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Di vergence of Bact erial Li popol ysaccharide P ro- Apopt oti c S ignal ing Down stream of IRAK-1

Douglas D. Bannerm an*, Joan C. Tupper‡, Ryan D. Er wert¶ , Robert K. Winn*, and John M.

Harl an‡

Departm ent s of Sur gery*, Medicine‡, and Pathol ogy¶, Universi ty of Washington School of Medi cine, Seatt le WA 98104

Running Ti tl e: LP S- Induced Endothel ial Apoptosis

Corr espondence:

Douglas D. Bannerm an, P h.D.Di vi sion of Hem atology, Box 359756Harborview R&T Bldg. , Room 521University of Washington, Seatt le325 9th Ave. Seat tle, WA 98104-2499Tel: ( 206) 341-5319Fax: ( 206) 341-5322em ai l: dbanner m@u.washington.edu

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on January 2, 2002 as Manuscript M111249200 by guest on A

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SUMMARY

The vascul ar endot helium is a key target of cir cul at ing bact eri al li popol ysacchar ide (LP S) .

LP S eli cit s a wide array of endothel ial responses, incl udi ng the upregul ati on of cytokines, adhesion

molecul es, and tissue fact or , many of which are dependent on NF -κB activati on. In addit ion, LP S

has been dem onstrated to induce endothelial apoptosis both in vitro and in vivo. Although the

mechani sm by which LP S act ivates NF-κB has been well el ucidated, the si gnali ng pathway( s)

involved in LPS -induced apoptosis remai n unknown. Using a vari ety of domi nant negat ive

constructs, we have identi fi ed a rol e for MyD88 and IRAK-1 in medi at ing LP S pro-apoptot ic

si gnali ng in human endotheli al cel ls. We al so dem onstr ate that LP S- induced endothel ial NF -κB

acti vat ion and apoptosis occur independent of one anot her . Toget her, these data suggest that the

pr oximal signal ing molecul es invol ved in LPS -induced NF -κB activati on have a requisit e

involvement in LPS -i nduced apoptosis, and that the pathways leading to NF- κB activati on and

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INTRODUCTION

Gr am -negat ive bact er ial sepsis is a lif e-t hr eat eni ng event t hat af fl ict s approximately 200,000

pati ent s annual ly in the Uni ted St at es (1). A delet er ious out com e of sepsis is the devel opm ent of

sept ic shock. Endot hel ial cell (E C)1 inj ury and/ or dysfunct ion is a comm onalit y among sever al key

complicati ons associ ated wit h sept ic shock, including syst em ic vascular coll apse, di sseminat ed

intr avascular coagul ati on, and vascular leak syndr om es (2,3). T he pat hogenesi s of septi c shock and

it s att endant vascul ar com pl ications is beli eved to be mediated, at least in part, by

li popol ysacchar ide ( LP S) , a component of the out er envel ope of all Gram- negati ve bacteri a (4-6).

LP S dir ect ly activat es the vascular endothel ium and eli cit s an arr ay of EC responses

incl udi ng the upregul ati on of pr o- inf lam mator y cytokines, adhesion mol ecules, and tissue factor.

Many of these responses ar e mediat ed by NF -κB (7). T he signaling pat hway by whi ch LP S

acti vat es NF -κB in EC has onl y recent ly been eluci dat ed (8). Circulati ng LP S binds to the acute

phase prot ei n, LPS -binding prot ein (LBP ), and this complex is recognized by sol ubl e CD- 14

(sCD14) (9). Although the exact mechanism of interacti on rem ai ns unknown, this LP S-L BP -

sCD14 complex is recognized by the tr ansmembr ane recept or, Toll -li ke receptor (Tl r) -4 (10) . T he

extr acellular domain of Tl r- 4 cont ai ns repeating leucine-ri ch motif s, and the cytoplasmi c por tion

cont ains a Toll receptor-i nt erl eukin-1 receptor (T IR) domain wi th homol ogy to the intracel lular

si gnali ng domai n of the type 1 int er leukin-1 receptor (11) . F oll owi ng Tl r-4 acti vat ion, another TIR

domain- containi ng pr otein, MyD88, is recruit ed to Tl r-4 through respect ive TIR- TIR interacti ons

(12, 13) . MyD88 also cont ai ns a death dom ai n (DD) , a highly conserved protein bindi ng dom ai n

that facil it ates its inter action wit h anot her DD-contai ning signal ing molecule, IL -1 receptor-

associated kinase- 1 (IRAK- 1) (11) . I RAK-1 subsequent ly autophosphor ylates, dissociates fr om

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MyD88, and interacts wi th TNF recept or- associat ed factor-6 (TRAF-6) (14, 15) . T his acti vat es a

downstr eam ki nase cascade invol ving NF- κB- inducing ki nase (NI K) and IκB ki nase (IKK) ,

resulti ng in the phosphoryl at ion and degradat ion of the NF- κB inhibitor, IκB, and the nucl ear

tr anslocat ion of NF- κB (11) .

