Differentiation Of Growth Plate Chondrocytes Research Articles

Type B γ-Aminobutyric Acid Receptors Modulate the Function of the Extracellular Ca2+-Sensing Receptor and Cell Differentiation in Murine Growth Plate Chondrocytes

Extracellular calcium-sensing receptors (CaRs) and metabotropic or type B gamma-aminobutyric acid receptors (GABA-B-Rs), two closely related members of family C of the G protein-coupled receptor superfamily, dimerize in the formation of signaling and membrane-anchored receptor complexes. We tested whether CaRs and two GABA-B-R subunits (R1 and R2) are expressed in mouse growth plate chondrocytes (GPCs) by PCR and immunocytochemistry and whether interactions between these receptors influence the expression and function of the CaR and extracellular Ca(2+)-mediated cell differentiation. Both CaRs and the GABA-B-R1 and -R2 were expressed in the same zones of the growth plate and extensively colocalized in intracellular compartments and on the membranes of cultured GPCs. The GABA-B-R1 co-immunoprecipitated with the CaR, confirming a physical interaction between the two receptors in GPCs. In vitro knockout of GABA-B-R1 genes, using a Cre-lox recombination strategy, blunted the ability of high extracellular Ca(2+) concentration to activate phospholipase C and ERK1/2, suppressed cell proliferation, and enhanced apoptosis in cultured GPCs. In GPCs, in which the GABA-B-R1 was acutely knocked down, there was reduced expression of early chondrocyte markers, aggrecan and type II collagen, and increased expression of the late differentiation markers, type X collagen and osteopontin. These results support the idea that physical interactions between CaRs and GABA-B-R1s modulate the growth and differentiation of GPCs, potentially by altering the function of CaRs.

Gradients in bone morphogenetic protein-related gene expression across the growth plate

In the growth plate, stem-like cells in the resting zone differentiate into rapidly dividing chondrocytes of the proliferative zone and then terminally differentiate into the non-dividing chondrocytes of the hypertrophic zone. To explore the molecular switches responsible for this two-step differentiation program, we developed a microdissection method to isolate RNA from the resting (RZ), proliferative (PZ), and hypertrophic zones (HZ) of 7-day-old male rats. Expression of approximately 29,000 genes was analyzed by microarray and selected genes verified by real-time PCR. The analysis identified genes whose expression changed dramatically during the differentiation program, including multiple genes functionally related to bone morphogenetic proteins (BMPs). BMP-2 and BMP-6 were upregulated in HZ compared with RZ and PZ (30-fold each, P < 0.01 and 0.001 respectively). In contrast, BMP signaling inhibitors were expressed early in the differentiation pathway; BMP-3 and gremlin were differentially expressed in RZ (100- and 80-fold, compared with PZ, P < 0.001 and 0.005 respectively) and growth differentiation factor (GDF)-10 in PZ (160-fold compared with HZ, P < 0.001). Our findings suggest a BMP signaling gradient across the growth plate, which is established by differential expression of multiple BMPs and BMP inhibitors in specific zones. Since BMPs can stimulate both proliferation and hypertrophic differentiation of growth plate chondrocytes, these findings suggest that low levels of BMP signaling in the resting zone may help maintain these cells in a quiescent state. In the lower RZ, greater BMP signaling may help induce differentiation to proliferative chondrocytes. Farther down the growth plate, even greater BMP signaling may help induce hypertrophic differentiation. Thus, BMP signaling gradients may be a key mechanism responsible for spatial regulation of chondrocyte proliferation and differentiation in growth plate cartilage.

