Raina Devi Ramnath, Jia Sun, and Madhav Bhatia. Department of Pharmacology, National University of Singapore, Singapore

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1 -3565/9/ $. THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 39, No. Copyright 9 y The Amerin Soiety for Phrmology nd Experimentl s 48684/ JPET 39:48 48, 9 Printed in U.S.A. Involvement of Sr Fmily Kinses in Sustne P-Indued Chemokine Prodution in Mouse Pnreti Ainr Cells nd Its Signifine in Aute Pnretitis Rin Devi Rmnth, Ji Sun, nd Mdhv Bhti Deprtment of Phrmology, Ntionl University of Singpore, Singpore Reeived Novemer, 8; epted Ferury, 9 ABSTRACT Sustne P is known to ply key role in the pthogenesis of ute pnretitis. Sr fmily kinses (SFKs) re known to e involved in ytokine signling. However, the involvement of SFKs in sustne P-indued hemokine prodution nd its role in ute pnretitis hve not een investigted yet. To tht end, we hve used primry preprtions of mouse pnreti inr ells s our model to show tht sustne P/neurokinin reeptor (NKR) indued tivtion of SFKs. SFKs medited the tivtion of mitogen-tivted protein kinses [extrellulr signl-regulted kinse (ERK), -Jun NH -terminl kinse (JNK)], trnsription ftors [signl trnsduer nd tivtor of trnsription (STAT) 3, nuler ftor (NF) B, tivtor protein- (AP-)], nd prodution of hemokines in pnreti inr ells. We further tested the signifine of the SFK signling pthwy in ute pnretitis. Our results show, for the first time, tht tretment of mie with the potent nd seletive SFK inhiitor PP [4-mino-5-(4-hlorophenyl)-7-(t-utyl) Aute pnretitis is inresing in inidene nd is often ftl humn disese, in whih the pnres digests itself nd its surroundings (Bhti et l.,, ; Bhti, ; Bhti nd Moohhl, 4). Aumulting experimentl evidene hs suggested tht sustne P/NKR nd hemokines ply ritil roles in the pthogenesis of ute pnretitis. We hve shown previously tht sustne P y itself used n inresed synthesis in CC nd CXC hemokines in pnreti inr ells (Rmnth nd Bhti, 6). Moreover, lokde of sustne P reeptor, NKR, ttenuted This work ws supported y the Ntionl Medil Reserh Counil [Grnt R ]; nd y the Biomedil Reserh Counil [Grnt R ]. Artile, pulition dte, nd ittion informtion n e found t doi:.4/jpet pyrzolo [3,4-D] pyrimidine], ut not its negtive inhiitor PP3 (4-mino-7-phenylpyrzol [3,4-D] pyrimidine), redued the severity of pnretitis. This ws proven y signifint ttenution of hypermylsemi, pnreti myeloperoxidse tivity, hemokines, nd wter ontent. Histologil evidene of diminished pnreti injury lso onfirmed the protetive effet of the inhiition of SFKs. Moreover, tretment with the sustne P reeptor ntgonist [(S,3S)-is--(diphenylmethyl)- N-((-methoxyphenyl)-methyl)--ziylo(...)-otn-3- mine] ttenuted ute pnretitis-indued tivtion of SFKs, ERK, JNK, STAT3, NF B, nd AP-. The proposed signling pthwy through whih sustne P medites ute pnretitis is through sustne P/NKR-SFKs-(ERK, JNK)- (STAT3, NF B, AP-) hemokines. In light of our study, we propose tht drugs trgeting the sustne P-medited signling pthwys ould prove enefiil in improving tretment effiy in ute pnretitis. hemokine prodution in pnreti inr ells nd proteted mie from ute pnretitis (Lu et l., 5; Rmnth et l., 7; Sun nd Bhti, 7). SFKs, speifilly Sr, hve een widely studied in tumorigenesis. However, reent evidene hs reveled tht SFKs re mong the most importnt fmilies for the intrellulr signl trnsdution relted to ute inflmmtory responses (Yun, ; Armstrong, 4; Lowell, 4). Severl niml studies hve shown tht inhiition of SFKs with smll hemil inhiitors prevented ishemi-reperfusion-indued injury in the rin nd hert. Moreover, lokde of SFKs ttenuted sepsis, ute lung injury, nd other orgn dmge (Pul et l., ; Akiym et l., 4; Khdroo et l., 4; Kusk et l., 4; Lennmyr et l., 4; Weis et l., 4; Severgnini et l., 5). SFKs re tivted in response Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 9, 8 ABBREVIATIONS: NKR, neurokinin reeptor; SFK, Sr fmily kinse; STAT, signl trnsduer nd tivtor of trnsription; PP, 4-mino- 5-(4-hlorophenyl)-7-(t-utyl) pyrzolo [3,4-D] pyrimidine; PP3, 4-mino-7-phenylpyrzol [3,4-D] pyrimidine; NF, nuler ftor; AP-, tivtor protein-; ERK, extrellulr signl-regulted kinse; JNK, -Jun NH -terminl kinse; 96345, (S,3S)-is--(diphenylmethyl)-N-((-methoxyphenyl)-methyl)--ziylo(...)-otn-3-mine; PBST,.5% Tween in phosphte-uffered sline; HPRT, hypoxnthine-gunine phosphoriosyl trnsferse; ELISA, enzyme-linked immunosorent ssy; M, monoyte hemottrtnt protein; MIP, mrophge inflmmtory protein; MPO, myeloperoxidse; MAP, mitogen-tivted protein; SP, sustne P. 48

