Identification of Secreted Proteins during Protease- Activated …abap.co.in/sites/default/files/Paper-1-Corrected.pdf · The aim of the present study was to determine the secretory

Current Trends in Biotechnology and PharmacyVol. 10 (3) 194-221 July 2016, ISSN 0973-8916 (Print), 2230-7303 (Online)

194

Identification of Secreted Proteins during Protease-Activated Receptor 2 Activation in Gastrointestinal

Smooth Muscle Cells

Sorratod Bosuwan1, Sittiruk Roytrakul2, Karnam S. Murthy3 and Wimolpak Sriwai4*

1Department of Medical Technology, School of Allied Health Sciences, Thammasat University,PathumThani, 12121, Thailand

2Proteomics Research Laboratory, National Center for Genetic Engineering and Biotechnology,PathumThani, 12120, Thailand

3Department of Physiology, VCU Program in Enteric Neuromuscular Sciences, Virginia CommonwealthUniversity, Richmond, Virginia, United States of America

4Department of Medical Technology, School of Allied Health Sciences, Thammasat University,PathumThani, 12121, Thailand

*For Correspondence - [email protected]

Activation in Gastrointestinal Smooth Muscle Cells

AbstractProtease-activated receptor 2 is a

member of G protein-coupled receptor andexpressed in a wide variety of cells. PAR2 isinvolved in several physiological andpathophysiological processes, including growthand development, inflammation, migration, andapoptosis depending on cell types. However, therole of PAR2 in the context of secretory functionof inflammatory proteins in smooth muscle cellsremains unknown. The aim of the present studywas to determine the secretory proteins involvedin inflammatory process upon PAR2 activationin smooth muscle cells. Gastric smooth musclecells were left untreated or treated with PAR2activating peptide (PAR2-AP) SLIGKV (1 µM) forup to 48 hours in serum-free medium. Untreatedmedia were used as negative controls.Secretome were collected at 48 hours andsubjected to proteomic analysis by LC-MS/MS.A total of 310 peptides were detected and foundto correspond to 275 proteins. This approachidentified 160 significant changes in proteinlevels. These proteins were expressed atsignificantly different levels in treated sample ascompared with untreated sample. These proteinscould be classified by their functions ininflammation, proliferation and apoptosis, and

response to stress. It resulted in the first LC-MS/MS based secretome profiles of gastrointestinalsmooth muscle cells. Such secretory protein maybe of pathological significance in the role of PAR2in regulating the inflammation, proliferation, andapoptotic process and may offer protection ofcells against physiological stress.

Keyword: Protease-activated receptor 2, PAR2,Proteomics, LC-MS, Inflammation

IntroductionThe proteinase-activated receptors

(PARs) belong to the family of G protein-coupledreceptors. PARs consist of four members, PAR1,PAR2, PAR3 and PAR4, which can be cleavedby certain serine proteases. These serineproteases derive from leukocyte, coagulationfactors, pathogen, and many different sources.The unique mechanism of activation involvesirreversible enzymatic cleavage of the N-terminusdomains at a specific site and unmasking atethered ligand that triggers intracellular signaling(1-4). Signaling by activated PARs is terminatedby receptor phosphorylation and association withβ-arrestins. PARs also can be activated withoutenzymatic cleavage by synthetic activatingpeptides (PAR-APs), as short as six amino acids.


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Sorratod Bosuwan et al

PAR-APs correspond to the tethered liganddomain that can directly bind to and activatePARs (5, 6).

