ATDC5 Chondroprogenitor Research Articles

TET1 Directs Chondrogenic Differentiation by Regulating SOX9 Dependent Activation of Col2a1 and Acan In Vitro.

ABSTRACTSkeletal development is a tightly orchestrated process in which cartilage and bone differentiation are intricately intertwined. Recent studies have highlighted the contribution of epigenetic modifications and their writers to skeletal development. Methylated cytosine (5mC) can be oxidized to 5‐hydroxymethylcytosine (5hmC) by the Ten‐eleven‐translocation (TET) enzymes leading to demethylation. We have previously demonstrated that 5hmC is stably accumulated on lineage‐specific genes that are activated during in vitro chondrogenesis in the ATDC5 chondroprogenitors. Knockdown (KD) of Tet1 via short‐hairpin RNAs blocked ATDC5 chondrogenic differentiation. Here, we aimed to provide the mechanistic basis for TET1 function during ATDC5 differentiation. Transcriptomic analysis of Tet1 KD cells demonstrated that 54% of downregulated genes were SOX9 targets, suggesting a role for TET1 in mediating activation of a subset of the SOX9 target genes. Using genome‐wide mapping of 5hmC during ATDC5 differentiation, we found that 5hmC is preferentially accumulated at chondrocyte‐specific class II binding sites for SOX9, as compared with the tissue‐agnostic class I sites. Specifically, we find that SOX9 is unable to bind to Col2a1 and Acan after Tet1 KD, despite no changes in SOX9 levels. Finally, we compared this KD scenario with the genetic loss of TET1 in the growth plate using Tet1 −/− embryos, which are approximately 10% smaller than their WT counterparts. In E17.5 Tet1 −/− embryos, loss of SOX9 target gene expression is more modest than upon Tet1 KD in vitro. Overall, our data suggest a role for TET1‐mediated 5hmC deposition in partly shaping an epigenome conducive for SOX9 function. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Aggrecan is required for chondrocyte differentiation in ATDC5 chondroprogenitor cells.

Aggrecan is an integral component of the extracellular matrix in cartilaginous tissues, including the growth plate. Heterozygous defects in the aggrecan gene have been identified as a cause of autosomal dominant short stature, bone age acceleration, and premature growth cessation. The mechanisms accounting for this phenotype remain unknown. We used ATDC5 cells, an established model of chondrogenesis, to evaluate the effects of aggrecan deficiency. ATDC5 aggrecan knockdown cell lines (AggKD) were generated using lentiviral shRNA transduction particles. Cells were stimulated with insulin/transferrin/selenium for up to 21 days to induce chondrogenesis. Control ATDC5 cells showed induction of Col2a1 starting at day 8 and induction of Col10a1 starting at day 12. AggKD cells had significantly reduced expression of Col2a1 and Col10a1 (p<0.0001) with only minimal increases in expression over time, indicating that chondrogenesis was markedly impaired. The induction of Col2a1 and Col10a1 was not rescued by culturing of AggKD cells in wells pre-conditioned with ATDC5 extracellular matrix or in co-culture with wild-type ATDC5 cells. We interpret our studies as indicating that aggrecan has an integral role in chondrogenesis that may be mediated through intracellular mechanisms.

MON-262 Aggrecan Is Required for Chondrocyte Differentiation in ATDC5 Chondroprogenitor Cells