PTHrP regulates growth plate chondrocyte differentiation and proliferation in a Gli3 dependent manner utilizing hedgehog ligand dependent and independent mechanisms

Growth plate chondrocytes undergo a tightly regulated process of differentiation, allowing for the longitudinal growth of bones. Although it is known that parathyroid hormone related protein (PTHrP) and Indian hedgehog regulate the differentiation of growth plate chondrocytes, how these pathways interact to regulate chondrocyte development is not fully elucidated. We examined how the interaction between PTHrP and the hedgehog activated transcription factors, Gli2 and Gli3, regulates growth plate chondrocyte differentiation and proliferation. Analysis of fetal limbs showed that Gli2 is a negative regulator and Gli3 a positive regulator of type X collagen expression. Limb explant cultures showed that PTHrP treatment inhibited type X collagen expression and increased chondrocyte proliferation. This effect was substantially enhanced in Gli2−/− limbs, was blocked in Gli3−/− limbs, and was only partially inhibited by hedgehog ligand blockade. PTHrP negatively regulated Gli mediated transcription in cell cultures, and regulated the level of the repressor form of Gli3 in a PKA dependent manner. These results show that PTHrP regulates growth plate chondrocyte proliferation and differentiation in part through the activity of Gli3, suggesting a crucial role for Gli3 in growth plate chondrocyte development.

Distinct developmental changes in the distribution of calcium, phosphorus and sulphur during fetal growth‐plate development

Gradients in the concentrations of free phosphate (Pi) and calcium (Ca) exist in fully developed growth zones of long bones and ribs, with the highest concentrations closest to the site of mineralization. As high concentrations of Pi and Ca induce chondrocyte maturation and apoptosis, it has been hypothesized that Ca and Pi drive chondrocyte differentiation in growth plates. This study aimed to determine whether gradients in the important spectral elements phosphorus (P), Ca and sulphur (S) are already present in early stages of development, or whether they gradually develop with maturation of the growth zone. We quantified the concentration profiles of Ca, P, S, chloride and potassium at four different stages of early development of the distal growth plates of the porcine femurs, using particle-induced X-ray emission and forward- and backward-scattering spectrometry with a nuclear microprobe. A Ca concentration gradient towards the mineralized area and a stepwise increase in S was found to develop slowly with tissue maturation. The increase in S co-localizes with the onset of proliferation. A P gradient was not detected in the earliest developmental stages. High Ca levels, which may induce chondrocyte maturation, are present near the mineralization front. As total P concentrations do not correspond with former free Pi measurements, we hypothesize that the increase of free Pi towards the bone-forming site results from enzymatic cleavage of bound phosphate.

Changes in perlecan during chondrocyte differentiation in the fetal bovine rib growth plate

Perlecan is a heparan sulfate proteoglycan present in the growth plate and essential for endochondral ossification. We evaluated the synthesis and structure of perlecan in the different zones of the growth plate. The growth plates from fetal bovine ribs were isolated and sequentially sliced into 1-mm sections containing the hypertrophic zone, lower proliferative zone, upper proliferative zone, intermediate zone, and resting zone, respectively. The slices were then either incubated in culture medium with 35SO4 to measure total sulfated proteoglycan synthesis and perlecan synthesis, extracted for perlecan core protein analysis by Western blot, or extracted for perlecan isolation and subsequent characterization of glycosaminoglycan size and disaccharide composition. 35SO4 incorporation into perlecan was three-fourfold higher in the proliferating/hypertrophic zone than the resting zone. Western blot showed perlecan content was greatest in the lower and upper proliferating zones and that a perlecan fragment lacking portions of the N- and C-terminal domains containing heparan sulfate was also present in all zones. Purified perlecan from the hypertrophic/lower proliferative zone had larger chondroitin sulfate chains and a different composition of CS and HS disaccharides than the perlecan isolated from the resting zone. These results indicate perlecan deposition is increased and is turned over during proliferation to be replaced by a perlecan with a different sulfation pattern.

