Nitric oxide inhibits growth of glomerular mesangial cells: Role of the transcription factor EGR-1

Kidney International, Vol. 57 (2000), pp. 70–82

Nitric oxide inhibits growth of glomerular mesangial cells: Role of the transcription factor EGR-1 HARALD D. RUPPRECHT, YOSHITAKA AKAGI, ANNETTE KEIL, and GERHARD HOFER Medizinische Klinik IV, University Erlangen-Nu¨rnberg, Erlangen, Germany; First Department of Medicine, University of Medicine, Osaka University, Osaka, Japan

Nitric oxide inhibits growth of glomerular mesangial cells: Role of the transcription factor Egr-1. Background. In previous studies, we found a close link of early growth response gene-1 (Egr-1) expression to mesangial cell (MC) proliferation. Antiproliferative agents inhibited mitogen-induced Egr-1 expression. Here we investigated the effect of S-nitrosoglutathione (GSNO) on the proliferation of MCs, specifically asking how GSNO regulates the transcription factor Egr-1, which we have previously shown to be critical for the induction of MC mitogenesis. Methods. The proliferation of MCs was measured by thymidine incorporation and cell counting. Egr-1 mRNA and protein levels were detected by Northern and Western blots. Electrophoretic mobility shift assays (EMSAs) and chloramphenicol acetyltransferase (CAT) assays were performed to test whether GSNO modulates DNA binding and transcriptional activation of Egr-1. Results. GSNO strongly inhibited serum-induced MC proliferation (284% at 1 mmol/L). A mild inhibition of serum-induced Egr-1 mRNA was observed at GSNO concentrations from 50 to 200 mmol/L, whereas mRNA levels increased again at concentrations above 500 mmol/L. This increased mRNA expression, however, was not translated into Egr-1 protein. Instead, Egr-1 protein induction was inhibited (240%). EMSAs indicated that GSNO inhibited specific binding of Egr-1 to its DNA consensus sequence. Moreover, transcriptional activation by Egr-1 in CAT assays using a reporter plasmid bearing three Egr-1 binding sites was strongly suppressed by GSNO. Conclusions. Our data identify GSNO as a potent inhibitor of MC growth with potential beneficial effects in proliferative glomerular diseases. This antimitogenic property is mediated at least in part by inhibitory effects of GSNO on Egr-1 protein levels and by reducing the ability of Egr-1 to activate transcription by impairing its DNA binding activity.

toxic effects, and the control of growth-regulatory and apoptotic events [1–5]. These multiple biological effects of NO are mediated through various mechanisms [6–11]: (a) activation of the soluble guanylate cyclase with the liberation of cGMP and the induction of cGMP-dependent protein kinases (smooth muscle relaxation and vasodilation, inhibition of platelet aggregation, inhibition of cell proliferation), (b) complexation of central iron atoms with consecutive inactivation of enzymes of the respiratory chain like aconitase or cytochrome (cytotoxicity, unspecific immune defense against various microbial pathogens), (c) reaction as radical or reaction with oxygen radicals to peroxynitrite (direct DNA damage and cytotoxicity), (d) nitrosylation of protein thiol groups involved in complexation of Zn21 or Cd21 with subsequent formation of disulfide bonds [12], and (e) direct modulation of signal transduction pathways, for example, the induction of p53 protein synthesis, activation of iron-response elements, induction of p21ras, and so forth [13–15]. The early growth response gene-1 (Egr-1), also known as zif268, Krox 24, TIS 8, or NGFI-A, is a member of the family of immediate early genes. It is rapidly and transiently induced after a variety of mitogenic signals [16, 17], but also during fetal development in the mouse [18], following differentiation signals [19, 20], upon depolarization of PC12 cells [19], after ionizing radiation [21], or in response to stretch/relaxation [22]. Egr-1 mRNA is superinduced in the presence of cycloheximide [17]. The induction has been shown to occur mainly at the transcriptional level [23–25]. The gene encodes a 75 to 80 kd nuclear phosphoprotein [26, 27]. It has been shown to bind DNA at the consensus sequence GCGGGGGCG in a zinc-dependent fashion through three zinc-finger domains [27–29] and to activate transcription [27, 30–32]. In recent studies employing antisense-oligonucleotide technology, we were able to demonstrate that Egr-1 induction is a necessary requirement for induction of mitogenesis in cultured rat mesangial cells (MCs). Antisense oligonucleotides directed against Egr-1 potently inhibited Egr-1 mRNA and protein induction as well as serum-,

Several studies suggest that nitric oxide (NO) exerts complex regulatory actions in the kidney. These include the regulation of renal hemodynamics, radical-like cytoKey words: early growth response gene-1, mesangial cell mitogenesis, hemodynamics, S-nitrosoglutathione. Received for publication January 28, 1999 and in revised form August 6, 1999 Accepted for publication August 23, 1999