Background Aggrecan is an integral part of the extracellular matrix in cartilaginous tissue, including that of the growth plate. Heterozygous defects in the aggrecan gene have been identified as a cause of autosomal dominant short stature, bone age acceleration, and premature growth cessation, all of which are consistent with a role for aggrecan in the cartilaginous growth plate. Previous animal studies of aggrecanopathies have shown disruption of growth plate architecture and acceleration of chondrocyte hypertrophy, although the mechanisms accounting for this phenotype remains unknown. We undertook studies to elucidate the mechanism by which aggrecan loss-of-function mutations lead to impaired long bone growth in concert with bone age acceleration. Methods We used ATDC5 chondroprogenitor cells, an established model of chondrogenesis, to evaluate the effects of aggrecan deficiency. ATDC5 aggrecan knock down cell lines (AggKD) were generated using shRNA lentiviral transduction particles. Cells were stimulated with insulin to induce chondrogenesis for 21 days and studied. Aggrecan qPCR and Alcian blue staining were used to assess levels of aggrecan knock down. Control and AggKD cells were cultured in media containing 10 μg/ml insulin to stimulate chondrogenesis. Cells were studied at multiple time points over 21 days using qPCR, hemocytometer counting, and flow cytometry with propidium iodide staining for cell cycle analysis. Extracellular matrix (ECM) pre-conditioning experiments and co-culture experiments of AggKD cells with wild-type (WT) ATDC5 cells were also used to evaluate effects on chondrogenesis. Results Control ATDC5 cells showed induction of col2a1 starting at day 8 and induction of col10a1 starting at day 12. AggKD cells had significantly reduced expression of col2a1 and col10a1 (p<0.0001) with only minimal increases in expression over time, indicating that chondrogenesis was markedly impaired. This reduction of col2a1 and col10a1 was not rescued by culturing AggKD cells in wells pre-conditioned with WT ATDC5 ECM, or even by co-culturing with WT ATDC5 cells. Sox9 and ihh levels were similar between control and AggKD cell lines. Hemocytometer counts showed increased cells in the control line compared to AggKD lines at Day 8 (p<0.0005), but there was no difference in cell cycle analysis using flow cytometry. Conclusion Our studies indicate that AggKD cells do not undergo normal chondrogenesis, suggesting for the first time that aggrecan has an integral role in this cell development process. Experiments that reintroduced aggrecan via extracelluar and pericellular matrices from pre-conditioning wells and co-culturing cells were unsuccessful at rescuing chondrogenesis in AggKD cells. This suggests that the role of aggrecan in chondrogenesis appears to be mediated through intracellular mechanisms.

Hypergravity down-regulates c-fos gene expression via ROCK/Rho-GTP and the PI3K signaling pathway in murine ATDC5 chondroprogenitor cells.

Chondrocytes are known to be physiologically loaded with diverse physical factors such as compressive stress, shear stress and hydrostatic pressure. Although the effects of those mechanical stimuli onto various cell models have been widely studied, those of hypergravity have not yet been revealed clearly. Hereby, we hypothesized that the hypergravity affects relative positions of intracellular elements including nucleus and cytoskeletons due to their density differences, triggering mechanotransduction in the cell. The aim of this study was to investigate the effect of hypergravity on c-fos expression in the murine ATDC5 chondroprogenitor cells, as c-fos is a well known key regulator of cell proliferation and differentiation, including in chondrocytes. We first found that hypergravity down-regulated c-fos expression transiently via ROCK/Rho-GTP and PI3K signaling, and the down-regulation was suppressed by inhibition of actin polymerization.

Stable 5-Hydroxymethylcytosine (5hmC) Acquisition Marks Gene Activation During Chondrogenic Differentiation.

Regulation of gene expression changes during chondrogenic differentiation by DNA methylation and demethylation is little understood. Methylated cytosines (5mC) are oxidized by the ten-eleven-translocation (TET) proteins to 5-hydroxymethylcytosines (5hmC), 5-formylcytosines (5fC), and 5-carboxylcytosines (5caC), eventually leading to a replacement by unmethylated cytosines (C), ie, DNA demethylation. Additionally, 5hmC is stable and acts as an epigenetic mark by itself. Here, we report that global changes in 5hmC mark chondrogenic differentiation in vivo and in vitro. Tibia anlagen and growth plate analyses during limb development at mouse embryonic days E 11.5, 13.5, and 17.5 showed dynamic changes in 5hmC levels in the differentiating chondrocytes. A similar increase in 5hmC levels was observed in the ATDC5 chondroprogenitor cell line accompanied by increased expression of the TET proteins during in vitro differentiation. Loss of TET1 in ATDC5 decreased 5hmC levels and impaired differentiation, demonstrating a functional role for TET1-mediated 5hmC dynamics in chondrogenic differentiation. Global analyses of the 5hmC-enriched sequences during early and late chondrogenic differentiation identified 5hmC distribution to be enriched in the regulatory regions of genes preceding the transcription start site (TSS), as well as in the gene bodies. Stable gains in 5hmC were observed in specific subsets of genes, including genes associated with cartilage development and in chondrogenic lineage-specific genes. 5hmC gains in regulatory promoter and enhancer regions as well as in gene bodies were strongly associated with activated but not repressed genes, indicating a potential regulatory role for DNA hydroxymethylation in chondrogenic gene expression.