Several reports indi cat e that LP S eli cit s vascul ar endot helial apoptosis both in vitro (2,16-

20) and in vivo (21- 23) . We have previousl y elucidated an ant i-apoptot ic si gnaling pat hway

involvi ng FL ICE -li ke inhibit ory pr ot ein (F LI P) that protects human EC from LPS- induced

apoptosis (17) . F LIP is a cytoprotecti ve protein, which is rapidl y degraded by the pr ot easome. In

the absence of new prot ein synt hesis, F LIP l evels decrease r esulti ng in sensiti zat ion of hum an EC to

LP S- induced apoptosis. Recent ly, Tlr- 4 has been repor ted to have a requisi te invol vem ent in LP S

pr o- apoptot ic si gnaling (24) , si mil ar to it s rol e in mediat ing LPS- induced NF- kB acti vat ion.

However , the intracellular signali ng pathway through which LP S act ivates apoptosis remai ns

unknown. We, ther ef ore, decided to invest igate whet her downstr eam signali ng molecul es invol ved

in T lr- 4-m ediat ed activati on of NF -κB were sim il arl y i nvolved in mediati ng apoptosis.

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EXPERIMENTAL PROCEDURES

Materials. LP S from Escheri chi a coli serotype 0111:B4 and Polymyxin B wer e pur chased from

Si gm a Chem ical Co. , (St . Louis, MO). Recombinant human TNF- α was purchased from R&D

Syst ems, Inc., (Mi nneapoli s, MN). T he caspase inhibitor peptide, z- VAD-FMK (zVAD) and the

pr ot ein synt hesis inhibitor, cycl ohexim ide (CHX), were purchased from Calbiochem -Novabiochem

Corp., (La Joll a, CA) .

Cell Culture. The hum an dermal mi cr ovascular EC line (devel oped and generously provided by

F. J. Candal and Dr . E. Ades, Centers for Disease Cont rol, and Dr. T. Lawl ey, Em ory University,

At lanta, GA) (25) was cultured in RPMI medi um (Bi owhit taker , Inc., Walkersvil le, MD) enri ched

wi th 10% fet al bovine serum (FBS) (Hycl one Laborator ies, Logan, UT), endotheli al cel l growth

fact or prepared fr om bovine hypothal amus, L- gl ut ami ne (2 mM), sodi um pyruvat e (1 mM), and

nonessenti al am ino acids, in the presence of penicil lin (100 U/ ml) and str eptom yci n (100 µg/ ml ) ( all

purchased fr om Bi owhit taker ).

Cl oning and Stable Expression of cDNA Const ructs. cDNA encoding either the DD or TIR domain

of MyD88, the DD of IRAK-1 (generous gi fts of Dr. Marta Muzi o, Mar io Negr i Inst it ute, Mil an,

It al y), or the DD of Fas- associ at ed death domai n (FADD) (generous gi ft of Dr . Vi shva Di xi t,

Genentech, Inc. , South San Francisco, CA) was cloned into the bi ci str oni c ret rovir al expressi on

pl asmid, pBMN-IRES- enhanced green fluorescent pr otein (E GFP ) (ki ndl y provided by Dr . Gar y

Nolan, Stanf ord University, Stanford, CA) (26) . High-ti ter retr ovirus was pr epared from the

Phoenix am photr opi c packaging cell line (ATCC, Manassas, VA) tr ansfected wit h 24 µg of the

expr ession pl asmid by calcium phosphate pr eci pi tat ion. Recom bi nant ret roviral supernat ant s wer e

coll ect ed 48 h aft er tr ansfecti on and fil ter ed through a Mi ll ex- HV 0. 45 µM fi lter (Mi lli por e Cor p.,

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Bedf ord, MA) . For infecti on, 4x105 EC wer e seeded per wel l of a 6-well pl ate for 24 h to achieve

~80% confl uence. The growth medium was repl aced wit h 2.5 ml of retr ovi ral supernatant

supplem ent ed wi th 32 µg/ ml polybrene and 10 mM HEPE S, and t he pl ate cent rif uged for 2 h (1430

x g; 32oC) . The cel ls wer e then incubated for 10 h (5% CO2, 37oC) after whi ch the retr ovi ral

supernatant was repl aced wit h norm al gr owt h medium . Cells were anal yzed and sorted on the basi s

of EGFP expr ession using a FACVant age SE cell sort er (Bect on Di cki nson Cor p., Frankli n Lakes,

NJ).

