrograms/ml) induced chondrogenic differentiation of the cells in a postconfluent phase through a cellular condensation process, resulting in the formation of cartilage nodules, as evidenced by expression of type II collagen and aggrecan genes. We found that differentiated cultures of ATDC5 cells abundantly expressed the high affinity receptor for PTH (Mr approximately 80 kD; Kd = 3.9 nM; 3.2 x 10(5) sites/cell). The receptors on differentiated cells were functionally active, as evidenced by a PTH-dependent activation of adenylate cyclase. Specific binding of PTH to cells markedly increased with the formation of cartilage nodules, while undifferentiated cells failed to show specific binding of PTH. Northern blot analysis indicated that expression of the PTH/PTHrP receptor gene became detectable at the early stage of chondrogenesis of ATDC5 cells, preceding induction of aggrecan gene expression. Expression of the PTH/PTHrP receptor gene was undetectable in undifferentiated cells. The level of PTH/PTHrP receptor mRNA was markedly elevated parallel to that of type II collagen mRNA. These lines of evidence suggest that the expression of functional PTH/PTHrP receptor is associated with the onset of chondrogenesis. In addition, activation of the receptor by exogenous PTH or PTHrP significantly interfered with cellular condensation and the subsequent formation of cartilage nodules, suggesting a novel site of PTHrP action.