PAR2 signaling has been implicated in thedevelopment of inflammatory bowel diseases.PAR2 are found on the surface of several celltypes (7) including epithelial cell (1), fibroblasts,endothelial cells (8), keratinocytes (9, 10),sensory neurons (11), inflammatory cells (12),and smooth muscle cell (13). The primaryfunction of smooth muscle cells is to alter thestiffness or diameter of hollow organs bycontracting and relaxing. It has been reportedthat gastric smooth muscle cells constitutivelyexpress high levels of PAR2 and are able tomediate PAR2-AP-induced contraction (13).Growing evidence has shown that smoothmuscle cells secrete highly active signalingproteins, which have a regulatory role in themuscles and other organs via endocrine,autocrine, or paracrine actions (14). It hassignificant capacity to synthesize and secretevarious signaling proteins including cytokines,chemokines, and peptide growth factors (15-17).These active signaling proteins are suggestedto mediate the course of several common andhighly debilitating chronic diseases, such asatherosclerosis (18,19), asthma (20,21), andinflammatory bowel diseases (22). Despiteextensive research on the secretory function ofvascular and airway smooth muscle, there isconsiderably less study regarding gastrointestinalsmooth muscle.

Characterization of the sets of proteinssecreted from smooth muscle cells andsubsequent analysis of their functions are acritical first step in better understanding theregulation of muscle during normal andpathological processes. Here, we sought toidentify the secreted proteome induced by PAR2-AP, SLIGKV.This study uses the rabbit gastricsmooth muscle cell as a model system and aproteomics approach to analyze the secretomeof smooth muscle during PAR2 activation. Weidentified 172 significant changes in protein levelscompared to control and associated with wide

range of biological processes. Among theseproteins, inflammatory proteins such as IL-25,IL-17RE, and TLR2 were found to be secreted.This work expands our knowledge of thesecretory function and potential roles of PAR2on smooth muscle cell. Mediators released fromsmooth muscle may serve as autocrine andparacrine factors that are involved in progressionof an inflammatory process.

Materials and MethodsCell culture : Rabbit gastric smooth muscle cells(a kind gift from Murthy KS., the VCU Programin Enteric Neuromuscular Sciences, VirginiaCommonwealth University, Richmond, Virginia,United States of America) were cultured in DMEMcontaining 200 units/milliliter (U/ml) penicillin, 200milligrams/milliliter(mg/ml)streptomycin, 100 mg/ml gentamycin, 2.5 mg/ml amphotericin B and10% fetal bovine serum (DMEM-10)(13). Thecultured plate was incubated at 37°C in aCO

2incubator. DMEM-10 medium was replaced

every three days for 2–3 weeks until the cellsattained confluence. All experiments were doneon cells in the first passage.

PAR2 activation of smooth muscle cells : ForPAR2 activation experiment, cells were dividedinto two groups: control and PAR2-AP (SLIGKV)(Bachem, Torrance, CA) stimulated smoothmuscle cells. Smooth muscle cells were washedtwice with sterile PBS and then exposed to serumfree medium for 18 - 24 h. PAR2-AP was directlydiluted in cell culture medium of treatment groupto achieve a final concentration of 1 μm. After48-h treatment the serum free conditionedmedium was collected. The resulting supernatantcontains secreted proteins. The supernatant weresubjected to lyophilization and stored at -80°Cuntil experiment was performed. An experimentwas repeated three independent times.

Determination of protein concentration byLowry method : The freeze dry media wereresuspended in 0.5% sodium dodecyl sulfate andprotein concentration was determined by Lowrymethod (23). Absorbance of the solution wasmeasured at 750 nm using a spectrophotometer.


196


The protein concentration was be calculatedusing a standard curve of BSA solution.

Prefractionation protein by sodium dodecylsulfate polyacrylamide gel electrophoresis(SDS-PAGE) : Proteins were separated on SDS-PAGE mini gel with the HoeferMiniVEelectrophoresis system (Amersham Biosciences,UK) (8 x 9 x 0.1 cm). The polyacrylamide gelwas prepared according to the method aspreviously described (24). For PAGE, thestacking gel had 5% acrylamide while theseparating gel contained 12.5% in Tris-HCl pH8.3. Fifty micrograms of each protein samplewas then mixed with equal volume of 5Xconcentrated sample buffer (0.125M Tris-HCl pH6.8, 20% glycerol, 5% SDS, 0.2 M dithiothreitol(DTT), 0.02% bromophenol blue) and heat at95oC for 10 min before placingon the gel. Lowmolecular weight protein standard marker(Amersham Biosciences, UK) was used toestimate size of polypeptides.Electrophoresiswas performed in SDS electrophoresis buffer(25mMTris-HCl pH 8.3, 192mM glycine, 0.1%SDS) until the tracking dye reached the bottomof the gel. Performed gels were stained bysilver (25).