PTHrP and Skeletal Development

Parathyroid hormone-related protein (PTHrP) participates in the regulation of endochondral bone development. After the cartilage mold is established in fetal life, perichondrial cells and chondrocytes at the ends of the mold synthesize PTHrP. This ligand then acts on PTH/PTHrP receptors on chondrocytes. As chondrocytes go through a program of proliferation and then further differentiation into post-mitotic, hypertrophic chondrocytes, PTHrP action keeps chondrocytes proliferating and delays their further differentiation. Indian hedgehog (Ihh) is synthesized by chondrocytes that have just stopped proliferating and is required for synthesis of PTHrP. The feedback loop between PTHrP and Ihh serves to regulate the pace of chondrocyte differentiation and the sites at which perichondrial cells first differentiate into osteoblasts. Activation of the PTH/PTHrP receptor leads to stimulation of both Gs and Gq family heterotrimeric G proteins. Genetic analyses demonstrate that Gs activation mediates the action of PTHrP to keep chondrocytes proliferating, while Gq activation opposes this action. Downstream from Gs activation, synthesis of the cyclin-cdk inhibitor, p57, is suppressed, thereby increasing the pool of proliferating chondrocytes. PTHrP's actions to delay chondrocyte differentiation are mediated by the phosphorylation of the transcription factor, SOX9, and by suppression of synthesis of mRNA encoding the transcription factor, Runx2. These pathways and undoubtedly others cooperate to regulate the pace of differentiation of growth plate chondrocytes in response to PTHrP.

Matrix and gene expression in the rat cranial base growth plate

Recent data have shown that the proliferation and differentiation of the cranial base growth plate (CBGP) chondrocytes are modulated by mechanical stresses. However, little is known about the expression of genes and matrix molecules in the CBGP during development or under mechanical stresses. The objective of the present study was to determine whether several cartilage- and bone-related molecules are expressed in the CBGP and whether their expression is modulated by cyclic loading. The CBGP of normal 8-day-old rats (n=8) were isolated immediately after death, followed by extraction of total RNA and reverse transcription/polymerase chain reaction (RT-PCR) analysis. All studied genes, including type II and X collagens, biglycan, versican, osteocalcin, osteopontin, and fetal liver kinase 1, were expressed in the CBGP with a reproducible absence of decorin mRNA. In age- and sex-matched rats (n=10), exogenous cyclic forces were applied to the maxilla at 500 mN and 4 Hz for 20 min/day over 2 days, followed by RNA isolation and RT-PCR analysis. This exogenous cyclic loading consistently induced the expression of the decorin gene, which was non-detectable, by the current RT-PCR approach, in control neonatal CBGPs without loading. Immunolocalization of several of the above-studied gene products demonstrated their remarkable site-specific expression. Decorin proteoglycan was primarily expressed in the perichondrium instead of various cartilage growth zones, especially upon mechanical loading. These findings serve as baseline data for the expression of several genes and gene products in the neonatal CBGP. Mechanical modulation of decorin expression is consistent with recent reports of its susceptibility to mechanical loading in several connective tissues.

Peroxisome Proliferator-Activated Receptor-γPromotes Adipogenic Changes in Growth Plate Chondrocytes In Vitro

Chondrocytes and adipocytes are two differentiated cell types which are both derived from mesenchymal cells. The purpose of this study was to investigate whether peroxisome proliferator-activated receptor-γ (PPARγ), a transcription factor involved in lineage determination during adipogenesis, is able to induce adipogenic differentiation in growth plate chondrocytes. Isolated epiphyseal chondrocytes were infected with a PPARγ adenovirus or treated with the PPARγ agonist ciglitazone. Both of these treatments resulted in lipid droplet accumulation and expression of the adipogenic markers aP2, lipoprotein lipase, and adipsin in chondrocytes. Proteoglycan matrix synthesis was decreased in the PPARγ-infected cells, as was the expression of the chondrogenic genes Col2a1 and aggrecan. Growth plate cells transfected with a PPARγ expression plasmid under the control of the collagen α1(II) promoter also demonstrated a similar adipogenic changes. Terminal differentiation of growth plate chondrocytes induced by thyroid hormone was also inhibited by overexpression of PPARγ and ciglitazone treatment, with decreased expression of alkaline phosphatase and Runx2/Cbfa1 genes. These in vitro data suggest that PPARγ is able to promote adipogenic differentiation in growth plate chondrocytes, while negatively regulating chondrogenic differentiation and terminal differentiation.

