UV-B induced alteration in purinergic receptors and signaling on HaCaT keratinocytes

1 1. In oduc ion The inc easing incidence o skin cance indica es he impo ance o unde s anding he pa hogenesis o hese diseases. Epidemiological s udies ha e e ealed a s ong associa ion be ween skin cance o ma ion and exposu e o sunligh [1]. While he sunligh has a wide spec um o elec omagne ic adia ion, om he h ee componen s o UV ligh (UV-A, UV-B, and UV-C) UV-A is biologically less e ec i e, UV-C – al hough highly e ec i e – is abso bed by he a mosphe e, he e o e, UV-B has he mos se e e biological e ec s [2]. UV ligh has been implica ed in he genesis o se e al o ms o cu aneous malignancies including melanoma, basal cell ca cinoma, and squamous cell ca cinoma [3]. UV i adia ion is assumed o lead o mu a ions in he DNA [4]. Excessi e DNA damage o de ec i e DNA epai mechanisms can lead o cell dea h o , e en wo se, o malignan ans o ma ion o he cells. Unde no mal condi ions, howe e , cells wi h damaged DNA should be ecognized and elimina ed. Indeed, a ious epai and sa e y mechanisms exis o main ain he in eg i y o he skin epide mis. Fo example, i UV-ligh -induced DNA damage is un epa able, cells con aining modi ied DNA can be elimina ed by apop osis [5]. An impo an e en in he p ocess o UV-B- induced apop osis is he inc ease o he in acellula calcium concen a ion ([Ca 2+ ] i ; see e.g. [6]) as he cy osolic Ca 2+ signal seems o be in ol ed in egula ing UV-induced apop osis in HeLa cells [7]. The e a e a numbe o possible ways o a cell o inc ease [Ca 2+ ] i , including ol age- and ligand-ga ed calcium channels in he su ace and endoplasmic e iculum (ER) memb anes. In ke a inocy es di e en pu ine gic ecep o s ha e been implica ed o play essen ial oles in deciding he a e o he cells h ough egula ing p oli e a ion and di e en ia ion. While P2Y ecep o s seem o con ol he p oli e a ion o hese cells, P2X ecep o s a e likely o be esponsible o hei di e en ia ion [8]. Among he la e P2X 7 ecep o is an apop osis-inducing ecep o [9], composed o h ee iden ical subuni s [10]. In ke a inocy es, he P2Y 2 ecep o akes pa in he egula ion o mig a ion as a nega i e egula o [11]. In hese expe imen s he connec ion be ween UV-B i adia ion and he pa e n o he exp ession o pu ine gic ecep o s is desc ibed. We demons a e a clea educ ion in P2X 7 ecep o exp ession ollowing UV-B i adia ion. In line wi h he al e ed exp ession pa e n, di e ences in es ing [Ca 2+ ] i and in he way how cells espond o ex acellula ATP was also 2 ecognized. These modi ica ions in pu ine gic signaling could con ibu e o an al e ed apop o ic endency and hus in pa explain he inc eased p obabili y o su i al o malignan ly ans o med cells. 2. Ma e ials and me hods 2.1 Cul u ing HaCaT ke a inocy es HaCaT ke a inocy es [12] we e cul u ed in Dulbecco’s modi ied Eagle’s medium (DMEM, Sigma, Budapes , Hunga y) supplemen ed wi h 10% e al cal se um (Sigma), 2 mmol/L L- glu amine, and an ibio ics (50 NE/ml penicillin, 50 µg/ml s ep omicin and 1,25 µg/ml Fungizon) a 37°C empe a u e in a 5% CO 2 a mosphe e as desc ibed in ou p e ious epo s [13, 14]. 