Oxygen radicals as second messengers for expression of the monocyte chemoattractant protein, JE/MCP 1, and the monocyte colony stimulating factor, CSF 1, in response to tumor necrosis factor alpha and immunoglobulin G Evidence for involvement of reduced

Oxygen radicals as second messengers for

expression of the monocyte chemoattractant

protein, JE/MCP-1, and the monocyte

colony-stimulating factor, CSF-1, in response to tumor

necrosis factor-alpha and immunoglobulin G.

Evidence for involvement of reduced

nicotinamide adenine dinucleotide phosphate

(NADPH)-dependent oxidase.

J A Satriano, … , Z Shan, D Schlondorff

J Clin Invest. 1993;92(3):1564-1571. https://doi.org/10.1172/JCI116737.

The potential involvement of reactive oxygen species in the expression of genes involved in immune response was examined in mesangial cells. Tumor necrosis factor (TNF-alpha) and aggregated (aggr.) IgG increased mRNA levels for the monocyte chemoattractant protein, JE/MCP-1, and the colony-stimulating factor, CSF-1. Scavengers for free radicals such as di- and tetra-methylthiourea (DMTU and TMTU) attenuated the increase in mRNA levels in response to TNF-alpha and aggr. IgG. Generation of superoxide anion by xanthine oxidase and hypoxanthine increased mRNA levels of these genes, but exogenous H2O2 did not. Addition of NADPH to activate a membrane-bound NADPH-oxidase generated superoxide and caused a dose-dependent increase in mRNA levels and further enhanced the stimulation by TNF-alpha or aggr. IgG. An inhibitor of NADPH-dependent oxidase 4'-hydroxy-3'-methoxy-acetophenone attenuated the rise in mRNA levels in response to TNF-alpha and aggr. IgG. By nuclear run-on experiments TNF-TNF-alpha, aggr. IgG and NADPH increased the transcription rates for JE/MCP-1 and CSF-1, effects inhibited by TMTU. We conclude that generation of reactive oxygen species, possibly by NADPH-dependent

oxidase, are involved in the induction of the JE/MCP-1 and CSF-1 genes by TNF-alpha and

Research Article

Find the latest version:

Rapid

Publication

Oxygen Radicals

as

Second

Messengers

for

Expression

of

the

Monocyte

Chemoattractant Protein, JE/MCP-1,

and the

Monocyte

Colony-stimulating

Factor, CSF-1,

in

Response

to

Tumor Necrosis

Factor-a and

Immunoglobulin

G

Evidence for Involvement of Reduced NicotinamideAdenine Dinucleotide Phosphate

(NADPH)-dependent

Oxidase

JosephA.Satriano,MarcShuldiner,KazuhikoHora,YunXing,ZiheShan,andDetlefSchiondorff

Department ofMedicine,Albert EinsteinCollegeofMedicine, Bronx,New York 10461

Abstract

The potential involvementof reactive oxygen species in the

expressionof genes involved in immune responsewasexamined in

mesangial

cells.Tumor necrosis factor(TNF-a)and aggre-gated (aggr.) IgG increased mRNA levels for the monocyte chemoattactant protein,JE/MCP-1,and the colony-stimulat-ingfactor, CSF-1.Scavengersforfree radicals suchasdi- and

tetra-methylthiourea(DMTUandTMTU)attenuated the

in-creasein mRNA levels in responsetoTNF-a and aggr.IgG. Generation ofsuperoxide anion by xanthineoxidase and hypo-xanthine increasedmRNAlevelsofthese genes, but exogenous

H202 didnot. Addition ofNADPH toactivate a membrane-bound NADPH-oxidase generated

superoxide

and caused a

dose-dependent increaseinmRNAlevels andfurtherenhanced the stimulation by TNF-a or aggr.

IgG.

An inhibitor of NADPH-dependent oxidase

4'-hydroxy-3'-methoxy-aceto-phenone attenuated the rise in mRNA levels in response to

TNF-aand aggr.IgG. Bynuclearrun-on

experiments

TNF-a,

aggr.IgG and NADPH increased the

transcription

rates for

JE/MCP-1 andCSF-1, effects inhibited byTMTU. We

con-clude thatgeneration ofreactive oxygen

species,

possibly

by NADPH-dependent

oxidase,

are involved in the induction of the JE/MCP-1 and CSF-1 genes byTNF-a and

IgG

com-plexes.Theconcerted

expression

of

leukocyte-directed

cyto-kines representsageneral responsetotissue

injury.

(J. Clin. Invest.1993. 92:1564-1571.) Keywords: gene

regulation.

kid-ney.

mesangialcell

Introduction

Cytokines play

a

major

role intissue

injury

and

repair.

These processes alsoinvolve the

participation

of monocyte-macro-phages. Recently a number of

monocyte-specific cytokines

have beendescribedandincludethe monocyte

colony-stimulat-AddressreprintrequeststoDr.DetlefSchlondorff,UllmannBuilding,

Room617, AlbertEinstein College ofMedicine, 1300 MorrisPark Avenue,Bronx, NY 10461.

