Chondrogenic Cell Line Research Articles

Fibronectin regulates proteoglycan production balance in transforming growth factor-β1-induced chondrogenesis

Transforming growth factor (TGF)-β and bone morphogenetic protein (BMP) induce a cartilage-specific extracellular matrix (ECM) gene, aggrecan, in a chondrogenic cell line, ATDC5. The results of our recent study show that TGF-β1, but not BMP-4, strongly induces an ECM gene, fibronectin, during chondrogenesis. However, the role of fibronectin in chondrogenesis is unclear. In the current study, our results showed that TGF-β1, but not BMP-4, led to versican-dominant proteoglycan production during chondrogenesis of ATDC5 cells. siRNA-mediated reduction of fibronectin and interference in the liaison between fibronectin and integrins by the Arg-Gly-Asp-Ser (RGDS) peptide increased aggrecan expression, and decreased versican expression by TGF-β1 stimulation. These data suggest that fibronectin is a critical mediator for TGF-β-specific production balance of 2 major proteoglycans, aggrecan and versican, during chondrogenesis.

Marcaje de células madre mesenquimales derivadas de tejido adiposo para su localización y seguimiento mediante RM en terapias de regeneración ósea

Objective The aim of this study was to study the viability and multipotential capacity of adipose derived mesenchymal stem cells, after being labelled with superparamagnetic iron oxide particles. The final aim is to monitor stem cells after being implanted by cellular therapy and tissue engineering techniques in the field of Orthopaedics and Traumatology. Method Ten biopsies of adipose tissue were processed in order to isolate adipose derived mesenchymal stem cells, which were cultured under standard in vitro culture conditions until pre-confluent state. Cells in 1P and 2P were labelled with superparamagnetic iron oxide particles (SPIO), using protamine sulphate as a transfection agent. After confirming labelling efficiency with Prussian blue staining, we studied the labelling effect on the growth and multipotency of stem cells by analysing their in vitro growth curves and their capacity to differentiate into adipogenic, osteogenic and chondrogenic cell lines. We also tried to view the labelled stem cells using 3 Tesla magnetic resonance imaging. Results and conclusion Adipose derived stem cells labelled with SPIO showed a proliferation and differentiation capacity similar to control stem cells. These cells were visible on MR images after been implanted in a bone piece. Therefore, in vitro cell labelling of adipose derived mesenchymal stem cells with SPIO could be useful for in vivo cell tracking by MRI in bone tissue engineering.

Thermoresponsive poly(N‐isopropylacrylamide)‐g‐methylcellulose hydrogel as a three‐dimensional extracellular matrix for cartilage‐engineered applications

Recent advances in tissue engineering and regenerative medicine fields can offer alternative solutions to the existing techniques for cartilage repair. In this context, a variety of materials has been proposed, and the injectable hydrogels are among the most promising alternatives. The aim of this work is to explore the ability of poly(N-isopropylacrylamide)-g-methylcellulose (PNIPAAm-g-MC) thermoreversible hydrogel as a three-dimensional support for cell encapsulation toward the regeneration of articular cartilage through a tissue engineering approach. The PNIPAAm-g-MC copolymer was effectively obtained using ammonium-persulfate and N,N,N',N'-tetramethylethylenediamine as initiator as confirmed by Fourier transform infrared spectroscopy and (1) H NMR results. The copolymer showed to be temperature responsive, becoming a gel at temperatures above its lower critical solution temperature (~ 32 °C) while turning into a liquid below it. Results obtained from the MTS test showed that extracts of the hydrogel were clearly noncytotoxic to L929 fibroblast cells. ATDC5 cells, a murine chondrogenic cell line, were used as the in vitro model for this study; they were encapsulated at high cell density within the hydrogel and cultured for up to 28 days. PNIPAAm-g-MC did not affect the cell viability and proliferation, as indicated by both MTS and DNA assays. The results also revealed an increase in synthesis of glycosoaminoglycans within culture time measured by the dimethylmethylene blue quantification assay. These results suggest the viability of using PNIPAAm-g-MC thermoresponsive hydrogel as a three-dimensional scaffold for cartilage tissue engineering using minimal-invasive strategies.

