Reactive Oxygen Species Generated by NADPH Oxidase 2 and 4 Are Required for Chondrogenic Differentiation

Although generation of reactive oxygen species (ROS) by NADPH oxidases (Nox) is thought to be important for signal transduction in nonphagocytic cells, little is known of the role ROS plays in chondrogenesis. We therefore examined the possible contribution of ROS generation to chondrogenesis using both ATDC5 cells and primary chondrocytes derived from mouse embryos. The intracellular level of ROS was increased during the differentiation process, which was then blocked by treatment with the ROS scavenger N-acetylcysteine. Expression of Nox1 and Nox2 was increased upon differentiation of ATDC5 cells and primary mouse chondrocytes, whereas that of Nox4, which was relatively high initially, was decreased gradually during chondrogenesis. In developing limb, Nox1 and Nox2 were highly expressed in prehypertrophic and hypertrophic chondrocytes. However, Nox4 was highly expressed in proliferating chondrocytes and prehypertrophic chondrocytes. Depletion of Nox2 or Nox4 expression by RNA interference blocked both ROS generation and differentiation of ATDC5 cells, whereas depletion of Nox1 had no such effect. We also found that ATDC5 cells depleted of Nox2 or Nox4 underwent apoptosis. Further, inhibition of Akt phosphorylation along with subsequent activation of ERK was observed in the cells. Finally, depletion of Nox2 or Nox4 inhibited the accumulation of proteoglycan in primary chondrocytes. Taken together, our data suggest that ROS generated by Nox2 or Nox4 are essential for survival and differentiation in the early stage of chondrogenesis.     

Tumour necrosis factor-alpha up-regulates the expression of BMP-4 mRNA but inhibits chondrogenesis in mouse clonal chondrogenic EC cells, ATDC5

Tumour necrosis factor (TNF)-alpha causes the degradation of articular cartilage in arthritis via direct actions on chondrocytes. However, it remains unknown whether TNF-alpha affects chondrogenesis in chondroprogenitors. In the present study, we assessed the effects of TNF-alpha in vitro on chondrogenesis using mouse clonal chondrogenic EC cells, ATDC5. TNF-alpha (10 ng/ml) stimulated [3H] thymidine incorporation in undifferentiated ATDC5 cells, and suppressed cartilaginous nodule formation and the accumulation of cartilage-specific proteoglycan. We recently showed that undifferentiated ATDC5 cells express BMP-4 and that exogenously administered BMP-4 promotes chondrogenesis in these cells. Interestingly, TNF-alpha up-regulated the expression of BMP-4 mRNA in undifferentiated ATDC5 cells in time- and dose-dependent manners. However, exogenously administered BMP-4 was not capable of reversing the inhibitory action of TNF-alpha on chondrogenesis in ATDC5 cells. These results indicate that TNF-alpha stimulates both cell proliferation and BMP-4 expression but inhibits chondrogenesis in chondroprogenitor-like ATDC5 cells.     

Chondrogenic differentiation of clonal mouse embryonic cell line ATDC5 in vitro: differentiation-dependent gene expression of parathyroid hormone (PTH)/PTH-related peptide receptor

The regulatory role of parathyroid hormone (PTH)/PTH-related peptide (PTHrP) signaling has been implicated in embryonic skeletal development. Here, we studied chondrogenic differentiation of the mouse embryonal carcinoma-derived clonal cell line ATDC5 as a model of chondrogenesis in the early stages of endochondral bone development. ATDC5 cells retain the properties of chondroprogenitor cells, and rapidly proliferate in the presence of 5% FBS. Insulin (10 micrograms/ml) induced chondrogenic differentiation of the cells in a postconfluent phase through a cellular condensation process, resulting in the formation of cartilage nodules, as evidenced by expression of type II collagen and aggrecan genes. We found that differentiated cultures of ATDC5 cells abundantly expressed the high affinity receptor for PTH (Mr approximately 80 kD; Kd = 3.9 nM; 3.2 x 10(5) sites/cell). The receptors on differentiated cells were functionally active, as evidenced by a PTH-dependent activation of adenylate cyclase. Specific binding of PTH to cells markedly increased with the formation of cartilage nodules, while undifferentiated cells failed to show specific binding of PTH. Northern blot analysis indicated that expression of the PTH/PTHrP receptor gene became detectable at the early stage of chondrogenesis of ATDC5 cells, preceding induction of aggrecan gene expression. Expression of the PTH/PTHrP receptor gene was undetectable in undifferentiated cells. The level of PTH/PTHrP receptor mRNA was markedly elevated parallel to that of type II collagen mRNA. These lines of evidence suggest that the expression of functional PTH/PTHrP receptor is associated with the onset of chondrogenesis. In addition, activation of the receptor by exogenous PTH or PTHrP significantly interfered with cellular condensation and the subsequent formation of cartilage nodules, suggesting a novel site of PTHrP action.     

