Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Pathway Is Induced by Mechanical Load and Reduces the Activity of Hedgehog Signaling in Chondrogenic Micromass Cell Cultures

In . J. Mol. Sci. 2015, 16, 17344-17367; doi:10.3390/ijms160817344 In e na ional Jou nal o Molecula Sciences ISSN 1422-0067 www.mdpi.com/jou nal/ijms A icle Pi ui a y Adenyla e Cyclase Ac i a ing Polypep ide (PACAP) Pa hway Is Induced by Mechanical Load and Reduces he Ac i i y o Hedgehog Signaling in Chond ogenic Mic omass Cell Cul u es Tamás Juhász 1,*, Esz e Szen léleky 1, Csilla Szűcs Somogyi 1, Roland Takács 1, Nó a Dob osi 1, Má é Engle 1, And ea Tamás 2, Dó a Reglődi 2 and Róza Zákány 1 1 Depa men o Ana omy, His ology and Emb yology, Uni e si y o Deb ecen, Medical and Heal h Science Cen e, Nagye dei k . 98, H-4032 Deb ecen, Hunga y; E-Mails: [email p o ec ed] (E.S.); som[email p o ec ed] (C.S.S.); [email p o ec ed] (R.T.); [email protected] (N.D.); engle .ma [email protected] (M.E.); [email p o ec ed] (R.Z.) 2 Depa men o Ana omy, MTA-PTE “Lendüle ” PACAP Resea ch Team, Uni e si y o Pécs, Medical School, Szige i ú 12, H-7624 Pécs, Hunga y; E-Mails: and ea [email protected] (A.T.); [email p o ec ed] (D.R.) * Au ho o whom co espondence should be add essed; E-Mail: juha[email p o ec ed]; Tel.: +36-52-255-567; Fax: +36-52-255-115. Academic Edi o : Alan C. Leona d Recei ed: 30 Ap il 2015 / Accep ed: 18 June 2015 / Published: 29 July 2015 Abs ac : Pi ui a y adenyla e cyclase ac i a ing polypep ide (PACAP) is a neu oho mone exe ing p o ec i e unc ion du ing a ious s ess condi ions ei he in ma u e o de eloping issues. P e iously we p o ed he p esence o PACAP signaling elemen s in chicken limb bud-de i ed chond ogenic cells in mic omass cell cul u es. Since no da a can be ound i PACAP signaling is playing any ole du ing mechanical s ess in any issues, we aimed o in es iga e i s con ibu ion in mechano ansduc ion du ing chond ogenesis. Exp essions o he mRNAs o PACAP and i s majo ecep o , PAC1 inc eased, while ha o o he ecep o s, VPAC1, VPAC2 dec eased upon mechanical s imulus. Mechanical load enhanced he exp ession o collagen ype X, a ma ke o hype ophic di e en ia ion o chond ocy es and PACAP addi ion a enua ed his ele a ion. Mo eo e , exogenous PACAP also p e en ed he mechanical load e oked ac i a ion o hedgehog signaling: p o ein le els o Sonic and Indian Hedgehogs and Gli1 ansc ip ion ac o we e lowe ed while exp essions o Gli2 and Gli3 OPEN ACCESS In . J. Mol. Sci. 2015, 16 17345 we e ele a ed by PACAP applica ion du ing mechanical load. Ou esul s sugges ha mechanical load ac i a es PACAP signaling and exogenous PACAP ac s agains he hype ophy inducing e ec o mechanical load. Keywo ds: mechano ansduc ion; chond ocy e di e en ia ion; Sonic hedgehog; Indian hedgehog; hype ophy 1. In oduc ion Cells o skele al issues such as bone and a icula ca ilage change hei de elopmen al p og am, no mal li e cycle and exp ession o ex acellula ma ix componen s upon mechanical s imula ion. Mechanical load plays a c ucial ole in he de elopmen and egene a ion o he a icula ca ilage [1–3] sugges ing he p esence o cellula mechanisms o mechano ansduc ion ei he in de eloping o adul chond ocy es. Va ious cell su ace ecep o s can se e o mechanosensa ion o chond ocy es, such as ce ain pu ino ecep o s [4], in eg ins [5], s e ch ac i a ed Ca2+ channels [6], TRPV4 [7] and NMDA ecep o s [8]. Downs eam a ge s o hese ecep o s can egula e he mo phology, me abolism, p oli e a ion and Ca2+ homeos asis o chond ocy es. Ou g oup demons a ed a PKA egula ed mechanosensi i e ac i a ion o Sox9 and PP2A [9] and he in ol emen o he hedgehog signaling in mechano ansduc ion has also been p o en by Wu and colleagues [10]. Pi ui a y adenyla e cyclase ac i a ing pep ide (PACAP) is one o he membe s o VIP-Sec e in-GHRH-Glucagon supe amily and has been ex ac ed om o ine hypo halamus o e 25 yea s ago [11]. A e desc ibing he neu opep ide in he egula ion o de elopmen and no mal unc ion o cen al ne ous sys em, mo e and mo e pe iphe al issues we e demons a ed o exp ess and be in luenced by his neu oho mone [12–14]. PACAP is a sho pep ide exis ing in wo biological ac i e o ms; PACAP 1–38 and PACAP 1–27 [11]. Bo h o ms ha e a e y sho li e span [15]. Th ee main G-p o ein coupled ecep o s o PACAP ha e been iden i ied; PAC1, VPAC1 and VPAC2, he la e wo ha e lowe a ini y o he neu opep ide [16]. Ac i a ion o hese ecep o s can egula e di e s signaling pa hways [17–21], om which he canonical signaling connec ion induces he ac i a ion o PKA and/o MAPK sys em [19,21]. The exp ession o PACAP and i s ecep o s in chond ogenic di e en ia ion has been demons a ed by ou g oup [22]. We ha e also shown ha exogenous addi ion o PACAP exe s posi i e e ec s bo h on chond o- and os eogenesis [22,23]. Mul i ac o ial o pleio ophic e ec s o he neu opep ide ha e been obse ed in a ious biological p ocesses, and PACAP was p o en o p e en apop osis, ischemic condi ions, and oxida i e s ess [24–28]. We ha e demons a ed ha addi ion o PACAP escued he ca ilage o ma ions du ing oxida i e s ess in a high densi y cell cul u e model [22] and an iin lamma o y unc ions o he neu opep ide we e ound in o he skele al elemen s [26,29,30]. Chond i ying mic omass cell cul u es (HDC), es ablished om cells o chicken limb buds o ou -day-old chicken emb yos, ep esen a well ep oducible expe imen al model o ca ilage o ma ion [22,31]. In hese cell cul u es, chond ogenic cells di e en ia e spon aneously o chond ocy es on days wo and h ee and a conside able amoun o hyaline ca ilage is p oduced by day six o cul u ing. Signal ansduc ion p ocesses go e ning consecu i e s eps o chond ogenesis can easily be ollowed o modula ed ex e nally in his sys em. Responsi eness o chond ogenic cells o uniaxial cyclic In . J. Mol. Sci. 2015, 16 17346 comp essi e load gene a ed by ou cus om-made mechanical s imula o was demons a ed by he obse a ion ha his in e en ion lead o he inc ease o ca ilage di e en ia ion and modula ion o PKA egula ed signaling pa hways [9]. On he basis o his PKA-dependence o he mechano ansduc ion in his cul u e, we hypo hesized he ole o PACAP signaling in his p ocess. The e o e, he majo goal o he p esen s udy was o cla i y he ole o PACAP-signaling in mechanosensi i i y o chicken chond ogenic cells. We p esen e idence ha exp ession o PACAP and PAC1 ecep o is ele a ed by mechanical load. We p o e ha mechanical s imulus enhances he hedgehog signaling, while exogenous PACAP a enua es he ac i i y o his pa hway ei he wi h o wi hou mechanical load. Ou esul s sugges ha PACAP educes he endency o hype ophic ans o ma ion o chond ogenic cells e oked by mechanical load