Reactive oxygen species (ROS) have been proposed to serve

Transcription

1 Protein Cronyltion s Novel Mechnism in Redox Signling Chi Ming Wong, mrit K. Cheem, Lihu Zhng, Yuichiro J. Suzuki strct Rective oxygen species serve s second messengers for signl trnsduction; however, moleculr trgets of oxidnt signling hve not een defined. Here, we show tht lignd receptor medited signling promotes rective oxygen species dependent protein cronyltion. Tretment of pulmonry rtery smooth muscle cells with endothelin-1 incresed protein cronyls. Cronyltion of the mjority of proteins occurred trnsiently, suggesting tht there is lso mechnism for decronyltion induced y endothelin-1. Decronyltion ws suppressed y inhiition of thioredoxin reductse, nd cellulr thioredoxin ws upregulted during the decronyltion phse. These results indicte tht endothelin-1 promotes oxidnt signling s well s thioredoxin-medited reductive signling to regulte cronyltion nd decronyltion mechnisms. In cells treted with endothelin receptor ntgonists, hydrogen peroxide scvengers, or n iron cheltor, we identified, vi mss spectrometry, proteins tht re cronylted in receptor- nd Fenton rection dependent mnner, including nnexin 1, which promotes poptosis nd suppresses cell growth. Cronyltion of nnexin 1 y endothelin-1 ws followed y protesome-dependent degrdtion of this protein. We propose tht cronyltion nd susequent degrdtion of nnexin 1 my ply role in endothelin-medited cell growth nd survivl, importnt events in pulmonry vsculr remodeling. Protein cronyltion in response to lignd receptor interctions represents novel mechnism in redox signling. (Circ Res. 28;12:-8.) Key Words: endothelin-1 protein cronyltion oxidnt signling pulmonry hypertension smooth muscle Downloded from y on Septemer 28, 218 Rective oxygen species (ROS) hve een proposed to serve s second messengers for signl trnsduction processes. 1 5 Numerous studies demonstrted tht (1) lignd receptor interctions produce ROS; (2) ntioxidnts lock signl trnsduction; nd (3) ROS cn stimulte signling events ROS signling is thought to ply importnt roles in vrious diseses, including cncer, neurologicl disorders, immune diseses, nd crdiovsculr diseses. lthough mechnisms of ROS ctions during oxidnt signling hve not een defined, protein thiols eing the oxidtion trgets hve een populr concept Recently, Lee nd Helmnn 15 descried regultory mechnism for the cillus sutilis PerR trnscription fctor y metl-ctlyzed oxidtion. Thus, other types of protein oxidtion such s metlctlyzed protein cronyltion my lso e importnt for cell signling. Endothelin (ET)-1 is produced y vsculr endothelil cells nd exerts potent vsoconstrictive 16 nd mitogenic ctions on vsculr smooth muscle cells (SMCs). In pulmonry circultion, contriutes to vsoconstriction nd vsculr remodeling, which occur in pulmonry hypertension. The expression is incresed in the lungs of ptients with pulmonry hypertension, 18 nd ET receptor ntgonists hve een used to tret humn pulmonry hypertension, 19,2 indicting the clinicl importnce of signl trnsduction. is mitogen of pulmonry rtery SMCs vi the ctivtion of either ET or ET receptor. 24 Signl trnsduction pthwys induced y in pulmonry rtery SMCs, however, re not well understood. cn ctivte extrcellulr signl-regulted mitogen-ctivted protein kinse 25 s well s GT-4 trnscription fctor. 26 hs lso een shown to generte ROS, which promote pulmonry vsculr SMC prolifertion. 2 Thus, ROS my ply importnt roles in medited pulmonry rtery SMC growth during the development of pulmonry hypertension. The present study demonstrtes tht promotes protein cronyltion in pulmonry rtery SMCs in n ET receptor nd Fenton rection dependent fshion. We lso identified the decronyltion mechnism tht is ctivted y vi thioredoxin. Proteins tht re cronylted in response to were identified using 2D gel electrophoresis nd mss spectrometry., which inhiits cell growth, ws found to e mong the proteins tht were cronylted nd susequently degrded in response to in receptor nd metl-ctlyzed oxidtion dependent mnner. Mterils nd Methods Cell Culture Lrge to midsized ovine pulmonry rtery nd thorcic ort were otined from locl ttoir. ovine pulmonry rtery SMCs Originl received July 12, 2; revision received Novemer 1, 2; ccepted Novemer 29, 2. From the Deprtment of Phrmcology (C.M.W., Y.J.S.) nd Lomrdi Comprehensive Cncer Center (.K.C., L.Z.), Georgetown University Medicl Center, Wshington, DC. Correspondence to Dr Yuichiro J. Suzuki, Deprtment of Phrmcology, Georgetown University Medicl Center, NW43 Medicl Dentl uilding, 39 Reservoir Rd NW, Wshington, DC 25. E-mil ys@georgetown.edu 28 mericn Hert ssocition, Inc. Circultion Reserch is ville t DOI: /CIRCRESH