Robust Circadian Rhythm and Parathyroid Hormone-Induced Resetting during Hypertrophic Differentiation in ATDC5 Chondroprogenitor Cells.

Cartilage tissues possess intrinsic circadian oscillators, which influence chondrocyte function and chondrocyte specific gene expression. However, it is not fully understood how chondrogenesis influences the circadian clock, and vice versa. Thus, we established ATDC5 cells which were stably transfected with the Bmal1:luc reporter and revealed robust circadian rhythms in ATDC5 cells during differentiation. Moreover, the circadian clock in ATDC5 cells was strongly reset by PTH in a circadian time-dependent manner. This assay system is expected to be useful for investigating the role of the circadian clock in chondrogenic differentiation and the precise molecular mechanisms underlying PTH action on the chondrocyte circadian clock.

Midkine-deficiency delays chondrogenesis during the early phase of fracture healing in mice.

The growth and differentiation factor midkine (Mdk) plays an important role in bone development and remodeling. Mdk-deficient mice display a high bone mass phenotype when aged 12 and 18 months. Furthermore, Mdk has been identified as a negative regulator of mechanically induced bone formation and it induces pro-chondrogenic, pro-angiogenic and pro-inflammatory effects. Together with the finding that Mdk is expressed in chondrocytes during fracture healing, we hypothesized that Mdk could play a complex role in endochondral ossification during the bone healing process. Femoral osteotomies stabilized using an external fixator were created in wildtype and Mdk-deficient mice. Fracture healing was evaluated 4, 10, 21 and 28 days after surgery using 3-point-bending, micro-computed tomography, histology and immunohistology. We demonstrated that Mdk-deficient mice displayed delayed chondrogenesis during the early phase of fracture healing as well as significantly decreased flexural rigidity and moment of inertia of the fracture callus 21 days after fracture. Mdk-deficiency diminished beta-catenin expression in chondrocytes and delayed presence of macrophages during early fracture healing. We also investigated the impact of Mdk knockdown using siRNA on ATDC5 chondroprogenitor cells in vitro. Knockdown of Mdk expression resulted in a decrease of beta-catenin and chondrogenic differentiation-related matrix proteins, suggesting that delayed chondrogenesis during fracture healing in Mdk-deficient mice may be due to a cell-autonomous mechanism involving reduced beta-catenin signaling. Our results demonstrated that Mdk plays a crucial role in the early inflammation phase and during the development of cartilaginous callus in the fracture healing process.

Sulfation patterns of exogenous chondroitin sulfate affect chondrogenic differentiation of ATDC5 cells

Background Chondroitin sulfate (CS) has been used in cartilage tissue engineering techniques as a positive modulator of scaffolds. CS is a linear polysaccharide consisting of variously sulfated repeating disaccharides. The sulfation patterns of CS are closely related to their biological functions, but only monosulfated CS has been applied to scaffolds. In this study, we investigated the effects of various sulfation patterns of CS on chondrogenic differentiation using ATDC5 chondroprogenitor cells.Methods Disaccharide composition analysis of CS produced by ATDC5 cells at various differentiation steps was performed using high-performance liquid chromatography. ATDC5 cells were cultured with exogenously added, variously sulfated CS. Cell proliferation was analyzed by the 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8) assay. Extracellular matrix production was evaluated by Alcian blue staining. Alkaline phosphatase (ALP) activity was evaluated using an ALP assay kit. Expression of chondrogenic markers was evaluated by real-time reverse transcription polymerase chain reaction (RT-PCR) or an enzyme-linked immunosorbent assay (ELISA) using a Type II Collagen Detection kit.Results The major components of CS produced by ATDC5 cells were 4-O-monosulfated disaccharides throughout chondrogenic differentiation. Low proportions of 4,6-O-disulfated disaccharides were also detected. Compared to the control group, which did not contain GAGs, the WST-8 assay indicated fewer viable cells when treated with CS-E, which are rich in 4,6-O-disulfated disaccharides. CS-E significantly enhanced Alcian blue staining in a dose-dependent manner and decreased ALP activity after 21 days of culture. Real-time RT-PCR showed that CS-E significantly enhanced all chondrogenic markers, col2a1, aggrecan, and sox9, either at day 4 or day 14 of culture. The results of ELISA analysis confirmed that CS-E significantly enhanced the production of type II collagen.Conclusions ATDC5 cells produced four different monosulfated or disulfated disaccharides in their extracellular matrices. The sulfation patterns of exogenously added CS affected chondrogenic differentiation of ATDC5 cells. In particular, CS-E rich in disulfated disaccharides significantly promoted chondrogenic differentiation of ATDC5 cells. Thus, CS containing this disulfated structure may be a useful scaffold component for enhancing chondrogenesis in cartilage tissue engineering.