Homeoprotein Hex is expressed in mouse developing chondrocytes

Endochondral ossification is a complex process involving the formation of cartilage and the subsequent replacement by mineralized bone. Although the proliferation and differentiation of chondrocytes are strictly regulated, the molecular mechanisms involved are not completely understood. Here, we show that a divergent-type homeobox gene, hematopoietically expressed homeobox gene (HEX), is expressed in mouse chondrogenic cell line ATDC5. The expression of Hex protein drastically increased during differentiation. The chondrogenic differentiation-enhanced expression of Hex protein was also observed in chondrocytes in the tibia of embryonic day 15.5 (E15.5) mouse embryos. The localization of Hex protein in the chondrocytes of the tibia changed in association with maturation; namely, there was Hex protein in the cytoplasm near the endoplasmic reticulum (ER) in resting chondrocytes, which moved to the nucleus in prehypertrophic chondrocytes, and thereafter entered the ER in hypertrophic chondrocytes. These results suggest Hex expression and subcellular localization are associated with chondrocyte maturation.

Interaction of TGFβ and BMP Signaling Pathways during Chondrogenesis

TGFβ and BMP signaling pathways exhibit antagonistic activities during the development of many tissues. Although the crosstalk between BMP and TGFβ signaling pathways is well established in bone development, the relationship between these two pathways is less well defined during cartilage development and postnatal homeostasis. We generated hypomorphic mouse models of cartilage-specific loss of BMP and TGFβ signaling to assess the interaction of these pathways in postnatal growth plate homeostasis. We further used the chondrogenic ATDC5 cell line to test effects of BMP and TGFβ signaling on each other's downstream targets. We found that conditional deletion of Smad1 in chondrocytes resulted in a shortening of the growth plate. The addition of Smad5 haploinsufficiency led to a more severe phenotype with shorter prehypertrophic and hypertrophic zones and decreased chondrocyte proliferation. The opposite growth plate phenotype was observed in a transgenic mouse model of decreased chondrocytic TGFβ signaling that was generated by expressing a dominant negative form of the TGFβ receptor I (ΔTβRI) in cartilage. Histological analysis demonstrated elongated growth plates with enhanced Ihh expression, as well as an increased proliferation rate with altered production of extracellular matrix components. In contrast, in chondrogenic ATDC5 cells, TGFβ was able to enhance BMP signaling, while BMP2 significantly reduces levels of TGF signaling. In summary, our data demonstrate that during endochondral ossification, BMP and TGFβ signaling can have antagonistic effects on chondrocyte proliferation and differentiation in vivo. We also found evidence of direct interaction between the two signaling pathways in a cell model of chondrogenesis in vitro.

Alleviating Effects of AS1892802, a Rho Kinase Inhibitor, on Osteoarthritic Disorders in Rodents

The effects of AS1892802, a selective Rho-associated coiled coil kinase (ROCK) inhibitor, on knee cartilage damage and pain behavior were examined in a rat model of osteoarthritis (OA). Monoiodoacetate (MIA) was intraarticularly injected into the right knee joints of rats. ROCK I and II mRNA levels increased in knee joints of MIA-injected rats. Our newly synthesized ROCK inhibitor, AS1892802, was injected into the ipsilateral knee or administered p.o. for 3 weeks. The compound dose-dependently and significantly inhibited of cartilage damage in the tibial plateau in a dose-dependent manner and decreased the weight distribution deficit associated with MIA injection. In addition, the compound also inhibited bradykinin induced pain responses in normal rats. In vitro, the compound could induce chondrocyte differentiation in a chondrogenic cell line and significantly inhibited IL-1β– or bradykinin-induced prostaglandin E2 production in a synovial cell line. AS1892802 prevents cartilage damage induced by MIA and has analgesic effects in rat pain models, suggesting that AS1892802 may be clinically useful for the treatment of OA.[Supplementary Figure: available only at http://dx.doi.org/10.1254/jphs.10319FP]

Overexpression of Hematopoietically Expressed Homeoprotein Induces Nonapoptotic Cell Death in Mouse Prechondrogenic ATDC5 Cells