2.2 I adia ing he cells Ke a inocy es we e cul u ed in 10 cm diame e Pe i dishes. A e media emo al and wash wi h phospha e based saline (PBS; Sigma) 0.5 ml o PBS was pu on he cells o p e en he exsicca ion o he monolaye cul u e. Fo i adia ion, a b oadband UV-B i adia ion was applied using a Philips TL-12 (57.5% o UV ou pu in UV-B, 280–315 nm) in 40 mJ/cm 2 dose as desc ibed in ou p e ious epo s [15]. P ope UV-B dosage was de e mined by a UVX Digi al Radiome e (UVP Inc., San Gab iel, CA, USA); egula ly. Mock ea ed samples we e handled he same way as i adia ed ones. Un ea ed con ols cells we e aken up a he ime o he expe imen , while UV-B i adia ed and mock ea ed cells we e supplied wi h 37 °C cul u e media and we e used o expe imen s a 8, 12, 16, 24, 30, 36 and 48 h as indica ed. 2.3 De e mina ion o iable cell numbe s The numbe o iable cells was de e mined by measu ing he con e sion o he e azolium sal MTT o o mazan (Sigma). Cells we e pla ed in 96-well mul i i e pla es (5000 cells/well densi y) and we e ea ed wi h UV-B and, a app op ia e imes, he cells we e incuba ed wi h 0.5mg/ml MTT o 2 h, and he concen a ion o o mazan c ys als (as he indica o o numbe o iable cells) was de e mined using a colo ime ic assay acco ding o he manu ac u e ’s p o ocol. Da a we e exp essed as mean ± SEM. 3 2.4 De e mina ion o apop osis To de ec he a e o apop osis, he Dead Cell Apop osis Ki wi h Annexin V FITC and PI (In i ogen, Ca lsbad, CA, USA) was used. Cells we e pla ed in 96 mul i i e pla es wi h black walls and clea bo oms (5000 cells/well densi y) a e he UV-B i adia ion. To de ec he o al cell numbe CyQUANT® Cell P oli e a ion Assays (In i ogen) was used, acco ding o he manu ac u e ’s p o ocol. To e oke apop osis 180 µM ATP was adminis e ed o he cells. Solu ion was changed h ee imes a e e e y 12 hou s. The apop o ic cell numbe was no malized o he o al cell numbe . 2.4 RT-PCR analysis. Fo RT-PCR analysis, cells we e washed h ee imes wi h CMF-PBS (calcium magnesium ee PBS), sc aped, snap- ozen in liquid ni ogen and s o ed a –70 °C. To al RNA was isola ed om cells using Quiagen RNeasy® Mic o Ki acco ding o he ins uc ions o he manu ac u e (Quiagen, Budapes , Hunga y). The assay mix u e (20 µL) o e e se ansc ip ase eac ion (Omnisc ip , Quiagen) con ained 500 ng RNA, 0.25 µL RNase inhibi o , 0.25 µL oligo(dT), 1 µL dNTP (200 µM), 1µl M-MLV RT in 1× RT bu e . Ampli ica ions o speci ic cDNA sequences we e pe o med wi h speci ic p ime s (In eg a ed DNA Technologies, Co al ille, IA, USA) ha we e designed based on published pu ino ecep o nucleo ide sequences ( o sequences o p ime pai s, see Supplemen a y Da a). PCR eac ions we e allowed o p oceed in a inal olume o 50 µL (con aining 1 µL o wa d and e e se p ime s, 1 µL dNTP [200 µM], and 5 uni s P omega GoTaq® DNA polyme ase in 1× eac ion bu e ) in a p og ammable he mocycle (Eppendo Mas e cycle, Ne hele , Hinz GmbH, Hambu g, Ge many) wi h he ollowing se ings: 2 min a 95 °C o ini ial dena u a ion ollowed by epea ed cycles o dena u a ion a 94 °C o 1 min, p ime annealing o 60 sec a an op imized empe a u e, and ex ension a 72 °C o 1 min 30 sec. A e he inal cycle, u he ex ension was allowed o p oceed o ano he 10 min a 72 °C. PCR p oduc s we e analyzed using a 1.5 % aga ose gel. The gel was s ained wi h 0.01% e hidium b omide solu ion o 30 minu es, and obse ed in UV ligh . Image J p og am (Wayne Rasband, Na ional Ins i u es o Heal h, USA) was used o densi ome y analysis. Toge he wi h he pu ino ecep o s GAPDH (glyce aldehyde-3-phospha e dehyd ogenase) mRNA exp ession was also de e mined and used as in e nal con ol. 4 2.5 Wes e n blo analysis To de e mine he exp ession o di e en pu ino ecep o s in cul u ed cells, he Wes e n blo echnique was applied [16]. Cells in cul u es we e washed wi h ice-cold PBS, ha es ed in homogeniza ion bu e (in mmol/L, 20 TRIS-Cl, 5 EGTA, 1 4-(2-aminoe