Receivedfor publication8September1992andinrevisedform 11

May 1993.

ingfactor(CSF- 1),1 and the monocyte chemoattractant pro-tein(MCP-l1) (1-4).MCP-l was identified as the product of a genebelongingtothe small, inducible cytokine family, known in the murine system as the JE gene (5-9). CSF-l is a cytokine required for the proliferation, maturation, activation, and sur-vivalofmonocytes ( 1, 10). Expression of the JE/MCP-l and

CSF-l genes can be rapidlyinduced by a number of agents,

including LPS, PDGF, TNF-a, I1-1, and

IFN-,y

(2, 4, 6, 1

1-21 ). We recently described that occupancy of Fc receptors by IgG aggregates resulted in rapid expression of mRNA for and secretion of JE/MCP-I and CSF-l in cultured mouse

mesan-gialcells(22).Mesangial cells (MC) represent modified vascu-lar smooth muscle cells that fulfill the function of specialized pericytes within the glomerulus of the kidney (23). MC also playarole in glomerular injury, where influx and activation of

monocyte-macrophages isimportant and local generation of MCP-l and CSF-I could contribute to this phenomenon (22,

24, 25).

The cellular signaling mechanisms for induction of JE/ MCP-l and CSF-l remain to be fully defined. While we have described that cAMP suppresses both basal and stimulated

ex-pression of both CSF-l and JE/MCP-1 (26), the second mes-senger systems for the upregulation of these genes remain un-clear. Recently it has been proposed that oxygen radicals may

serve as second messenger system for gene activation, espe-cially for genes under theregulation ofthetranscriptionfactor NF-KB(27-29). The potential for generation of oxygen radi-calsby membrane-boundNADPH-dependentoxidase systems maybe present in many cell types, among them MC (30, 31). Both TNF-a and IgG complexes are known stimulators of reactive oxygenspecies generationin MC (32-34). We there-fore examined whether generation of free radicals could

repre-sent asecond messenger system forinductionof JE/MCP-1 and CSF-1 in mouse MC. Our results show that addition of scavengers for free radicalscan attenuateexpressionof mRNA levels for these genes under basal conditions and after stimula-tion with TNF-a or IgG aggregates. In contrast, exogenous NADPHenhances basal and stimulated mRNA levels forJE/

1.Abbreviationsused inthispaper: aggr., aggregated (IgG); CSF, col-ony-stimulating factor; DMTU, dimethyl-thiourea; HMAP,

4'-hy-droxy-3'-methoxy-acetophenone; MC, mesangialcell; MCP, mono-cyte chemoattractant protein; NAC, N-acetyl-L-cysteine; PDTC, pyrrolidone-thiocarbamate; TES, N-Tris (hydroxymethyl) methyl-2-aminoethanesulfonicacid,pH7.4;TMTU,tetramethylthiourea.

J.Clin. Invest.

© TheAmericanSociety for Clinical Investigation, Inc. 0021-9738/93/09/1564/08 $2.00

MCP-1 and CSF-l effects attenuated by inhibition of NADPH-dependent oxidase. Thesechanges in mRNA levels

are atleast in part due to alteredtranscriptional rates. Thus reactive oxygen species generation, in part

NADPH-depen-dent, may represent a second messenger system for induction of JE/MCP-l and CSF-l expression notonly in response to TNF-a but also to Fcreceptor occupancybyIgG. These find-ings providefurther evidencefor thehypothesisthatfree oxy-genradicals can serve as second messenger systems,findings that may be ofsignificanceforcytokine induction

during

tissue

injury.

Methods

Materials.[32p]dCTP,wasobtainedfrom NewEngland Nuclear, Bos-ton,MA. RPMI 1640medium, penicillin (0.66 mg/ml)/streptomycin

(60mg/ml)solution, FCS,and Dulbecco's PBSwerepurchasedfrom FlowLaboratoriesInc.,McLean,VA.Nu-Serumwasfrom Collabora-tive Research Inc., Bedford, MA. Recombinant mouse TNF-a,

NADPH, NADP, andmultiprime DNA labelingsystemswere pur-chased from Boehringer Mannheim, Indianapolis, IN. Mouse IgG

(Cappel OrganonTeknikaCorp.,WestChester,PA)wasaggregatedby

heating to630Cfor 30 min. Insoluble aggregates wereremoved by centrifugation at 1l,000gfor 5 min and the supernate servedas ag-gregated (aggr.) IgG (22). 4'-Hydroxy-3'-methoxy-acetophenone (HMAP) was purchased from AldrichChemical Co., Milwaukee,WI. All otherchemicalswerefrom Sigma Chemical Co.,St.Louis, MO,

unlessotherwise specified.The cDNAprobe forthe murine CSF-Iwas

kindly provided byDr.Martha Ladnerof Chiron Corp., Emeryville,

CA(35),thatfor JE/MCP-1 byDr.J.Stiles (7).