Immunoblot ti ng. Cell monol ayers were washed once wit h phosphate-buff ered sal ine (P BS ), lysed

wi th ice-col d modi fi ed radi oim munoprecipi tation assay lysi s buf fer [50 mM Tr is-HCl (pH 7. 4), 1%

Noni det P -40, 0.25% sodium deoxycholate, 150 mM NaCl , 1 mM et hylenedi am inetet ra-acetic acid,

pr ot ease inhibi tor cocktai l tablet (Roche Mol ecular Bi ochem icals, Indianapol is, IN), 1 mM

vanadat e, 50 mM NaF], scraped, transf er red to mi cr ocentr if uge tubes, and centri fuged (16, 000 x g,

10 min, 4oC) . Total prot ein was det er mined using the BCA pr ot ein assay (Pierce Chem ical Co. ,

Rockfor d, IL ). The supernat ant s wer e combined wit h 5x sam pl e buff er (Genomic Solutions Inc.,

Chel msf ord, MA) , boi led for 3 m in, and 20 µg of pr otein/lane were resol ved by SDS- PAGE on a 4-

20% Tr is-Gl yci ne gr adi ent gel (Novex Inc. , San Di ego, CA) . Pr otein was subsequentl y transf err ed

for 1 h at 100 v to polyvinyli dene fluor ide membrane (Mi lli por e Cor p.) . Bl ot s were blocked with

5% dry mil k and then incubat ed wit h ant i-MyD88 (1. 0 µg/ ml ; Evot ech Biologics, Kent, WA), ant i-

human FADD (0.5 µg/ ml ; Tr ansducti on Laborator ies Inc. , Lexington, KY) , ant i-AU1 (1.0 µg/ ml ;

Berkeley Ant ibody Co., Richm ond, CA) , or ant i-HA.11 (1. 0 µg/ ml ; Berkel ey Ant ibody Co.)

anti bodies for 1 h at room temperature. T he bl ots were incubat ed wi th hor seradish-peroxidase-

conj ugated anti -mouse (0.2 µg/ ml ; Sant a Cruz Biotechnology) or anti -rabbit im munoglobul in G

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(0.13 µg/ ml ; Tr ansducti on Labs) , developed with enhanced chem iluminescence (Am er sham Lif e

Sciences, Ar lington Hei ght s, IL ), and exposed to Kodak X-Om at Bl ue fi lm (NE N Lif e Sci ences,

Inc. , Bost on, MA).

Luci ferase A ssay. A recom bi nant adenovi rus ( KZ142) syst em (gift of Dr. Jam es Kelly,

ZymoGeneti cs Inc., S eat tle, WA) was used t o tr ansfect cells wi th a luci fer ase r eporter constr uct.

The adenovi ral construct was created as fol lows: an ol igonucleot ide encoding a consensus NF -κB

bi nding si te, t he tandem NF- κB bi ndi ng si tes of t he HIV-1 long termi nal r epeat (27) , two copi es of

the coll agenase AP- 1 element and a singl e copy of t he c- jun TRE (28) wer e li gated into a luci fer ase

reporter casset te, and then placed i n t he pACCMV. pLpA adenovi ral shut tle vect or for constr uct ion

of r ecombi nant adenovi rus as descri bed (29) . For tr ansfecti on of the luci fer ase r eporter constr uct,

EC were seeded int o 96- wel l black vi ew plates ( Cor ni ng Inc., Corni ng, NY) at a densi ty of 50,000

cell s/well f or 24 h, and subsequentl y i ncubated for 16 h at a m ult iplicity of i nfect ion (m. o. i.) of 2000

in RPMI supplem ent ed wi th 1% FBS. F oll owi ng infecti on, EC were exposed to experim ental

tr eatment in Ham's F 12 medium supplemented with 2. 5% FBS, 20 mM HEPE S, and 0.5% BSA for

4 h at 37oC. Luci fer ase acti vit y was determi ned using a comm ercially avai lable assay ki t and a

TopCount NXT luminescence count er (both fr om Packard Instr um ent Co., Meri den, CT) .

Caspase Assay. EC were seeded i nt o 96-wel l plat es at a densi ty of 60,000 cel ls/ well, cult ured f or

24 h, t reated, and caspase acti vit y measur ed wi th the Homogenous Caspases Assay accor ding t o the

manufactur er 's instr uct ions (Roche Molecular Bi ochem icals). The plates were anal yzed on a

Cytofluor Series 4000 f luorescence plat e r eader (Persept ive Bi osyst ems I nc. , F ramingham, MA) at

485 nm exci tat ion and 530 nm emission, and caspase act ivi ty expressed rel ati ve to si multaneous

medi um contr ol. S ince hum an EC sensiti zat ion t o LP S- and T NF -α-i nduced apoptosis is dependent

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upon the i nhibi tion of new prot ein synt hesis by a mechanism that we have previousl y characterized

(17) , tr eat ment wit h t hese agent s was performed in the presence of 40 µg/ ml of CHX.

