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-Nurnberg, Erlangen, Germany; First Department of Medicine,University of Medicine, Osaka University, Osaka, Japan

Nitric oxide inhibits growth of glomerular mesangial cells: Role toxic effects, and the control of growth-regulatory andof the transcription factor Egr-1. apoptotic events [1–5]. These multiple biological effects

Background. In previous studies, we found a close link of of NO are mediated through various mechanisms [6–11]:early growth response gene-1 (Egr-1) expression to mesangial(a) activation of the soluble guanylate cyclase with thecell (MC) proliferation. Antiproliferative agents inhibited mi-liberation of cGMP and the induction of cGMP-depen-togen-induced Egr-1 expression. Here we investigated the ef-

fect of S-nitrosoglutathione (GSNO) on the proliferation of dent protein kinases (smooth muscle relaxation and vaso-MCs, specifically asking how GSNO regulates the transcription dilation, inhibition of platelet aggregation, inhibition of cellfactor Egr-1, which we have previously shown to be critical proliferation), (b) complexation of central iron atoms withfor the induction of MC mitogenesis.

consecutive inactivation of enzymes of the respiratoryMethods. The proliferation of MCs was measured by thymi-dine incorporation and cell counting. Egr-1 mRNA and protein chain like aconitase or cytochrome (cytotoxicity, unspe-levels were detected by Northern and Western blots. Electro- cific immune defense against various microbial patho-phoretic mobility shift assays (EMSAs) and chloramphenicol gens), (c) reaction as radical or reaction with oxygenacetyltransferase (CAT) assays were performed to test whether

radicals to peroxynitrite (direct DNA damage and cyto-GSNO modulates DNA binding and transcriptional activationtoxicity), (d) nitrosylation of protein thiol groups involvedof Egr-1.

Results. GSNO strongly inhibited serum-induced MC prolif- in complexation of Zn21 or Cd21 with subsequent forma-eration (284% at 1 mmol/L). A mild inhibition of serum-induced tion of disulfide bonds [12], and (e) direct modulationEgr-1 mRNA was observed at GSNO concentrations from 50 of signal transduction pathways, for example, the induc-to 200 mmol/L, whereas mRNA levels increased again at con-

tion of p53 protein synthesis, activation of iron-responsecentrations above 500 mmol/L. This increased mRNA expres-elements, induction of p21ras, and so forth [13–15].sion, however, was not translated into Egr-1 protein. Instead,

Egr-1 protein induction was inhibited (240%). EMSAs indi- The early growth response gene-1 (Egr-1), also knowncated that GSNO inhibited specific binding of Egr-1 to its DNA as zif268, Krox 24, TIS 8, or NGFI-A, is a member ofconsensus sequence. Moreover, transcriptional activation by

the family of immediate early genes. It is rapidly andEgr-1 in CAT assays using a reporter plasmid bearing threetransiently induced after a variety of mitogenic signalsEgr-1 binding sites was strongly suppressed by GSNO.

Conclusions. Our data identify GSNO as a potent inhibitor [16, 17], but also during fetal development in the mouseof MC growth with potential beneficial effects in proliferative [18], following differentiation signals [19, 20], upon depo-glomerular diseases. This antimitogenic property is mediated larization of PC12 cells [19], after ionizing radiation [21],at least in part by inhibitory effects of GSNO on Egr-1 protein

or in response to stretch/relaxation [22]. Egr-1 mRNAlevels and by reducing the ability of Egr-1 to activate transcrip-is superinduced in the presence of cycloheximide [17].tion by impairing its DNA binding activity.The induction has been shown to occur mainly at thetranscriptional level [23–25]. The gene encodes a 75 to

Several studies suggest that nitric oxide (NO) exerts 80 kd nuclear phosphoprotein [26, 27]. It has been showncomplex regulatory actions in the kidney. These include to bind DNA at the consensus sequence GCGGGGGCGthe regulation of renal hemodynamics, radical-like cyto- in a zinc-dependent fashion through three zinc-finger

domains [27–29] and to activate transcription [27, 30–32].In recent studies employing antisense-oligonucleotideKey words: early growth response gene-1, mesangial cell mitogenesis,

hemodynamics, S-nitrosoglutathione. technology, we were able to demonstrate that Egr-1 in-duction is a necessary requirement for induction of mito-Received for publication January 28, 1999genesis in cultured rat mesangial cells (MCs). Antisenseand in revised form August 6, 1999

Accepted for publication August 23, 1999 oligonucleotides directed against Egr-1 potently inhib-ited Egr-1 mRNA and protein induction as well as serum-, 2000 by the International Society of Nephrology

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Rupprecht et al: NO inhibits MC growth 71

platelet-derived growth factor–, or endothelin-1–mediated RNA extraction and Northern blot analysisgrowth of MCs [33, 34]. Mesangial cells were grown in 10 cm dishes until sub-

