Resting Zone Chondrocytes Research Articles

The G protein-coupled receptor ADGRG6 maintains mouse growth plate homeostasis through IHH signaling.

The cartilage growth plate is essential for maintaining skeletal growth; however, the mechanisms governing postnatal growth plate homeostasis are still poorly understood. Using approaches of molecular mouse genetics and spatial transcriptomics applied to formalin-fixed, paraffin-embedded tissues, we show that ADGRG6/GPR126, a cartilage-enriched adhesion G protein-coupled receptor (GPCR), is essential for maintaining slow-cycling resting zone cells, appropriate chondrocyte proliferation and differentiation, and growth plate homeostasis in mice. Constitutive ablation of Adgrg6 in osteochondral progenitor cells with Col2a1Cre leads to a shortened resting zone, formation of cell clusters within the proliferative zone, and an elongated hypertrophic growth plate, marked by limited expression of parathyroid hormone-related protein (PTHrP) but increased Indian Hedgehog (IHH) signaling throughout the growth plate. Attenuation of smoothened-dependent hedgehog signaling restored the Adgrg6 deficiency-induced expansion of hypertrophic chondrocytes, confirming that IHH signaling can promote chondrocyte hypertrophy in a PTHrP-independent manner. In contrast, postnatal ablation of Adgrg6 in mature chondrocytes with AcanCreERT2, induced after the formation of the resting zone, does not affect PTHrP expression but causes an overall reduction of growth plate thickness marked by increased cell death specifically in the resting zone cells and a general reduction of chondrocyte proliferation and differentiation. Spatial transcriptomics reveals that ADGRG6 is essential for maintaining chondrocyte homeostasis by regulating osteogenic and catabolic genes in all the zones of the postnatal growth plates, potentially through positive regulation of SOX9 expression. Our findings elucidate the essential role of a cartilage-enriched adhesion GPCR in regulating cell proliferation and hypertrophic differentiation by regulation of PTHrP/IHH signaling, maintenance of slow-cycle resting zone chondrocytes, and safeguarding chondrocyte homeostasis in postnatal mouse growth plates.

Hedgehog activation promotes osteogenic fates of growth plate resting zone chondrocytes through transient clonal competency.

The resting zone of the postnatal growth plate is organized by slow-cycling chondrocytes expressing parathyroid hormone-related protein (PTHrP), which include a subgroup of skeletal stem cells that contribute to the formation of columnar chondrocytes. The PTHrP-Indian hedgehog feedback regulation is essential for sustaining growth plate activities; however, molecular mechanisms regulating cell fates of PTHrP+ resting chondrocytes and their eventual transformation into osteoblasts remain largely undefined. Here, in a mouse model, we specifically activated Hedgehog signaling in PTHrP+ resting chondrocytes and traced the fate of their descendants using a tamoxifen-inducible Pthrp-creER line with patched-1-floxed and tdTomato reporter alleles. Hedgehog-activated PTHrP+ chondrocytes formed large, concentric, clonally expanded cell populations within the resting zone ("patched roses") and generated significantly wider columns of chondrocytes, resulting in hyperplasia of the growth plate. Interestingly, Hedgehog-activated PTHrP+ cell descendants migrated away from the growth plate and transformed into trabecular osteoblasts in the diaphyseal marrow space in the long term. Therefore, Hedgehog activation drives resting zone chondrocytes into transit-amplifying states as proliferating chondrocytes and eventually converts these cells into osteoblasts, unraveling a potentially novel Hedgehog-mediated mechanism that facilitates osteogenic cell fates of PTHrP+ skeletal stem cells.

A PTHrP Gradient Drives Mandibular Condylar Chondrogenesis via Runx2

The mandibular condylar cartilage (MCC) is an essential component of the temporomandibular joint, which orchestrates the vertical growth of the mandibular ramus through endochondral ossification with distinctive modes of cell differentiation. Parathyroid hormone–related protein (PTHrP) is a master regulator of chondrogenesis; in the long bone epiphyseal growth plate, PTHrP expressed by resting zone chondrocytes promotes chondrocyte proliferation in the adjacent layer. However, how PTHrP regulates chondrogenesis in the MCC remains largely unclear. In this study, we used a Pthrp-mCherry knock-in reporter strain to map the localization of PTHrP+ cells in the MCC and define the function of PTHrP in the growing mandibular condyle. In the postnatal MCC of Pthrp mCherry/+ mice, PTHrP-mCherry was specifically expressed by cells in the superficial layer immediately adjacent to RUNX2-expressing cells in the polymorphic layer. PTHrP ligands diffused across the polymorphic and chondrocyte layers where its cognate receptor PTH1R was abundantly expressed. We further analyzed the mandibular condyle of Pthrp mCherry/mCherry mice lacking functional PTHrP protein (PTHrP-KO). At embryonic day (E) 18.5, the condylar process and MCC were significantly truncated in the PTHrP-KO mandible, which was associated with a significant reduction in cell proliferation across the polymorphic layer and a loss of SOX9+ cells in the chondrocyte layers. The PTHrP-KO MCC showed a transient increase in the number of Col10a1+ hypertrophic chondrocytes at E15.5, followed by a significant loss of these cells at E18.5, indicating that superficial layer–derived PTHrP prevents premature chondrocyte exhaustion in the MCC. The expression of Runx2, but not Sp7, was significantly reduced in the polymorphic layer of the PTHrP-KO MCC. Therefore, PTHrP released from cells in the superficial layer directly acts on cells in the polymorphic layer to promote proliferation of chondrocyte precursor cells and prevent their premature differentiation by maintaining Runx2 expression, revealing a unique PTHrP gradient-directed mechanism that regulates MCC chondrogenesis.

