Chondrogenic Differentiation Of ATDC5 Cells Research Articles

Activation of genes for growth factor and cytokine pathways late in chondrogenic differentiation of ATDC5 cells

The mouse embryonal carcinoma cell line ATDC5 provides an excellent model system for chondrogenesis in vitro. To understand better the molecular mechanisms of endochondral bone formation, we investigated gene expression profiles during the differentiation course of ATDC5 cells, using an in-house microarray harboring full-length-enriched cDNAs. For 28 days following chondrogenic induction, 507 genes were up- or down-regulated at least 1.5-fold. These genes were classified into five clusters based on their expression patterns. Genes for growth factor and cytokine pathways were significantly enriched in the cluster characterized by increases in expression during late stages of chondrocyte differentiation. mRNAs for decorin and osteoglycin, which have been shown to bind to transforming growth factors-β and bone morphogenetic proteins, respectively, were found in this cluster and were detected in hypertrophic chondrocytes of developing mouse bones by in situ hybridization analysis. Taken together with assigned functions of individual genes in the cluster, interdigitated interaction between a number of intercellular signaling molecules is likely to take place in the late chondrogenic stage for autocrine and paracrine regulation among chondrocytes, as well as for chemoattraction and stimulation of progenitor cells of other lineages.

Involvement of phosphoinositide 3-kinase signaling pathway in chondrocytic differentiation of ATDC5 cells: application of a gene-trap mutagenesis.

Gene-trap mutagenesis is based on the notion that the random insertion of a trapping vector may disturb the function of inserted genes. Here, we applied this method to murine mesenchymal ATDC5 cells, which differentiate into mature chondrocytes in the presence of insulin. As the trap vector we used pPT1-geo, which lacks its own promoter and enhancer, but contains a lacZ-neo fusion gene as a reporter and selection marker driven by the promoter of the trapped gene. After pPT1-geo was introduced into ATDC5 cells by electroporation, the neomycin-resistant clones were screened for beta-galactosidase activity. The selected clones were cultured in differentiation medium to evaluate the chondrogenic phenotype. The clones no. 6-30 and 6-175, which exhibited impaired and accelerated mineralization, respectively, were subjected to further analysis. In clone no. 6-30 in which the gene coding for the p85alpha subunit of phosphoinositide 3-kinase (PI3K) was trapped, the expression of marker genes of early chondrocytes including collagen type II, aggrecan, and PTH/PTHrP receptor was delayed. The insulin-induced stimulation of growth was reduced in clone no. 6-30 compared with the parental ATDC5 cells. Moreover, treatment of parental ATDC5 cells with a specific inhibitor of PI3K, LY294002, phenocopied clone no. 6-30, suggesting the involvement of PI3K signaling in the chondrogenic differentiation of ATDC5 cells. Clone no. 6-175 with accelerated mineralization was revealed to have a gene homologous to human KIAA0312 trapped, whose function remains unclear. Taken together, the gene-trap in ATDC5 cells might be useful to identify the molecules involved in chondrogenic differentiation.

P21Cip-1/SDI-1/WAF-1 expression via the mitogen-activated protein kinase signaling pathway in insulin-induced chondrogenic differentiation of ATDC5 cells

The embryonal carcinoma-derived cell line, ATDC5, differentiates into chondrocytes in response to insulin or insulin-like growth factor-I stimulation. In this study, we investigated the roles of mitogen-activated protein (MAP) kinases in insulin-induced chondrogenic differentiation of ATDC5 cells. Insulin-induced accumulation of glycosaminoglycan and expression of chondrogenic differentiation markers, type II collagen, type X collagen, and aggrecan mRNA were inhibited by the MEK1/2 inhibitor (U0126) and the p38 MAP kinase inhibitor (SB203580). Conversely, the JNK inhibitor (SP600125) enhanced the synthesis of glycosaminoglycan and expression of chondrogenic differentiation markers. Insulin-induced phosphorylation of ERK1/2 and JNK but not that of p38 MAP kinase. We have previously clarified that the induction of the cyclin-dependent kinase inhibitor, p21Cip-1/SDI-1/WAF-1, is essential for chondrogenic differentiation of ATDC5 cells. To assess the relationship between the induction of p21 and MAP kinase activity, we investigated the effect of these inhibitors on insulin-induced p21 expression in ATDC5 cells. Insulin-induced accumulation of p21 mRNA and protein was inhibited by the addition of U0126 and SB203580. In contrast, SP600125 enhanced it. Inhibitory effects of U0126 or stimulatory effects of SP600125 on insulin-induced chondrogenic differentiation were observed when these inhibitors exist in the early phase of differentiation, suggesting that MEK/ERK and JNK act on early phase differentiation. SB202580, however, is necessary not only for early phase but also for late phase differentiation, indicating that p38 MAP kinase stimulates differentiation by acting during the entire period of cultivation. These results for the first time demonstrate that up-regulation of p21 expression by ERK1/2 and p38 MAP kinase is required for chondrogenesis, and that JNK acts as a suppressor of chondrogenesis by down-regulating p21 expression.