 2000 by the International Society of Nephrology

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platelet-derived growth factor–, or endothelin-1–mediated growth of MCs [33, 34]. In this study, we wanted to address the following issues: (a) Because conflicting data exist in the literature on the effect of different NO donors on the MC proliferative phenotype, we tested whether the NO donor S-nitrosoglutathione (GSNO) interfered with MC growth. (b) Egr-1 expression was in our previous studies closely linked to MC proliferation and antiproliferative agents like heparin or dexamethason inhibited mitogen-induced Egr-1 expression. We therefore tested whether GSNO influenced transcript or protein levels of Egr-1. (c) Because some of the actions of NO are mediated through the liberation of cGMP, we investigated the impact of cGMP on MC proliferation and Egr-1 induction. (d) NO has been shown to nitrosylate protein thiol groups and to thus destroy zinc-sulfur clusters in proteins such as metallothionein or the yeast transcriptional activator LAC9. We therefore analyzed whether GSNO directly interfered with the DNA-binding activity or the transcriptional-activating capability of Egr-1. METHODS Reagents The Hoechst dye 33258 and 8br-cGMP were from Sigma Chemical Co. (Deisenhofen, Germany). Pyruvate and nicotinamide adenine dinucleotide (NADH) were from Boehringer Mannheim (Mannheim, Germany). GSNO synthesis S-nitrosoglutathione was synthesized as described previously [35]. Briefly, glutathione was dissolved in 0.625 N HCl at 48C to a final concentration of 625 mmol/L. An equimolar amount of NaNO2 was added and the mixture was stirred for 40 minutes. After the addition of 2.5 volumes of acetone, stirring was continued for another 20 minutes, followed by filtration of the precipitate. GSNO was washed once with 80% acetone, two times with 100% acetone, and finally, three times with diethylether and was dried under vacuum. Freshly synthesized GSNO was characterized by high-performance liquid chromatography analysis and ultraviolet spectroscopy. Mesangial cell isolation and culture Glomeruli from rat kidneys were isolated, and glomerular outgrowth and subsequent subculturing of MCs were performed as previously described [16]. MCs were kept in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated (508C, 30 min) fetal calf serum (FCS), 50 U/mL penicillin, 50 mg/mL streptomycin, 2 mmol/L glutamin, 5 mg/mL insulin in a 95% air/5% CO2 humidified atmosphere at 378C. MCs were used for experiments between passages 5 and 20.

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RNA extraction and Northern blot analysis Mesangial cells were grown in 10 cm dishes until subconfluency and were growth arrested for 72 hours in a medium containing 0.4% FCS. After adequate stimulation, cells were washed twice in phosphate-buffered saline (PBS), and total RNA was extracted by the method of Chomczynski and Sacchi [36]. RNA was size fractionated on a 1% agarose formaldehyde gel and transferred onto Hybond nylon membrane (Amersham, Little Chalfont, Buckinghamshire, UK). The Northern blot was baked at 808C for two hours, prehybridized with 5 3 Denhardt’s, 5 3 SSC, 50% formamide, 50 mmol/L Na3PO4, 0.1% sodium dodecyl sulfate (SDS), 0.25 mg/mL salmon sperm DNA at 398C for four hours. DNA hybridization probes were labeled with [a-32P]-dCTP using a random primed labeling kit (Boehringer). The blots were hybridized in prehybridization solution containing 2 3 106 cpm/mL of probe at 398C for 20 hours. The blot was washed twice for 15 minutes with 2 3 SSC containing 0.1% SDS and then 30 minutes with 0.1 3 SSC containing 0.1% SDS. Blots were exposed to Kodak XAR-2 films with intensifying screens at 2808C. Protein extraction and Western blot analysis Mesangial cells were grown in 3.5 cm dishes until subconfluency, growth arrested for 72 hours in medium containing 0.4% FCS, and harvested after adequate stimulation in 100 mL RIPA solution [1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 mol/L NaCl, 50 mmol/L Tris-HCl, pH 7.2, 10 mmol/L ethylenediaminetetraacetic acid (EDTA), pH 7.2, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), leupeptin 2 mg/mL]. Protein concentration was determined using the Bradford assay (Bio-Rad, Hercules, CA, USA). Protein samples containing 20 mg of total protein were denatured by boiling for five minutes and separated on a 7.5% denaturing SDS-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the gels were electroblotted onto NC membranes, and the transfer was controlled by Ponceau-S staining. Blots were incubated in PBS containing 0.1% Tween-20 and 5% nonfat dry milk powder to block unspecific binding, washed in PBS containing 0.1% Tween-20, and incubated with the primary anti–Egr-1 antibody R5232-2 (1:3000), described in detail elsewhere [26]. Egr-1 was visualized with a secondary horseradish peroxidase-conjugated antirabbit IgG antibody using the ECL system (Amersham). Determination of lactate dehydrogenase release Following incubations, the medium of approximately 2.5 3 105 MCs was collected, and cells were supplemented with 0.2% (vol/vol) Triton-X 100 in PBS. Cells were lysed for four hours at 48C. A total of 500 mL of reaction mix containing 50 mmol/L triethanolamine