Adenovi ral Transducti on. The IκBα mut ant (IκBαM) and β-galactosidase recombinant

adenovi ruses were gi fts of Dr. C. B. Wi lson (University of Washington, Seatt le, WA). T he IκBαM

(S 32A/S 36A) cDNA was generousl y provided by Dr s. J. Di Donat o and M. Kari n (Uni versi ty of

Cali for nia, San Di ego, CA) (30) . Construct ion and pur ifi cation of IκBαM and cont rol (β-

galactosidase) recom binant adenovi rus were perf orm ed as pr evi ously described (20) . F or

adenovi ral tr ansducti on, EC wer e seeded into 96-well pl ates at a densit y of 50, 000 cell s/well for 24

h, washed once wit h medium , and incubat ed for 48 h at a mult ipl ici ty of infecti on (m. o. i.) of 1000

wi th contr ol or IκBαM adenovi rus in complete EC medium. E C were washed once wit h com plete

medi um and subj ect ed to experim ent al tr eat ment.

St at ist ical Met hods. A t-t est or analysis of variance ( ANOVA) was used to compare the m ean

responses between a single experim ental gr oup or m ult ipl e experi mental groups, r espectivel y, and

the control group. For experim ent s analyzed by ANOVA, the Tukey post hoc com par ison test was

used to determi ne between which gr oups signi ficant diff erences exi st ed. All st ati st ical analyses

were perform ed usi ng Gr aphPad P ri sm ver si on 3.00 for Maci ntosh (Gr aphPad S of tware, I nc. , S an

Di ego, CA) . A p-val ue of <0.05 was consider ed signi ficant .

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RESULTS

Polymyxin B Inhibi ts LP S-I nduced E C Apoptosis. As the nam e i mpl ies, LP S is com pr ised of bot h a

polysacchari de and a li pid moiety. It is well est ablished that the lipid A moi ety of LP S is

responsibl e for it pro- inf lammator y proper ti es (31) . F urt her , thi s por tion of LP S alone activates the

Tl r- 4 recept or, resulti ng in NF -κB activati on and the upregul ati on of pr o- inf lam mator y cytokines

(32) . T o determ ine whet her LP S- induced apoptosis is si mil ar ly mediated by lipi d A, EC wer e

exposed to medi um or LP S pre-incubat ed wi th or wi thout Polymyxin B (Fi g. 1) . Polymyxin B,

deri ved fr om the bacter ium Baci llus polymyxa, bi nds and neutrali zes the lipid A moi ety of LP S

(33, 34) . Consist ent with a role for lipi d A in cel l acti vation, neutr ali zation wit h Polymyxin B

complet ely i nhi bit ed LP S-i nduced NF- κB activati on (F ig. 1A). S im ilarly, Polymyxin B was able to

complet ely block LPS -induced apoptosis (F ig. 1B), suggest ing that the pro-apoptot ic pr operti es of

LP S ar e locali zed t o t he li pid A region as wel l.

Expression of Domi nant Negat ive (D/N) MyD88 Inhibi ts LP S-I nduced Apoptosis in EC. MyD88,

by virt ue of it s dir ect recr uit ment to Tlr -4, is one of the most proxim al intracel lular si gnali ng

molecul es invol ved in LPS- induced activati on of NF -κB. MyD88 contains two disti nct pr ot ein

interaction dom ains, a TIR domain and a DD, whi ch mediate it s abil it y to bind to Tlr -4 and IRAK,

respect ively (12) . T he expressi on of ei ther dom ain alone blocks Tl r- 4 m ediat ed NF- κB activati on in

a dominant negative fashion (8,12). T o determ ine whet her MyD88 medi at es LPS -i nduced

apoptosis, EC were st ably tr ansfected wit h cDNA encoding either the TIR domai n or the DD.

Expr ession of these two D/ N for ms of MyD88 was confi rmed by Wester n blot analysis (F ig. 2A).

Furt her , t he funct ional ef fi cacy of these D/ N constr uct s was confi rm ed by assaying for the abil ity of

each to inhi bit LP S- induced NF- κB activati on (F ig. 2B). E xpression of eit her the TI R domain or

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the DD alone inhibit ed LPS -i nduced NF-κB activati on by >80% . In exper iment s conducted in

parallel, expressi on of ei ther MyD88 D/ N also inhi bi ted LP S- induced caspase act ivi ty, albeit to a

lesser ext ent (Fig. 2C) . EC expressing ei ther the TIR dom ai n or the DD demonst rat ed a >30%

reducti on in caspase activit y f oll owing LP S exposure than EC expressing vect or alone.

Si mi lar to LP S, TNF-α is a well -described activat or of EC NF -κB (35) . Dist inct recept or

membrane com plexes and proxi mal intr acellular signal ing molecul es mediate LP S- and TNF -α-

induced NF -κB activati on; further downstream , however, the signali ng pathways leading to NF- κB

acti vat ion converge at the level of NIK and IKK (36) . Consist ent with the rol e of MyD88 in

medi ati ng si gnaling eli cit ed by LP S, but not TNF-α, expressi on of the MyD88 D/ N encodi ng the

DD had no ef fect on TNF -α-i nduced apoptosis (F ig. 2C). E C expressing the MyD88 D/N encoding

the TIR domain, however , dem onstrated ~15% l ess caspase acti vit y f ol lowing TNF- α exposure than

EC expr essing vect or al one. Al though this decr ease in caspase act ivity was subtle, it was

st at ist icall y signif icant.