Loss-of-function variant in SPIN4 causes an X-linked overgrowth syndrome.

Overgrowth syndromes can be caused by pathogenic genetic variants in epigenetic writers, such as DNA and histone methyltransferases. However, no overgrowth disorder has previously been ascribed to variants in a gene that acts primarily as an epigenetic reader. Here, we studied a male individual with generalized overgrowth of prenatal onset. Exome sequencing identified a hemizygous frameshift variant in Spindlin 4 (SPIN4), with X-linked inheritance. We found evidence that SPIN4 binds specific histone modifications, promotes canonical WNT signaling, and inhibits cell proliferation in vitro and that the identified frameshift variant had lost all of these functions. Ablation of Spin4 in mice recapitulated the human phenotype with generalized overgrowth, including increased longitudinal bone growth. Growth plate analysis revealed increased cell proliferation in the proliferative zone and an increased number of progenitor chondrocytes in the resting zone. We also found evidence of decreased canonical Wnt signaling in growth plate chondrocytes, providing a potential explanation for the increased number of resting zone chondrocytes. Taken together, our findings provide strong evidence that SPIN4 is an epigenetic reader that negatively regulates mammalian body growth and that loss of SPIN4 causes an overgrowth syndrome in humans, expanding our knowledge of the epigenetic regulation of human growth.

Effect of light intensity on growth performance and bone development of tibia in broilers.

Light management affects the health outcomes and growth performance of broiler chickens. However, the effects of different light intensities on growth performance and its association with tibia development of broilers remain unclear. In the present study, 462 Ross male broilers were divided into seven treatment groups with 6 replicates (11 birds per replicate), and then were subjected to different light intensity levels (0.5, 2, 5, 7, 9, 13 or 19 Lx) for 42days. The results demonstrated that broilers under lower light intensity (2, 5Lx) obtained higher body weight (p<0.05) and feed conversion ratio (p<0.05). Lower light intensity exposure had no effects on the length, width, weight, breaking strength and the mineral density of the tibia (p>0.05), but led to increased ash content and phosphorus during the starter phase (p<0.05). Also, plasma levels of calcium (Ca), phosphorus (P) and alkaline phosphatase were increased in response to lower light intensity conditions (p<0.05), but decreased under higher light intensity (p<0.05), indicating dynamic mineral metabolic and depositional activity to light intensity. In addition, broilers exposed to lower intensity (0.5 Lx, 2 Lx and 5 Lx) during the starter phase had decreased hypertrophic chondrocytes (p<0.05), but did not affect resting zone chondrocytes and proliferative chondrocytes of the growth plate (p>0.05). In contrast, the light intensity did not affect the growth performance and the development of the tibia of broilers during the finishing phase. In summary, we demonstrated that lower light intensity promoted the growth performance and the bone development of broilers. Application of lower light intensity at the starter phase might be a management strategy for broiler industries.

Pro-inflammatory signaling by 24,25-dihydroxyvitamin D3 in HepG2 cells.

The vitamin D metabolite 24,25-dihydroxyvitamin D3 (24, 25[OH]2D3) was shown to induce nongenomic signaling pathways in resting zone chondrocytes and other cells involved in bone remodeling. Recently, our laboratory demonstrated that 24,25-[OH]2D3 but not 25-hydroxyvitamin D3, suppresses apolipoprotein A-I (apo A-I) gene expression and high-density lipoprotein (HDL) secretion in hepatocytes. Since 24,25-[OH]2D3 has low affinity for the vitamin D receptor (VDR) and little is known with regard to how 24,25-[OH]2D3 modulates nongenomic signaling in hepatocytes, we investigated the capacity of 24,25-[OH]2D3 to activate various signaling pathways relevant to apo A-I synthesis in HepG2 cells. Treatment with 24,25-[OH]2D3 resulted in decreased peroxisome proliferator-activated receptor alpha (PPARα) expression and retinoid-X-receptor alpha (RXRα) expression. Similarly, treatment of hepatocytes with 50 nM 24,25-[OH]2D3 for 1-3 h induced PKCα activation as well as c-jun-N-terminal kinase 1 (JNK1) activity and extracellular-regulated kinase 1/2 (ERK1/2) activity. These changes in kinase activity correlated with changes in c-jun phosphorylation, an increase in AP-1-dependent transcriptional activity, as well as repression of apo A-I promoter activity. Furthermore, treatment with 24,25-[OH]2D3 increased IL-1β, IL-6, and IL-8 expression by HepG2 cells. These observations suggest that 24,25-[OH]2D3 elicits several novel rapid nongenomic-mediated pro-inflammatory protein kinases targeting AP1 activity, increasing pro-inflammatory cytokine expression, potentially impacting lipid metabolism and hepatic function.

