Transient receptor potential ankyrin 1 (TRPA1) is functionally expressed in primary human osteoarthritic chondrocytes

RESEARCH ARTICLE Open Access T ansien ecep o po en ial anky in 1 (TRPA1) is unc ionally exp essed in p ima y human os eoa h i ic chond ocy es Elina Nummenmaa 1 , Ma i Hämäläinen 1 , Lau i J. Moilanen 1 , E ja-Leena Paukke i 1 , Riina M. Nieminen 1 , Teemu Moilanen 1,2 , Ka iina Vuol eenaho 1 and Ee a Moilanen 1* Abs ac Backg ound: T ansien ecep o po en ial anky in 1 (TRPA1) is a memb ane-associa ed ca ion channel, widely exp essed in neu onal cells and in ol ed in nocicep ion and neu ogenic in lamma ion. We showed ecen ly ha TRPA1 media es ca ilage deg ada ion and join pain in he MIA-model o os eoa h i is (OA) sugges ing a hi he o unknown ole o TRPA1 in OA. The e o e, we aimed o in es iga e whe he TRPA1 is exp essed and unc ional in human OA chond ocy es. Me hods: Exp ession o TRPA1 in p ima y human OA chond ocy es was assessed by qRT-PCR and Wes e n blo . The unc ionali y o he TRPA1 channel was assessed by Ca 2+ -in lux measu emen s. P oduc ion o MMP-1, MMP-3, MMP-13, IL-6, and PGE 2 subsequen o TRPA1 ac i a ion was measu ed by immunoassay. Resul s: We show he e o he i s ime ha TRPA1 is exp essed in p ima y human OA chond ocy es and i s exp ession is inc eased ollowing s imula ion wi h in lamma o y ac o s IL-1β, IL-17, LPS, and esis in. Fu he , he TRPA1 channel was ound o be unc ional, as s imula ion wi h he TRPA1 agonis AITC caused an inc ease in Ca 2+ in lux, which was a enua ed by he TRPA1 an agonis HC-030031. Gene ic deple ion and pha macological inhibi ion o TRPA1 down egula ed he p oduc ion o MMP-1, MMP-3, MMP-13, IL-6, and PGE 2 in os eoa h i ic chond ocy es and mu ine ca ilage, espec i ely. Conclusions: The TRPA1 ca ion channel was ound o be unc ionally exp essed in p ima y human OA chond ocy es, which is an o iginal inding. The p esence and in lamma o y and ca abolic e ec s o TRPA1 in human OA chond ocy es p opose a highly in iguing ole o TRPA1 as a pa hogenic ac o and d ug a ge in OA. Keywo ds: Os eoa h i is, Chond ocy e, TRPA1, In lamma ion, Ma ix me allop o einase Backg ound T ansien ecep o po en ial anky in 1 (TRPA1) is a memb ane-associa ed ca ion channel which media es pain and hype algesia [1, 2] and unc ions as a chemo- senso o noxious compounds [3–5]. TRPA1 was i s disco e ed in 1999 [6] and has since hen been ound o be widely exp essed in a e en senso y neu ons, espe- cially in Aδand C ibe s o nocicep o s [7, 8]. In addi ion o pain, TRPA1 also has a ole in media ing neu ogenic in lamma ion [9, 10]. Mo e ecen ly, TRPA1 has been ound o be exp essed also in some nonneu o- nal cells such as ke a inocy es [11] and syno iocy es [12] bu he unc ional oles o nonneu onal exp ession e- main o be s udied. TRPA1 is ac i a ed by nume ous exogenous pungen compounds such as allyl iso hiocyana e (AITC) om mus a d oil [5], ac olein om exhaus umes and o- bacco smoke [9], and allicin om ga lic [3]. In e es - ingly, TRPA1 is also ac i a ed and sensi ized by agen s o med endogenously in in lamma o y eac ions, such as ni ic oxide [13], hyd ogen pe oxide [14] and ni o-oleic acid [15]. The ac i a ion o TRPA1 causes an in lux o ca ion ions, pa icula ly Ca 2+ , in o he ac i a ed cells [16] and his ele a ion o in acellula Ca 2+ has been * Co espondence: [email p o ec ed] 1 The Immunopha macology Resea ch G oup, Uni e si y o Tampe e School o Medicine and Tampe e Uni e si y Hospi al, Tampe e, Finland Full lis o au ho in o ma ion is a ailable a he end o he a icle © 2016 The Au ho (s). Open Access This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0 In e na ional License (h p://c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons license, and indica e i changes we e made. The C ea i e Commons Public Domain Dedica ion wai e (h p://c ea i ecommons.o g/publicdomain/ze o/1.0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed. Nummenmaa e al. A h i is Resea ch & The apy (2016) 18:185 DOI 10.1186/s13075-016-1080-4 shown o igge an ac ion po en ial in neu onal cells [16, 17]. In e es ingly, among he many egula o y e ec s o he al e a ions o in acellula Ca 2+ concen a ion, i s inc ease has also been shown o a ec he gene exp es- sion o in lamma o y media o s [18–20]. Recen e idence sugges s TRPA1 o ha e a ole in in- lamma ion h ough exogenous ac i a ion by TRPA1 ag- onis s and also h ough endogenous mechanisms. TRPA1 has been shown o media e ca ageenan-induced in lamma o y edema [21], umo nec osis ac o (TNF)- igge ed hype algesia [22], ai way hype eac i i y and in lamma ion [23, 24], and o ela e o acu e gou y a h- i is [25, 26]. Ve y ecen ly we ound ha TRPA1 has a ole in media ing acu e in lamma ion, ca ilage des uc- ion, and join pain in monosodium iodoace a e (MIA)- induced in lamma ion and os eoa h i is in he mouse [27]. Os eoa h i is (OA) is he mos common cause o musculoskele al disabili y and pain wo ldwide and i s p e alence is cons an ly inc easing as he popula ion ages. OA is a degene a i e disease o he join s, which is cha ac e ized by in lamma ion and hypoxia wi hin he join , leading o ca ilage deg ada ion, join de o mi y, disabili y, and pain [28, 29]. OA- ela ed ca ilage deg ad- a ion is caused by a g owing imbalance be ween he p o- duc ion o ca abolic, anabolic, and in lamma o y media o s wi hin he join d i en by he inc eased ex- p ession o ma ix-deg ading me allop o einases and p oin lamma o y media o s such as in e leukin (IL)-6 and p os aglandin E 2 (PGE 2 ) [28]. TRPA1 has no p e iously been in es iga ed in chon- d ocy es. Howe e , ac o s in ol ed in hypoxia and in- lamma ion, such as hyd ogen pe oxide (H 2 O 2 ), ni ic oxide (NO), and IL-6 ha e been shown o up egula e he exp ession o ac i a ion o TRPA1 in some o he cells [12–14]. Fu he mo e, he ac i a ion o TRPA1 has been epo ed o enhance he p oduc ion o in lamma o y ac- o s [12, 21, 26, 30]. Since he e is a hypoxic and in lam- ma o y s a e in OA join s [28, 31], and TRPA1 has been shown o be in ol ed in he media ion o acu e in lam- ma ion and ca ilage deg ada ion in MIA-induced os eo- a h i is [27], we hypo hesized ha TRPA1 is exp essed in he chond ocy es in os eoa h i ic join s, whe e i s ac- i a ion could play a i al pa in he in lamma ion and pa hogenesis o OA. In he p esen s udy, we es ed ha hypo hesis by measu ing he exp ession and unc ion o TRPA1 in p ima y human OA chond ocy es. Me hods Cell cul u e P ima y chond ocy e cul u es we e ca ied ou as p e i- ously desc ibed [32]. Le o e pieces o OA ca ilage om knee join eplacemen su ge y we e used unde ull pa ien consen . The pa ien s in his s udy ul illed he Ame ican College o Rheuma ology classi ica ion c i- e ia o OA [33] and he s udy was app o ed by he E hics Commi ee o Tampe e Uni e si y Hospi al, Tam- pe e, Finland ( e e ence numbe R09116), and ca ied ou in acco dance wi h he Decla a ion o Helsinki. The p ocedu es o isola e and cul u e he p ima y chond o- cy es a e desc ibed in he supplemen a y da a (Add- i ional ile 1). Du ing expe imen s he cells we e ea ed wi h IL-1β(R&D Sys ems Eu ope L d, Abingdon, UK), IL-17 (R&D Sys ems Eu ope L d.), lipopolysaccha ide (LPS) (Millipo e Sigma, S . Louis, MO, USA), esis in (BioVision Inc., Milpi as, CA, USA), he TRPA1 an ag- onis HC-030031 (Millipo e Sigma) o wi h combina- ions o hese compounds as indica ed. Immo alized human T/C28a2 chond ocy es [34] we e cul u ed as desc ibed in he