IL-1ra Secreted by ATP-Induced P2Y2 Negatively Regulates … · 2019-09-04 · IL-1ra c + ATP Positive control P2Y 2si-P2Y (a) GAPDH Mock + ATP IL-1ra P2Y 2 si-P2Y 2 (b) 0 2 4 6 8

Research ArticleIL-1ra Secreted by ATP-Induced P2Y2 NegativelyRegulates MUC5AC Overproduction via PLC1205733 duringAirway Inflammation

Jee-Yeong Jeong12 Jiwook Kim3 Bokyoum Kim3 Joowon Kim3 Yusom Shin3

Judeok Kim3 Siejeong Ryu3 Yu-Mi Yang4 and Kyoung Seob Song56

1Department of Biochemistry Kosin University College of Medicine Busan 49267 Republic of Korea2Cancer Research Institute Kosin University College of Medicine Busan 49267 Republic of Korea3Department of Anesthesiology and Pain Medicine Kosin University College of Medicine Busan 49267 Republic of Korea4Department of Oral Biology BK21 PLUS Project Yonsei University College of Dentistry Seoul 03722 Republic of Korea5Department of Physiology Kosin University College of Medicine Busan 49267 Republic of Korea6Institute of Medicine Kosin University College of Medicine Busan 49267 Republic of Korea

Correspondence should be addressed to Kyoung Seob Song kssongkosinackr

Received 15 September 2015 Revised 29 January 2016 Accepted 4 February 2016

Academic Editor Yona Keisari

Copyright copy 2016 Jee-Yeong Jeong et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Mucus secretion is often uncontrolled in many airway inflammatory diseases of humans Identifying the regulatory pathway(s) ofmucus gene expression mucus overproduction and hypersecretion is important to alleviate airway inflammation in these diseasesHowever the regulatory signaling pathway controlling mucus overproduction has not been fully identified yet In this study wereport that the ATPP2Y

2complex secretes many cytokines and chemokines to regulate airway inflammation among which IL-

1 receptor antagonist (IL-1ra) downregulates MUC5AC gene expression via the inhibition of G120572q-induced Ca2+ signaling IL-1rainhibited IL-1120572 protein expression and secretion and vice versa Interestingly ATPP2Y

2-induced IL-1ra and IL-1120572 secretion were

both mediated by PLC1205733 A dominant-negative mutation in the PDZ-binding domain of PLC1205733 inhibited ATPP2Y2-induced IL-

1ra and IL-1120572 secretion IL-1120572 in the presence of the ATPP2Y2complex activated the ERK12 pathway in a greater degree and

for a longer duration than the ATPP2Y2complex itself which was dramatically inhibited by IL-1ra These findings suggest that

secreted IL-1ra exhibits a regulatory effect on ATPP2Y2-inducedMUC5AC gene expression through inhibition of IL-1120572 secretion

to maintain the mucus homeostasis in the airway Therefore IL-1ra could be an excellent modality for regulating inflamed airwaymicroenvironments in respiratory diseases

1 Introduction

Mucus overexpression and hypersecretion in the humanrespiratory track are a critical characteristic of a numberof pulmonary diseases including rhinitis sinusitis asthmaCOPD and cystic fibrosis [1] A family of highly glycosylatedproteins named mucins has been identified as importantconstituents of mucus However the physiological functionsof mucins have not been fully understood yet In thehuman body several innate immune systems downregulatemucin overexpression and hypersecretion thus decreaseinflamed microenvironments and maintain homeostasis

Identification of the regulatory mechanisms which lead tocontrol mucin overexpression and hypersecretion in pul-monary diseases is essential for developing new therapeuticdrugs Since MUC5AC (for human Muc5ac for nonhumans)is one of the major mucins in human airway diseases themechanisms regulatingMUC5AC gene expression have beenconsidered critical to identify strategies to prevent airwaymucus overproduction

The IL-1 family of molecules consists of two agonistsIL-1120572 and IL-1120573 and a specific receptor antagonist calledIL-1ra which interact with two different receptors IL-1Rtype I (IL-1RI) and IL-1R type II (IL-1RII) [2 3] The IL-1

Hindawi Publishing CorporationMediators of InflammationVolume 2016 Article ID 7984853 10 pageshttpdxdoiorg10115520167984853

2 Mediators of Inflammation

family plays an important role in maintaining homeostasisagainst microorganisms air pollutants exotoxins and air-borne particulate matters in the human respiratory track Italso takes an important part in the innate immune systemwhich further regulates functions of the adaptive immunesystem [4]The balance between IL-1 and IL-1ra in the humanlung may affect the development of inflammatory diseasesVoelkel et al reported that treatment with IL-1ra reducedpulmonary hypertension generated by monocrotaline in rats[5] but IL-1120573 one of the earliest mediators secreted duringa proinflammatory response is present at high levels in thelungs of patients with chronic lung diseases [6] Howeverother secreted inflammatory mediators can also regulatetheir intracellular expressions thereby further modulatingcytokine-regulated MUC5AC overproduction

The purinergic P2Y2receptor (P2Y

2R) is highly expressed

in the apical membrane of the respiratory track [7ndash9] Extra-cellular nucleotides bound to the P2Y

2R have been shown to

induce a number of physiological phenomena in a variety ofhuman cell types and tissues [10] Recently P2Y

2R deficiency

was shown to attenuate IFN120574 IL-17 and TNF120572 secretionin mice [11] P2Y couples to the G-protein subtype G120572qand the G120572q protein regulates Ca2+ influx upon stimulationwith ligands [12] Next elevated [Ca2+]i increases PLC1205733PLC120573 isotypes have a long C-terminal region (sim400 residues)with relatively low homology among family members [13]This C-terminal region (residues 903ndash1142 of PLC1205731) isrequired for binding and stimulation by G120572q [14] MoreoverPLC120573 isotypes possess short consensus sequences knownas postsynaptic density- (PSD-) 95disc largeZO-1- (PDZ-)binding motifs The PDZ-binding motif is a well-knownshort linear motif that interacts with other PDZ proteins[15]The regulatorymechanism of PLC1205733-mediated cytokinesecretion to controlMUC5AC gene expression and the signalmolecules involved especially those involved in downstreamsignaling of PLC1205733 has not yet been demonstrated in thehuman airway epithelium

Here we report that ATP induces IL-1ra overexpressionand secretion via the P2Y

2receptor in human airway epithe-

lial cells IL-1ra secretion was associated with the P2Y2-G120572q-

[Ca2+]i-PLC1205733-ERK12 pathway sequentially Secreted IL-1ra inhibits ATPP2Y

2-induced MUC5AC gene expression

but IL-1120572 significantly increases the expression ofMUC5ACThe results of the present study reveal a novel mechanismfor intra- and intercellular signaling pathways that controlairway inflammation thus providing an opportunity forthe development of novel therapeutic modalities to treatrespiratory diseases

2 Materials and Methods

21 Materials ATP and Apyrase were purchased from SigmaAldrich (St LouisMO)The IL-1ra and IL-1120572ELISA kits werepurchased form RampD Systems (Minneapolis MN) siRNAtargeting P2Y

2was synthesized by Bioneer (Daejeon Korea)

P2Y2 GAGGAAGGUGGCUUACCAA (dTdT) and negative

control CCUACGCCACCAAUUUCGU (dTdT) All PLC1205733isotypes were kindly provided by Dr Pann-Ghill Suh (School

of Life Sciences Ulsan National Institute of Science andTechnology Ulsan Korea)

22 Cytokine Assay The cytokine assay was performed usinga Human Cytokine Array Panel A kit (RampD Systems) accord-ing to the manufacturerrsquos instructions Briefly cells wereplated in 6-well plates one day prior to transfection witheither P2Y

2or siRNA-P2Y

2using FuGENE 6 (Roche Indi-

anapolis IN) Twenty-four hours after transfection serum-starved cells were treated with ATP for 4 hours Aftertreatment the supernatant was assayed according to the kitinstructions

23 RT-PCR Real-time PCR was performed using a BioRadiQ iCycler Detection System (BioRad Laboratories HerculesCA) with iQ SYBR Green Supermix Reactions were per-formed in a total volume of 20 120583L including 10 120583L 2x SYBRGreen PCR Master Mix 300 nM of each primer and 1120583Lof previously reverse-transcribed cDNA template The fol-lowing primers were used MUC5AC forward 51015840-CAGCCA-CGTCCCCTTCAATA-31015840 and reverse 51015840-ACCGCATTT-GGGCATCC-31015840 120573

2-microglobulin used as a reference for

normalization forward 51015840-CGCTCCGTGGCCTTAGC-31015840and reverse 51015840-GAGTACGCTGGATAGCCTCCA-31015840 Param-eters were 95∘C for 10m followed by 40 cycles of 95∘C for 15 s60∘C for 30 s and 72∘C for 30 s All reactions were performedin triplicate Relative quantity of MUC5AC mRNA wasobtained using a comparative cycle threshold method andwas normalized using 120573