Evidence That Estrogen Hastens Epiphyseal Fusion and Cessation of Longitudinal Bone Growth by Irreversibly Depleting the Number of Resting Zone Progenitor Cells in Female Rabbits

With age, growth plate cartilage undergoes programmed senescence, eventually causing cessation of bone elongation and epiphyseal fusion. Estrogen accelerates this developmental process. We hypothesized that senescence occurs because progenitor cells in the resting zone are depleted in number and that estrogen acts by accelerating this depletion. To test this hypothesis, juvenile ovariectomized rabbits received injections of estradiol cypionate or vehicle for 5 weeks, and then were left untreated for an additional 5 weeks. Exposure to estrogen accelerated the normal decline in growth plate height and in the number of proliferative and hypertrophic chondrocytes. Five weeks after discontinuation of estrogen treatment, these structural parameters remained advanced, indicating an irreversible advancement in structural senescence. Similarly, transient estrogen exposure hastened epiphyseal fusion. Estrogen also caused a more rapid decline in functional parameters of growth plate senescence, including growth rate, proliferation rate, and hypertrophic cell size. However, in contrast to the structural parameters, once the estrogen treatment was discontinued, the growth rate, chondrocyte proliferation rate, and hypertrophic cell size all normalized, suggesting that estrogen has a reversible, suppressive effect on growth plate function. In addition, estrogen accelerated the normal loss of resting zone chondrocytes with age. This decrease in resting zone cell number did not appear to be due to apoptosis. However, it was maintained after the estrogen treatment stopped, suggesting that it represents irreversible depletion. The findings are consistent with the hypothesis that estrogen causes irreversible depletion of progenitor cells in the resting zone, thus irreversibly accelerating structural senescence and hastening epiphyseal fusion. In addition, estrogen reversibly suppresses growth plate function.

Mice with global deletion of mitogen inducible gene 6 display rapid and severe cartilage and subchondral bone damage after ligament and meniscus injury