In-gel digestion : The gel pieces were subjectedto in-gel digestion using an in-house methoddeveloped by Proteomics Laboratory, GenomeInstitute, National Center for Genetic Engineeringand Biotechnology (BIOTEC), National Scienceand Technology Development Agency (NSTDA),Thailand. In short, protein bands were cut into15 segments according to size and transferredto 96-well plate. The gel pieces were dehydratedwith 100% acetonitrile (ACN), reduced with 10milimolars (mM) DTT in 10mM ammoniumbicarbonate at room temperature for 1 h andalkylated with 100mMiodoacetamide (IAA) in10mM ammonium bicarbonate at roomtemperature for 1 h in the dark. The gel pieceswere dehydrated twice with 100% ACN for 5 minand 10 microliters (µl) of trypsin solution (10 ng/µl trypsin in 50% ACN / 10mM ammoniumbicarbonate) was added for in-gel digestion ofproteins followed by incubation for 20 min at room

temperature. To keep the gels immersedthroughout digestion, 20 µl of 30% ACN wasadded and incubation continued for a few hoursto over night at 37ºC.The digested peptides wereextracted from the gel using 30 µl of 50% ACN in0.1% formic acid and incubation continued whileshaking for 10 min at room temperature.Extracted peptides were collected andtransferred into the new tube. The extractedpeptides were dried by using a speed-vacconcentrator and stored the vial at -80 ºC formass spectrometric analysis.

Identification of protein by liquidchromatography coupled to massspectrometry (LC/MS-MS) configuration :Quantitative LC/MS-MS was performed on 1 μgof protein digest per sample, using ananoACQUITY UPLC system (Waters Corp.,Milford, MA, USA) coupled to a WatersSYNAPT™ HDMS™ system (Waters Corp.,Manchester, UK) via a nanoelectrosprayionization source. Briefly, the sample wasseparated using ananalytical reversed phasecolumn (75 μm × 250 mm, Waters Corp., Milford,MA, USA) packed with 1.7 μm ethylene bridgedhybrid (BEH) C

18 stationary phase. The A solvent

is HPLC grade water with 0.1% formic acid. TheB solvent is acetonitrile with 0.1% formic acid.The sample was initially transferred with a solventto the trap column with a flow rate of 15microliters/minute (μl/min) for 1 min. After whichthe analytical separation was performed using a60-min gradient of 2 to 40% acetonitrile with 0.1%formic acid at a flow rate of 350 nl/min followedby a 15 min rinse of 80% acetonitrile. The columntemperature was maintained at 35 ºC. Theanalysis of tryptic peptides was performed usinga SYNAPT™ HDMS™ mass spectrometer whichwas operated in the V-mode of analysis with aresolution of at least 10,000 full-width half-maximum, using positive nanoelectrospray ionmode. The time-of-flight analyzer of the massspectrometer was externally calibrated with a(Glu1) fibrinopeptide B mixture from m/z 50 to1600. The collision gas used was argon,maintained at a constant pressure of 2.0×10-3


197


mbar in the collision cell. The lock mass, (Glu1)fibrinopeptide B, was delivered from the auxiliarypump of the nanoACQUITY system with aconcentration of 100 fmol/μl at 0.5 μl/min to thereference sprayer of the nanolock spray source.The data were post-acquisition lock-masscorrected using the monoisotopic mass of thedoubly charged precursor of (Glu1) fibrinopeptideB, delivered through the reference sprayer, whichwas sampled every 20 s. Accurate massprecursor and fragment ion LC-MS data werecollected with data direct acquisition mode. TheMS/MS survey was over range from 50 to 1990.The intact mass spectra were deconvoluted tothe zero charge state (Ensemble 1, Iterations 50,auto peak width determination) using MaxEnt 3,a deconvolution software available withinMassLynx 4.0.