Estrogen Signaling Is Active in Cartilaginous Tumors: Implications for Antiestrogen Therapy as Treatment Option of Metastasized or Irresectable Chondrosarcoma

Chondrosarcoma is a malignant cartilaginous matrix-producing tumor that can be lethal in 10% to 50% of the patients. Surgery is the only effective treatment known as these tumors are notorious refractory to all types of conventional chemotherapy or radiotherapy. To identify a target for therapy, we want to determine whether estrogen signaling is active in chondrosarcoma because estrogen is important in the regulation of longitudinal growth that is initiated by chondrocyte proliferation and differentiation in the epiphyseal growth plate of long bones. We studied protein expression of the estrogen receptor in 35 cartilaginous tumors as well as mRNA levels for the estrogen receptor and for aromatase, an enzyme for estrogen synthesis and another potential therapeutic target. Furthermore, the activity of aromatase was determined in vitro by the tritiated water release assay. Dose-response experiments with chondrosarcoma cultured cells were done with estrogen, androstenedione, and exemestane. All chondrosarcomas tested showed mRNA and nuclear protein expression of the estrogen receptor. Also, aromatase mRNA was detected. The aromatase activity assay showed a functional aromatase enzyme in primary chondrosarcoma cultures and in a cell line. Growth of chondrosarcoma cell cultures can be stimulated by adding estrogen or androstenedione, which can be inhibited by exemestane. These results show, on the RNA, protein, and cell biological levels, that the ligand and the receptor are active in estrogen-mediated signal transduction. This observation implicates potential use of targeted drugs that interfere with estrogen signaling, such as those applied for treating breast cancer.

Exogenous PTH-Related Protein and PTH Improve Mineral and Skeletal Status in 25-Hydroxyvitamin D-1α-Hydroxylase and PTH Double Knockout Mice

We examined the effect of NH2-terminal fragments of PTHrP and PTH in young mutant mice deficient in both PTH and 1,25-dihydroxyvitamin D. Both proteins prolonged murine survival by increasing serum calcium, apparently by enhancing renal calcium transporter expression. The dominant effect on the skeleton was an increase in both endochondral bone and appositional formation without increased bone resorption. PTH-related protein (PTHrP) was discovered as a hypercalcemic agent responsible for the syndrome of humeral hypercalcemia of malignancy, and PTH is the major protein hormone regulating calcium homeostasis. Both proteins have skeletal anabolic actions when administered intermittently. We examined effects of exogenous PTHrP(1-86) and PTH(1-34) in double null mutant mice deficient in both PTH and 25-hydroxyvitamin D-1alpha-hydroxylase [1alpha(OH)ase] to determine the action of these proteins in the absence of the two major regulators of calcium and skeletal homeostasis. Mice heterozygous for the PTH null allele and for the 1alpha(OH)ase null allele were mated to generate pups homozygous for both null alleles. PTHrP(1-86) and PTH(1-34) were administered subcutaneously starting 4 days after birth. Serum biochemistry and skeletal radiology, histology, and histomorphometry were performed, and indices of bone formation, resorption, and renal calcium transport were determined by real time RT-PCR, Western blot, and immunohistochemical approaches. In the double mutant mice, which die within 3 weeks after birth with severe hypocalcemia, tetany, and skeletal defects, exogenous PTHrP and PTH enhanced survival of the animals by improving serum calcium. Both proteins increased renal calcium transporter expression and long bone length and augmented growth plate chondrocyte proliferation, differentiation, and cartilage matrix mineralization. Cortical and trabecular bone mass was increased with augmented osteoblast number and activity; however, bone resorption was not increased. PTHrP and PTH reduced hypocalcemia by enhancing renal calcium reabsorption but not by increasing bone resorption. The major skeletal effects of exogenous PTHrP and PTH were to increase bone anabolism.