Culture ofmouse MC. Mouse MCtransformedwith nonreplicat-ing, non-capsid formingSV-40virus (strainRh911)werekindly pro-vided by Dr. E. Neilson, University ofPennsylvania, Philadelphia.

These cells have been characterized (36).Inpreliminary experiments

weestablishedthat the responsesofthesetransformedmouseMC to TNF-a and IgG in terms ofexpressionof JE/MCP-IandCSF- I were comparabletoprimarycultures of mouseMC,used in ourprevious

experiments(22,26).The mMCweremaintainedin DMEmedium with 1%Pen-Strepand2.5% FCS withtwiceweeklyfeedingsin a 95%

air, 5% CO2 atmosphere. Cellswerepassagedatweeklyintervals by harvestingwith 0.25%trypsin, I mM EDTA solution.

Measurementofsuperoxidegeneration. For measurementof super-oxidegeneration,MCwereculturedin 96-wellplatesandsuperoxide generationwasdetermined asdescribedby Pick andMizel(37). In

brief,culturemediawereremoved and replacedby Earle'sbalanced

saltsolution (0.1 ml) containing ferrichromec(50;1M)with or

with-outtheexperimentalagentsindicatedin Results. Parallel wellsreceived

inaddition superoxide dismutase(300 U/ml). Appropriateblanks,

i.e.,wellswithoutcellsbutcontainingthebuffer withorwithout experi-mental agents, wererunfor each assay. Absorbancewasreadat550-nm wavelength in a Multiskan reader (FlowLaboratories) after0, 15, and 30 minof incubation and,aftersubtractionof the respective blanks, wereconverted tosuperoxidegenerated asdescribed(37).

Northern blots. Total RNA was extracted from cultured cells by the

methodofChomczynski and Sacchi (38). Aliquots (20 ug) of total RNA were treated withglyoxal/DMSO,electrophoresed in 1.0% aga-rosegel along with RNA size standards (GIBCO/BRL, Gaithersburg, MD), and then transferred to nylon filters (gene screen, New England Nuclear, Boston,MA)(39). The cDNA probes were used for hybridiza-tion after [32p] dCTP labeling by random oligonucleotide priming

(40).Thefilterswerewashed toafinalhigh stringencyof 0. Ix SSPE, 1.0% SDS for2x 30 min at 65°C. Autoradiographywas performed withanintensifyingscreen at -70°C for appropriate time periods. The size of the respective RNA wasidentifiedby comparison with the ethi-dium bromide-stainedRNAstandards.

Nuclear

runofftranscription

assay.ConfluentMC(2XI0'cells per

condition)

wereincubatedwithexperimentalagentsat370Cfor 1 h.

Cultureswerethen washed with ice-cold PBS and the nucleiprepared

by

lysis

in 0.5% NP-40

lysis

buffer_(41).Nucleiwerecollectedby

cen-trifugation

and stored in 50 mMTris, pH 8.3,40% glycerol, 5 mM

MgCl2,

0.1 mMEDTAat-150'C._{Upon thawing,}nucleiwere

resus-pendedandincubated inan_equalvolume of reaction buffercontaining

300 mM KCI, 0.5 mM each of_ATP,GTP,andCTP, and 100

_MCi

(a-32P]UTP (760

Ci/mmol, NewEnglandNuclear) at30Cfor 30 min. The reactionwasterminated_bythe addition of 600

MlI

ofRNase free DNase 1

(200

U/ml),for 5 minat30'C,followed_by200MlSDS/ Tris buffer_{(5% SDS,}0.5 M_{Tris-Cl, pH}7.4,0.125MEDTA)and101

at20mg/mlProteinase K for 30 minat_420C.Thesampleswerethen extracted with_{phenol/chloroform/isoamyl}alcohol(25:24:1 )and

pre-cipitatedonice in 5%TCA/30mM sodiumpyrophosphatesolution. The

_precipitates

were_trappedonaMillipore-typeHA(0.45 MUm)filter and treated with 200 U RNase free DNase I(BoehringerMannheim)at

370C

for30 min. The reactionwas_{quenchedwith 45 gl of EDTA(0.5}

M),and68Mlof 20%SDS,eluted from the filtersat65°C anddigested with_proteinaseK_(4.5Ml;20mg/ml)for 30min. Afterafinalphenol/ chloroform/isoamyl alcohol_extraction, the RNAwasdenatured by NaOH

(750

Ml,1M)onice for 10min,thenquenched bythe addition

of1.5 mlof 1 MHepes (free acid)and ethanolprecipitated(0.53ml 3 Msodiumacetate + 14.5 ml 100%ethanol)overnightat-20°C. The RNAwasspun downat_10,000gfor 30min and thepellet resuspended in 1 ml ofTES _(N-Tris

[hydroxymethyl]methyl-2-aminoethanesul-fonic

acid),

pH 7.4,solution(10mMTES, 10 mMEDTA,0.2%

SDS).