IR AK-1 D/N Inhi bit s LPS-Induced EC Apoptosis. Following Tlr -4 activati on and MyD88

recr uit ment, IRAK- 1 transi ently bi nds MyD88 and undergoes an autophosphor ylation step leading

to its act ivati on (12, 14, 15) . Opti mal acti vat ion of NF- κB by LP S is dependent upon functi onal

IRAK-1 (8,12,15). T o determ ine whet her IRAK-1 has a si mil ar invol vement in promot ing LP S

apoptot ic si gnaling, a truncated for m of IRAK-1, report ed to funct ion as a D/N, was expressed in

EC. Consi st ent wi th pr evi ous report s (12, 20) , expressi on of the DD alone of IRAK-1 signi ficant ly

bl ocked LP S- induced NF- kB acti vat ion (Fig. 3A) . Expressi on of this IRAK- 1 D/N construct

bl ocked >60% of the NF- kB acti vat ion elicit ed by LP S. S imi larly, the IRAK- 1 D/N was abl e to

abrogat e L PS -induced caspase activat ion by ~50% (F ig. 3B). In contr ast , t he IRAK- 1 D/N fail ed to

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bl ock T NF- α-i nduced apoptosis (F ig. 3B).

FA DD Is Not Required for LP S Pro-Apoptot ic Si gnaling. FADD is a pro-apoptot ic si gnaling

molecul e that couples deat h recept or s to ini tiator caspases. F ADD has been clearl y est abl ished to

medi ate apoptosis initi ated by sever al receptor s, including those for TNF (37, 38) , TRAIL (39, 40) ,

and CD95L (37, 41) . F ADD has also been impl icated in mediat ing Tlr- 2- induced apoptosis (42) .

Both Tl r-2 and Tlr -4, which recognize bact er ial li poprotei ns and LP S, respect ively, are highl y

homologous receptors belongi ng to the larger famil y of Tol l- like receptors (43) . Although each

recogni zes dist inct li gands, Tlr -2 and Tlr- 4 uti lize a comm on intracel lular si gnali ng pathway that

acti vat es NF -κB. To det er mine whether FADD is requir ed for LPS- induced apoptosis, EC were

tr ansfected wit h cDNA encoding a tr uncat ed form of FADD that has pr eviously been demonst rat ed

to function in a D/N manner (37, 38) . West ern blot anal ysi s confir med effi cient expression of the

FADD D/ N constr uct (Fig. 4A) . EC expressi ng the FADD D/N di spl ayed equivalent amounts of

caspase acti vit y fol lowing LP S sti mul at ion as EC expr essing vector al one (Fig. 4B) . Consi stent

wi th pr evi ous studies (37, 38) , expressi on of the FADD D/N const ruct signi ficant ly inhibit ed TNF -

α-i nduced apoptosis.

LP S- Induced EC Apoptosis Is Independent of NF- κB Activati on. To deter mi ne whether NF- κB

acti vat ion is requir ed for LPS- induced apoptosis, EC were tr ansduced with a gene encoding

mutations in the inhibi tor of NF-κB, IκBα, that render it resist ant to phosphoryl at ion and

degr adation (20, 30) . Accordi ngly, expr ession of the IκBα mut ant (IκBαM) signifi cantl y inhibi ted

LP S- induced NF- κB activati on compared to expression of a vector cont rol encoding β-

galactosidase (Fig. 5A) . EC expressing the IκBαM, however , dem onstr ated equivalent levels of

caspase acti vit y fol lowing LP S exposure as those EC expressing vector alone (Fi g. 5B) . To

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determi ne whether caspase activati on contr ibutes to NF- κB si gnaling, EC were exposed to LP S in

the presence or absence of the caspase inhibitor, zVAD. At a concentr ati on that could bl ock 100%

of the LPS -i nduced caspase acti vit y (Fi g. 5C), zVAD failed to inhibit LPS -i nduced NF-κB

acti vat ion (Fig. 5D) . Int er est ingly, zVAD actuall y enhanced LPS -i nduced NF-kB acti vat ion by

~40% .

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DISCUSS ION

We have pr eviously establi shed that LPS -induces human EC apoptosis by sever al di fferent

cr it eri on including: P ARP cl eavage; nuclear hi st one release, and DNA ladderi ng (17, 44) . T o

quantif y rel ati ve changes in apoptosis, we have used a caspase acti vit y assay. Caspases are hi ghl y

specifi c ef fector proteases that ar e act ivated duri ng apoptosis and cleave cellular substrates (45) .

The observed increase in caspase act ivi ty following LP S exposure is consist ent with pr evious

reports that LP S-i nduced E C apoptosis is mediated, in part, by caspase-dependent pr oteol ysi s of key

EC substrates (16, 17, 44) .

