Differentiation Of Cartilage Cells Research Articles

MicroRNA-138: an emerging regulator of skeletal development, homeostasis, and disease.

Noncoding microRNAs are powerful epigenetic regulators of cellular processes by their ability to target and suppress expression of numerous protein-coding mRNAs. This multitargeting function is a unique and complex feature of microRNAs. It is now well-described that microRNAs play important roles in regulating the development and homeostasis of many cell/tissue types, including those that make up the skeletal system. In this review, we focus on microRNA-138 (miR-138) and its effects on regulating bone and cartilage cell differentiation and function. In addition to its reported role as a tumor suppressor, miR-138 appears to function as an inhibitor of osteoblast differentiation. This review provides additional information on studies that have attempted to alter miR-138 expression in vivo as a means to dampen ectopic calcification or alter bone mass. However, a review of the published literature on miR-138 in cartilage reveals a number of contradictory and inconclusive findings with respect to regulating chondrogenesis and chondrocyte catabolism. This highlights the need for more research in understanding the role of miR-138 in cartilage biology and disease. Interestingly, a number of studies in other systems have reported miR-138-mediated effects in dampening inflammation and pain responses. Future studies will reveal if a multifunctional role of miR-138 involving suppression of ectopic bone, inflammation, and pain will be beneficial in skeletal conditions such as osteoarthritis and heterotopic ossification.

Heterotopic ossification ‐ replacement of one tissue with another

Our body is composed of multiple organ systems that use specific types of cells for specialized functions. Bone and cartilage cells are normally restricted to the skeleton, however in heterotopic ossification, osteogenesis and chondrogenesis occurs within tissues such as skeletal muscle, tendons, and ligaments, and leads to the formation of bone tissue outside of the skeleton. Heterotopic ossification is most commonly associated with severe trauma such as following hip arthroplasty, traumatic brain and spinal cord injury, and high impact blast injury. It is difficult to predict who will respond to these traumas by forming heterotopic bone, and there are currently no effective preventions or treatments. However, genetic forms of heterotopic ossification are providing new insights into the cellular and molecular mechanisms that control postnatal de novo formation of bone. Our investigations of a rare genetic disorder of heterotopic ossification, fibrodysplasia ossificans progressive (FOP), have not only identified key cell signaling pathways that regulate bone and cartilage cell differentiation, but also revealed the importance of the tissue microenvironment in supporting and directing aberrant cell differentiation. Our data support that heterotopic ossification involves more than mis‐directed cell differentiation, but is a multi‐step, multi‐cell type process leading to new tissue formation.

Non-Coding RNAs in Cartilage Development: An Updated Review.

In the development of the skeleton, the long bones are arising from the process of endochondral ossification (EO) in which cartilage is replaced by bone. This complex process is regulated by various factors including genetic, epigenetic, and environmental elements. It is recognized that DNA methylation, higher-order chromatin structure, and post-translational modifications of histones regulate the EO. With emerging understanding, non-coding RNAs (ncRNAs) have been identified as another mode of EO regulation, which is consist of microRNAs (miRNAs or miRs) and long non-coding RNAs (lncRNAs). There is expanding experimental evidence to unlock the role of ncRNAs in the differentiation of cartilage cells, as well as the pathogenesis of several skeletal disorders including osteoarthritis. Cutting-edge technologies such as epigenome-wide association studies have been employed to reveal disease-specific patterns regarding ncRNAs. This opens a new avenue of our understanding of skeletal cell biology, and may also identify potential epigenetic-based biomarkers. In this review, we provide an updated overview of recent advances in the role of ncRNAs especially focus on miRNA and lncRNA in the development of bone from cartilage, as well as their roles in skeletal pathophysiology.

PPARD is an Inhibitor of Cartilage Growth in External Ears.