2 Wong et l Protein Cronyltion nd Redox Signling 1 Downloded from y on Septemer 28, 218 (PSMCs) nd ortic SMCs were isolted s descried previously 28 nd cultured in RPMI medium 164 supplemented with 1% FS, 1% penicillin/streptomycin, nd.5% fungizone (Invitrogen, Crlsd, Clif). Two to 8 pssges were used for experiments. Cells were growth-rrested for 24 hours in medium supplemented with.1% FS nd then treted with (Sigm-ldrich, St Louis, Mo), H 2 O 2 (Fisher Scientific, Hmpton, NH), or 1-chloro-2,4- dinitroenzene (DNC) (Sigm-ldrich) for, 5, 1, 15, 2, nd 3 minutes. In some experiments, cells were pretreted for 3 minutes with Q123, Q88, eselen, deferoxmine, MG132, or DNC (Sigm-ldrich). Orgn Culture For orgn culture of rt pulmonry rtery, lungs, nd herts were otined from mle Sprgue Dwley rts (25 to 3 g) nd plced in ice-cold PS. Mid-to-lrge pulmonry rteries were dissected, nd surrounding connective tissues were removed under dissecting microscope. lood vessels were cut into smll pieces of ring segments nd incuted in RPMI medium 164 contining.1% FS for 3 minutes t 3 C efore treting with. Sttisticl nlysis Comprisons etween 2 groups were nlyzed y 2-tiled Student s t test, nd comprisons mong 3 or more groups were nlyzed y NOV with Student Newmn Keuls post hoc test. P.5 ws considered to e significnt. Dt re presented s mens SEM. n expnded mterils nd methods section, s well s supplementl figures, cn e found in the online dt supplement t Results Promotes Protein Cronyltion Tretment of PSMCs with (3 nmol/l) resulted in the production of ROS, s monitored y detecting green dichlorofluorescein fluorescence (supplementl Figure I). The production of ROS ws pprent fter 5 minutes of tretment, nd the level of tht produced dichlorofluorescein fluorescence ws comprle to the level produced y dding 5 mol/l hydrogen peroxide (H 2 O 2 ) extrcellulrly (supplementl Figure I). Thus, produces ROS in PSMCs. Immunolotting of 2,4-dinitrophenylhydrzine (DNPH)- derivtized proteins reveled tht vrious proteins were cronylted in PSMCs (Figure 1). Without DNPH derivtiztion, no nds were detected (dt not shown). Tretment of cells with for 1 minutes incresed protein cronyltion of specific proteins, s determined y 1D (Figure 1) nd 2D gel electrophoresis (supplementl Figure II). Coomssie lue stining of the memrnes showed no chnges in totl protein content (Figure 1, ottom). We ssigned numers for ech of cronylted protein (CP) nds from 1D gel electrophoresis experiments to e CP1 to CP (Figure 1). The medited promotion of cronyltion of these proteins ws lso found in the tissue culture of ovine pulmonry rtery smooth muscle (supplementl Figure II) nd in the orgn culture of rt pulmonry rtery (supplementl Figure IIC). Roles of ET Receptors To determine whether medited protein cronyltion is dependent on ET receptors, PSMCs were treted with Q123 (ET receptor ntgonist) 29 or Q88 (ET receptor kd 4 kd 4 MW (kd) Immunolot Coomssie stining 1 5 min exposure Cronyl content Cronylted proteins CP1 (28 kd) CP2 ( kd) CP3 (129 kd) CP4 ( 9 kd) CP5 ( 4 kd) CP6 ( 61 kd) CP ( 52 kd) CP8 ( 4 kd) CP9 ( 39 kd) Cronylted proteins CP1 (33 kd) CP11 (3 kd) CP12 (2 kd) CP13 (24 kd) CP14 (21 kd) CP15 (14 kd) CP16 (11 kd) CP ( 9 kd) 3 min exposure Figure 1. promotes protein cronyltion in PSMCs., Growth-rrested PSMCs were treted with (3 nmol/l) nd hrvested t the indicted time points. Cell lystes were prepred nd derivtized with DNPH. CP levels were monitored y Western lot with the DNP ntiody. Coomssie lue stining of the memrne shows the equl protein loding for ech smple. The