and i implies ha PACAP signaling may ake pa in he egula ion o e minal di e en ia ion o chond ocy es in i o. 2. Resul s 2.1. Mechanical S imula ion and PACAP (Pi ui a y Adenyla e Cyclase Ac i a ing Polypep ide) Adminis a ion Enhance Collagen P oduc ion Adminis a ion o PACAP 1–38 and/o applica ion o mechanical load only on day-2 o cul u ing did no signi ican ly al e he ma ix p oduc ion o di e en ia ing chond ocy es (Figu e 1A,C). In line wi h ou p e iously epo ed esul s, ei he in e mi en load [9] o addi ion o PACAP [22] signi ican ly ele a ed he amoun o me ach oma ic ca ilage ma ix p oduced in chicken mic omass cul u es by day six as i was e ealed by dime hylme hylene blue (DMMB) and oluidine blue (TB) s ainings (Figu e 1B). We we e able o e i y hese da a wi h Sa anin O s aining as well (Figu e 1D). In e es ingly he combined applica ion o mechanical load and PACAP did no ha e addi i e e ec , he p oduc ion o me ach oma ic ca ilage nodules emained a a le el simila o ha o a single in e en ion (125% o con ol cul u es; Figu e 1A,D). Collagen ibe s play an essen ial ole in he es ablishmen and main enance o p ope biomechanics o ca ilage. We ollowed up he in luence o mechanical load and PACAP addi ion on collagen syn hesis wi h he measu emen o he inco po a ion o 3H-p oline in o mic omass cul u es. Mechanical load alone caused an almos h ee old ele a ion o 3H-p oline-inco po a ion, while applica ion o PACAP alone did no cause any signi ican change o his pa ame e (Figu e 2A). When PACAP 1–38 was adminis a ed du ing mechanical load, lowe 3H-p oline-inco po a ion was de ec ed in compa ison o ha o measu ed du ing mechanical load alone bu s ill an ele a ion was demons a ed compa ing wi h he un ea ed con ol (Figu e 2A). Nex , we moni o ed he mRNA and p o ein exp ession o a ious ypes o collagens, speci ic o ca ilage, du ing mechanical load and PACAP adminis a ion. Upon mechanical s imula ion, he mRNA exp ession o collagen ype II (Col2a1) and collagen ype IX (Col9a1) inc eased (Figu e 2B,C), while collagen ype X (Col10a1) esponded wi h a signi ican dec ease. PACAP 1–38 ea men also enhanced he mRNA exp ession o collagen ype II (Col2a1) bu i dec eased he mRNA exp ession o collagen ype IX, while ype X collagen mRNA exp ession did no show any signi ican change (Figu e 2B,C). Simila ly o he esul s obse ed in ela ion o he me ach oma ic ca ilage ma ix, he p esence o PACAP 1–38 du ing mechanical s imula ion sligh ly a enua ed he e ec s o mechanical load In . J. Mol. Sci. 2015, 16 17347 on he exp ession all o he in es iga ed collagens (Figu e 2D,E). Rega ding he changes o he collagens a p o ein le el, p e iously we demons a ed ha collagen ype II p oduc ion was ele a ed du ing PACAP addi ion [22]. Mechanical s imula ion did no ele a e he collagen ype II p o ein le el, while he combined applica ion o PACAP and mechanical load caused a signi ican dec ease o i (Figu e 2D,E). PACAP 1–38 signi ican ly educed collagen ype IX p o ein le el (Figu e 2D,E). On he con a y, mechanical load caused a signi ican ele a ion o his collagen ype, bu i did no compensa e he educing e ec o he neu opep ide du ing he combined applica ion (Figu e 2D,E). In con as o he mRNA exp ession p o ile, ea men wi h PACAP 1–38 did no in luence he amoun o collagen ype X p o ein, while he mechanical s imulus inc eased i (Figu