NAD(P)H Oxidase Activity of Nox4 in Chondrocytes Is Both Inducible and Involved in Collagenase Expression

Abstract

Reactive oxygen species (ROS) are regulators of redox-sensitive cell signaling pathways. In osteoarthritis, human interleukin-1beta is implicated in cartilage destruction through an ROS-dependent matrix metalloproteinase production. To determine the molecular source of ROS production in the human IL-1beta (hIL-1beta)-sensitive chondrocyte immortalized cell line C-20/A4, transfected cells were constructed that overexpress NAD(P)H oxidases. First, RT-PCR analysis showed that the C-20/A4 cell line expressed Nox2, Nox4, p22( phox ), and p67( phox ), but not p47( phox ). It was found that ROS production by C-20/A4 chondrocytes does not depend on PMA and ionomycin activation. This indicates that Nox2 was not involved in the production of ROS. In C- 20/A4 cells that overexpress Nox4, hIL-1beta stimulated ROS production three times more than the normal production of C-20/A4 cells. Moreover, there was a fourfold increase in the production of collagenase (MMP-1) by chondrocytes that overexpress Nox4. Interestingly, MMP-1 production in cells that overexpress Nox2 was not sensitive to hIL-1beta. These data suggest that under hIL-1beta stimulation, C-20/A4 chondrocytes produce MMP-1 through a Nox4-mediated, ROS-dependent pathway.

Quinone compounds regulate the level of ROS production by the NADPH oxidase Nox4

“…access NP_001020605.1). Nox4A, Nox4B, NQO1A, and NQO1C were cloned into pEF6V5/HisB for mammalian expression as described [18].…”

“…One or 5 mg of RNA were converted to cDNA by reverse transcription with 20 U of AMV reverse transcriptase or SuperScriptIII first-strand synthesis (Life Technologies). Real time PCRs were performed with the LightCycler FastStart DNA Master plus SYBR Green I kit (Roche) or QuantiTect SYBR Green RT-PCR kit (Qiagen) as described [18]. Real time RT-PCR was conducted using the LightCycler 1 CarouselBased System (Roche) or a Stratagene Mx3005P (Stratagene).…”

“…ROS production was measured as previously described [18]. Cells were washed twice with PBS, detached with trypsin, and collected by centrifugation (250 g, 5 min at 20 8C).…”

“…To confirm that the observed effect of AA-861 and tBuBHQ was specifically related to Nox4 proteins, we used two other characterized cell lines that over-expressed Nox4: human chondrocyte C-20/A4 [18] and Nox4 T-REx TM in which Nox4 expression could be temporally induced by the addition of tetracycline [27]. Consistently, the constitutive ROS production in Nox4 C-20/A4 cells or in 4 h-induced Nox4 T-REx cells was enhanced after the addition of AA-861 or tBuBHQ (Fig.…”

“…Primary discovered in kidney tissue [13], Nox4 appears to be ubiquitously distributed. Its dysfunction has been linked to several pathologies including hypertension [14], diabetes [15], atherosclerosis [16], cancer [17], osteoarthritis [18] and inflammation [19], and Nox4 represent a potential therapeutic target [20]. Despite its wide distribution, its activation mechanisms at the molecular level are unclear.…”