r grph shows mens SEM (n 6) of the percentge of totl cronyl content reltive to untreted control, s determined y densitometry. The symols nd denote vlues tht re significntly different from untreted control nd the vlue from cells treted with for 1 minutes, respectively, t P.5., Numers were ssigned for ech of the CP nds to e CP1 to CP. The moleculr weights for CPs were clculted using the DNP-conjugted stndrd proteins. Five nd 3 minutes of exposure of the film re shown. ntgonist) 3 efore tretment with. Results show tht Q123 inhiited medited cronyltion of CP8, -1, -11, -12, nd - (Figure 2 nd supplementl Figure III); nd Q88 inhiited tht of CP3, -5, -, -1, -11, -12, -13, -15, nd - (Figure 2 nd supplementl Figure III). Neither of the ET receptor ntgonists influenced sl levels of protein cronyltion. This is the first demonstrtion of receptor-medited signling promoting protein cronyltion. Roles of H 2 O 2 nd Metl-Ctlyzed Oxidtion CPs re often formed vi peroxide-dependent Fenton rection, nd H 2 O 2 hs een shown to ply integrl roles in oxidnt-medited signl trnsduction. 9,32 Consistently, pretretment of PSMCs with eselen, 33 mimetic of glutthione peroxidse tht decomposes H 2 O 2, effectively inhiited ll of the ET receptor dependent protein cronyltion (Figure 2C nd supplementl Figure IIIC). Similr results were otined y ctlse overexpression vi denovirusmedited gene trnsfer (dt not shown). Furthermore, H 2 O 2 t the concentrtion s low s.5 mol/l mimicked

3 2 Circultion Reserch Ferury 15, 28 kd Immunolot kd Immunolot C kd Immunolot Downloded from y on Septemer 28, kd 4 D kd 4 kd 4 Untreted 1 µm Q µm Q µm Q123 1 µm Q123 Coomssie stining Immunolot µm H 2 O 2 (min) Coomssie stining 4 kd 4 E Untreted kd 1 µm Q88+ 1 µm Q88+ 1 µm Q88 1 µm Q88 Coomssie stining medited protein cronyltion (Figure 2D nd supplementl Figure IIID). These results suggest tht promotes protein cronyltion vi H 2 O 2. Recently, trnscription fctor PerR ws found to e regulted y metl-ctlyzed oxidtion. 15 Thus, we tested the effects of deferoxmine, nd we found tht this iron cheltor inhiited medited cronyltion of CP3, -, -13, -15, nd - (Figure 2E nd supplementl Figure IIIE), suggesting the role of metl-ctlyzed oxidtion. 4 kd 4 Immunolot Untreted Deferoxmine+ Deferoxmine Coomssie stining 4 kd 4 Untreted 1 µm Eselen+ 2 µm Eselen+ 1 µm Eselen 2 µm Eselen Coomssie stining Figure 2. ctivtes protein cronyltion vi ET receptors, H 2 O 2, nd metl-ctlyzed oxidtion. Growth-rrested PSMCs were pretreted with Q123 (), Q88 (), eselen (C), nd 5 mol/l deferoxmine (E) for 3 minutes nd then treted with (3 nmol/l) for 1 minutes. D, PSMCs were treted with H 2 O 2 (.5 mol/l) nd hrvested t the indicted time points. Cell lystes were prepred nd derivtized with DNPH. CP levels were monitored y Western lot. Representtive results re shown here, nd mens SEM with sttisticl nlyses re shown in supplementl Figure III. Kinetics of Medited Protein Cronyltion s shown in Figure 1, the increses in protein cronyls y pper trnsient in cell culture of PSMCs (Figure 1) nd in tissue culture of ovine pulmonry rtery smooth muscle (supplementl Figure II). nlyses of individul nds further reveled tht the levels of protein cronyls were trnsiently incresed. In PSMCs, cronyltion of CP1, -11, -12, -13, nd -15 incresed y 5 minutes, peked t 1 minutes, nd decresed to the sl level y 3 minutes of tretment (Figure 3). In contrst, cronyltion of these CPs in ovine ortic SMCs incresed rpidly y 5 minutes nd ws sustined for 2 to 3 minutes (Figure 3), differing from trnsient kinetics s seen in SMCs of pulmonry circultion. These experiments identified tht the medited stimultion of protein cronyltion is followed y normliztion of CPs (we termed decronyltion ) in pulmonry rtery SMCs, wheres in ortic SMCs, such decronyltion mechnism is slower. Mechnism of Decronyltion decrese in CPs my e ttriutle to the protesoml degrdtion of oxidtively modified proteins. 34,35 To test this, PSMCs were pretreted with MG132 (protesome inhiitor) for 3 minutes efore the tretment. Figure 4 shows tht medited increses in the mjority of CPs occurred trnsiently even fter the tretment with MG132 (3 mol/l) for 3 minutes. MG132 cn effectively inhiit proteolytic degrdtion in PSMCs, ecuse it inhiited the degrdtion of I - induced y tumor necrosis fctor- (supplementl Figure IV). Thus, the decronyltion mechnism ppers to e independent of the protesoml degrdtion of oxidtively modified proteins. We hypothesized tht reductive signling my e promoted y, susequently to oxidnt signling, perhps to serve s negtive-feedck mechnism. Consistently, CPs in PSMCs re reduced y -mercptoethnol (Figure 5). In