Proteins quantitation and identification :DeCyder MS differential analysis software(DeCyderMS, GE Healthcare) was used foridentification and quantitation of peptides basedon MS signal intensities of individual LC-MS.Quantitation of peptides was performed usingthe PepDetect module. Peptides were matchedacross different signal intensity maps betweenthe control and treated samples using thePepMatch module. The relative abundances ofpeptides were expressed as log

2 intensities. All

log2 intensities of the sample were normalized

with the ion intensity distribution of a referencestandard, bovine serum albumin (BSA).

The analyzed MS/MS data fromDeCyderMS were submitted to database searchusing the Mascot software version 2.2 (MatrixScience, London, UK). The data was searchedagainst the NCBI database for proteinidentification. Search parameters were taxonomy(Oryctolagus cuniculus); enzyme (trypsin);variable modifications (carbamidomethyl,oxidation of methionine residues); mass values(monoisotopic); protein mass (unrestricted);peptide mass tolerance (1 Da); fragment masstolerance (±0.4 Da); peptide charge state (1+,2+ and 3+); max missed cleavages (1) andinstrument = ESI-QUAD-TOF. Proteins

considered as identified proteins had at least onepeptides with an individual mascot scorecorresponding to p<0.05.

To assign proteins to gene symbols, proteinlists are mapped to corresponding entries inUniProt Knowledgebase (UniProtKB) of all knownproteins based on protein names and sequences.Searches were performed against the Homosapiens protein data sets.

Bioinformatic analysis : Normalizedquantitative data sets were used for subsequentanalyses. A 2-fold cutoff value was set to identifyproteins whose expression was significantlyincreased or decreased. To assign biologicalfunctions and processes to the entire data set ofover-expressed or highly suppressed proteins,Gene ontologies of identified and quantifiedproteins were determined with PANTHERsoftware (http://www.pantherdb.org) andUniprotKB databases (http://www.uniprot.org).Proteins with 2-fold or greater changes inexpression upon treatment were used for gene-pathway annotations using Reactome tools(Reactome V57) (http://www.reactome.org/) andto create protein-protein interaction networks viaSTRING database (Ingenuity Systems, version10.0 (http://string-db.org/). Networks representa highly interconnected set of proteins derivedfrom the input data set. To predict which pathwaysare being affected by the changes in proteinexpression, a Fisher’s exact test was applied tothe mapping of significantly over-expressedproteins in the data set to each pathway todetermine the significance of any over-representation of the proteins to that pathway.

Results

Protein identification and characterization :To identify secretory proteins upon PAR2activation in gastric smooth muscle cell, wesystematically assessed the supernatantcontains secreted proteins or secretome ofsmooth muscle cell activated with specificpeptide, SLIGKV, for 48 hours compared tountreated cells. After LC-MS/MS analysis of theisolated peptides, all MS/MS spectra were


198


searched against rabbit protein database. MSanalysis results are summarized in Table 1. A totalof 300 peptides were detected from supernatantacross all samples. The analysis of thesesupernatant samples resulted in the identificationof 271 and 256 peptides under untreated andPAR2-induced conditions, respectively. Weassigned protein names and sequences tohuman gene symbols using UniProtKB based ona target reference of human proteins. Total 300GI numbers obtained from the MS data wereconverted to UniProtKB accession numbers andfound to correspond to275genes.From theseproteins, 212 proteins were common to bothcontrol and treatment sample.

Smooth muscle secretome composition ischanged by PAR2-AP treatment : Functionalclassification of secreted proteins revealed achange in secretome composition after PAR-2AP treatment. Protein expression profiles werecompared between untreated and PAR2-inducedsample. Of the 275 differentially expressedproteins we found that 160 proteins hadexpression levels that differed by more than 2.0fold in treatment sample when compared tountreated sample, comprising 61 up-regulatedand 99 down-regulated proteins (Figure 2A). Weselected the 160 differentially expressed proteinsas candidate secreted proteins associated withPAR2 activation. Details of these proteins arepresented in Table 1. Those proteins weresubjected to K-means clustering having the K-means performs optimally for 6 clusters asdetermined by Figures of Merit (FOM) method(Figure 2B). Cluster analysis classified samplesinto a small number of groups based on thesimilarities of expression. Clearly each of the 6clusters shows proteins specifically expressedin one of the conditions.