Runx2 induces osteoblast and chondrocyte differentiation and enhances their migration by coupling with PI3K-Akt signaling.

Runx2 and phosphatidylinositol 3-kinase (PI3K)–Akt signaling play important roles in osteoblast and chondrocyte differentiation. We investigated the relationship between Runx2 and PI3K-Akt signaling. Forced expression of Runx2 enhanced osteoblastic differentiation of C3H10T1/2 and MC3T3-E1 cells and enhanced chondrogenic differentiation of ATDC5 cells, whereas these effects were blocked by treatment with IGF-I antibody or LY294002 or adenoviral introduction of dominant-negative (dn)–Akt. Forced expression of Runx2 or dn-Runx2 enhanced or inhibited cell migration, respectively, whereas the enhancement by Runx2 was abolished by treatment with LY294002 or adenoviral introduction of dn-Akt. Runx2 up-regulated PI3K subunits (p85 and p110β) and Akt, and their expression patterns were similar to that of Runx2 in growth plates. Treatment with LY294002 or introduction of dn-Akt severely diminished DNA binding of Runx2 and Runx2-dependent transcription, whereas forced expression of myrAkt enhanced them. These findings demonstrate that Runx2 and PI3K-Akt signaling are mutually dependent on each other in the regulation of osteoblast and chondrocyte differentiation and their migration.

Human Crossveinless-2 is a novel inhibitor of bone morphogenetic proteins

Drosophila Crossveinless-2 (dCV-2) is required for local activation of Mad phosphorylation in the fruit fly wing and has been postulated to be a positive regulator of BMP-mediated signaling. In contrast, the presence of 5 Chordin-like cysteine-rich domains in the CV-2 protein suggests that CV-2 belongs to a family of well-established inhibitors of BMP function that includes Chordin and Sog [Development 127 (2000) 3947]. We have identified a human homolog of Drosophila CV-2 (hCV-2). Here we show that purified recombinant hCV-2 protein inhibits BMP-2 and BMP-4 dependent osteogenic differentiation of W-20-17 cells, as well as BMP dependent chondrogenic differentiation of ATDC5 cells. Interestingly, hCV-2 messenger RNA is expressed at high levels in human primary chondrocytes, whereas expression in primary human osteoblasts is low. These results suggest that hCV-2 may regulate BMP responsiveness of osteoblasts and chondrocytes in vivo. Taken together we have shown that contrary to the function predicted from the fruit fly, Crossveinless-2 is a novel inhibitor of BMP function.

FK506 induces chondrogenic differentiation of clonal mouse embryonic carcinoma cells, ATDC5

FK506 (Tacrolimus) and cyclosporin A exert their immunosuppressive effects via a common mechanism, calcineurin inhibition, after binding to intracellular proteins termed immunophilins: FK506-binding protein (FKBP) and cyclophilin. In this study, FK506 was found to induce chondrogenic differentiation of ATDC5 cells (clonal mouse embryonal carcinoma cells) in a concentration-dependent manner (0.1–1000 ng/ml). Immunohistochemical staining showed that ATDC5 cells induced to differentiate by FK506 produced proteoglycan and type II collagen, main components of the extracellular matrix of cartilage. Rapamycin, an immunosuppressant that binds to FKBP, antagonized the effect of FK506. Cyclosporin A did not induce chondrogenesis at concentrations up to 1000 ng/ml. Taken together, these results suggest that FK506 induces chondrogenic differentiation of ATDC5 cells via a calcineurin-independent mechanism, after binding to FKBP.