Peroxisome proliferator-activated receptor beta/delta (PPARD) is an important determinant of multiple biological processes. Our previous studies identified a missense mutation in the PPARD gene that significantly reduces its transcription activity, and consequently causes enlarged external ears in pigs. However, the mechanisms underlying the causality has remained largely unknown. Here, we show that PPARD retards the development of auricular cartilage by accelerating the apoptosis of cartilage stem/progenitor cells (CSPCs), the terminal differentiation of cartilage cells and the degradation of cartilage extracellular matrix in the auricle. At the transcription level, PPARD upregulates a set of genes that are associated with CSPCs apoptosis and chondrogenic differentiation, chondroblast differentiation and extracellular matrix degradation. ChIP-seq identified direct target genes of PPARD, including a well-documented gene for cartilage development: PPARG. We further show that compared to wild-type PPARD, the G32E mutant up-regulates the expression of PPARG and subsequently leads to the downregulation of critical genes that inhibit cartilage growth. These findings allow us to conclude that PPARD is an inhibitor of auricular cartilage growth in pigs. The causative mutation (G32E) in the PPARD gene attenuates the PPARD-mediated retardation of cartilage growth in the auricle, contributing to enlarged ears in pigs. The findings advance our understanding of the mechanisms underlying auricular development in mammals, and shed insight into the studies of innate pinna disorders and cartilage regeneration medicine in humans.

Expression of genes encoding matrilin-3 and cyclin-I during the impairment and recovery of chicken growth plate in tibial dyschondroplasia.

Tibial dyschondroplasia (TD) is a skeletal disease characterized by the disruption ofendochondral bone formation in avian species. The aim of this study was to determine the expression of Matrilin-3 and Cyclin-I genes in chicken growth plate during impairment and recovery from TD. Gene expressions were analyzed by polymerase chain reaction, and proteins by immunohistochemistry and in situ hybridizations. Expression of genes encoding Matrilin-3 and Cyclin-I were diminished with parallel decrease in proteins during TD, with fewer signs of cartilage cell differentiation. In contrast, there was an increase in mRNA expressions and protein levels of both genes during the recovery phase. These findings suggest that the Matrilin-3 and Cyclin-I genes also play a role in chondrocyte differentiation during the impairment and recovery of growth plate in TD.

Osteogenic and chondrogenic potential of biomembrane cells from the PMMA‐segmental defect rat model

A layer of cells (the "biomembrane") has been identified in large segmental defects between bone and surgically placed methacrylate spacers or antibiotic-impregnated cement beads. We hypothesize that this contains a pluripotent stem cell population with potential valuable applications in orthopedic tissue engineering. Objectives using biomembranes harvested from rat segmental defects were to: (1) Culture biomembrane cells in specialized media to direct progenitor cells along bone or cartilage cell differentiation lineages; (2) evaluate harvested biomembranes for mesenchymal stem cell markers, and (3) define relevant gene expression patterns in harvested biomembranes using microarray analysis. Culture in osteogenic media produced mineralized nodules; culture in chondrogenic media produced masses containing chondroitin sulfate/sulfated proteoglycans. Molecular analysis of biomembrane cells versus control periosteum showed significant upregulation of key genes functioning in mesenchymal stem cell differentiation, development, maintenance, and proliferation. Results identified significant upregulation of WNT receptor signaling pathway genes and significant upregulation of BMP signaling pathway genes. Findings confirm that the biomembrane has a pluripotent stem cell population. The ability to heal large bone defects is clinically challenging, and novel tissue engineering uses of the biomembrane hold great promise in treating non-unions, open fractures with large bone loss and/or infections, and defects associated with tumor resection.

Nfatc2 is a primary response gene of Nell-1 regulating chondrogenesis in ATDC5 cells.

Nell-1 is a growth factor required for normal skeletal development and expression of extracellular matrix proteins required for bone and cartilage cell differentiation. We identified the transcription factor nuclear factor of activated T cells (Nfatc2) as a primary response gene of Nell-1 through a microarray screen, with validation using real-time polymerase chain reaction (PCR). We investigated the effects of recombinant Nell-1 protein on the chondrogenic cell line ATDC5 and primary mouse chondrocytes. The osteochondral transcription factor Runx2 was investigated as a possible intermediary between Nell-1 and Nfatc2 using adenoviral overexpression of wild-type and dominant-negative Runx2. Nell-1 transiently induced both transcription and translation of Nfatc2, an effect inhibited by transduction of dominant-negative Runx2, suggesting that Runx2 was necessary for Nfatc2 induction. Differentiation assays revealed inhibitory effects of Nell-1 on ATDC5 cells. Although proliferation was unaffected, expression of chondrocyte-specific genes was decreased, and cartilage nodule formation and proteoglycan accumulation were suppressed. siRNA knockdown of Nfatc2 significantly reversed these inhibitory effects. To elucidate the relationship between Nell-1, Runx2, and Nfatc2 in vivo, their presence and distribution were visualized in femurs of wild-type and Nell1-deficient mice at both neonatal and various developmental stages using immunohistochemistry. All three proteins colocalized in the perichondrium of wild-type femurs but stained weakly or were completely absent in Nell1-deficient femurs at neonatal stages. Thus Nfatc2 likely plays an important role in Nell-1-mediated osteochondral differentiation in vitro and in vivo. To our knowledge, this is the first demonstration that Nfatc2 is a primary response gene of Nell-1.