supplemen a y da a (Add- i ional ile 1). Du ing he expe imen s T/C28a2 chond o- cy es we e ea ed wi h IL-1β(R&D Sys ems Eu ope L d), IL-17 (R&D Sys ems Eu ope L d.), LPS (Millipo e Sigma), HC-030031 (Millipo e Sigma) o wi h combina- ions o hese compounds as indica ed. HEK 293 human emb yonic kidney cells (Ame ican Type Cul u e Collec ion, Manassas, VA, USA) we e cul- u ed as desc ibed in he supplemen a y da a (Addi ional ile 1). The cells we e ans ec ed using 0.42 mg/cm 2 o human TRPA1 plasmid DNA (pCMV6-XL4 by O igene, Rock ille, MD, USA) wi h lipo ec amine 2000 (In i o- gen, Li e Technologies, Ca lsbad, CA, USA) acco ding o he manu ac u e ’s ins uc ions. Animals Wild- ype (WT) and TRPA1 knockou (KO) male B6;129P-T pa1( m1Kykw)/J mice (Cha les Ri e Labo a- o ies, Sulz eld, Ge many) aged 19–22 days we e used in mouse ca ilage cul u e expe imen s. Mice we e housed unde s anda d condi ions (12–12 h ligh –da k cycle, 22 ± 1 °C) wi h ood and wa e p o ided ad libi um. Animal expe imen s we e ca ied ou in acco dance wi h he le- gisla ion o he p o ec ion o animals used o scien i ic pu poses (Di ec i e 2010/63/EU) and he expe imen s we e app o ed by The Na ional Animal Expe imen Boa d ( e e ence numbe UTA 845/712-86). Animals we e sac i iced by ca bon monoxide ollowed by c anial disloca ion. Mouse ca ilage cul u e A e mice we e eu hanized, ull- hickness a icula ca - ilage om emo al heads we e emo ed and cul u ed as desc ibed in he supplemen a y da a (Addi ional ile 1). The ca ilage pieces we e exposed o IL-1β(R&D Sys- ems Eu ope L d.) o i s ehicle o 42 h and he ea e cul u e media we e collec ed and ma ix me allop o ein- ase (MMP)-3, IL-6, and PGE 2 concen a ions we e mea- su ed by immunoassay. Nummenmaa e al. A h i is Resea ch & The apy (2016) 18:185 Page 2 o 11 Wes e n blo measu emen s A e he cell cul u e expe imen s, o al p o ein was ex- ac ed, and TRPA1 was immunop ecipi a ed and ana- lyzed wi h Wes e n blo as desc ibed in he supplemen a y da a (Addi ional ile 1). TRPA1 an ibody NB110-40763 (No us Biologicals, LCC, Li le on, CO, USA) was used as he p ima y an ibody and goa an i- abbi HRP-conjuga e (sc-2004, San a C uz Bio echnol- ogy, Inc., Dallas, TX, USA) as he seconda y an ibody in he Wes e n blo analysis. Immunoassay Concen a ions o IL-6, MMP-1, MMP-3, MMP-13 and PGE 2 in medium samples we e de e mined by enzyme- linked immunoso ben assay (ELISA) wi h comme cial eagen s (PGE 2 : Cayman Chemical Co., Ann A bo , MI, USA; human IL-6: eBioscience Inc. San Diego, CA, USA; MMP-1, MMP-3, MMP-13 and mouse IL-6: R&D Sys- ems Eu ope L d). RNA ex ac ion and quan i a i e RT-PCR A he indica ed ime poin s, o al RNA was ex ac ed and analyzed by quan i a i e e e se ansc ip ion poly- me ase chain eac ion (qRT-PCR) o he exp ession o TRPA1 mRNA as desc ibed in he supplemen a y da a (Addi ional ile 1). Ca 2+ -in lux measu emen s TRPA1-media ed Ca 2+ in lux was measu ed in HEK293 cells [35] ans ec ed wi h human TRPA1 plasmid, in human T/C28a2 chond ocy es, and in p ima y human OA chond ocy es as desc ibed p e iously [36]. B ie ly, he cells we e loaded wi h 4 μM luo-3-ace oxyme hyl es e (Fluo-3-AM, Millipo e Sigma) and 0.08 % Plu onic F-127® (Millipo e Sigma in Hanks’balanced sal solu ion (HBSS, Lonza, Ve ie s, Belgium) con aining 1 mg/ml o bo ine se um albumin, 2.5 mM p obenecid and 25 mM HEPES pH 7.2 (all om Millipo e Sigma) o 30 min a oom empe a u e. The in acellula - ee Ca 2+ le els we e assessed by Vic o 3 1420 mul ilabel coun e (Pe - kin Elme , Wal ham, MA, USA) a exci a ion/emission wa eleng hs o 485/535 nm. In he expe imen s, he cells we e i s p eincuba ed wi h he TRPA1 an agonis HC- 030031 (100 μM, Millipo e Sigma) o he ehicle o 30 min a +37 °C. The ea e , he TRPA1 agonis allyl iso hiocyana e (AITC, 50 μM, Millipo e Sigma) was added and he measu emen s we e con inued o 30 s a e which a obus Ca 2+ in lux was induced by applica- ion o he con ol ionopho e compound ionomycin (1 μM, Millipo e Sigma). S a is ical analysis Da a we e analyzed using G aph-Pad InS a e sion 3.00 so wa e (G aphPad So wa e, San Diego, CA, USA). The esul s a e p esen ed as mean + s anda d e o o he mean (SEM) unless o he wise indica ed. Unpai ed es , pai ed es , one-way analysis o a iance (ANOVA) o epea ed-measu es ANOVA, ollowed by Dunne ’s es we e used in he s a is ical analysis. Di e ences we e conside ed signi ican a p< 0.05, p< 0.01, and p< 0.001. Resul s TRPA1 is exp essed in p ima y human OA chond ocy es and in immo alized human T/C28a2 chond ocy e cell line P ima y human OA chond ocy es and immo alized hu- man T/C28a2 chond ocy e cell line exp essed TRPA1. The exp ession was measu ed by quan i a i e RT-PCR on isola ed o al mRNA using a speci ic TaqMan assay. The p oin lamma o y cy okine IL-1βwas ound o in- c ease TRPA1 exp ession in a ime-dependen manne : in p ima y chond ocy es he exp ession o TRPA1 in- c eased up o 48 hou s and declined he ea e (Fig. 1a), whe eas in he human T/C28a2 chond ocy es he ex- p ession maximum was a 6 hou s (Fig. 1b). In addi ion, TRPA1 exp ession was also enhanced by in lamma o y ac o s IL-17, LPS, and esis in (Fig. 2). To e i y he ansla ion o TRPA1 mRNA in o p o- ein, we ex ac ed o al p o ein om p ima y human OA chond ocy es and human T/C28a2 chond ocy es and pe o med Wes e n blo analysis. HEK293 cells ansi- en ly ans ec ed wi h TRPA1 plasmid we e used as posi- i e con ol and he p o ein was de ec ed wi h a speci ic human TRPA1 an ibody. Rema kably, bo h cell ypes we e ound o exp ess TRPA1 p o ein as seen in Fig. 3. Human chond ocy es exp ess a unc ional TRPA1 channel To con i m ha TRPA1 mRNA and he subsequen p o- ein exp essed by human chond ocy es p oduces a unc- ional channel, Ca 2+ -in lux measu emen s we e ca ied ou . P ima y human chond ocy es and T/C28a2 chon- d ocy es we e cul u ed wi h IL-1β, which was ound o s imula e TRPA1 exp ession, o wi h i s ehicle o 24 h, and he ea e TRPA1 was ac i a ed wi h he TRPA1 agonis AITC. IL-1βs imula ion esul ed in an inc eased esponsi eness o AITC as seen as an enhanced Ca 2+ in- lux, and he selec i e TRPA1 an agonis HC-030031 was shown o p e en his e ec (Fig. 4). MMP, IL-6 and PGE 2 p oduc ion is down egula ed by gen- e ic deple ion and pha macological inhibi ion o TRPA1 A e inding ha unc ional TRPA1 was indeed exp essed in chond ocy es, we aimed o u he examine he possible a h i ogenic ole o he TRPA1 channel. We in es iga ed he e ec o gene ic deple ion o TRPA1 on he p oduc ion o OA- ela ed ac o s MMP-3, IL-6, and PGE 2 by using a icula ca ilage samples om TRPA1-de icien (knockou , KO) and co esponding wild- ype (WT) mice. IL-1β ea men inc eased MMP- Nummenmaa e al. A h i is Resea ch & The apy (2016) 18:185 Page 3 o 11 3, IL-6, and PGE 2 p oduc ion in ca ilage as expec ed. Rema kably, his esponse was signi ican ly a enua ed in he ca ilage om he TRPA1 KO mice as compa ed o he co esponding WT mice (Fig. 5). Fu he , we ea ed p ima y human chond ocy es wi h IL-1βalone and o- ge he wi h he selec i e TRPA1 an agonis HC-030031 o 24 h. In e es ingly, he selec i e TRPA1 an agonis HC-030031 down egula ed IL-1β-enhanced MMP-1, MMP-3, MMP-13, IL-6, and PGE 2 p oduc ion by 25– 45 % (Fig. 6), sugges ing ha TRPA1 plays a ole in he up egula ion o hese ca abolic and in lamma o y ac o s in OA ca ilage. Discussion The indings o he p esen s udy sugges a hi he o un- known ole o TRPA1 in he pa hogenesis o OA. We ha e shown o he i s ime he exp