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dissolved in 5 mmol/L EDTA, pH 7.6, 127 mmol/L pyruvate, and 14 mmol/L NADH in 1% NaHCO3 was added to 300 mL cell medium or lysed cells. Lactate dehydrogenase (LDH) activity was monitored by the oxidation of NADH following the decrease in absorbance at 334 nm. The percentage of LDH released was defined as the ratio of LDH activity in the supernatant to the sum of the LDH amount released plus the activity measured in the cell lysate. Staining of nuclei with Hoechst dye 33258 Mesangial cells were seeded in 35 mm culture dishes and serum starved for three days in medium containing 0.4% FCS. MCs were then stimulated with GSNO 500 mmol/L in the presence or absence of FCS in a final concentration of 2% for 20 hours. Cells were then scraped off the culture dish and pelleted by centrifuging for two minutes at 1000 g. The cell pellet was resuspended in 50 mL paraformaldehyde (3% in PBS) and transferred onto a glass slide. After air drying for 10 to 15 minutes, cells were stained with 100 mL of Hoechst dye 33258 (8 mg/ mL) for five minutes, washed three times for five minutes in PBS, and mounted in Mowiol. Nuclei were visualized using a fluorescence microscope with an excitation wavelength of 340 to 380 nm. Three hundred cells were counted, and results are given as a percentage of cells with condensed nuclei compared with total cell number. Determination of 3H-thymidine uptake Mesangial cells were subcultured in 96-well plates in medium supplemented with 10% FCS until subconfluency and growth arrested for 72 hours in medium supplemented with 0.4% FCS. Quiescent MCs were then exposed to fresh medium containing 0.4% FCS with or without GSNO for six hours before stimulation with FCS in a final concentration of 2%. Cells were pulsed with 1 mCi/mL [3H-methyl]-thymidine (specific activity, 5 mCi/mmol/L; ICN, Costa Mesa, CA, USA) from 0 to 24 hours after the addition of FCS. The cells were washed twice with PBS, lysed with distilled water, and harvested onto filters by an automated cell harvester (Dunn, Asbach, Germany). Incorporated counts were measured by a liquid scintillation counter (Beckmann, Fullerton, CA, USA). Determination of cell number Mesangial cells were seeded in 96-well plates at a density of 3 3 103 cells per well and were growth arrested for 48 hours in medium supplemented with 0.4% FCS and an additional 24 hours in medium without FCS. When cells were preincubated with GSNO, it was added 20 hours before serum stimulation. The cell number was determined 48 hours after growth stimulation. Monolayers were washed twice in PBS. Cells were trypsinized and transferred into 10 mL of Isoton for counting in a Coulter counter (Coulter, Luton, England).

Nuclear extract preparation and electrophoretic mobility shift assay Two 3 107 MCs were harvested on ice in PBS, resuspended in 300 mL hypotonic buffer A (10 mmol/L HEPES, pH 7.9, 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1 mmol/L dithiothreitol, 0.5 mmol/L PMSF) and kept on ice for 15 minutes. Twenty microliters of 10% Nonidet P40 were added to lyze cells by vortexing. Nuclei were collected by centrifugation and suspended in 40 mL ice-cold buffer C (20 mmol/L HEPES, pH 7.9, 25% glycerol, 0.4 mol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA). The suspension was vigorously agitated at 48C to extract nuclear proteins. Extracts were stored at 2808C. Five micrograms nuclear extract were incubated with 1 ng radiolabeled Egr-1 binding site (59-AGCTTC GCGGGGGCGAGG-39, 39-AGCGCCCCCGCTCCT TAA-59) or AP-1 binding site (59-AGCTAAAGCAT GAGTCAGACAGCCT, 39-TTTCGTACTCAGTCT GTCGGATCGA-59) in 25 mL binding buffer (10 mmol/L Tris-HCl, pH 7.5, 50 mmol/L NaCl, 0.5 mmol/L EDTA, 0.5 mmol/L dithiothreitol, 1 mmol/L MgCl2, 4% glycerol, 0.05 mg/mL poly dIdC). Nuclear extracts were incubated for 30 minutes at 48C in binding buffer. The labeled binding site was added, and reactions were incubated for 30 minutes at 48C and separated on a 5% polyacrylamide gel. The dried gel was exposed to x-ray films. Assay for chloramphenicol acetyltransferase activity Growing MCs were transfected at a confluency of approximately 60% with 1 mg chloramphenicol acetyltransferase (CAT)-reporter plasmid pEBS3CAT and 0.2 mg lacZ-expressing plasmid pCMVbgal using Superfect transfection reagent (Qiagen, Du¨sseldorf, Germany). Sixteen hours after transfection cells were growth stimulated with FCS (10%) and treated with GSNO (500 mmol) and incubated for a further 24 hours. Instead of FCS treatment, MCs were alternatively cotransfected with 4 mg of Egr-1-expression plasmid pRSVEgr-1 [31]. Cells were harvested in cold PBS, resuspended in 100 mL 0.25 mol/L Tris-HCl (pH 7.8) and freeze-thaw extracts were prepared. Fifty microliters of extract were mixed with CAT-reaction mixture (50 mL 1 mol/L TrisHCl, pH 7.8, 10 mL 14C-chloramphenicol, 0.1 mCi/mL, 20 mL 3.5 mg/mL acetyl coenzyme A). Reactions were incubated for 16 hours at 378C. Chloramphenicol and its acetylated derivates were extracted with ethylacetate and separated by thin-layer chromatography. Plates were exposed to x-ray films, and radioactive spots were cut out and measured in a liquid scintillation counter to calculate percentage of acetylated forms of chloramphenicol. b-Galactosidase assay was performed as described [37], and activities were used to correct for differences in transfection efficiency.