2 to the stimultion of vriety of ell surfe reeptors (Thoms nd Brugge, 997). One suh reeptor type is the G protein-oupled reeptor. One tivted, Sr is then ple of interting with nd tivting the trnsription ftor STAT3 (Brown, 996). The STAT fmily of proteins hs een implited in the funtions of wide rnge of ells (Akir, ; Bromerg nd Drnell, ), nd it is known to tivte vrious key inflmmtory meditors; for exmple, the ytokine signling pthwy (Severgnini et l., 4). Limited knowledge is ville on the role of SFKs in hemokine prodution in ute pnretitis. Therefore, the im ws to investigte the role of SFKs in mediting sustne P-indued hemokine prodution in pnreti inr ells nd lso the underlying signl trnsdution mehnisms involved. Moreover, we sought to test the signifine of our in vitro findings in n in vivo model of ute pnretitis. Mterils nd Methods Animl Welfre. All niml experiments were pproved y the Animl Ethis Committee of Ntionl University of Singpore nd rried out in ordne with estlished Interntionl Guiding Priniples for Animl Reserh. Mle Swiss lino mouse (5 3 g) were mintined in the Animl Housing Unit of the Ntionl University of Singpore in n environment with ontrolled temperture ( 4 C) nd lighting (-/-h light/drk yle). Stndrd lortory how nd drinking wter were provided d liitum. A period of dys ws llowed for nimls to limtize efore ny experimentl mnipultions were undertken. Test System Used. Pnreti inr ells were otined from mouse pnres y ollgense tretment s desried previously (Bhti et l., ; Rmnth et l., 8). In rief, pnreses from three Swiss mie ( 5 g) were removed, infused with uffer A (4 mm NCl, 4.7 mm KCl,.3 mm MgCl, mm CCl, mm gluose, mm HEPES, ph 7.) ontining IU/ml ollgense (Worthington Biohemils, Freehold, NJ) nd.5 mg/ml soyen trypsin inhiitor (Sigm-Aldrih, St. Louis, MO), nd inuted in shking wter th for min t 37 C. The digested tissue ws entrifuged through 5 mg/ml ovine serum lumin nd wshed twie with uffer A for further experiments. A ell suspension (in uffer A) onsisting of only smll lumps, round three to five inr ells, ws used to rry out the following experiments. Viility of Mouse Pnreti Ainr Cells. Viility of the pnreti inr ells ws determined y trypn lue dye exlusion ssy. One drop of.4% trypn lue dye ws dded to one drop of the isolted inr ells. The viility ws determined under the light mirosope (Crl Zeiss GmH, Jen, Germny). The numer of unstined ells/lumps ws expressed s perentge of the totl numer of ells/lumps. This proess ws repeted for different fields, nd the verge ws then lulted. In ll experiments, ell viility ws greter thn 95%. In Vitro Experimentl Design. Pnreti inr ells (5 l of ell suspension) were treted with sustne P (Sigm-Aldrih) t onentrtion of M for, 3, 5,, 5, 3, nd 45 min t 37 C. After tretment with sustne P, the ells were lysed to detet for SFK tivtion y Western lot nlysis. In some experiments, ells were either pretreted with potent nd seletive inhiitor of the Sr fmily of protein tyrosine kinses, PP (Cliohem, Sn Diego, CA) t nd M, or negtive ontrol for the Sr fmily protein tyrosine kinse inhiitor, PP3 (Cliohem) t M, for 3 min followed y stimultion with M sustne P or vehile for or 45 min t 37 C. The superntnt ws used susequently for hemokine detetion, nd the pellet ws used for either nuler extrt to detet STAT3, NF B (p65), nd AP-(-Jun) tivtion or ell lysis for Role of SFKs in Aute Pnretitis 49 Western lot nlysis to detet SFKs, ERK, nd JNK. In nother experiment, isolted pnreti inr ells were preinuted with the seletive NKR ntgonist, 96345, t M (Pfizer Centrl Reserh, Groton, CT) for 3 min followed y tretment with M sustne P or vehile for min t 37 C. The ells were lysed susequently nd used for Western lot nlysis to detet SFKs. Preprtion of Totl Cell Lystes for Western Blot Anlysis. After tretment, pnreti inr ells or pnreti tissue were homogenized on ie in rdioimmunopreipittion ssy uffer supplemented with mm phenylmethylsulfonyl fluoride nd the protese inhiitor oktil (Sigm-Aldrih) ontining pepsttin, leupeptin, hymosttin, ntipin, nd protinin (5 g/ml eh) nd entrifuged t 4 C for 5 min t 4,g. The superntnts were olleted nd stored t 8 C until use. Protein onentrtions were determined y using the Bio-Rd (Herules, CA) protein ssy. Five miroliters of smple ws dded to 5 l of Brdford regent (Bio- Rd) nd red t 595 nm fter short inution (5 min) t room temperture. Western Blot Anlysis. Cell lystes (5- g protein) were seprted on % SDS-polyrylmide gel nd eletrophoretilly trnsferred to nitroellulose memrnes (Bio-Rd). Nonspeifi inding ws loked y -h inution of the memrnes, t room temperture, in 5% nonft dry milk in phosphte-uffered sline Tween (PBST) (.5% Tween in phosphte-uffered sline, ph 7.4). The lots were then inuted overnight t 4 C with the primry ntiodies (t : dilutions in the uffer ontining.5% nonft dry milk in PBST), phospho-sr fmily, phospho-erk/, phosphostress-tivted protein kinse/jnk (Cell Signling Tehnology In., Dnvers, MA), nd HPRT, purhsed from Snt Cruz Biotehnology, In. (Snt Cruz, CA). HPRT ws used s the housekeeping protein. The phospho-sr fmily (Tyr46) ntiody detets endogenous levels of Sr only when phosphorylted t Tyr46. The ntiody my ross-ret with other Sr fmily memers (Lyn, Fyn, Lk, Yes, nd Hk) when phosphorylted t equivlent sites. It does not rossret with Sr phosphorylted t Tyr57. Afterwrd, the memrnes were wshed four times with PBST nd finlly inuted for h t room temperture with got nti-rit horserdish peroxidse-onjugted seondry ntiody (Snt Cruz