Reactive Oxygen Species Generated by NADPH Oxidase 2 and 4 Are Required for Chondrogenic Differentiation
Laboratory of Cellular and Molecular Biochemistry, School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea. Journal of Biological Chemistry
(Impact Factor: 4.57).
10/2010; 285(51):40294-302. DOI: 10.1074/jbc.M110.126821
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.
Available from: David Moulin
- "The potency of chondrocytes to synthesize ROS has been reported through the detection of high levels of O 2 d À , H 2 O 2 , or OH d  in response to cytokines or phorbol ester. In addition, NADPH oxidase/Nox-mediated ROS production has been reported to contribute both to chondrogenic differentiation  and to expression of MMP-1  or -13 . Therefore, we next investigated whether 15d-PGJ 2 -induced expression of HSP70, in both resting and cytokine-stimulated chondrocytes, was supported or not by the generation of ROS. "
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ABSTRACT: The inhibitory effect of 15-deoxy-Δ(12, 14)-prostaglandin J2 (15d-PGJ2) on pro-inflammatory genes expression has been extensively documented and frequently ascribed to its ability to prevent NF-κB pathway activation. We and others have previously demonstrated that it was frequently independent of the peroxisome proliferator activated receptor (PPAR)γ activation. Here, we provide evidence that induction of intracellular Heat Shock Protein (HSP)70 by oxidative stress is an additional regulatory loop supporting the anti-inflammatory effect of 15d-PGJ2 in chondrocytes. Using real-time quantitative PCR and western blotting, we showed that 15d-PGJ2 stimulated HSP70, but not HSP27 expression while increasing oxidative stress as measured by spectrofluorimetry and confocal spectral imaging. Using N-acetylcysteine (NAC) as an antioxidant, we demonstrated further that oxidative stress was thoroughly responsible for the increased expression of HSP70. Finally, using HSP70 antisense strategy, we showed that the inhibitory effect of 15d-PGJ2 on IL-1-induced activation of NF-κB pathway, COX-2 and mPGES-1 expression, and PGE2 synthesis was partly supported by HSP70. These data provide a new anti-inflammatory mechanism to support the PPARγ-independent effect of 15d-PGJ2 in chondrocyte and suggest a possible feedback regulatory loop between oxidative stress and inflammation via intracellular HSP70 up-regulation. This cross-talk is consistent with 15d-PGJ2 as a putative negative regulator of the inflammatory reaction.
Free Radical Biology and Medicine 08/2014; 76. DOI:10.1016/j.freeradbiomed.2014.07.028 · 5.74 Impact Factor
Available from: Francis Rousset
- "Indeed, previous works have reported a role for ROS in mediating chondrocyte hypertrophy during endochondral ossification. However, hypertrophic phenotype is also observed among osteoarthritic chondrocytes which die by apoptosis  and NADPH oxidase Nox4 may contribute to this phenomenon . Furthermore, pseudoachondroplasia, a genetic pathology caused by a mutation on the protein COMP (Cartilage Oligo Matrix Protein) is characterised by abnormal joint architecture, joint erosion and osteoarthritis. "
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ABSTRACT: Interleukin-1β (IL-1β) activates the production of reactive oxygen species (ROS) and secretion of MMPs as well as chondrocyte apoptosis. Those events lead to matrix breakdown and are key features of osteoarthritis (OA). We confirmed that in human C-20/A4 chondrocytes the NADPH oxidase Nox4 is the main source of ROS upon IL-1β stimulation. Since heme molecules are essential for the NADPH oxidase maturation and activity, we therefore investigated the consequences of the modulation of Heme oxygenase-1 (HO-1), the limiting enzyme in heme catabolism, on the IL-1β signaling pathway and more specifically on Nox4 activity. Induction of HO-1 expression decreased dramatically Nox4 activity in C-20/A4 and HEK293 T-REx™ Nox4 cell lines. Unexpectedly, this decrease was not accompanied by any change in the expression, the subcellular localization or the maturation of Nox4. In fact, the inhibition of the heme synthesis by succinylacetone rather than heme catabolism by HO-1, led to a confinement of the Nox4/p22(phox) heterodimer in the endoplasmic reticulum with an absence of redox differential spectrum highlighting an incomplete maturation. Therefore, the downregulation of Nox4 activity by HO-1 induction appeared to be mediated by carbon monoxide (CO) generated from the heme degradation process. Interestingly, either HO-1 or CO caused a significant decrease in the expression of MMP-1 and DNA fragmentation of chondrocytes stimulated by IL-1β. These results all together suggest that a modulation of Nox4 activity via heme oxygenase-1 may represent a promising therapeutic tool in osteoarthritis.
PLoS ONE 06/2013; 8(6):e66478. DOI:10.1371/journal.pone.0066478 · 3.23 Impact Factor
Available from: Swati Choksi
- "NADPH oxidase (NOX)-mediated superoxide production is the main non-mitochondrial source of ROS. Some reports have described the relevance of NOX-mediated superoxide production in the differentiation of cell types25,29. To check the role of NOX-generated superoxide in monocyte-macrophage differentiation and macrophage polarization, we knocked down the expression of the small GTPase RAC1, which is an essential component of both NOX1 and NOX2 enzyme complexes30, in monocytes. "
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ABSTRACT: Differentiation to different types of macrophages determines their distinct functions. Tumor-associated macrophages (TAMs) promote tumorigenesis owing to their proangiogenic and immune-suppressive functions similar to those of alternatively activated (M2) macrophages. We report that reactive oxygen species (ROS) production is critical for macrophage differentiation and that inhibition of superoxide (O(2-)) production specifically blocks the differentiation of M2 macrophages. We found that when monocytes are triggered to differentiate, O(2-) is generated and is needed for the biphasic ERK activation, which is critical for macrophage differentiation. We demonstrated that ROS elimination by butylated hydroxyanisole (BHA) and other ROS inhibitors blocks macrophage differentiation. However, the inhibitory effect of ROS elimination on macrophage differentiation is overcome when cells are polarized to classically activated (M1), but not M2, macrophages. More importantly, the continuous administration of the ROS inhibitor BHA efficiently blocked the occurrence of TAMs and markedly suppressed tumorigenesis in mouse cancer models. Targeting TAMs by blocking ROS can be a potentially effective method for cancer treatment.Cell Research advance online publication 11 June 2013; doi:10.1038/cr.2013.75.
Cell Research 06/2013; 23(7). DOI:10.1038/cr.2013.75 · 12.41 Impact Factor
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