4 Wong et l Protein Cronyltion nd Redox Signling 3 Cronyl content CP1 (33kD) Cronyl content CP11 (3kD) Cronyl content CP12 (2kD) Cronyl content Cronyl content CP13 (24kD) CP1 (33kD) Cronyl content Cronyl content CP15 (14kD) CP11 (3kD) Cronyl content 2 1 CP12 (2kD) Figure 3. Kinetics of induced protein cronyltion. Growth-rrested PSMCs () nd ovine ortic SMCs () were treted with (3 nmol/l) nd hrvested t the indicted time points. Cell lystes were prepred nd derivtized with DNPH. CP levels were monitored y Western lot with the DNP ntiody. r grphs show mens SEM (n 6) of the percentge of cronyl content reltive to untreted control, s determined y densitometry. The symols nd denote vlues tht re significntly different from untreted control nd the vlue from cells treted with for 1 minutes, respectively, t P.5. Downloded from y on Septemer 28, 218 Cronyl content 2 1 CP13 (24kD) Cronyl content CP15 (14kD) these experiments, -mercptoethnol ws dded to PSMC lystes efore derivtiztion with DNPH. lso, n inhiitor of thioredoxin reductse, DNC 36 effectively promoted protein cronyltion (Figure 6 nd supplementl Figure V), suggesting the possile role of thioredoxin in reductive signling. Unlike trnsient cronyltion induced y in PSMCs, DNC-medited cronyltion ws sustined for t lest 3 minutes without exhiiting decronyltion. Tretment of PSMCs with fter DNC pretretment further promoted some degree of protein cronyltion; however, signling did not elicit decronyltion mechnism under these conditions (Figure 6 nd supplementl Figure V), demonstrting tht the thioredoxin system is required for medited decronyltion. Western lotting reveled tht time-dependently incresed the levels of thioredoxin protein in PSMCs (Figure 6C). Interestingly, not only did fil to increse thioredoxin in ortic SMCs, ut the sl level of ortic SMC thioredoxin ws found to e sustntilly lower compred with tht in pulmonry rtery SMCs (Figure 6C), providing possile mechnism for sustined kinetics of medited protein cronyltion nd the lck of the decronyltion mechnism in ortic SMCs. ctinomycin D, generl inhiitor of gene trnscription, inhiited medited increse in thioredoxin expression (supplementl Figure VC), suggesting tht ctivtes thioredoxin gene trnscription. These 2 1 results suggest tht, in pulmonry rtery SMCs, thioredoxin my ply role in signl trnsduction nd the regultion of decronyltion. To provide direct evidence tht thioredoxin regultes protein cronyltion in pulmonry rtery SMCs, the hypothesis tht promotes protein protein interctions etween thioredoxin nd CPs ws tested. Lyste smples of PSMCs with or without tretment were derivtized with DNPH nd immunoprecipitted with the 2,4- dinitrophenylhydrzone (DNP) ntiody, electrophoresed, nd lotted with the thioredoxin ntiody. Thioredoxin CP interctions pper intct even in the presence of DNPH derivtiztion. Thioredoxin interctions with CP were found to e incresed during the decronyltion phse (ie, 2 to 3 minutes fter tretment) (Figure 6D). Thioredoxin itself is not cronylted, ecuse the thioredoxin nd resides etween CP16 nd CP (dt not shown). These results suggest tht thioredoxin cronyl group interctions my regulte decronyltion. Furthermore, surprisingly, we found tht tretment initilly decresed thioredoxin CP interctions during the period when CPs re incresed (1 to 15 minutes), reveling the possiility tht the mechnism of the promotion of cronyltion my involve the dissocition of thioredoxin from the trget proteins. These results support experiments using DNC, which provided evidence for the role of thioredoxin system in the regultion of decronyltion.