Regulator of G-protein signaling (RGS) proteins differentially control chondrocyte differentiation

Control of chondrocyte differentiation is attained, in part, through G-protein signaling, but the functions of the RGS family of genes, well known to control G-protein signaling at the Galpha subunit, have not been studied extensively in chondrogenesis. Recently, we have identified the Rgs2 gene as a regulator of chondrocyte differentiation. Here we extend these studies to additional Rgs genes. We demonstrate that the Rgs4, Rgs5, Rgs7, and Rgs10 genes are differentially regulated during chondrogenic differentiation in vitro and in vivo. To investigate the roles of RGS proteins during cartilage development, we overexpressed RGS4, RGS5, RGS7, and RGS10 in the chondrogenic cell line ATDC5. We found unique and overlapping effects of individual Rgs genes on numerous parameters of chondrocyte differentiation. In particular, RGS5, RGS7, and RGS10 promote and RGS4 inhibits chondrogenic differentiation. The identification of Rgs genes as novel regulators of chondrogenesis will contribute to a better understanding of both normal cartilage development and the etiology of chondrodysplasias and osteoarthritis.     

p21(Cip-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, p21(Cip-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.     

Effects of transforming growth factor-beta signaling on chondrogenesis in mouse chondrogenic EC cells, ATDC5

Cellular condensation of chondroprogenitors is a distinct cellular event in chondrogenesis. During this process, N-cadherin mediates cell-cell interactions responsible for the initial stage of cellular condensation and subsequently fibronectin contributes to cell-matrix interactions mediating a progression of chondrogenesis. We previously showed that chondrogenesis in mouse chondrogenic EC cells, ATDC5, was induced, at a high incidence in the presence of insulin, through formation of cellular condensation. In this study, we took advantage of the sequential progression of chondrogenesis in ATDC5 cells and evaluated, in vitro in these cells, the role of endogenous transforming growth factor (TGF)-beta in chondrogenesis. ATDC5 cells expressed TGF-beta2 mRNA at a cellular condensation stage. The treatment of undifferentiated ATDC5 cells with anti-TGF-beta32 neutralizing antibody inhibited the accumulation of Alcian blue stainable proteoglycan in a dose-dependent manner. Transfection of a dominant-negative mutant of mouse TGF-beta type II receptor to undifferentiated ATDC5 cells completely inhibited cellular condensation. Moreover, exogenously administered TGF-beta2 upregulated the expression of fibronectin and type II collagen (a phenotypic marker gene of chondrogenesis) mRNAs and downregulated that of N-cadherin mRNA in time- and dose-dependent manners. These results indicate that TGF-beta stimulates chondrogenesis via initiation of cellular condensation by transition from an initial N-cadherin-contributing stage to a fibronectin-contributing stage during processes of chondrogenesis in ATDC5 cells.     

Positive and negative regulation of chondrogenesis by splice variants of PEBP2αA/CBFα1 in clonal mouse EC cells,ATDC5

The αA type of the α subunit of the polyomavirus enhancer binding protein 2 (PEBP2αA), also called the core binding factor α1 (CBFα1) or til-1, plays crucial roles in osteogenesis. Little is known, however, about the function of PEBP2αA in chondrogenesis. Here, we examined the role of PEBP2αA in chondrogenesis of clonal mouse embryonal carcinoma cells, ATDC5, which are committed as chondroprogenitors. We found that as ATDC5 cells condensed and formed cartilaginous nodules, PEBP2αA increased, and the level was maintained throughout the process of chondrocytic maturation. When an established dominant negative form of PEBP2αA was introduced in undifferentiated ATDC5 cells, the cellular condensation and the subsequent processes were inhibited. This inhibition was overcome with BMP-4 treatment, which increased the endogenous expression of PEBP2αA. Thus, the process of chondrogenesis is regulated by the level of PEBP2αA activity. Along with the wild-type PEBP2αA, a splice variant form, til-1 G2, is naturally expressed in ATDC5 cells. In luciferase reporter assays, til-1 G2 not only exhibited a limited ability to transactivate the osteocalcin promoter but also inhibited the activity achieved by the wild-type PEBP2αA. When til-1 G2 was overexpressed by stable transfection in undifferentiated ATDC5 cells, it inhibited the progression of chondrogenesis. Therefore, we conclude that PEBP2αA acts as a positive regulator of chondrogenesis, and that this positive effect may be finely tuned by the opposing effect of the til-1 G2 isoform. J. Cell. Physiol. 181:169–178, 1999. © 1999 Wiley-Liss, Inc.     