An impact of masticatory muscle function on IL-1β and Sox9 expression in condyle

The importance of masticatory muscle function on the growth and differentiation of condyle was examined. The aim of this study was to investigate how reduced masticatory muscle function influences intrinsic regulatory factors which govern growth and differentiation of condylar cartilage. Masseter muscles of 3-week-old Wistar rats were resected bilaterally. Masseteric resected animals and corresponding control animals were sacrificed at 3, 6, 12 and 21 days post-resection. The condyles were then processed for histological and immunohistochemical analysis. The expression patterns of an inhibitory regulator (IL-1beta) and a master regulator (Sox9) of chondrogenesis in condylar cartilage of growing rats were investigated. Quantitative analysis shows that masseteric resection significantly increased the number of IL-1beta positive cells in proliferative layer. In contrast, the number of Sox9 positive cells was significantly decreased compared to the control animals. It can be concluded that the reduced articular function due to masseteric resection decreased condylar cartilage cell differentiation which led to the decrease in the thickness of condylar cartilage.

Interaction of AP-1 and the ctgf gene: a possible driver of chondrocyte hypertrophy in growth cartilage.

The expression of the connective tissue growth factor ( ctgf) gene increases along with the differentiation of growth cartilage cells, and the highest expression is observed in the hypertrophic stage. Similarly, recent reports demonstrated c- fos expression in chondrocytes in the early hypertrophic zone of growth cartilage, and suggested that the c- fos gene may play a crucial role in the regulation of hypertrophic differentiation. A chondrocytic human cell line, HCS-2/8, is known to retain a variety of chondrocytic phenotypes. When such cells were kept overconfluent, they expressed increasing levels of c- fos transcripts along a time course phenotypically similar to that of hypertrophic differentiation. Moreover, by using a competitive electromobility-shift assay, we found that AP-1, a Fos/Jun heterodimer, in HCS-2/8 was capable of binding not only to a typical AP-1-binding DNA fragment but also to the enhancer fragment of the ctgf gene. Based on the findings above, we hypothesize that, prior to hypertrophic differentiation, AP-1-related oncogenes are activated and that their gene products subsequently activate ctgf gene expression, which might eventually induce hypertrophy.

Expression of connective tissue growth factor in cartilaginous tumors

Connective tissue growth factor (CTGF) predominantly is expressed in hypertrophic chondrocytes and its specific receptors are demonstrated on chondrocytic cells. Therefore, CTGF may be involved in the proliferation and/or differentiation of cartilage cells. In the current study, CTGF expression was examined both in chondrosarcoma and enchondroma to clarify the relation between the expression of CTGF and the grade of malignancy. The expression of CTGF and proliferating cell nuclear antigen (PCNA) were analyzed immunohistochemically in 34 cartilaginous tumor specimens. Eighteen tumors were determined to be chondrosarcoma including 8 Grade 1 tumors, 6 Grade 2 tumors, and 4 Grade 3 tumors. The percentage of CTGF positive and PCNA positive cells was quantified using at least 500 cells. CTGF was expressed in 70.1% of enchondroma cells, 84.0% of Grade 1 chondrosarcoma cells, 53.7% of Grade 2 tumor cells, and 26.8% of Grade 3 tumor cells (rho = -0.501; P = 0.0053). In chondrosarcoma cases, CTGF expression was correlated closely with tumor grade (rho = -0.920; P = 0.0001). There was a strong correlation between PCNA expression and tumor grade (rho = 0.907; P < 0.0001) and a strong negative correlation between CTGF and PCNA expression (rho = -0.493; P = 0.0061). In chondrosarcoma cases, patients with high expression of CTGF (>/= 30%) showed higher overall survival compared with those with low expression (< 30%) (P = 0.004). The current study revealed a correlation between the histologic grade of chondrosarcoma and prognosis, and the concomitant association between CTGF immunostaining and tumor grade and prognosis. Therefore, immunohistochemical staining with CTGF is a useful procedure for assessing the tumor grade and clinical course in patients with chondrosarcoma.