Biotehnology, In.) t : dilutions in the uffer ontining.5% nonft dry milk in PBST. The lots were developed for visuliztion using n enhned hemiluminesene detetion kit (Piere Chemil, Rokford, IL). The densities of the nds were quntified using UVP GelDo-It Imging Systems. Nuler Extrt Preprtion. Nuler extrts were prepred y employing kit (Ative Motif In., Crlsd, CA), s desried previously (Rmnth et l., 8). Nuler extrt ws prepred from oth isolted pnreti inr ells nd pnreti tissue fter erulein-indued ute pnretitis. STAT3/NF B/AP- DNA-Binding Ativity. The inding of STAT3/NF B/AP- to DNA ws mesured in nuler extrts with ELISA-sed TrnsAM STAT3/NF B p65/ap- -jun ssy kits (Ative Motif, SiMed, Asi). This ssy uses multiwell pltes oted with n unleled oligonuleotide ontining the onsensus inding site for STAT [5 -TTCCCGGAA-3 )/NF B (5 -GGGACTTTCC-3 )/ AP- (5 -TGAGTCA-3 ]. Nuler proteins (5 g) were dded to eh well nd inuted for h t room temperture to llow STAT3/ NF B/AP- DNA inding. Susequently, y using n ntiody tht is direted ginst STAT3/NF B p65/ap- -jun suunit, the STAT3/ NF B/AP- omplex ound to the oligonuleotide is deteted. Addition of the seondry ntiody onjugted to horserdish peroxidse provides sensitive olorimetri redout tht is esily quntified y spetrophotometry. Asorne ws red t 45 nm within 5 min y using 96-well miroplte reder (Ten, Durhm, NC). The wildtype onsensus oligonuleotide is provided s ompetitor for STAT3/NF B/AP- inding to monitor the speifiity of the ssy. Results were expressed s -fold inrese over the ontrol group. Chemokine Detetion. Pnreti inr ell superntnt nd pnreti homogente were ssyed for M-, MIP-, nd MIP- Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 9, 8

3 4 Rmnth et l. Time Control SP (Tyr 46) (Tyr 46) HPRT HPRT d Time (min).5.5 Control SP Fig.. Sustne P (SP)/NKR indues time-dependent inrese nd derese in phosphoryltion of Sr fmily (Tyr46) in mouse pnreti inr ells. Freshly isolted pnreti ini were inuted with either M SP/vehile (sline) for, 3, 5,, 5, 3, nd 45 min t 37 C or preinuted with M for 3 min followed y stimultion with M SP for min. The ells were lysed, nd ell proteins were sujeted to Western lot nlysis using ntiodies ginst phospho-sr fmily (Tyr46) nd HPRT ( nd ). Densitometri nlysis of Western lot experiments were performed, nd the group dt from three independent preprtions (n 3) re presented in nd d. The results re representtive of three independent (n 3) experiments. Results shown re the mens S.E., P.5 ompred with -min ontrol;, P.5 ompred with SP. (Tyr 46) p-erk p-jnk HPRT PP SP (µm) C SP P 44 P 4 P 54 P 46 d p-44 ERK Control SP µm µm p-54 JNK PP SP p-4 ERK Control SP µm µm p-46 JNK PP SP Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 9, Control SP µm µm Control SP µm µm Control SP µm µm PP SP PP SP PP SP Fig.. Sustne P (SP)-indued tivtion of ERK nd JNK is medited y SFKs in pnreti inr ells. Freshly isolted pnreti ini were preinuted with PP t or M or vehile (dimethyl sulfoxide) for 3 min t 37 C followed y stimultion with M SP for min t 37 C. Cells were susequently lysed, nd ell proteins were sujeted to Western lot nlysis using ntiodies ginst phospho-sr fmily (Tyr46), phospho- ERK, phospho-jnk, nd HPRT (). The phosphorylted suunits, suh s the p-sr fmily, p-44 ERK, p-4 ERK, p-54 JNK, nd p-46 JNK, hve een quntified. Densitometri nlyses of Western lot experiments were performed, nd the group dt from three independent preprtions (n 3) re presented in to d. Results shown re the mens S.E., P.5 ompred with ontrol;, P.5 ompred with SP.

4 Role of SFKs in Aute Pnretitis 4 STAT3 AP Control SP µm µm Control SP µm µm NFkB PP SP Control SP µm µm PP SP PP SP Fig. 3. SFKs re involved in SP-indued STAT3, NF B, nd AP- tivtion in pnreti inr ells. Freshly isolted pnreti ini were preinuted with PP t nd M or vehile (dimethyl sulfoxide) followed y stimultion with M SP for 45 min. The ells were seprted from inution medium y entrifugtion. The pellet (ells) ws used for STAT3 (), NF B (), nd AP- () nuler extrtion. STAT3, NF B, nd AP- DNA-inding ssys were then rried out s desried under Mterils nd Methods. The results re representtive of three independent (n 3) experiments. Results shown re the mens SE., P.5 ompred with ontrol;, P.5 ompred with SP. Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 9, 8 using sndwih ELISA, ording to the mnufturer s instrutions (Duoset kit; R&D Systems, Minnepolis, MN). For the M- ssy, the nti-m- primry ntiody ws liquoted onto ELISA pltes nd inuted t room temperture overnight. Smples ( l of ell superntnt) nd stndrds were inuted t room temperture for h, the pltes were wshed, nd iotinylted nti- M- ntiody ws dded nd left t room temperture for h. Pltes were wshed gin, nd streptvidin ound to horserdish peroxidse ws dded for min t room temperture. After further wsh, tetrmethylenzidine ws dded for olor development, nd the retion ws terminted with MH SO 4. Asorne ws red t 45 nm within 5 min y using 96-well miroplte reder (Ten). The sme proedure ws followed for the detetion