2-microglobulin as an endogenous

control

24 Western Blot Analysis NCI-H292 cells were grown toconfluence in 6-well plates After treatment with ATP cellswere lysed with 2x lysis buffer (250mM Tris-Cl (pH 65)2 SDS 4 120573-mercaptoethanol 002 bromphenol blueand 10 glycerol) Equal amounts of whole cell lysateswere resolved by 10ndash15 SDS-PAGE and transferred topolyvinylidene difluoride membranes (Millipore BedfordMA) Membranes were blocked with 5 skim milk in Tris-buffered saline (50mM Tris-Cl (pH 75) 150mM NaCl) for2 h at room temperature Blots were then incubated overnightwith primary antibodies in TTBS (05 Tween 20 in Tris-buffered saline) After washing with TTBS blots were furtherincubated for 45min at room temperature with anti-rabbitor anti-mouse antibody (Cell Signaling Danvers MA) inTTBS and visualized using the ECL system (GE HealthcareUppsala Sweden)

25 Measurement of Intracellular Ca2+ Concentration([Ca2+]i) NCI-H292 cells were seeded on cover glasses in35-mm dishes (5 times 104 cells) After 24 h cells were transfectedwith P2Y

2-expressing construct The cells expressing P2Y

2

in physiological salt solution [140mM NaCl 5mM KCl1mM MgCl

2 1 mM CaCl

2 10mM HEPES 10mM glucose

and 310mOsm (pH 74)] were incubated with 5 120583M Fura-2AM (TEFLabs Inc Austin TX) and 005 pluronicF-127 for 60min at room temperature Fura-2 fluorescencewas measured at the appropriate excitation wavelengths

Mediators of Inflammation 3

(340380 nm) and emission at 510 nm wavelengths (ratio =F340F380) using a Molecular Devices (Downingtown PA)imaging system The emitted fluorescence was monitoredwith a charge-coupled device camera (Photometrics TucsonAZ) attached to an invertedmicroscope Fluorescence imageswere obtained at 2 sec intervals All data were analyzed usingMetaFlour software (Molecular Devices)

26 Statistical Analysis Data are presented as the mean plusmnSD of at least three independent experimentsWhere appro-priate statistical differences were assessed by the WilcoxonMann-Whitney test 119901 values less than 005 were consideredstatistically significant

3 Results

31 ATP Secretes IL-1ra Extracellularly via the G120572q-CoupledP2Y2Receptor in NCI-H292 Cells In our previous study

[12] ATP was shown to induce MUC5AC gene expres-sion via the P2Y

2receptor and the MAPK pathway in

NCI-H292 (mucoepidermoid pulmonary carcinoma) cellsTo determine whether ATP treatment induces or inhibitssecretion of cytokines that may regulate the respiratorymicroenvironment to maintain homeostasis we performedthe cytokine array with cell culture medium after treatmentof NCI-H292 cells with ATP for 4 hours (Figure 1(a)) Thesecretion of IL-1 receptor antagonist (IL-1ra) was increasedin cells overexpressing P2Y

2 but not in cells transfected

with P2Y2-specific siRNA (Figure 1(a)) To examine whether

P2Y2is essential for IL-1ra secretion and overproduction

we performedWestern blot analysis (Figure 1(b)) and IL-1ra-specific ELISA (Figure 1(c)) in the presence or absence oftransfectionwith the constructs forwild-type P2Y

2or siRNA-

P2Y2expression Treatment with ATP alone for four hours

did not induce either IL-1ra secretion or overproductionhowever ATP did induce IL-1ra secretion and overpro-duction in cells overexpressing P2Y

2 Conversely siRNA-

mediated knockdown of P2Y2dramatically decreased ATP-

mediated induction of IL-1ra secretion and overproductionAfter showing that IL-1ra secretion was accelerated early bythe inflammatory response we examined whether ATPP2Y

2

signaling could induce IL-1ra overproduction We previ-ously showed that IL-1120572 secretion and overproduction wereupregulated by the ATPP2Y

2signaling complex in airway

epithelium [16] Extracellular secretion of IL-1ra reached itsmaximum level after 4 hours of treatment with ATPP2Y

2

and decreased thereafter (Figure 1(d)) Next to investigatewhether extracellular ATP secreted by exogenous ATP-induced P2Y

2activation is required for this phenomenon

cells were incubated with Apyrase an ATPaseADPase inorder to block the action of secreted ATP followed by incu-bation with ATP120574S which is not hydrolyzed by Apyrase IL-1ra secretion induced by ATP120574S treatment was decreased inan Apyrase dose-dependent manner (Figure 1(e)) indicatingthat the secreted ATP rather than the treated ATP is criticalfor increased IL1-ra secretion In addition we also comparedthe stimulatory activities of various nucleotides for IL-1rasecretion Only ATP or UTP could dramatically increase

the secretion of IL-1ra in cells expressing P2Y2(Figure 1(f))

The effects of nucleotides on IL-1ra secretion were as followsATP gt UTP gt GTP = ADP = UDP These results suggestthat intracellular secretion of IL-1ra is stimulated by theATPP2Y

2complex which plays an important immuno-

logical role to control inflammatory signaling in airwaycells These results demonstrated that cells activated bythe ATPP2Y

2complex could promote secretion of ATP

which may trigger signal transduction pathways in eitheran autocrine or paracrine manner resulting in subsequentsecretion of IL-1ra from the cells ATPP2Y

2is appeared to

be essential for extracellular secretion and overproduction ofIL-1ra in airway epithelial cells

32 IL-1ra Antagonizes IL-1120572 Secretion and Overproduction toInhibit IL-1120572-Induced MUC5AC Gene Expression We previ-ously showed thatMUC5AC gene expressionwas upregulatedby LPS-induced secretion of ATP [17] In addition IL-1120572secretionwas alsomediated by ATPP2Y

2signaling to induce

MUC8 gene expression [16] Thus we hypothesized that IL-1ra secreted by ATPP2Y

2might regulate IL-1120572 secretion and

overproduction to antagonize IL-1120572 function Moreover wespeculated that IL-1120572 secretion could alter IL-1ra secretionand overproduction reciprocally First we confirmed thatthe P2Y

2complex was essential for ATP-induced MUC5AC

gene expression in human airway epithelial NCI-H292cells (Figure 2(a)) [12] Second P2Y

2-transfected cells were

treated with either ATP only or ATP and IL-1ra (50 ngmL)for 4 hours We found that IL-1ra treatment inhibitedATPP2Y

2-induced IL-1120572 overproduction by Western blot

analysis (Figure 2(b) upper panel) and its secretion by ELISA(Figure 2(b) lower panel) In the same way IL-1120572 treatmentalso inhibited IL-1ra secretion and overproduction recipro-cally (Figure 2(c)) Next we investigated whether secretedIL-1120572 or IL-1ra by ATPP2Y

2could increase MUC5AC gene

expression in airway epithelial cells Interestingly whereasIL-1ra treatment dramatically decreasedATPP2Y

2-mediated

induction of MUC5AC gene expression IL-1120572 treatmentslightly increased expression of MUC5AC (Figure 2(d)) Toexamine the role of IL-1ra in IL-1120572 secretion by cells express-ing P2Y

2 we cotreated cells with both IL-1120572 (50 ngmL) and

IL-1ra in a dose-dependent manner (0 100 and 150 ngmL)as a competitive inhibitor reciprocally Because IL-1ra hasthe same binding strength for the IL-1 receptor as IL-1120572 the intrinsic agonist activity of IL-1120572 may abolish itsown function High dosages (100 and 150 ngmL) of IL-1ra markedly reduced IL-1120572 (50 ngmL)-induced MUC5ACgene expression however IL-1120572 dosages (100 and 150 ngmL)dramatically increased IL-1ra (50 ngmL)-reduced expression(Figure 2(e)) These results indicate that IL-1ra secretionantagonizes physiological phenomena induced by IL-1120572Therefore IL-1ra secreted by the ATPP2Y

2complex can

regulate airway inflammation to decrease MUC5AC geneexpression in airway epithelial cells

33 IL-1ra Abolishes G120572q-Mediated PLC1205733 Activation andCa2+ Secretion To determine whether IL-1ra or IL-1120572 canaffect phospholipase C (PLC) 1205733 activation we performed


IL-1ra

c +ATP

Positivecontrol

P2Y2 si-P2Y2

(a)

GAPDH

Mock +ATP

IL-1raP2Y2 si-P2Y2

(b)

0

2

4

6

8

10

12

Mock +

ATP

IL-1

ra se

cret

ion

(pg

mL)

lowastlowast

lowast

P2Y2 si-P2Y2

(c)

Time (hr) 2 4 6 8

ATP + + + + + + + ++ + + +

0

2

4

6

8

10

12

IL-1

ra se

cret

ion

(pg

mL)

lowast

lowast

P2Y2 minusminusminusminus

(d)

Apyrase (UmL)

IL-1

ra se

cret

ion

(pg

mL)

0

2

4

6

8

10

12

lowastlowastlowast

lowastlowast

lowast

5025100c

ATP120574S + P2Y2

ATP120574Sonly

(e)

IL-1

ra se

cret

ion

(pg

mL)

Moc

k

ATP

UTP

AD

P

UD

P

GTP AT

P

UTP

AD

P

UD

P

GTP

pcDNA3

0

2

4

6

8

10

12 lowast

P2Y2

(f)

Figure 1 ATP induces IL-1ra secretion and overexpression through a P2Y2-dependent pathway inNCI-H292 cells (a) A construct expressing

wild-type P2Y2or siRNA-P2Y

2was transiently transfected into NCI-H292 cells The cells were washed and serum-starved overnight They

were subsequently treated with ATP for 4 hours and a cytokine assay was performed (b) Cells were transfected with either a construct drivingthe expression of wild-type P2Y

2or P2Y

2-specific siRNA Cells were then treated with ATP for four hours and their supernatants collected