s / Osteoarthritis and Cartilage 21 (2013) S9–S62 S13 protein subunits was investigated in growth plates of 6 week old mice by immunohistochemistry. Expression of RMRP, its protein subunits and important chondrogenic differentiation markers (Col2a1, Acan, Col10a1, Sox9, Runx2) was also determined by RT-qPCR and immunoblotting in chondrogenically differentiating ATDC5 cells and human bone marrow stem cells (hBMSCs). In addition, RMRP was functionally investigated during chondrogenic differentiation by targeting RMRP expression using a specific siRNA duplex and analysing the outcome on chondrogenic gene expression. Furthermore, chondrogenic induction of dermal fibroblasts was used as a model to test the effect of CHH-associated mutations in RMRP on chondrogenic differentiation of CHH patient-derived fibroblasts. Results: To determine expression of RNase MRP during chondrogenic differentiation in vivo, mouse growth plate sections were immunohistochemically stained for RNaseMRP protein subunits.Without exception, all proteins showed identical growth plate distribution patterns: Resting zone chondrocytes express RNaseMRPproteins,whereas nooronlyweak expression levelswere observed inproliferative chondrocytes. In contrast, expression of RNase MRP proteins was clearly detectable in hypertrophic chondrocytes in the growth plate. This differentiation phase-specific expression of RNase MRP protein subunits was confirmed for Rpp25, Rpp30 and Rpp40 on ATDC5 and hBMSC differentiation models (mRNA); their expressionwas upregulated from day 14 in differentiation onwards, simultaneously with hypertrophic genes. Knockdown of RMRP expression by RNAi resulted in upregulation of RNase MRP mRNA substrates Clb2 and viperin and decreased chondrogenic marker expression (Sox9, Col2a1, Runx2, Col10a1), suggesting a role for RMRP in chondrogenic differentiation. In addition, knockdown of RMRP also resulted in altered Bapx1/Nkx3.2 expression, further substantiating a role in chondrocyte hypertrophy. To verify our results we transdifferentiated CHH patientderived fibroblasts and matched controls to acquire a chondrocyte-like phenotype. Chondrogenic capacity of the CHH patients was clearly disrupted and showed a more than 50% reduced Col10a1 expression as compared to matched control cultures. Conclusions: Our data show that expression of RNase MRP and its subunits is regulated during chondrogenic differentiation, is predominantly associated with chondrocyte hypertrophy and shows functional cross-talk with chondrogenic pathways. These data are the first to show the association of RNase MRP with skeletal development and may provide an explanation for the CHH-associated skeletal phenotype as well as unveiling a potential novel regulator of chondrocyte hypertrophic differentiation in skeletal development. 10 MICE WITH GLOBAL DELETION OF MITOGEN INDUCIBLE GENE 6 DISPLAY RAPID AND SEVERE CARTILAGE AND SUBCHONDRAL BONE DAMAGE AFTER LIGAMENT AND MENISCUS INJURY D.M. Joiner, K.D. Less, E. VanWieren, B.O. Williams. Van Andel Res. Inst., Grand Rapids, MI, USA Purpose: The Epidermal Growth Factor Receptor (EGFR) Signaling pathway plays important roles in osteochondral metabolism and joint disease, which can be triggered by damage to ligaments andmenisci. The goal of this study was to examine the early effect of global deletion of mitogen inducible gene 6 (MIG6), a negative regulator of EGFR, on cartilage and subchondral bone in mice after ligament and meniscus injury. Methods: Global loss of function MIG6 knock-out mice (MIG6-/-) and wild-type (WT) littermates were anesthetized and ligament transections and meniscus removal were performed on the right knee using an established surgical destabilization method. Subchondral bone from surgery knees and untouched contralateral control knees was assessed with x-ray. HE arrows point to bone erosion). Bone damage in surgery knees was greater for MIG6-/animals compared to the control knee and to surgery knees from WT littermates (Figure 2). Ectopic cartilaginous growth pockets were present inMIG6-/mice 4 weeks after ligament and meniscus damage (Figure 1). MIG6-/mice also had increased EGFR expression 4 weeks after surgery (Figure 3) and increased osteoclasts (Figure 4; arrows point to osteoclasts) in the knee joint compared to WT animals. Conclusions: MIG6-/mice displayed rapid and more severe bone and cartilage damage after knee injury compared to WT mice. MIG6 and EGFR could be potential therapeutic targets for preventing cartilage and bone damage and subsequent joint disease development after ligament and meniscus injury.

Sex-specific response of rat costochondral cartilage growth plate chondrocytes to 17β-estradiol involves differential regulation of plasma membrane associated estrogen receptors

Both male and female rat growth plate chondrocytes express estrogen receptors (ERs); however 17β-estradiol (E2) induces membrane responses leading to activation of phospholipase A2 (PLA2), phospholipase C (PLC), prostaglandin E2 (PGE2) production, protein kinase C (PKC), and ultimately mitogen protein kinase (MAPK) only in female cells. This study investigated if these sex-specific responses are due to differences in the actual ERs or in downstream signaling. Western blots and flow cytometry of costochondral cartilage resting zone chondrocytes (RCs) showed 2–3 times more ERα in plasma membranes (PMs) from female cells than male cells. Tunicamycin blocked E2-dependent ER-translocation to the PM, indicating palmitoylation was required. Co-immunoprecipitation showed E2 induced complex formation between ER isoforms only in female RCs. To examine if the lack of response in PKC and PGE2 in males is due to differences in signaling, we examined involvement of ERs and the role of PLC and PLA2. Selective ERα (propylpyrazole triol, PPT) and ERβ (diarylproprionitrile, DPN) agonists activated PKC in female RCs only. The PLC inhibitor, U73122 blocked E2's effect on PKC and the cytosolic PLA2 inhibitor, AACOCF3 inhibited the effect on PGE2 in female RCs, confirming involvement of PLC and PLA2 in the mechanism. The PLC activator, m-3M3FβS activated PKC and PLAA peptide increased PGE2 levels in male and female RCs, showing that the signaling pathways are present. These data indicate that differences in membrane ER amount, localization, translocation and interaction are responsible for the sexual dimorphic response to E2.

Monocarboxylate Transporter 10 Functions as a Thyroid Hormone Transporter in Chondrocytes

Thyroid hormone is essential for normal proliferation and differentiation of chondrocytes. Thus, untreated congenital hypothyroidism is marked by severe short stature. The monocarboxylate transporter 8 (MCT8) is a highly specific transporter for thyroid hormone. The hallmarks of Allan-Herndon-Dudley syndrome, caused by MCT8 mutations, are severe psychomotor retardation and elevated T(3) levels. However, growth is mostly normal. We therefore hypothesized that growth plate chondrocytes use transporters other than MCT8 for thyroid hormone uptake. Extensive analysis of thyroid hormone transporter mRNA expression in mouse chondrogenic ATDC5 cells revealed that monocarboxylate transporter 10 (Mct10) was most abundantly expressed among the transporters known to be highly specific for thyroid hormone, namely Mct8, Mct10, and organic anion transporter 1c1. Expression levels of Mct10 mRNA diminished with chondrocyte differentiation in these cells. Accordingly, Mct10 mRNA was expressed most abundantly in the growth plate resting zone chondrocytes in vivo. Small interfering RNA-mediated knockdown of Mct10 mRNA in ATDC5 cells decreased [(125)I]T(3) uptake up to 44% compared with negative control (P < 0.05). Moreover, silencing Mct10 mRNA expression abolished the known effects of T(3), i.e. suppression of proliferation and enhancement of differentiation, in ATDC5 cells. These results suggest that Mct10 functions as a thyroid hormone transporter in chondrocytes and can explain at least in part why Allan-Herndon-Dudley syndrome patients do not exhibit significant growth impairment.