In this study, we wanted to address the following is- confluency and were growth arrested for 72 hours in asues: (a) Because conflicting data exist in the literature on medium containing 0.4% FCS. After adequate stimula-the effect of different NO donors on the MC proliferative tion, cells were washed twice in phosphate-buffered sa-phenotype, we tested whether the NO donor S-nitroso- line (PBS), and total RNA was extracted by the methodglutathione (GSNO) interfered with MC growth. (b) of Chomczynski and Sacchi [36]. RNA was size fraction-Egr-1 expression was in our previous studies closely ated on a 1% agarose formaldehyde gel and transferredlinked to MC proliferation and antiproliferative agents onto Hybond nylon membrane (Amersham, Little Chal-like heparin or dexamethason inhibited mitogen-induced font, Buckinghamshire, UK). The Northern blot wasEgr-1 expression. We therefore tested whether GSNO baked at 808C for two hours, prehybridized with 5 3influenced transcript or protein levels of Egr-1. (c) Be- Denhardt’s, 5 3 SSC, 50% formamide, 50 mmol/Lcause some of the actions of NO are mediated through Na3PO4, 0.1% sodium dodecyl sulfate (SDS), 0.25 mg/mLthe liberation of cGMP, we investigated the impact of salmon sperm DNA at 398C for four hours. DNA hybrid-cGMP on MC proliferation and Egr-1 induction. (d) NO ization probes were labeled with [a-32P]-dCTP using ahas been shown to nitrosylate protein thiol groups and random primed labeling kit (Boehringer). The blots wereto thus destroy zinc-sulfur clusters in proteins such as hybridized in prehybridization solution containing 2 3metallothionein or the yeast transcriptional activator 106 cpm/mL of probe at 398C for 20 hours. The blot wasLAC9. We therefore analyzed whether GSNO directly washed twice for 15 minutes with 2 3 SSC containinginterfered with the DNA-binding activity or the tran- 0.1% SDS and then 30 minutes with 0.1 3 SSC containingscriptional-activating capability of Egr-1. 0.1% SDS. Blots were exposed to Kodak XAR-2 films

with intensifying screens at 2808C.METHODS

Protein extraction and Western blot analysisReagents

Mesangial cells were grown in 3.5 cm dishes until sub-The Hoechst dye 33258 and 8br-cGMP were from

confluency, growth arrested for 72 hours in medium con-Sigma Chemical Co. (Deisenhofen, Germany). Pyruvate

taining 0.4% FCS, and harvested after adequate stimula-and nicotinamide adenine dinucleotide (NADH) weretion in 100 mL RIPA solution [1% Triton X-100, 1%from Boehringer Mannheim (Mannheim, Germany).sodium deoxycholate, 0.1% SDS, 0.15 mol/L NaCl, 50mmol/L Tris-HCl, pH 7.2, 10 mmol/L ethylenediamine-GSNO synthesistetraacetic acid (EDTA), pH 7.2, 1 mmol/L phenylmeth-S-nitrosoglutathione was synthesized as described pre-ylsulfonyl fluoride (PMSF), leupeptin 2 mg/mL]. Proteinviously [35]. Briefly, glutathione was dissolved in 0.625concentration was determined using the Bradford assayN HCl at 48C to a final concentration of 625 mmol/L.(Bio-Rad, Hercules, CA, USA). Protein samples con-An equimolar amount of NaNO2 was added and thetaining 20 mg of total protein were denatured by boilingmixture was stirred for 40 minutes. After the additionfor five minutes and separated on a 7.5% denaturingof 2.5 volumes of acetone, stirring was continued forSDS-polyacrylamide gel electrophoresis (SDS-PAGE).another 20 minutes, followed by filtration of the precipi-After electrophoresis, the gels were electroblotted ontotate. GSNO was washed once with 80% acetone, twoNC membranes, and the transfer was controlled by Pon-times with 100% acetone, and finally, three times withceau-S staining. Blots were incubated in PBS containingdiethylether and was dried under vacuum. Freshly syn-0.1% Tween-20 and 5% nonfat dry milk powder to blockthesized GSNO was characterized by high-performanceunspecific binding, washed in PBS containing 0.1%liquid chromatography analysis and ultraviolet spectros-Tween-20, and incubated with the primary anti–Egr-1copy.antibody R5232-2 (1:3000), described in detail elsewhere

Mesangial cell isolation and culture [26]. Egr-1 was visualized with a secondary horseradishperoxidase-conjugated antirabbit IgG antibody using theGlomeruli from rat kidneys were isolated, and glomer-ECL system (Amersham).ular outgrowth and subsequent subculturing of MCs

were performed as previously described [16]. MCs wereDetermination of lactate dehydrogenase releasekept in Dulbecco’s modified Eagle’s medium (DMEM)

Following incubations, the medium of approximatelysupplemented with 10% heat-inactivated (508C, 30 min)2.5 3 105 MCs was collected, and cells were supple-fetal calf serum (FCS), 50 U/mL penicillin, 50 mg/mLmented with 0.2% (vol/vol) Triton-X 100 in PBS. Cellsstreptomycin, 2 mmol/L glutamin, 5 mg/mL insulin in awere lysed for four hours at 48C. A total of 500 mL95% air/5% CO2 humidified atmosphere at 378C. MCs

were used for experiments between passages 5 and 20. of reaction mix containing 50 mmol/L triethanolamine