The total cell lysates were analyzed byWestern blot analysis Mock transfection was performed with parent pcDNA3 only (c)The levels of IL-1ra in the cell supernatants were measured by ELISA lowast119901 lt 005 compared with ATP only lowastlowast119901 lt 005 compared with ATPP2Y

2-treated cells

(d) Cells were transfected with pcDNA31P2Y2 or pcDNA31 and were treated with ATP (10120583M) for the indicated times The concentrationsof IL-1ra in the resultant supernatants were measured by ELISA Values shown are the means plusmn SD of three technical replicates from asingle experiment lowast119901 lt 005 compared with the ATP only treatment each time (e) After transfection with wild-type P2Y

2 the cells were

subsequently treated with Apyrase for 4 hours prior to treatment with ATP120574S (10 120583M) for 4 hours IL-1ra secretion was measured by ELISAValues shown represent themeansplusmn SDs of three technical replicates from a single experiment lowast119901 lt 005 compared to the control lowastlowast119901 lt 005compared to ATP only and lowastlowastlowast119901 lt 005 compared to ATPP2Y

2-treated cells (f) Cells transfected with either pcDNA3 or wild-type P2Y

2

were treated with nucleotides (10 120583M each) for 4 hours IL-1ra secretion was measured by ELISA Values shown represent the means plusmn SDsof three technical replicates from a single experiment lowast119901 lt 005 compared with control Data shown are representative of three independentexperiments

Western blot analysis with a phospho-specific PLC1205733 anti-body (Figure 3(a)) IL-1ra dramatically inhibited PLC1205733phosphorylation whereas IL-1120572 slightly increased phospho-rylation of PLC1205733 PLC1205733 has short consensus sequencesknown as postsynaptic density-95discs largeZO-1- (PDZ-)

bindingmotifs that consist of the amino acids X(ST)X(VL)-COOH at the immediate C-terminus [12 15] The PDZdomain of PLC1205733 is critical for binding to the four aminoacids at the C-terminus of target proteins [18ndash20] To inves-tigate whether the PDZ domain of the PLC1205733 C-terminus


c +ATP

0

2

4

6

8

gene

expr

essio

n (fo

ld o

ver b

asal

)

lowastlowastlowast

lowastlowast

lowast

P2Y2 si-P2Y2

Nor

mal

ized

rela

tiveM

UC5

AC(a)

c ATP only +IL-1120572

Tubulin

IL-1ra

IL-1120572

secr

etio

n (p

gm

L)

0

5

10

15

lowast

lowastlowast

lowastlowastlowast

ATP + P2Y2

(b)

c ATP only + IL-1120572

Tubulin

IL-1ra

IL-1

ra se

cret

ion

(pg

mL)

0

3

6

9

12

lowastlowast

ATP + P2Y2

lowast

(c)

c ATP only + IL-1120572 IL-1ra0

2

4

6

8

gene

expr

essio

n (fo

ld o

ver b

asal

)

lowast

lowastlowast

ATP + P2Y2

lowastlowastlowast

lowastlowastlowast

Nor

mal

ized

rela

tiveM

UC5

AC

(d)

c +ATP IL-1120572IL-1ra

gene

expr

essio

n

IL-1120572IL-1ra

0

3

6

9

ATP + P2Y2

lowastlowastlowast

lowastlowast

lowast

onlyNor

mal

ized

rela

tive M

UC5

AC

(fold

ove

r bas

al)

(e)

Figure 2 IL-1ra inhibits IL-1120572-induced MUC5AC gene expression via inhibiting IL-1120572 secretion and overproduction (a) Cells weretransfected with either a construct driving the expression of wild-type P2Y

2or siRNA specific for P2Y

2 Cells were then treated with ATP

for 24 hours prior to the generation of total cell lysates and then qRT-PCR forMUC5AC transcript was performed lowast119901 lt 005 compared tothe control lowastlowast119901 lt 005 compared to ATP only and lowastlowastlowast119901 lt 005 compared to ATPP2Y

2-treated cells (b) After transfection with wild-type

P2Y2or siRNA-P2Y

2 the cells were subsequently treated with IL-1ra (50 ngmL) for 4 hours prior to treatment with ATP for 4 hours IL-1120572

productionsecretion was measured by Western blot and ELISA respectively using the identical set of samples Values shown represent themeans plusmn SDs of three technical replicates from a single experiment Two additional experiments showed similar results lowast119901 lt 005 comparedto the control lowastlowast119901 lt 005 compared to ATP only and lowastlowastlowast119901 lt 005 compared to ATPP2Y

2-treated cells (c) Cells were transfected with either

a construct driving the expression of wild-type P2Y2or siRNA specific for P2Y

2 The cells were subsequently treated with IL-1120572 (50 ngmL)

for 4 hours prior to treatment with ATP for 4 hours IL-1ra productionsecretion was measured by Western blot and ELISA Values shownrepresent the means plusmn SDs of three technical replicates from a single experiment lowast119901 lt 005 compared to ATP only and lowastlowast119901 lt 005 comparedto ATPP2Y

2-treated cells (d) After transfection with wild-type P2Y

2 the cells were subsequently treated with either IL-1ra (50 ngmL) or IL-

1120572 (50 ngmL) for 4 hours prior to treatment with ATP for 24 hours qRT-PCR forMUC5AC transcript was performed lowast119901 lt 005 comparedto the control lowastlowast119901 lt 005 compared to ATP only and lowastlowastlowast119901 lt 005 compared to ATPP2Y

2-treated cells (e) After transfection with wild-type

P2Y2 the cells were treated with both IL-1120572 (50 ngmL) and IL-1ra (0 100 150 ngmL) or both IL-1ra (50 ngmL) and IL-1120572 (0 100 150 ngmL)

for 4 hours prior to treatment with ATP for 24 hours qRT-PCR forMUC5AC transcript was performed lowast119901 lt 005 compared to the controllowastlowast119901 lt 005 compared to ATP only and lowastlowastlowast119901 lt 005 compared to ATPP2Y

2-treated cells Data shown are representative of three independent

experiments


plays a role in ATPP2Y2-activated IL-1ra and IL-1120572 secre-

tion and overproduction each of the last four amino acidresidues of PLC1205733 (1231NTQL1234-COOH) was mutated toAla [12]When treated with ATP for 4 hours cells transfectedwith the construct overexpressing wild-type PLC1205733 (NTQL)showed an increase in IL-1120572 and IL-1ra secretion whereas thedominant-negative mutant PLC1205733 NTQA (L1234A) signif-icantly inhibited their secretion In contrast PLC1205733 ATQL(N1231A) NAQL (T1232A) and NTAL (Q1233A) had noinhibitory effect on IL-1120572 and IL-1ra secretion (Figure 3(b))These results indicate that the fourth amino acid (Leucine)of the PDZ domain in PLC1205733 is critical for binding someregulatory protein(s) to control IL-1ra and IL-1120572 secretionand overexpression by the ATPP2Y

2signaling complex

These results show that PLC1205733 may have a major physiolog-ical function in the pathway mediating ATPP2Y

2-induced

IL-1ra and IL-1120572 secretion thus regulating MUC5AC geneexpression in NCI-H292 cells P2Y

2is a G120572q-coupled G-

protein receptor therefore we investigated whether IL-1rahas an effect on P2Y

2-induced Ca2+ influx after treatment

with ATP IL-1ra significantly decreased [Ca2+]i but IL-1120572slightly increased [Ca2+]i (Figures 3(a) and 3(b))

34 Downstream Signaling by PLC1205733 of the ERK12-CREBPathway Is Controlled by IL-1ra and IL-1120572 ERK12 signalingis critical for ATPP2Y


in airway epithelial cells [12] therefore we hypothesizedthat IL-1ra or IL-1120572 may affect ERK12 MAPK activationPhosphorylation of ERK12 was increased by ATP treat-ment for 5min but then decreased rapidly ERK12 phos-phorylation activated by IL-1120572 was prolonged comparedto phosphorylation by the ATPP2Y

2signaling pathway

(Figure 4(a)) However IL-1ra treatment for 10min inhibitedERK12 phosphorylation increased by ATPP2Y

2signaling

Treatment with IL-1120572 or ATP for 10 and 15min respec-tively had an additive effect on ERK12 phosphorylationwhich was dramatically inhibited by IL-1ra (Figure 4(b))Interestingly treatment with both ATP and IL-1120572 resulted inprolonged activity to maintain ERK12 activation whereasIL-1ra remarkably abolishedATPP2Y

2-induced ERK12 acti-

vation These results suggest that IL-1120572 prolonged ERK12activation but IL-1ra had a negative effect on activationNext to examine whether ERK12 MAPK is essential for IL-1ra- or IL-1120572-modulated MUC5AC gene expression U0126a MEK12 specific inhibitor was evaluated (Figure 4(c)) Asexpected IL-1ra- and IL-1120572-mediated gene expression ofMUC5AC were downregulated by U0126 Thus ERK12 maybe associated with IL-1ra- and IL-1120572-modulated MUC5ACgene expression in human airway epithelial cells

4 Discussion

MUC5AC known as tracheobronchial mucin [21] is a majormucin of lung mucus and is abundantly expressed duringacute and chronic lung diseases [22] and thereby greatly con-tributes to disease morbidity and mortality [6] As describedabove studies regarding MUC5AC have focused on the

signaling pathway for gene expression itself by different stim-ulants such as proinflammatory cytokines LPS endotoxinscigarette smoking yellow dust or airborne particulatematterin several lung cell types However there are few reports onregulatory pathways that controlMUC5AC secretion or over-production An understanding of the regulatory mechanismsthat control mucus hypersecretion in respiratory diseases isessential for improving future therapies