5 4 Circultion Reserch Ferury 15, 28 Downloded from y on Septemer 28, 218 Cronyl content Cronyl content Cronyl content 2 1 CP (52kD) -MG132 +MG CP11 (3kD) 3 -MG132 +MG CP13 (24kD) -MG132 +MG Cronyl content Cronyl content Cronyl content CP15 (14kD) Identifiction of Cronylted Proteins To determine the identities of proteins tht re cronylted in response to, we further nlyzed 2D gels. Using informtion descried in supplementl Figure II, in which the first dimension ws seprted with ph rnge of 3 to 1 nd 4% to 2% grdient gels were used for the second dimension, ph 6 to 8 isoelectric focusing with 1.5% to 14% gels were chosen for the mplifiction of the region with undnt responsive CPs. We first identified 26 spots with intensities tht were incresed y tretment for 1 minutes (supplementl Figure VI). We then performed series of experiments to refine the spots to inducile CPs tht were inhiitle y ET receptor ntgonists eselen nd deferoxmine to define ET receptor nd Fenton rection dependent protein cronyltion. Supplementl Figure VI shows tht spot 1 is upregulted y, nd this upregultion ws ttenuted y Q123, Q88, eselen, nd deferoxmine. ll of the 26 spots were nlyzed, nd the results show tht spots 8, 1, 11, 12, 13, 14, 16,, 2, 21, nd 26 stisfied these criteri. The results of these experiments re shown in supplementl Figure VI nd VIC, nd the loctions of these 11 spots re summrized in supplementl Figure VID. ll of these 11 spots were excised from the gels nd underwent mss spectrometric nlyses. Mss spectrometric results re shown in supplementl Figure VII. Peptide mss fingerprinting nd sequence nlyses identified these proteins s het shock protein -1 (spot 8 nd 16), peroxiredoxin 6 (spot 1 nd 11), nnexin 2 (spot 12 nd 13), phosphoglycerte mutse 1 (spot 14), phosphoglycerte dehydrogense (spot ), cofilin-1 (spot 2), nnexin 1 (spot 21), nd DJ CP1 (33kD) -MG132 -MG132 +MG CP12 (2kD) 4 -MG132 +MG MG132 Figure 4. Effects of protesome inhiitor on decronyltion. PSMCs were pretreted with MG132 (3 mol/l) for 3 minutes nd then treted with. Cell lystes were prepred nd derivtized with DNPH. CP content ws monitored y Western lot. r grphs show mens SEM (n 6) of the percentge of cronyl content reltive to untreted control. The symols nd denote vlues tht re significntly different from untreted control nd the vlue from cells treted with for 1 minutes, respectively, t P.5. protein (spot 26) with high confidence ( 95%) nd n verge sequence coverge of 4%. We thus identified 8 proteins tht re cronylted in response to in receptor- nd Fenton rection dependent mnner, s summrized in Figure. To confirm these mss spectrometric results, immunoprecipittion/immunolot experiments were performed for peroxiredoxin 6 nd nnexin 1. Cell lystes from untreted nd treted cells were derivtized with DNPH, immunoprecipitted with DNP ntiody, nd lotted with either peroxiredoxin 6 (Figure ) or nnexin 1 (Figure C). Similrly, cronyltion of nnexin 2 nd cofilin-1 were lso confirmed (supplementl Figure VIII). The results demonstrte tht cronyltion of these proteins re promoted y. Consequences of Cronyltion hs een shown to inhiit prolifertion nd to promote poptosis in vrious cell types Similrly, in pulmonry rtery SMCs, overexpression of nnexin 1 reduced cell numer nd inhiited cell prolifertion induced y pltelet-derived growth fctor nd FS (Figure 8). We found tht cronyltion of nnexin 1 induced y ws followed y decresed nnexin 1 protein expression (Figure 8), wheres the protein expression of peroxiredoxin 6 ws not ltered (dt not shown). The medited reduction of nnexin 1 protein ws inhiited y protesome inhiitor, MG132 (Figure 8C). These results suggest tht, wheres mny of proteins cronylted in response to signling undergo decronyltion, cronyltion of nnexin 1 is followed y protesome-dependent degrdtion. The degrdtion of nnexin 1 should llevite the ctions of this protein to inhiit cell prolifertion nd promote poptosis, nd these events my e involved in the mechnism of medited pulmonry rtery SMC growth. Discussion In summry, here, we descrie novel redox signling events tht occur in response to lignd receptor interctions. In