Title: Reactive Oxygen Species Generated by NADPH Oxidase 2 and 4 Are Required for Chondrogenic Differentiation
Abstract: 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. 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. IntroductionThe formation of cartilage is characterized by the condensation of mesenchymal cells and the subsequent differentiation of chondrocytes, which includes cell proliferation, maturation, and hypertrophy (1Kronenberg H.M. Nature. 2003; 423: 332-336Crossref PubMed Scopus (2090) Google Scholar). Chondrocyte differentiation is accompanied by the secretion of a matrix rich in collagen type II and aggrecan, and this process is regulated by transforming growth factor-β, fibroblast growth factors, bone morphogenetic proteins, and the transcription factor Sox9 (2Kawakami Y. Rodriguez-León J. IzpisúaBelmonte J.C. Curr. Opin. Cell Biol. 2006; 18: 723-729Crossref PubMed Scopus (130) Google Scholar, 3de Crombrugghe B. Lefebvre V. Nakashima K. Curr. Opin. Cell Biol. 2001; 13: 721-727Crossref PubMed Scopus (401) Google Scholar). The chondrogenic cell line ATDC5 has been studied as an in vitro model of chondrocyte differentiation (4Shukunami C. Shigeno C. Atsumi T. Ishizeki K. Suzuki F. Hiraki Y. J. Cell Biol. 1996; 133: 457-468Crossref PubMed Scopus (343) Google Scholar). Specifically, exposure of ATDC5 cells to insulin induces a process reminiscent of chondrocyte differentiation (5Phornphutkul C. Wu K.Y. Gruppuso P.A. Mol. Cell. Endocrinol. 2006; 249: 107-115Crossref PubMed Scopus (68) Google Scholar). Insulin stimulation elicits the rapid production of H2O2 in various cell types, resulting in oxidative inhibition of protein-tyrosine phosphatases, followed by the enhancement of protein tyrosine phosphorylation (6Mahadev K. Motoshima H. Wu X. Ruddy J.M. Arnold R.S. Cheng G. Lambeth J.D. Goldstein B.J. Mol. Cell. Biol. 2004; 24: 1844-1854Crossref PubMed Scopus (436) Google Scholar, 7Goldstein B.J. Mahadev K. Wu X. Zhu L. Motoshima H. Antioxid. Redox Signal. 2005; 7: 1021-1031Crossref PubMed Scopus (191) Google Scholar). ROS 3The abbreviations used are: ROSreactive oxygen speciesDCFH-DA2′,7′-dichlorohydrofluorescein diacetateDPIdiphenyleneiodonium chlorideNACN-acetylcysteineNoxNADPH oxidaseqPCRquantitative PCR. such as H2O2, superoxide anion (O2⨪), and hydroxyl radical (•OH) are classically considered to be harmful to cells and tissues. However, more recent studies have revealed that ROS function as important signaling molecules in the regulation of cell growth and differentiation (8Lambeth J.D. Nat. Rev. Immunol. 2004; 4: 181-189Crossref PubMed Scopus (2427) Google Scholar).ROS are generated in cells by several pathways. The major sources of intracellular ROS production include mitochondria, various metabolic and detoxifying enzymes, and Nox. Nox isoforms generate ROS in a variety of cells and tissues in response to stimulation with various growth factors or cytokines (9Bedard K. Krause K.H. Physiol. Rev. 2007; 87: 245-313Crossref PubMed Scopus (4811) Google Scholar). To date, Nox isoforms include Nox1, Nox2, Nox3, Nox4, Nox5, Duox1, and Duox2 (10Cheng G. Cao Z. Xu X. van Meir E.G. Lambeth J.D. Gene. 2001; 269: 131-140Crossref PubMed Scopus (693) Google Scholar). Although they share structural features, each member of the Nox family is thought to play a specific biological role depending on the type of cell or tissue (11Krause K.H. Jpn. J. Infect. Dis. 2004; 57: S28-S29PubMed Google Scholar, 12Sumimoto H. FEBS J. 2008; 275: 3249-3277Crossref PubMed Scopus (527) Google Scholar). The classic Nox isoform Nox2 (also known as gp91phox) was first identified in phagocytes (8Lambeth J.D. Nat. Rev. Immunol. 2004; 4: 181-189Crossref PubMed Scopus (2427) Google Scholar, 13Babior B.M. Lambeth J.D. Nauseef W. Arch. Biochem. Biophys. 