e 2D,E). Combina ion o PACAP and mechanical load esul ed in an a enua ed bu s ill signi ican ele a ion o collagen ype X p o ein le el (Figu e 2D,E). Figu e 1. E ec s o PACAP and/o mechanical load on ma ix p oduc ion o HDC. PACAP 1–38 a 100 nM was applied on day wo o cul u ing (A,C) and con inuously om day one (B,D). MS, mechanical s imula ion applied on day wo (A,C) and days 2–3 (B,D) o 30 min. (A,B) Me ach oma ic ca ilage a eas in six-day-old cul u es we e isualized wi h DMMB dissol ed in 3% ace ic acid. Me ach oma ic (pu ple) s uc u es ep esen ca ilaginous nodules o med by many cells and ca ilage ma ix ich in sulpha ed glycosaminoglycans (GAGs) and p o eoglycans (PGs). Op ical densi y (OD625) was de e mined in samples con aining TB ex ac ed wi h 8% HCl dissol ed in absolu e e hanol. S a is ically signi ican di e ence o he ex ac ed TB in cul u es ha ecei ed he loading egime and/o PACAP 1–38 s. con ol cul u es is ma ked by as e isk (* p < 0.05); (C,D) Sa anin O s aining o isualiza ion o ca ilage nodules o HDC. O iginal magni ica ion was 4×. Scale ba , 500 µm. Rep esen a i e da a o h ee independen expe imen s a e shown. (P1–38, PACAP 1–38; MS; mechanical s imulus). In . J. Mol. Sci. 2015, 16 17348 Figu e 2. Con . In . J. Mol. Sci. 2015, 16 17349 Figu e 2. PACAP and/o mechanical load al e ed he collagen p oduc ion o HDC. PACAP 1–38 a 100 nM was applied con inuously om day one. Sho ime, ansien MS, was applied on days wo and h ee o 30 min on bo h days. (A) Collagen p oduc ion o HDC was de e mined by 3H-collagen inco po a ion; (B) RNA exp ession o collagen ype II (Col2a1), collagen ype IX (Col9a1) and collagen ype X (Col10a1) on day h ee. GAPDH was used as a con ol; (D) P o ein exp ession o collagen ype II, collagen ype IX, collagen ype X on day h ee. Ac in was used as a con ol. Fo panels (B,D) numbe s below signals ep esen in eg a ed densi ies o signals de e mined by ImageJ so wa e. Rep esen a i e da a o h ee independen expe imen s a e shown; (C,E) S a is ical analysis o RT-PCR and Wes e n blo da a. All da a p esen ed a e he a e ages o a leas h ee di e en expe imen s. S a is ical analysis was pe o med by S uden ’s - es . All da a we e no malized on GAPDH/Ac in and da a a e exp essed as mean ± SEM. (C, con ol; P1–38, PACAP 1–38; MS; mechanical s imulus). S a is ically signi ican di e ence be ween collagen p oduc ion a e o cells in cul u es ha ecei ed he loading egime and/o PACAP 1–38 s. con ol cul u es is ma ked by as e isk (* p < 0.05) in all panels. 2.2. PACAP and PAC1 Recep o Exp essions A e Ele a ed by Mechanical Loading In he ligh o he gene al ophic e ec o PACAP i could be a ques ion o in e es whe he he neu opep ide plays a ole in he cellula s ess managemen o chond ocy es upon mechanical s imulus. The e o e, we in es iga ed he exp ession o PACAP and i s ecep o s ollowing he applica ion o mechanical load. The mRNA exp ession o p ep oPACAP was inc eased du ing mechanical s imula ion and a modes dec ease was de ec ed a he p esence o exogenous PACAP du ing he s imula ion p ocess (Figu e 3A,B). The mRNA and he p o ein exp ession o PAC1 ecep o was also ele a ed by mechanical load which e ec was pa ly compensa ed by he PACAP adminis a ion (Figu e 3A–D). When PACAP was added o he medium o cell cul u es wi hou mechanical load, i did no cause any signi ican al e a ions