Fibrin Glue

Cartilage-based treatments have vast applications in plastic and reconstructive surgery, especially craniofacial constructs. Current techniques in craniofacial cartilage reconstructions greatly rely on autologous donor site harvest. Whole cartilage grafts are wrought with complications of warping, resorption, extrusion, and donor site morbidity. Percutaneous delivery of expanded chondrocytes would have the potential to expand a small quantity of autologous cells to deliver cell therapy. To deliver chondrocytes effectively, there must be a reliable medium in which chondrocytes can be kept. The purpose of this work is to highlight the utility of fibrin glue sealant, Evicel, as a suitable chondrocyte carrier in the treatment of a critical-sized defect model of nonarticular cartilage previously developed in our laboratory. Athymic rats were separated into 2 groups: fibrin glue (n = 3) and fibrin glue + rat chondrocytes (n = 6). The animals with an empty defect were used to ensure that they responded normally to the procedure. All animals received a 3-mm full-thickness xiphoid cartilage defect characterized previously as a critical-sized defect in our laboratory (Moyer HR, Wang Y, Farooque T, et al. Tissue Eng Part A. 2010;16:2321-2330). A control animal received no xiphoid defect creation procedure. The fibrin glue group was treated with 0.5 mL of fibrin glue placed directly into the 3-mm defect. The fibrin glue/rat chondrocyte group received a mixture of 1 × 10 resting zone chondrocytes mixed with 0.5 mL of fibrin glue. Rats were euthanized at 5 weeks (35 days) and their xiphoid cartilages harvested. The xiphoids were analyzed with morphometrics through histology and microcomputed tomography. In the fibrin glue vehicle group, there was minimal evidence of wound healing. Xiphoid defects treated with resting zone chondrocytes in a fibrin glue carrier were significantly smaller (P = 0.002) at harvest and had significantly more glycosaminoglycan content on microcomputed tomography analysis. Thus, there was significant healing in the chondrocyte/fibrin glue group. Human fibrin sealant is an effective chondrocyte carrier and retains viable cells. Treatment of a nonarticular critical-size defect with resting zone chondrocytes embedded in a fibrin glue polymer demonstrates tissue healing.

Inorganic Phosphate Induces Mammalian Growth Plate Chondrocyte Apoptosis in a Mitochondrial Pathway Involving Nitric Oxide and JNK MAP Kinase

Chondrocytes in the hypertrophic zone of the growth plate undergo apoptosis during endochondral bone development via mechanisms that involve inorganic phosphate (Pi) and nitric oxide (NO). Recent evidence suggests that Pi-dependent NO production plays a role in apoptosis of cells in the resting zone as well. This study examined the mechanism by which Pi induces NO production and the signaling pathways by which NO mediates its effects on apoptosis in these cells. Pi decreased the number of viable cells based on MTT activity; the number of TUNEL-positive cells and the level of DNA fragmentation were increased, indicating an increase in apoptosis. Blocking NO production using the NO synthase (NOS) inhibitor L: -NAME or cells from eNOS(-/-) mice blocked Pi-induced chondrocyte apoptosis, indicating that NO production is necessary. NO donors NOC-18 and SNOG both induced chondrocyte apoptosis. SNOG also upregulated p53 expression, the Bax/Bcl-2 expression ratio, and cytochrome c release from mitochondria, as well as caspase-3 activity, indicating that NO induces apoptosis via a mitochondrial pathway. Inhibition of JNK, but not of p38 or ERK1/2, MAP kinase was able to block NO-induced apoptosis, indicating that JNK is necessary in this pathway. Pi elevates NO production via eNOS in resting zone chondrocytes, which leads to a mitochondrial apoptosis pathway dependent on JNK.