Proteins in cluster 2 were highlyexpressed at control condition (Figure 2C),whereas proteins in cluster 3 were highlyexpressed at PAR2 treatment condition (Figure2D). Proteins in cluster 1 and 6 were highlyexpressed in both treated and untreated

conditions but the expressions of proteins incluster 4 and 5 were relatively repressed in bothconditions. In cluster 2, results showed severalproteins involved in metabolic processes, cellularprocesses, and biological regulation.Interestingly, proteins related to immune systemprocess, apoptotic process, and response tostimulus were found. Immune-related protein wasincluded complement component C1q receptor(CD93). Apoptotic-related protein was includedapoptosis-stimulating of p53 protein 2(TP53BP2).In addition, the expressions of dualspecificity mitogen-activated protein kinasekinase 5 (MAP2K5), leucine-rich repeat-containing protein 16A (LRRC16A), low-densitylipoprotein receptor-related protein 6 (LRP6),collagen alpha-1(VII) chain (COL7A1), andpleckstrin homology domain-containing family Gmember 4B (PLEKHG4B) were also observedin this cluster. In cluster 3, immune-relatedproteins (interleukin-25) was identified.

Protein annotation and ontology enrichmentanalysis : To investigate the properties ofidentified proteins, we then performed functionalenrichment of secreted proteins usingPANTHER. The biological characterization ofsecreted proteins could be classified accordingto molecular function, biological process, andcellular component (Figure 1). Gene setenrichment analysis revealed that all thedifferentially expressed proteins were enrichedin 31Gene Ontology (GO) terms (p<0.05),including 10 molecular functions, 13 biologicalprocesses, and 8 cellular components. The geneontology analysis of our secretome suggestedthese genes were associated with GO molecularfunction such as binding (33%), catalytic activity(29%), and receptor activity (10%). The GObiological process results revealed severalproteins involved in metabolic processes (24%),cellular processes (22%), and biologicalregulation (13%). In addition, the GO cellularcomponent results revealed several proteinslocated in cell part (42%), organelle (23%), andextracellular region (11%), and. Proteins thatwere not described or unspecified in the


199


Fig. 1. Analysis of functional distribution of smooth muscle phosphoproteins. Gene ontology characterizationof identified phosphoproteins whose expression found to be increased or decreased by 2-fold or greatercompared to control, according to (A) GO molecular functions, (B) GO biological processes, and (C) GOcellular component.

B

C


200


A

B

C


201

Fig. 2. Heat map of hierarchical clustering analysis of proteins differentially regulated by PAR2.Clustering was done using MeV tools. Analysis was performed based on log2 intensities of 160proteins differentially expressed, between the 1 µM PAR2 treatment and control groups. Rows rep-resent genes and columns represent samples. Color bar indicates log2-intensity value and corre-sponding colors (red, black, and green for up-regulated, not changing and down regulated, respec-tively). The color scale is shown by the bar at the top. Protein genes grouped into 10 clusters on thebasis of the similarity of expression (clustering type: K-means clustering, Distance metric: Euclideandistance). The number of the expressed genes and the percentage in each cluster are indicated. (A)Total, (B) protein clusters, (C) cluster 2, and (D) cluster 3.


D


202


UniProtKB and GO database were screened outand not further assessed for this report.

Functional classification of secretedproteins revealed a change in secretomecomposition after treatment. Protein expressionprofiles were compared between untreated andPAR2-induced sample. Of the 275 differentiallyexpressed proteins we found that 160 proteinshad expression levels that differed by more than2.0 fold in treatment sample when compared tountreated sample, comprising 61 upregulatedand 99 downregulated proteins (Table 1). Weselected the 160 differentially expressed proteinsas candidate secreted proteins associated withPAR2 activation. Details of these proteins arepresented in Table 1.