in he abo e exp ession le els (Figu e 3A,B). We de ec ed mRNA and p o ein exp ession o bo h VPAC1 and VPAC2 ecep o s in high densi y cul u es om which only he p o ein exp ession o In . J. Mol. Sci. 2015, 16 17350 VPAC1 dec eased signi ican ly du ing he adminis a ion o PACAP 1–38 and mechanical load exe ed he same e ec (Figu e 3C,D). Combina ion o he wo in e en ions did no esul in any change o hese lowe ed exp essions o VPAC1 (Figu e 3A–D). VPAC2 ecep o appea ed wi h a low p o ein exp ession p o ile which u he dec eased du ing mechanical load and he combined ea men s (Figu e 3C,D). Figu e 3. Con . In . J. Mol. Sci. 2015, 16 17351 Figu e 3. Exp ession o p ep oPACAP and PACAP ecep o s a e PACAP adminis a ion and/o mechanical load in chond i ying mic omass cul u es. Fo RT-PCR (A) eac ions GAPDH and o Wes e n blo (C) eac ions Ac in was used as con ol. Op ical densi y o signals was measu ed and esul s we e no malised o he op ical densi y o con ols. Fo panels (A,C) numbe s below signals ep esen in eg a ed densi ies o signals de e mined by ImageJ so wa e. Rep esen a i e da a o h ee independen expe imen s; (B,D) S a is ical analysis o RT-PCR and Wes e n blo da a. All da a a e he a e age o a leas h ee di e en expe imen s. S a is ical analysis was pe o med by S uden ’s - es . All da a we e no malized on GAPDH/Ac in and da a a e exp essed as mean ± SEM. As e isks indica e signi ican (* p < 0.05) al e a ion o exp ession as compa ed o he espec i e con ol in all panels (C, con ol; P1–38, PACAP 1–38; MS; mechanical s imulus). 2.3. Hedgehog Signaling Pa hways Become Up egula ed upon Mechanical Load, While PACAP A enua es he Ac i i y o This Pa hway The enhanced p o ein le els o collagen ype IX and X imply ha chond ogenic cells a e shi ed owa d a p ehype ophic di e en ia ion. As PAC1 ecep o ac i a ion is known modula ing hedgehog (Hh) signaling [32], we in es iga ed he exp ession p o ile o his pa hway. Indeed, we ound a complex al e a ion o he exp ession o he membe s o Hh signaling in ou expe imen s. Al hough he mRNA exp ession o Sonic hedgehog (SHH) was no al e ed by any o he applied ea men s (Figu e 4A,B), he SHH p o ein became almos unde ec able upon PACAP 1–38 adminis a ion (Figu e 4C,D). On he con a y, he mechanical s imula ion esul ed in a signi ican ele a ion o SHH p o ein exp ession which was no malized by he applica ion o exogenous PACAP (Figu e 4C,D). The mRNA exp ession o Indian hedgehog (IHH) was dec eased, mo eo e , he p o ein exp ession educed o an almos unde ec able le el a e PACAP adminis a ion (Figu e 4A–D). In con as wi h his, he mechanical load enhanced he mRNA and p o ein exp ession o IHH (Figu e 4A–D). Simila ly o he SHH bo h he mRNA and p o ein exp ession o IHH we e educed by he combined ea men In . J. Mol. Sci. 2015, 16 17352 (Figu e 4A–D). Exp ession o PTH P, a signaling molecule known o wo k hand-in-hand wi h IHH, was dec eased signi ican ly du ing mechanical s imula ion, while we ailed o de ec any al e a ion in i s exp ession caused by PACAP 1–38 addi ion (Figu e 4A–D). Combined adminis a ion o he neu opep ide wi h he mechanical load p e en ed he educ ion o PTH P exp ession (Figu e 4A–D). Smoo hened, he co-ac i a o o hedgehog ecep o s did no show any mRNA o p o ein exp ession al e a ion upon any o he ea men s (Figu e 4A–D). Exp ession pa e n o he downs eam