Physiological cell death is an essential event in normal development and maintenance of homeostasis. Recently, the morphological and pharmacological characteristics of programmed cell death, which are distinct from those of apoptosis under physiological and pathological conditions, have been reported. However, the molecular mechanism and executioner of this type of cell death are unknown. We show that overexpression of hematopoietically expressed homeoprotein (Hex), a homeoprotein of divergent type, and enhanced green fluorescent protein (EGFP) fusion protein (Hex-EGFP) induces cell death in mouse chondrogenic cell line ATDC5. The expression rate of Hex-EGFP decreased more rapidly than that of EGFP 96 h after transfection. The time-lapse image of living cells revealed the Hex-EGFP-positive cells rapidly died in a necrosis-like fashion. The nuclei of Hex-EGFP-expressing cells were rarely fragmented; however, these cells were negative for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining. The expression rate of Hex-EGFP clearly increased by treatment with radical scavengers, propyl gallate and butylated hydroxyanisole, slightly increased with a caspase inhibitor, zVAD-fmk, and was not affected by N-acetyl cysteine in ATDC5 cells. A fluorescent probe indicated that reactive oxygen species (ROS) were localized near the nuclei in Hex-EGFP-positive cells. In differentiated ATDC5 cells, as hypertrophic chondrocyte-like cells, the expression rate of Hex-EGFP increased above that in uninduced ATDC5 cells. These results suggest that Hex induces nonapoptotic cell death through local accumulation of reactive oxygen species, and mature chondrocytes, which express Hex, might be able to escape cell death induced by Hex in cartilage.

MEPE regulates growth plate mineralisation through its cleavage to the ASARM peptide

Event Abstract Back to Event MEPE regulates growth plate mineralisation through its cleavage to the ASARM peptide KA Staines1*, VE Macrae1 and C Farquharson1 1 the University of Edinburgh, the Roslin Institute and Royal (Dick) School of Veterinary Studies, United Kingdom MEPE is mainly expressed in mineralising tissues, and has been implicated in osteoblast matrix mineralisation directly by releasing an ASARM peptide. Although the growth plates of MEPE transgenic mice display severe morphological disruption, the expression and function of MEPE in growth plate mineralisation remains largely undefined. {BR}Proximal tibiae from 3-week-old wild-type mice were analysed for Mepe expression by in situ hybridisation and growth plate microdissection. Gene expression by the ATDC5 chondrogenic cell line was examined by RT-qPCR over a 20-day culture period under calcifying conditions. 20µM phosphorylated (pASARM) and non-phosphorylated ASARM (npASARM) peptides were added to ATDC5 cultures, and to 17-day-old and 15-day-old embryonic metatarsal explants. {BR}Mepe expression was abundant throughout the growth plate as shown by in situ hybridisation. Microdissection of the growth plate confirmed an increased expression of Mepe in the hypertrophic chondrocytes (9-fold increase compared to proliferative chondrocytes, P<0.05). ATDC5 cells showed an initial decrease in Mepe expression at day-10 of culture, after which expression increased at day-15. Treatment of ATDC5 cells with pASARM peptide caused an inhibition of mineralisation, determined by alizarin red staining (P<0.01). Treatment with npASARM promoted mineralisation (P<0.01). The growth rate of 15 and 17-day-old embryonic metatarsals and the proliferation of chondrocytes within were not affected by treatment with 20µM pASARM or npASARM peptides. This indicates that the peptides were not toxic and the bones were still viable after 7 days of culture. However, in 17-day-old embryonic metatarsals, the growth of the central diaphyseal mineralisation zone was inhibited in bones treated with 20µM pASARM (P<0.001). This was further examined in 15-day-old embryonic metatarsals in which the central mineralised core only forms after 7 days in culture. Treatment with 20µM pASARM peptides completely prevented mineralisation. Alkaline phosphatase activity was unchanged between the treated and untreated bones, suggesting that the observed inhibition of mineralisation is not a result of decreased enzyme activity. {BR}Our findings indicate that MEPE is expressed by growth plate chondrocytes and that it is likely to have a developmental role in the inhibition of cartilage matrix mineralisation through its cleavage and subsequent phosphorylation of the ASARM peptide. Keywords: Bones, Bone Research Conference: 2011 joint meeting of the Bone Research Society & the British Orthopaedic Research Society, Cambridge, United Kingdom, 27 Jun - 29 Jun, 2011. Presentation Type: Oral Topic: Abstracts Citation: Staines K, Macrae V and Farquharson C (2011). MEPE regulates growth plate mineralisation through its cleavage to the ASARM peptide. Front. Endocrinol. Conference Abstract: 2011 joint meeting of the Bone Research Society & the British Orthopaedic Research Society. doi: 10.3389/conf.fendo.2011.02.00063 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Sep 2011; Published Online: 30 Sep 2011. * Correspondence: Prof. KA Staines, the University of Edinburgh, the Roslin Institute and Royal (Dick) School of Veterinary Studies, United Kingdom, k.staines@brighton.ac.uk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers KA Staines VE Macrae C Farquharson Google KA Staines VE Macrae C Farquharson Google Scholar KA Staines VE Macrae C Farquharson PubMed KA Staines VE Macrae C Farquharson Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