The important proinflammatory role of IL-1 in manyhuman diseases has been described over the past a decadeThe balance between IL-1 and IL-1ra has been extensivelystudied in a variety of experimental animalmodels of diseasesincluding arthritis inflammatory bowel disease (IBD) gran-ulomatous and fibrotic lung disorders infectious diseasesand arterial diseases [4] Extracellular ATP is known toregulate a number of inter- and intracellular functions inmany cells and tissues [23] ATP is extracellularly secreted inresponse to LPS-induced airway inflammation in the respi-ratory system [12] In several studies ATPP2Y

2signaling in

the airway is a critical sensor for airway exposure to airborneallergens and secretes the IL-33 cytokine into the airwaylumen [24] We hypothesized that ATPP2Y

2signaling may

function in maintaining homeostasis by abolishing inflamedmicroenvironments in the airway or by creating amore severeinflamed condition Therefore we tried to identify whichproteins or signaling molecules control these processesWe speculated that IL-1ra and IL-1120572 which have oppositeregulatory roles may also be important in human airwayepithelial cells We found that IL-1ra or IL-1120572 has a negativeeffect on production and secretion of each other reciprocallyThis is consistent with the results of studies on leukocytesshowing that binding of IL-1120572 to IL-1RI results in inductionof bioactive IL-1120573 [25 26] and sIL-1ra [27] whereas sIL-1rainhibits both the activity and synthesis of IL-1120572 and IL-1120573in monocytes [28] These results suggest that overexpressionof IL-1120572 andor underproduction of IL-1ra predisposes oneto the development of disease therefore the therapeuticadministration of IL-1ra is efficacious in preventing tissuedamage [4]

P2Y2is a G120572q-coupled receptor therefore an increase

in the concentration of Ca2+ influx was not surprising resultof this study However it is notable that secreted IL-1ra orIL-1120572 regulated the concentration of Ca2+ influx induced byATPP2Y

2(Figures 3(a) and 3(b)) More surprisingly IL-

1ra inhibited PLC1205733 activation (Figure 3(a)) These resultssuggest that Ca2+-PLC1205733 may be essential for IL-1ra- or IL-1120572-regulated MUC5AC gene expression in airway epithelialcells Understanding the physiological features of the PDZdomain in PLC1205733 will provide additional insight into themolecular signalingmechanism that leads to protein complexformation [12] The PDZ domain is critical for signalingbecause it depends heavily on the PDZ-binding partnerprotein(s) to modulate positivelynegatively its own signaltransduction pathway Our previous study reported that thePDZ domain in PLC1205733 is associated with ATP-inducedMUC5AC gene expression [12] In the present study wefound that the binding activity of the PDZ domain andconsequently the function of PLC1205733 is determined by the


c ATP only + IL-1120572 IL-1ra

Tubulin

ATP + P2Y2

p-PLC1205733

(a)

PLC120573

3-N

TQL

PLC120573

3-AT

QL

PLC120573

3-N

AQL

PLC120573

3-N

TAL

PLC120573

3-N

TQA

ATP

only +c

PLC120573

3-N

TQL

PLC120573

3-AT

QL

PLC120573

3-N

AQL

PLC120573

3-N

TAL

PLC120573

3-N

TQA

ATP

only +c

0

10

20

30

0

10

20

30

IL-1120572

secr

etio

n (p

gm

L)

IL-1

ra se

cret

ion

(pg

mL)

ATP + P2Y2 ATP + P2Y2

lowastlowastlowast

lowastlowast

lowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowast

lowast

(b)

01Ratio

ATP ATP + IL-1ra

ATP + IL-1120572

ATP

ATP

ATP

2min(c)

Nor

mal

ized

pea

k va

lue

IL-1120572+ IL-1ra0

30

60

90

120

ATP

lowastlowast

lowast

(d)

Figure 3 Effect of PLC1205733 activation on IL-1ra and IL-1120572 secretion (a) After transfection with wild-type P2Y2 the cells were subsequently

treated with IL-1ra or IL-1120572 for 4 hours prior to treatment with ATP for 5min PLC1205733 activation was measured byWestern blot Tubulin wasused as a loading control (b) Cells were transiently transfected with both wild-type P2Y

2and wild-type (1231NTQL1234) dominant-negative

PLC1205733ATQL (N1231A) NAQL (T1232A) NTAL (Q1233A) orNTQA (L1234A) construct Each of the individual residues of the PDZ-bindingmotif (NTQL) of PLC1205733 was mutated to Ala respectively Cells were serum-starved overnight and then treated with ATP for 4 hours afterwhich supernatants were harvested for ELISA For IL-1120572 ELISA (left panel) lowast119901 lt 005 compared to the control lowastlowast119901 lt 005 compared toATP only lowastlowastlowast119901 lt 005 compared to ATPP2Y

2-treated cells and lowastlowastlowastlowast119901 lt 005 compared to ATPP2Y

2wild-type PLC1205733-treated cells For

IL-1ra ELISA (right panel) lowast119901 lt 005 compared to ATP only lowastlowast119901 lt 005 compared to ATPP2Y2-treated cells and lowastlowastlowast119901 lt 005 compared

to ATPP2Y2wild-type PLC1205733-treated cells Data shown are representative of three independent experiments (c) After transfection with

wild-type P2Y2 the cells were treated with ATP IL-1120572 or IL-1ra Ca2+ release was measured by adding increasing concentrations of Fura-2

See in Section 2 (d) Normalized peak value of the results in Figure 4(a) lowast119901 lt 005 compared to ATP-treated cells lowastlowast119901 lt 005 compared toATPP2Y

2IL-1120572-treated cells


0 5 10 15 30 60+ minus minus minus

+ + minus minus

+ + + minus

+ + minus +

p-ERK12

Tubulin

ATP

IL-1120572

IL-1

ra

P2Y 2

(a)

ATP

only

+ IL-1120572

IL-1

ra

p-ERK12

Tubulin

p-ERK12

Tubulin

cTreatment

time

10m

15m

ATP + P2Y2

(b)

0

2

4

6

8

gene

expr

essio

n (fo

ld o

ver b

asal

)

c

ATP

only +

IL-1120572

IL-1

raU

IL-1120572

+ U

IL-1

ra +

U

lowastlowast

lowastlowastlowast

lowast

ATP + P2Y2

Nor

mal

ized

rela

tiveM

UC5

AC

(c)

GDP120572 GTP

ATP

120572 GTP

ERK12

IL-1ra

IL-1ra IL-1120572

IL-1120572

MUC5AC

PLC1205733

P2Y2 120573120574

120572

[Ca2+]uarr

uarr

(d)

Figure 4 IL-1ra decreases ATPP2Y2-inducedMUC5AC gene expression by inhibiting ERK12 activation After transfection with wild-type

P2Y2 the cells were treated with either IL-1ra or IL-1120572 for 4 hours prior to treatment with ATP for different times (a) or either 10 or 15min (b)

ERK12 phosphorylation was analyzed byWestern blot Tubulin was used as a loading control (c) After transfection with wild-type P2Y2 the

cells were treated with IL-1ra IL-1120572 or U0126 (10 120583M) for 4 hours prior to treatment with ATP for 24 hours qRT-PCR forMUC5AC transcriptwas performed lowast119901 lt 005 compared to the control lowastlowast119901 lt 005 compared to ATP only and lowastlowastlowast119901 lt 005 compared to ATPP2Y

2-treated cells

Data shown are representative of three independent experiments (d) A schematic diagram is presented to show the potential mechanismsfor secretion of IL-1ra and IL-1120572 and their physiological roles during inflammatory responses

ability of L1234 leading to secretion of IL-1ra or IL-1120572which in turn controls MUC5AC gene expression in airwayepithelial cells This is a noteworthy result which suggeststhat the balance between IL-1ra and IL-1120572 can contribute toan inflamed microenvironment in the airway For the samereason PDZ-binding partner protein(s) that can be recruitedto increase inflammatory conditions ormaintain homeostasisin the respiratory track should further be investigated

The molecule mediating PLC1205733 signaling to control IL-1120572- or IL-1ra-regulatedMUC5AC gene expression is yet to beelucidated In our previous studies ERK12 MAPK was iden-tified as a molecule involved in cytokine-inducedmucin geneexpression [12 13 29] Since this protein is tightly regulatedby PLC1205733 in NHNE and NCI-H292 cells [12] its effect onATP-mediated cytokine signaling also needed to be clarifiedThe activation of ERK12 by IL-1120572 was more prolonged thanthat of ATP alone (Figures 4(a) and 4(b)) suggesting thatERK12 may be essential for ATPP2Y

2-induced MUC5AC

gene expressionThis is not surprising since ERK12 played animportant role as a protein kinase to increaseMUC5AC gene

expression by various cytokines in our system ATPP2Y2-

induced ERK12 phosphorylation was inhibited by IL-1raresulting in downregulation of ATPP2Y

2-inducedMUC5AC

gene expression Consequently IL-1ra secretion was affectedby PLC1205733 activation thus the secretion of IL-1ra couldcontrol MUC5AC gene expression by regulating ERK12activity (Figure 4(c))