Human Immunodeficiency Virus Type 1 Enhancer-binding Protein 3 Is Essential for the Expression of Asparagine-linked Glycosylation 2 in the Regulation of Osteoblast and Chondrocyte Differentiation

Human immunodeficiency virus type 1 enhancer-binding protein 3 (Hivep3) suppresses osteoblast differentiation by inducing proteasomal degradation of the osteogenesis master regulator Runx2. In this study, we tested the possibility of cooperation of Hivep1, Hivep2, and Hivep3 in osteoblast and/or chondrocyte differentiation. Microarray analyses with ST-2 bone stroma cells demonstrated that expression of any known osteochondrogenesis-related genes was not commonly affected by the three Hivep siRNAs. Only Hivep3 siRNA promoted osteoblast differentiation in ST-2 cells, whereas all three siRNAs cooperatively suppressed differentiation in ATDC5 chondrocytes. We further used microarray analysis to identify genes commonly down-regulated in both MC3T3-E1 osteoblasts and ST-2 cells upon knockdown of Hivep3 and identified asparagine-linked glycosylation 2 (Alg2), which encodes a mannosyltransferase residing on the endoplasmic reticulum. The Hivep3 siRNA-mediated promotion of osteoblast differentiation was negated by forced Alg2 expression. Alg2 suppressed osteoblast differentiation and bone formation in cultured calvarial bone. Alg2 was immunoprecipitated with Runx2, whereas the combined transfection of Runx2 and Alg2 interfered with Runx2 nuclear localization, which resulted in suppression of Runx2 activity. Chondrocyte differentiation was promoted by Hivep3 overexpression, in concert with increased expression of Creb3l2, whose gene product is the endoplasmic reticulum stress transducer crucial for chondrogenesis. Alg2 silencing suppressed Creb3l2 expression and chondrogenesis of ATDC5 cells, whereas infection of Alg2-expressing virus promoted chondrocyte maturation in cultured cartilage rudiments. Thus, Alg2, as a downstream mediator of Hivep3, suppresses osteogenesis, whereas it promotes chondrogenesis. To our knowledge, this study is the first to link a mannosyltransferase gene to osteochondrogenesis.     

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.     

FLRT2 promotes cellular proliferation and inhibits cell adhesion during chondrogenesis

One of the earliest events during chondrogenesis is the formation of condensations, a necessary pre‐requisite for subsequent differentiation of a chondrogenic phenotype. Members of the Fibronectin Lecucine Rich Transmembrane (FLRT) proteins have been shown to be involved in cell sorting and neurite outgrowth. Additionally, FLRT2 is highly expressed at putative sites of chondrogenic differentiation during craniofacial development. In this study, we demonstrate that FLRT2 plays a role in mediating cell proliferation and cell–cell interactions during early chondrogenesis. Clones of stable transfectants of a murine chondroprogenitor cell line, ATDC5, were established in which FLRT2 was knocked down or overexpressed. Cells in which FLRT2 was knocked down proliferated at a slower rate compared to control wild‐type ATDC5 cells or those containing a non‐coding shRNA. In addition, FLRT2 knockdown cells formed numerous lectin peanut agglutinin (PNA) stained aggregates and exhibited higher expression of the cell adhesion molecule, N‐cadherin. In an in vitro wound healing assay, fewer FLRT2 knockdown cells appeared to migrate into the defect. Surprisingly, the FLRT2 knockdown cells demonstrated increased formation of Alcian blue‐stainable extracellular matrix, suggesting that their reduced aggregate formation did not inhibit subsequent chondrogenic differentiation. The opposite trends were observed in ATDC5 clones that overexpressed FLRT2. Specifically, FLRT overexpressing cells proliferated faster, formed fewer PNA‐positive aggregates, accumulated increased Alcian blue‐positive matrix, and migrated faster to close a wound. Collectively, our findings provide evidence for a role of FLRT2 in enhancing cell proliferation and reducing intercellular adhesion during the early stages of chondrogenesis. J. Cell. Biochem. 112: 3440–3448, 2011. © 2011 Wiley Periodicals, Inc.     

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-beta 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.