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Statistical analysis Statistical analysis was performed using Student’s ttest for unpaired samples. RESULTS GSNO is a potent inhibitor of serum-induced mesangial cell growth Because conflicting data exist in the literature concerning the effects of NO on MC growth using various NO donors, we tested whether the potent NO-donor GSNO affected serum-induced MC proliferation. Synthesis of DNA, assessed by the incorporation of 3H-thymidine, and a serum-induced increase in total cell number were monitored after preincubation of MCs with increasing doses of GSNO. GSNO led to a dose-dependent inhibition of serum-stimulated 3H-thymidine incorporation into MCs (Fig. 1A) and to a prominent suppression of serum-induced increase in cell number (inhibition of serum-induced increase in cell number: GSNO 50 mmol/L, 219 6 23%; GSNO 100 mmol/L, 247 6 20%; GSNO 1 mmol/L, 284 6 23%; data were obtained from four independent experiments each consisting of quintuplicate samples; a representative experiment is shown in Fig. 1B). Because it has been demonstrated that NO can induce detachment of MCs cultured in the absence of serum [38], cells in the supernatant were counted in addition to cells adhering to the culture dish. As indicated in Figure 1B, GSNO did not induce detachment of serum-stimulated MCs. We further addressed the question of whether other NO donors like spermine-NO (SpNO) or S-nitroso-Nacetylpenicillamine (SNAP) would affect serum-stimulated (FCS 2%) MC growth. SpNO (25 mmol/L, 50 mmol/L, and 100 mmol/L) led to a dose-dependent inhibition of MC thymidine uptake (213 6 10%, 236 6 9%, and 288 6 57%). Higher concentrations appeared to be toxic, decreasing thymidine uptake below control levels. In contrast, SNAP exhibited a less pronounced inhibitory effect on FCS-induced MC growth (growth inhibition at 25 mmol/L, 215%; at 100 mmol/L, 244%). Data were obtained from four independent experiments each consisting of quintuplicate samples (SpNO). For SNAP, results given are means of two independent experiments consisting of quintuplicate samples (data not shown). GSNO does not induce toxic or apoptotic death of serum-stimulated mesangial cells To exclude toxicity of the GSNO concentrations used, we monitored the release of the cytoplasmic enzyme LDH from cultured MCs during varying preincubation periods with GSNO. As indicated in Figure 2A, GSNO did not induce the release of LDH over control cells in concentrations of up to 1 mmol/L and during preincuba-

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tion periods of up to 48 hours. At a concentration of 2 mmol/L, GSNO induced LDH release when preincubated with MCs for 24 hours or longer. Ethanol served as a positive control. It has been reported that exogenously added NO donors, like GSNO, can induce apoptosis in serum-starved cultured rat MCs [39, 40]. To address the question of whether the reduction in the serum-stimulated increase in MC number by GSNO was in part due to apoptotic processes, we looked for condensation and fragmentation of nuclei by staining with the Hoechst dye 33258. As reported previously by others [39, 40], we could demonstrate that approximately 20% of serum-starved MCs were rendered apoptotic by incubation with 500 mmol/L GSNO. GSNO had, however, only marginal effects on the induction of apoptosis when added to serum-stimulated MCs (Fig. 2B). We therefore conclude that GSNO in the concentrations used did not induce necrotic or apoptotic cell death of serum-stimulated MCs. The decrease in 3H-thymidine uptake or cell number is therefore due to specific antiproliferative effects of GSNO. To further eliminate the possibility that the glutathion (GSH) component of GSNO per se was responsible for the antiproliferative effects observed, MCs were incubated with equimolar amounts of either GSH alone, GSNO that was kept at room temperature for 24 hours, or fresh GSNO. As can be seen in Figure 3, GSH had no effect on 3H-thymidine uptake. Twenty-four-hour old GSNO had significant but weak growth inhibitory actions, whereas fresh GSNO retained its full antiproliferative activity. Effects of GSNO on Egr-1 mRNA and protein levels in serum-stimulated mesangial cells In previous studies, we demonstrated that the induction of Egr-1 was necessary for the induction of MC growth [33, 34]. Furthermore, we could show that antiproliferative substances such as heparin or dexamethason inhibited the mitogen-induced increase in Egr-1 mRNA and protein in MCs [33]. The degree of inhibition of Egr-1 correlated with the degree of inhibition of growth. To test whether the antimitogenic effects of GSNO were due to a down-regulation of Egr-1, transcript and protein levels of Egr-1 were measured in MCs preincubated with GSNO for six hours and stimulated with FCS 2% for one or two hours, respectively. Incubation of MCs with FCS for one hour led to a prominent increase in Egr-1 mRNA. Preincubation with increasing doses of GSNO had a biphasic effect on Egr-1 mRNA levels. At concentrations from 50 to 200 mmol/L an inhibition of serum-induced Egr-1 mRNA levels was observed. The average inhibition of Egr-1 mRNA at 200 mmol/L GSNO was 29 6 14% in three independent experiments. In contrast, at higher concentrations of GSNO, Egr-1 mRNA returned to, and in some experi-