2002; 397: 342-344Crossref PubMed Scopus (707) Google Scholar). Nox isoforms including Nox2 have been shown to be expressed in a wide variety of nonphagocytic as well as phagocytic cells and to function in signal transduction (8Lambeth J.D. Nat. Rev. Immunol. 2004; 4: 181-189Crossref PubMed Scopus (2427) Google Scholar, 11Krause K.H. Jpn. J. Infect. Dis. 2004; 57: S28-S29PubMed Google Scholar).The receptor activator of nuclear factor-κB ligand (RANKL) induces osteoclast differentiation in a manner dependent on Nox-derived ROS (14Ha H. Kwak H.B. Lee S.W. Jin H.M. Kim H.M. Kim H.H. Lee Z.H. Exp. Cell Res. 2004; 301: 119-127Crossref PubMed Scopus (260) Google Scholar, 15Sasaki H. Yamamoto H. Tominaga K. Masuda K. Kawai T. Teshima-Kondo S. Rokutan K. J. Med. Invest. 2009; 56: 33-41Crossref PubMed Scopus (75) Google Scholar). The production of superoxide by Nox4 in osteoclasts also contributes to bone resorption (16Grange L. Nguyen M.V. Lardy B. Derouazi M. Campion Y. Trocme C. Paclet M.H. Gaudin P. Morel F. Antioxid. Redox Signal. 2006; 8: 1485-1496Crossref PubMed Scopus (63) Google Scholar). Furthermore, intracellular ROS were found to inhibit the proliferation of immature chondrocytes and promote the induction of chondrocyte hypertrophy in cells derived from ataxia telangiectasia mutated knock-out mice, whereas ROS produced by the up-regulation of Nox1 expression were found to be associated with chondrocyte differentiation (17Morita K. Miyamoto T. Fujita N. Kubota Y. Ito K. Takubo K. Miyamoto K. Ninomiya K. Suzuki T. Iwasaki R. Yagi M. Takaishi H. Toyama Y. Suda T. J. Exp. Med. 2007; 204: 1613-1623Crossref PubMed Scopus (143) Google Scholar).The role of ROS generation in chondrogenesis remains to be fully characterized. We show here that intracellular ROS generated by Nox2 and Nox4 are required for differentiation of ATDC5 cells and primary chondrocytes.DISCUSSIONWe investigated the association of ROS generated by Nox and chondrogenesis in ATDC5 cells. Among Nox family members, Nox1, Nox2, and Nox4 were found to be expressed in ATDC5 cells during chondrogenesis (Fig. 2). Depletion of either Nox2 or Nox4, but not that of Nox1, inhibited ROS generation, suppressed differentiation, and induced apoptosis in ATDC5 cells (FIGURE 5, FIGURE 6). These results suggest that ROS generation by Nox2 and Nox4 was required for chondrogenic differentiation of ATDC5 cells.A previous study on ATDC5 cells revealed a role for ROS in chondrocyte hypertrophy and showed that induction of chondrogenic differentiation is associated with the up-regulation of Nox1 gene expression (17Morita K. Miyamoto T. Fujita N. Kubota Y. Ito K. Takubo K. Miyamoto K. Ninomiya K. Suzuki T. Iwasaki R. Yagi M. Takaishi H. Toyama Y. Suda T. J. Exp. Med. 2007; 204: 1613-1623Crossref PubMed Scopus (143) Google Scholar). We now have shown the expression of Nox1 along with that of Nox2 and Nox4 in ATDC5 cells as well as in primary chondrocytes isolated from mouse embryonic limb buds (Fig. 2). Using RNA interference, we also found that Nox2 and Nox4 were required in the early phase of chondrogenic differentiation in ATDC5 cells. The level of intracellular ROS was increased early and maintained during ATDC5 cell differentiation. The expression of the Nox1 and Nox2 genes was increased as differentiation progressed, whereas that of the Nox4 gene, which was initially high, was gradually decreased during differentiation (Fig. 2). By immunohistochemical analysis of developing limbs obtained from mouse embryos, we found that both Nox1 and Nox2 were strongly expressed in prehypertrophic and hypertrophic chondrocytes, whereas the Nox4 signal was observed in proliferating chondrocytes and prehypertrophic chondrocytes (Fig. 3). We also found that Nox2 and Nox4 were important for the condensation and differentiation of primary chondrocytes derived from mouse embryos (Fig. 6).Nox1 did not appear to contribute to ROS generation during the early phase of ATDC5 cell differentiation, suggesting that the level of intracellular ROS was maintained by Nox2 and Nox4 and that the relative importance of these enzymes increases and decreases, respectively, with changes in their relative levels of expression. It was recently shown that Nox2 and Nox4 each compensate for the deficiency of the other in lung endothelial cells (37Pendyala S. Gorshkova I.A. Usatyuk P.V. He D. Pennathur A. Lambeth J.D. Thannickal V.J. Natarajan V. Antioxid. Redox Signal. 