Rupprecht et al: NO inhibits MC growth72

dissolved in 5 mmol/L EDTA, pH 7.6, 127 mmol/L pyr- Nuclear extract preparation and electrophoreticuvate, and 14 mmol/L NADH in 1% NaHCO3 was added mobility shift assayto 300 mL cell medium or lysed cells. Lactate dehydroge- Two 3 107 MCs were harvested on ice in PBS, resus-nase (LDH) activity was monitored by the oxidation of pended in 300 mL hypotonic buffer A (10 mmol/LNADH following the decrease in absorbance at 334 nm. HEPES, pH 7.9, 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1The percentage of LDH released was defined as the ratio mmol/L EGTA, 1 mmol/L dithiothreitol, 0.5 mmol/Lof LDH activity in the supernatant to the sum of the PMSF) and kept on ice for 15 minutes. Twenty microlitersLDH amount released plus the activity measured in the of 10% Nonidet P40 were added to lyze cells by vortexing.cell lysate.

Nuclei were collected by centrifugation and suspendedin 40 mL ice-cold buffer C (20 mmol/L HEPES, pH 7.9,Staining of nuclei with Hoechst dye 3325825% glycerol, 0.4 mol/L NaCl, 1 mmol/L EDTA, 1 mmol/LMesangial cells were seeded in 35 mm culture dishesEGTA). The suspension was vigorously agitated at 48Cand serum starved for three days in medium containingto extract nuclear proteins. Extracts were stored at0.4% FCS. MCs were then stimulated with GSNO 5002808C. Five micrograms nuclear extract were incubatedmmol/L in the presence or absence of FCS in a final con-with 1 ng radiolabeled Egr-1 binding site (59-AGCTTCcentration of 2% for 20 hours. Cells were then scrapedGCGGGGGCGAGG-39, 39-AGCGCCCCCGCTCCToff the culture dish and pelleted by centrifuging for twoTAA-59) or AP-1 binding site (59-AGCTAAAGCATminutes at 1000 g. The cell pellet was resuspended in 50GAGTCAGACAGCCT, 39-TTTCGTACTCAGTCTmL paraformaldehyde (3% in PBS) and transferred ontoGTCGGATCGA-59) in 25 mL binding buffer (10 mmol/La glass slide. After air drying for 10 to 15 minutes, cellsTris-HCl, pH 7.5, 50 mmol/L NaCl, 0.5 mmol/L EDTA,were stained with 100 mL of Hoechst dye 33258 (8 mg/0.5 mmol/L dithiothreitol, 1 mmol/L MgCl2, 4% glycerol,mL) for five minutes, washed three times for five minutes0.05 mg/mL poly dIdC). Nuclear extracts were incubatedin PBS, and mounted in Mowiol. Nuclei were visualizedfor 30 minutes at 48C in binding buffer. The labeledusing a fluorescence microscope with an excitation wave-binding site was added, and reactions were incubatedlength of 340 to 380 nm. Three hundred cells were

counted, and results are given as a percentage of cells for 30 minutes at 48C and separated on a 5% poly-with condensed nuclei compared with total cell number. acrylamide gel. The dried gel was exposed to x-ray films.

Determination of 3H-thymidine uptake Assay for chloramphenicol acetyltransferase activityMesangial cells were subcultured in 96-well plates in Growing MCs were transfected at a confluency of ap-

medium supplemented with 10% FCS until subconfluency proximately 60% with 1 mg chloramphenicol acetyltrans-and growth arrested for 72 hours in medium supple- ferase (CAT)-reporter plasmid pEBS3CAT and 0.2 mgmented with 0.4% FCS. Quiescent MCs were then exposed lacZ-expressing plasmid pCMVbgal using Superfectto fresh medium containing 0.4% FCS with or without transfection reagent (Qiagen, Dusseldorf, Germany).GSNO for six hours before stimulation with FCS in a final Sixteen hours after transfection cells were growth stimu-concentration of 2%. Cells were pulsed with 1 mCi/mL lated with FCS (10%) and treated with GSNO (500[3H-methyl]-thymidine (specific activity, 5 mCi/mmol/L;

mmol) and incubated for a further 24 hours. Instead ofICN, Costa Mesa, CA, USA) from 0 to 24 hours after FCS treatment, MCs were alternatively cotransfectedthe addition of FCS. The cells were washed twice with

with 4 mg of Egr-1-expression plasmid pRSVEgr-1 [31].PBS, lysed with distilled water, and harvested onto filters