Extracellular Calcium and Parathyroid Hormone-Related Peptide Signaling Modulate the Pace of Growth Plate Chondrocyte Differentiation

An adequate supply of Ca2+ is critical for normal growth plate development. Previous studies suggest that changes in extracellular [Ca2+] ([Ca2+]e) modulate the function of chondrocytes with high [Ca2+]e promoting cell differentiation. In contrast, signal transduction by the PTH/PTHrP type I receptor (PTH1R) slows down chondrocyte differentiation. This study addressed whether changes in [Ca2+]e modulate the differentiation of mouse growth plate chondrocytes by interacting with PTHrP/PTH1Rs. Raising [Ca2+]e from 0.5-3.0 mM dose-dependently promoted the development of mouse growth plate chondrocytes as indicated by decreases in proteoglycan accumulation and in the expression of early differentiation marker genes and by increases in mineral deposition and in the expression of markers of terminal differentiation. The effects of high [Ca2+]e on gene expression and matrix synthesis were blunted by incubating cells with PTHrP and vice versa. High [Ca2+]e also suppressed the expression of PTH1Rs. Chronic stimulation of PTHrP/PTH1R signaling by adenoviral expression of constitutively active human PTH1Rs (223hPTH1Rs) reduced the effects of high [Ca2+]e on proteoglycan synthesis and gene expression. Similar results were seen when we treated cells with forskolin or 8-bromo-cAMP. Taken together, these data support the idea that the pace of chondrocyte differentiation depends on a balance of interactions between PTHrP/PTH1R and extracellular Ca2+ signaling and that high [Ca2+]e promote cell differentiation potentially by reducing the availability of PTH1Rs and the level of cAMP-dependent signal transduction.

Peroxisome proliferator activated receptor-γ (PPARγ) represses thyroid hormone signaling in growth plate chondrocytes

Peroxisome proliferator activated receptors (PPARs) are DNA-binding nuclear hormone receptors that are upregulated in response to high fat diets. PPARs are structurally related to the type II nuclear receptors, including the thyroid hormone receptors (TRs). To investigate if PPARs modulate TR-mediated terminal differentiation of growth plate chondrocytes, primary cultures of epiphyseal chondrocytes transiently transfected with TRα and PPARγ expression vectors were treated with the PPAR ligands ciglitazone or troglitazone. Forced overexpression of PPARγ decreased TRα1-mediated transcriptional activity and suppressed T3-induced increases in alkaline phosphatase activity and type X collagen expression. Similar effects were observed when the cells were treated with the PPARγ activator ciglitazone or troglitazone. Overexpression of retinoid X receptor-α (RXRα) partially restored not only the inhibition of transcriptional activation by PPARγ but also T3-induced hypertrophic differentiation. These data demonstrate that activation of PPARγ signaling by either addition of PPARγ ligands or overexpression of PPARγ in growth plate chondrocytes inhibits TR-mediated gene transcription and inhibits the biological effects of thyroid hormone on terminal differentiation. The molecular mechanism involved in this inhibition appears to be competition between PPARγ and TRα for limiting amounts of the heterodimeric partner RXR.

ADAMTS‐1–Knockout mice do not exhibit abnormalities in aggrecan turnover in vitro or in vivo