Taken together we suggest that the interaction betweenATP and the P2Y

2receptor led to the secretion of various

cytokines regulating airway inflammation Not only can ATPinduce inflammation but also it can also amplify and transferinflammatory signals to nearby epithelial cells in the airwayThe G120572q-PLC1205733-Ca2+ pathway modulated IL-1ra and IL-1120572secretion to maintain homeostasis Therefore the balancebetween these cytokines up- or downregulated MUC5ACgene expression In this signaling pathway the PDZ domainin PLC1205733 may play a crucial role in the developmentof inflammation in the airway In addition secreted IL-1ra diminished IL-1120572 productionsecretion and MUC5ACgene expression (Figure 4(d)) Thus the development of a


protein or small molecule as a novel therapeutic drug toinhibit ATPP2Y

2signaling could be useful to control airway

inflammation

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Jee-Yeong Jeong and Jiwook Kim contributed equally to thiswork

Acknowledgment

This study was supported by a grant from the NationalResearch Foundation of Korea (NRF) funded by the Koreangovernment (MEST NRF-2015R1D1A1A01058387 for Jee-Yeong Jeong and NRF-2014R1A1A2055774 for Kyoung SeobSong)

References

[1] M C Rose and J A Voynow ldquoRespiratory tract mucin genesand mucin glycoproteins in health and diseaserdquo PhysiologicalReviews vol 86 no 1 pp 245ndash278 2006

[2] C A Dinarello ldquoInterleukin-1 and interleukin-1 antagonismrdquoBlood vol 77 no 8 pp 1627ndash1652 1991

[3] W P Arend ldquoInterleukin-1 receptor antagonistrdquo Advances inImmunology vol 54 pp 167ndash227 1993

[4] W P Arend ldquoThe balance between IL-1 and IL-1Ra in diseaserdquoCytokine and Growth Factor Reviews vol 13 no 4-5 pp 323ndash340 2002

[5] N F Voelkel R M Tuder J Bridges and W P ArendldquoInterleukin-1 receptor antagonist treatment reduces pul-monary hypertension generated in rats by monocrotalinerdquoAmerican Journal of Respiratory Cell andMolecular Biology vol11 no 6 pp 664ndash675 1994

[6] Y Chen L M Garvin T J Nickola A M Watson A MColberg-Poley and M C Rose ldquoIL-1120573 induction of MUC5ACgene expression is mediated by CREB andNF-120581B and repressedby dexamethasonerdquo American Journal of PhysiologymdashLungCellular and Molecular Physiology vol 306 no 8 pp L797ndashL807 2014

[7] E R Lazarowski and R C Boucher ldquoPurinergic receptors inairway epitheliardquo Current Opinion in Pharmacology vol 9 no3 pp 262ndash267 2009

[8] T Muller B Robaye R P Vieira et al ldquoThe purinergic receptorP2Y2 receptor mediates chemotaxis of dendritic cells andeosinophils in allergic lung inflammationrdquo Allergy vol 65 no12 pp 1545ndash1553 2010

[9] K B Adler M J Tuvim and B F Dickey ldquoRegulated mucinsecretion from airway epithelial cellsrdquo Frontiers in Endocrinol-ogy vol 4 article 129 2013

[10] J-Y Jeong H-J Cha and K S Song ldquoATP significantlyincreases P2Y2-dependent RANTES secretion and overexpres-sion in human airway epithelial cellsrdquo Genes amp Genomics vol36 no 5 pp 655ndash659 2014

[11] L J Relvas M Makhoul R Dewispelaere et al ldquoP2Y2R

deficiency attenuates experimental autoimmune uveitis devel-opmentrdquo PLOS ONE vol 10 no 2 Article ID e0116518 2015

[12] S S Kyoung T-J Lee K Kim C C Kwang and J-HYoon ldquocAMP-responding element-binding protein and c-Ets1interact in the regulation of ATP-dependent MUC5AC geneexpressionrdquoThe Journal of Biological Chemistry vol 283 no 40pp 26869ndash26878 2008

[13] D-Y Jhon H-H Lee D Park et al ldquoCloning sequencingpurification and Gq-dependent activation of phospholipase C-1205733rdquo Journal of Biological Chemistry vol 268 no 9 pp 6654ndash6661 1993

[14] C G Kim D Park and S G Rhee ldquoThe role of carboxyl-terminal basic amino acids in 119866q120572-dependent activation par-ticulate association and nuclear localization of phospholipaseC-1205731rdquo The Journal of Biological Chemistry vol 271 no 35 pp21187ndash21192 1996

[15] H-C Kornau L T SchenkerM B Kennedy and PH SeeburgldquoDomain interaction betweenNMDAreceptor subunits and thepostsynaptic density protein PSD-95rdquo Science vol 269 no 5231pp 1737ndash1740 1995

[16] H J Cha M S Jung D W Ahn et al ldquoSilencing of MUC8by siRNA increases P2Y

2-induced airway inflammationrdquo The

American Journal of PhysiologymdashLung Cellular and MolecularPhysiology vol 308 no 6 pp L495ndashL502 2015

[17] S S Kyoung H J Kim K Kim G L Jeung and J-H YoonldquoRegulator of G-protein signaling 4 suppresses LPS-inducedMUC5AC overproduction in the airwayrdquo American Journal ofRespiratory Cell and Molecular Biology vol 41 no 1 pp 40ndash492009

[18] J-I Hwang K Heo K-J Shin et al ldquoRegulation of phospho-lipase C-1205733 activity by Na+H+ exchanger regulatory factor 2rdquoJournal of Biological Chemistry vol 275 no 22 pp 16632ndash166372000

[19] M Sheng and C Sala ldquoPDZ domains and the organizationof supramolecular complexesrdquo Annual Review of Neurosciencevol 24 pp 1ndash29 2001

[20] J-I Hwang H S Kim R L Jae E Kim H R Sungand P-G Suh ldquoThe interaction of phospholipase C-1205733 withShank2 regulates mGluR-mediated calcium signalrdquoThe Journalof Biological Chemistry vol 280 no 13 pp 12467ndash12473 2005

[21] D Meerzaman P Charles E Daskal M H PolymeropoulosB M Martin and M C Rose ldquoCloning and analysis of cDNAencoding amajor airway glycoprotein human tracheobronchialmucin (MUC5)rdquo The Journal of Biological Chemistry vol 269no 17 pp 12932ndash12939 1994

[22] S Kirkham J K Sheehan D Knight P S Richardson andD J Thornton ldquoHeterogeneity of airways mucus variations inthe amounts and glycoforms of the major oligomeric mucinsMUC5AC andMUC5Brdquo Biochemical Journal vol 361 no 3 pp537ndash546 2002

[23] C D Douillet W P Robinson III P M Milano R CBoucher and P B Rich ldquoNucleotides induce IL-6 release fromhuman airway epithelia via P2Y 2 and p38 MAPK-dependentpathwaysrdquo The American Journal of PhysiologymdashLung Cellularand Molecular Physiology vol 291 no 4 pp L734ndashL746 2006

[24] H Kouzaki K Iijima T Kobayashi S M OrsquoGrady and HKita ldquoThe danger signal extracellular ATP is a sensor for anairborne allergen and triggers IL-33 release and innateTh2-typeresponsesrdquoThe Journal of Immunology vol 186 no 7 pp 4375ndash4387 2011


[25] C A Dinarello T Ikejima S J C Warner et al ldquoInterleukin 1induces interleukin 1 I Induction of circulating interleukin 1 inrabbits in vivo and in human mononuclear cells in vitrordquo TheJournal of Immunology vol 139 no 6 pp 1902ndash1910 1987

[26] J C Manson J A Symons F S Di Giovine S Poole and GW Duff ldquoAutoregulation of interleukin 1 productionrdquo EuropeanJournal of Immunology vol 19 no 2 pp 261ndash265 1989

[27] R Dziak ldquoBiochemical and molecular mediators of bonemetabolismrdquo Journal of Periodontology vol 64 no 5 pp 407ndash415 1993

[28] E V Granowitz B D Clark E Vannier M V Callahan andC A Dinarello ldquoEffect of interleukin-1 (IL-1) blockade oncytokine synthesis I IL-1 receptor antagonist inhibits IL-1-induced cytokine synthesis and blocks the binding of IL-1 to itstype II receptor on human monocytesrdquo Blood vol 79 no 9 pp2356ndash2363 1992

[29] K S Song W-J Lee K C Chung et al ldquoInterleukin-1120573 and tumor necrosis factor-120572 induce MUC5AC overex-pression through a mechanism involving ERKp38 mitogen-activated protein kinases-MSK1-CREB activation in humanairway epithelial cellsrdquoThe Journal of Biological Chemistry vol278 no 26 pp 23243ndash23250 2003


family plays an important role in maintaining homeostasisagainst microorganisms air pollutants exotoxins and air-borne particulate matters in the human respiratory track Italso takes an important part in the innate immune systemwhich further regulates functions of the adaptive immunesystem [4]The balance between IL-1 and IL-1ra in the humanlung may affect the development of inflammatory diseasesVoelkel et al reported that treatment with IL-1ra reducedpulmonary hypertension generated by monocrotaline in rats[5] but IL-1120573 one of the earliest mediators secreted duringa proinflammatory response is present at high levels in thelungs of patients with chronic lung diseases [6] Howeverother secreted inflammatory mediators can also regulatetheir intracellular expressions thereby further modulatingcytokine-regulated MUC5AC overproduction

The purinergic P2Y2receptor (P2Y

2R) is highly expressed

in the apical membrane of the respiratory track [7ndash9] Extra-cellular nucleotides bound to the P2Y

