Nitric oxide down‐regulates the expression of the catalytic NADPH oxidase subunit Nox1 in rat renal mesangial cells

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Abstract
SPECIFIC AIM Over the past years there has been growing body of evidence indicating that reactive oxygen species (ROS) play an important role in cellular signaling. The discovery of new NAD(P)H-oxidase (Nox) subunits that are involved in crucial cell responses newly inspired the research on biologically active radicals. It seems probable that, similar to protein phosphorylation, protein oxidation by Nox-derived ROS provides a general form of signaling that triggers many cell responses. Since the balance between ROS and nitric oxide (NO) determines cell responses and we and others found that ROS amplify NO formation by co-inducing the transcription of the inducible NO synthase, we investigated the action of NO on ROS production in mesangial cells (MC). PRINCIPAL FINDINGS 1. Nox1 mRNA steady-state levels are diminished in MC exposed to the NO donor diethylenetriamine nitric oxide (DETA-NO) MC are able to produce ROS in a NADPH-dependent manner and most of the components of a functional NADPH oxidase complex are expressed in these cells. However, the active component, namely the glycoprotein gp91phox (Nox2), has never been characterized in MC. In preliminary experiments, we detected mRNA encoding for Nox1 and Nox4 but not Nox2 by RT-PCR. To analyze the effects of NO on Nox1 mRNA levels, MC were stimulated with DETA-NO (500 μM) and the mRNA was subjected to real-time RT-PCR. A significant down-regulation of Nox1 mRNA levels to 50% compared with untreated controls was observed after 4–12 h treatment (Fig. 1 ⤻ ). Similar results were obtained when SNAP was used as a NO donor. Figure 1. Time and dose dependency of NO-modulated Nox1 mRNA expression. Quiescent mesangial cells were treated for the indicated times with DETA-NO (500 μM). Total RNA (each 1 μg) was reverse transcribed and each 50 ng were analyzed for Nox1 mRNA, and each 5 ng for 18S rRNA by real-time PCR using a GeneAmp 7700 System (Applied Biosystems). A)Results of 3 independent experiments displayed as % of reduction vs. control (means±sd; *PPPB). Download figure Download PowerPoint 2. Analysis of NO-modulated Nox1 protein expression To evaluate whether DETA-NO-induced changes in mRNA expression are followed by changes in Nox1 protein levels, we analyzed Nox1 protein expression by Western blot. An immunoreactive band was obtained at a molecular mass of 75 kDa (Fig. 2 ⤻ ). The specificity of the antibody was demonstrated by affinity chromatography, by blocking experiments with the immunizing peptide and by control experiments using the pre-immune serum. Treatment of mesangial cells with DETA-NO resulted in a marked reduction of Nox1 protein levels that was significant already at a DETA-NO concentration of 125 μM (47.8±10.7% vs. unstimulated controls). Figure 2. Influence of the cGMP signaling pathway on NO-modulated Nox1 expression. Quiescent mesangial cells were treated for 12 h with DETA-NO (100 μM), in the absence or presence of the sGC inhibitors ODQ (200 μM) or NS2028 (5 μM), or with the NO-independent sGC activator YC-1, as indicated. Thereafter, total RNA and protein were isolated. Nox1 mRNA was analyzed by real-time RT-PCR (A). Each 40 μg protein were subjected to Western blot analysis against a polyclonal anti-Nox1 antibody. A representative blot is displayed in panel B. The results of 3 independent experiments are shown in panel C (**PP§§PG-monomethyl-l-arginine (L-NMMA). Nox1 protein expression was not affected by IL-1β compared with unstimulated control cells, but cotreatment of IL-1β-stimulated MC with L-NMMA resulted in a 2.6-fold increase in Nox1 protein levels, indicating a down-regulation of Nox1 protein expression by endogenously produced NO. Cloning and analysis of the nox1 promoter revealed a down-regulation of Nox1 transcription by NO and an up-regulation by IL-1β. The use of inhibitors and activators of the cGMP signaling pathway indicated that the effect of NO on Nox1 transcription occurs in a cGMP-independent manner. 3. Involvement of soluble guanylyl cyclase (sGC) in NO-induced down-regulation of Nox1 To test whether the activation of sGC is responsible for the NO-induced decrease in Nox1 expression, mesangial cells were stimulated with DETA-NO and sGC was blocked by coadministration of the sGC inhibitors ODQ or NS2028. The effects of cGMP on Nox1 expression were further analyzed by exposing MC to the NO independent sGC activator YC-1. ODQ and NS2028 clearly prevented and YC-1 mimicked the effects of DETA-NO on Nox1 mRNA expression. Similar results were obtained for Nox1 protein (Fig. 2 ⤻ ). The membrane-permeable cGMP analog 8-Br-PET-cGMP dose-dependently reduced Nox1 protein levels. Our findings show that the inhibition of Nox1 expression by cGMP is probably due to post-transcriptional effects. 4. Effect of DETA-NO and silencing of Nox1 and Nox4 expression on superoxide formation in mesangial cells To test whether the NO-induced decrease of Nox1 expression translates into a corresponding decrease in NADPH oxidase activity, we analyzed the effect of DETA-NO treatment on O2– production in mesangial cells. To...
Funding Information
  • Deutsche Forschungsgemeinschaft (SFB553, Mu 900/4-1, PF 361/1-2, BR1839/2-2)