To determine the role of the proteinase ADAMTS-1 in normal and accelerated catabolism of aggrecan in articular and growth plate cartilage of mice. Expression of ADAMTS-1 was determined using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of RNA isolated from microdissected chondrocytes from different zones of mouse growth plate and articular cartilage. Real-time RT-PCR for ADAMTS-4, ADAMTS-5, and ADAMTS-9 was performed on femoral head cartilage of wild-type (WT) and ADAMTS-1-knockout (KO) mice. Histologic and immunohistologic evaluation of growth plate and articular cartilage was performed in WT and KO mice from birth to 12 weeks of age. The effect of ADAMTS-1 ablation on cartilage proteoglycan loss was studied in antigen-induced arthritis (AIA). Aggrecan catabolism in WT and KO mice was studied in an in vitro model of cartilage degradation, by quantitation of glycosaminoglycan loss and histologic, immunohistologic, and Western immunoblot analyses. ADAMTS-1 messenger RNA (mRNA) was expressed in normal mouse articular and growth plate cartilage and was up-regulated in terminal hypertrophic differentiation of growth plate chondrocytes. There was no difference in mRNA levels in the cartilage of WT compared with KO mice for the other potential aggrecanases ADAMTS-4, ADAMTS-5, or ADAMTS-9. ADAMTS-1-KO mice were significantly smaller than their WT littermates; however, no morphologic differences between the genotypes were evident in growth plate or articular cartilage from birth to skeletal maturity (12-16 weeks). Similarly, no difference in cartilage aggrecan content or presence of aggrecan degradation products was detected between WT and KO mice. There was no difference between WT and KO mice in the degree of synovial inflammation or depletion of cartilage aggrecan in AIA. There was no difference between WT and KO cartilage in either basal or stimulated aggrecan loss in vitro; however, subtle changes in the aggrecanase-generated aggrecan catabolites were observed in interleukin-1-treated cartilage. Although ADAMTS-1 is expressed in articular and growth plate cartilage and is able to cleave aggrecan at physiologically relevant sites, our results indicate that it does not play a significant nonredundant role in normal cartilage and bone development and growth. Similarly, ablation of ADAMTS-1 offered no protection from accelerated aggrecanolysis in an inflammatory model of arthritis or in an in vitro model of early cartilage degradation. ADAMTS-1 does not appear to be a viable target for treatment of cartilage destruction in arthritis.

Fibroblast Growth Factor (FGF) 18 Signals through FGF Receptor 3 to Promote Chondrogenesis

Signaling by fibroblast growth factor (FGF) 18 and FGF receptor 3 (FGFR3) have been shown to regulate proliferation, differentiation, and matrix production of articular and growth plate chondrocytes in vivo and in vitro. Notably, the congenital absence of either FGF18 or FGFR3 resulted in similar expansion of the growth plates of fetal mice and the addition of FGF18 to human articular chondrocytes in culture enhanced proliferation and matrix production. Based on these and other experiments it has been proposed that FGF18 signals through FGFR3 to promote cartilage production by chondrocytes. Its role in chondrogenesis remains to be defined. In the current work we used the limb buds of FGFR3(+/+) and FGFR3(-/-) embryonic mice as a source of mesenchymal cells to determine how FGF18 signaling affects chondrogenesis. Confocal laser-scanning microscopy demonstrated impaired cartilage nodule formation in the FGFR3(-/-) cultures. Potential contributing factors to the phenotype were identified as impaired mitogenic response to FGF18, decreased production of type II collagen and proteoglycan in response to FGF18 stimulation, impaired interactions with the extracellular matrix resulting from altered integrin receptor expression, and altered expression of FGFR1 and FGFR2. The data identified FGF18 as a selective ligand for FGFR3 in limb bud mesenchymal cells, which suppressed proliferation and promoted their differentiation and production of cartilage matrix. This work, thus, identifies FGF18 and FGFR3 as potential molecular targets for intervention in tissue engineering aimed at cartilage repair and regeneration of damaged cartilage.

Chondrocyte-Specific Knockout of the G Protein Gsα Leads to Epiphyseal and Growth Plate Abnormalities and Ectopic Chondrocyte Formation