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Fig. 1. S-nitrosogluthathione (GSNO) inhibits serum-induced mesangial cell (MC) growth. (A) 3H-thymidine uptake was measured between 0 and 24 hours after fetal calf serum (FCS) stimulation (2%) of MCs growth arrested for 72 hours. GSNO was added in the indicated concentrations six hours before FCS addition. (B) The cell number was determined 48 hours after FCS stimulation (2%) of MCs growth arrested for 72 hours. GSNO was added in increasing concentrations 20 hours before stimulation with FCS. Cells attached to the culture plates (j) as well as cells in the supernatant ( ) were counted. Data are given as mean 6 sd of quadruplicate determinations. *P , 0.05; ***P , 0.001 compared with serum-stimulated cells.

ments even exceeded, the levels reached with FCS-stimulation alone (Fig. 4). Surprisingly, this enhanced Egr-1 mRNA expression at high GSNO concentrations was not translated into elevated protein levels. GSNO induced a dose-dependent suppression of serum-stimulated Egr-1 protein levels. Inhibition was 24 6 32% at 200 mmol/L GSNO and 40 6 24% at 1 mmol/L GSNO in four independent experiments (a representative experiment is shown in Fig. 5). A similar inhibition was observed when spermine NO was used as a donor of NO. Effect of the stable cGMP analogue 8br-cGMP on mesangial cell proliferation and Egr-1 induction Because at least some of the actions of NO are mediated by generation of cGMP, we tested for the effects of

the stable cGMP-analogue, 8br-cGMP, on serum-induced MC growth and Egr-1 induction. 8br-cGMP led to a dosedependent inhibition of serum-stimulated MC growth. At concentrations of 100 mmol/L and 1 mmol/L, MC proliferation, as assessed by 3H-thymidine uptake, was suppressed by 15 6 4% and 35 6 12%, respectively (data from 3 independent experiments each consisting of quintuplicate samples; a representative experiment is shown in Fig. 6A). At the same time, 8br-cGMP inhibited the induction of Egr-1 protein by FCS by 15 6 11% (100 mmol/L) or 33 6 13% (1 mmol/L; data from 3 independent experiments; a representative experiment is shown in Fig. 6B). The decrease in serum-stimulated Egr-1 protein levels therefore paralleled the decrease in 3 H-thymidine uptake elicited by 8br-cGMP.

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Fig. 2. GSNO does not induce toxic or apoptotic death of serum-induced MCs. (A) LDH-release from serum-stimulated MCs preincubated for 8 (j, ), 24 (j, ), or 48 (m) hours with increasing concentrations of GSNO was determined as described in the Methods section. The percentage of LDH released was determined as the ratio of LDH activity in the supernatant to the total LDH activity. GSNO had no toxic effects up to a concentration of 1 mmol/L. Ten percent ethanol served as a positive control. (B) The proportion of apoptotic cells was determined by staining paraformaldehyde fixed cells with Hoechst dye 33258. Serum-starved control cells or serum-stimulated cells were either left untreated or were incubated with GSNO (500 mmol/L) for 20 hours. An increase in apoptotic cells was only observed in serum-starved MCs. Results are given as percentage of cells with condensed nuclei compared with total cell number.

Fig. 3. GSNO but not glutathion (GSH) inhibits serum-stimulated 3Hthymidine uptake by MCs. 3H-thymidine uptake was measured between 0 and 24 hours after FCS stimulation (2%) of MCs that have been growth arrested for 72 hours. Fresh GSNO, GSNO that was kept at room temperature for 24 hours, or GSH were each added at concentrations of 500 mmol/L six hours before serum stimulation. ***P , 0.001 compared with serum-stimulated MCs. NS, not significant.