Matrilin-3 chondrodysplasia mutations cause attenuated chondrogenesis, premature hypertrophy and aberrant response to TGF-β in chondroprogenitor cells.

Studies have shown that mutations in the matrilin-3 gene (MATN3) are associated with multiple epiphyseal dysplasia (MED) and spondyloepimetaphyseal dysplasia (SEMD). We tested whether MATN3 mutations affect the differentiation of chondroprogenitor and/or mesenchymal stem cells, which are precursors to chondrocytes. ATDC5 chondroprogenitors stably expressing wild-type (WT) MATN3 underwent spontaneous chondrogenesis. Expression of chondrogenic markers collagen II and aggrecan was inhibited in chondroprogenitors carrying the MED or SEMD MATN3 mutations. Hypertrophic marker collagen X remained attenuated in WT MATN3 chondroprogenitors, whereas its expression was elevated in chondroprogenitors expressing the MED or SEMD mutant MATN3 gene suggesting that these mutations inhibit chondrogenesis but promote hypertrophy. TGF-β treatment failed to rescue chondrogenesis markers but dramatically increased collagen X mRNA expression in mutant MATN3 expressing chondroprogenitors. Synovium derived mesenchymal stem cells harboring the SEMD mutation exhibited lower glycosaminoglycan content than those of WT MATN3 in response to TGF-β. Our results suggest that the properties of progenitor cells harboring MATN3 chondrodysplasia mutations were altered, as evidenced by attenuated chondrogenesis and premature hypertrophy. TGF-β treatment failed to completely rescue chondrogenesis but instead induced hypertrophy in mutant MATN3 chondroprogenitors. Our data suggest that chondroprogenitor cells should be considered as a potential target of chondrodysplasia therapy.

FLRT2 Interacts With Fibronectin in the ATDC5 Chondroprogenitor Cells

Expression studies have implicated FLRT2 in cranial neural crest cell migration and prechondrogenic cell condensation during craniofacial skeletogenesis. We aimed to determine whether FLRT2 was involved in mediating cell-matrix interactions in the ATDC5 chondroprogenitor cell line. Immunolocalization experiments of ATDC5 cells revealed that FLRT2 was present on the cell membrane as well as extracellularly, where it colocalized with Fibronectin (Fn). After cell extraction of the matrix, FLRT2 was identified in the ATDC5-derived extracellular matrix (ECM) and was further found to be associated with Fn-coated beads in cell cultures. Blockage of Fn fibril formation via a blocking peptide resulted in a concomitant decrease in extracellular FLRT2 accumulation. Over a 7-day period following the replenishment of the Fn blocking peptide to the cultures, there was a partial rebound in Fn fibril formation that was accompanied by a concomitant reappearance of FLRT2 co-expression. Co-immunoprecipitation confirmed that FLRT2 and Fn interacted, either directly or indirectly. Immunoprecipitation and Western blot analyses with antibodies recognizing epitopes located on the extra- and intracellular domains of FLRT2 further revealed the presence of different sized bands, suggesting that FLRT2 may exist in both membrane-bound and shed forms. Our data therefore provide evidence that FLRT2 and/or its cleavage products may be cooperating with Fn and other ECM proteins to regulate critical cellular events. Further studies will be necessary in delineate more precisely the roles of FLRT2 in mediating cell- and cell-matrix interactions during normal development.