of the remining hemokines MIP- nd MIP-. Indution of Aute Pnretitis. Swiss mie ( 5 g) were rndomly ssigned to ontrol or experimentl groups using or more nimls for eh group. Animls were given hourly intrperitonel injetions of norml sline or sline ontining erulein (5 g/kg) for h. PP t doses of.5,., nd.5 mg/kg, PP3 t dose of. mg/kg, nd t dose of.5 mg/kg were dministered to mie intrperitonelly either h efore or h fter the first erulein injetion. One hour fter the lst erulein injetion, nimls were srified y n intrperitonel injetion of lethl dose of pentoritone. Hrvested heprinized lood ws entrifuged, nd the plsm ws removed nd stored t 8 C. Rndom ross-setions of the hed, ody, nd til of the pnres were fixed in 4% neutrl phosphte-uffered formlin, then emedded in prffin wx. Freshly hrvested smples of pnres were weighed nd then dried nd reweighed to determine pnreti wter ontent (Bhti et l., 998). Smples of pnres were stored t 8 C for susequent mesurement of tissue myeloperoxidse (MPO) tivity nd for ELISA nd Western lot nlysis. Amylse Estimtion. Amylse tivity ws mesured using kineti spetrophotometri ssy. Plsm smples were inuted with the sustrte, 4,6-ethylidene (G 7 )-p-nitrophenyl (G )--D-mltoheptoside (Sigm-Aldrih), for min t 37 C nd sorne me-

5 4 Rmnth et l. M- MIP MIP-α Control SP µm µm µm PP SP Control SP µm µm µm PP SP PP3 SP PP3 SP Control SP µm µm µm PP SP PP3 SP Fig. 4. SFKs re involved in the seretion of CC nd CXC hemokines in pnreti inr ells. Freshly isolted pnreti ini were preinuted with either PP t nd M or PP3 t M for 3 min followed y stimultion with M SP for 45 min. The superntnt ws used to mesure M- (), MIP- (), nd MIP- () levels y ELISA s desried under Mterils nd Methods. The results re representtive of three independent (n 3) experiments. Results shown re the mens S.E., P.5 ompred with ontrol;, P.5 ompred with SP. Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 9, 8 sured every minute for the susequent min t 45 nm (Pierre et l., 976; Bhti et l., 998, ). The hnge in sorne ws used to lulte the mylse tivity. Myeloperoxidse Estimtion. Neutrophil sequestrtion in pnres ws quntified y mesuring tissue MPO tivity (Bhti et l., 998, ). Tissue smples were thwed, homogenized in mm phosphte uffer, ph 7.4, entrifuged (,g, min, 4 C), nd the resulting pellet ws resuspended in 5 mm phosphte uffer, ph 6., ontining.5% hexdeyltrimethylmmonium romide (Sigm-Aldrih). The suspension ws sujeted to four yles of freezing nd thwing nd further disrupted y sonition (4 s). The smple ws then entrifuged (,g, 5 min, 4 C), nd the superntnt ws used for the MPO ssy. The retion mixture onsisted of the superntnt,.6 mm tetrmethylenzidine (Sigm-Aldrih), 8 mm sodium phosphte uffer, ph 5.4, nd.3 mm hydrogen peroxide. This mixture ws inuted for s, the retion ws terminted with MH SO 4, nd the sorne ws mesured t 45 nm. This sorne ws then orreted for the protein ontent of the tissue smple. Morphologil Exmintion. Prffin-emedded pnres smples were setioned (5 m), stined with hemtoxylin/eosin, nd exmined under light mirosopy t 4 mgnifitions. For these studies, eight rndomly hosen mirosopi fields ( 5) were exmined for eh tissue smple. Pnreti dmge ws ssessed sed on the presene of inr ell ghosts, vuoliztion, swelling of inr ells, edem, nd infiltrtion of neutrophils. Sttistis. Dt re expressed s the men S.E.M. The signifine of hnges ws evluted y using nlysis of vrine. If nlysis of vrine indited signifint differene, the dt were nlyzed y using Tukey s method s post ho test for the differene etween groups. A P vlue of.5 ws onsidered to indite signifint differene.

6 Role of SFKs in Aute Pnretitis 43 Amylse U/L Pnres Wter Content (%) Control Cerulein Control Cerulein.5.5 Amylse U/L Results PP ( PP3 ( PP ( PP3 ( d Pnres Wter Content (%) PP ( PP3 ( PP ( Sustne P/NKR Indues Rpid nd Trnsient Inrese in Phosphoryltion of Sr Fmily (Tyr46) in Mouse Pnreti Ainr Cells. Pnreti inr ells were stimulted with M sustne P or vehile (sline) for, 3, 5,, 5, 3, nd 45 min t 37 C. Cells were then lysed, nd ell proteins were sujeted to Western lot nlysis. As shown in Fig., nd, sustne P indued phosphoryltion of the Sr fmily (Tyr46) in time-dependent mnner up to min, followed y time-dependent derese. Densitometri nlysis of Western lot experiments reveled signifint inrese in phosphoryltion Sr fmily (Tyr46) t nd 5 min ompred with -min ontrol. No signifint hnge ws oserved when pnreti inr ells were treted with the vehile t different time points. Pnreti ini were lso pretreted with M 96345, seletive NKR ntgonist, for 3 min followed y stimultion with M sustne P for min. Cells were then lysed, nd ell proteins were sujeted to Western lot nlysis. As demonstrted in Fig., nd d, signifintly redued sustne P-indued phosphoryltion of Sr fmily (Tyr46) in pnreti inr ells. No signifint hnge ws deteted in the housekeeping proteins HPRT. This shows tht sustne P-indued phosphoryltion of Sr fmily is medited through sustne P/NKR in pnreti inr ells. Involvement of SFKs in Sustne P-Indued MAP Kinses in