In order to identify significantly enrichedpathways and biological processes within thegene sets, pathway and gene ontology wereanalyzed using 160 differentially expressedproteins via DAVID tools. Functional annotationin DAVID showed that10 clusters (cluster 1, 2, 3,4, 5, 6, 8, 9, 13, and 15) were found to besignificantly enriched (p-value < 0.05) for 31 GOterms (Table 2).The majority of enriched clustersof pathways and GObiological processes arerelated to regulation of signal transduction(cluster 1 and 2), developmental process andsegmentation (cluster 3 and 4), cytokinesis andcell cycle (cluster 5 and 6), response to bioticstimulus (cluster 8 and 15), programmed celldeath (cluster 9), nitrogen compound and nucleicacid metabolic process (cluster 13).

To further interpret the likely roles ofdifferentially expressed proteins involved in PAR2activation, these proteins were categorized byReactome cellular pathway. Of the 160differentially expressed proteins we found that99 proteins were mapped into 20 pathways asbeing significantly (p < 0.05) overrepresentedafter PAR2 stimulation. Twenty four biologicalpathways from the Reactome database relevantfor the PAR2 signaling were investigated, andthe most significantly enriched functional pathwayaccording to their p-value and FDR values

wasinterleukin-17 signaling pathway representedin Table 3. The up-regulated protein interleukin-17 receptor E (IL17RE) and interleukin-25 (IL25)(Table 3) was predicted to be involved in thisfunction. The differentially expressed proteinswhich included in top 20 enriched functionalpathways comprised 11 up-regulated and 14down-regulated proteins upon PAR2 activation.

DiscussionIn the present study, we determined the

secretory proteins involved in inflammatoryprocess upon PAR2 activation by PAR2-AP insmooth muscle cells using LC-MS/MS basedmethod. Rabbit gastric smooth muscle was usedbecause human tissue was not readily available.It is best known that smooth muscle can changeits appearance and its function in response to awide array of stimuli. This is referred to as thephenotypic plasticity (26). Smooth muscle canlose its contractile apparatus and become asecretory cell in response to the right stimuli. Ithas been shown that smooth muscle cellssecrete highly active signaling proteins (14),indicating the validity of the approach taken. Wedemonstrate that the PAR2 activation inducesrobust protein secretion in smooth muscle cells.A total of 310 peptides were detected and foundto correspond to 275 proteins. This approachidentified 160 proteins with a 2-fold change intreated sample as compared with untreatedsample. These proteins could be classified bytheir functions in inflammation, cytokinesis andcell cycle, regulation of apoptosis, developmentalprocess and response to stress.

Although many of the altered proteins weremainly related to regulation of small G-proteinsignaling, developmental process, cell cycle andapoptosis, and response to stress, more recentwork has focused on the ability of muscle toinfluence immune function through cytokine andgrowth factor production. Growing evidence hasshown that smooth muscle cells secrete highlyactive signaling proteins (14). Nonetheless,results are limited at this point to a few cytokinesand chemokines such as IL-1 beta (27), IL-8 andIL-6 (20), and monocyte chemotactic protein


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(MCP) (18). These active signaling proteins aresuggested to be very important in cell proliferation(28) and the development of non-immunediseases with etiological origins in inflammatoryprocesses such as atherosclerosis (18) as wellas that of immune system disorders like asthma(20) and inflammatory bowel diseases (29,30).However, considering the variety of secretoryproteins that are available much less is knownconcerning the proteins that synthesize andsecrete from gastrointestinal smooth musclecells and their significance of these proteins inthe context of inflammatory processes, and thepresent discussion will therefore be restricted toproteins involved in inflammatory responses,growth and cell survival.

