| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 27, 2005 as doi:10.1096/fj.05-3791fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ková*,1
,2,2
* Pharmazentrum Frankfurt (ZAFES), Klinikum der Johann Wolfgang-Goethe-Universität, Frankfurt am Main, Germany; and
Institut für Kardiovaskuläre Physiologie, Johann Wolfgang-Goethe-Universität, Frankfurt am Main, Germany
3Correspondence: Pharmazentrum Frankfurt, Theodor-Stern-Kai 7, Frankfurt am Main D-60590, Germany. E-mail: pfeilschifter{at}em.uni-frankfurt.de
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.
|
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).
|
To assess the influence of endogenously produced NO on Nox1 protein levels, mesangial cells were treated for 24 h with IL-1ß (1 nM) in order to induce iNOS expression. In some experiments, cells were also treated with the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA). Nox1 protein expression was not affected by IL-1ß compared with unstimulated control cells, but cotreatment of IL-1ß-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-1ß. 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 this end, DETA-NO-treated or untreated (control) MC were incubated with or without platelet-derived growth factor BB (PDGF-BB; 50 ng/mL), in the absence or presence of polyethylene glycol-conjugated superoxide dismutase (PEG-SOD). After 5 min, O2– was trapped by the spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine hydrochloride (CMH) for 10 min. The cell suspension was quickly transferred into an EPR capillary tube, and EPR recordings started immediately at room temperature, either by field scan, or for real-time recording of superoxide formation by continuous monitoring of the low-field line of the triplet EPR signal. In the presence of nonstimulated MC, CM• nitroxide radical generation proceeded for 10 min at a constant rate, as indicated by the steady increase in EPR signal intensity. This rate exceeded spontaneous CM• nitroxide radical formation in HEPES-Tyrode's solution in the absence of cells. After stimulation with PDGF, cells generated CM• radicals at a higher rate. Addition of SOD decreased this rate to the rate observed with nonstimulated cells in the presence of SOD. Basal superoxide formation was not different between DETA-NO-exposed and control cells. Silencing of Nox1 expression by a siRNA approach abolished the effect of DETA-NO on ROS formation, indicating that changes in the expression of Nox1 by DETA-NO are responsible for the decreased ROS formation. siRNA directed against Nox4 had no significant effect.
CONCLUSIONS AND SIGNIFICANCE
Inflammation is a complex and tightly regulated sequence of events that starts with an initial formation of proinflammatory factors that recruit immune cells to clear the offending trigger. A second anti-inflammatory phase follows in which resident cells, located mainly in the renal glomerulus, acquire the potential for protecting themselves from further activation and injury. NO and O2– are simple molecules with unpaired electrons that are generated as a host defense mechanism in various cell types in inflammatory diseases. These radicals not only act as cytotoxic agents but also as important physiological or pathophysiological mediators of proliferative, apoptotic, vasoregulatory, or inflammatory signaling cascades. NO antagonizes O2– at different levels: it neutralizes O2– by forming peroxynitrite, and it reduces O2– production by directly inhibiting the NADPH oxidase enzyme activity. Under certain conditions, NO has been reported to render cells resistant to oxidative stress. This also underscores the need for an intricate balance between NO and O2– for cellular fidelity (Fig. 3
).
|
We tested the effects of NO on the expression of the NADPH-oxidase subunit Nox1 as well as the activity of NADPH-oxidases. Treatment of mesangial cells with the NO donor DETA-NO resulted in a time- and dose-dependent, cGMP-mediated down-regulation of Nox1 mRNA and protein steady-state levels and a reduced ROS production.
The bioactivity of O2– reflects its rates of production by NAD(P)H-oxidases, xanthine oxidase, or other pathways and its inactivation by superoxide dismutases (SODs). Based on our observations and the reports of others, we assume that in an inflammatory setting mesangial cells shift the balance of reactive oxygen and nitrogen species to the NO side to circumvent deleterious effects of O2–. They do so by two mechanisms, a down-regulation of O2– formation by suppression of Nox1 expression and by simultaneous up-regulation of O2– dismutating SODs. In the inflammatory state, this could result in a protective pathway even when only low amounts of NO are available within the inflamed tissue. The fine-tuning of the spatial and temporal O2– and NO production will determine the outcome of an inflammatory process and may offer therapeutic intervention strategies.
Since both Nox1 and Nox4 are expressed in mesangial cells, we performed experiments using siRNAs directed against Nox1 and Nox4, we identified Nox1 as the relevant enzyme. Our conclusions are corroborated by recent publications, which describe that Nox4, in contrast to Nox1 and Nox2, needs no activation by PKC or other kinases and is therefore constitutively active. In our experiments, only PDGF-induced formation of ROS is blocked by NO, suggesting that down-regulation of Nox1 but not Nox4 expression is responsible for the reduced ROS formation observed.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3791fje;
1 These authors contributed equally to this work. 
2 Present address: II. Medizinische Klinik und Poliklinik, Klinikum der Johannes Gutenberg-Universität Mainz, Langenbeckstr. 1, Mainz 55131, Germany
This article has been cited by other articles:
|
|
 |
|
  B. Wagner, J. M. Ricono, Y. Gorin, K. Block, M. Arar, D. Riley, G. G. Choudhury, and H. E. Abboud Mitogenic Signaling via Platelet-Derived Growth Factor beta in Metanephric Mesenchymal Cells J. Am. Soc. Nephrol., November 1, 2007; 18(11): 2903 - 2911. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Orient, A. Donko, A. Szabo, T. L. Leto, and M. Geiszt Novel sources of reactive oxygen species in the human body Nephrol. Dial. Transplant., May 1, 2007; 22(5): 1281 - 1288. [Full Text] [PDF]
|
|
|
|
 |
|
 K. Bedard and K.-H. Krause The NOX Family of ROS-Generating NADPH Oxidases: Physiology and Pathophysiology Physiol Rev, January 1, 2007; 87(1): 245 - 313. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Jiang, S. J. Roberts, S. r. Datla, and G. J. Dusting NO Modulates NADPH Oxidase Function Via Heme Oxygenase-1 in Human Endothelial Cells Hypertension, November 1, 2006; 48(5): 950 - 957. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S. Wolin and N. G. Abraham Heme Oxygenase-1 Inhibition of Nox Oxidase Activation Is a Microvascular Endothelial Antioxidant Effect of NO Hypertension, November 1, 2006; 48(5): 826 - 827. [Full Text] [PDF]
|
|
|
|
 |
|
 M. Geiszt NADPH oxidases: New kids on the block Cardiovasc Res, July 15, 2006; 71(2): 289 - 299. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