Involvement of the phosphoinositide 3-kinase/protein kinase B signaling pathway in insulin/IGF-I-induced chondrogenesis of the mouse embryonal carcinoma-derived cell line ATDC5

The embryonal carcinoma-derived cell line, ATDC5, differentiates into chondrocytes in response to insulin/insulin-like growth factor-I (IGF-I) stimulation. In the present study, we examined whether insulin/IGF-I stimulation caused activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB) pathway in ATDC5 cells. We also determined whether the insulin-stimulated differentiation of ATDC5 cells into chondrocytes could be mimicked by activation of the PKB pathway alone. ATDC5 cells produced phosphatidylinositol 3,4,5-trisphosphate and the pleckstrin homology domain of PKB was recruited to the plasma membrane in response to insulin stimulation. This was probably a result of activation of PI3K because the PI3K inhibitors, wortmannin and LY294002, inhibited both responses, although the effective concentrations were as high as 10 μM. Insulin stimulation caused the chondrogenic differentiation of ATDC5 cells as assessed by chondrogenic nodule staining with alcian blue. The addition of wortmannin or LY294002, PI3K inhibitors, suppressed the staining, and the suppression was reversible, indicating the effect of the inhibitors is not toxic. Finally, we exogenously expressed a constitutively-activated from of PKB (myristoylated PKB, myr-PKB) in ATDC5 cells, and found the chondrogenic differentiation of ATDC5 cells to form nodules occurred in the absence of insulin stimulation. The kinase-negative mutant of myr-PKB did not caused differentiation, indicating that kinase activity is required. These results support the hypothesis that the PI3K/PKB signaling pathway is involved in the chondrogenic differentiation of ATDC5 cells in response to insulin/IGF-I stimulation. This is the first report that demonstrates the involvement of phosphoinositide signaling in the induction of chondrogenesis from undifferentiated cells.

P21Cip-1/SDI-1/WAF-1 gene is involved in chondrogenic differentiation of ATDC5 cells in vitro.

Development of skeletal cartilage is characterized with coupling growth arrest and cell differentiation. Here, to understand the cyclin-dependent kinase inhibitors involved in the progression of chondrogenic differentiation, we examined changes in the expression levels of cyclin-dependent kinase inhibitor members using mouse ATDC5 prechondrocytes as a widely used in vitro model of cartilage differentiation. Up-regulation of p21 and p27 mRNA was observed following a decrease in growth rate of prechondrocytes, and both transcripts subsequently accumulated during chondrogenic differentiation; p15, p18, and p19 mRNA, in contrast, did not change during differentiation. Only the up-regulation of p21 mRNA during differentiation was prevented by the continuous treatment of early chondrogenic inhibitor, parathyroid hormone, indicating a close correlation between differentiation and p21 induction in ATDC5 cells. Therefore, to examine the role of p21 during chondrogenesis, we established stable cell lines overexpressing full-length p21 antisense RNA in ATDC5. The reduction of endogenous p21 in these cell lines caused inhibition of early chondrogenic differentiation in ATDC5, indicating that p21 gene plays an important role in this process of the cells in vitro. Furthermore, the level of p21 protein and p21.CDK2 complexes transiently increased during differentiation, but not in undifferentiated cells, leading to a decrease in CDK2-associated kinase. However, differentiation-dependent expressed p21 protein was degraded by a proteasome-dependent pathway. Thus, the progression of chondrogenic differentiation requires down-regulation of CDK2-associated kinase with an increase in p21 protein and subsequent degradation of this protein by a proteasomal pathway.

Molecular Cloning and Biological Activity of a Novel Lysyl Oxidase-related Gene Expressed in Cartilage

We cloned a cDNA encoding a novel lysyl oxidase-related protein, named LOXC, by suppression subtractive hybridization between differentiated and calcified ATDC5 cells, a clonal mouse chondrogenic EC cell line. The deduced amino acid sequence of mouse LOXC consists of 757 amino acids and shows 50% identity with that of mouse lysyl oxidase. Northern blot analysis showed a distinct hybridization band of 5.4 kilobases, and Western blot analysis showed an immunoreactive band at 82 kilodaltons. Expression of LOXC mRNA was detected in osteoblastic MC3T3-E1 cells and embryonic fibroblast C3H10T1/2 cells, whereas none of NIH3T3 fibroblasts and myoblastic C2C12 cells expressed LOXC mRNA in vitro. Moreover, the LOXC mRNA and protein levels dramatically increased throughout a process of chondrogenic differentiation in ATDC5 cells. In vivo, LOXC gene expression was localized in hypertrophic and calcified chondrocytes of growth plates in adult mice. The conditioned media of COS-7 cells transfected with the full-length LOXC cDNA showed the lysyl oxidase activity in both type I and type II collagens derived from chick embryos, and these activities of LOXC were inhibited by beta-aminopropionitrile, a specific inhibitor of lysyl oxidase. Our data indicate that LOXC is expressed in cartilage in vivo and modulates the formation of a collagenous extracellular matrix.