Effect of metabolic acidosis on the growth hormone/IGF-I endocrine axis in skeletal growth centers

Chronic metabolic acidosis (CMA) adversely affects bone metabolism and skeletal growth. Given the cardinal role played by the local growth hormone (GH)/insulin-like growth factor-I (IGF-I) in promoting cell proliferation and differentiation in growth plates, we tested the effect of CMA on the GH/IGF-I axis in a skeletal growth center. We employed an in vitro organ culture system using the murine mandibular condyle as a model for endochondral active growth center. Condyles from six-day-old ICR mice were cultured in BGJb medium of either neutral pH (pH approximately 7.4) or acidic pH (pH approximately 7.15). After 24, 48, 72, and 96 hours of culture, the condyles were washed, fixed in formaldehyde, and processed for paraffin embedding. We assessed histologic markers of the growth center. In addition, the protein level and mRNA expression for the different components of the GH/IGF-I axis were evaluated by immunohistochemistry and in situ hybridization, respectively. Finally, we evaluated the effect of acidosis on the biological functions mediated by GH and IGF-I (namely, proliferation and differentiation of cartilage cells in the active growth center). Following three to four days in acidic conditions, there was a marked reduction in the size of young chondrocytic population, suggesting a defect in the process of endochondral differentiation. Immunohistochemistry and in situ hybridization analyses revealed a marked reduction in the expression of the IGF-I receptor, as well as in the GH receptor. These changes were already evident after 48 hours of incubation in acidic conditions. At 48 hours of acidosis, there was also a marked reduction in the expression of IGF-I both under basal conditions (nonstimulated) and following stimulation with GH. The expression of IGF binding protein 2 (IGFBP-2) and IGFBP-4, which serve as negative modulators of IGF-I, was enhanced in CMA. IGF-I markedly stimulated chondrocytic proliferation (assessed by BrdU incorporation into DNA) and differentiation (assessed as cartilage specific proteoglycan expression). These responses were markedly attenuated in acidic conditions. CMA exerts an anti-anabolic effect in bone growth centers, which is partly related to a state of resistance to GH and IGF-I, created by CMA. This phenomenon may underlie the disturbance in longitudinal bone growth in CMA (that is, renal tubular acidosis) and may contribute to renal osteodystrophy in patients suffering from chronic renal failure.

Expression of Trk receptors during cartilage differentiation.

The products of the tyrosine kinase trk family of proto-oncogenes bind neurotrophins (NTFs) and are components of their high-affinity receptors. The trk gene encodes for the TrkA transmembrane glycoprotein, which functions as a receptor for nerve growth factor (NGF). Brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4) are the preferred ligands for TrkB. and neurotrophin-3 (NT-3) for TrkC (reviewed in reference 1). The presence of Trk receptors in nonneuronal cells of developing tissues and organs indicates that these molecules may play a role in embryonic development that would be distinct from a neurotrophic function. Cartilage development involves mesoderm-derived cells (chondrocytes) that contribute to appositional growth of the limb skeleton. Chondrocytes synthesize macromolecules characteristic of cartilage such as collagen type I1 and sulfated proteoglycans. The majority of studies dealing with cartilage cell differentiation have been carried out on mesenchymal cells isolated from limb buds.* However, the molecular mechanisms regulating cartilage formation are not filly understood. In the present study we analyzed the expression of Trk receptors during mouse limb development in vivo and in micromass cultures in vitro. Because little is known about their regulation, we studied the effects of various growth factors on expression of Trk receptors in vitro. F 1 (CBA x NMRI) and Balb C mouse embryos (E 10-El 8) were used. Immunohistochemistry was performed according to Mitsiadis et aL3 Fivepm sections were incubated with TrkA (B. B. Rudkin), TrkB (Santa Cruz Biotechnology, USA), and TrkC (D. Martin-Zanca) affinity purified antibodies against the extracellular domain of the receptors, and cTrk (Santa Cruz Biotechnology, USA) an-

Treatment of resting zone chondrocytes with 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] induces differentiation into a 1,25-(OH)2D3-responsive phenotype characteristic of growth zone chondrocytes