ansc ip ion ac o s o hedgehog signaling pa hways such as Gli1, 2 and 3 we e also moni o ed. The mRNA exp ession o Gli1 was no al e ed upon ei he PACAP ea men o mechanical s imula ion al hough he combined applica ion ele a ed i s mRNA exp ession (Figu e 4A,B). The p o ein exp ession o Gli1 du ing PACAP addi ion educed (Figu e 4C,D) bu signi ican ele a ion was de ec ed a e mechanical load (Figu e 4C,D). When he mechanical s imula ion was applied pa allel wi h PACAP addi ion i signi ican ly ele a ed he mRNA o Gli1 al hough i s p o ein exp ession emained a a le el simila o ha o de ec ed when PACAP was applied alone (Figu e 4A–D). The mRNA exp essions o Gli3 and Gli2, he ansc ip ion ac o s which can ac as ep esso s o IHH o special localiza ion is needed o hei ac i a ion [33,34], we e no al e ed unde he e ec o PACAP and/o mechanical s imula ion (Figu e 4A,B). Al hough p o ein exp essions o bo h ansc ip ion ac o s ele a ed in he p esence o PACAP 1–38 and educed by mechanical load, hey we e no signi ican ly al e ed du ing he combined ea men (Figu e 4C,D). The sho e ep esso o m o Gli3 p o ein (i.e., 83 kD) showed a p ominen inc ease du ing PACAP adminis a ion bu a signi ican educ ion was de ec ed du ing mechanical load (Figu e 4C,D). Figu e 4. Con . In . J. Mol. Sci. 2015, 16 17359 4.6. RT-PCR Analysis Cell cul u es we e dissol ed in T izol (Applied Biosys ems, Fos e Ci y, CA, USA), 20% RNase ee chlo o o m was added and he samples we e cen i uged a 4 °C on 10,000 pm o 15 min. Samples we e incuba ed in 500 µL o RNase ee izop opanol in −20 °C o 1 h hen o al RNA was ha es ed in RNase ee wa e and s o ed a −20 °C. The assay mix u e o e e se ansc ip ase eac ion con ained 2 µg RNA, 0.112 µM oligo(dT), 0.5 mM dNTP, 200 uni s High Capaci y RT (Applied Bio-Sys ems, Fos e Ci y, CA, USA) in 1× RT bu e . Fo he sequences o p ime pai s and u he de ails o polyme ase chain eac ions, see Table 1. Ampli ica ions we e pe o med in a he mal cycle (Labne Mul iGene™ 96-well G adien The mal Cycle ; Labne In e na ional, Edison, NJ, USA) in a inal olume o 11 μL (con aining 0.5 μL o wa d and e e se p ime s (0.4 μM), 0.25 μL dNTP (200 μM), and i e uni s o P omega GoTaq® DNA polyme ase in 1× eac ion bu e ) as ollows: 95 °C, 2 min, ollowed by 35 cycles (dena u a ion, 94 °C, 1 min; annealing a op imized empe a u es as gi en in Table 1 o 1 min; ex ension, 72 °C, 90 s) and hen 72 °C, 10 min. PCR p oduc s we e analyzed by elec opho esis in 1.2% aga ose gel con aining e hidium b omide. GAPDH was used as an in e nal con ol. Op ical densi y o signals was measu ed by using ImageJ 1.40g eewa e (h p:// sbweb.nih.go /ij/) and esul s we e no malized o he op ical densi y o un ea ed con ol cul u es. 4.7. Wes e n Blo Analysis Abou 10 µg o p o ein was sepa a ed by 7.5% SDS-PAGE gel o de ec ion o PAC1, VPAC1, VPAC2, Ac in, Collagen ype II, Collagen ype IX, Collagen ype X, PTH P, SHH, IHH, Smoo hened (Smo), Gli1, Gli2, and Gli3. P o eins we e ans e ed elec opho e ically o ni ocellulose memb anes. A e blocking wi h 5% non- a d y milk in phospha e bu e ed saline (PBST) wi h 0.1% Tween 20, memb anes we e washed and exposed o he p ima y an ibodies o e nigh a 4 °C in he dilu ion as gi en in Table 2. A e washing o 30 min in PBST, memb anes we e incuba ed wi h an i- abbi IgG (Bio-Rad Labo a o ies, He cules, CA, USA) in 