P2Y2 receptors and GRK2 are involved in oscillatory fluid flow induced ERK1/2 responses in chondrocytes

Mechanical loading is an important factor regulating cartilage metabolism maintained by chondrocytes. However, some of its underlying mechanisms remain poorly understood. In this study, we employed a chondrogenic cell line ATDC5 to investigate roles of P2Y(2) and GRK2 in chondrocyte mechanotransduction. We first confirmed the expression of chondrocyte markers in differentiated ATDC5 cells. We then exposed both differentiated and undifferentiated ATDC5 cells to oscillatory fluid flow, and found that differentiated ATDC5 cells responded to oscillatory fluid flow by increasing COX-2 and aggrecan expressions. More importantly, fluid flow induced ERK1/2 response in differentiated cells was increased more than 10 times compared to those in undifferentiated cells. Furthermore, we found that P2Y(2) mRNA and protein levels in differentiated ATDC5 cells were significantly higher than those in undifferentiated cells. In contrast, GRK2 protein levels in differentiated cells were significantly lower than those in undifferentiated cells. Finally, overexpressions of P2Y(2) and GRK2 in differentiated ATDC5 cells result in a 34% increase and a 21% decrease of the ERK1/2 phosphorylation, respectively, in response to oscillatory fluid flow, suggesting important roles of P2Y(2) and GRK2 in chondrocyte mechanotransduction.

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.

The transcription factor Znf219 regulates chondrocyte differentiation by assembling a transcription factory with Sox9

Sox9 is an essential transcription factor for chondrogenesis by regulating the expression of chondrogenic genes. However, its regulatory mechanism is not fully understood. To address this, we attempted to identify the transcriptional partners of Sox9 by screening the cDNA library of the chondrogenic cell line ATDC5 using the collagen 2α1 (Col2α1) gene promoter fused to a luciferase reporter gene. One of the positive clones encoded the Znf219 gene. Whole mount in situ hybridization experiments indicated that Znf219 mRNA was specifically expressed in the developing limb buds where Col2α1 and Sox9 were strongly expressed. Znf219 markedly enhanced the transcriptional activity of Sox9 on the Col2a1 gene promoter. In addition, Znf219 is physically associated with Sox9 and is colocalized with Sox9 in the nucleus. We also found that overexpression of Znf219 profoundly increased Sox9-induced mRNA expression of Col2a1, aggrecan and Col11a2. Consistently, knockdown of Znf219 decreased the Sox9-induced mRNA expression of these genes. Furthermore, a dominant-negative mutant Znf219 inhibited Bmp2-induced chondrocyte differentiation. Our results suggest that Znf219 plays an important role in the regulation of chondrocyte differentiation as a transcriptional partner of Sox9.

Expression of Ten‐m/Odz3 in the fibrous layer of mandibular condylar cartilage during postnatal growth in mice

It has been speculated that the mandibular condyle develops via the differentiation of the fibroblast-like cells covering the condyle into chondrocytes; however, the developmental mechanisms behind this process have not been revealed. We used laser-capture microdissection and cDNA microarray analysis to elucidate the genes that are highly expressed in these fibroblast-like cells. Among these genes, the transcription of Ten-m/Odz3 was significantly increased in the fibroblast-like cells compared with other cartilage tissues. For the first time, we describe the temporal and spatial expression of Ten-m/Odz3 mRNA in relation to the expression of type I, II, and X collagen mRNA, as determined by in-situ hybridization in mouse mandibular condylar cartilage and mouse femoral cartilage during the early stages of development. Ten-m/Odz3 was expressed in the fibrous layer and the proliferating and mature chondrocyte layers, which expressed type I and II collagen, respectively, but was not detected in the hypertrophic chondrocyte layer. Furthermore, we evaluated the in-vitro expression of Ten-m/Odz3 using ATDC5 cells, a mouse chondrogenic cell line. Ten-m/Odz3 was expressed during the early stage of the differentiation of mesenchymal cells into chondrocytes. These findings suggest that Ten-m/Odz3 is involved in the differentiation of chondrocytes and that it acts as a regulatory factor in the early stages of the development of mandibular condylar cartilage.