ession o he TRPA1 channel in p ima y human OA chond ocy es and in he human T/C28a2 chond ocy e cell line. We showed he exp ession o TRPA1 mRNA and p o ein by Fig. 1 P ima y human OA chond ocy es (a) and human T/C28a2 chond ocy e cell line (b) exp ess TRPA1 mRNA and i s exp ession is enhanced by IL-1 in a ime-dependen manne . Cul u es o p ima y human OA chond ocy es (a) and human T/C28a2 chond ocy es (b) we e s imula ed wi h IL-1β(100 pg/ml) o 0–96 h and 0–24 h, espec i ely, and he ea e o al RNA was ex ac ed. TRPA1 mRNA le els we e measu ed by qRT-PCR, and he esul s we e no malized agains GAPDH mRNA. The mRNA le els a e exp essed as a bi a y uni s wi h he le els measu ed a 24 h (a; p ima y OA chond ocy es) o 6 h (b; T/C28a2 chond ocy es) se as 100 %; and he alues a he o he ime poin s a e ela ed o hose alues. P ima y chond ocy e samples we e ob ained om h ee o i e di e en dono s and he expe imen s we e ca ied ou in duplica e. Human T/C28a2 chond ocy e expe imen s we e ca ied ou in quad uplica e. Resul s a e exp essed as mean + SEM. One-way ANOVA ollowed by Dunne ’s pos - es was used in he s a is ical analysis; ** indica es p< 0.01 compa ed o he con ol (0 h) sample. OA os eoa h i is, TRPA1 ansien ecep o po en ial anky in 1 Nummenmaa e al. A h i is Resea ch & The apy (2016) 18:185 Page 4 o 11 qRT-PCR and Wes e n blo , espec i ely. We we e also able o show ha he exp essed TRPA1 was unc ional, as e idenced by Ca 2+ -in lux measu emen s. Fu he , we ound TRPA1 o ha e a ole in media ing he p oduc ion o OA- ela ed ac o s MMP-1, MMP-3, MMP-13, IL-6, and PGE 2 as e idenced by pha macological inhibi ion and gene ic deple ion o TRPA1. TRPA1 was i s disco e ed in 1999 in e al lung ib o- blas s [6]. Since hen i has been mainly s udied in di e - en a e en senso y neu ons such as Aδand C ibe s o --- - - - - - - - - - - - - - -- - Fig. 2 TRPA1 mRNA exp ession is inc eased ollowing s imula ion wi h in lamma o y ac o s IL-1β, IL-17, LPS, and esis in in p ima y human OA chond ocy es (a-d) and in human T/C28a2 chond ocy e cell line (e). Isola ed p ima y human OA chond ocy es we e s imula ed wi h IL-1β(1–100 pg/ ml) (a), IL-17 (10–100 ng/ml) (b), LPS (10 ng/ml) (c). and esis in (0.3–3μg/ml) (d); and human T/C28a2 chond ocy es wi h IL-1β(1–100 pg/ml), IL-17 (100 ng/ml) and LPS (10 ng/ml) (e) o 24 h; and he ea e o al RNA was ex ac ed. TRPA1 mRNA le els we e measu ed by qRT-PCR, and he esul s we e no malized agains GAPDH mRNA le els. The esul s a e exp essed as a pe cen age in compa ison o un ea ed con ol samples, which was se as 100 %. P ima y chond ocy e samples we e ob ained om ou di e en dono s and he expe imen s we e pe o med in duplica e. Human T/C28a2 chond ocy e expe imen s we e ca ied ou in quad uplica e. Resul s a e exp essed as mean + SEM. Repea ed measu es ANOVA (a,b,d) and one-way ANOVA (e) ollowedbyDunne ’s pos - es o pai ed es (c) was used in he s a is ical analysis; * p<0.05and ** p< 0.01, compa ed o he un ea ed con ol samples. IL in e leukin, LPS lipopolysaccha ide, OA os eoa h i is, TRPA1 ansien ecep o po en ial anky in 1 Nummenmaa e al. A h i is Resea ch & The apy (2016) 18:185 Page 5 o 11 nocicep o s [7, 8]. Mo e ecen ly, howe e , TRPA1 has also been ound o be exp essed in some nonneu onal cells such as ke a inocy es [11, 37, 38], syno iocy es [12, 39] and ai way epi helial and smoo h muscle cells [30]. I is no ewo hy, ha no all o hese s udies ha e shown unc ionali y o he TRPA1 ion channel and some ha e only epo ed he exp ession o TRPA1 a he mRNA le el. In he p esen s udy, we ha e comp ehensi ely shown he exp ession and ac i a ion o TRPA1 in hu- man chond ocy es, o suppo he c i e ia se by Fe - nandes e al. [40]. We we e able o show o he i s