Cells were harvested in cold PBS, resuspended in 100by an automated cell harvester (Dunn, Asbach, Ger-mL 0.25 mol/L Tris-HCl (pH 7.8) and freeze-thaw ex-many). Incorporated counts were measured by a liquidtracts were prepared. Fifty microliters of extract werescintillation counter (Beckmann, Fullerton, CA, USA).mixed with CAT-reaction mixture (50 mL 1 mol/L Tris-HCl, pH 7.8, 10 mL 14C-chloramphenicol, 0.1 mCi/mL,Determination of cell number20 mL 3.5 mg/mL acetyl coenzyme A). Reactions wereMesangial cells were seeded in 96-well plates at a den-incubated for 16 hours at 378C. Chloramphenicol andsity of 3 3 103 cells per well and were growth arrestedits acetylated derivates were extracted with ethylacetatefor 48 hours in medium supplemented with 0.4% FCSand separated by thin-layer chromatography. Plates wereand an additional 24 hours in medium without FCS.exposed to x-ray films, and radioactive spots were cutWhen cells were preincubated with GSNO, it was addedout and measured in a liquid scintillation counter to20 hours before serum stimulation. The cell number wascalculate percentage of acetylated forms of chloram-determined 48 hours after growth stimulation. Mono-phenicol. b-Galactosidase assay was performed as de-layers were washed twice in PBS. Cells were trypsinizedscribed [37], and activities were used to correct for differ-and transferred into 10 mL of Isoton for counting in a

Coulter counter (Coulter, Luton, England). ences in transfection efficiency.


Statistical analysis tion periods of up to 48 hours. At a concentration of 2mmol/L, GSNO induced LDH release when preincu-Statistical analysis was performed using Student’s t-bated with MCs for 24 hours or longer. Ethanol servedtest for unpaired samples.as a positive control.

It has been reported that exogenously added NO do-RESULTS nors, like GSNO, can induce apoptosis in serum-starved

cultured rat MCs [39, 40]. To address the question ofGSNO is a potent inhibitor of serum-inducedwhether the reduction in the serum-stimulated increasemesangial cell growthin MC number by GSNO was in part due to apoptoticBecause conflicting data exist in the literature concern-processes, we looked for condensation and fragmenta-ing the effects of NO on MC growth using various NOtion of nuclei by staining with the Hoechst dye 33258.donors, we tested whether the potent NO-donor GSNOAs reported previously by others [39, 40], we could dem-affected serum-induced MC proliferation. Synthesis ofonstrate that approximately 20% of serum-starved MCsDNA, assessed by the incorporation of 3H-thymidine,were rendered apoptotic by incubation with 500 mmol/Land a serum-induced increase in total cell number wereGSNO. GSNO had, however, only marginal effects onmonitored after preincubation of MCs with increasingthe induction of apoptosis when added to serum-stimu-doses of GSNO. GSNO led to a dose-dependent inhibi-lated MCs (Fig. 2B). We therefore conclude that GSNOtion of serum-stimulated 3H-thymidine incorporationin the concentrations used did not induce necrotic orinto MCs (Fig. 1A) and to a prominent suppression ofapoptotic cell death of serum-stimulated MCs. The de-serum-induced increase in cell number (inhibition of se-crease in 3H-thymidine uptake or cell number is thereforerum-induced increase in cell number: GSNO 50 mmol/L,due to specific antiproliferative effects of GSNO.

219 6 23%; GSNO 100 mmol/L, 247 6 20%; GSNOTo further eliminate the possibility that the glutathion1 mmol/L, 284 6 23%; data were obtained from four

(GSH) component of GSNO per se was responsible forindependent experiments each consisting of quintupli-the antiproliferative effects observed, MCs were incu-

cate samples; a representative experiment is shown inbated with equimolar amounts of either GSH alone,

Fig. 1B). Because it has been demonstrated that NO canGSNO that was kept at room temperature for 24 hours,

induce detachment of MCs cultured in the absence ofor fresh GSNO. As can be seen in Figure 3, GSH had

serum [38], cells in the supernatant were counted in no effect on 3H-thymidine uptake. Twenty-four-hour oldaddition to cells adhering to the culture dish. As indi- GSNO had significant but weak growth inhibitory ac-cated in Figure 1B, GSNO did not induce detachment tions, whereas fresh GSNO retained its full antiprolifera-of serum-stimulated MCs. tive activity.

We further addressed the question of whether otherNO donors like spermine-NO (SpNO) or S-nitroso-N- Effects of GSNO on Egr-1 mRNA and protein levelsacetylpenicillamine (SNAP) would affect serum-stim- in serum-stimulated mesangial cellsulated (FCS 2%) MC growth. SpNO (25 mmol/L, 50 In previous studies, we demonstrated that the induc-mmol/L, and 100 mmol/L) led to a dose-dependent inhibition of Egr-1 was necessary for the induction of MCtion of MC thymidine uptake (213 6 10%, 236 6 9%, growth [33, 34]. Furthermore, we could show that anti-and 288 6 57%). Higher concentrations appeared to be proliferative substances such as heparin or dexametha-toxic, decreasing thymidine uptake below control levels. son inhibited the mitogen-induced increase in Egr-1In contrast, SNAP exhibited a less pronounced inhibitory mRNA and protein in MCs [33]. The degree of inhibitioneffect on FCS-induced MC growth (growth inhibition at of Egr-1 correlated with the degree of inhibition of25 mmol/L, 215%; at 100 mmol/L, 244%). Data were growth. To test whether the antimitogenic effects ofobtained from four independent experiments each con- GSNO were due to a down-regulation of Egr-1, tran-sisting of quintuplicate samples (SpNO). For SNAP, re- script and protein levels of Egr-1 were measured in MCssults given are means of two independent experiments preincubated with GSNO for six hours and stimulatedconsisting of quintuplicate samples (data not shown). with FCS 2% for one or two hours, respectively. Incuba-