Inorganic phosphate (Pi) modulates the expression of key regulatory proteins of the inorganic pyrophosphate (PPi) metabolism in TGF-β1-stimulated chondrocytes

The balance between extracellular inorganic phosphate (ePi) and extracellular inorganic pyrophosphate (ePPi) is controlled by four membrane proteins: the transporters ANK (exporting PPi outside the cells) and PiT-1 (importing ePi into the cells), and the enzymes PC-1 (generating ePPi from nucleotides) and Tissue Non-specific Alkaline Phosphatase (TNAP, hydrolyzing ePPi into ePi). TGF-β1 was shown to stimulate ANK and PC-1 expression in articular chondrocytes, and subsequent ePPi level, as well as to increase ePi uptake by inducing PiT-1 expression in a chondrogenic cell line. Thus, we investigated the ability of ePi to modulate the effect of TGF-β1 on the regulatory proteins of the ePi/ePPi balance in chondrocytes. In the pathophysiological range of 0.01-1 mM, ePi was inactive by itself but potentiated the stimulatory effects of TGF-β1 on ANK, PC-1 or PiT-1 mRNA (RT-qPCR) and protein (Western blot) levels. PC-1 activity was also increased by TGF-β1 and further potentiated by ePi supplementation. TNAP mRNA and activity became undetectable in response to TGF-β1. These data suggest that ePi could increase ePPi level by changing the control of ANK and PC-1 expression by TGF-β1, further highlighting an adaptative regulation of the Pi/PPi balance to prevent basic calcium phosphate deposition into the joints.

Cytokine Receptor-Like Factor 1 is Highly Expressed in Damaged Human Knee Osteoarthritic Cartilage and Involved in Osteoarthritis Downstream of TGF-β

Osteoarthritis (OA) is the most prevalent joint disease and is characterized by pain and functional loss of the joint. However, the pathogenic mechanism of OA remains unclear, and no drug therapy for preventing its progress has been established. To identify genes related to the progress of OA, the gene expression profiles of paired intact and damaged cartilage obtained from OA patients undergoing joint substitution were compared using oligo microarrays. Using functional categorization combined with gene ontology and a statistical analysis, five genes were found to be highly expressed in damaged cartilage (HBEGF, ASUS, CRLF1, LOX, CDA), whereas three genes were highly expressed in intact tissues (CHST2, PTPRD, CPAN6). Among these genes, the upregulated expression of CRLF1 was reconfirmed using real-time PCR, and the in vivo expression of CRLF1 was detected in clusters of chondrocytes and fibrocartilage-like cells in damaged OA cartilages using in situ hybridization. In vitro, the transcriptional level of CRLF1 was positively regulated by TGF-beta1 in the mouse chondrogenic cell line ATDC5. Additionally, the CRLF1/CLC complex promoted the proliferation of ATDC5 cells and suppressed the expression level of aggrecan and type II collagen. Our data suggest that the CRLF1/CLC complex disrupts cartilage homeostasis and promotes the progress of OA by enhancing the proliferation of chondrocytes and suppressing the production of cartilage matrix. A component of the complex, CRLF1, may be useful as a biomarker of OA; and the corresponding receptor is a potential new drug target for OA.