A Novel Alternatively Spliced Fibroblast Growth Factor Receptor 3 Isoform Lacking the Acid Box Domain Is Expressed during Chondrogenic Differentiation of ATDC5 Cells

To determine the role of fibroblast growth factor (FGF).FGF receptor (FGFR) signaling in chondrogenesis, we analyzed the gene expression of alternatively spliced FGFRs during chondrogenic differentiation of ATDC5 cells in vitro. Two isoforms of FGFR3 were expressed in these cells. One was the complete form of FGFR3 (FGFR3) already reported, and the other was a novel one that lacks the acid box domain (FGFR3DeltaAB). The gene of FGFR3DeltaAB was expressed in undifferentiated ATDC5 cells. In contrast, the transcripts of FGFR3 were not detectable in undifferentiated cells but increased during cellular condensation, which is an obligatory step for chondrogenic differentiation. FGFR1 and FGFR2 expression was higher than that of FGFR3 in undifferentiated cells. The gene expression of cell cycle inhibitor p21 was induced during cell condensation and correlated best with the expression of FGFR3 among the FGFR isoforms expressed. The differential expression of FGFR3 isoforms during chondrogenesis suggests that these isoforms may play different roles in the regulation of growth and differentiation in chondrocytes. To define the mitogenic response of FGFR3DeltaAB and FGFR3 to FGFs, their cDNAs were stably transfected into mouse BaF3 pro-B cells. FGFR3 preferentially mediates the mitogenic response to FGF1 and poor response to FGF2. In contrast, FGFR3DeltaAB mediated a higher mitogenic response to FGF2 as well as to FGF1. In addition, FGFR3DeltaAB responds to FGF1 at lower concentrations of heparin than FGFR3 does. These results suggest that the acid box plays an important role in the regulation of FGFR3 to mediate biological activities in response to FGFs.

Requirement of autocrine signaling by bone morphogenetic protein-4 for chondrogenic differentiation of ATDC5 cells

Mouse EC cell line ATDC5 undergoes differentiation to form cartilage nodules via the cellular condensation stage in the presence of insulin. ATDC5 cells expressed transcripts for bone morphogenetic protein-4 (BMP-4), and type IA and type II BMP receptors. Moreover, cells retained responsiveness to BMP-4, which induced the formation of chondrocytes in the culture. When transfected with a kinase domain-truncated type IA BMP receptor construct, cells failed to undergo differentiation beyond the condensation stage even in the presence of insulin. The soluble form of type IA BMP receptor also blocked the formation of chondrocytes in a dose dependent manner. These lines of evidence suggested that autocrine BMP-4 signaling is required for the conversion of chondrogenic precursor cells into chondrocytes.

Differential expressions of BMP family genes during chondrogenic differentiation of mouse ATDC5 cells.

Clonal cell line ATDC5 enables the monitoring of the early- and late-phase chondrogenic differentiation in a single culture. Undifferentiated ATDC5 cells differentiate into type II collagen expressing chondrocytes through a cellular condensation stage (early-phase differentiation) and then to type X collagen-expressing hypertrophic chondrocytes (late-phase differentiation). Progression of cellular differentiation was accelerated by the activation of bone morphogenetic protein (BMP) signaling. ATDC5 cells expressed transcripts for at least four members of the BMP family. The BMP-4 transcripts were expressed in all stages of differentiation, as were transcripts for BMP type IA receptor (ALK-3) and BMP type II receptor. In contrast, transcripts for Growth/ Differentiation factor-S (GDF-5) were induced during a cellular condensation, and those for BMP-6 were induced during the formation of cartilage nodules, and declined as the differentiated ATDC5 cells became hypertrophic, and BMP-7 transcripts were only detected after cells became calcified. Exogenously added BMP-4 indeed promoted the early-phase differentiation. Late-phase differentiation of cells was also stimulated by BMP-4 and BMP-6. Thus, the cumulative increase in BMP signaling promoted the sequential transitions of differentiation steps of cells. These results indicate that the coordinated expressions of endogenous BMPs are involved in the progression of chondrogenic differentiation in ATDC5 cells.