1:1500 o an i-mouse IgG (Bio-Rad Labo a o ies) in 1:1500 dilu ion. Signals we e de ec ed by enhanced chemiluminescence (Pie ce™, The mo Fishe Scien i ic Inc., Wal ham, MA, USA) acco ding o he ins uc ions o he manu ac u e . Signals we e de eloped wi h gel documen a y sys em (Fluo chem E, P o einSimple, San Jose, CA, USA). Op ical densi y o Wes e n blo signals was measu ed by using ImageJ 1.40g eewa e and esul s we e no malized o ha o un ea ed con ol cul u es. In . J. Mol. Sci. 2015, 16 17360 Table 1. Nucleo ide sequences, ampli ica ion si es, GenBank accession numbe s, amplime sizes and PCR eac ion condi ions o each p ime pai a e shown. Gene P ime Nucleo ide Sequence (5′→3′) GenBank ID Annealing Tempe a u e Amplime Size (bp) Collagen II (Col2a1) sense GGA CCC AAA GGA CAG ACG G (1191–1210) NM_204426 59 °C 401 an isense TCG CCA GGA GCA CCA GTT (1573–1591) Collagen IX (Col9a1) sense GGG ACAA GAG GAA TAA ACG (1732–1750) NM_001100911.1 52 °C 163 an isense CTG GTA AAC CTG GCA ATC (1877–1894) Collagen X (Colxa1) sense TCT GGG ATG CCG CTT GTC (1681–1698) NM_009925.4 56 °C 261 an isense CGT AGG CGT GCC GTT CTT (1924–1941) PACAP (ADCYAP1) sense CTT CGC ACT ACG AGC AGG (156–163) NM_001001291 52.5 °C 198 an isense TTG ACA GCC ATT TGT TTC C (335–363) PAC1 (ADCYAP1R1) sense GTC AGA CAA CCA GGA TTA C (435–453) NM_001098606 49 °C 141 an isense TGG ATA AAG TTC CGA GTG (559–575) VPAC1 (VIPR1) sense GTT CTA TGG CAC GGT CAA (376–393) NM_001097523 52 °C 216 an isense AGC AAT GTT CGG GTT CTC (573–590) VPAC2 (VIPR2) sense TCG GAA CTA CAT CCA TCT (477–497) NM_001014970 48 °C 177 an isense TTT GCC ATA ACA CCA TAC (636–653) SHH (Shh) sense TCA GTG GCA GCG AAA TCA (787–804) NM_204821.1 56 °C 168 an isense CAT CCG GTC GAG GAA GGT (937–954) IHH (Ihh) sense TCG CCT ACA AGC AGT TCA GCC (455–475) NM_204957.1 60 °C 155 an isense GCC GGT GTT CTC CTC GTC CT (590–609) In . J. Mol. Sci. 2015, 16 17361 Table 1. Con . Gene P ime Nucleo ide Sequence (5′→3′) GenBank ID Annealing Tempe a u e Amplime Size (bp) PTH P (P hlh) sense TAC GGA AGA TCA GTA GAG G (155–173) NM_001174106.1 46 °C 191 an isense GTA GCA GGC TTA GGG TTA (328–345) Smoo hened (SMO) sense TCT GCT TCG TGG GTT ACA AG (843–862) XM_414970.4 56 °C 350 an isense TGG GAT GGG TTT ATT GGT CT (1173–1192) Gli1 (Gli1) sense CTC ACC CAC CCA GCA TCA G (2604–2622) XM_004950861.1 58 °C 244 an isense AAT CCC TCC TCC ATC TCC CT (2828–2847) Gli2 (Gli2) sense TTG CTC CAA GGC TTA CTC (1553–1570) M_001271901.1 50 °C 174 an isense TTA CAG ACA TAG GGT TTC TCA T (1705–1726) Gli3 (Gli3) sense TCA CCC GTA CAT TAA CCC (579–596) NM_001271903.1 52 °C 270 an isense CTT GGA CTC GGA AAC CTG (831–848) GAPDH (GAPDH) sense GAG AAC GGG AAA CTT GTC AT (238–258) NM_204305 54 °C 556 an isense GGC AGG TCA GGT CAA CAA (775–793) In . J. Mol. Sci. 2015, 16 17362 Table 2. Tables o an ibodies used in he expe imen s. An ibody Hos Animal Dilu ion Dis ibu o and Ca No. An i-PAC1 abbi , polyclonal, 1:600 Sigma-Ald ich, S . Louis, MO, USA; P8872 An i-VPAC1 abbi , polyclonal, 1:800 Alomone Labs., Je usalem, Is ael; AVR-001 An i-VPAC2 abbi , polyclonal, 1:600 Abcam, Cam idge, UK; ab28624 An i-Coll. II. abbi , polyclonal, 1:100 No us Biologicals, Li le on, CO, USA; NB600-844 An i-Coll. IX. abbi , polyclonal, 1:800 Abcam, Cam idge, UK; ab134568 An i-Coll. X. abbi , polyclonal, 1:800 Sigma-Ald