Stable subclones of the chondrogenic murine cell line MC615 mimic distinct stages of chondrocyte differentiation

Fourteen stable subclones derived from the murine chondrogenic cell line MC615 were established and characterised regarding their differentiation stages and responsivity to BMP2. Based on their gene expression profiles which revealed remarkable variances in Col2a1 and Col10a1 expression, subclones could be grouped into at least three distinct categories. Three representative subclones (4C3, 4C6 and 4H4) were further characterised with respect to gene expression pattern and differentiation capacity. These subclones resembled (i) weakly differentiated chondrogenic precursors, strongly responding to BMP2 stimulation (4C3), (ii) collagen II expressing chondrocytes which could be induced to undergo maturation (4C6) and (iii) mature chondrocytes expressing Col10a1 and other markers of hypertrophy (4H4). Interestingly, BMP2 administration caused Smad protein phosphorylation and stimulated Col10a1 expression in all clones, but induced Col2a1 expression only in precursor-like cells. Most remarkably, these clones maintained a stable gene expression profile at least until the 30th passage of subconfluent culture, but revealed reproducible changes in gene expression and differentiation pattern in long term high density cultures. Thus, the newly established MC615 subclones may serve as a potent new tool for investigations on the regulation of chondrocyte differentiation and function.

Interleukin-6 inhibits early differentiation of ATDC5 chondrogenic progenitor cells

Interleukin (IL)-6 is a causative agent of systemic juvenile idiopathic arthritis (sJIA), a chronic inflammatory disease complicated with severe growth impairment. Recent trials of anti-IL-6 receptor monoclonal antibody, tocilizumab, indicated that tocilizumab blocks IL-6/IL-6 receptor-mediated inflammation, and induces catch-up growth in children with sJIA. This study evaluates the effects of IL-6 on chondrogenesis by ATDC5 cells, a clonal murine chondrogenic cell line that provides an excellent model for studying endochondral ossification at growth plate. ATDC5 cells were examined for the expression of IL-6 receptor and gp130 by fluorescence-activated cell sorting analysis. Recombinant murine IL-6 was added to ATDC5 cultures to observe cell differentiation, using a quantitative RT-PCR for the chondrogenic differentiation markers type II collagen, aggrecan, and type X collagen. To block IL-6, the anti-mouse IL-6 receptor monoclonal antibody MR16-1 was added. As a result, the cells expressed IL-6 receptor and gp130. The expression of chondrogenic differentiation marker gene was reduced by IL-6, but this was abrogated by MR16-1. We conclude that IL-6 inhibits early chondrogenesis of ATDC5 cells suggesting that IL-6 may affect committed stem cells at a cellular level during chondrogenic differentiation of growth plate chondrocytes, and that IL-6 may be a cellular-level factor in growth impairment in sJIA.

Expression and modulation of ghrelin O‐acyltransferase in cultured chondrocytes

To use reverse transcription-polymerase chain reaction to detect ghrelin O-acyltransferase (GOAT) transcripts in both murine and human chondrocytes, to evaluate the effect of pharmacologic in vitro treatments with lipopolysaccharide (LPS), growth hormone, ghrelin, and dexamethasone on GOAT messenger RNA (mRNA) expression, and to study the GOAT mRNA profile during chondrocyte differentiation. Murine and human GOAT and ghrelin mRNA levels were determined by the SYBR Green-based quantitative real-time polymerase chain reaction method. GOAT mRNA was expressed in murine cartilage explants as well as in the cultured murine chondrogenic ATDC-5 cell line. GOAT was also expressed in human immortalized chondrocyte cell lines and in human cultured primary chondrocytes. In addition, GOAT mRNA expression in differentiating ATDC-5 cells was lower at the early stage of differentiation (days 3-7), whereas GOAT mRNA levels increased progressively at the late stages. Finally, among the drugs and hormones tested, only LPS was able to strongly decrease GOAT mRNA expression. These data indicate that chondrocytes are equipped with biochemical machinery for the synthesis of acylated ghrelin and suggest a novel role of the ghrelin axis in prehypertrophic and hypertrophic chondrocyte differentiation during endochondral ossification.

