TRPA1 expression is downregulated by dexamethasone and aurothiomalate in human chondrocytes : TRPA1 as a novel factor and drug target in arthritis

1 Nummenmaa E, e al. RMD Open 2017;3:e000556. doi:10.1136/ mdopen-2017-000556 T ansien ecep o po en ial anky in 1 (TRPA1) is a ligand-ga ed memb ane-bound ca ion channel. TRPA1 has been la gely s udied in neu ons, whe e i media es pain and neu ogenic in lamma ion and ac s as a chemosenso o ha m ul exogenous compounds.1 2 Mo e ecen ly, TRPA1 has been ound o be ac i a ed also by endoge- nous compounds o med in in lamma o y condi ions cha ac e is ic o a h i is, such as eac i e oxygen and ni ogen species.3 We ha e ecen ly disco e ed ha TRPA1 is unc ionally exp essed in p ima y human os eoa h i ic chond ocy es,4 whe e i up egula ed he p oduc ion o media o s ela ed o a h i is: in e leukin (IL)-6, p os- aglandin E2 and ma ix me allop o einases 1, 3 and 13.4 Fu he mo e, we showed in monosodium iodoace a e-induced expe i- men al a h i is ha TRPA1 ac i a ion medi- a es in lamma ion, ca ilage deg ada ion and pain.5 TRPA1 hus eme ges as a no el ac o associa ed wi h a h i is. The e o e, we in es iga ed he e ec s o disease-mod- i ying an i heuma ic d ugs me ho exa e, sul asalazine, hyd oxychlo oquine and au o hiomala e, glucoco icoid dexame h- asone and non-s e oidal an i-in lamma o y d ug ibup o en on he exp ession o TRPA1 in human chond ocy es. Human T/C28a2 chond ocy es6 we e cul u ed wi h he a o emen ioned d ugs, along wi h IL-1β, which was ecen ly ound o up egula e TRPA1 exp ession in chon- d ocy es.4 Dexame hasone and au o hioma- la e inhibi ed TRPA1 mRNA exp ession in a dose-dependen manne ( igu e 1A), while me ho exa e (10 µM), sul asalazine (10 µM), hyd oxychlo oquine (10 µM) and ibup o en (10 µM) had no e ec . Dexame hasone and au o hiomala e also dec eased TRPA1 p o ein le els ( igu e 1C). Nex , we examined i he obse ed d ug e ec s on TRPA1 exp ession le els a e unc- ional, ha is, i hey a e ansla ed o changes in TRPA1-media ed calcium in lux. In chon- d ocy es, which had been cul u ed in he p esence o IL-1β, TRPA1-media ed calcium in lux was inc eased; while ha e ec was e e sed in cells which had been cul u ed wi h a combina ion o IL-1β and dexame hasone o au o hiomala e ( igu e 1D,E), con i ming Sho epo TRPA1 exp ession is down egula ed by dexame hasone and au o hiomala e in human chond ocy es: TRPA1 as a no el ac o and d ug a ge in a h i is Elina Nummenmaa,1 Ma i Hämäläinen,1 Lau i J Moilanen,1 Teemu Moilanen,1,2 Ka iina Vuol eenaho,1 Ee a Moilanen1 To ci e: Nummenmaae, hämäläinenM, MoilanenLJ, e al. pA1 exp ession is down egula ed by dexame hasone and au o hiomala e in human chond ocy es: pA1 as a no el ac o and d ug a ge in a h i is. RMD Open 2017;3:e000556. doi:10.1136/ mdopen-2017-000556 ►p epublica ion his o y o his pape is a ailable online. o iew hese iles please isi he jou nal online (h p:// dx. doi. o g/ 10. 