G(s)alpha is a ubiquitously expressed G protein alpha-subunit that couples receptors to adenylyl cyclase. Mice with chondrocyte-specific ablation of the G(s)alpha gene had severe epiphyseal and growth plate abnormalities and ectopic cartilage formation within the metaphyseal region of the tibia. These results show that G(s)alpha negatively regulates chondrocyte differentiation and is the critical signaling mediator of the PTH/PTH-rP receptor in growth plate chondrocytes. G(s)alpha is a ubiquitously expressed G protein alpha-subunit that mediates signaling through G protein-coupled receptors to activate the cAMP/protein kinase A signaling pathway. Although studies suggest an important role for G(s)alpha in regulating growth plate development, direct in vivo results examining this role are lacking. The G(s)alpha gene was ablated in murine cartilage by mating mice with loxP sites surrounding the G(s)alpha promoter and first exon with collagen 2a1 promoter-Cre recombinase transgenic mice. Skeletal tissues were studied by gross and microscopic pathology, and gene expression was determined by in situ hybridization. Mice with complete chondrocyte-specific G(s)alpha deficiency (homozygotes) died within minutes after birth and had severe epiphyseal and growth plate defects with shortening of the proliferative zone and accelerated hypertrophic differentiation of growth plate chondrocytes, a phenotype similar to that of PTH/PTH-related peptide (PTHrP) receptor knockout mice. Indian hedgehog and PTH/PTHrP receptor expression in prehypertrophic chondrocytes was unaffected in mutant mice. PTHrP expression in periarticular cartilage was increased in the mutant mice, probably because of the closer proximity of Ihh-secreting chondrocytes to the periarticular zone. In addition, these mice developed ectopic cartilage at the anterior side of the metaphyseal region in the tibia. Mice with partial G(s)alpha deficiency (heterozygotes) exhibited no phenotype. These results show that G(s)alpha negatively regulates chondrocyte differentiation and is the critical signaling mediator of the PTH/PTHrP receptor in epiphyseal and growth plate chondrocytes.

RhoA/ROCK Signaling Regulates Sox9 Expression and Actin Organization during Chondrogenesis

Endochondral ossification is initiated by the differentiation of mesenchymal precursor cells to chondrocytes (chondrogenesis). This process is characterized by a strong interdependence of cell shape, cytoskeletal organization, and the onset of chondrogenic gene expression, but the molecular mechanisms mediating these interactions are not known. Here we investigated the role of the RhoA/ROCK pathway, a well characterized regulator of cytoskeletal organization, in chondrogenesis. We show that pharmacological inhibition of ROCK signaling by Y27632 resulted in increased glycosaminoglycan synthesis and elevated expression of the chondrogenic transcription factor Sox9, whereas overexpression of RhoA in the chondrogenic cell line ATDC5 had the opposite effects. Suppression of Sox9 expression by ROCK signaling was achieved through repression of Sox9 promoter activity. These molecular changes were accompanied by reorganization of the actin cytoskeleton, where RhoA/ROCK signaling suppressed cortical actin organization, a hallmark of differentiated chondrocytes. This led us to analyze the regulation of Sox9 expression by drugs affecting cytoskeletal dynamics. Both inhibition of actin polymerization by cytochalasin D and stabilization of existing actin filaments by jasplakinolide resulted in increased Sox9 mRNA levels, whereas inhibition of microtubule polymerization by colchicine completely blocked Sox9 expression. In conclusion, our data suggest that RhoA/ROCK signaling suppresses chondrogenesis through the control of Sox9 expression and actin organization.

Annexin V and terminal differentiation of growth plate chondrocytes

Terminal differentiation and mineralization are the final events in endochondral bone formation and allow the replacement of cartilage by bone. Retinoic acid (RA) stimulates these events, including upregulation of expression and activity of alkaline phosphatase (APase), expression of annexins II, V, and VI proteins, which bind to membranes and form Ca 2+ channels, expression of osteocalcin and runx2, another mineralization-related protein and terminal differentiation-related transcription factor, and ultimately mineralization. Chelating cytosolic Ca 2+ with BAPTA-AM, interfering with annexin Ca 2+ channel activities using K-201, a specific annexin Ca 2+ channel blocker, or suppression of annexin V expression using siRNA inhibited these events. Overexpression of annexin V in embryonic chicken growth plate chondrocytes resulted in an increase of cytoplasmic Ca 2+ concentration, [Ca 2+] i similar to [Ca 2+] i increase in RA-treated cultures. Overexpression of annexin V also resulted in upregulation of annexin II, annexin VI, osteocalcin, and runx2 gene expression, expression and activity of APase, and ultimately stimulation of mineralization. K-201 inhibited upregulation of osteocalcin and runx2 gene expression, APase expression and activity, and mineralization in annexin V-overexpressing growth plate chondrocytes. These findings indicate that annexins II, V, and VI alter Ca 2+ homeostasis in growth plate chondrocytes thereby regulating terminal differentiation and mineralization events. Overexpression of annexin V is sufficient to stimulate these terminal differentiation events in growth plate chondrocytes, whereas suppression of annexin V expression inhibits these events.