Chondrogenic cell proliferation and signaling pathways induced by bioassayable growth hormone

Bioassayable growth hormone (BGH) was previously shown to stimulate growth of the tibial epiphyseal plate in rats in vivo, but the mechanisms contributing to this response have not been elucidated. The objective of this study, therefore, was to investigate the ability of BGH to induce cartilage proliferation in vitro, and examine the cellular signaling pathways responsible for this effect. The RCJ3.1C5.18 chondrogenic cell line was cultured to 10 days post‐confluence and stimulated with BGH‐containing plasma or conditioned media samples. Proliferation was measured by immunocytochemical staining for bromodeoxyuridine incorporation and quantified by cell counting. Intracellular protein fractions from control and stimulated cultures were isolated and subjected to Western blotting for phosphotyrosine, phosphoERK‐1/2 and phosphoMEK‐1/2. RCJ3.1C5.18 cell proliferation was significantly and dose‐dependently increased by BGH. BGH also increased the overall level of protein tyrosine phosphorylation, and elevated the cellular concentration of phosphorylated ERK‐1 and ERK‐2 in a dose‐dependent fashion. In addition, BGH enhanced the Ser/Thr phosphorylation of MEK‐2. The results demonstrate that BGH directly stimulates cartilage proliferation in vitro, and may utilize multiple signaling molecules to achieve this effect.Support: NIH/NCRR 5G12 RR008124, NIH/NIGMS 5R25GM0490, NSF DBI‐0353887

Paraspeckle protein p54nrb links Sox9-mediated transcription with RNA processing during chondrogenesis in mice

The Sox9 transcription factor plays an essential role in promoting chondrogenesis and regulating expression of chondrocyte extracellular-matrix genes. To identify genes that interact with Sox9 in promoting chondrocyte differentiation, we screened a cDNA library generated from the murine chondrogenic ATDC5 cell line to identify activators of the collagen, type II, alpha 1 (Col2a1) promoter. Here we have shown that paraspeckle regulatory protein 54-kDa nuclear RNA-binding protein (p54nrb) is an essential link between Sox9-regulated transcription and maturation of Sox9-target gene mRNA. We found that p54nrb physically interacted with Sox9 and enhanced Sox9-dependent transcriptional activation of the Col2a1 promoter. In ATDC5 cells, p54nrb colocalized with Sox9 protein in nuclear paraspeckle bodies, and knockdown of p54(nrb) suppressed Sox9-dependent Col2a1 expression and promoter activity. We generated a p54nrb mutant construct lacking RNA recognition motifs, and overexpression of mutant p54nrb in ATDC5 cells markedly altered the appearance of paraspeckle bodies and inhibited the maturation of Col2a1 mRNA. The mutant p54nrb inhibited chondrocyte differentiation of mesenchymal cells and mouse metatarsal explants. Furthermore, transgenic mice expressing the mutant p54nrb in the chondrocyte lineage exhibited dwarfism associated with impairment of chondrogenesis. These data suggest that p54nrb plays an important role in the regulation of Sox9 function and the formation of paraspeckle bodies during chondrogenesis.

Krüppel-like Factor 5 Causes Cartilage Degradation through Transactivation of Matrix Metalloproteinase 9

Although degradation of cartilage matrix has been suggested to be a rate-limiting step for endochondral ossification during skeletal development, little is known about the transcriptional regulation. This study investigated the involvement of KLF5 (Krüppel-like factor 5), an Sp/KLF family member, in the skeletal development. KLF5 was expressed in chondrocytes and osteoblasts but not in osteoclasts. The heterozygous deficient (KLF5+/-) mice exhibited skeletal growth retardation in the perinatal period. Although chondrocyte proliferation and differentiation were normal, cartilage matrix degradation was impaired in KLF5+/- mice, causing delay in replacement of cartilage with bone at the primary ossification center in the embryonic limbs and elongation of hypertrophic chondrocyte layer in the neonatal growth plates. Microarray analyses identified MMP9 (matrix metalloproteinase 9) as a transcriptional target, since it was strongly up-regulated by adenoviral transfection of KLF5 in chondrogenic cell line OUMS27. The KLF5 overexpression caused gelatin degradation by stimulating promoter activity of MMP9 without affecting chondrocyte differentiation or vascular endothelial growth factor expression in the culture of chondrogenic cells; however, in osteoclast precursors, it affected neither MMP9 expression nor osteoclastic differentiation. KLF5 dysfunction by genetic heterodeficiency or RNA interference was confirmed to cause reduction of MMP9 expression in cultured chondrogenic cells. MMP9 expression was decreased in the limbs of KLF5+/- embryos, which was correlated with suppression of matrix degradation, calcification, and vascularization. We conclude that KLF5 causes cartilage matrix degradation through transcriptional induction of MMP9, providing the first evidence that transcriptional regulation of a proteinase contributes to endochondral ossification and skeletal development.