2R have been shown to

induce a number of physiological phenomena in a variety ofhuman cell types and tissues [10] Recently P2Y

2R deficiency

was shown to attenuate IFN120574 IL-17 and TNF120572 secretionin mice [11] P2Y couples to the G-protein subtype G120572qand the G120572q protein regulates Ca2+ influx upon stimulationwith ligands [12] Next elevated [Ca2+]i increases PLC1205733PLC120573 isotypes have a long C-terminal region (sim400 residues)with relatively low homology among family members [13]This C-terminal region (residues 903ndash1142 of PLC1205731) isrequired for binding and stimulation by G120572q [14] MoreoverPLC120573 isotypes possess short consensus sequences knownas postsynaptic density- (PSD-) 95disc largeZO-1- (PDZ-)binding motifs The PDZ-binding motif is a well-knownshort linear motif that interacts with other PDZ proteins[15]The regulatorymechanism of PLC1205733-mediated cytokinesecretion to controlMUC5AC gene expression and the signalmolecules involved especially those involved in downstreamsignaling of PLC1205733 has not yet been demonstrated in thehuman airway epithelium

Here we report that ATP induces IL-1ra overexpressionand secretion via the P2Y

2receptor in human airway epithe-

lial cells IL-1ra secretion was associated with the P2Y2-G120572q-

[Ca2+]i-PLC1205733-ERK12 pathway sequentially Secreted IL-1ra inhibits ATPP2Y


but IL-1120572 significantly increases the expression ofMUC5ACThe results of the present study reveal a novel mechanismfor intra- and intercellular signaling pathways that controlairway inflammation thus providing an opportunity forthe development of novel therapeutic modalities to treatrespiratory diseases

2 Materials and Methods

21 Materials ATP and Apyrase were purchased from SigmaAldrich (St LouisMO)The IL-1ra and IL-1120572ELISA kits werepurchased form RampD Systems (Minneapolis MN) siRNAtargeting P2Y

2was synthesized by Bioneer (Daejeon Korea)

P2Y2 GAGGAAGGUGGCUUACCAA (dTdT) and negative

control CCUACGCCACCAAUUUCGU (dTdT) All PLC1205733isotypes were kindly provided by Dr Pann-Ghill Suh (School

of Life Sciences Ulsan National Institute of Science andTechnology Ulsan Korea)

22 Cytokine Assay The cytokine assay was performed usinga Human Cytokine Array Panel A kit (RampD Systems) accord-ing to the manufacturerrsquos instructions Briefly cells wereplated in 6-well plates one day prior to transfection witheither P2Y

2or siRNA-P2Y

2using FuGENE 6 (Roche Indi-

anapolis IN) Twenty-four hours after transfection serum-starved cells were treated with ATP for 4 hours Aftertreatment the supernatant was assayed according to the kitinstructions

23 RT-PCR Real-time PCR was performed using a BioRadiQ iCycler Detection System (BioRad Laboratories HerculesCA) with iQ SYBR Green Supermix Reactions were per-formed in a total volume of 20 120583L including 10 120583L 2x SYBRGreen PCR Master Mix 300 nM of each primer and 1120583Lof previously reverse-transcribed cDNA template The fol-lowing primers were used MUC5AC forward 51015840-CAGCCA-CGTCCCCTTCAATA-31015840 and reverse 51015840-ACCGCATTT-GGGCATCC-31015840 120573

2-microglobulin used as a reference for

normalization forward 51015840-CGCTCCGTGGCCTTAGC-31015840and reverse 51015840-GAGTACGCTGGATAGCCTCCA-31015840 Param-eters were 95∘C for 10m followed by 40 cycles of 95∘C for 15 s60∘C for 30 s and 72∘C for 30 s All reactions were performedin triplicate Relative quantity of MUC5AC mRNA wasobtained using a comparative cycle threshold method andwas normalized using 120573

2-microglobulin as an endogenous

control

24 Western Blot Analysis NCI-H292 cells were grown toconfluence in 6-well plates After treatment with ATP cellswere lysed with 2x lysis buffer (250mM Tris-Cl (pH 65)2 SDS 4 120573-mercaptoethanol 002 bromphenol blueand 10 glycerol) Equal amounts of whole cell lysateswere resolved by 10ndash15 SDS-PAGE and transferred topolyvinylidene difluoride membranes (Millipore BedfordMA) Membranes were blocked with 5 skim milk in Tris-buffered saline (50mM Tris-Cl (pH 75) 150mM NaCl) for2 h at room temperature Blots were then incubated overnightwith primary antibodies in TTBS (05 Tween 20 in Tris-buffered saline) After washing with TTBS blots were furtherincubated for 45min at room temperature with anti-rabbitor anti-mouse antibody (Cell Signaling Danvers MA) inTTBS and visualized using the ECL system (GE HealthcareUppsala Sweden)

25 Measurement of Intracellular Ca2+ Concentration([Ca2+]i) NCI-H292 cells were seeded on cover glasses in35-mm dishes (5 times 104 cells) After 24 h cells were transfectedwith P2Y

2-expressing construct The cells expressing P2Y

2

in physiological salt solution [140mM NaCl 5mM KCl1mM MgCl

2 1 mM CaCl

2 10mM HEPES 10mM glucose

and 310mOsm (pH 74)] were incubated with 5 120583M Fura-2AM (TEFLabs Inc Austin TX) and 005 pluronicF-127 for 60min at room temperature Fura-2 fluorescencewas measured at the appropriate excitation wavelengths




3 Results









2or siRNA-



2 Conversely siRNA-



2




2










2is appeared to















2-mediated




2complex can




IL-1ra

c +ATP

Positivecontrol

P2Y2 si-P2Y2

(a)

GAPDH

Mock +ATP

IL-1raP2Y2 si-P2Y2

(b)

0

2

4

6

8

10

12

Mock +

ATP

IL-1

ra se

cret

ion

(pg

mL)

lowastlowast

lowast

P2Y2 si-P2Y2

(c)

Time (hr) 2 4 6 8

ATP + + + + + + + ++ + + +

0

2

4

6

8

10

12

IL-1

ra se

cret

ion

(pg

mL)

lowast

lowast


(d)

Apyrase (UmL)

IL-1

ra se

cret

ion

(pg

mL)

0

2

4

6

8

10

12

lowastlowastlowast

lowastlowast

lowast

5025100c

ATP120574S + P2Y2

ATP120574Sonly

(e)

IL-1

ra se

cret

ion

(pg

mL)

Moc

k

ATP

UTP

AD

P

UD

P

GTP AT

P

UTP

AD

P

UD

P

GTP

pcDNA3

0

2

4

6

8

10

12 lowast

P2Y2

(f)





2or P2Y



2-treated cells


2 the cells were



2





c +ATP

0

2

4

6

8

gene

expr

essio

n (fo

ld o

ver b

asal

)

lowastlowastlowast

lowastlowast

lowast

P2Y2 si-P2Y2

Nor

mal

ized

rela

tiveM

UC5

AC(a)


Tubulin

IL-1ra

IL-1120572

secr

etio

n (p

gm

L)

0

5

10

15

lowast

lowastlowast

lowastlowastlowast

ATP + P2Y2

(b)


Tubulin

IL-1ra

IL-1

ra se

cret

ion

(pg

mL)

0

3

6

9

12

lowastlowast

ATP + P2Y2

lowast

(c)


2

4

6

8

gene

expr

essio

n (fo

ld o

ver b

asal

)

lowast

lowastlowast

ATP + P2Y2

lowastlowastlowast

lowastlowastlowast

Nor

mal

ized

rela

tiveM

UC5

AC

(d)


gene

expr

essio

n

IL-1120572IL-1ra

0

3

6

9

ATP + P2Y2

lowastlowastlowast

lowastlowast

lowast

onlyNor

mal

ized

rela

tive M

UC5

AC

(fold

ove

r bas

al)

(e)






P2Y2or siRNA-P2Y














experiments




2signaling complex


2-induced









2signaling pathway


2signaling




4 Discussion




2signaling in


2signaling may








Tubulin

ATP + P2Y2

p-PLC1205733

(a)

PLC120573

3-N

TQL

PLC120573

3-AT

QL

PLC120573

3-N

AQL

PLC120573

3-N

TAL

PLC120573

3-N

TQA

ATP

only +c

PLC120573

3-N

TQL

PLC120573

3-AT

QL

PLC120573

3-N

AQL

PLC120573

3-N

TAL

PLC120573

3-N

TQA

ATP

only +c

0

10

20

30

0

10

20

30

IL-1120572

secr

etio

n (p

gm

L)

IL-1

ra se

cret

ion

(pg

mL)


lowastlowastlowast

lowastlowast


lowastlowast

lowastlowastlowast

lowast

(b)

01Ratio

ATP ATP + IL-1ra

ATP + IL-1120572

ATP

ATP

ATP

2min(c)

Nor

mal

ized

pea

k va

lue

IL-1120572+ IL-1ra0

30

60

90

120

ATP

lowastlowast

lowast

(d)














+ + minus minus

+ + + minus

+ + minus +

p-ERK12

Tubulin

ATP

IL-1120572

IL-1

ra

P2Y 2

(a)

ATP

only

+ IL-1120572

IL-1

ra

p-ERK12

Tubulin

p-ERK12

Tubulin

cTreatment

time

10m

15m

ATP + P2Y2

(b)