Effects of GSNO on Egr-1 DNA binding capability and transcriptional activating capacity The growth inhibitory effects of GSNO (84% inhibition of serum-stimulated increase in cell number at 1 mmol/L GSNO) were far more pronounced than the effects of 8br-cGMP on MC growth (35% inhibition at 1 mmol/L) or the effects of GSNO on Egr-1 protein levels (40% inhibition at 1 mmol/L GSNO). Therefore, mechanisms other than liberation of cGMP or inhibition of Egr-1 protein levels must be active. Because Egr-1 activ-

ity is intricately linked to MC growth [33, 34], we tested whether GSNO exerted additional effects on DNA binding or transcriptional activating capacity of Egr-1. First, we tested the influence of NO on the ability of Egr-1 to bind DNA. As shown in Figure 7A, serum induced Egr-1 binding to a DNA fragment containing an Egr-1 consensus sequence (closed arrow). The specificity of this binding was demonstrated by the addition of an Egr-1 antibody (supershift, open arrow) or the addition of an excess of cold competitor to the binding reaction. GSNO, when added to the binding reaction, drastically inhibited the binding of FCS-induced Egr-1 protein to its binding site (Fig. 7B). We also asked whether GSH (500 mmol/L) per se would interfere with Egr-1 binding when added to the binding reaction, but no inhibiting effect was observed (data not shown). To test whether the presence of GSNO in the binding reaction would inhibit the binding of other transcription factors, we added 500 mmol/L GSNO to an electrophoretic mobility shift assay (EMSA) for activator protein-1 (AP-1; Fig. 7C). GSNO led to an enhanced binding of AP-1 to its binding sequence. In the experiment shown in Figure 8, GSNO was added to the cells before serum stimulation and preparation of nuclear extracts. There was a dose-dependent decrease in Egr-1 binding activity, and near complete inhibition to levels seen in controls was observed at 500 mmol/L GSNO. To demonstrate that GSNO not only inhibited DNA binding but also transcriptional activation by Egr-1, CAT

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Fig. 4. Biphasic effect of increasing concentrations of GSNO on serum-induced early growth response gene-1 (Egr-1) mRNA expression. MCs were growth arrested for 72 hours in medium containing 0.4% FCS. After preincubation for six hours with increasing concentrations of GSNO, cells were serum stimulated (2%) for 60 minutes. RNA was extracted and 20 mg total RNA per lane were size fractionated on a 1% agarose gel. Blots were probed with cDNAs for Egr-1 and GAPDH, radiolabeled by random priming.

Fig. 5. GSNO inhibits serum-induced Egr-1 protein expression. MCs were growth arrested for 72 hours in medium containing 0.4% FCS. After preincubation with GSNO or SpNO for six hours, cells were stimulated with FCS (2%), and two hours later, protein extracts were prepared. Twenty micrograms of protein per lane were separated on a 7.5% denaturing SDS-PAGE gel. Blots were probed with the primary Egr-1 antibody R5232-2 (1:3000). Egr-1 was visualized with a secondary horseradish peroxidase-conjugated antirabbit IgG antibody using the ECL system (upper panel). Expression was quantitated using video densitometry (lower panel).

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Fig. 6. 8br-cGMP inhibits serum-induced 3H-thymidine uptake (A) and Egr-1 protein expression (B) in cultured rat MCs. Serum-starved MCs were preincubated for three hours with the indicated concentrations of 8br-cGMP and stimulated with FCS (2%). 3H-thymidine uptake was measured between 0 and 24 hours after FCS addition. Protein extracts for Western Blots were prepared two hours after FCS stimulation. *P , 0.05; ***P , 0.001 compared with serum-stimulated MCs.

assays were performed. In Figure 9A, MCs were transfected with 1 mg of a CAT-reporter construct bearing 3 Egr-1 binding sites (pEBS3CAT). MCs were then stimulated with FCS in the presence or absence of 500 mmol/L GSNO. GSNO led to a marked inhibition of CAT activity elicited by stimulation of MCs with 10% FCS (reduction from 71% to 21% acetylated chloramphenicol). In Figure 9B, Egr-1 expression was achieved by cotransfection of a plasmid expressing Egr-1 under the control of the constitutively active RSV promoter (pRSVEgr-1). GSNO also led to a near complete inhibition of CAT activity (reduction from 79% to 9% acetylated chloramphenicol). In Figure 9C, GSNO (500 mmol/L) was added to MCs transfected with 0.5 mg or 1 mg of pRR55, which leads to constitutive CAT expression. No reduction of acetylated chloramphenicol by treatment with GSNO was detectable, demonstrating that GSNO did not exhibit unspecific effects on CAT activity. DISCUSSION Nitric oxide has multiple actions in the kidney. The most prominent is regulation of glomerular hemodynamics. NO potently antagonizes the vasoconstrictor effects of angiotensin II at the efferent arteriole [41]. Synthesis of NO by eNOS (endothelial NO synthase) in glomerular endothelial cells leads to increases in effective renal plasma flow and glomerular filtration rate and decreases