Pnreti Ainr Cells. Stimultion of pnreti inr ells with M sustne P signifintly upregulted phosphoryltion of the Sr fmily (Tyr46), ERK, nd JNK. Pnreti inr ells were pretreted for 3 min with the seletive inhiitor of the Sr fmily of protein tyrosine kinses, PP, t nd M, followed y stimultion with M sustne P for min. Cells were then lysed, nd ell proteins were sujeted to Western lot nlysis. As shown in Fig., PP signifintly deresed sustne P- indued phosphoryltion of the Sr fmily (Tyr46), hene onfirming its inhiitory effet. Moreover, PP dose-dependently ttenuted sustne P-indued phosphoryltion of ERK nd JNK in pnreti inr ells (Fig., nd d). No signifint hnge ws deteted in the housekeeping proteins HPRT. Sustne P-Indued SFK Is Involved in Ativtion of STAT3, NF B, nd AP- in Pnreti Ainr Cells. Pnreti inr ells were preinuted for 3 min with PP t nd M followed y stimultion with M sustne P for 45 min. This time point ws estlished from PP3 ( Fig. 5. Effets of prophylti nd therpeuti PP dministrtion on the severity of ute pnretitis. Mie (n in eh group) were given hourly injetions of erulein (5 g/kg i.p.). PP or PP3 ws dministered to mie intrperitonelly either prophyltilly ( h efore) or therpeutilly h fter the first erulein injetion. One hour fter the lst erulein injetion, mie were srified y n intrperitonel injetion of lethl dose of pentoritone. Plsm mylse tivity ( nd ) nd pnreti edem (wter ontent; nd d) were determined s desried under Mterils nd Methods. Results shown re the mens S.E., P.5 when erulein- or PP3-treted nimls were ompred with pleo-treted nimls., P.5 when PP-treted nimls were ompred with erulein-treted nimls. Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 9, 8

7 44 Rmnth et l. erlier dt (Rmnth nd Bhti, 6). STAT3, NF B, nd AP- DNA-inding ssy reveled tht tretment with sustne P led to notle inrese in the tivity of STAT3, NF B, nd AP- in pnreti inr ell. As shown in Fig. 3, to, pretretment with PP ttenuted sustne P-indued DNA-inding tivity of STAT3, NF B, nd AP-. SFKs Medite Sustne P-Indued Prodution of CC nd CXC Chemokines in Pnreti Ainr Cells. Tretment of pnreti ini with M sustne P resulted in inresed prodution of CC hemokine M-, MIP-, nd CXC hemokine MIP- (Fig. 4). Pnreti inr ells were pretreted with either PP t nd M orits negtive ontrol PP3 t M for 3 min followed y stimultion with M sustne P for 45 min. The protein levels of these hemokines were determined y ELISA. Figure 4, to, shows tht pretretment with PP mrkedly deresed M-, MIP-, nd MIP- prodution. However, tretment with the negtive ontrol PP3 hd no signifint effet on hemokine prodution ompred with sustne P-only treted ells. Effet of nd Tretment with PP on the Severity of Cerulein-Indued Aute Pnretitis. Aute pnretitis ws indued y intrperitonel dministrtion of erulein t dose of 5 g/kg hourly for h. Evidene of pnreti injury in ute pnretitis mie ws onfirmed y rise in plsm mylse, s shown in Fig. 5, nd. There ws lso n inrese in pnreti edem s evidened y elevted pnreti wter ontent, s shown in Fig. 5, nd d. Animls were dministered either PP or PP3 h efore or fter strting erulein injetion. Both prophylti nd therpeuti tretment with PP signifintly ttenuted plsm mylse levels nd pnreti wter ontent ompred with nimls treted with erulein lone (Fig. 5, nd ). Qulittive histologil exmintion of pnres setions onfirmed protetion y PP tretment on ute pnretitis in terms of inr ell injury/nerosis, neutrophil infiltrtion, nd edem (Fig. 6). However, prophylti nd therpeuti tretment with PP3 hd no protetive effet on pnreti injury in ute pnretitis ompred with mie treted with erulein lone. Involvement of SFKs in the Moiliztion of Neutrophils nd Chemokines in Aute Pnretitis. Indution of ute pnretitis y erulein hyperstimultion resulted in heightened pnreti MPO, mesure of neutrophil infiltrtion (Fig. 7). Aute pnretitis lso resulted in inresed levels of pnreti CC hemokine M-, MIP-, nd CXC hemokine MIP- ompred with pleo-treted nimls. tretment with PP resulted in derese in MPO tivity, M-, MIP-, nd MIP- levels in the pnres ompred with erulein-treted nimls. Moreover, mie dministered with PP therpeutilly hd signifint redution in pnreti MPO tivity nd hemokines M-, MIP-, nd MIP- levels ompred with erulein-treted mie (Fig. 7). Inhiition of SFKs Attenuted the Ativtion of Pnreti STAT3, NF B, AP-, nd MAP Kinses in Aute Pnretitis. Cerulein-indued ute pnretitis resulted in signifint tivtion of trnsription ftors STAT3, NF B, nd AP- in pnres ompred with pleo-treted nimls (Fig. 8). Tretment with PP oth prophyltilly nd therpeutilly signifintly ttenuted the tivtion of Fig. 6. Morphologil hnges in mouse pnres on indution of ute pnretitis with/without prophylti nd therpeuti tretment with PP or PP3. Representtive mirogrphs from eh group were shown ( 4 mgnifitions). A, ontrol, no pnretitis. B, erulein-indued ute pnretitis. C, erulein-indued ute pnretitis in mie dministered mg/kg PP3 (prophylti). D, erulein-indued ute pnretitis in mie dministered mg/kg PP3 (therpeuti). E, eruleinindued ute pnretitis in