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.

Female mice lacking p47phoxhave altered adipose tissue gene expression and are protected against high fat-induced obesity

“…Some authors have suggested that constitutively active NOX4 acts as an oxygen sensor and that NOX4-mediated ROS signaling results in downstream activation of NOX1 and NOX2 and amplification of the ROS signal through recruitment of the cytosolic phox activator proteins and the Rac1 GTPase (29). Moreover, there is evidence in some cell types, such as preosteoclasts and chondrocytes, that multiple NOX enzymes can compensate for each other in the same pathway (15,33). However, in other cells, NOX4 and NOX2 appear to regulate separate pathways via different sets of MAP kinases (1).…”

WISP3 - IGF1 interaction regulates chondrocyte hypertrophy

“…Additionally, we demonstrate that WISP3 inhibits IGF1 induced collagen X induction, ROS accumulation and alkaline phosphatase activity, all of which are associated with the induction of chondrocyte hypertrophy (Böhme et al, 1992;Wang et al, 1999;Kronenberg, 2003;Zheng et al, 2003;Morita et al, 2007;Mueller and Tuan, 2008;Wu et al, 2008;Kim et al, 2010). Moreover, both IGF1 and ROS in turn stimulate WISP3 expression.…”

ATRA transcriptionally induces nSMase2 through CBP/p300-mediated histone acetylation

“…Additional analysis of the literature suggested ATRA could also induce Sox9 at early nSMase2 induction by cyclopamine (21), was used. Cells were pretreated for 3 h with 500 M NAC prior to stimulation with ATRA for 24 h, with previous studies demonstrating this is sufficient time for NAC to exert its antioxidant effects (26,27). Results indicated that ATRA induction of nSMase2 was comparable in the presence and absence of NAC (Fig.…”