in glomerular filtration pressure [1, 42]. These effects are predominantly mediated by cGMP. Increasing glomerular NO production by l-arginine substitution has been shown to be protective in various renal diseases models, like 5/6 nephrectomy or salt-sensitive hypertension [42, 43]. The protective effects are most likely due to a decrease in glomerular capillary pressure. NO is also synthesized by brain NO synthase (bNOS) in the macula densa and regulates secretion of renin [1, 44]. Thus, NO has important functions in the autoregulation of renal perfusion. Another source of glomerular NO is mesangial cells and infiltrated macrophages inducible NO synthase (iNOS). NO production by iNOS is not only important for defense mechanisms against infectious pathogens but plays pathophysiologically important roles in immunologic diseases, such as in glomerulonephritis [45, 46]. Cattell, Cook, and Moncada demonstrated that glomeruli isolated from nephritic rats secreted large amounts of nitrite into the culture supernatant [47]. Increased NO synthesis was shown in various glomerulonephritis models, such as rat nephrotoxic serum nephritis [47], Heymann nephritis [48], anti-Thy1.1 nephritis [49, 50], and in MRL-lpr/lpr mice [51]. Infiltrating macrophages seem to be the main source of iNOS expression [48, 49]. Different effects of NO on the course of these nephritis models have been described.

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Fig. 7. DNA-binding of Egr-1 is inhibited by GSNO added to the binding reaction. Nuclear extracts were prepared as described in the Methods section from growth arrested MCs or from MCs stimulated with FCS for two hours. Five micrograms of nuclear extract were incubated with 1 ng radiolabeled Egr-1 binding site for 30 minutes at 48C. (A) To demonstrate specificity of Egr-1 binding, Egr-1 antibody (R5232-2, 1:250) or a 100fold molar excess of cold competitor DNA was added to the binding reaction. Open arrow indicates supershift of Egr-1–DNA-complexes by addition of Egr-1 antibody. (B) GSNO was added to the binding reaction to a final concentration of 500 mmol/L. Closed arrow indicates specific DNA-binding of Egr-1 (C) MC extracts used for the AP-1 gelshift were obtained six hours after stimulation with FCS. GSNO (500 mmol/L) was added directly to the binding reaction.

In anti-Thy1.1 nephritis, an application of the NOSinhibitor L-NNMA (NG-monomethyl-l-arginine) shortly before induction of nephritis prevented mesangiolysis and proteinuria [50]. Similar, albeit weaker effects were obtained with a diet poor in l-arginine [50]. Positive effects of systemic NO inhibition have also been described in the mouse model of lupus nephritis (MRL-lpr/lpr

mice) [51]. In contrast, NOS inhibition led to increased proteinuria and aggravated disease in rat nephrotoxic serum nephritis [52]. Because many different pathomechanic processes are at play during development of glomerulonephritis, such as proliferation of intrinsic glomerular cells, infiltration and proliferation of inflammatory cells, altered matrix

Rupprecht et al: NO inhibits MC growth

Fig. 8. Prestimulation of MCs with GSNO inhibits binding of seruminduced Egr-1 protein to its DNA-binding site. Growth arrested MCs were preincubated with increasing doses of GSNO for one hour before stimulation with FCS (4%) for two hours. Nuclear extracts were prepared as described in the Methods section. Five micrograms nuclear extract were incubated with 1 ng radiolabeled Egr-1 binding site for 30 minutes at 48C. Specificity of binding was demonstrated by addition of Egr-1 antibody or a 100-fold molar excess of cold competitor DNA to the binding reaction.

metabolism or apoptotic events, NO might have different effects, depending on the predominant feature in the respective models. We investigated in this study the effects of NO on one aspect of many glomerular diseases, that is, MC proliferation, and tried to elucidate signaling pathways involved. We found that the NO-donor GSNO was a potent inhibitor of MC growth and could confirm studies in the literature using other NO donors [53, 54]. No toxic effects or effects on apoptosis were observed on MCs cultured