1136/ mdopen- 2017- 000556). ecei ed 10 Augus 2017 Accep ed 14 Augus 2017 1 he Immunopha macology esea ch G oup, Facul y o Medicine and Li e Sciences, Uni e si y o ampe e and ampe e Uni e si y hospi al, ampe e, Finland 2Coxa hospi al o Join eplacemen , ampe e, Finland Co espondence o p o esso ee a Moilanen; ee a. moilanen@ u a. i In lamma o y a h i is Key messages Wha is al eady known abou his subjec ? ► ansien ecep o po en ial anky in 1 ( pA1) is a memb ane-associa ed ca ion channel p ima ily s udied in senso y neu ons, whe e i ac s as a chemosenso o pungen compounds and media es pain. ► pA1 has ecen ly eme ged as a po en ial ac o / media o in a h i is; we ha e shown pA1 o media e in lamma o y and ca abolic esponses in p ima y human chond ocy es, as well as ca ilage deg ada ion, in lamma ion and pain in an expe imen al model o a h i is. Wha does his s udy add? ► his s udy shows ha an i-in lamma o y d ugs dexame hasone and au o hiomala e down egula e he exp ession o unc ional pA1 in human chond ocy es. How migh his impac on clinical p ac ice? ► hese esul s show a p e iously unknown mechanism o ac ion o dexame hasone and au o hiomala e ia down egula ion o pA1, and hus p o ides a no el concep o he de elopmen o d ugs o a h i is wi h analgesic and disease modi ying p ope ies. g oup.bmj.com on Oc obe 9, 2017 - Published by h p:// mdopen.bmj.com/Downloaded om 2Nummenmaa e, e al. RMD Open 2017;3:e000556. doi:10.1136/ mdopen-2017-000556 RMD Open Figu e 1 Dexame hasone and au o hiomala e inhibi TRPA1 exp ession in human chond ocy es. Human chond ocy es (p ima y human chond ocy es o T/C28a2 chond ocy e cell line) we e cul u ed wi h IL-1β alone o in combina ion wi h he an i- in lamma o y compound dexame hasone o au o hiomala e, o wi h he selec i e NF-κB inhibi o PDTC a concen a ions gi en in he igu e. (A, B) Fo TRPA1 mRNA exp ession analysis, human T/C28a2 chond ocy es (A) we e incuba ed o 6 hou s and he expe imen s we e ca ied ou in quad uplica e; p ima y chond ocy es (B) we e incuba ed o 24 hou s and he expe imen s we e ca ied ou in duplica e and epea ed wi h cells om six dono pa ien s. To al RNA was ex ac ed and TRPA1 mRNA le els we e measu ed by quan i a i e RT-PCR (TaqMan Gene Exp ession Assay o human TRPA1, Hs00175798_m1), and he esul s we e no malised agains GAPDH mRNA le els. (C) Fo TRPA1 p o ein analysis human T/C28a2 chond ocy es we e incuba ed o 24 hou s a e which p o eins we e ex ac ed and immunop ecipi a ed wi h TRPA1 an ibody (SAB2105082, Sigma-Ald ich), and TRPA1 was de ec ed wi h Wes e n blo (wi h p ima y an ibody: NB110-40763, No usBiologicals). The igu e shows one ep esen a i e blo o six independen expe imen s wi h simila esul s. (D, E) Fo Ca2+in lux analysis human T/C28a2 chond ocy es we e incuba ed wi h IL-1β alone o in combina ion wi h dexame hasone o au o hiomala e o 24 hou s. The ea e , he cells we e loaded wi h Fluo-3-AM and he TRPA1-media ed Ca2+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. 