tion of MCs with FCS for one hour led to a prominentGSNO does not induce toxic or apoptotic death of increase in Egr-1 mRNA. Preincubation with increasingserum-stimulated mesangial cells doses of GSNO had a biphasic effect on Egr-1 mRNA

To exclude toxicity of the GSNO concentrations used, levels. At concentrations from 50 to 200 mmol/L an inhi-we monitored the release of the cytoplasmic enzyme bition of serum-induced Egr-1 mRNA levels was ob-LDH from cultured MCs during varying preincubation served. The average inhibition of Egr-1 mRNA at 200periods with GSNO. As indicated in Figure 2A, GSNO mmol/L GSNO was 29 6 14% in three independent ex-did not induce the release of LDH over control cells in periments. In contrast, at higher concentrations of

GSNO, Egr-1 mRNA returned to, and in some experi-concentrations of up to 1 mmol/L and during preincuba-


Fig. 1. S-nitrosogluthathione (GSNO) inhib-its serum-induced mesangial cell (MC) growth.(A) 3H-thymidine uptake was measured be-tween 0 and 24 hours after fetal calf serum(FCS) stimulation (2%) of MCs growth ar-rested for 72 hours. GSNO was added in theindicated concentrations six hours before FCSaddition. (B) The cell number was determined48 hours after FCS stimulation (2%) of MCsgrowth arrested for 72 hours. GSNO wasadded in increasing concentrations 20 hoursbefore stimulation with FCS. Cells attachedto the culture plates (j) as well as cells in thesupernatant ( ) were counted. Data are givenas mean 6 sd of quadruplicate determina-tions. *P , 0.05; ***P , 0.001 compared withserum-stimulated cells.

ments even exceeded, the levels reached with FCS-stimu- the stable cGMP-analogue, 8br-cGMP, on serum-inducedMC growth and Egr-1 induction. 8br-cGMP led to a dose-lation alone (Fig. 4). Surprisingly, this enhanced Egr-1

mRNA expression at high GSNO concentrations was not dependent inhibition of serum-stimulated MC growth.At concentrations of 100 mmol/L and 1 mmol/L, MCtranslated into elevated protein levels. GSNO induced a

dose-dependent suppression of serum-stimulated Egr-1 proliferation, as assessed by 3H-thymidine uptake, wassuppressed by 15 6 4% and 35 6 12%, respectivelyprotein levels. Inhibition was 24 6 32% at 200 mmol/L

GSNO and 40 6 24% at 1 mmol/L GSNO in four inde- (data from 3 independent experiments each consistingof quintuplicate samples; a representative experiment ispendent experiments (a representative experiment is

shown in Fig. 5). A similar inhibition was observed when shown in Fig. 6A). At the same time, 8br-cGMP inhibitedthe induction of Egr-1 protein by FCS by 15 6 11%spermine NO was used as a donor of NO.(100 mmol/L) or 33 6 13% (1 mmol/L; data from 3

Effect of the stable cGMP analogue 8br-cGMP on independent experiments; a representative experimentmesangial cell proliferation and Egr-1 induction is shown in Fig. 6B). The decrease in serum-stimulated

Egr-1 protein levels therefore paralleled the decrease inBecause at least some of the actions of NO are medi-ated by generation of cGMP, we tested for the effects of 3H-thymidine uptake elicited by 8br-cGMP.


Fig. 2. GSNO does not induce toxic or apoptotic death of serum-induced MCs. (A) LDH-release from serum-stimulated MCs preincubated for8 (j, ), 24 (j, ), or 48 (m) hours with increasing concentrations of GSNO was determined as described in the Methods section. Thepercentage of LDH released was determined as the ratio of LDH activity in the supernatant to the total LDH activity. GSNO had no toxic effectsup to a concentration of 1 mmol/L. Ten percent ethanol served as a positive control. (B) The proportion of apoptotic cells was determined bystaining paraformaldehyde fixed cells with Hoechst dye 33258. Serum-starved control cells or serum-stimulated cells were either left untreated orwere incubated with GSNO (500 mmol/L) for 20 hours. An increase in apoptotic cells was only observed in serum-starved MCs. Results are givenas percentage of cells with condensed nuclei compared with total cell number.