Chondrocyte‐intrinsic Smad3 represses Runx2‐inducible matrix metalloproteinase 13 expression to maintain articular cartilage and prevent osteoarthritis

To identify mechanisms by which Smad3 maintains articular cartilage and prevents osteoarthritis. A combination of in vivo and in vitro approaches was used to test the hypothesis that Smad3 represses Runx2-inducible gene expression to prevent articular cartilage degeneration. Col2-Cre;Smad3(fl/fl) mice allowed study of the chondrocyte-intrinsic role of Smad3 independently of its role in the perichondrium or other tissues. Primary articular cartilage chondrocytes from Smad3(fl/fl) mice and ATDC5 chondroprogenitor cells were used to evaluate Smad3 and Runx2 regulation of matrix metalloproteinase 13 (MMP-13) messenger RNA (mRNA) and protein expression. Chondrocyte-specific reduction of Smad3 caused progressive articular cartilage degeneration due to imbalanced cartilage matrix synthesis and degradation. In addition to reduced type II collagen mRNA expression, articular cartilage from Col2-Cre;Smad3(fl/fl) mice was severely deficient in type II collagen and aggrecan protein due to excessive MMP-13-mediated proteolysis of these key cartilage matrix constituents. Normally, transforming growth factor β (TGFβ) signals through Smad3 to confer a rapid and dynamic repression of Runx2-inducible MMP-13 expression. However, we found that in the absence of Smad3, TGFβ signals through p38 and Runx2 to induce MMP-13 expression. Our findings elucidate a mechanism by which Smad3 mutations in humans and mice cause cartilage degeneration and osteoarthritis. Specifically, Smad3 maintains the balance between cartilage matrix synthesis and degradation by inducing type II collagen expression and repressing Runx2-inducible MMP-13 expression. Selective activation of TGFβ signaling through Smad3, rather than p38, may help to restore the balance between matrix synthesis and proteolysis that is lost in osteoarthritis.

Regulatory role of stromal cell-derived factor-1 in bone morphogenetic protein-2-induced chondrogenic differentiation in vitro

Chemokine stromal cell-derived factor-1 (SDF-1) signals via binding to its primary transmembrane receptor, cysteine (C)-X-C chemokine receptor-4 (CXCR4). We previously reported that SDF-1 regulates osteogenic differentiation of mesenchymal stem/progenitor cells (MPCs) induced by bone morphogenetic protein-2 (BMP2). Although BMP2 is also capable of inducing chondrogenic differentiation of MPCs, the involvement of SDF-1 signaling in this function of BMP2 remains unknown. In this study, we aimed to test the role of SDF-1 signaling involved in BMP2-induced chondrogenic differentiation, using ATDC5 chondroprogenitors and mouse bone marrow-derived mesenchymal stromal cells (BMSCs). Our data showed that blocking of the SDF-1/CXCR4 pathway inhibits the differentiation of these cells into the chondrocytic lineages in response to BMP2 stimulation, evidenced by the reduced expression of type II collagen α1 (Col2α1), aggrecan, and type X collagen α1 (Col10α1), markers for chondrogenic differentiation. This effect of blocking SDF-1 signaling on BMP2-chondrogenic differentiation is associated with suppressed Sox9 and Runx2 expression (key transcription factors required for early and late stages of chondrogenic differentiation, respectively) and mediated via inhibiting intracellular Smad and Erk activation. Moreover, we found that addition of exogenous SDF-1 protein synergistically enhances the BMP2-induced chondrogenic differentiation in a dose-dependent manner. Collectively, our results demonstrated a novel role of SDF-1 signaling in regulating BMP2-induced chondrogenic differentiation in vitro. These data provide new insights into molecular mechanisms underlying BMP2-osteo/chondrogenesis, and may lead to the identification of new therapeutic targets and strategies to improve cartilage repair and regeneration in broad orthopaedic situations.