5

_,ul

ofeach

_sample

wascounted and

samples subsequently

werediluted

withTEStonormalizeintersamplevariationsinradioactivity. 1 mlof TES-NaCI(10mMTES, 10 mM_EDTA,0.2%SDS,0.6 MNaCl)was

addedto1 mlofthe above RNAsample/TESsolution₍I0'cpm)and

placedinto a5-ml_plastic scintillation vialcontaining 5 Mg of

dena-tured,linearizedDNAimmobilizedon_{stripsof gene-screen(DuPont/}

NEN, Wilmington,DE) nylonmembrane and allowedtohybridizefor 36 hat65°C(41).

Afterhybridization,the membraneswerewashed twice in 20 ml of

2xSSCfor1 hat65°C.UnhybridizedRNAwasthendigestedin 8 ml of2xSSCcontaining8Mlof 10mg/mlRNaseAfor 30 minat37°C.

Increasinglystringentwasheswereperformedasfollows:once2x SSC in 0.1%SDSat65°C for30min,once lXSSCwith0.1% SDSat65°C

for30_min,twice 0.IxSSC with0.1%SDSat65°C for 30min,twice 0.1XSSCatroom_temperaturefor 30 min. The filterswerethen blotted

dryonWhatman Inc.(Clifton, NJ)3 MM paper, andexposedto_X-ray filmat-70°Cusing intensifyingscreens.

Results

Radicalscavengers decreasemRNA levels

for

JE/MCP-I and CSF-1

after

stimulation with TNF-a or

IgG.

Previously we have shown that exposure of MCtoTNF-aoraggr.IgGresults inincreased levels ofmRNA for JE/MCP-l and CSF-1

(22,

26).

BothTNF-aand

IgG

complexesalsoenhance free oxygen

radical formation

by

cultured MC(

32-34).

Wetherefore

exam-inedwhether scavengers for free radicalssuchasdimethyland

tetra-methylthiourea

(DMTU and TMTU) (42) could

influ-ence mRNA levels. As shown in Fig. 1, both DMTU and

TMTU decreasedmRNA levelsfor CSF-1 and JE/MCP-1 in responsetoeither_{TNF-a or aggr. IgG. Equimolar urea, used as}

anonscavengercontrol,hadnoeffect. TMTUalsosuppressed the _{induction by cycloheximide} (Fig. 1). Neither DMTU or TMTU influenced expression of GAPDH, a housekeeping

gene,

indicating

that these agents didnotsuppressmRNA

ex-pression nonspecifically.

Reducedgentathioneandglutathione peroxidase represent

a

major

intracellular defense system against oxygen-derived

free radicals (43). Addition of N-acetylcysteine (NAC) tocells

increases intracellular glutathione levels (44). We have

re-portedthatNAC (10 mM) increased glutathione contentof

CSF-1

ak

" : :.._ .,

JE/MCP-1

GAPDH _w j i

-J

O l_ J

LL

Z D +

8

,,mL

z

..

_

...

.. ..._{.... ....}

._ _k,

_ _w

GAPDH

4

*J Lu

LL + +

z z

t t

z z

-J

0

z

0

U)

.0

LI) ' XU

cn 0)

-4.5Kb -3.8

-2.3 Figure 1. Northern blot analysis for mRNA

lev--1.6 _{els of CSF-1, JE/MCP- 1, and GAPDH.}

Mesan-gialcells wereincubated for4hbeforeRNA ex-- 0.8 traction under control conditions or in the

pres-enceof TNF-a (100 U/ml or 1.7 x 10-10M), aggregated IgG (200,g/ml), cycloheximide

(CHX; 15 g/ml),urea(10 mM),

dimethylth--1.4 ioruea(DMTU; 10mM),tetramethylthiourea (TMTU; 1OmM) orcombinationsthereof. Po-sitionofRNAsize markersareindicatedin ki-lobases on theright. Results are from represen-tativeexperimentsand wereconfirmedin two tofourexperimentalsetseach.

MC by 40% from 89±6 nmol/mg protein to 137±20 (45).

Addition of NAC consistently blunted the increase inmRNA

for JE/MCP-I andCSF-l inresponse toTNF-a(Fig. 2 A).In contrast, NACdidnotdecrease mRNAlevels inresponse to aggr. IgG (Fig. 2 A).

Anotherthiol compound and radical scavenger, pyrroli-done-dithio-carbamate (PDTC)has been showntoinhibit the TNF-a-induced activationofthetranscriptionfactorNF-KBin several celllines (27-29).InMC,addition of PDTC(0.1 mM) reduced mRNA levels forJE/MCP-1 in response to TNF-a

andIgG(Fig. 2 B). Howeverattheconcentration of 0.1mM

PDTCdidnotdecrease mRNA levels for CSF-1 inresponse to

CSF-1

-4.5Kb

-3.8 -2.3

-1.6

CSF-1

eitherTNF-a orIgG (Fig.2B). Evenat 1 mM PDTCdidnot

influence mRNAlevelsfor CSF-1 (resultsnotshown). Thus

regulation of the CSF-1 gene appears to differ in respect to

susceptibilitytoPDTC inhibition.