ime he exp ession o bo h TRPA1 mRNA and p o ein and he unc ionali y o he TRPA1 channel in p ima y human OA chond ocy es and in human T/C28a2 chon- d ocy e cell line. This inding is pa icula ly in e es ing as in OA join s he e is a hypoxic [31] and in lamma o y [28, 41] s a e and ela ed ac o s, H 2 O 2 , NO, and IL-6, ha e p e iously been shown o up egula e he exp ession and ac i a ion o TRPA1 [12–14]. Acco ding o Ha ano e al. [12] he human TRPA1 p omo e has a leas six pu a i e nuclea ac o kappa B (NF-kB) binding si es and en co e hypoxia esponse elemen s (HREs), which a e binding si es o hypoxia-inducible ac o (HIF) an- sc ip ion ac o s. HIFs a e known o media e adap i e esponses o hypoxia as well as o be ac i a ed by in- lamma ion [42, 43] and he binding o HIFs o consen- sus HREs on hei a ge genes egula es gene ansc ip ion. A e disco e ing TRPA1 exp ession in chond ocy es, we aimed o in es iga e whe he in lamma o y ac o s/ mechanisms ela ed o he pa hogenesis o OA [28, 29] egula e exp ession o TRPA1, which would indica e a ole o TRPA1 as a media o in OA. IL-1βis conside ed as a majo playe in OA associa ed wi h ca ilage de- s uc ion. IL-1βis ele a ed in OA join s and i sup- p esses ype II collagen and agg ecan exp ession, s imula es he elease o MMP-1, MMP-3, and MMP-13, and induces he p oduc ion o IL-6 and some o he cy- okines as well as PGE 2 [28]. In pa IL-17 eeds o wa d hese mechanisms as i u he induces IL-1β, TNF, and IL-6 p oduc ion, up egula es NO and MMPs and down- egula es p o eoglycan le els ela ed o he pa hogenesis o OA [28]. Based on ou esul s, IL-1βand IL-17 bo h also induce TPRA1 exp ession and in iguingly, some o he IL-1β-induced in lamma o y and ca abolic e ec s a e pa ly media ed by TRPA1. In OA he inna e immune sys em and in pa icula oll-like ecep o s (TLRs) ac i- a ed by ca ilage ma ix deg ada ion p oduc s, also play a signi ican pa in disease p og ession. Chond ocy es exp ess TLRs, which igge majo in lamma o y pa h- ways and a e ac i a ed by bac e ial lipopolysaccha ide (LPS) and damage-associa ed molecula pa e ns [29], and also he adipocy okine esis in known o be exp essed in OA join s [44] has been shown o ans- duce i s e ec s h ough oll-like ecep o 4 [45]. In he p esen s udy, we ound ha bo h LPS and esis in in- c eased exp ession o TRPA1 in human chond ocy es, sugges ing a TLR-media ed mechanism o enhance TRPA1 exp ession in OA ca ilage. In suppo o he p esen esul s, Ha ano e al. showed ha TRPA1 gene exp ession was enhanced in syno iocy es by in lamma- o y ac o s TNF-αand IL-1 [12], and he p esen s udy oge he wi h ha o Ha ano e al. [12] sugges s a p e i- ously un ecognized mechanism ha links TRPA1 as an inducible ac o o join in lamma ion. Ac i a ion o TRPA1 esul s in a subs an ial in lux o Ca 2+ in o he s imula ed cells [46]. He e we e i ied he unc ionali y and ac i a ion o he TRPA1 channel in hu- man chond ocy es by measu ing Ca 2+ in lux using he TRPA1 agonis AITC as well as he TRPA1 an agonis HC-030031. As shown p e iously, ele a ed in acellula Ca 2+ concen a ion may a ec he exp ession o in lam- ma o y genes bo h in a di ec o indi ec manne [20]. In he p esen s udy, we ound ha TRPA1 egula ed he p oduc ion o in lamma o y and ca abolic ac o s, namely MMP enzymes, IL-6, and PGE 2 in chond ocy es. IL-1-induced MMP-3, IL-6, and PGE 2 p oduc ion in he ca ilage om TRPA1-de icien mice was less han hal β β Fig. 3 TRPA1 p o ein is exp essed in p ima y human OA chond ocy es and human T/C28a2 chond ocy e cell line. Chond ocy e cul u es we e s imula ed wi h IL-1β(100 pg/ml) o 24 h. Ex ac ed p o eins we e immunop ecipi a ed and TRPA1 was de ec ed wi h Wes e n blo analysis. HEK293 cells ansien ly ans ec ed wi h human TRPA1 