Vitamin D3 metabolites affect the proliferation and differentiation of cartilage cells. Previous reports have shown that rat costochondral cartilage chondrocytes isolated from the growth zone (GC) respond to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], whereas those from the resting zone (RC) respond to 24,25-(OH)2D3. The aim of the present study was to determine whether 24,25-(OH)2D3 induces differentiation of RC cells into a 1,25-(OH)2D3-responsive GC phenotype. To do this, confluent, fourth passage RC chondrocytes were pretreated for 24, 36, 48, 72, and 120 h with 10(-7) M 24,25-(OH)2D3. The medium was then replaced with new medium containing 10(-10) to 10(-8) M 1,25-(OH)2D3, and the cells were incubated for an additional 24 h. At harvest, DNA synthesis was measured as a function of [3H]thymidine incorporation; cell maturation was assessed by measuring alkaline phosphatase (ALPase) specific activity. Incorporation of [3H]uridine was used as a general indicator of RNA synthesis. Matrix protein synthesis was assessed by measuring incorporation of [3H]proline into collagenase-digestible protein (CDP) and collagenase-nondigestible protein (NCP) as well as 35SO4 incorporation into proteoglycans. When RC cells were pretreated for 24 h with 24,25-(OH)2D3, they responded like RC cells that had received no pretreatment; further treatment of these cells with 1,25-(OH)2D3 had no effect on ALPase, proteoglycan, or NCP production, but CDP production was inhibited. However, when RC cells were pretreated for 36-120 h with 24,25-(OH)2D3, treatment with 1,25-(OH)2D3 caused a dose-dependent increase in ALPase, CDP, and proteoglycan synthesis, with no effect on NCP production. RC cells pretreated with 1,25-(OH)2D3 responded like RC cells that had not received any pretreatment. To determine whether these responses were specific to chondrocytes in the endochondral pathway, cells were isolated from the xiphoid process, a hyaline cartilage. In these cells, 1,25-(OH)2D3 inhibited ALPase, whereas 36 h of pretreatment with 24,25-(OH)2D3 caused these cells to lose their response to 1,25-(OH)2D3. These results indicate that 24,25-(OH)2D3 can directly regulate the differentiation and maturation of RC chondrocytes into GC chondrocytes, as evidenced by increased responsiveness to 1,25-(OH)2D3. 24,25-(OH)2D3 also promotes differentiation of cells derived from xiphoid cartilage, resulting in the loss of 1,25-(OH)2D3 responsiveness. These observations support the hypothesis that 24,25-(OH)2D3 plays a significant role in cartilage development.

Treatment of resting zone chondrocytes with 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] induces differentiation into a 1,25-(OH)2D3-responsive phenotype characteristic of growth zone chondrocytes.

Vitamin D3 metabolites affect the proliferation and differentiation of cartilage cells. Previous reports have shown that rat costochondral cartilage chondrocytes isolated from the growth zone (GC) respond to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], whereas those from the resting zone (RC) respond to 24,25-(OH)2D3. The aim of the present study was to determine whether 24,25-(OH)2D3 induces differentiation of RC cells into a 1,25-(OH)2D3-responsive GC phenotype. To do this, confluent, fourth passage RC chondrocytes were pretreated for 24, 36, 48, 72, and 120 h with 10(-7) M 24,25-(OH)2D3. The medium was then replaced with new medium containing 10(-10) to 10(-8) M 1,25-(OH)2D3, and the cells were incubated for an additional 24 h. At harvest, DNA synthesis was measured as a function of [3H]thymidine incorporation; cell maturation was assessed by measuring alkaline phosphatase (ALPase) specific activity. Incorporation of [3H]uridine was used as a general indicator of RNA synthesis. Matrix protein synthesis was assessed by measuring incorporation of [3H]proline into collagenase-digestible protein (CDP) and collagenase-nondigestible protein (NCP) as well as 35SO4 incorporation into proteoglycans. When RC cells were pretreated for 24 h with 24,25-(OH)2D3, they responded like RC cells that had received no pretreatment; further treatment of these cells with 1,25-(OH)2D3 had no effect on ALPase, proteoglycan, or NCP production, but CDP production was inhibited. However, when RC cells were pretreated for 36-120 h with 24,25-(OH)2D3, treatment with 1,25-(OH)2D3 caused a dose-dependent increase in ALPase, CDP, and proteoglycan synthesis, with no effect on NCP production. RC cells pretreated with 1,25-(OH)2D3 responded like RC cells that had not received any pretreatment. To determine whether these responses were specific to chondrocytes in the endochondral pathway, cells were isolated from the xiphoid process, a hyaline cartilage. In these cells, 1,25-(OH)2D3 inhibited ALPase, whereas 36 h of pretreatment with 24,25-(OH)2D3 caused these cells to lose their response to 1,25-(OH)2D3. These results indicate that 24,25-(OH)2D3 can directly regulate the differentiation and maturation of RC chondrocytes into GC chondrocytes, as evidenced by increased responsiveness to 1,25-(OH)2D3. 24,25-(OH)2D3 also promotes differentiation of cells derived from xiphoid cartilage, resulting in the loss of 1,25-(OH)2D3 responsiveness. These observations support the hypothesis that 24,25-(OH)2D3 plays a significant role in cartilage development.