pulmonry rtery SMCs, promotes protein cronyltion vi n ET receptor dependent mechnism, followed y the reduction of CPs. We demonstrte tht receptor-dependent signling for protein cronyltion is medited y H 2 O 2 - nd iron-dependent Fenton rection. We lso show tht the susequent reductive event termed decronyltion ppers to involve thioredoxin nd thioredoxin reductse. lthough the mjority of proteins tht re cronylted in response to undergo reductiondependent decronyltion, some CPs re degrded y protesomes. ws found to e cronylted y in receptor- nd Fenton rection dependent mnner nd ecomes degrded y protesomes. ecuse nnexin 1 functions to promote poptosis nd inhiit cell growth, the cronyltiondriven degrdtion of this protein my serve s mechnisms for cell growth nd survivl signling y. These results show tht lignd receptor interctions cn promote protein cronyltion events, which my ply importnt functionl roles. Protein Cronyltion s Novel Mechnism in Redox Signling The field of ROS hd undergone prdigm shift when the ide hd emerged tht these species re not merely dmge-cusing

6 Wong et l Protein Cronyltion nd Redox Signling 5 Downloded from y on Septemer 28, 218 Cronyl content kd kd Immunolot Coomssie stining Cronyl content Cronyl content Cronyl content Cronyl content Cronyl content CP3 (129kD) ut cn lso serve s second messengers for signl trnsduction. 1,2,4 Susequent to the reports y Herzenerg nd collegues showing tht ntioxidnts such s N-cetylcysteine cn inhiit nucler fctor nd HIV ctivtion, 42,43 euerle nd collegues proposed tht H 2 O 2 is widely used second messenger for nucler fctor ctivtion in T cells. 8 In vsculr smooth muscle, erly work demonstrted tht ROS promoted cell growth, protooncogene expression, 44 nd C 2 signling. 1 Susequently, ROS were reported to medite signl trnsduction induced y ngiotensin II 6 nd pltelet-derived growth fctor 9 in ortic SMCs. In pulmonry rtery SMCs, Fnurg nd collegues found tht serotonin ctivtes the production of superoxide 45,46 nd H 2 O 2, 4 presumly vi ND(P)H oxidse. We hve shown tht GT-4 trnscription fctor plys n importnt role in pulmonry rtery SMC growth nd tht ntioxidnts cn inhiit serotonin-induced GT-4 ctivtion. 26 Lwrie et l 48 reported tht, in humn pulmonry rtery SMCs, serotonin ctivtes GT-4 vi ROS produced y monomine oxidse. hs lso een shown to produce ROS in fetl sheep pulmonry rtery SMCs vi ND(P)H oxidse, nd ntioxidnts CP (52kD) CP1 (33kD) CP12 (2kD) CP15 (14kD) Cronyl content Cronyl content Cronyl content Cronyl content Cronyl content CP5 (4kD) CP8 (4kD) CP11 (3kD) CP13 (24kD) CP (9kD) Figure 5. Effects of thiol reductnt on protein cronyltion. PSMCs were treted with, nd cell lystes were prepred with uffer with or without 2% -mercptoethnol ( ). Lystes were then derivtized with DNPH to monitor CP content. The r grph represents mens SEM (n 4) of the percentge of totl cronyl content reltive to untreted control without -mercptoethnol tretment. The symol * denotes tht 2 groups re significntly different from ech other. lock induced cell prolifertion, 2 suggesting tht ROS my ply role in medited pulmonry vsculr thickening. The trgets of ROS produced y s well s other meditors of pulmonry hypertension, however, hve not een defined. For the mechnisms of ROS signling, lignd receptor interctions producing ROS vi ND(P)H oxidse 6,49 nd ROS trgeting protein thiols 12,14 re currently populr proposed mechnisms. During vrious oxidtive stress conditions, protein oxidtion results in the inctivtion of protein functions. Cronyltion is one of oxidtion processes, which cn occur on protein molecules. Protein cronyls re quite stle products formed on proline, rginine, lysine or threonine residues, often in response to metl-ctlyzed Fenton rection. Protein cronyl groups rect with DNPH, nd vrious techniques hve een developed to detect their interctions; these groups lso hve een used effectively s mrkers of oxidtive stress. To our knowledge, the present study is the first demonstrtion of the protein cronyl formtion in response to lignd receptor interctions nd, therefore, the first dem-