plasmid we e used as a posi i e con ol. Rep esen a i e blo o h ee independen expe imen s wi h simila esul s. IL in e leukin, TRPA1 ansien ecep o po en ial anky in 1 Nummenmaa e al. A h i is Resea ch & The apy (2016) 18:185 Page 6 o 11 o ha ound in he ca ilage om wild- ype mice. Ac- co dingly, he selec i e TRPA1 an agonis HC-030031 educed IL-1-induced MMP-1, MMP-3, MMP-13, IL-6, and PGE 2 p oduc ion by 25–45 % in p ima y human OA chond ocy es. In he la e expe imen , he cells we e incuba ed in he p esence o IL-1 and HC-030031 o 24 h; he e o e he esul may be an unde es ima e o he e ec o o al inhibi ion o TRPA1 in OA chond o- cy es because HC-030031 is a e e sible TRPA1 an ag- onis wi h a ela i ely sho hal -li e [47]. These indings Fig. 4 The TRPA1 ion channel is unc ional in p ima y human OA chond ocy es (a,b) and human T/C28a2 chond ocy e cell line (c) as shown by TRPA1-media ed Ca 2+ in lux. P ima y human chond ocy es (a,b) and human T/C28a2 chond ocy es (c) we e cul u ed wi h o wi hou (con ol) IL-1β(100 pg/ml) o 24 h. HEK293 cells ans ec ed wi h plasmids encoding human TRPA1 we e used as posi i e con ol cells (d). The cells we e loaded wi h Fluo-3-AM and he TRPA1-media ed Ca 2+ in lux was measu ed by Vic o 3 mul ilabel coun e a exci a ion/emission wa eleng hs o 485/535 nm a 1/s equency. The cells we e i s p eincuba ed wi h he TRPA1 an agonis HC-030031 (100 μM) o he ehicle o 30 min a +37 ° C. In he measu emen s, basal luo escence was i s eco ded o 15 s and he ea e he selec i e TRPA1 agonis allyl iso hiocyana e (AITC; 50 μM) was added and he measu emen was con inued o 30 s a e which he con ol ionopho e compound ionomycin (1 μM) was in oduced o he cells. IL-1βs imula ion esul ed in an ele a ion in AITC-induced Ca 2+ in lux compa ed o uns imula ed con ol cells, and i was a enua ed by he selec i e TRPA1 an agonis HC-030031. The esul s we e no malized agains he backg ound and exp essed as mean o eigh simul aneous measu emen s. Cu es in A,Cand Dexp ess esul s om one ep esen a i e expe imen . In (b) a ea unde he cu e (AUC) om 15 o 45 s was calcula ed om measu emen s o p ima y chond ocy e om ou dono s (each wi h eigh epea s). Resul s a e exp essed as mean + SEM. Repea ed measu es ANOVA ollowed by Dunne ’s pos - es was used in he s a is ical analysis; ** p< 0.01 compa ed o he IL-1β- ea ed samples. IL in e leukin, OA os eoa h i is, TRPA1 ansien ecep o po en ial anky in 1 Nummenmaa e al. A h i is Resea ch & The apy (2016) 18:185 Page 7 o 11 a e suppo ed by p e ious s udies indica ing ha TRPA1 ac i a ion egula es he p oduc ion o IL-1 in ke a inocy es [38], IL-6 and IL-8 in syno iocy es [12], and PGE 2 along wi h leuko iene B 4 in ib oblas s and ke a inocy es [48]. We ha e ecen ly ound ha TRPA1 also egula es he ex- p ession o cyclooxygenase-2 (COX-2) [21, 27] and he p o- duc ion o monocy e chemo ac ic p o ein-1 (MCP-1), IL-6, IL-1β, myelope oxidase (MPO), MIP-1αand MIP-2 in in- lamma o y condi ions [26]. The de ailed molecula mecha- nisms o his egula ion emain, howe e , o be s udied. TRPA1 is shown o be in ol ed in pain, hype algesia, and neu ogenic in lamma ion [10, 16, 49, 50]. In OA- ela ed pain, he ole o TRPA1 has been in es iga ed in s udies by Moilanen e al. [27] McGa augh y e al. [51] and Okun e al. [52] using he MIA-model o OA. The wo i s - men ioned s udies [27, 51] concluded TRPA1 o con ibu e o join pain in expe imen al OA. In addi ion, Moilanen e al. [27] epo ed ha TRPA1-de icien mice de eloped less se e e ca ilage changes ollowing MIA injec ions. Acco d- ingly, we showed he e ha TRPA1 is unc ionally exp essed in chond ocy es. We also examined he possible unc ions o he channel by ea ing p ima y chond ocy e cul u es wi h IL-1βand he