A New Avian Fibroblast Growth Factor Receptor in Myogenic and Chondrogenic Cell Differentiation

We studied the expression of FREK (fibroblast growth factor receptor-like embryonic kinase), a new receptor recently cloned from quail embryo, during the differentiation of skeletal muscle satellite cells and epiphyseal growth-plate chondrocytes. Although FREK mRNA was expressed in both cell types, satellite cells expressed higher levels of this mRNA than chondrocytes. FREK gene expression was found to be modulated by b-FGF in a biphasic manner: low concentrations increased expression, whereas high concentrations attenuated it. In both cell cultures, the levels of FREK mRNA declined during terminal differentiation. Moreover, retinoic acid (RA), which induces skeletal muscle satellite cells to differentiate, also caused a reduction in FREK gene expression in these cells. Induction of chondrocyte differentiation with ascorbic acid was monitored by a decrease in collagen type II gene expression and an increase in alkaline phosphatase activity. Satellite cell differentiation was marked by morphological changes as well as by increased sarcomeric myogenin content and creatine kinase activity and changes in the expression of the regulatory muscle-specific genes, MyoD and myogenin. DNA synthesis in both cell types was stimulated by b-FGF. However, in satellite cells, the response was bell-shaped, peaking at 1 ng/ml b-FGF, whereas in chondrocytes, higher levels of b-FGF were needed. b-FGF-dependent DNA synthesis in satellite cells was decreased by RA at concentrations over 10 -7 M . The observed correlation between the level of FREK gene expression and various stages of differentiation, its modulation by b-FGF and RA, as well as the correlation between FREK gene expression and the physiological response to b-FGF, suggest that this specific FGF receptor plays an important role in muscle and cartilage cell differentiation.

Modulation of tensin and vimentin expression in chick embryo developing cartilage and cultured differentiating chondrocytes.

It has been proposed that tensin, in association with several other proteins, mediates the micro-filament-integrin link. Here we describe the isolation of clones spanning about 5 kb from the 3' end of tensin mRNA from cultured chick embryo chondrocyte and embryonic heart cDNA libraries. Tensin expression was investigated in cultured chick embryo cells. It was observed that tensin expression is dependent upon substrate adhesion and it is turned off after 7 days of suspension culture. This process is reversible. Tensin expression is also regulated during cartilage cell differentiation in vivo; at Hamburger and Hamilton stage 39-40, non-hypertrophic tibial chondrocytes express both RNA and protein while hypertrophic chondrocytes do not. In the culture system the expression of vimentin, a major component of intermediate filaments, showed an opposite behaviour since the suspension culture enhances the accumulation of both vimentin and its mRNAs. Therefore in chick embryo cultured chondrocytes and in vivo, during cartilage development, cell shape changes and/or integrin-extracellular matrix protein interactions may be involved in the regulation of these two genes coding for cytoskeletal proteins.

Heat-shock response in cultured chick embryo chondrocytes. Osteonectin is a secreted heat-shock protein.

We investigated the induction of specific protein expression by heat shock in dedifferentiated and hypertrophic chick embryo chondrocytes in a culture system that allows 'in vitro' differentiation of cartilage cells [Castagnola, P., Moro, G., Descalzi-Cancedda, F. and Cancedda, R. (1986) J. Cell. Biol. 102, 2310-2317]. As control, we used cultures of embryonic fibroblasts from the whole body and from the skin. In the cell lysates of all cultures we identified four major heat-shock proteins (HSP), with a molecular size corresponding to HSP families previously described (HSP 90, HSP 70, HSP 47 and HSP 26). Some of these proteins were constantly induced when the temperature was raised, others were expressed in a more variable manner. Differences also existed in the relative amount of the HSP synthesized by the four cultures. When we specifically investigated HSP species released into the culture medium, we observed a 43-45 kDa protein constantly expressed and secreted in large amount by the cells. On the basis of its biochemical characteristic and its precipitation by specific antibodies, this protein has been identified as osteonectin (SPARC, BM-40).