hyl) benzenesulphonyl luo ide, 0,02 leupep in, pH 7.4; all om Sigma) and dis up ed by sonica ion on ice. P o ein con en o samples was measu ed by a modi ied bicinchoninic acid (BCA) p o ein assay (Pie ce, Rock o d, IL, USA). To al cell lysa es we e mixed wi h sodium dodecyl sulpha e-polyac ylamide gel elec opho esis (SDS-PAGE) sample bu e and boiled o 10 min a 100 °C. The samples we e subjec ed o SDS-PAGE (8% gels we e loaded wi h 40–50 µg p o ein pe lane) and ans e ed o ni ocellulose memb anes (BioRad, Wien, Aus ia). Memb anes we e hen blocked wi h 5% d y milk in PBS and p obed wi h he app op ia e, p ima y an ibodies o e nigh (see Supplemen a y Da a). Pe oxidase-conjuga ed goa an i-mouse immunoglobulin G (IgG; BioRad) we e used as seconda y an ibodies, and he immuno eac i e bands we e isualized by an enhanced chemiluminescence (ECL) wes e n blo ing de ec ion ki (Ame sham, Li le Chal on , England). Image J p og am was used o densi ome y analysis 2.6 Measu ing he changes in in acellula calcium concen a ion Changes in [Ca 2+ ] i we e moni o ed using Fu a-2, as desc ibed in ou ea lie epo s [17, 18]. In b ie , isola ed cells we e placed on a co e slip and loaded wi h 5 µM Fu a-2 AM (ace oxy me hyl es e ) o 60 minu es. Cells we e hen equilib a ed in Ty ode’s solu ion (in mmol/L, 137 NaCl, 5.4 KCl, 0.5 MgCl 2 , 1.8 CaCl 2 , 11.8 Hepes-NaOH, 1 g/L glucose, pH 7.4, all om Sigma) o a hal an hou a oom empe a u e. Co e slips wi h Fu a-2-loaded cells we e placed on he s age o an in e ed luo escence mic oscope (Diapho ; Nikon, Tokyo, Japan). The exci a ion wa eleng h was al e ed be ween 340 and 380 nm by a mic ocompu e -con olled dual-wa eleng h monoch oma o (Del ascan; Pho on Technology In e na ional, New B unswick, NJ), whe eas he emission was moni o ed a 510 nm using a pho omul iplie a 10 Hz acquisi ion a e o he a ios. [Ca 2+ ] i was calcula ed om he a io o measu ed luo escence in ensi ies (F 340 /F 380 ) as desc ibed [19]. Cells we e con inuously washed by Ty ode’s solu ion using a backg ound pe usion sys em, whe eas he in es iga ed agen s we e applied h ough a local pe usion sys em, which is posi ioned in close p oximi y o he measu ed cell. 5 3. Resul s In ou case a 1 minu e long, 40 mJ/cm 2 UV-B i adia ion dec eased he numbe o iable cells as assessed using he MTT assay. Figu e 1A shows ha , as measu ed a se e al poin s om 8 hou s up o 2 days a e i adia ion, he numbe o iable cell – exp essed as he pe cen o con ol – dec eased subs an ially. Albei no ye signi ican he dec ease was al eady p esen 8 hou s a e he i adia ion. I became signi ican a e 16 hou s and eached mo e han 50% a e 24 hou s. No only did he numbe o cells dec ease bu hei mo phology was also al e ed as shown on he phase con as images o he cells (Fig. 1B). Compa ing wi h he non-i adia ed cells, he i adia ed cells sh ank and los hei ex ensions. Cells, ne e heless, esponded o challenges o ex acellula ATP by ele a ing hei in acellula Ca 2+ le el (Fig. 2). We measu ed he e ec o 180 µM ATP on [Ca 2+ ] i one day a e he i adia ion by adminis e ing i o he close p oximi y o cells. Figu e 2A and B demons a e ha he adminis a ion o ATP induced a ansien inc ease in [Ca 2+ ] i in bo h con ol and in i adia ed cells ba hed in Ca 2+ -con aining solu ion. Figu e 2A and B also demons a e ha in he non-i adia ed cells he ampli ude o he esponse o he ATP challenge ( he change in [Ca 2+ ] i ) was highe han in i adia ed cells. In addi ion, in case o he i adia ed cells, a e he esponse o ATP, [Ca 2+ ] i