7 6 Circultion Reserch Ferury 15, 28 Downloded from y on Septemer 28, 218 Cronyl content kd 4 kd 4 C Thioredoxin Thioredoxin expression DNC (min) Immunolot Coomssie stining ctin 2 1 PSMC Cronyl content CP1 (33kD) DNC+ DNC onstrtion of such formtion in signl trnsduction processes. Protein cronyltion could offer specific trgeting mechnism for oxidnt-medited signl trnsduction, ecuse sitedirected Fenton rection my occur t certin metl-inding sites within protein molecules. Further understnding of the roles of protein cronyltion in signl trnsduction pthwys my yield invlule informtion for the identifiction of trgeting mechnisms for ROS signling. Discovery of the Decronyltion Mechnism It ws interesting to note tht cronyltion of the mjority of the proteins were formed trnsiently in response to in pulmonry rtery SMCs. This led us to discover process in which cronyltion cn e eliminted in the cell. We termed this process decronyltion. pprent decronyltion could e oserved if CPs were degrded, ecuse oxidized proteins hve een shown to e susceptile to proteolytic degrdtion. 35 However, our experiments show tht decronyltion occurs PSMC SMC SMC D Time (min) IP: DNP lot: Thioredoxin Thioredoxin-cronylted protein interction Thioredoxin-cronylted protein interction/totl thioredoxin content Rte of cronyltion (.u.) Rte of de-cronyltion (.u.) CP1 (33kD) DNC DNC + CP1 (33kD) DNC DNC Figure 6. Role of thioredoxin in decronyltion mechnism., PSMCs were treted with DNC (3 mol/l). Cell lystes were prepred nd derivtized with DNPH. CP levels were monitored y Western lot with the DNP ntiody. The r grph shows mens SEM (n 5) of the percentge of totl cronyl content reltive to untreted control. The symol denotes vlues tht re significntly different from untreted control. Representtive dt nd results of totl cronyl nlyses re shown here. Results of nlyses of individul CPs re shown in supplementl Figure V., PSMCs were pretreted with DNC (3 mol/l) for 3 minutes efore tretment nd hrvested t the indicted time points. Groups treted with lone or DNC lone ct s controls. Cell lystes were prepred nd derivtized with DNPH. CP levels were monitored y Western lot with the DNP ntiody. The line grph shows mens SEM (n 5) of the percentge of cronyl content reltive to the level t minutes. The r grphs show mens SEM (n 5) of rtes of cronyltion ( to 1 minutes of tretment) nd decronyltion (1 to 3 minutes of tretment). The symol denotes vlues tht re significntly different from the group of lone. Representtive results of CP1 re shown here, nd results of other CPs re shown in supplementl Figure V. C, PSMCs nd ovine ortic SMCs were treted with. Cell lystes were prepred, nd thioredoxin expression ws monitored y Western lot. The r grph represents mens SEM (n 6) of the percentge of thioredoxin expression reltive to untreted control. The symol denotes vlues tht re significntly different from untreted control. D, PSMCs were treted with. Cell lystes were prepred, derivtized with DNPH, immunoprecipitted with DNP ntiody, nd lotted with thioredoxin ntiody. The r grph shows mens SEM (n 6) of the percentge of thioredoxin nd density reltive to untreted control. The symols nd denote vlues tht re significntly different from untreted control nd vlues from cells treted with for 15 minutes, respectively. even when protesomes re inhiited, suggesting tht the degrdtion mechnism my not explin our oservtions. It is plusile tht CPs my e ggregted; however, ecuse our cell lysis solution contins detergent, it is expected tht these proteins remin in the superntnt fter centrifugtion. We propose tht decronyltion is dependent on reduction rections through severl lines of evidence. First, CPs re sensitive to nd cn e eliminted y rections with reductnts such s -mercptoethnol. Second, thioredoxin reductse inhiitor cn promote protein cronyltion without the occurrence of decronyltion. Thirdly, when thioredoxin reductse is inhiited, promotes protein cronyltion in sustined fshion without the occurrence of decronyltion. lso, it is interesting to note tht thioredoxin is upregulted in pulmonry rtery SMCs in response to, nd its expression is sustntilly lower in ortic SMCs, where the decronyltion event is not pprent. These results indicte tht thioredoxin, which hs een shown to ply integrl roles in redox signling, 5 my lso