ich, S . Louis, MO, USA; C7974 An i-SHH abbi , polyclonal, 1:600 Cell Signaling, Dan e s, MA, USA; C9C5 An i-IHH abbi , polyclonal, 1:600 Millipo e, Bille ica, MA, USA; MABF23 An i-PTH P mouse, monoclonal, #677939 1:300 R&D Sys ems, Minneapolis, MN, USA; MAB6734 An i-Smoo hened mouse, monoclonal, #2D10 1:500 Sigma-Ald ich, S . Louis, MO, USA; SAB1412475 An i-Gli1 abbi , polyclonal, 1:600 Cell Signaling, Dan e s, MA, USA; V812 An i-Gli2 abbi , polyclonal, 1:500 Sigma-Ald ich, S . Louis, MO, USA; SAB2900411 An i-Gli3 abbi , polyclonal, 1:500 Sigma-Ald ich, S . Louis, MO, USA; HAP005534 An i-Ac in mouse, monoclonal, #AC-15 1:10,000 Sigma-Ald ich, S . Louis, MO, USA; A5441 4.8. S a is ical Analysis All da a a e ep esen a i e o a leas h ee independen expe imen s. A e ages a e exp essed as mean ± SEM (s anda d e o o he mean). S a is ical analysis was pe o med by unpai ed S uden ’s - es . Th eshold o s a is ically signi ican di e ences as compa ed o con ol cul u es was se a * p < 0.05). S a is ical analysis and compa ison o Wes e n blo and PCR esul s can be seen in Figu es 2C,E, 3B,D, and 4B,D. Acknowledgmen s The au ho s a e g a e ul o K isz ina Bí ó o excellen echnical assis ance and o And ea Pál i and Vince Szegecki medical s uden s o hei skil ul help du ing he s udies. This wo k was suppo ed by g an s om Aki a A imu a Founda ion Resea ch G an , he Hunga ian Science Resea ch Fund (OTKA CNK80709 and OTKA K 104984), Bolyai Schola ship and he Hunga ian Minis y o Educa ion (MTA “Lendüle ” P og am) and om he New Széchenyi Plan (TÁMOP-4.2.2.A-11/1/KONV-2012-0053; The p ojec is co- inanced by he Eu opean Union and he Eu opean Social Fund). This esea ch and Tamás Juhász was suppo ed by Bolyai Janos Schola ship, Szodo ay Lajos and Magya y Zol án Funds by he Eu opean Union and he S a e o Hunga y, co- inanced by he Eu opean Social Fund in he amewo k o TÁMOP 4.2.4. A/2-11-1-2012-0001 “Na ional Excellence P og am”. Tamás Juhász and Róza Zákány a e suppo ed by GOP-1.1.1-11-2012-0197 inanced by he Hunga ian go e nmen and he EU. Au ho Con ibu ions Concei ed and designed he expe imen s: Tamás Juhász, Dó a Reglődi, And ea Tamás, Róza Zákány. Pe o med he expe imen s: Tamás Juhász, Esz e Szen léleky, Csilla Szűcs Somogyi, Nó a Dob osi, Roland Takács, Má é Engle . Analyzed he da a: Tamás Juhász, Esz e Szen léleky, In . J. Mol. Sci. 2015, 16 17363 Róza Zákány. Con ibu ed eagen s/ma e ials/analysis ools: Dó a Reglődi, And ea Tamás. W o e he manusc ip : Tamás Juhász, Dó a Reglődi, And ea Tamás, Róza Zákány. Con lic s o In e es The au ho s decla e no con lic o in e es . Abb e ia ions cAMP, cyclic adenosine monophospha e; CREB, cAMP esponse elemen -binding p o ein; DMMB, dime hylme hylene blue; ECM, ex acellula ma ix; HH, hedgehog; IHH, Indian hedgehog; MAPK, mi ogen-ac i a ed p o ein kinase; NMDA, N-Me hyl-D-aspa a e; PAC1, pi ui a y adenyla e cyclase-ac i a ing polypep ide ype I ecep o ; PACAP, pi ui a y adenyla e cyclase polypep ide; PKA, p o ein kinase A; PTH P, pa a hy oid ho mone ela ed pep ide; Runx2, Run - ela ed ansc ip ion ac o 2; SHH, Sonic hedgehog; TB, oluidine blue; TGFβ, ans o ming g ow h ac o -β; TRPV, ansien ecep o po en ial channels o anilloid; VIP, asoac i e in es inal pep ide; VPAC, asoac i e in es inal pep ide ecep o . 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