e u ned o i s es ing le el slowe han in con ol cells. Figu e 2C p esen s pooled da a o con ol and i adia ed cells. Apa om he abo e men ioned changes, Fig. 2C also demons a es ha he es ing calcium le el and he maximal a e o ise o he calcium ansien s we e highe in con ol cells. All hese di e ences we e ound o be signi ican . These obse a ions clea ly indica e ha he pu ine gic signaling mus ha e been al e ed in i adia ed cells. To explain hese al e a ions he changes in ecep o exp ession was assessed in bo h RT- PCR and Wes e n blo expe imen s. P e ious esul s om ou lab iden i ied he P2X 1 , P2X 3 , P2X 7 and P2Y 2 pu ine gic ecep o s as he mos impo an on no mal HaCaT cells [14], hus in he ollowings he changes in he exp ession pa e ns o hese ecep o s, especially in ha o he p oapop o ic P2X 7 ecep o , we e pu in ocus. Figs. 3 and 4 demons a e ha a e seeding he exp ession o hese ecep o s ollowed a cha ac e is ic pa e n. Wi h Wes e n blo , again, he con ol and i adia ed cells we e compa ed. Bo h iono opic and me abo opic pu ino ecep o s we e examined a e 8, 12, 24, 36h o UV-B i adia ion (Fig. 3). Fi s he changes in he exp ession le el o he iono opic 6 pu ino ecep o s we e checked. Figu e 3 demons a es ha excep o 8h a e i adia ion, whe e he p o ein le els seemed o be he same, in e e y o he case he P2X 1 p o ein le el was less in he i adia ed cells as compa ed o con ol (mock- ea ed) cells a he same ime. In he case o P2X 3 ecep o , a e he i adia ion, an ex a band appea ed below he P2X 3 p o ein band. This migh indica e ha UV-B i adia ion damaged some o hese ecep o s. Examining he P2X 7 p o ein le els we could de ec he cha ac e is ic (80 kDa) band o he in ac ecep o only in he non-i adia ed cells. In he i adia ed cells we could only de ec an app oxima ely 20 kDa band, which could ep esen he monome ic o m o his ecep o [10]. Densi ome ic analysis was ca ied ou on he abo e men ioned Wes e n blo s (Fig. 4). The p o ein le els o P2X 1 ecep o we e signi ican ly dec eased in he i adia ed cells 12 and 36 hou s a e he i adia ion (Fig. 4A). The le el o he P2X 3 ecep o was signi ican ly highe han in he con ol cells 8, 12 and 24 hou s a e i adia ion (Fig. 4B). In case o P2X 7 ecep o , he densi ome ic analysis was sepa a ely made om bo h 80 kDa and 20 kDa bands. In case o he i adia ed cells he 80 kDa band o P2X 7 ecep o could no be ound a all (Fig. 4Ca). 12, 24 and 36 hou s a e he i adia ion he 20 kDa bands o P2X 7 ecep o we e signi ican ly highe in i adia ed cells compa ed o con ol cells (Fig. 4Cb). In case o he P2Y 2 ecep o 8 hou s a e he i adia ion he UV-B exposed cells had signi ican ly mo e P2Y 2 p o ein han did he con ol cells. 24 and 36 hou s a e he i adia ion, he P2Y 2 p o ein le el was signi ican ly less in he i adia ed as compa ed o con ol cells (Fig. 4D). The mRNA le els o he ecep o s we e checked wi h RT-PCR (Fig. 5). Excep om 24 h a e i adia ion, whe e he mRNA le els seemed o be he same in bo h con ol and i adia ed cells, we de ec ed less P2X 7 ecep o mRNA a e i adia ion. RT-PCR also indica ed ha a e i adia ion he P2Y 2 mRNA le els dec eased, and a e 36 hou s om he i adia ion, he mRNA o his ecep o disappea ed om he UV-B ea ed cells (Fig 5A). Densi ome ic analysis o hese ecep o s we e made by no malizing he bands o ha o GAPDH. Figu e 5B demons a es ha he mRNA le el o he P2X 7 ecep o was signi ican ly highe in he con ol as compa ed o he i adia ed cells 8, 12 and 36 hou s a e he i adia ion. The P2Y 2 mRNA le el was signi ican ly lowe in