Generationofsuperoxide does, but H202doesnot increase mRNAlevelsforJE/MCP-I and CSF-1. Taken together, these results show thatscavengersoffree radicalscan attenuatethe

expressionofJE/MCP-l and CSF-1 inresponse to TNF-aand

aggr.IgG,which could indicate that free radicalsareinvolved

in theintracellular signaling. We therefore examined whether

exogenous reactive oxygenspecies could also enhancemRNA levels for thesegenes. Direct addition of 50 or 100 ,uM H202 to

-4.5

Kb

ss -~~~~3.8

-2.3

-1.6

-0.8

!W

1W

-1.4

-0 L ox 0m

L <

z Z Z

z + +

o

A y O

z

A

I--JE/MCP-1 :w

GAPDH _. _{_m*}

B

o o o,

0 C. C

0

F +

+~~~~~~~~~0

Figure2. Effects of (A)

N-ace-- 0.8 tylcysteine(NAC;10mM)or

(B) pyrrolidone-dithio-carba--1.4 mate(PDTC;0.1 mM)on

mRNAlevels forCSF-l,JE/

MCP-1,orGAPDHin

mesan-gial cells incubated for4hwith TNF-a (100 U/ml)or

aggre-gated IgG (200Ag/ml).

-4.5 Kb -3.8

-2.3 -1.6

-0.8

-1.4

CSF-1

JE/MCP-1

JE/MCP-1

GAPDH

"am,

W.

W.W.

_,wr

_W

.,,

_l

-4.5 Kb

: 3.8

-2.3

*! 1

-1.6 JE/MCP-1

E E

O o o

z ,

N N

C.) x x

-0.8

GAPDH - 1.4

o o C

8i x x 0

z 0 0 0.

x +

0

B

Figure 3. Effectsofreactive oxygen

specieson mRNAlevels for CSF- 1,

JE/MCP-1,orGAPDH.Mesangial

cells wereincubated for4 hwith(A)

H202 or (B)hypoxanthine(HX; 0.5 mM) together with eitherxanthine

oxidase (XO; 10 mU/ml) or

heat-in-activated xanthineoxidase (iXO)in

the presence or absence of pyrroli-done-dithio-carbamate (PDTC; 0.1 mM).

MC had no effect on mRNA levels (Fig. 3 A). In contrast

generation of superoxide by the addition of xanthine oxidase andhypoxantine increased mRNA levels for JE/MCP-l and CSF- (Fig. 3 B). Heat-inactivation of xanthine oxidase

atten-uated thisresponse. PDTC decreased the increase in mRNA forJE/MCP- butnotCSF-1inresponseto xanthine-oxidase-hypoxanthine (Fig. 3 B), comparabletoPDTC's effectonthe TNF-a andaggr. IgGresponse(Fig. 2 B).

NADPHincreasesmRNAlevelsforJE/MCP-J andCSF-I

effects attenuated by radical scavengers andan inhibitor of

NADPH-dependentoxidase. The generation ofoxygenfree rad-icals by MC has been linkedtoamembrane-bound

NADPH-dependent oxidase system(31 ). Thissystemcould be stimu-latedby addition ofNADPHtothe extracellular medium (31 ), findings confirmed byus(Table I). We therefore examined the effect ofincreasing concentrations ofNADPH inthe medium on mRNA levels. As showninFig.4A, addition ofNADPH

resulted inadose-dependent increase ofmRNA for JE/MCP-l and CSF- 1. The effect ofNADPHwasmaximalat5mMand showedadeclineat10mM.Thereasonsfor thedeclineat 10 mMis unclearatpresent.NADPH(5 mM)notonly increased mRNAlevels under basal conditions, but also after stimulation with TNF-aoraggr.IgG (Fig. 4 B). Consistent with NADPH acting via enhancement of free radical formation, the radical

scavengers DMTU and TMTU preventedthe rise in mRNA

levels in thepresenceof NADPH (Fig. 4C).NADP,which is

not a substrate for the NADPH-dependent oxidase, had no

effect on the mRNA levels. These resultsareconsistent with the proposal that free radical generation by a membrane-bound NADPH-dependent oxidasesystemcontributesas a sec-ond messenger system to the induction of mRNA for JE/ MCP-1 and CSF-1. To further examine this hypothesis we used HMAP, an agent reported to be an inhibitor of the NADPH-dependent oxidase (46). In orderto verify that MC generated superoxide underour experimental conditions we determined superoxide production. As shown in Table I,

NADPH increased superoxide production under basal condi-tions and after stimulation by TNF-aor aggr. IgG, consistent with publishedreports(32-34). HMAPinhibited stimulation of superoxide generation. HMAP also decreased mRNA levels forJE/MCP-1andCSF-Iduring incubation of MC with

TNF-aoraggr.IgG (Fig. 4 D), furthersupportingamediator rolefor superoxide anion generated by NADPH-dependent oxidase in the activation of thesegenes.