ity is intricately linked to MC growth [33, 34], we testedwhether GSNO exerted additional effects on DNA bind-ing or transcriptional activating capacity of Egr-1. First,we tested the influence of NO on the ability of Egr-1 tobind DNA. As shown in Figure 7A, serum induced Egr-1binding to a DNA fragment containing an Egr-1 consen-sus sequence (closed arrow). The specificity of this bind-ing was demonstrated by the addition of an Egr-1 anti-body (supershift, open arrow) or the addition of an excessof cold competitor to the binding reaction. GSNO, whenadded to the binding reaction, drastically inhibited the

Fig. 3. GSNO but not glutathion (GSH) inhibits serum-stimulated 3H-binding of FCS-induced Egr-1 protein to its binding sitethymidine uptake by MCs. 3H-thymidine uptake was measured between

0 and 24 hours after FCS stimulation (2%) of MCs that have been (Fig. 7B). We also asked whether GSH (500 mmol/L) pergrowth arrested for 72 hours. Fresh GSNO, GSNO that was kept at room se would interfere with Egr-1 binding when added to thetemperature for 24 hours, or GSH were each added at concentrations of

binding reaction, but no inhibiting effect was observed500 mmol/L six hours before serum stimulation. ***P , 0.001 comparedwith serum-stimulated MCs. NS, not significant. (data not shown). To test whether the presence of GSNO

in the binding reaction would inhibit the binding of othertranscription factors, we added 500 mmol/L GSNO to an

Effects of GSNO on Egr-1 DNA binding capability electrophoretic mobility shift assay (EMSA) for activatorand transcriptional activating capacity protein-1 (AP-1; Fig. 7C). GSNO led to an enhanced

binding of AP-1 to its binding sequence. In the experimentThe growth inhibitory effects of GSNO (84% inhibi-shown in Figure 8, GSNO was added to the cells beforetion of serum-stimulated increase in cell number at 1serum stimulation and preparation of nuclear extracts.mmol/L GSNO) were far more pronounced than theThere was a dose-dependent decrease in Egr-1 bindingeffects of 8br-cGMP on MC growth (35% inhibition atactivity, and near complete inhibition to levels seen in1 mmol/L) or the effects of GSNO on Egr-1 protein levelscontrols was observed at 500 mmol/L GSNO.(40% inhibition at 1 mmol/L GSNO). Therefore, mecha-

To demonstrate that GSNO not only inhibited DNAnisms other than liberation of cGMP or inhibition ofEgr-1 protein levels must be active. Because Egr-1 activ- binding but also transcriptional activation by Egr-1, CAT


Fig. 4. Biphasic effect of increasing concen-trations of GSNO on serum-induced earlygrowth response gene-1 (Egr-1) mRNA ex-pression. MCs were growth arrested for 72hours in medium containing 0.4% FCS. Afterpreincubation for six hours with increasingconcentrations of GSNO, cells were serumstimulated (2%) for 60 minutes. RNA was ex-tracted and 20 mg total RNA per lane were sizefractionated on a 1% agarose gel. Blots wereprobed with cDNAs for Egr-1 and GAPDH,radiolabeled by random priming.

Fig. 5. GSNO inhibits serum-induced Egr-1protein expression. MCs were growth arrestedfor 72 hours in medium containing 0.4% FCS.After preincubation with GSNO or SpNO forsix hours, cells were stimulated with FCS(2%), and two hours later, protein extractswere prepared. Twenty micrograms of proteinper lane were separated on a 7.5% denaturingSDS-PAGE gel. Blots were probed with theprimary Egr-1 antibody R5232-2 (1:3000).Egr-1 was visualized with a secondary horse-radish peroxidase-conjugated antirabbit IgGantibody using the ECL system (upper panel).Expression was quantitated using video densi-tometry (lower panel).


Fig. 6. 8br-cGMP inhibits serum-induced 3H-thymidine uptake (A) and Egr-1 protein expression (B) in cultured rat MCs. Serum-starved MCswere preincubated for three hours with the indicated concentrations of 8br-cGMP and stimulated with FCS (2%). 3H-thymidine uptake wasmeasured 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 trans- in glomerular filtration pressure [1, 42]. These effectsfected with 1 mg of a CAT-reporter construct bearing 3 are predominantly mediated by cGMP. Increasing glo-Egr-1 binding sites (pEBS3CAT). MCs were then stimu- merular NO production by l-arginine substitution haslated with FCS in the presence or absence of 500 mmol/L been shown to be protective in various renal diseasesGSNO. GSNO led to a marked inhibition of CAT activ- models, like 5/6 nephrectomy or salt-sensitive hyperten-ity elicited by stimulation of MCs with 10% FCS (reduc- sion [42, 43]. The protective effects are most likely duetion from 71% to 21% acetylated chloramphenicol). In to a decrease in glomerular capillary pressure. NO isFigure 9B, Egr-1 expression was achieved by cotransfec- also synthesized by brain NO synthase (bNOS) in thetion of a plasmid expressing Egr-1 under the control of macula densa and regulates secretion of renin [1, 44].the constitutively active RSV promoter (pRSVEgr-1). Thus, NO has important functions in the autoregulationGSNO also led to a near complete inhibition of CAT of renal perfusion.activity (reduction from 79% to 9% acetylated chloram- Another source of glomerular NO is mesangial cellsphenicol). In Figure 9C, GSNO (500 mmol/L) was added and infiltrated macrophages inducible NO synthaseto MCs transfected with 0.5 mg or 1 mg of pRR55, which (iNOS). NO production by iNOS is not only importantleads to constitutive CAT expression. No reduction of for defense mechanisms against infectious pathogens butacetylated chloramphenicol by treatment with GSNO plays pathophysiologically important roles in immuno-was detectable, demonstrating that GSNO did not ex- logic diseases, such as in glomerulonephritis [45, 46].hibit unspecific effects on CAT activity. Cattell, Cook, and Moncada demonstrated that glomer-