mie dministered mg/kg PP (prophylti). F, erulein-indued ute pnretitis in mie dministered mg/kg PP (therpeuti). pnreti STAT3, NF B, nd AP- ompred with erulein-treted nimls, s shown in Fig. 8, to. To further explore the signling pthwys medited y SRC tyrosine kinses in ute pnretitis, we exmined the role of MAP kinses. Aute pnretitis inresed phosphoryltion of MAP kinses ERK nd JNK in the pnres ompred with pleo-treted nimls. Both prophylti nd therpeuti tretment with PP signifintly ttenuted tivtion of ERK nd JNK ompred with erulein-treted mie (Fig. 8, d to f). No signifint hnge ws oserved in the housekeeping protein HPRT in the pnres. Sustne P/NKIR-Medited Ativtion of Pnreti SFKs nd Its Downstrem Signling Pthwy in Aute Pnretitis. Tretment with oth prophyltilly nd therpeutilly ttenuted the tivtion of pnreti SFKs nd MAP kinses ERK nd JNK ompred with erulein-treted mie s shown in Fig. 9. No signifint hnge ws oserved in the housekeeping protein HPRT in the pnres. Moreover, s shown in Fig. 8, to, loked the up-regultion of pnreti STAT3, NF B, nd AP- in ute pnretitis. Disussion SFKs hve een shown to ply key role in ytokine signling nd inflmmtory responses (Chturvedi et l., Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 9, 8

8 Role of SFKs in Aute Pnretitis 45 Pnres MPO Pnres MIP Pnres M Control Cerulein.5.5 PP ( Control Cerulein.5.5 PP ( PP ( PP ( d Pnres MIP Control Cerulein.5.5 PP ( Control Cerulein.5.5 PP ( PP ( PP ( Fig. 7. Involvement of SFKs in the moiliztion of pnreti neutrophils nd hemokine in ute pnretitis. Mie (n in eh group) were given hourly injetions of erulein (5 g/kg i.p.). PP ws dministered in mie t doses of.5,, nd.5 mg/kg i.p. h efore or t dose of mg/kg h fter the first erulein injetion. One hour fter the lst erulein injetion, mie were srified y n intrperitonel injetion of lethl dose of pentoritone, nd pnreti MPO (), M- (), MIP- (), nd MIP- (d) levels were mesured s desried under Mterils nd Methods. Results shown re the mens S.E., P.5 when erulein-treted nimls were ompred with pleo-treted nimls., P.5 when PP-treted nimls were ompred with erulein-treted nimls. Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 9, 8 998; Song et l., 3). However, the involvement of SFKs in sustne P-indued hemokine prodution nd lso its role in ute pnretitis hve not een investigted yet. To tht end, we hve demonstrted tht sustne P/NKR indued tivtion of the Sr fmily in pnreti inr ells. SFKs lso medited sustne P-indued tivtion of MAP kinses ERK nd JNK. We lso illustrted tht sustne P-indued phosphoryltion of Sr tyrosine kinses ws involved in the tivtion of STAT3, NF B, nd AP- nd prodution of hemokines M-, MIP-, nd MIP- in pnreti inr ells. Our results re in greement with severl other studies, wherey sustne P ws found to indue Sr tivtion, whih ws lso implited in the tivtion of MAP kinses (Dell Ro et l., 997; DeFe et l., ; Ymguhi et l., 5). Moreover, MAP kinses were found to modulte the trnsriptionl tivity of STAT3 (Chung et l., 997; Turkson et l., 999). We hve demonstrted previously the involvement of ERK, JNK, NF B, nd AP- in sustne P indued hemokine synthesis (Rmnth et l., 7). Sustne P-indued tivtion of SFKs most likely is mediting hemokine prodution through the ERK- nd JNK-indued trnsription ftors NF B nd AP-. However, whether sustne P-indued STAT3 tivtion is diretly involved in the prodution of hemokines remined to e investigted. On the one hnd, studies hve suggested tht STAT3 plys pivotl role in orhestrting inflmmtory responses y inresing the expression of inflmmtory meditors, ytokines, nd hemokines (Shumnn et l., 996; Severgnini et l., 4). However, on the other hnd, studies hve reveled tht STAT3 modultes nti-inflmmtory responses (Tked et l., 999; Mtsukw et l., 3). Hene, more work needs to e rried

9 46 Rmnth et l. d PP ( Pnres STAT PP PP p-erk p-jnk HPRT Control Ce Pro The P 44 P 4 P 54 P 46 Pnres NF B Pnres AP PP PP PP PP out to eluidte the omplexity of STAT3 signling mehnisms in ute pnretitis. We further sought to investigte the in vivo relevne of the in vitro findings. Our results show tht tretment of nimls with the potent nd seletive inhiitor of the Sr fmily of protein tyrosine kinses, PP, ut not its negtive inhiitor, PP3 (either prophylti or therpeuti), redues the severity of pnretitis s evidened y signifint ttenution of hypermylsemi, pnreti MPO tivity, hemokine prodution, nd wter ontent, whih is mesure of edem. Moreover, histologil evidene of diminished pnreti injury, suh s inr ell injury/nerosis, neutrophil infiltrtion, nd edem, lso onfirmed the protetive effet of the inhiition of SFKs on ute pnretitis. In line with our study, Sr tyrosine kinse inhiitors were found to suppress inflmmtory responses in vivo y loking the funtion of inflmmtory ells inluding neutrophils, monoytes, nd mrophges (Okutni et l., 6). Using the in vivo model of ute pnretitis, we further explored the moleulr mehnisms through whih SFKs proteted ginst ute pnretitis, nd we found tht SFKs medite their protetive effets through the sme