Alteration of cGMP metabolism during chondrogenic differentiation of chondroprogenitor-like EC cells, ATDC5

Guanosine 3′,5′-cyclic monophosphate (cGMP) has been recently reported to be involved in bone formation. ATDC5 cells were used to investigate cGMP metabolism during chondrogenic differentiation. Natriuretic peptide receptor (NPR)-A and NPR-B coupled with guanylate cyclase (GC) mediate biological functions of NPs, whereas NPR-C uncoupled with GC is thought to be the clearance receptor for NPs. The amounts of NPR-A, NPR-B, and CNP transcripts were increased but the amount of NPR-C transcripts was decreased in association with the chondrogenic differentiation of ATDC5 cells. CNP, a specific ligand for NPR-B lets ATDC5 cells accumulate great amounts of cGMP, revealing NPR-B as a dominant biological receptor through differentiation. cGMP hydrolytic activities of PDE1 and PDE5 existed in ATDC5 cells, and the activity of PDE1, which is stimulated by Ca 2+ and calmodulin (CaM) was major of them. Total cGMP hydrolytic activities as well as the amounts of PDE1 and PDE5 transcripts were enhanced during chondrogenic differentiation. Therefore, cGMP production and hydrolysis, cGMP metabolism was considered to be activated in association with chondrogenic differentiation of ATDC5 cells. These observations may lead to a better understanding of cGMP in the chondrocytes where bone formation occurs.

Roles of bone morphogenetic protein type I receptors and Smad proteins in osteoblast and chondroblast differentiation.

The biological effects of type I serine/threonine kinase receptors and Smad proteins were examined using an adenovirus-based vector system. Constitutively active forms of bone morphogenetic protein (BMP) type I receptors (BMPR-IA and BMPR-IB; BMPR-I group) and those of activin receptor-like kinase (ALK)-1 and ALK-2 (ALK-1 group) induced alkaline phosphatase activity in C2C12 cells. Receptor-regulated Smads (R-Smads) that act in the BMP pathways, such as Smad1 and Smad5, also induced the alkaline phosphatase activity in C2C12 cells. BMP-6 dramatically enhanced alkaline phosphatase activity induced by Smad1 or Smad5, probably because of the nuclear translocation of R-Smads triggered by the ligand. Inhibitory Smads, i.e., Smad6 and Smad7, repressed the alkaline phosphatase activity induced by BMP-6 or the type I receptors. Chondrogenic differentiation of ATDC5 cells was induced by the receptors of the BMPR-I group but not by those of the ALK-1 group. However, kinase-inactive forms of the receptors of the ALK-1 and BMPR-I groups blocked chondrogenic differentiation. Although R-Smads failed to induce cartilage nodule formation, inhibitory Smads blocked it. Osteoblast differentiation induced by BMPs is thus mediated mainly via the Smad-signaling pathway, whereas chondrogenic differentiation may be transmitted by Smad-dependent and independent pathways.

Noggin and Bone Morphogenetic Protein-4 Coordinately Regulate the Progression of Chondrogenic Differentiation in Mouse Clonal EC Cells, ATDC5

Here we report the gene expression and regulation and the function of noggin in clonal mouse chondrogenic EC cells, ATDC5. In ATDC5 cells, the expression of Noggin mRNA increased in parallel with the progression of chondrogenic differentiation. The treatment with conditioned medium of noggin-transfected COS-7 cells decreased the levels of type II and type X collagen gene transcripts of differentiated ATDC5 cells in a dose-dependent manner, and this inhibitory action was reversed by exogenously administered BMP-4 in a dose-dependent manner. The steady-state level of noggin gene transcripts was markedly upregulated by exogenously administered BMP-4 in time- and dose-dependent manners. Furthermore, this stimulatory effect of BMP-4 was attenuated by treatment with actinomycin D, but not with cycloheximide. These results indicate that noggin and BMP-4 coordinately regulate the progression of chondrogenic differentiation in ATDC5 cells.