uli isolated from nephritic rats secreted large amountsDISCUSSION of nitrite into the culture supernatant [47]. Increased

NO synthesis was shown in various glomerulonephritisNitric oxide has multiple actions in the kidney. Themodels, such as rat nephrotoxic serum nephritis [47],most prominent is regulation of glomerular hemodynam-Heymann nephritis [48], anti-Thy1.1 nephritis [49, 50],ics. NO potently antagonizes the vasoconstrictor effectsand in MRL-lpr/lpr mice [51]. Infiltrating macrophagesof angiotensin II at the efferent arteriole [41]. Synthesisseem to be the main source of iNOS expression [48, 49].of NO by eNOS (endothelial NO synthase) in glomerularDifferent effects of NO on the course of these nephritisendothelial cells leads to increases in effective renal

plasma flow and glomerular filtration rate and decreases models have been described.


Fig. 7. DNA-binding of Egr-1 is inhibited by GSNO added to the binding reaction. Nuclear extracts were prepared as described in the Methodssection from growth arrested MCs or from MCs stimulated with FCS for two hours. Five micrograms of nuclear extract were incubated with 1 ngradiolabeled 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 100-fold molar excess of cold competitor DNA was added to the binding reaction. Open arrow indicates supershift of Egr-1–DNA-complexes byaddition of Egr-1 antibody. (B) GSNO was added to the binding reaction to a final concentration of 500 mmol/L. Closed arrow indicates specificDNA-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) wasadded directly to the binding reaction.

In anti-Thy1.1 nephritis, an application of the NOS- mice) [51]. In contrast, NOS inhibition led to increasedproteinuria and aggravated disease in rat nephrotoxicinhibitor L-NNMA (NG-monomethyl-l-arginine) shortly

before induction of nephritis prevented mesangiolysis serum nephritis [52].Because many different pathomechanic processes areand proteinuria [50]. Similar, albeit weaker effects were

obtained with a diet poor in l-arginine [50]. Positive at play during development of glomerulonephritis, suchas proliferation of intrinsic glomerular cells, infiltrationeffects of systemic NO inhibition have also been described

in the mouse model of lupus nephritis (MRL-lpr/lpr and proliferation of inflammatory cells, altered matrix


in the presence of serum. Under low serum culture condi-tions (0.5% FCS), GSNO also induced apoptosis, as pre-viously described [39, 40]. We obtained similar resultson proliferation and Egr-1 protein levels with SpNO, adifferent NO donor. SNAP did moderately inhibit se-rum-induced MC growth. Because some of the effectsof NO are mediated through cGMP, we tested the anti-proliferative effects of 8br-cGMP on MCs. 8br-cGMPhad, as described by others [54, 55], weak but significantgrowth inhibitory actions at higher concentrations. Theactions of cGMP could, however, not entirely accountfor the strong growth-inhibiting effects of GSNO. There-fore, other mechanisms must be at play. We were ableto identify two such mechanisms when investigating theeffects of GSNO on the transcription factor and immedi-ate early gene Egr-1. We chose to investigate Egr-1 be-cause we had previously shown that its induction wascritical for MC proliferation to occur in response to vari-ous mitogens [33, 34]. Antisense oligonucleotides againstEgr-1 inhibited the induction of Egr-1 mRNA as well asEgr-1 protein and nearly completely abolished serumplatelet-derived growth factor or endothelin-1–inducedMC proliferation.