signling pthwy tht we hve shown in our in vitro model of isolted e f p-44 ERK PP ( p-54 JNK PP ( PP ( PP ( p-4 ERK inr ells. It hs een shown tht tretment with the NKR ntgonist proteted mie ginst ute pnretitis y ttenuting hemokine prodution (Lu et l., 5; Sun nd Bhti, 7). In the present study, we hve further shown tht ttenuted tivtion of SFKs, ERK, JNK, STAT3, NF B, nd AP- in ute pnretitis. Bsed on our results, we proposed tht elevted level of sustne P, whih is produed s result of ute pnretitis, inds to NKR to tivte severl signling moleules to medite hemokine prodution. One suh signling omplex is SFK, nd its lokde ttenuted MAP kinses, STAT3, NF B, nd AP-, nd hemokines, thus resulting in protetion ginst ute pnretitis. The proposed signling pthwy through whih sustne P medites ute pnretitis is through sustne P/NKR-SFKs-(ERK, JNK)- (STAT3, NF B, AP-)-(M-, MIP-, MIP-). The mehnism of tivtion of SFKs in ute pnretitis is most likely to e multiftoril; however, one of the ftors involved is the neuropeptide sustne P. Using isolted pnreti inr ell preprtions, we were le to show tht sustne P/NKR indues tivtion of SRC tyrosine kinses. Blokde of SFKs ttenuted hemokine prodution oth in vitro nd in vivo nd proteted the mie ginst ute PP ( p-46 JNK PP ( PP ( PP ( Fig. 8. Inhiition of SFKs ttenuted the tivtion of pnreti STAT3, NF B, AP-, nd MAP kinses in ute pnretitis. Mie (n in eh group) were given hourly injetions of erulein (5 g/kg i.p.). PP ws dministered to mie t dose of mg/kg i.p. h efore or h fter the first erulein injetion. One hour fter the lst erulein injetion, mie were srified y n intrperitonel injetion of lethl dose of pentoritone. The tivtion of pnreti STAT3 (), NF B (), AP- (), nd MAP kinses (d f) ws quntified s desried under Mterils nd Methods. Results shown re the mens S.E., P.5 when erulein-treted nimls were ompred with pleo-treted nimls., P.5 when PP-treted nimls were ompred with erulein-treted nimls. Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 9, 8

10 Role of SFKs in Aute Pnretitis (.5 Control Ce Pro The (Tyr 46) p-erk p-jnk HPRT p-44 ERK p-4 ERK pnretitis. This study gives us deeper insight into the mehnisms y whih sustne P ontriutes to ute pnretitis. Inresed understnding of the signl trnsdution mehnisms involved in ute pnretitis would filitte the disovery of novel therpeuti pprohes tht n trget seletive pthwys to prevent disese progression in ute pnretitis nd/or improve tretment effiy. Aknowledgments We thnk Mei Leng Shoon for tehnil ssistne. Referenes Akir S () Roles of STAT3 defined y tissue-speifi gene trgeting. Onogene 9:67 6. Akiym C, Yuguhi T, Nishio M, Tomishim T, Fujink T, Tniguhi M, Nkjim Y, Kohmur E, nd Yoshimine T (4) Sr fmily kinse inhiitor PP redues seondry dmge fter spinl ord ompression in rts. J Neurotrum : Armstrong SC (4) Protein kinse tivtion nd myordil ishemi/ reperfusion injury. Crdiovs Res 6: Bhti M () Novel therpeuti trgets for ute pnretitis nd ssoited multiple orgn dysfuntion syndrome. Curr Drug Trgets Inflmm Allergy : Bhti M, Brdy M, Kng YK, Costello E, Newton DJ, Christms SE, Neoptolemos JP, nd Slvin J () M- ut not CINC synthesis is inresed in rt pnreti ini in response to erulein hyperstimultion. Am J Physiol Gstrointest Liver Physiol 8:G77 G85. Bhti M, Brdy M, Shokuhi S, Christms S, Neoptolemos JP, nd Slvin J () Inflmmtory meditors in ute pnretitis. J Pthol 9:7 5. Bhti M, Brdy M, Zgorski J, Christms SE, Cmpell F, Neoptolemos JP, nd Slvin J () Tretment with neutrlizing ntiody ginst ytokine indued neutrophil hemottrtnt (CINC) protets rts ginst ute pnretitis ssoited lung injury. Gut 47: Bhti M nd Moohhl S (4) Role of inflmmtory meditors in the pthophysiology of ute respirtory distress syndrome. J Pthol : Bhti M, Neoptolemos JP, nd Slvin J () Inflmmtory meditors s therpeuti trgets in ute pnretitis. Curr Opin Investig Drugs : Bhti M, Sluj AK, Hofuer B, Frossrd JL, Lee HS, Cstgliuolo I, Wng CC, Gerrd N, Pothoulkis C, nd Steer ML (998) Role of sustne P nd the d p54-jnk p46-jnk neurokinin reeptor in ute pnretitis nd pnretitis ssoited lung injury. Pro Ntl Ad Si U S A 95: Bromerg J nd Drnell JE Jr () The role of STATs in trnsriptionl ontrol nd their impt on ellulr funtion. Onogene 9: Brown MT nd Cooper JA (996) Regultion, sustrtes nd funtions of Sr. Biohim Biophys At 87: 49. Chturvedi P, Reddy MV, nd Reddy EP (998) Sr kinses nd not JAKs tivte STATs during IL-3 indued myeloid ell prolifertion. Onogene 6: Chung J, Uhid E, Grmmer TC, nd Blenis J (997) STAT3 serine phosphoryltion y ERK-dependent nd -independent pthwys negtively modultes its tyrosine phosphoryltion. Mol Cell Biol 7: DeFe KA, Vughn ZD, O Bryn EM, Nishijim D, Déry O, nd Bunnett NW () The prolifertive nd ntipoptoti effets of sustne P re filitted y formtion of et-rrestin-dependent sffolding omplex. Pro Ntl Ad Si U S A 97:86 9. Dell Ro GJ, vn Biesen T, Dk Y, Luttrell DK, Luttrell LM, nd Lefkowitz RJ (997) Rs-dependent mitogen-tivted protein kinse tivtion y G proteinoupled reeptors: onvergene of Gi- nd Gq-medited pthwys on