A study of the local RAS in ATDC5 chondroprogenitor cells and epiphyseal plates of mice

A study of the local RAS in ATDC5 chondroprogenitor cells and epiphyseal plates of mice

BMP-2 and BMP-7: differential regulation of chondrogenic differentiation

BMP-2 and BMP-7: differential regulation of chondrogenic differentiation

Stimulation of chondrogenesis in ATDC5 chondroprogenitor cells and hypertrophy in mouse by Genkwadaphnin

The growth in height of the bone plate is a result of endochondral proliferation in epiphyseal growth plates and the conversion of chondrocytes into new bone. The control of chondrogenic differentiation and hypertrophy is critical for these processes. The present study was aimed to demonstrate the chondromodulating activity of Genkwadaphnin. ATDC5 cultures treated with Genkwadaphnin produced cartilaginous nodules that were greater in number and larger in size than control cultures. Genkwadaphnin treated ATDC5 cells also stained more intensely with Alcian blue than control cells, suggesting greater synthesis of matrix proteoglycans in the former. Genkwadaphnin markedly induced the activation of alkaline phosphatase, as well as the expression of chondrogenic marker genes such as type II collagen, aggrecan, type I collagen, type X collagen, osteocalcin, and bone sialoprotein in ATDC5 cells. The expression of signaling molecules involved in chondrogenesis including Smad4, Sox9, and β-catenin was also induced by treatment of ATDC5 cells with Genkwadaphnin. Furthermore, Genkwadaphnin induced the activation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). To analyze the role of Genkwadaphnin in growth plate chondrocyte in vivo, we analyzed chondrogenesis in mice treated with Genkwadaphnin. The significant expansion in growth plate and hypertrophic zone and numerous numbers of chondrocyte positive cells in hypertrophic and proliferative bone areas were observed. These observations provide the first evidence that Genkwadaphnin has chondromodulating activity and may open new therapeutic avenues to treat a variety of skeletal diseases, such as dwarfism.

Stimulatory actions of lysophosphatidic acid on mouse ATDC5 chondroprogenitor cells

Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are bioactive lysophospholipids that affect various cellular processes through G protein-coupled receptors. In our current study, we found by in situ hybridization that E11.5 mouse embryos strongly expressed the LPA receptor subtype LPA(1) in cartilaginous bone primordia and the surrounding mesenchymal cells. However, despite their wide-ranging actions, the roles of lysophospholipids in chondrogenesis remain poorly understood. The mouse clonal cell line ATDC5 undergoes a sequential differentiation of chondroprogenitor cells in vitro. Undifferentiated and differentiated ATDC5 cells express LPA(1) and other lysophospholipid receptors including S1P receptor S1P(1) and S1P(2). Taking advantage of this cell model, we studied the effects of LPA on the activities of chondroprogenitor cells. LPA markedly stimulates both DNA synthesis and the migration of ATDC5 chondroprogenitor cells in culture, whereas S1P suppresses the migration of these cells. Treatment with Ki16425, an LPA(1)- and LPA(3)-specific receptor antagonist, suppressed the fetal bovine serum-stimulated migration of ATDC5 cells by almost 80%. These results indicate that LPA plays an important role in the activation of chondroprogenitor cells.

Inhibitory effect of lactoferrin on hypertrophic differentiation of ATDC5 mouse chondroprogenitor cells

The skeleton is formed by two different mechanisms. In intramembranous ossification, osteoblasts form bone directly, whereas in endochondral ossification, chondrocytes develop a cartilage template, prior to osteoblast-mediated skeletogenesis. Lactoferrin is an iron-binding glycoprotein belonging to the transferrin family. It is known to promote the growth and differentiation of osteoblasts. In this study, we investigated the effects of bovine lactoferrin on the chondrogenic differentiation of ATDC5 chondroprogenitor cells. This mouse embryonic carcinoma-derived clonal cell line provides an in vitro model of chondrogenesis. Lactoferrin treatment of differentiating ATDC5 cells promoted cell proliferation in the initial stage of the differentiation process. However, lactoferrin treatment resulted in inhibition of hypertrophic differentiation, characterized by suppression of alkaline phosphatase activity, aggrecan synthesis and N-cadherin expression. This inhibitory effect was accompanied by sustained Sox9 expression, as well as increased Smad2/3 expression and phosphorylation, suggesting that lactoferrin regulates chondrogenic differentiation by up-regulating the Smad2/3-Sox9 signaling pathway.