NADPH, TNF-a, andIgG enhancetranscription ofJE/ MCP-J and CSF-J, effects blocked by the radicalscavenger TMTU. Levels of mRNA for both JE/MCP-l and CSF-1are regulated by transcriptional andpost transcriptional mecha-nisms(2, 6, 11-20). We used nuclear run-offassaytechnique

to examine the transcriptional ratesafterincubation of MC

TableLGeneration ofSuperoxide by MC

Basal HMAP TNF-a TNF-a+HMAP IgG IgG+HMAP

nmol/J06cells

-NADPH

30 minincubation 7.3±0.6 6.0±0.4 14.3±1.6* 8.9±1.6 18.4±2.8* 9.2±1.3

+NADPH

15minincubation 11.1±0.6 6.9±1.2 21.6±2.5* 7.6±0.9 27.9±2.2* 13.0±1.5

MCwereincubatedwith theexperimentalagents andsuperoxide generation was determined as described in Methods.Analysisof variancewas

performedusingtheFisher PLSD and Scheffe-F test to evaluate differences between groups. Resultsaremeans±SEMof sixtoeightseriesof incubationsforeachcondition. Because of the lower ratesof02generation,incubationswereperformedfor 30 min in the absence of NADPH. Inthe presence of NADPH (5 mM)generationofsuperoxidetendedtoleveloffafter 20 min and incubations+NADPHweretherefore carried outfor 15 minonly. *P<0.05ascomparedtorespectivebasal valuesbyeithertest.

OxygenRadicals and GeneExpression 1567

CSF-1

JE/MCP-1

-4.5Kb -3.8

-2.3

-1.6

CSF-1

- 0.8

GAPDH

_{so_.}

_-1.4

A

"I"':

1.:-..J;

:I _6.,' .,...1s, .,

CSF-1I

JE/MCP-1

GAPDH

dow

-0.8

-l

0

z 0

U

0 10 5 2.5 1.0 0.5 0.25

0

49

z

B NADPHmM

-4.5Kb

-3.8

-2.3

-1.6

-0.8

-1.4

IL x D M a IL

a CZ

*.Z

z

g-U. I.

z a

I- 4

z

t!

z

C, Z

Cal

_ a

4c z

Cal

O

w_ _.

T -.

CSF-1

JE/MCP-1 e

GAPDH _ _t

_a

_m

.~~ ~ ~ ~ ~ ~

22

D

Figure4.(A)Effects ofaddition of increasing concentrations of

NADPH(from0 to 10 mM)to the incubation mediumonmRNA levelsforCSF-l,JE/MCP-l, and

GAPDH extracted frommesangial .4.5 Kb cells after 4 h of incubation.

(B)

-3.8 NADPH (5 mM) alone or in 2.3 combination with TNF-a

(100

U/

-*

ml)oraggregatedIgG (200

Ag/

- 1.6 ml) on RNA levels ofCSF-l, JE/

MCP-1,orGAPDH.(C) Com-parisonof the effects of NADP(5

- 0.8 mM) or NADPH (5 mM) alone

orincombination with DMTUor

- 1.4 TMTU

(10

mM) on mRNA lev-els.(D)Effects of NADPH(5

mM)and/or hydroxy-methoxy-acetophenone (HMAP; 100o_,g/

ml) incombination withTNF-a

(100 U/ml)oraggregated IgG (200,g/ml)onmRNA levels.

withNADPH, TNF-aor aggr.IgG in thepresenceand absence of the radical scavenger TMTU. TNF-a and IgG both

in-creased transcription for JE/MCP-l and CSF-1 (Fig. 5).

Pre-treatment of the cells with 5 mM NADPH also resulted in enhanced transcriptional activity for both genes (Fig. 5). TMTUdecreased basal transcriptional activity forJE/MCP-I

and CSF-l and attenuated the increaseinresponsetoNADPH,

TNI

NA

CONTROL _

TMTU

TNF-a

F-a+ TMTU 4:

NADPH

,DPH+TMTU

IgG _ _; ,,

IgG+TMTU _

rRNA GAPDH CSF-1 MCP-1 pGEM

Figure 5. Nuclearrun-off analysis for transcriptional activity in nuclei isolated from mesangial cellsafter1h of incubation under the

condi-tions indicated. Labeled transcripts (I0'cpm) generated by the nuclei

werehybridized with cDNA for either ribosomal RNA (asa

nonspe-cific, positive control), GAPDH, CSF-l, JE/MCP-l,orthe plasmid vectorpGEM-4Z (asanegative control).

TNF-aor aggr.IgG(Fig. 5). These resultsareconsistentwith

changes in transcriptional activity contributingtotheobserved

changes in mRNA levels. None ofthe experimentalagentshad

anaffectontranscription for ribosomal RNAorGAPDH.