The Role of the Growth Plate in Longitudinal Bone Growth

The epiphyseal growth plate is the main site of longitudinal growth of the long bones. At this site, cartilage is formed by the proliferation and hypertrophy of cells and synthesis of the typical extracellular matrix. The formed cartilage is then calcified, degraded, and replaced by osseous tissue. Proliferation and differentiation of cartilage cells (i.e., chondrocytes) as studied mostly in culture, is regulated by various endocrine, paracrine, and autocrine agents such as growth hormone, insulin-like growth factor-I (IGF-I), transforming growth factor (TGE-β), and vitamin D metabolites (1,25-dihydroxycholecalciferol and 24,25-dihydroxycholecalciferol). Avian chondrocyte proliferation is enhanced by agents which use adenosine 3′:5′-cyclic monophosphate as a second messenger, such as parathyroid hormone or prostaglandin-E2, and is depressed by guanosine 3′:5′-cyclic monophosphate agonists, such as atrial natriuretic peptide. Several of the regulating agents also affect synthesis of the main extracellular components (i.e., collagen and proteoglycans) and their transfer to the extracellular space. Cartilage calcification involves matrix vesicles secreted by the chondrocytes at a specific stage. Calcification probably involves some initial nucleation agent and participation of phosphatases. During sexual maturation, the growth plate closes by an unknown mechanism and longitudinal bone growth ceases. Disorders in the metabolism of the controlling agents or the cellular responses in growth plate may lead to several deformities classified as dysplasias. In poultry, this class of disorders is represented by chondrodystrophy and dyschondroplasia.

The determination of myogenic and cartilage cells in the early chick embryo and the modifying effect of retinoic acid.

The time of determination of cartilage and skeletal muscle was studied by making chimeric grafts or explants of small tissue pieces from several stages of early chick or quail embryos. Chondrogenesis was assessed by histology or with antibodies directed against type II collagen or cartilage proteoglycan, while myogenesis was detected immunohistochemically with antibodies directed against 3 different muscle markers, including muscle myosin. Grafts from Hensen's node, primitive streak and segmental plate of donor embryos of Stage 3-5 (Hamburger and Hamilton) were transplanted under the ectoderm in the extraembryonic area of Stage 12 host embryos. In addition, explants and mesodermal cells were cultured on glass in DMEM+F12 medium supplemented with 10% FCS. The results showed that determined myogenic cells could first be detected in Hensen's node and the primitive streak at Stage 3+-4 and that they developed from mesodermal cells located between the epiblast and hypoblast. Myogenic cells also appeared in grafted and explanted segmental plate with or without notochord from Stage 5 embryos. On the other hand, cartilage cells only formed in grafted and explanted segmental plate that also contained notochord. RA (1 ng/ml) could induce the formation of cartilage cells in the explanted primitive streak without Hensen's node or notochord taken from Stage 3-5 embryos and could also promote the differentiation of myogenic cells in primitive streak from Stage 3 embryo. Thus RA can substitute for Hensen's node or the notochord in the induction of cartilage cells and has some stimulatory effects on the differentiation of myogenic cells. Additional evidence indicates that the hypoblast might play an "inductive" role in the formation of the notochord which may subsequently promote the differentiation of cartilage cells.

Immunocytochemical detection of S-100 protein in rat mandibular condylar cartilage

Light and electron microscopic immunocytochemistry was used to localize S-100 protein. It was demonstrated in chondrocytes of the proliferative and hypertrophic zones but none could be found in the fibrous articular layer. The staining intensity for S-100 protein was stronger in the hypertrophic cells than in chondrocytes from the proliferative zone. Ultrastructurally, immunoreactive gold particles were detected in the cytosolic region, closely apposed to the profiles of endoplasmic reticulum with occasional dense aggregations. A few gold particles were seen on the nuclear chromatin and on condensing vacuoles of Golgi complexes. Because of the calcium-binding and possibly fatty acid-binding properties of the protein molecule, the immunocytochemical expression of S-100 protein in the chondrocytes may be involved in cartilage cell differentiation, metabolism and mineralization