he i adia ed cells as compa ed o con ol a e 12, 24 and 36 hou s o he i adia ion (Fig 5C). As a nex s ep he a e o apop osis was measu ed in con ol and UV-B i adia ed cells using es ablished luo escence me hods. Al hough equal numbe o cells we e pla ed, he numbe 7 o cells a he ime o he measu emen we e di e en as expec ed om he esul s p esen ed in Fig. 1. To co ec o his di e ence he a e o apop osis was i s no malized o cell coun . The a e o apop osis in i adia ed cells – in he absence and p esence o ATP – was hen exp essed as pe cen age o he alues ob ained o non-i adia ed cells (Fig 6). Al hough al eady he basal a e o apop osis (in he absence o ATP) was sligh ly smalle in UV-B i adia ed han in con ol cells (78±19%, n=8), his di e ence did no p o e o be s a is ically signi ican (p>0.2). On he o he hand, i apop osis was induced by he addi ion o ATP in o he ba hing medium ( o bo h non- i adia ed and i adia ed cells) he a e o apop osis was signi ican ly smalle in UV-B i adia ed cells as compa ed o non-i adia ed (mock- ea ed) cells (61±22%, n=8; p<0.02), in line wi h he absence o unc ional P2X 7 ecep o s on hese cells. 4. Discussion In ou expe imen s, we examined how ke a inocy es espond o UV-B i adia ion in espec o hei pu ine gic calcium signaling. One minu e long, 40 mJ/cm 2 UVB i adia ion – co esponding o app oxima ely 2-4 minimal e y hemal dose in an in ac skin [20, 21] – was used o check he accompanying changes in he exp ession le els o pu ino ecep o s, and he changes in he esponse o he cells o he ex acellula applica ion o ATP. A one minu e long 40 mJ/cm 2 UVB exposu e was selec ed because longe i adia ion imes esul ed in he dea h o essen ially e e y cell. Simila i adia ion ime and ligh in ensi y was used in ou p e ious measu emen s conce ning gene exp ession [15], he e o e hese da a a e di ec ly compa able o hose esul s. Impo an ly hough, e en his low ene gy exposu e o UV-B ligh dec eased he iable cell numbe signi ican ly as compa ed o con ol cells. In he e en s o p og ammed cell dea h one o he i s s eps is he inc ease in he in acellula calcium concen a ion o he cell. Se e al s udies ha e linked he ac i a ion o pu ino ecep o s especially ha o he P2X 7 ecep o [9], [22] o he ini ia ion o apop o ic cell dea h. In i o hese pu ino ecep o s may be ac i a ed by ATP coming om o he dead cells, o physiologically eleased om ne es [23], u oepi hel [24] and endo hel [25]. In he human squamosus cell ca cinoma line A431 mic omola concen a ions o ATP we e mi ogenic [26] and inc eased in acellula calcium le els [27]. ATP has also been shown o ac i a e cell p oli e a ion in MCF-7 b eas cance cells [28] and in human o a ian umo cells [29]. Impo an ly ough high 8 concen a ions o ATP and BzATP (speci ic agonis o he P2X 7 ecep o ) caused a signi ican dec ease in A431 cell numbe [30]. In ou expe imen s, a e i adia ion, he P2X 7 ecep o s we e diminished in he cells, and we de ec ed less P2X 7 ecep o mRNA. This migh indica e ha cells which we e damaged o some ex en bu su i ed he i adia ion we e no as capable o ini ia ing apop o ic cell dea h. This in u n could gi e ise o a popula ion o cells wi h damaged DNA bu low apop o ic capabili ies hus enabling malignan ly ans o med cells o pe sis . I should be no ed, howe e , ha while DNA damage in he skin is commonly desc ibed in associa ion wi h UV-B i adia ion [31] he di ec deg ada ion o p o eins ollowing exposu e o UV-B ligh is a less equen ly obse ed [32]. O he P2X and P2Y ecep o s also ha e impo an unc