TGF-b signaling in chondrocytes

Transforming growth factor-beta (TGF-beta) regulates a large variety of cellular activities. Binding of TGF- beta to its cell surface receptor triggers several signaling cascades, among which the TGF- beta -Smad pathway is the most extensively studied. TGF- beta also activates protein kinases, including MAPK, PKA and PKC, and modulates gene expression via its delicate interaction with other signaling pathways. During endochondral bone formation, TGF- beta acts as a potent inhibitor of the terminal differentiation of epiphyseal growth plate chondrocytes. This effect appears to be primarily mediated by Smad molecules, although MAPK-ATF2 signaling is also involved. The rate of chondrocyte maturation is tightly regulated through the interactions of Smad-mediated signaling, the Wnt signaling pathway, and the transcription factor Runx2. Improving our understanding of the exact mechanisms underlying TGF- beta -mediated signaling pathways and their effects may greatly impact the diagnosis and treatment of many common orthopaedic diseases.

PGE2 inhibits chondrocyte differentiation through PKA and PKC signaling

Prostaglandins are ubiquitous metabolites of arachidonic acid, and cyclooxygenase inhibitors prevent their production and secretion. Animals with loss of cyclooxygenase-2 function have reduced reparative bone formation, but the role of prostaglandins during endochondral bone formation is not defined. The role of PGE2 as a regulator of chondrocyte differentiation in chick growth plate chondrocytes (GPCs) was examined. While PGE2, PGD2, PGF2α, and PGJ2 all inhibited colX expression, approximately 80% at 10 −6 M, PGE2 was the most potent activator of cAMP response element (CRE)-mediated transcription. PGE2 dose-dependently inhibited the expression of the differentiation-related genes, colX, VEGF, MMP-13, and alkaline phosphatase gene, and enzyme activity with significant effects at concentrations as low as 10 −10 M. PGE2 induced cyclic AMP response element binding protein (CREB) phosphorylation and increased c-Fos protein levels by 5 min, and activated transcription at CRE-Luc, AP-1-Luc, and c-Fos promoter constructs. The protein kinase A (PKA) inhibitor, H-89, completely blocked PGE2-mediated induction of CRE-Luc and c-Fos promoter-Luc promoters, and partially inhibited induction of AP-1-Luc, while the protein kinase C (PKC) inhibitor Go-6976 partially inhibited all three promoters, demonstrating substantial cross-talk between these signaling pathways. PGE2 inhibition of colX gene expression was dependent upon both PKA and PKC signaling. These observations demonstrate potent prostaglandin regulatory effects on chondrocyte maturation and show a role for both PKA and PKC signaling in PGE2 regulatory events.

Cell-cycle control and the cartilage growth plate.

The longitudinal growth of endochondral bones is governed by proliferation and hypertrophic differentiation of growth plate chondrocytes. Numerous growth factors and hormones have been implicated in the regulation of these processes, but the intracellular mechanisms involved remain much less understood. We had suggested a role of cell-cycle genes in the integration of these diverse extracellular signals and their translation into coordinated proliferation and differentiation of chondrocytes. Numerous recent studies have provided support for such a scenario and provide novel insights into the regulation and function of cell-cycle genes in chondrocytes. This review article summarizes recent progress in the field.