PAR2 influences the secretion of inflamma-tory molecules : Role of PAR2 in the regulationof inflammation is supported by several studies.Inflammatory mediators such as interleukin 1alpha, tumor necrosis factor alpha andlipopolysaccharide increased the expression ofPAR2 in cultured human umbilical veinendothelial cells (31). PAR2 activation alsostimulates the release of prostanoids inendothelial cells (32) as well as the release ofIL-6, IL-8 and PGE2 in fibroblasts (33). In thegastrointestinal tract, PAR2 has been reportedto be constitutively expressed in various cell typessuch as smooth muscle cells, interstitial cells ofCajal, and enterocytes (1,13, 34). Recentevidence has shown that activation of PAR2increase impairment of the epithelial barrier,neutrophil infiltration, and pro-inflammatorycytokines secretion (22, 35) which areparameters of inflammation.

In this study, the immune-related proteinsof smooth muscle cell in response to PAR2 havebeen characterized. In our quantitative secretomeanalysis, the secreted proteins released fromsmooth muscle cells during PAR2 activationincluding cytokine (IL-25) and receptors (IL-17REand TLR2).

IL-25 (also known as IL-17E) can stimulatethe production of IL-10 and inhibits production ofmucosal IFN-gamma in DSS-induced colitis (36).

IL-25 also promotes type 2 helper T (Th2) cellresponses (37). Type 2 responses assist with theresolution of cell-mediated inflammation.Recombinant IL-25 is able to significantly inhibitthe production of IL-1 beta, IL-6, and TNF-alphaby peripheral blood mononuclear cells from activeVogt-Koyanagi-Harada syndrome patients (38).Contrary to these findings, it has been shownthat IL-17E induces activation of NF-kB andstimulates production of the proinflammatorychemokine IL-8 in renal cell carcinoma cell lines(39).

IL-17RE is a specific receptor for IL-17C(40). Activation of IL-17RE stimulates toproduction of antibacterial peptides as well aspro-inflammatory molecules such as TNF and IL-1 betavia NF-kB and MAPK signaling pathway(40-43). In contrast to that IL-17 may play aprotective role depending on the state of disease.There is evidence shown that IL-17R-deficientmice are more susceptible to alveolar bone lossinduced by P. gingivalis (23). These protectiveeffects of IL-17 could be attributed to its ability toregulate COX-2 and PGE2 production.

In addition, we showed that TLR2 is presentin culture supernatant of smooth muscle cells,its levels is increased after PAR2 activation.Inagreement with our results, recent evidence hasidentified a linkage between PAR2 and TLR2.PAR2 is needed for TLR2 mRNA expression asdetermined by silencing PAR2 in culturedepithelial cells with subsequent bacterialstimulation (44).TLR over-activation may lead toend organ damage and serious acute and chronicinflammatory conditions. Therefore, TLRresponses must be tightly regulated to controldisease outcomes. A soluble form of TLR2(sTLR2) has recently been identified as aregulator of TLR2-mediated inflammatoryresponses to bacteria. sTLR2 acts as a decoymicrobial receptor and disrupts the interactionof TLR2 with its co-receptor, CD14. Therefore, itis capable of blunting immune responses withoutabrogating microbial recognition (45). Thepresent study shows that TLR2 is identified inculture supernatant of smooth muscle cell and



214

assumed to be in its soluble from which mayparticipate in regulating the inflammatoryresponse. Taken together, PAR2 may contributeto protective role during inflammation sincePAR2-AP stimulates secretion of IL-25, IL-17RE,and TLR2 from smooth muscle cells.

Protein expression profiling of culturesupernatant revealed down-regulation ofprostaglandin G/H synthase 2 (PTGS2), sCD93,and CD163 during PAR2 activation.PTGS2 (alsoknown as cyclooxygenase-2 or COX-2) convertsarachidonate to prostaglandin H2 (PGH2) whichcan then be metabolized to several products withdiffering biologic activities, including prostacyclin,thromboxane, prostaglandin D2, prostaglandinE2, and prostaglandin F2a. Prostanoids exertboth pro-inflammatory and anti-inflammatoryactions not only by acting as mediators of acuteinflammation but also by regulating geneexpression in mesenchymal and epithelial cellsat inflammatory site. The present study showsthat the smooth muscle cells secrete COX-2either constitutively or after stimulation withPAR2-AP. Cox-2 is an endoplasmic reticulum-resident enzyme and its expression is inducible(46). In agreement with our results, COX-2 iscapable of secreting from goblet cells into theintestinal lumen along with mucins. High mucinCOX-2 from goblet cells may increase luminalprostaglandin synthesis, alter epithelialpermeability, and modulate intestinal immuneresponses (47). Thus reduced level of COX-2after stimulation with PAR2-AP may lead to theanti-inflammatory effects.