Stimulation of the side population fraction of ATDC5 chondroprogenitors by hypoxia

The influence of oxygen tension on the side population (SP) fraction sorted from ATDC5 chondroprogenitor cells was investigated. ATDC5 cells cultured in normoxia (20%) or hypoxia (1% oxygen) were compared. The SP fraction was significantly higher in the cells cultured in hypoxia. The gene expression of 3 ABC transporters, abcb1a/b (mdr1a/b) and abcg2 (bcrp1) was quantified by RT-PCR. SP cells were characterized by the higher expression of abcb1a. The expression levels of abcb1b and abcg2 were higher than abcb1a. However, there was no significant difference between SP and non-SP fractions in the expression of abcb1b and abcg2. The telomeric repeat amplification protocol assay showed that SP cells tended to show lower telomerase activity than non-SP cells. Chondrogenic properties of ATDC5 cells derived from SP or non-SP were assessed by micromass culture. There were not significant differences between SP and non-SP derived cells in Alcian blue staining and sox9, Aggrecan, Col2a1 and SZP mRNA expression. The results demonstrate that the SP fraction was stimulated by hypoxia and chondrogenic properties of SP and non-SP fraction of ATDC5 cells were similar.

Proper expression of helix–loop–helix protein Id2 is important to chondrogenic differentiation of ATDC5 cells

The process of chondrogenesis can be mimicked in vitro by insulin treatment of mouse ATDC5 chondroprogenitor cells. To identify novel factors that are involved in the control of chondrogenesis, we carried out a large-scale screening through retroviral insertion mutagenesis and isolated a fast-growing ATDC5 clone incapable of chondrogenic differentiation. Inverse-PCR analysis of this clone revealed that the retroviral DNA was inserted into the promoter region of mouse Id2 (inhibitor of DNA-binding protein 2) gene. This retroviral insertion increased Id2 protein levels to twice those found in normal ATDC5 cells. To investigate whether an elevated level of Id2 protein was responsible for inhibition of chondrogenic differentiation, ATDC5 cells were infected with a retrovirus to stably express Id2. ATDC5 cells expressing ectopic Id2 exhibited signs of de-differentiation, such as rapid growth, and insulin failed to induce expression of Sox9 (Sry-type high-mobility-group box 9) or matrix genes such as type II collagen (COL2) in these cells. When endogenous Id2 was knocked down by siRNA (small interfering RNA) in ATDC5 cells, expression of Sox9 and COL2 was increased and chondrogenic differentiation was accelerated. To examine how Id2 is expressed in chondrocytes in vivo, we carried out immunostaining of E16.5 mouse embryos and found that Id2 is expressed in articular chondrocytes and proliferating chondrocytes, but barely detectable in hypertrophic chondrocytes. Our results suggest that proper expression of Id2 is important to achieving a fine balance between growth and differentiation during chondrogenesis.

Rab23 Regulates Differentiation of ATDC5 Chondroprogenitor Cells

Insulin treatment of mouse ATDC5 chondroprogenitors induces these cells to differentiate into mature chondrocytes. To identify novel factors that are involved in this process, we carried out mutagenesis of ATDC5 cells through retroviral insertion and isolated two mutant clones incapable of differentiation. Inverse PCR analysis of these clones revealed that the retroviral DNA was inserted into the promoter region of the Rab23 gene, resulting in increased Rab23 expression. To investigate whether an elevated level of Rab23 protein led to inhibition of chondrogenic differentiation, we characterized ATDC5 cells that either overexpress endogenous Rab23 or stably express ectopic Rab23. Our results revealed that up-regulation of Rab23 can indeed inhibit chondrogenic differentiation with a concomitant down-regulation of matrix genes such as type II collagen and aggrecan. In addition, stable small interfering RNA knockdown of Rab23 also resulted in inhibition of chondrogenic differentiation as well as down-regulation of Sox9, a master regulator of chondrogenesis. Interestingly, Sox9 expression has recently been linked to Gli1, and we found that Rab23 knockdown decreased Gli1 expression in chondrocytes. Because the phenotypes of Rab23 mutations in mice and humans include defects in cartilage and bone development, our study suggests that Rab23 is involved in the control of Sox9 expression via Gli1 protein.