17β-Estradiol Regulates Rat Growth Plate Chondrocyte Apoptosis Through a Mitochondrial Pathway Not Involving Nitric Oxide or MAPKs

Estrogens cause growth plate closure in both males and females, by decreasing proliferation and inducing apoptosis of postproliferative growth plate chondrocytes. In vitro studies using 17β-estradiol (E(2)) conjugated to bovine serum albumin (E(2)-BSA) show that rat costochondral growth plate resting zone chondrocytes also respond to E(2). Moreover, they are regulated by E(2)-BSA via a protein kinase C and ERK MAPK signaling pathway that is functional only in female cells. To better understand how E(2) regulates apoptosis of growth plate chondrocytes, rat resting zone chondrocytes cells were treated with E(2) or E(2)-BSA. E(2) caused apoptosis in male and female resting zone and growth zone chondrocytes in a dose-dependent manner, based on elevated DNA fragmentation, terminal deoxynucleotidyl transferase dUTP nick end labeling staining and caspase-3 activation. E(2) also up-regulated p53 and Bax protein (Bcl-2-associated X protein) levels and induced release of cytochrome C from the mitochondria, indicating a mitochondrial apoptotic pathway. The apoptotic effect of E(2) did not involve elevated nitric oxide production or MAPKs. It was reduced by ICI 182780, which is an estrogen receptor (ER) antagonist and blocked by antibodies to Erα36, a membrane-associated ER. E(2)-BSA reduced cell viability and increased caspase-3 activity; ICI 182780 had no effect, but anti-ERα36 antibodies blocked the effect. The results indicate that estrogen is able to directly affect the cell population kinetics of growth plate chondrocytes by regulating apoptosis, as well as proliferation and differentiation in both resting zone and growth zone cells. They also have provided further information about the physiological functions of estrogen on longitudinal bone growth.

24R,25-Dihydroxyvitamin D3, lysophosphatidic acid, and p53: A signaling axis in the inhibition of phosphate-induced chondrocyte apoptosis

Maintenance of the pool of chondrocytes in the resting zone of the growth plate in the presence of the physiological apoptogen inorganic phosphate (Pi) is crucial for skeletal development. Costochondral resting zone chondrocytes are regulated by the vitamin D metabolite 24R,25-dihydroxyvitamin D3 [24R,25(OH) 2D 3], with increased production of sulfated glycosaminoglycan-rich extracellular matrix, and reduced matrix metalloproteinase activity. The effects of 24R,25(OH) 2D 3 are mediated by activation of phospholipase D (PLD), resulting in increased production of lysophosphatidic acid (LPA) and LPA-mediated proliferation, maturation, inhibition of Pi-induced apoptosis, and reduction of p53. However, the exact mechanism by which 24R,25(OH) 2D 3 and LPA exert their effects is not fully understood. It was found that both 24R,25(OH) 2D 3 and LPA attenuate Pi-induced caspase-3 activity. The actions of 24R,25(OH) 2D 3 and LPA were dependent upon G αi, LPA receptor(s) 1 and/or 3, PLD, phospholipase C (PLC), and intracellular calcium, phosphoinositide 3-kinase (PI 3K) signaling, and nuclear export. 24R,25(OH) 2D 3 decreased both p53 abundance and p53-medaited transcription and inhibited Pi-induced cytochrome c translocation. Moreover, LPA induced increased mdm2 phosphorylation, a negative regulator of p53. Taken together, these data show that 24R,25(OH) 2D 3 inhibits Pi-induced apoptosis through Ca 2+, PLD, and PLC signaling and through LPA-LPA1/3-G αi-PI 3K-mdm2-mediated p53 degradation, resulting in decreased cytochrome c translocation and caspase-3 activity.

457 Lysophosphatidic acid induces cell-cell adhesion disassembly and actin cytoskeleton disorganization through an event that requires RhoA-Rock and Src signaling in colon cancer cells

Maintenance of the pool of chondrocytes in the resting zone of the growth plate in the presence of the physiological apoptogen inorganic phosphate (Pi) is crucial for skeletal development. Costochondral resting zone chondrocytes are regulated by the vitamin D metabolite 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3], with increased production of sulfated glycosaminoglycan-rich extracellular matrix, and reduced matrix metalloproteinase activity. The effects of 24R,25(OH)2D3 are mediated by activation of phospholipase D (PLD), resulting in increased production of lysophosphatidic acid (LPA) and LPA-mediated proliferation, maturation, inhibition of Pi-induced apoptosis, and reduction of p53. However, the exact mechanism by which 24R,25(OH)2D3 and LPA exert their effects is not fully understood. It was found that both 24R,25(OH)2D3 and LPA attenuate Pi-induced caspase-3 activity. The actions of 24R,25(OH)2D3 and LPA were dependent upon Gαi, LPA receptor(s) 1 and/or 3, PLD, phospholipase C (PLC), and intracellular calcium, phosphoinositide 3-kinase (PI3K) signaling, and nuclear export. 24R,25(OH)2D3 decreased both p53 abundance and p53-medaited transcription and inhibited Pi-induced cytochrome c translocation. Moreover, LPA induced increased mdm2 phosphorylation, a negative regulator of p53. Taken together, these data show that 24R,25(OH)2D3 inhibits Pi-induced apoptosis through Ca2+, PLD, and PLC signaling and through LPA-LPA1/3-Gαi-PI3K-mdm2-mediated p53 degradation, resulting in decreased cytochrome c translocation and caspase-3 activity.