In the present repor t, we have est ablished that the lipid A moi ety of LP S is responsi ble for

it s pro-apoptot ic si gnaling proper ti es. Consistent wit h its role in activat ion, neutralizat ion of the

li pi d A moiety of LP S wit h Polymyxin B inhibi ted LPS- induced NF -κB activati on (F ig. 1A).

Si mi lar ly, Polymyxin B was able to complet ely abrogat e LPS -induced apoptosis (F ig. 1B),

suggest ing that the lipid A por tion of the molecul e is responsi ble for its pro- apoptot ic si gnaling

pr opert ies. It has previously been report ed that LP S-i nduced apoptosis is dependent on Tl r-4

si gnali ng (24) . I n that study, the author s demonstrated that macr ophages der ived from C3H/HeJ

mi ce, which have a mi ssense m ut ati on in the t hi rd exon of Tl r- 4 (46) , were resistant t o LPS -induced

apoptosis. It has furt her been establi shed that the domai n of LP S recognized by Tl r-4 is the lipid A

moiety (32) . T ogether, these data im pl y that the lipid A moi et y conf er s pro- apoptot ic si gnaling

through a Tlr-4-dependent pathway. Thi s i s consistent wit h our fi nding that neutr al ization of lipid A

complet ely prot ect s against LPS -induced apoptosis.

Ther e has been som e controversy in t he past regarding whet her T lr- 2 or Tlr -4 mediates L PS-

induced si gnali ng. The genetic evidence clearl y i ndicates that Tl r- 4 i s the putat ive recept or for LP S

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(46) . Other studies, using over expressi on, have concl uded that Tlr -2 recognizes LP S (47, 48) . I t

was lat er ascer tai ned that cert ain comm ercial prepar ati ons of LP S wer e contam inated with bact eri al

li poprotei ns, the latter of whi ch activate Tlr- 2 signal ing (49) . I n the pr esent repor t, hi ghl y pur ifi ed

LP S, which was phenol extr acted and pur ifi ed by ion exchange chrom at ogr aphy, was used for al l

experim ent s. T he fi ndi ng that Polymyxin B com pletely blocks LPS- induced NF -κB activati on and

apoptosis rules out that t he EC responses st udi ed were inf luenced by contami nat ing l ipoproteins.

To investi gate whether signaling mol ecules that li nk Tl r-4 to NF-κB activati on ar e also

involved i n mediat ing L PS- induced apoptosis, D/N ver si ons of MyD88 and IRAK- 1 wer e expressed

in EC, and apoptosis assayed. LPS -induced apoptosis was signif icantl y inhibi ted by expression of

ei ther one of two MyD88 D/ N constr ucts (Fi g. 2C) or an IRAK- 1 D/N (F ig. 3B). T hese constr ucts

had minimal or no inhibitory ef fect on TNF -α-i nduced apoptosis, consi stent wi th pr evi ous reports

that TNF-α signal ing occurs independently of MyD88 and IRAK- 1 (50, 51) . E xpr ession of a D/N

form of FADD, a pr o- apoptot ic adapt er molecule that li nks deat h recept or s to ini tiator caspases,

fail ed to bl ock LP S- induced apoptosis (F ig. 4B). I n accordance wit h FADD's wel l- descri bed r ole i n

pr om oti ng TNF-α-i nduced apoptosis (37) , FADD D/N expr ession signif icantl y inhibi ted TNF- α-

induced caspase acti vat ion. It has previously been report ed that both MyD88 and F ADD cont ri but e

to Tlr- 2-i nduced apoptosis. Since LP S has been shown to init iate apoptosis vi a Tlr- 4 (24) , the

pr esent data suggest s diff er ences in pr o-apoptot ic si gnaling bet ween dif fer ent member s of the Tl r

fami ly. S im ilar to Tlr -2, MyD88 promot es Tl r-4-induced apoptosis; in cont rast, Tl r- 4-i nduced

apoptosis occur s i ndependent ly of FADD.

The MyD88 and IRAK-1 D/ N constr uct s wer e abl e to inhibi t >30% and 50% of the LPS-

induced caspase acti vat ion, respecti vel y. E xpr ession of the FADD D/ N, whi ch has been

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demonst rat ed to have a requi sit e involvement in TNF- α-i nduced apoptosis (37) , inhibited ~50% of

the caspase act ivi ty el ici ted by TNF -α. T he inabi lit y to com pletely inhibit apoptosis using any of

the D/N constructs is not surpr isi ng, consideri ng endogenously expressed ful l-l ength MyD88,

IRAK-1, and FADD are st il l expr essed. There is evidence suggesti ng that Tlr -4 activati on of NF -

κB can occur thr ough a MyD88 and IRAK-1- independent pathway. Fi rst , LPS -induced NF -κB

DNA binding act ivi ty in macr ophages der ived from eit her MyD88 or IRAK-1 knockout mice is

delayed, but not inhibi ted, indicati ng that cel lul ar activat ion by LP S can occur in the absence of

these signal ing molecul es (15, 52) . S econdl y, a MyD88-li ke pr otein has been recentl y descri bed by

two independent gr oups, named MyD88- adpater- like protei n (MAL) or TI R domain-containing

adapter pr ot ein (T IRAP) , whi ch can prom ote LPS- induced NF- κB si gnaling thr ough IRAK-2

(53, 54) . Whet her MAL/ TIRAP and/or IRAK-2 medi ate LPS- induced apoptosis remai ns unknown.