lium/ lmodulin, Pyk, nd Sr kinse. J Biol Chem 7: Khdroo RG, He R, Prodo J, Powers KA, Mrshll JC, Kpus A, nd Rotstein OD (4) The role of the Sr fmily of tyrosine kinses fter oxidnt-indued lung injury in vivo. Surgery 36: Kusk G, Ishikw M, Nnd A, Grnger DN, nd Zhng JH (4) Signling pthwys for erly rin injury fter surhnoid hemorrhge. J Cere Blood Flow Met 4: Lu HY, Wong FL, nd Bhti M (5) A key role of neurokinin reeptors in ute pnretitis nd ssoited lung injury. Biohem Biophys Res Commun 37: Lennmyr F, Erisson A, Gerwins P, Akterin S, Ahlström H, nd Terént A (4) Sr fmily kinse-inhiitor PP redues fol ishemi rin injury. At Neurol Snd : Lowell CA (4) Sr-fmily kinses: rheostts of immune ell signling. Mol Immunol 4: Mtsukw A, Tked K, Kudo S, Med T, Kgym M, nd Akir S (3) Aerrnt inflmmtion nd lethlity to septi peritonitis in mie lking STAT3 in mrophges nd neutrophils. J Immunol 7: Okutni D, Lodyg M, Hn B, nd Liu M (6) Sr protein tyrosine kinse fmily nd ute inflmmtory responses. Am J Physiol Lung Cell Mol Physiol 9:L9 L4. Pul R, Zhng ZG, Elieiri BP, Jing Q, Boi AD, Zhng RL, Chopp M, nd Cheresh DA () Sr defiieny or lokde of Sr tivity in mie provides ererl protetion following stroke. Nt Med 7: 7. Pierre KJ, Tung KK, nd Ndj H (976) A new enzymti kineti method for the determintion of serum nd urine mylse. Clin Chem :9. Rmnth RD nd Bhti M (6) Sustne P tretment stimultes hemokine Fig. 9. Blokde of NKR redued ute pnretitis-indued tivtion of MAP kinses nd trnsription ftors STAT3, NF B, nd AP- in the pnres. Mie (n 6 in eh group) were given hourly injetions of erulein (5 g/kg i.p.) ws dministered to mie t dose of.5 mg/kg i.p., h efore or h fter the first erulein injetion. One hour fter the lst erulein injetion, mie were srified y n intrperitonel injetion of lethl dose of pentoritone. The tivtion of pnreti STAT3 nd MAP kinses () nd the densitometry nlysis ( nd d) were desried under Mterils nd Methods. Results shown re the mens S.E., P.5 when erulein-treted nimls were ompred with pleo-treted nimls., P.5 when treted nimls were ompred with erulein-treted nimls. Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 9, 8

11 48 Rmnth et l. synthesis in pnreti inr ells vi the tivtion of NF-kppB. Am J Physiol Gstrointest Liver Physiol 9:G3 G9. Rmnth RD, Sun J, Adhikri S, nd Bhti M (7) Effet of MAP kinses on hemokine synthesis indued y sustne P in mouse pnreti inr ells. J Cell Mol Med : Rmnth RD, Sun J, nd Bhti M (8) Role of lium in sustne P-indued hemokine synthesis in mouse pnreti inr ells. Br J Phrmol 54: Shumnn RR, Kirshning CJ, Unehun A, Aerle HP, Knope HP, Lmping N, Ulevith RJ, nd Herrmnn F (996) The lipopolyshride-inding protein is seretory lss ute-phse protein whose gene is trnsriptionlly tivted y APRF/STAT/3 nd other ytokine-induile nuler proteins. Mol Cell Biol 6: Severgnini M, Tkhshi S, Rozo LM, Homer RJ, Kuhn C, Jhung JW, Perides G, Steer M, Hssoun PM, Fnurg BL, et l. (4) Ativtion of the STAT pthwy in ute lung injury. Am J Physiol Lung Cell Mol Physiol 86:L8 L9. Severgnini M, Tkhshi S, Tu P, Perides G, Homer RJ, Jhung JW, Bhvsr D, Cohrn BH, nd Simon AR (5) Inhiition of the Sr nd Jk kinses protets ginst lipopolyshride-indued ute lung injury. Am J Respir Crit Cre Med 7: Song L, Turkson J, Krrs JG, Jove R, nd Hur EB (3) Ativtion of Stt3 y reeptor tyrosine kinses nd ytokines regultes survivl in humn non-smll ell rinom ells. Onogene : Sun J nd Bhti M (7) Blokde of neurokinin reeptor ttenutes CC nd CXC hemokine prodution in experimentl ute pnretitis nd ssoited lung injury. Am J Physiol Gstrointest Liver Physiol 9:G43 G53. Tked K, Clusen BE, Kisho T, Tsujimur T, Terd N, Förster I, nd Akir S (999) Enhned Th tivity nd development of hroni enteroolitis in mie devoid of Stt3 in mrophges nd neutrophils. Immunity : Thoms SM nd Brugge JS (997) Cellulr funtions regulted y Sr fmily kinses. Annu Rev Cell Dev Biol 3: Turkson J, Bowmn T, Adnne J, Zhng Y, Djeu JY, Sekhrm M, Frnk DA, Holzmn LB, Wu J, Seti S, et l. (999) Requirement for Rs/R-medited p38 nd -Jun N-terminl kinse signling in Stt3 trnsriptionl tivity indued y the Sr onoprotein. Mol Cell Biol 9: Weis S, Shintni S, Weer A, Kirhmir R, Wood M, Crvens A, MShrry H, Iwkur A, Yoon YS, Himes N, et l. (4) Sr lokde stilizes Flk/ dherin omplex, reduing edem nd tissue injury following myordil infrtion. J Clin Invest 3: Ymguhi K, Kugimiy T, nd Miyzki T (5) Sustne P reeptor in U373 MG humn stroytom ells tivtes mitogen-tivted protein kinses ERK/ through Sr. Brin Tumor Pthol : 8. Ymguhi K, Rihrdson MD, Bigner DD, nd Kwtr MM (5) Signl trnsdution through sustne P reeptor in humn gliolstom ells: roles for Sr nd PKCdelt. Cner Chemother Phrmol 56: Yun SY () Protein kinse signling in the modultion of mirovsulr permeility. Vsul Phrmol 39:3 3. Address orrespondene to: Dr. Mdhv Bhti, Deprtment of Phrmology, Ntionl University of Singpore, Yong Loo Lin Shool of Mediine, Centre for Life Sienes, 8 Medil Drive, Singpore E-mil: mhti@nus.edu.sg Downloded from jpet.spetjournls.org t ASPET Journls on Septemer 9, 8