Discussion

Our results show thatscavengersoffree radicalscansuppress

the expression of theJE/MCP-I and CSF-Igenesinresponse

toTNF-aoraggr.IgG in MC, whereas addition ofexogenous NADPH increased their expression under basal andstimulated

conditions. The stimulation of thegeneexpression is also

at-tenuatedby radicalscavengers.Furthermore,aputative

inhibi-torof NADPH-dependent oxidase alsoattenuatesthe increase in mRNA for CSF- and JE/MCP-l inresponsetoaggr.IgGor TNF-a. We therefore conclude that free radicals generated-at least in part-byanNADPH-dependent oxidase systemcan serve as second messengers foractivation of the JE/MCP-1

and CSF-1 genes inresponse toTNF-aandaggr. IgG. Thus generation of reactiveoxygenspecies inresponsetooccupancy of Fc receptorby aggr. IgG and TNF-a receptoroccupancy

doesnotonly resultintheoxygenburst, butmayalsoserve as a signaling system forcytokine generation. The concerted ex-pression of the leukocyte chemoattractant and activating

cyto-kines MCP-I and CSF-Imayregulateleukocyte influxtosites of tissue injury.

Recently it has been proposed that free radicalsmay serve as intracellular signals forgene activation involving specific CSF-1

m;S

-'

q-_we d__W_W -4.5Kb

...~~~~~ -3.8

J-¢ 2 _{.- 2.3}

.1.6

JE/MCP-1 _go..

-4.5Kb

-3.8 -2.3

-1.6

-0.8

-1.4

GAPDH

la

i

a

-1.4

A

CSF-1

JF/MCP-1

GAPDH

-J

0

I-.

z

0

C.)

C

transcription factors suchas NF-KB(27-29, 44). This hypothe-sis is supported by datashowingthat theactivation ofNF-KB by,e.g., TNF-a is blunted by NAC and PDTC (27, 28, 44).

NAC increases the intracellular glutathione content which

serves as amajor protectant against free radicals (43). The

thiol-containing PDTC alsoserves as aradicalscavenger(27). Trans-activation by the phorbol responsive transcription factor

AP-1 couldalso beinduced by oxygen radicals, butwas not

blockedby NAC (27). The mechanism(s) of the free radical generation may involveamembrane-bound

NADPH-depen-dentoxidase thatappears tobepresentinmanycelltypes(30).

Furthermore, a membrane-bound NADPH-oxidase system

wasdescribed in MC and addition ofexogenous NADPH

en-hancedsuperoxide generation inresponse tostimuli (31). Gen-erationof superoxideinresponse to,e.g.,TNF-aandIgG

com-plexes has also been reported for MC (32, 34), results

con-firmed in our presentstudies with mouse MC. Furthermore,

our studies show that HMAP, a putative inhibitor ofthe

NADPH-dependent oxidasesystem (46), alsoinhibits

super-oxidegenerationin MC under basal conditions andafter

stimu-lation with NADPH, TNF-a, or aggr. IgG. These changesin

superoxidegeneration areassociated withparallel changes in

mRNAlevels forinduciblecytokines. For example,exogenous NADPH, but not NADP,increasesmRNAlevelsfor

JE/MCP-1andCSF-1under basalconditions and inresponse to TNF-a oraggr.IgG. Inbothourstudy and that of Radekeetal.( 31 ), extracellularaddition ofNADPHincreasedsuperoxide produc-tion,especiallyin thepresenceof IgGorTNF-a.Thusithasto

beconsidered thatexogenous NADPH maygainaccess tothe cell membrane-bound oxidase system. A mediator role for

NADPH-dependentsuperoxide generation in the alteredgene expression is further supported by the data with HMAP. HMAPhas beenreportedtoinhibit NADPH-dependent oxi-daseandtosuppressendothelial leukocyte adhesion

molecule-1 (ELAM-1) mRNA expression in endothelial cells (46). In ourstudies suppression ofsuperoxidegeneration by HMAP

also resulted inanattenuation of the rise inmRNAlevelsfor CSF-l and

JE/MCP-l

inresponse to NADPHwith TNF-aor aggr. IgG. Thus generation ofoxygen radicals and levels of mRNAforthesegeneschange inaconcordantmannerunder theexperimentalconditions used. The fact that the NADPH-dependent responses could beabrogated by addition of

cell-permeable freeradicalscavengers,suchasPDTC,DMTU, and TMTU,but notby thenonscavenger ureais consistent with the

NADPHeffectbeingmediated by freeradical generation.The

free radical involved may represent superoxide anion,

inas-muchasgeneration of superoxide by addition of xanthine

oxi-daseandhypoxanthinealsoincreased themRNAlevel,effects inhibitableby PDTC. In contrast, exogenousH202 hadno ef-fecton mRNAlevels. These resultsarenotnecessarilyatodds with thoseof Schrecket al. (27), whoreportedactivation of

NF-KBbyextracellular H202. These authors noted that H202

may not have had a direct effect but probably involved an

additional cellular reaction induced byH202.