2009; 11: 747-764Crossref PubMed Scopus (150) Google Scholar).Down-regulation of Nox4 expression is associated with adipocyte differentiation (38Mouche S. Mkaddem S.B. Wang W. Katic M. Tseng Y.H. Carnesecchi S. Steger K. Foti M. Meier C.A. Muzzin P. Kahn C.R. Ogier-Denis E. Szanto I. Biochim. Biophys. Acta. 2007; 1773: 1015-1027Crossref PubMed Scopus (66) Google Scholar), and Nox4 has been proposed to act as a switch that controls the transition between proliferation and differentiation of preadipocytes (39Schröder K. Wandzioch K. Helmcke I. Brandes R.P. Arterioscler. Thromb. Vasc. Biol. 2009; 29: 239-245Crossref PubMed Scopus (200) Google Scholar). Our results now suggest that increasing the expression of Nox1 and Nox2 and decreasing the expression of Nox4 are characteristics of chondrogenesis. Depletion of Nox2 or Nox4 resulted in the down-regulation of Sox9 and collagen type II expression in ATDC5 and primary chondrocytes (Fig. 6). Sox9 is an essential chondrogenic transcription factor, and N-cadherin is an important downstream target of Sox9 that modulates cell adhesion (40Panda D.K. Miao D. Lefebvre V. Hendy G.N. Goltzman D. J. Biol. Chem. 2001; 276: 41229-41236Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Signaling by Rac1, a member of the Rho family of small GTPases, is also thought to regulate N-cadherin expression during chondrogenesis (20Woods A. Wang G. Dupuis H. Shao Z. Beier F. J. Biol. Chem. 2007; 282: 23500-23508Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Rac1 is also an important cytosolic activator of Nox enzymes (9Bedard K. Krause K.H. Physiol. Rev. 2007; 87: 245-313Crossref PubMed Scopus (4811) Google Scholar, 12Sumimoto H. FEBS J. 2008; 275: 3249-3277Crossref PubMed Scopus (527) Google Scholar). Although our results suggest that Nox1 does not contribute to the early phase of differentiation in ATDC5 cells, observations that Nox1 expression gradually increased as differentiation progressed suggest that Nox1 may play a role in the later stages of differentiation.We found that apoptosis was induced in ATDC5 cells depleted of Nox2 or Nox4 (Fig. 5). Apoptosis, a redox-regulated event, is triggered by disruption of the extracellular matrix in adherent cells. Matrix-cell contacts result in the generation of ROS as well as activation of pro-survival signaling (41Giannoni E. Buricchi F. Grimaldi G. Parri M. Cialdai F. Taddei M.L. Raugei G. Ramponi G. Chiarugi P. Cell Death Differ. 2008; 15: 867-878Crossref PubMed Scopus (113) Google Scholar). Nox2- or Nox4-depleted cells also did not undergo condensation or differentiation. Suppression of ROS generation by depletion of Nox2 or Nox4 inhibited the insulin-induced phosphorylation of Akt. Further, ERK was phosphorylated in Nox2 or Nox4 knockdown cells. It is reported that a balance between phosphatidylinositol 3-kinase-Akt and MEK-ERK activity regulates chondrocyte matrix synthesis, and this balance is modulated by ROS (33Yin W. Park J.I. Loeser R.F. J. Biol. Chem. 2009; 284: 31972-31981Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). ROS generated by Nox have been shown to be important for the survival, proliferation, and differentiation of various cell types, including pancreatic adenocarcinoma, glioma, endothelial, vascular smooth muscle, and embryonic stem cells (37Pendyala S. Gorshkova I.A. Usatyuk P.V. He D. Pennathur A. Lambeth J.D. Thannickal V.J. Natarajan V. Antioxid. Redox Signal. 