ions in egula ing he p oli e a ion and di e en ia ion o cells. The me abo opic P2Y 1 and P2Y 2 ecep o s we e shown o con ol he p oli e a ion o ke a inocy es [8]. When a skin cance e ol es om heal hy ke a inocy es, he pa e n o hese pu ino ecep o s may change. We ound ha a 1 minu e long 40 mJ/cm 2 UV-B i adia ion dec eased he amoun o bo h P2X 1 and P2Y 2 ecep o s, while essen ially des oyed he P2X 7 ecep o s. In pa icula , P2Y 2 ecep o s ha e been associa ed wi h p omo ing p oli e a ion in bo h no mal human ke a inocy es and in HaCaT cells [33]. In ou expe imen s, we demons a ed ha he iable cell numbe was dec eased a e he 1 minu e long 40 mJ/cm 2 UV-B i adia ion, in which he dec eased amoun o P2Y 2 ecep o s migh also ha e played a ole. Indeed, he expe imen s e ealed ha he mRNA o he P2Y 2 ecep o was less in he UV-B ea ed han in con ol cells, and 36 and 48 hou s a e he i adia ion he mRNA o his ecep o disappea ed om he i adia ed cells. Al hough 8 hou s a e he i adia ion he p o ein le el o he P2Y 2 ecep o was highe in he UV-B ea ed han in he con ol cells, he amoun o his ecep o was dec eased in he UV-B ea ed cells a e e y o he poin in ime. By loosing much o hese ecep o s, cells could lose he con ol o e he p oli e a ion, di e en ia ion o bo h, and hus be shi ed owa ds a malignan ans o ma ion. In line wi h he al e ed exp ession o pu ino ecep o s, di e ences in he calcium ansien s ob ained in esponse o challenges wi h ATP we e also obse ed. The ampli ude and he a e o ise o hese calcium ansien s we e lowe in he i adia ed cells, which may e lec he lowe amoun o pu ine gic ecep o s on hese cells. The la e migh also be caused by he shi om mos ly iono opic (P2X) o pa ly me abo opic (P2Y) esponse in he i adia ed cells. Fo [Ca 2+ ] i o e u n o i s es ing le el ook longe imes in he i adia ed cells. This could indica e ha o he 9 p o eins, o example he calcium pump was also damaged by UV-B i adia ion. This no ion should, howe e , be aken wi h cau ion since es ing [Ca 2+ ] i was lowe in i adia ed han in con ol cells. Acknowledgemen s The au ho s wish o hank Va gáné Kiss Ibolya o he excellen echnical assis ance. This wo k was suppo ed by g an s om he Hunga ian Science Fund (OTKA NK78398 and K68401), ETT (186/09), and TÁMOP 4.2.2.-08/1-2008-0019. Re e ences 1. S.D. Wo swick, M. Cockbu n, D. Peng, Measu emen o ul a iole exposu e in epidemiological s udies o skin and skin cance s, Pho ochem Pho obiol, 84 (2008) 1462- 1472 2. W. Ambach, M. Blum hale , Biological e ec i eness o sola UV adia ion in humans, Expe ien ia, 49 (1993) 747-753 3. C. Pe lis, M. He lyn, Recen ad ances in melanoma biology, Oncologis 9 (2004) 182- 187 4. D. Be ega, P. Calzola i, F. Belloni, F. Di Lena, S. Genchi, M. Lupi, P. Massa iello, S. O sini, L. Tallone, D. Tomasoni, P. Ubezio, J.L. Redpa h, Sola UV adia ion: di e en ial e ec i eness o UVB subcomponen s in causing cell dea h, mic onucleus induc ion and delayed exp ession o he i able damage in human hyb id cells, In J Radia Biol. 77 (2001) 963-970 5. P. E b, J. Ji, E. Kump, A. Mielgo, M. We nli, Apop osis and pa hogenesis o melanoma and nonmelanoma skin cance , Ad Exp Med Biol 624 (2008) 283-295 6. F.M. Lyng, C.B. Seymou , C. Mo he sill, Ea ly e en s in he apop o ic cascade ini ia ed in cells ea ed wi h medium om he p ogeny o i adia ed cells, Radia P o Dosime y 99 (2002) 169-172 7. Y. Pu, D.C. Chang, Cy osolic Ca2+ signal is in ol ed in egula ing UV-induced apop osis in HeLa cells, Biochem Biophys Res Commun, 282 (2001) 84-89