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in the presence of serum. Under low serum culture conditions (0.5% FCS), GSNO also induced apoptosis, as previously described [39, 40]. We obtained similar results on proliferation and Egr-1 protein levels with SpNO, a different NO donor. SNAP did moderately inhibit serum-induced MC growth. Because some of the effects of NO are mediated through cGMP, we tested the antiproliferative effects of 8br-cGMP on MCs. 8br-cGMP had, as described by others [54, 55], weak but significant growth inhibitory actions at higher concentrations. The actions of cGMP could, however, not entirely account for the strong growth-inhibiting effects of GSNO. Therefore, other mechanisms must be at play. We were able to identify two such mechanisms when investigating the effects of GSNO on the transcription factor and immediate early gene Egr-1. We chose to investigate Egr-1 because we had previously shown that its induction was critical for MC proliferation to occur in response to various mitogens [33, 34]. Antisense oligonucleotides against Egr-1 inhibited the induction of Egr-1 mRNA as well as Egr-1 protein and nearly completely abolished serum platelet-derived growth factor or endothelin-1–induced MC proliferation. GSNO at lower concentrations inhibited serum-induced Egr-1 mRNA induction, and at higher concentrations, Egr-1 mRNA levels rose again to serum-stimulated control levels. This rise in mRNA was, however, not translated into a rise in Egr-1 protein. Instead, Egr-1 protein levels were significantly suppressed with increasing doses of GSNO. Therefore, GSNO induced a translational blockade, preventing Egr-1 accumulation and thereby inhibiting MC growth. A similar translational control mechanism has been postulated in Sol8 mouse muscle cells by Maass et al [56]. The authors could show that only agents that induced proliferation led to a rise in Egr-1 mRNA and Egr-1 protein, whereas stimuli that induced differentiation up-regulated Egr-1 mRNA but led to a translational blockade preventing protein accumulation. A second cGMP-independent mechanism by which NO inhibits MC growth is by affecting binding properties of the transcription factor Egr-1. Binding of Egr-1 present in nuclear extracts from serum-stimulated MCs to its DNA-binding sequence was inhibited by addition of GSNO to the binding reaction, but not by glutathion per se. To test for nonspecific down-regulation of binding activities under these stringent conditions, we performed a gelshift for another transcription factor, AP-1. Here we found that GSNO enhanced the binding of the factor to its DNA binding site, indicating that GSNO does not unspecifically down-regulate DNA binding of transcription factors. Similar results have been reported previously in macrophages [57]. In this experimental setting, NO might have directly altered the DNA containing the Egr-1 consensus site, thereby affecting binding [58, 59]. Therefore, we also performed EMSA

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Fig. 9. GSNO reduces transcriptional activation of the chloramphenicol acetyltransferase (CAT) reporter gene by Egr-1. (A) MCs were transiently transfected with 1 mg CAT reporter plasmid (pEBS3CAT) containing 3 Egr-1 binding sites in front of the CAT gene using Superfect transfection reagent. Sixteen hours after transfection, cells were stimulated with FCS (2 or 10%) in the absence or presence of GSNO (500 mmol/L) and were incubated for another 24 hours. As a positive control, MCs were transfected with 1 mg plasmid pRR55 constitutively expressing CAT under the control of the human CMV promotor. Cell extracts were prepared and CAT assays performed as described in the Methods section. Chloramphenicol and its acetylated derivates were separated by thin layer chromatography (upper panel). Plates were exposed to x-ray films. Radioactive spots were excised and measured in a scintillation counter. CAT activity was expressed as the percentage of acetylated forms of chloramphenicol relative to the total amount of chloramphenicol. (B) MCs were cotransfected with 1 mg pEBS3CAT and 4 or 10 mg of the Egr-1 expression plasmid pRSVEgr-1. Sixteen hours after transfection cells were incubated in the absence or presence of GSNO (500 mmol/L) for an additional 24 hours before preparation of cell extracts. (C) MCs were transfected with 0.5 or 1 mg of the plasmid pRR55, which constitutively expresses CAT under the control of the CMV-promoter. Sixteen hours after transfection cells were incubated further 24 hours in the presence or absence of GSNO (500 mmol/L).

with nuclear extracts prepared from cells that were pretreated with GSNO and obtained similar results. With GSNO treatment, serum-induced binding of Egr-1 to its consensus sequence was almost completely suppressed to control levels. Only part of this suppression can be due to reduction in Egr-1 protein levels because GSNO led to a maximum 40% suppression of Egr-1 protein expression. In CAT assays, we demonstrated that reduced DNA binding was also translated into a reduced transcriptional activation by Egr-1. Whereas GSNO dramatically reduced the transcriptional activation of CAT via Egr-1, no effect was seen on the CMV promoterdriven CAT expression. A potential mechanism by which NO inhibits DNA binding of Egr-1 is by interfering with the zinc-finger domains that establish the DNA contacts [29]. It has been described that NO leads to metal ion release from the Zn21/Cd21-complexing protein metallothionein via nitrosylation of suohydroxyl or thiol groups and subsequent disulfide formation [12]. Similar results were obtained for the yeast zinc-finger containing transcriptional activator LAC9, and it is therefore conceivable that NO also destroys the Cys2/His2-type zinc-fingers of the Egr-1 protein and thus prevents DNA binding. We conclude that GSNO inhibits growth of glomerular

MCs by cGMP-dependent as well as, more importantly, by cGMP-independent pathways that include interference with the zinc-finger transcription factor Egr-1. Glomerular NO release or application of NO-donors at the appropriate time might prove beneficial in mesangioproliferative glomerular disorders. ACKNOWLEDGMENTS The work was supported by a grant by the Deutsche Forschungsgemeinschaft to H.D. Rupprecht (Klinische Forschergruppe, Teilprojekt IV). We gratefully acknowledge the technical assistance of Ms. Katja Bruch and Ms. Isabella Kolberg. Reprint requests to Harald D. Rupprecht, M.D., Nephrologische Forschungslabors, Medizinische Klinik IV, Universita¨t Erlangen-Nu¨rnberg, Loschgestr. 8, 91054 Erlangen, Germany.

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