2009; 11: 747-764Crossref PubMed Scopus (150) Google Scholar, 42Xiao Q. Luo Z. Pepe A.E. Margariti A. Zeng L. Xu Q. Am. J. Physiol. Cell Physiol. 2009; 296: C711-C723Crossref PubMed Scopus (156) Google Scholar, 43Mochizuki T. Furuta S. Mitsushita J. Shang W.H. Ito M. Yokoo Y. Yamaura M. Ishizone S. Nakayama J. Konagai A. Hirose K. Kiyosawa K. Kamata T. Oncogene. 2006; 25: 3699-3707Crossref PubMed Scopus (198) Google Scholar, 44Shono T. Yokoyama N. Uesaka T. Kuroda J. Takeya R. Yamasaki T. Amano T. Mizoguchi M. Suzuki S.O. Niiro H. Miyamoto K. Akashi K. Iwaki T. Sumimoto H. Sasaki T. Int. J. Cancer. 2008; 123: 787-792Crossref PubMed Scopus (77) Google Scholar, 45Clempus R.E. Sorescu D. Dikalova A.E. Pounkova L. Jo P. Sorescu G.P. Schmidt H.H. Lassègue B. Griendling K.K. Arterioscler. Thromb. Vasc. Biol. 2007; 27: 42-48Crossref PubMed Scopus (264) Google Scholar, 46Peshavariya H. Dusting G.J. Jiang F. Halmos L.R. Sobey C.G. Drummond G.R. Selemidis S. Naunyn-Schmiedebergs Arch. Pharmacol. 2009; 380: 193-204Crossref PubMed Scopus (86) Google Scholar).Although no bone abnormalities have been reported in Nox1−/− or Nox2−/− mice (14Ha H. Kwak H.B. Lee S.W. Jin H.M. Kim H.M. Kim H.H. Lee Z.H. Exp. Cell Res. 2004; 301: 119-127Crossref PubMed Scopus (260) Google Scholar, 47Koh J.M. Lee Y.S. Kim Y.S. Kim D.J. Kim H.H. Park J.Y. Lee K.U. Kim G.S. J. Bone Miner. Res. 2006; 21: 1003-1011Crossref PubMed Scopus (158) Google Scholar, 48Yang S. Madyastha P. Bingel S. Ries W. Key L. J. Biol. Chem. 2001; 276: 5452-5458Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar), it is clear that Nox-derived ROS are important for chondrogenesis. Nox2 and Nox4 were expressed in converse fashion (Fig. 2), but knockdown of each exerted a dramatic reductive effect on ROS generation even after 2 days of chondrogenesis (Fig. 4). This critical effect of Nox2 and Nox4 was also observed in the suppression of apoptosis (Fig. 5). However, expression of marker proteins was more reduced in Nox4 knockdown cells than in Nox2 knockdown cells (Fig. 6). This implies that Nox4 was important for the basal state, whereas Nox2 was important for differentiation. Thus, both Nox2 and Nox4 worked cooperatively for ROS generation by changing their expression. In summary, our findings suggest that ROS generated by both Nox2 and Nox4 played key roles in the early stage of chondrogenic differentiation in ATDC5 cells and primary chondrocytes. IntroductionThe formation of cartilage is characterized by the condensation of mesenchymal cells and the subsequent differentiation of chondrocytes, which includes cell proliferation, maturation, and hypertrophy (1Kronenberg H.M. Nature. 2003; 423: 332-336Crossref PubMed Scopus (2090) Google Scholar). Chondrocyte differentiation is accompanied by the secretion of a matrix rich in collagen type II and aggrecan, and this process is regulated by transforming growth factor-β, fibroblast growth factors, bone morphogenetic proteins, and the transcription factor Sox9 (2Kawakami Y. Rodriguez-León J. IzpisúaBelmonte J.C. Curr. Opin. Cell Biol. 2006; 18: 723-729Crossref PubMed Scopus (130) Google Scholar, 3de Crombrugghe B. Lefebvre V. Nakashima K. Curr. Opin. Cell Biol. 2001; 13: 721-727Crossref PubMed Scopus (401) Google Scholar). The chondrogenic cell line ATDC5 has been studied as an in vitro model of chondrocyte differentiation (4Shukunami C. Shigeno C. Atsumi T. Ishizeki K. Suzuki F. Hiraki Y. J. Cell Biol. 1996; 133: 457-468Crossref PubMed Scopus (343) Google Scholar). Specifically, exposure of ATDC5 cells to insulin induces a process reminiscent of chondrocyte differentiation (5Phornphutkul C. Wu K.Y. Gruppuso P.A. Mol. Cell. Endocrinol. 2006; 249: 107-115Crossref PubMed Scopus (68) Google Scholar). Insulin stimulation elicits the rapid production of H2O2 in various cell types, resulting in oxidative inhibition of protein-tyrosine phosphatases, followed by the enhancement of protein tyrosine phosphorylation (6Mahadev K. Motoshima H. Wu X. Ruddy J.M. Arnold R.S. Cheng G. Lambeth J.D. Goldstein B.J. Mol. Cell. Biol. 2004; 24: 1844-1854Crossref PubMed Scopus (436) Google Scholar, 7Goldstein B.J. Mahadev K. Wu X. Zhu L. Motoshima H. Antioxid. Redox Signal. 