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 AITC was added and he measu emen s we e con inued o 30 s. The esul s we e no malised agains he backg ound and exp essed as a mean o eigh measu emen s. In (E), AUC om 15 o 45 s was calcula ed. Resul s a e exp essed as mean+SEM, esul s in (A–C) a e exp essed as a pe cen age in compa ison o IL-1β- ea ed samples which we e se as 100%. S a is ical signi icance o he esul s was calcula ed wi h one-way o epea ed measu es ANOVA ollowed by Bon e oni pos - es . Da a we e analysed using G aphPad InS a V.3.0. ***p<0.001.AITC, allyl iso hiocyana e; ANOVA, analysis o a iance; AUC, a ea unde he cu e; GAPDH, glyce aldehyde- 3-phospha e dehyd ogenase; IL, in e leukin; NF-κB, nuclea ac o kappa B; OA, os eoa h i ic; PDTC,ammonium py olidinedi hioca bama e; RT-PCR, e e se ansc ip ion PCR; TRPA1, ansien ecep o po en ial anky in 1. g oup.bmj.com on Oc obe 9, 2017 - Published by h p:// mdopen.bmj.com/Downloaded om 3 Nummenmaa e, e al. RMD Open 2017;3:e000556. doi:10.1136/ mdopen-2017-000556 In lamma o y a h i is he unc ional down egula ion o TRPA1 exp ession by hese wo d ugs. To con i m he e ec s o dexame hasone and au o hiomala e in p ima y human chond ocy es, cells we e isola ed om ca ilage samples ob ained om join eplacemen su ge y and cul u ed as desc ibed p e i- ously.4 Dexame hasone and au o hiomala e down eg- ula ed IL-1β-induced TRPA1 exp ession also in hese p ima y chond ocy es ( igu e 1B). The cu en esul s show o he i s ime ha wo an i-in lamma o y d ugs which a e e ec i e in he ea men o a h i is and e a d ca ilage deg ada ion, namely dexame hasone and au o hiomala e, down- egula e he exp ession o TRPA1 in human chond o- cy es. No ably, inhibi ion o he ansc ip ion ac o nuclea ac o kappa B (NF-κB) also down egula ed TRPA1 exp ession ( igu e 1A,B). Acco dingly, Ha ano e al7 epo ed ecen ly ha he TRPA1 p omo e has a leas six pu a i e NF-κB binding si es; hey also showed NF-κB o be in ol ed in he induc ion o TRPA1 in syno - iocy es. Glucoco icoids8 as well as au o hiomala e9 ha e been shown o inhibi NF-κB ac i a ion. The e o e, he down egula ion o TRPA1 exp ession by hese d ugs may occu , a leas in pa , ia inhibi ion o NF-κB. The p esen indings, oge he wi h p e ious esul s,4 5 7 s ongly sugges TRPA1 as a no el ac o and d ug a ge in a h i is. Acknowledgemen s We wish o hank Ms e hi Salonen, heini B ande and ella Leh o o excellen echnical assis ance and Ms heli Mää ä o skil ul sec e a ial help. Con ibu o s eN, Mh, LJM, M, KV and eM con ibu ed o he design o he s udy and o he acquisi ion, analysis and in e p e a ion o da a. eM concei ed and supe ised he s udy. eN d a ed he manusc ip . All au ho s e ised he manusc ip c i ically o impo an in ellec ual con en and ha e app o ed i s inal e sion o submission. Funding he s udy was suppo ed by g an s om he Compe i i e esea ch Funding o he pi kanmaa hospi al Dis ic , Finland; Finnish Cul u al Founda ion, Finland; esea ch Founda ion o heuma ic Diseases, Finland; and pa ien o ganiza ion o heuma oid A h i is ( ampe een eumayhdis ys), Finland. Compe ing in e es s None decla ed. Pa ien consen W i en in o med consen was ob ained om all pa ien s. E hics app o al e hics Commi ee o he pi kanmaa hospi al Dis ic , Finland. P o enance and pee e iew No commissioned; ex e nally pee e iewed. Da a sha ing s a emen All he da a is epo ed in he manusc ip . Open Access his is an open Access a icle dis ibu ed in acco dance wi h he C ea i e Commons A ibu ion Non Comme cial (CC BY-NC 4.0) license, which pe mi s o he s o dis ibu e, emix, adap , build upon his wo k non-comme cially, and license hei de i a i e wo ks on di e en e ms, p o ided he o iginal wo k is p ope ly ci ed and he use is non-comme cial. See: h p:// c ea i ecommons. o g/ licenses/ by- nc/ 4. 