Lysophosphatidic acid signaling promotes proliferation, differentiation, and cell survival in rat growth plate chondrocytes

Growth plate cartilage is responsible for long bone growth in children and adolescents and is regulated by vitamin D metabolites in a cell zone-specific manner. Resting zone chondrocytes (RC cells) are regulated by 24,25-dihydroxyvitamin D3 via a phospholipase D-dependent pathway, suggesting downstream phospholipid metabolites are involved. In this study, we showed that 24R,25(OH) 2D 3 stimulates rat costochondral RC chondrocytes to release lysophosphatidic acid (LPA) and, therefore sought to determine the role of LPA signaling in these cells. RC cells expressed the G-protein coupled receptors LPA1–5 and peroxisome proliferator-activated receptor gamma (PPAR-γ). LPA and the LPA1/3 selective agonist OMPT increased proliferation and two maturation markers, alkaline phosphatase activity and [ 35S]-sulfate incorporation. LPA and 24R,25(OH) 2D 3's effects were inhibited by the LPA1/3 selective antagonist VPC32183(S). Furthermore, apoptosis induced by either inorganic phosphate or chelerythrine was attenuated by LPA, based on DNA fragmentation, TUNEL staining, caspase-3 activity, and Bcl-2:Bax protein ratio. LPA prevented apoptotic signaling by decreasing the abundance, nuclear localization, and transcriptional activity of the tumor-suppressor p53. LPA treatment also regulated the expression of the p53-target genes Bcl-2 and Bax to enhance cell survival. Collectively, these data suggest that LPA promotes differentiation and survival in RC chondrocytes, demonstrating a novel physiological function of LPA-signaling.

Thrombin peptide TP508 prevents nitric oxide mediated apoptosis in chondrocytes in the endochondral developmental pathway

TP508 is a 23-amino acid peptide derived from human prothrombin that increases cartilage matrix production and reduces alkaline phosphatase activity without changing chondrocyte proliferation. This study tested the hypothesis that TP508 acts by blocking the onset of apoptosis associated with hypertrophy. Rat costochondral resting zone chondrocytes and human auricular chondrocytes were cultured in DMEM containing 50μM ascorbic acid and 10% FBS. Apoptosis was induced by treatment of confluent cultures with chelerythrine, tamoxifen, or inorganic phosphate (Pi) for 24h. One half of the cultures received TP508 (0, 0.7, or 7μg/ml). Apoptosis was assessed as a function of DNA fragmentation ([3H]-thymidine labeled DNA fragments), TUNEL staining, and cell viability using the MTT assay, as well as by assessing the Bcl-2/Bax mRNA and protein ratios and caspase-3 activity. The universal NO synthase inhibitor l-NMMA was used to assess the effect of NO production on chondrocyte apoptosis and specific NO synthase subspecies were identified using iNOS inhibitor 1400W and nNOS inhibitor vinyl-l-NIO, as well as l-NAME, which inhibits both iNOS and eNOS. Finally, we assessed if TP508 would block NO production induced by the apoptogens. Chelerythrine, tamoxifen and Pi-induced apoptosis and this was reversed by TP508. All apoptogens increased NO production and this was reduced by TP508. TP508 reduced NO levels to the same extent as 1400W but not to the same extent as l-NAME, suggesting that its effects are mediated primarily by iNOS. In addition, TP508 reduced the effect of chelerythrine to the same extent as 1400W and l-NAME, again indicating that it acts via inhibition of an iNOS pathway. TP508 also regulated Bcl-2/Bax mRNA in a time and dose-dependent manner. The Bcl-2/Bax mRNA ratio was 0.11 in the absence of TP508 at 1h and 4.95 at 7μg/ml TP508; by 3h the ratio was approximately 1 in both groups. The Bcl-2/Bax protein ratio also increased by 63% at 1h. TP508 did not affect caspase-3 activity. TP508 also caused a dose-dependent increase in protein kinase C (PKC) activity within 9min that was maximal at 270min. These results show that TP508 prevents apoptosis in growth plate chondrocytes via inhibition of iNOS-dependent NO and suggest a possible role for PKC in the mechanism.