2005; 7: 1021-1031Crossref PubMed Scopus (191) Google Scholar). ROS 3The abbreviations used are: ROSreactive oxygen speciesDCFH-DA2′,7′-dichlorohydrofluorescein diacetateDPIdiphenyleneiodonium chlorideNACN-acetylcysteineNoxNADPH oxidaseqPCRquantitative PCR. such as H2O2, superoxide anion (O2⨪), and hydroxyl radical (•OH) are classically considered to be harmful to cells and tissues. However, more recent studies have revealed that ROS function as important signaling molecules in the regulation of cell growth and differentiation (8Lambeth J.D. Nat. Rev. Immunol. 2004; 4: 181-189Crossref PubMed Scopus (2427) Google Scholar).ROS are generated in cells by several pathways. The major sources of intracellular ROS production include mitochondria, various metabolic and detoxifying enzymes, and Nox. Nox isoforms generate ROS in a variety of cells and tissues in response to stimulation with various growth factors or cytokines (9Bedard K. Krause K.H. Physiol. Rev. 2007; 87: 245-313Crossref PubMed Scopus (4811) Google Scholar). To date, Nox isoforms include Nox1, Nox2, Nox3, Nox4, Nox5, Duox1, and Duox2 (10Cheng G. Cao Z. Xu X. van Meir E.G. Lambeth J.D. Gene. 2001; 269: 131-140Crossref PubMed Scopus (693) Google Scholar). Although they share structural features, each member of the Nox family is thought to play a specific biological role depending on the type of cell or tissue (11Krause K.H. Jpn. J. Infect. Dis. 2004; 57: S28-S29PubMed Google Scholar, 12Sumimoto H. FEBS J. 2008; 275: 3249-3277Crossref PubMed Scopus (527) Google Scholar). The classic Nox isoform Nox2 (also known as gp91phox) was first identified in phagocytes (8Lambeth J.D. Nat. Rev. Immunol. 2004; 4: 181-189Crossref PubMed Scopus (2427) Google Scholar, 13Babior B.M. Lambeth J.D. Nauseef W. Arch. Biochem. Biophys. 2002; 397: 342-344Crossref PubMed Scopus (707) Google Scholar). Nox isoforms including Nox2 have been shown to be expressed in a wide variety of nonphagocytic as well as phagocytic cells and to function in signal transduction (8Lambeth J.D. Nat. Rev. Immunol. 2004; 4: 181-189Crossref PubMed Scopus (2427) Google Scholar, 11Krause K.H. Jpn. J. Infect. Dis. 2004; 57: S28-S29PubMed Google Scholar).The receptor activator of nuclear factor-κB ligand (RANKL) induces osteoclast differentiation in a manner dependent on Nox-derived ROS (14Ha H. Kwak H.B. Lee S.W. Jin H.M. Kim H.M. Kim H.H. Lee Z.H. Exp. Cell Res. 2004; 301: 119-127Crossref PubMed Scopus (260) Google Scholar, 15Sasaki H. Yamamoto H. Tominaga K. Masuda K. Kawai T. Teshima-Kondo S. Rokutan K. J. Med. Invest. 2009; 56: 33-41Crossref PubMed Scopus (75) Google Scholar). The production of superoxide by Nox4 in osteoclasts also contributes to bone resorption (16Grange L. Nguyen M.V. Lardy B. Derouazi M. Campion Y. Trocme C. Paclet M.H. Gaudin P. Morel F. Antioxid. Redox Signal. 2006; 8: 1485-1496Crossref PubMed Scopus (63) Google Scholar). Furthermore, intracellular ROS were found to inhibit the proliferation of immature chondrocytes and promote the induction of chondrocyte hypertrophy in cells derived from ataxia telangiectasia mutated knock-out mice, whereas ROS produced by the up-regulation of Nox1 expression were found to be associated with chondrocyte differentiation (17Morita K. Miyamoto T. Fujita N. Kubota Y. Ito K. Takubo K. Miyamoto K. Ninomiya K. Suzuki T. Iwasaki R. Yagi M. Takaishi H. Toyama Y. Suda T. J. Exp. Med. 2007; 204: 1613-1623Crossref PubMed Scopus (143) Google Scholar).The role of ROS generation in chondrogenesis remains to be fully characterized. We show here that intracellular ROS generated by Nox2 and Nox4 are required for differentiation of ATDC5 cells and primary chondrocytes.