Reducedglutathione together with theglutathione peroxi-daserepresent themajor intracellular defensesystem against

oxidantstress(43). Addition of NACcanincreasethe intracel-lularglutathione levels and therebyenhance the radical

sca-venging potential of cells (27, 44). Previous studies had

re-portedthat NAC as wellastheradicalscavenger PDTCareable

toreduce theactivation ofNF-KB in responsetoTNF-a (27, 44). Consistent withthese reports andourresultswiththe

scav-engers TMTU andDMTU, addition of NAC and PDTC also

diminished the increase inmRNA for JE/MCP-l in response to TNF-a. However, PDTC did not alter mRNA levels for CSF- I, inspite of inhibition by TMTU and DMTU. This may indicatethatCSF-1 expression isregulatedinadifferent man-ner thanJE/MCP-1. Results with NAC were alsodivergent. NACdidnot reduce themRNAresponseofCSF-Ito aggr.IgG but suppressed thatin response to TNF-a.This would indicate that theintracellularsignaling inresponse to TNF-a or toaggr.

IgGinvolves,at least in part,differentpathways. Wespeculate that theeffect of aggr. IgG via Fc receptorsongeneactivation may involve several signaling systems and transcription fac-tors, thatare differentially influenced by NAC. For example, the AP-l transcription factors require reduced glutathione in order to effectively bind to and initiate DNA transcription (47), and phorbol esteractivation ofAP-1 was notinhibitedby NAC in thestudies of Schreck et al. (27). IgG receptor occu-pancy results inbothgeneration of oxygen radicals and activa-tionof protein kinase C pathway (34, 48), whiletheeffectof

TNF-aon geneactivation appears to bekinase Cindependent (49). Thus, in response to aggr.IgG, NAC will decrease the free radical pathway but have no effectormayevenincrease the protein kinase C and AP- 1 pathway. This couldexplain the absence ofan inhibitory effect of NAC on mRNA levels in response to IgG. In the caseof signaling via free radicals

with-outcontributionbykinaseC, asmightapplytoTNF-a, NAC would markedly reduce the mRNA response, as wasindeed observed. It is clear that additional experiments will be re-quiredtoaddress theseissues.

The mechanism for the increasein mRNA levels for JE/ MCP-l and CSF-l in response to TNF-a, aggr. IgG, and

NADPHinvolves enhancedtranscription as demonstrated by the nuclearrun-onexperiments.Therun-onexperimentsalso show that the decreases inmRNAfor JE/ MCP- 1 and CSF- 1 in the presence of the radical scavenger TMTUareprimarilydue

tolowertranscriptionalactivity.However, both theJE/MCP-1 and CSF-1 gene contain AUUUA richsequences in their 3' untranslatedregionsthat have been associated withinstability ofmRNA(50). Thus thepossibility of additional

post-tran-scriptional regulation ofthesemRNAspecieshastobe consid-ered. Our presentexperimentsdidnotaddressthispossibility.

Howeverthedown-regulation ofmRNAlevels by radical

scav-engers doesnotrequiredenovoprotein synthesisasitwasalso observed in thepresenceofcycloheximide, whichsuperinduces

both theCSF-I andJE/ MCP-I genes(1 1, 16). Thisfindingis consistent with the observation by Schreck et al. (27), who noted that NACalso decreased theeffect ofcycloheximideto

activateNF-KB.

Freeradicals couldinfluencetranscription by activation of

transcription factors. Forexample, activation ofNF-KB has been shownto occur inresponse to anumber of stimuli,

in-cludingfreeradicals,TNF-a, andcycloheximideand results in

theinducible expression ofa numberof genes (51).Both the

murine CSF-1 and the human MCP-1 gene contain NF-KB

bindingsites ( 18, 52). Furthermore, it has been proposedthat NF-KBmayact as asignal for cell distressinresponsetomany

adverse conditions (51 ). Activation ofNF-KBwould

coordi-natethegeneration ofnumerousproducts,includingcytokines and intercellular adhesion molecules, that are important for tissuedefense, remodeling,andinflammation(29,51). Gener-ation of MCP-1 would

certainly

fit into this overall

picture.

Thefactthatcytokinescanberegulated byfree radical

tion is of particular interest ashypoxia-reperfusionand inflam-matory injury are associated with increased generation of free radicals. Local generation of oxygen radicals could represent a factorresponsiblefor the expression ofJE/MCP-l in ischemic renal injury, reported by Safirstein et al. (53), and in immune-mediated in vivo MCglomerulonephritis(25,54). In the case ofglomerular immune injury, triggers for reactive oxygen spe-ciesgeneration could includeTNF-a(32), immune complexes (22), and complement (55). While generation of large amountsof free oxygenradicalsmay play amajorrole in tissue

injury, regulated generation oflow concentrations of oxygen radicals,possiblybyNADPH-oxidase, may represent a second messenger system forgenerationofcytokines involvedin tissue

injury

andrepair in general.

Acknowledgment

This workwassupported bygrantDK-22036from theNational Insti-tutesofHealth.

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