8 Wong et l Protein Cronyltion nd Redox Signling Downloded from y on Septemer 28, 218 IP-DNP with lot-peroxiredoxin 6/peroxiredoxin 6 kd 88 6 ph 8 Phosphoglycerte dehydrogense nnexin Phosphoglycerte Peroxiredoxin 6 mutse Het-shock 2 Het-shock protein et-1 18 protein et-1 DJ-1 protein Cofilin-1 IP-DNP lot-peroxiredoxin Peroxiredoxin 6 Untreted Untreted IP-DNP with lot-nnexin 1/nnexin 1 IP-DNP lot- Untreted regulte signl trnsduction mechnism involving protein cronyltion nd decronyltion. ecuse protein cronyltion is not chemiclly reversile, 51 the nture of decronyltion is not yet known. Clinicl Implictions ecuse ET receptor ntgonists re used for the tretment of humn pulmonry hypertension, these redox regultory mechnisms promoted y my offer importnt insights to therpeutic strtegies. Our lortory hs previously demonstrted tht cn promote nti-poptotic signling in pulmonry vsculr SMCs, 52 suggesting tht my exert multiple ctions to contriute to the development of pulmonry hypertension. ecuse proteins tht were found to e cronylted in response to signling include nnexin 1, which hs een shown to promote poptosis nd inhiit cell prolifertion, 3 41 protein cronyltion of this protein y my increse cell growth. In fct, we found tht medited cronyltion of nnexin 1 ws followed y protesome-dependent degrdtion, consistent with the ide tht llevites poptotic nd ntiprolifertive ctions of C 2 1 Untreted Figure. Identifictions of responsive CPs., Identified proteins through mss spectrometric nlyses re indicted in the immunolot of CPs in treted PSMCs., PSMCs were treted with 1 minutes of (3 nmol/l). Cell lystes were prepred, derivtized with DNPH, immunoprecipitted with the DNP ntiody, nd sujected to Western lotting with the peroxiredoxin 6 ntiody. The r grph represents mens SEM (n 6) of the percentge of nd density reltive to untreted control. The symol denotes the vlue tht is significntly different from untreted control. C, PSMC lystes were derivtized with DNPH, immunoprecipitted with the DNP ntiody, nd sujected to Western lotting with the nnexin 1 ntiody. Chnges in cell numer (% Control) nnexin 1 for promoting survivl signling nd incresing SMC numer. Further studies of cronylted nnexin 1, s well s other identified proteins, should provide importnt informtion for determining the mechnism of redox regultion of signl trnsduction, s well s for identifying effective therpeutic interventions ginst pulmonry hypertension. Sources of Funding This work ws supported in prt y NIH grnts HL634 nd HL2844 (to Y.J.S.). None trnsfection.1% FS 1ng/ml PDGF 1% FS expression ctin 2 1 trnsfection.1% FS 1ng/ml PDGF 1% FS expression Disclosures References 15 -MG132 +MG rcher SL, Hung J, Henry T, Peterson D, Weir EK. redox-sed O 2 sensor in rt pulmonry vsculture. Circ Res. 1993;3: Weir EK, rcher SL. The mechnism of cute hypoxic pulmonry vsoconstriction: the tle of two chnnels. FSE J. 1995;9: Sen CK, Pcker L. ntioxidnt nd redox regultion of gene trnscription. FSE J. 1996;1: Suzuki YJ, Formn HJ, Sevnin. Oxidnts s stimultors of signl trnsduction. Free Rdic iol Med. 199;22: C expression ctin ctin MG132 +MG Figure 8. Consequences of nnexin 1 cronyltion., PSMCs were trnsfected with nnexin 1 DN for 6 hours nd growth-rrested for 2 dys efore tretments. The vlues in the r grph represent mens SEM (n 4) of the cell numer. The symols *,, nd denote vlues significntly different from untrnsfected control, untrnsfected plus pltelet-derived growth fctor (PDGF), nd untrnsfected plus FS (1%), respectively. The representtive immunolot nd the r grph show the protein expression of nnexin 1., PSMCs were treted with. Cell lystes were prepred, nd the levels of nnexin 1 nd ctin proteins were monitored y Western lot. The line grph represents mens SEM (n 6). The symol * denotes vlues tht re significntly different from untreted control. C, Cells were pretreted with MG132 (3 mol/l) for 3 minutes efore tretment. The r grph represents mens SEM (n 6).

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