GSNO at lower concentrations inhibited serum-inducedEgr-1 mRNA induction, and at higher concentrations,Egr-1 mRNA levels rose again to serum-stimulated con-trol levels. This rise in mRNA was, however, not trans-lated into a rise in Egr-1 protein. Instead, Egr-1 proteinlevels were significantly suppressed with increasing dosesof GSNO. Therefore, GSNO induced a translationalblockade, preventing Egr-1 accumulation and therebyinhibiting MC growth. A similar translational controlmechanism has been postulated in Sol8 mouse musclecells by Maass et al [56]. The authors could show thatonly agents that induced proliferation led to a rise inEgr-1 mRNA and Egr-1 protein, whereas stimuli that

Fig. 8. Prestimulation of MCs with GSNO inhibits binding of serum-induced differentiation up-regulated Egr-1 mRNA butinduced Egr-1 protein to its DNA-binding site. Growth arrested MCs

were preincubated with increasing doses of GSNO for one hour before led to a translational blockade preventing protein accu-stimulation with FCS (4%) for two hours. Nuclear extracts were pre- mulation. A second cGMP-independent mechanism bypared as described in the Methods section. Five micrograms nuclear

which NO inhibits MC growth is by affecting bindingextract were incubated with 1 ng radiolabeled Egr-1 binding site for 30minutes at 48C. Specificity of binding was demonstrated by addition of properties of the transcription factor Egr-1. Binding ofEgr-1 antibody or a 100-fold molar excess of cold competitor DNA to Egr-1 present in nuclear extracts from serum-stimulatedthe binding reaction.

MCs to its DNA-binding sequence was inhibited by addi-tion of GSNO to the binding reaction, but not by glutath-ion per se. To test for nonspecific down-regulation ofbinding activities under these stringent conditions, wemetabolism or apoptotic events, NO might have different

effects, depending on the predominant feature in the performed a gelshift for another transcription factor,AP-1. Here we found that GSNO enhanced the bindingrespective models. We investigated in this study the ef-

fects of NO on one aspect of many glomerular diseases, of the factor to its DNA binding site, indicating thatGSNO does not unspecifically down-regulate DNAthat is, MC proliferation, and tried to elucidate signaling

pathways involved. binding of transcription factors. Similar results have beenreported previously in macrophages [57]. In this experi-We found that the NO-donor GSNO was a potent

inhibitor of MC growth and could confirm studies in the mental setting, NO might have directly altered the DNAcontaining the Egr-1 consensus site, thereby affectingliterature using other NO donors [53, 54]. No toxic effects

or effects on apoptosis were observed on MCs cultured binding [58, 59]. Therefore, we also performed EMSA


Fig. 9. GSNO reduces transcriptional activation of the chloramphenicol acetyltransferase (CAT) reporter gene by Egr-1. (A) MCs were transientlytransfected with 1 mg CAT reporter plasmid (pEBS3CAT) containing 3 Egr-1 binding sites in front of the CAT gene using Superfect transfectionreagent. Sixteen hours after transfection, cells were stimulated with FCS (2 or 10%) in the absence or presence of GSNO (500 mmol/L) and wereincubated for another 24 hours. As a positive control, MCs were transfected with 1 mg plasmid pRR55 constitutively expressing CAT under thecontrol of the human CMV promotor. Cell extracts were prepared and CAT assays performed as described in the Methods section. Chloramphenicoland its acetylated derivates were separated by thin layer chromatography (upper panel). Plates were exposed to x-ray films. Radioactive spotswere excised and measured in a scintillation counter. CAT activity was expressed as the percentage of acetylated forms of chloramphenicol relativeto the total amount of chloramphenicol. (B) MCs were cotransfected with 1 mg pEBS3CAT and 4 or 10 mg of the Egr-1 expression plasmidpRSVEgr-1. Sixteen hours after transfection cells were incubated in the absence or presence of GSNO (500 mmol/L) for an additional 24 hoursbefore preparation of cell extracts. (C) MCs were transfected with 0.5 or 1 mg of the plasmid pRR55, which constitutively expresses CAT underthe 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 pre- MCs by cGMP-dependent as well as, more importantly,by cGMP-independent pathways that include interfer-treated with GSNO and obtained similar results. With

GSNO treatment, serum-induced binding of Egr-1 to its ence with the zinc-finger transcription factor Egr-1. Glo-merular NO release or application of NO-donors at theconsensus sequence was almost completely suppressed

to control levels. Only part of this suppression can be appropriate time might prove beneficial in mesangio-proliferative glomerular disorders.due to reduction in Egr-1 protein levels because GSNO

led to a maximum 40% suppression of Egr-1 proteinACKNOWLEDGMENTSexpression. In CAT assays, we demonstrated that re-

duced DNA binding was also translated into a reduced The work was supported by a grant by the Deutsche Forschungsgem-einschaft to H.D. Rupprecht (Klinische Forschergruppe, Teilprojekttranscriptional activation by Egr-1. Whereas GSNO dra-IV). We gratefully acknowledge the technical assistance of Ms. Katjamatically reduced the transcriptional activation of CATBruch and Ms. Isabella Kolberg.

via Egr-1, no effect was seen on the CMV promoter-Reprint requests to Harald D. Rupprecht, M.D., Nephrologischedriven CAT expression.

Forschungslabors, Medizinische Klinik IV, Universitat Erlangen-Nurn-A potential mechanism by which NO inhibits DNAberg, Loschgestr. 8, 91054 Erlangen, Germany.

binding of Egr-1 is by interfering with the zinc-fingerdomains that establish the DNA contacts [29]. It has REFERENCESbeen described that NO leads to metal ion release from

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