selec i e an agonis HC-030031 [2, 53, 54]. Ou esul s sugges an in lamma o y and ca abolic ole o TRPA1 in human chond ocy es, as we ound inhibi ion o TRPA1 o supp ess he p oduc ion o OA- ela ed ac o s MMP-1, MMP-3, MMP-13, IL-6, and PGE 2 .These esul s we e suppo ed by expe imen s wi h ca ilage om WT and TRPA1-de icien mice: ollowing s imula ion wi h IL- 1βMMP-3, IL-6, and PGE 2 p oduc ion was lowe in he ca ilage om TRPA1-de icien mice han om WT ani- mals. These esul s oge he sugges ha TRPA1-ac i a ing Fig. 5 IL-1β-induced p oduc ion o MMP-3 (a), IL-6 (b), and PGE 2 (c) in he ca ilage is a enua ed by gene ic deple ion o TRPA1. Ca ilage samples we e ob ained om TRPA1-de icien (knockou , KO) and co esponding wild- ype (WT) mice. The samples we e cul u ed wi h and wi hou IL-1β (100 pg/ml) o 42 h and he ea e he cul u e medium was collec ed and analyzed o concen a ions o MMP-3, IL-6, and PGE 2 by immunoassay. The esul s a e exp essed as mean + SEM, n = 6–9. Unpai ed es was used in he s a is ical analysis; * p<0.05, ** p< 0.01, and *** p< 0.001 compa ed o he WT mice. IL in e leukin, MMP ma ix me allop o einase, PGE 2 p os aglandin E 2 ,TRPA1 ansien ecep o po en ial anky in 1 Nummenmaa e al. A h i is Resea ch & The apy (2016) 18:185 Page 8 o 11 ac o s a e p esen in OA join s, and ha TRPA1 media es, a leas pa ly, OA- ela ed pain, in lamma ion, and ca ilage des uc ion in neu onal and nonneu onal cells in he join . Conclusions In conclusion, we ound he TRPA1 ca ion channel o be unc ionally exp essed in p ima y human OA chond ocy es and in pa o media e in lamma o y and ca abolic e ec s, which a e bo h o iginal indings. The in lamma o y and hyp- oxic en i onmen in he OA join is conduci e o enhance he exp ession and ac i a ion o TRPA1. The p esence and e ec s o TRPA1 in human OA ca ilage as ound in he p esen s udy, oge he wi h he p e ious indings on TRPA1 in expe imen ally induced OA [27, 51] p opose an in iguing ole o TRPA1 as a media o and d ug a ge in OA. Addi ional ile Addi ional ile 1: Supplemen a y da a. Supplemen a y in o ma ion o he “Me hods”. (DOCX 23 kb) Abb e ia ions AITC, allyl iso hiocyana e; ANOVA, analysis o a iance; COX-2, cyclooxygenase-2; ELISA, enzyme-linked immunoso ben assay; H 2 O 2 , hyd ogen pe oxide; HIF, hypoxia-inducible ac o ; HRE, hypoxia esponse elemen ; IL, in e leukin; KO, knockou ; LPS, lipopolysaccha ide; MCP-1, monocy e chemo ac ic p o ein-1; MIA, monosodium iodoace a e; MIP, mac ophage in lamma o y p o ein; MMP, ma ix me allop o einase; MPO, myelope oxidase; NF-kB, nuclea ac o -kappa B; NO, ni ic oxide; OA, os eoa h i is; PGE 2 , p os aglandin E 2 ; qRT-PCR, quan i a i e e e se ansc ip ion polyme ase chain eac ion; SEM, s anda d e o o he mean; TLR, oll-like ecep o ; TNF, umo nec osis ac o ; TRPA1, ansien ecep o po en ial anky in 1; WT, wild- ype Fig. 6 IL-1β-enhanced exp ession o MMP-1 (a), MMP-3 (b), MMP-13 (c), IL-6 (d), and PGE 2 (e) in p ima y human OA chond ocy es is a enua ed by pha macological inhibi ion o TRPA1. P ima y human OA chond ocy es we e s imula ed wi h IL-1β(100 pg/ml) in he p esence and absence o he selec i e TRPA1 an agonis HC-030031 (100 μM) o 24 h. MMP-1, MMP-3, MMP-13, IL-6, and PGE 2 concen a ions in he cul u e media we e measu ed by immunoassay and he esul s a e exp essed as mean + SEM. Samples we e ob ained om eigh pa ien s and he expe imen s we e ca ied ou in duplica e. Pai ed es was used in he s a is ical analysis; * p< 0.05, ** p< 0.01, and *** p< 0.001 compa ed o he IL-1β- ea ed samples. IL in e leukin, MMP ma ix me allop o einase, OA os eoa h i is, PGE 2 p os aglandin E 2 ,TRPA1 ansien ecep o po en ial anky in 1 Nummenmaa e al. A h i is Resea ch & The apy (2016) 18:185 Page 9 o 11