T he exist ence of al ter nat ive acti vation pat hways, which m ay al so be invol ved i n LP S pro-apoptot ic

si gnali ng, coul d explai n the lower effi cacy of the MyD88 D/N in abrogat ing LPS- induced caspase

acti vat ion.

Si nce both MyD88 and IRAK- 1 contri bute to LP S-i nduced NF-κB activati on and apoptosis,

we investi gated whet her these two event s wer e mutual ly dependent. NF-κB activati on has been

cl assicall y viewed as a cytoprotecti ve event based on its abili ty to upregul ate anti -apoptot ic pr oteins

(55, 56) , however, ther e are repor ts that NF -κB si gnaling has a role in pr o-apoptot ic si gnaling as

well (57, 58) . I n the pr esent st udy, inhibi tion of NF- κB activati on had no eff ect on LPS- induced

apoptosis and inhi bi tion of caspases fai led to downregulate LP S-i nduced NF- κB activati on,

suggest ing that these two responses occur independent of one another (F ig. 5). Interestingl y,

caspase inhi bit ion augm ent ed LP S-i nduced NF- kB acti vat ion. Recentl y, the p65 subunit of NF-κB

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has been identi fied as a substr ate of E C caspases (59) . S ince zVAD exposed EC demonst rat ed 30%

less caspase activit y than EC exposed to medium al one, it is possi bl e that zVAD pr event s the

degr adation of NF- κB under basal condit ions, thus, incr easing the amount of functi onal NF- κB in

the cel l. T his may account for the enhanced LP S-i nduced NF- κΒ acti vat ion in EC pre-tr eat ed wi th

the caspase inhibi tor.

In summ ary, we have identi fi ed a pro-apoptot ic si gnaling pat hway involved in LP S- induced

apoptosis. Fir st, t he lipid A moi et y of LP S was identif ied as t he porti on of the m ol ecule that confers

pr o- apoptot ic si gnali ng. Second, a role for MyD88 and IRAK-1 in medi ati ng LP S-i nduced

apoptosis was establ ished. Thi rd, LPS- induced apoptosis was determ ined to occur through a

FADD-independent pat hway. F inally, despit e the involvement of MyD88 and IRAK-1 in

pr om oti ng both LPS -i nduced NF-kB signali ng and apoptosis, these cel lul ar responses wer e

determi ned to occur independent of one another. We propose that LPS -induced apoptosis is

medi ated thr ough Tlr -4 and involves the recr uit ment of MyD88 and IRAK-1. Further, the dat a

pr esent ed here suggest that the si gnali ng pathways leading to NF-κB activati on and apoptosis

di verge downstr eam of I RAK-1.

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ACKNOWLEDG EMENTS

This work was suppor ted by Nati onal Instit ut es of Healt h Grants GM07037, GM42686,

HL 18645, and HL 03174.

FO OTNOTES

1The abbreviations used are: E C, endot hel ial cell s, LPS, li popol ysacchar ide; sCD14, soluble

CD14; LBP, LPS- bindi ng protein; Tl r, toll- li ke receptor ; TIR, Toll receptor- int erl eukin-1

receptor dom ain; DD, death domain; IRAK, IL- 1 r ecept or- associat ed ki nase; TRAF -6, TNF

receptor-associ ated factor -6; NIK, NF-κB- inducing ki nase; IKK, IκB ki nase; FL IP , FLICE-

li ke inhibit ory pr ot ein; zVAD, z-VAD- FMK; CHX, cycl ohexim ide; FBS , fet al bovine

serum; EGF P, enhanced green fluorescent pr ot ein; IκBαM, IκBα mut ant ; ANOVA,

anal ysi s of var iance; D/N, domi nant negati ve; F ADD, fas- associ at ed death domai n.

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FIGURE LEG ENDS

Fi gure 1. Ef fect of Polymyxin B on LPS -Indu ced NF-κB Activati on an d Ap op tosis.

Medi um or medium contai ning LP S (100 ng/m l) was pre- incubated wit h or wit hout

Polymyxin B (100 µg/ ml ) for 1 h. EC were subsequent ly tr eat ed for 4 h or 8 h and assayed

for luci fer ase (A) or caspase (B) acti vi ty, respect ively. Ver ti cal bars represent mean (+S. E. )

NF -κB (A) or caspase (B) activit y relati ve to si mul taneous media contr ol s. * =

si gnifi cantl y decr eased compared t o LP S alone.