Gene Expression Profiling of Mouse Articular and Growth Plate Cartilage

Articular cartilage is recalcitrant to repair and regeneration. Tissue engineering and regenerative medicine are potential strategies to treat the damage to articular cartilage. A thorough understanding of the gene expression profiles in articular cartilage and growth plate chondrocytes will be an important prerequisite for tissue engineering of cartilage. Regeneration is a recapitulation of embryonic development and morphogenesis. We used laser capture microdissection to capture the surface articular chondrocytes and the resting zone chondrocytes of growth plate from 14-day-old C57BL/6J mice. Total RNA was individually purified, pooled, and amplified by two rounds of in vitro transcription. Labeled cRNA probes were analyzed using the Affymetrix GeneChip Mouse Genome 430 2.0 Array. We identified 107 genes that were highly expressed by the surface articular chondrocytes and 130 genes that were highly expressed by the resting zone chondrocytes of growth plate (> or = fivefold). The expression of major matrix proteins aggrecan and collagen II were similar, while several morphogens and growth factors were differentially expressed by the surface articular chondrocytes and the resting zone chondrocytes of growth plate. The results of this investigation will be of use in the evaluation of tissue engineered cartilage.

Regulation of Growth Plate Chondrocytes by 1,25-Dihydroxyvitamin D3 Requires Caveolae and Caveolin-1

We examined the role of caveolae and caveolin-1 in the mechanism of 1alpha,25(OH)(2)D(3) action in growth plate chondrocytes. We found that caveolae are required for rapid 1alpha,25(OH)(2)D(3)-dependent PKC signaling, and caveolin-1 must be present based on studies using chondrocytes from Cav-1(-/-) mice. 1,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] regulates endochondral ossification in part through membrane-associated mechanisms, including protein kinase C (PKC) signaling activated by a membrane-associated 1alpha,25(OH)(2)D(3)-binding protein, ERp60. We tested the hypothesis that caveolae are required for 1alpha,25(OH)(2)D(3) action and play an important role in regulating chondrocyte biology and growth plate physiology. Rat costochondral chondrocytes were examined for caveolae by transmission electron microscopy of cultured cells and of cells in situ. Western blots and confocal microscopy were used to detect caveolae proteins including caveolin-1 (Cav-1) and 1alpha,25(OH)(2)D(3) receptors. Caveolae cholesterol was depleted with beta-cyclodextrin (CD) and effects of 1alpha,25(OH)(2)D(3) on PKC, DNA synthesis, alkaline phosphatase, and proteoglycan production determined. Chondrocytes from Cav-1(-/-) and C57BL/6 wildtype mice were also treated with 1alpha,25(OH)(2)D(3). Epiphyses and costochondral junctions of 8-week-old male Cav-1(-/-) and wildtype mice (N = 8) were compared by histomorphometry and microCT. Data were analyzed by ANOVA and Bonferroni for posthoc comparisons. Growth zone chondrocytes had caveolae and Cav-1, -2, and -3. Resting zone chondrocytes, which do not exhibit a rapid 1alpha,25(OH)(2)D(3)-dependent increase in PKC activity, also had these caveolins, but caveolae were larger and fewer in number. ERp60 but not VDR co-localized with Cav-1 in plasma membranes and in lipid rafts. CD-treatment blocked 1alpha,25(OH)(2)D(3) effects on all parameters tested. The Cav-1(-/-) cells did not respond to 1alpha,25(OH)(2)D(3), although 1alpha,25(OH)(2)D(3) increased PKC, alkaline phosphatase, and [(35)S]-sulfate incorporation in wildtype C57BL/6 cells. Histology and microCT showed that Cav-1(-/-) growth plates were longer and had more hypertrophic cells in each column. Growth plate changes were reflected in the metaphysis. The membrane-mediated effects of 1alpha,25(OH)(2)D(3) require caveolae and Cav-1, and Cav-1 deficiency results in altered growth plate physiology.

Depletion of resting zone chondrocytes during growth plate senescence

With age, the growth plate undergoes senescent changes that cause linear bone growth to slow and finally cease. Based on previous indirect evidence, we hypothesized that this senescent decline occurs because growth plate stem-like cells, located in the resting zone, have a finite proliferative capacity that is gradually depleted. Consistent with this hypothesis, we found that the proliferation rate in rabbit resting zone chondrocytes (assessed by continuous 5-bromo-2'-deoxy-uridine labeling) decreases with age, as does the number of resting zone chondrocytes per area of growth plate. Glucocorticoid excess slows growth plate senescence. To explain this effect, we hypothesized that glucocorticoid inhibits resting zone chondrocyte proliferation, thus conserving their proliferative capacity. Consistent with this hypothesis, we found that dexamethasone treatment decreased the proliferation rate of rabbit resting zone chondrocytes and slowed the numerical depletion of these cells. Estrogen is known to accelerate growth plate senescence. However, we found that estradiol cypionate treatment slowed resting zone chondrocyte proliferation. Our findings support the hypotheses that growth plate senescence is caused by qualitative and quantitative depletion of stem-like cells in the resting zone and that growth-inhibiting conditions, such as glucocorticoid excess, slow senescence by slowing resting zone chondrocyte proliferation and slowing the numerical depletion of these cells, thereby conserving the proliferative capacity of the growth plate. We speculate that estrogen might accelerate senescence by a proliferation-independent mechanism, or by increasing the loss of proliferative capacity per cell cycle.