0

2

4

6

8

gene

expr

essio

n (fo

ld o

ver b

asal

)

c

ATP

only +

IL-1120572

IL-1

raU

IL-1120572

+ U

IL-1

ra +

U

lowastlowast

lowastlowastlowast

lowast

ATP + P2Y2

Nor

mal

ized

rela

tiveM

UC5

AC

(c)

GDP120572 GTP

ATP

120572 GTP

ERK12

IL-1ra

IL-1ra IL-1120572

IL-1120572

MUC5AC

PLC1205733

P2Y2 120573120574

120572

[Ca2+]uarr

uarr

(d)





2-treated cells




2-induced MUC5AC




2-inducedMUC5AC








inflammation





Acknowledgment


References





































3 Results









2or siRNA-



2 Conversely siRNA-



2




2










2is appeared to















2-mediated




2complex can




IL-1ra

c +ATP

Positivecontrol

P2Y2 si-P2Y2

(a)

GAPDH

Mock +ATP

IL-1raP2Y2 si-P2Y2

(b)

0

2

4

6

8

10

12

Mock +

ATP

IL-1

ra se

cret

ion

(pg

mL)

lowastlowast

lowast

P2Y2 si-P2Y2

(c)

Time (hr) 2 4 6 8

ATP + + + + + + + ++ + + +

0

2

4

6

8

10

12

IL-1

ra se

cret

ion

(pg

mL)

lowast

lowast


(d)

Apyrase (UmL)

IL-1

ra se

cret

ion

(pg

mL)

0

2

4

6

8

10

12

lowastlowastlowast

lowastlowast

lowast

5025100c

ATP120574S + P2Y2

ATP120574Sonly

(e)

IL-1

ra se

cret

ion

(pg

mL)

Moc

k

ATP

UTP

AD

P

UD

P

GTP AT

P

UTP

AD

P

UD

P

GTP

pcDNA3

0

2

4

6

8

10

12 lowast

P2Y2

(f)





2or P2Y



2-treated cells


2 the cells were



2





c +ATP

0

2

4

6

8

gene

expr

essio

n (fo

ld o

ver b

asal

)

lowastlowastlowast

lowastlowast

lowast

P2Y2 si-P2Y2

Nor

mal

ized

rela

tiveM

UC5

AC(a)


Tubulin

IL-1ra

IL-1120572

secr

etio

n (p

gm

L)

0

5

10

15

lowast

lowastlowast

lowastlowastlowast

ATP + P2Y2

(b)


Tubulin

IL-1ra

IL-1

ra se

cret

ion

(pg

mL)

0

3

6

9

12

lowastlowast

ATP + P2Y2

lowast

(c)


2

4

6

8

gene

expr

essio

n (fo

ld o

ver b

asal

)

lowast

lowastlowast

ATP + P2Y2

lowastlowastlowast

lowastlowastlowast

Nor

mal

ized

rela

tiveM

UC5

AC

(d)


gene

expr

essio

n

IL-1120572IL-1ra

0

3

6

9

ATP + P2Y2

lowastlowastlowast

lowastlowast

lowast

onlyNor

mal

ized

rela

tive M

UC5

AC

(fold

ove

r bas

al)

(e)






P2Y2or siRNA-P2Y














experiments




2signaling complex


2-induced









2signaling pathway


2signaling




4 Discussion




2signaling in


2signaling may








Tubulin

ATP + P2Y2

p-PLC1205733

(a)

PLC120573

3-N

TQL

PLC120573

3-AT

QL

PLC120573

3-N

AQL

PLC120573

3-N

TAL

PLC120573

3-N

TQA

ATP

only +c

PLC120573

3-N

TQL

PLC120573

3-AT

QL

PLC120573

3-N

AQL

PLC120573

3-N

TAL

PLC120573

3-N

TQA

ATP

only +c

0

10

20

30

0

10

20

30

IL-1120572

secr

etio

n (p

gm

L)

IL-1

ra se

cret

ion

(pg

mL)


lowastlowastlowast

lowastlowast


lowastlowast

lowastlowastlowast

lowast

(b)

01Ratio

ATP ATP + IL-1ra

ATP + IL-1120572

ATP

ATP

ATP

2min(c)

Nor

mal

ized

pea

k va

lue

IL-1120572+ IL-1ra0

30

60

90

120

ATP

lowastlowast

lowast

(d)














+ + minus minus

+ + + minus

+ + minus +

p-ERK12

Tubulin

ATP

IL-1120572

IL-1

ra

P2Y 2

(a)

ATP

only

+ IL-1120572

IL-1

ra

p-ERK12

Tubulin

p-ERK12

Tubulin

cTreatment

time

10m

15m

ATP + P2Y2

(b)

0

2

4

6

8

gene

expr

essio

n (fo

ld o

ver b

asal

)

c

ATP

only +

IL-1120572

IL-1

raU

IL-1120572

+ U

IL-1

ra +

U

lowastlowast

lowastlowastlowast

lowast

ATP + P2Y2

Nor

mal

ized

rela

tiveM

UC5

AC

(c)

GDP120572 GTP

ATP

120572 GTP

ERK12

IL-1ra

IL-1ra IL-1120572

IL-1120572

MUC5AC

PLC1205733

P2Y2 120573120574

120572

[Ca2+]uarr

uarr

(d)





2-treated cells




2-induced MUC5AC




2-inducedMUC5AC








inflammation





Acknowledgment


References



































IL-1ra

c +ATP

Positivecontrol

P2Y2 si-P2Y2

(a)

GAPDH

Mock +ATP

IL-1raP2Y2 si-P2Y2

(b)

0

2

4

6

8

10

12

Mock +

ATP

IL-1

ra se

cret

ion

(pg

mL)

lowastlowast

lowast

P2Y2 si-P2Y2

(c)

Time (hr) 2 4 6 8

ATP + + + + + + + ++ + + +

0

2

4

6

8

10

12

IL-1

ra se

cret

ion

(pg

mL)

lowast

lowast


(d)

Apyrase (UmL)

IL-1

ra se

cret

ion

(pg

mL)

0

2

4

6

8

10

12

lowastlowastlowast

lowastlowast

lowast

5025100c

ATP120574S + P2Y2

ATP120574Sonly

(e)

IL-1

ra se

cret

ion

(pg

mL)

Moc

k

ATP

UTP

AD

P

UD

P

GTP AT

P

UTP

AD

P

UD

P

GTP

pcDNA3

0

2

4

6

8

10

12 lowast

P2Y2

(f)





2or P2Y



2-treated cells


2 the cells were



2





c +ATP

0

2

4

6

8

gene

expr

essio

n (fo

ld o

ver b

asal

)

lowastlowastlowast

lowastlowast

lowast

P2Y2 si-P2Y2

Nor

mal

ized

rela

tiveM

UC5

AC(a)


Tubulin

IL-1ra

IL-1120572

secr

etio

n (p

gm

L)

0

5

10

15

lowast

lowastlowast

lowastlowastlowast

ATP + P2Y2

(b)


Tubulin

IL-1ra

IL-1

ra se

cret

ion

(pg

mL)

0

3

6

9

12

lowastlowast

ATP + P2Y2

lowast

(c)


2

4

6

8

gene

expr

essio

n (fo

ld o

ver b

asal

)

lowast

lowastlowast

ATP + P2Y2

lowastlowastlowast

lowastlowastlowast

Nor

mal

ized

rela

tiveM

UC5

AC

(d)


gene

expr

essio

n

IL-1120572IL-1ra

0

3

6

9

ATP + P2Y2

lowastlowastlowast

lowastlowast

lowast

onlyNor

mal

ized

rela

tive M

UC5

AC

(fold

ove

r bas

al)

(e)






P2Y2or siRNA-P2Y














experiments




2signaling complex


2-induced









2signaling pathway


2signaling




4 Discussion




2signaling in


2signaling may








Tubulin

ATP + P2Y2

p-PLC1205733

(a)

PLC120573

3-N

TQL

PLC120573

3-AT

QL

PLC120573

3-N

AQL

PLC120573

3-N

TAL

PLC120573

3-N

TQA

ATP

only +c

PLC120573

3-N

TQL

PLC120573

3-AT

QL

PLC120573

3-N

AQL

PLC120573

3-N

TAL

PLC120573

3-N

TQA

ATP

only +c

0

10

20

30

0

10

20

30

IL-1120572

secr

etio

n (p

gm

L)

IL-1

ra se

cret

ion

(pg

mL)


lowastlowastlowast

lowastlowast


lowastlowast

lowastlowastlowast

lowast

(b)

01Ratio

ATP ATP + IL-1ra

ATP + IL-1120572

ATP

ATP

ATP

2min(c)

Nor

mal

ized

pea

k va

lue

IL-1120572+ IL-1ra0

30

60

90

120

ATP

lowastlowast

lowast

(d)














+ + minus minus

+ + + minus

+ + minus +

p-ERK12

Tubulin

ATP

IL-1120572

IL-1

ra

P2Y 2

(a)

ATP

only

+ IL-1120572

IL-1

ra

p-ERK12

Tubulin

p-ERK12

Tubulin

cTreatment

time

10m

15m

ATP + P2Y2

(b)

0

2

4

6

8

gene

expr

essio

n (fo

ld o

ver b

asal

)

c

ATP

only +

IL-1120572

IL-1

raU

IL-1120572

+ U

IL-1

ra +

U

lowastlowast

lowastlowastlowast

lowast

ATP + P2Y2

Nor

mal

ized

rela

tiveM

UC5

AC

(c)