Fi gure 2. Expressi on of MyD88 D/N In hib its LPS- Ind uced Ap op tosis. EC were st ably

tr ansfected wit h eit her EGFP -vector alone or cDNA encoding either the TIR domai n or DD

of MyD88 (A- C). An ant ibody raised against MyD88, which recognizes the TI R dom ain of

MyD88, and an anti -AU1 ant ibody were used to confi rm expressi on of the TIR-cont aining

MyD88 D/N (A). An anti -HA anti body was used to detect HA- tagged MyD88 D/N

comprised of the DD alone (A). To conf irm the funct ional ef ficacy of the D/ N constr uct s,

these cell s wer e treated for 4 h wit h either medium or LP S (100 ng/m l), lysed, and assayed

for luci fer ase acti vit y (B) . In ot her experim ents, EC expr essing EGFP -vect or, the TIR

domain of MyD88, or the DD of MyD88 wer e treated for 8 h wit h medi um , LP S (100

ng/m l), or TNF- α (10 ng/m l), and assayed for caspase acti vit y (C) . Ver ti cal bars represent

mean (+S. E. ) NF-κB ( B) or caspase (C) acti vi ty rel at ive to simultaneous medi a cont rols. *

= si gnifi cantl y decr eased compared to EGF P-vector tr ansfected EC exposed to ident ical

tr eatment.

Fi gure 3. Expressi on of IRAK -1 D/N Inhi bit s LPS -In du ced Ap op tosis. EC were st ably

tr ansfected wit h eit her EGFP -vector alone or cDNA encoding the DD of IRAK-1 (IRAK-1-

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D/ N) (A, B). T he funct ional ef ficacy of thi s D/N const ruct to inhibit LPS -i nduced NF-κB

acti vat ion was assayed (A) . EC were tr eat ed for 4 h wi th ei ther medium or LP S (100

ng/m l), lysed, and assayed for luci fer ase acti vit y. I n other exper iments, EC expressi ng

EGFP -vector or IRAK- 1-D/N were treat ed for 8 h wit h medium , LP S (100 ng/m l), or TNF-

α (10 ng/m l), and assayed for caspase acti vit y (B) . Ver ti cal bars represent mean (+S. E. )

NF -κB ( A) or caspase (B) acti vi ty rel at ive to simultaneous medi a cont rols. * =

si gnifi cantl y decr eased compared to EGF P-vector tr ansfected EC exposed to ident ical

tr eatment.

Fi gure 4. Ef fect of FADD D/N Expression on LPS- Ind uced Ap op tosis. EC were st ably

tr ansfected wit h eit her EGFP -vector alone or cDNA encoding the DD of FADD (A, B). An

anti -AU1 ant ibody and an ant ibody raised against FADD that recogni zes its DD were used

to conf irm expr ession of the FADD D/ N ( A). In other exper im ent s, these cell s were treated

for 8 h wi th medium, LP S (100 ng/m l), or TNF- α (10 ng/m l), and assayed for caspase

acti vit y (B) . Ver ti cal bars represent mean (+S. E. ) caspase acti vi ty rel at ive to simultaneous

medi a cont rols. * = si gnifi cantl y decr eased compared to EGF P-vector tr ansfected EC

exposed to i dentical tr eat ment.

Fi gure 5. Ef fect of the In hi bit ion of NF-κB Activati on on LP S- ind uced Ap op tosis. EC

were infected with either contr ol (β-gal ) or IkBαM adenovi rus for 48 h, subsequent ly

tr eated wi th medium or LP S (100 ng/m l) for 4 h or 8 h, and assayed for luci fer ase (A) or

caspase acti vit y (B) , respectively. In ot her experi ments, EC were pret reated for 1 h with

Me2SO or the caspase inhibitor peptide, zVAD (100 µM) , treated as above, and assayed for

caspase (C) and luci fer ase acti vit y (D) . Verti cal bars repr esent mean (+S. E. ) NF-κB ( A, D)

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or caspase (B, C) activity relative to sim ul taneous media contr ols. * = si gnifi cantl y

decr eased compared t o β-gal vector control. ** = si gnifi cantl y diff er ent than LP S alone.

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A

B

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-18kDa

VE

CT

OR

My

D8

8-D

/N (

TIR

)

My

D8

8-D

/N (

DD

)

VE

CT

OR

IB: AU1 HAMyD88

VE

CT

OR

My

D8

8-D

/N (

TIR

)

35kDa-

18kDa-

B

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B

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CT

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FA

DD

-D/N

AU1

A

IB:

- 16 kDa

- 25 kDa

B

FA

DD

-D/N

VE

CT

OR

FADD

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HarlanDouglas D. Bannerman, Joan C. Tupper, Ryan D. Erwert, Robert K. Winn and John M.

IRAK-1Divergence of bacterial Lipopolysaccharide pro-apoptotic signaling downstream of

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