0/ © A icle au ho (s) (o hei employe (s) unless o he wise s a ed in he ex o he a icle) 2017. All igh s ese ed. No comme cial use is pe mi ed unless o he wise exp essly g an ed. Re eRences 1. Zygmun PM, Höges ä ED. TRPA1. Handb Exp Pha macol 2014;222:583–630. 2. Koi is o A, Chapman H, Jala a N, e al. TRPA1: a ansduce and ampli ie o pain and in lamma ion. Basic Clin Pha macol Toxicol 2014;114:50–5. 3. Bau is a DM, Pelleg ino M, Tsunozaki M. TRPA1: A ga ekeepe o in lamma ion. Annu Re Physiol 2013;75:181–200. 4. Nummenmaa E, Hämäläinen M, Moilanen LJ, e al. 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. A h i is Res The 2016;18:185. 5. Moilanen LJ, Hämäläinen M, Nummenmaa E, e al. Monosodium iodoace a e-induced in lamma ion and join pain a e educed in TRPA1 de icien mice--po en ial ole o TRPA1 in os eoa h i is. Os eoa h i is Ca ilage 2015;23:2017–26. 6. Gold ing MB, Bi khead JR, Suen LF, e al. In e leukin-1 be a- modula ed gene exp ession in immo alized human chond ocy es. J Clin In es 1994;94:2307–16. 7. Ha ano N, I oh Y, Suzuki H, e al. Hypoxia-inducible ac o -1α (HIF1α) swi ches on ansien ecep o po en ial anky in epea 1 (TRPA1) gene exp ession ia a hypoxia esponse elemen -like mo i o modula e cy okine elease. J Biol Chem 2012;287:31962–72. 8. Ha mann K, Koenen M, Schaue S, e al. Molecula ac ions o glucoco icoids in ca ilage and bone du ing heal h, disease, and s e oid he apy. Physiol Re 2016;96:409–47. 9. Vuol eenaho K, Kujala P, Moilanen T, e al. Au o hiomala e and hyd oxychlo oquine inhibi ni ic oxide p oduc ion in chond ocy es and in human os eoa h i ic ca ilage. Scand J Rheuma ol 2005;34:475–9. g oup.bmj.com on Oc obe 9, 2017 - Published by h p:// mdopen.bmj.com/Downloaded om d ug a ge in a h i is chond ocy es: TRPA1 as a no el ac o and humandexame hasone and au o hiomala e in TRPA1 exp ession is down egula ed by Moilanen, Ka iina Vuol eenaho and Ee a Moilanen Elina Nummenmaa, Ma i Hämäläinen, Lau i J Moilanen, Teemu doi: 10.1136/ mdopen-2017-000556 2017 3: RMD Open h p:// mdopen.bmj.com/con en /3/2/e000556 Upda ed in o ma ion and se ices can be ound a : These include: Re e ences #BIBLh p:// mdopen.bmj.com/con en /3/2/e000556 This a icle ci es 9 a icles, 2 o which you can access o ee a : Open Access h p://c ea i ecommons.o g/licenses/by-nc/4.0/non-comme cial. See: p o ided he o iginal wo k is p ope ly ci ed and he use is non-comme cially, and license hei de i a i e wo ks on di e en e ms, pe mi s o he s o dis ibu e, emix, adap , build upon his wo k Commons A ibu ion Non Comme cial (CC BY-NC 4.0) license, which This is an Open Access a icle dis ibu ed in acco dance wi h he C ea i e se ice Email ale ing box a he op igh co ne o he online a icle. Recei e ee email ale s when new a icles ci e his a icle. Sign up in he No es h p://g oup.bmj.com/g oup/ igh s-licensing/pe missions To eques pe missions go o: h p://jou nals.bmj.com/cgi/ ep in o m To o de ep in s go o: h p://g oup.bmj.com/subsc ibe/ To subsc ibe o BMJ go o: g oup.bmj.com on Oc obe 9, 2017 - Published by h p:// mdopen.bmj.com/Downloaded om