GDP120572 GTP

ATP

120572 GTP

ERK12

IL-1ra

IL-1ra IL-1120572

IL-1120572

MUC5AC

PLC1205733

P2Y2 120573120574

120572

[Ca2+]uarr

uarr

(d)





2-treated cells




2-induced MUC5AC




2-inducedMUC5AC








inflammation





Acknowledgment


References



































c +ATP

0

2

4

6

8

gene

expr

essio

n (fo

ld o

ver b

asal

)

lowastlowastlowast

lowastlowast

lowast

P2Y2 si-P2Y2

Nor

mal

ized

rela

tiveM

UC5

AC(a)


Tubulin

IL-1ra

IL-1120572

secr

etio

n (p

gm

L)

0

5

10

15

lowast

lowastlowast

lowastlowastlowast

ATP + P2Y2

(b)


Tubulin

IL-1ra

IL-1

ra se

cret

ion

(pg

mL)

0

3

6

9

12

lowastlowast

ATP + P2Y2

lowast

(c)


2

4

6

8

gene

expr

essio

n (fo

ld o

ver b

asal

)

lowast

lowastlowast

ATP + P2Y2

lowastlowastlowast

lowastlowastlowast

Nor

mal

ized

rela

tiveM

UC5

AC

(d)


gene

expr

essio

n

IL-1120572IL-1ra

0

3

6

9

ATP + P2Y2

lowastlowastlowast

lowastlowast

lowast

onlyNor

mal

ized

rela

tive M

UC5

AC

(fold

ove

r bas

al)

(e)






P2Y2or siRNA-P2Y














experiments




2signaling complex


2-induced









2signaling pathway


2signaling




4 Discussion




2signaling in


2signaling may








Tubulin

ATP + P2Y2

p-PLC1205733

(a)

PLC120573

3-N

TQL

PLC120573

3-AT

QL

PLC120573

3-N

AQL

PLC120573

3-N

TAL

PLC120573

3-N

TQA

ATP

only +c

PLC120573

3-N

TQL

PLC120573

3-AT

QL

PLC120573

3-N

AQL

PLC120573

3-N

TAL

PLC120573

3-N

TQA

ATP

only +c

0

10

20

30

0

10

20

30

IL-1120572

secr

etio

n (p

gm

L)

IL-1

ra se

cret

ion

(pg

mL)


lowastlowastlowast

lowastlowast


lowastlowast

lowastlowastlowast

lowast

(b)

01Ratio

ATP ATP + IL-1ra

ATP + IL-1120572

ATP

ATP

ATP

2min(c)

Nor

mal

ized

pea

k va

lue

IL-1120572+ IL-1ra0

30

60

90

120

ATP

lowastlowast

lowast

(d)














+ + minus minus

+ + + minus

+ + minus +

p-ERK12

Tubulin

ATP

IL-1120572

IL-1

ra

P2Y 2

(a)

ATP

only

+ IL-1120572

IL-1

ra

p-ERK12

Tubulin

p-ERK12

Tubulin

cTreatment

time

10m

15m

ATP + P2Y2

(b)

0

2

4

6

8

gene

expr

essio

n (fo

ld o

ver b

asal

)

c

ATP

only +

IL-1120572

IL-1

raU

IL-1120572

+ U

IL-1

ra +

U

lowastlowast

lowastlowastlowast

lowast

ATP + P2Y2

Nor

mal

ized

rela

tiveM

UC5

AC

(c)

GDP120572 GTP

ATP

120572 GTP

ERK12

IL-1ra

IL-1ra IL-1120572

IL-1120572

MUC5AC

PLC1205733

P2Y2 120573120574

120572

[Ca2+]uarr

uarr

(d)





2-treated cells




2-induced MUC5AC




2-inducedMUC5AC








inflammation





Acknowledgment


References





































2signaling complex


2-induced









2signaling pathway


2signaling




4 Discussion




2signaling in


2signaling may








Tubulin

ATP + P2Y2

p-PLC1205733

(a)

PLC120573

3-N

TQL

PLC120573

3-AT

QL

PLC120573

3-N

AQL

PLC120573

3-N

TAL

PLC120573

3-N

TQA

ATP

only +c

PLC120573

3-N

TQL

PLC120573

3-AT

QL

PLC120573

3-N

AQL

PLC120573

3-N

TAL

PLC120573

3-N

TQA

ATP

only +c

0

10

20

30

0

10

20

30

IL-1120572

secr

etio

n (p

gm

L)

IL-1

ra se

cret

ion

(pg

mL)


lowastlowastlowast

lowastlowast


lowastlowast

lowastlowastlowast

lowast

(b)

01Ratio

ATP ATP + IL-1ra

ATP + IL-1120572

ATP

ATP

ATP

2min(c)

Nor

mal

ized

pea

k va

lue

IL-1120572+ IL-1ra0

30

60

90

120

ATP

lowastlowast

lowast

(d)














+ + minus minus

+ + + minus

+ + minus +

p-ERK12

Tubulin

ATP

IL-1120572

IL-1

ra

P2Y 2

(a)

ATP

only

+ IL-1120572

IL-1

ra

p-ERK12

Tubulin

p-ERK12

Tubulin

cTreatment

time

10m

15m

ATP + P2Y2

(b)

0

2

4

6

8

gene

expr

essio

n (fo

ld o

ver b

asal

)

c

ATP

only +

IL-1120572

IL-1

raU

IL-1120572

+ U

IL-1

ra +

U

lowastlowast

lowastlowastlowast

lowast

ATP + P2Y2

Nor

mal

ized

rela

tiveM

UC5

AC

(c)

GDP120572 GTP

ATP

120572 GTP

ERK12

IL-1ra

IL-1ra IL-1120572

IL-1120572

MUC5AC

PLC1205733

P2Y2 120573120574

120572

[Ca2+]uarr

uarr

(d)





2-treated cells




2-induced MUC5AC




2-inducedMUC5AC








inflammation





Acknowledgment


References




































Tubulin

ATP + P2Y2

p-PLC1205733

(a)

PLC120573

3-N

TQL

PLC120573

3-AT

QL

PLC120573

3-N

AQL

PLC120573

3-N

TAL

PLC120573

3-N

TQA

ATP

only +c

PLC120573

3-N

TQL

PLC120573

3-AT

QL

PLC120573

3-N

AQL

PLC120573

3-N

TAL

PLC120573

3-N

TQA

ATP

only +c

0

10

20

30

0

10

20

30

IL-1120572

secr

etio

n (p

gm

L)

IL-1

ra se

cret

ion

(pg

mL)


lowastlowastlowast

lowastlowast


lowastlowast

lowastlowastlowast

lowast

(b)

01Ratio

ATP ATP + IL-1ra

ATP + IL-1120572

ATP

ATP

ATP

2min(c)

Nor

mal

ized

pea

k va

lue

IL-1120572+ IL-1ra0

30

60

90

120

ATP

lowastlowast

lowast

(d)














+ + minus minus

+ + + minus

+ + minus +

p-ERK12

Tubulin

ATP

IL-1120572

IL-1

ra

P2Y 2

(a)

ATP

only

+ IL-1120572

IL-1

ra

p-ERK12

Tubulin

p-ERK12

Tubulin

cTreatment

time

10m

15m

ATP + P2Y2

(b)

0

2

4

6

8

gene

expr

essio

n (fo

ld o

ver b

asal

)

c

ATP

only +

IL-1120572

IL-1

raU

IL-1120572

+ U

IL-1

ra +

U

lowastlowast

lowastlowastlowast

lowast

ATP + P2Y2

Nor

mal

ized

rela

tiveM

UC5

AC

(c)

GDP120572 GTP

ATP

120572 GTP

ERK12

IL-1ra

IL-1ra IL-1120572

IL-1120572

MUC5AC

PLC1205733

P2Y2 120573120574

120572

[Ca2+]uarr

uarr

(d)





2-treated cells




2-induced MUC5AC




2-inducedMUC5AC








inflammation





Acknowledgment


References




































+ + minus minus

+ + + minus

+ + minus +

p-ERK12

Tubulin

ATP

IL-1120572

IL-1

ra

P2Y 2

(a)

ATP

only

+ IL-1120572

IL-1

ra

p-ERK12

Tubulin

p-ERK12

Tubulin

cTreatment

time

10m

15m

ATP + P2Y2

(b)

0

2

4

6

8

gene

expr

essio

n (fo

ld o

ver b

asal

)

c

ATP

only +

IL-1120572

IL-1

raU

IL-1120572

+ U

IL-1

ra +

U

lowastlowast

lowastlowastlowast

lowast

ATP + P2Y2

Nor

mal

ized

rela

tiveM

UC5

AC

(c)

GDP120572 GTP

ATP

120572 GTP

ERK12

IL-1ra

IL-1ra IL-1120572

IL-1120572

MUC5AC

PLC1205733

P2Y2 120573120574

120572

[Ca2+]uarr

uarr

(d)





2-treated cells




2-induced MUC5AC




2-inducedMUC5AC








inflammation





Acknowledgment


References





































inflammation





Acknowledgment


References








































Documents

IL-1ra Secreted by ATP-Induced P2Y2 Negatively Regulates … · 2019-09-04 · IL-1ra c + ATP Positive control P2Y 2si-P2Y (a) GAPDH Mock + ATP IL-1ra P2Y 2 si-P2Y 2 (b) 0 2 4 6 8