sCD93 is a receptor for complement C1q,mannose-binding lectin (MBL2), and pulmonarysurfactant protein A (SPA) (48-50). The functionof sCD93 is involved in removal or destruction ofpathogens and immune complexes mediated bymonocyte and macrophage phagocytosis (50).A soluble form of CD93 (sCD93) has recentlybeen identified (51). It was demonstrated thatsCD93 level is increased during inflammation invivo (52). Serum level of sCD93 is increased inasthmatic patients compared to healthy controls(53). High level of sCD93 in peritoneal lavage

fluid from wild-type mice significantly enhancesengulfment of apoptotic cells in vitro whencompared to inflammatory fluid from sCD93-deficient mice[52].Consistent with the previousobservation that sCD93 induced monocyteadherence and enhanced phagocytosis (54) .Thus, reduced level of sCD93 after PAR2activation may lead to defective clearance ofapoptotic cells. To our knowledge, this is the firstreport to identify the smooth muscle cells as asource of sCD93.

CD163 is a cell surface glycoprotein thatis required for clearance and endocytosis ofapoptotic cells or hemoglobin/haptoglobincomplexes by macrophages (55). Recentlysoluble form of CD163 (sCD163) has beenidentified. Soluble CD163 is a normal plasmaprotein with normal level to be in the range of0.7–4.69 ìg/ml (56). sCD163 actively inhibitsphorbol-ester-induced proliferation oflymphocytes (57). Only the soluble form of theCD163 participates in anti-inflammation byinhibition of activated T-lymphocyte,whereas theclearance of hemoglobin uses CD163 in its cell-bound form (58). These observations confirm thatsCD163 participates in the anti-inflammatoryprocess, through the reduction of immuneresponse. Thus, reduced level of sCD163 afterPAR2 activation may lead to the reduction of anti-inflammatory response.

Taken together, our data support previousstudies that PAR2 is capable of regulatinginflammation through not only on upregulationof pro-inflammatory mechanisms but also onsuppression of the anti-inflammatory responseby smooth muscle cells.Activation of PAR2stimulates the release of cytokine and solubleproteins such as IL-25, IL-17RE and TLR2 whichfurther stimulatethe production of pro-inflammatory cytokines and immunoregulatingcytokines. In addition, activation of PAR2attenuates an anti-inflammatory program throughsuppression of sCD93,sCD163,and COX-2secretion.Further studies are needed to addresshow IL-25, IL-17RE, TLR2, COX-2, sCD93, andsCD163 fit into the existing network of the



215

numerous inflammatory and anti-inflammatoryproteins.

PAR2 positive regulation of apoptosis andcell cycle : Apoptosis is a physiologicalphenomenon. The significance of apoptosis isto remove senescent cells (59) and the overfunctional cells. Deregulation of apoptosis isassociated with the pathogenesis of a numberof disorders, such as tumor cell growth.Interestingly, differentially expressed proteinsrelated to apoptotic process were found uponactivation of PAR2. In our quantitative secretomeanalysis, the secreted proteins identified includedserine-protein kinase ataxia telangiectasiamutated (ATM), breast cancer type 2susceptibility protein (BRCA2), and apoptosis-stimulating of p53 protein 2 (ASPP2).

ATM is a serine protein kinase implicatedin cell cycle regulation and DNA repair (60).UponDNA damage, eukaryotic cells activate multipleevents such as ATM-Chk2 and/or ATR-Chk1checkpoint to arrest the cell cycle and initiate DNArepair (61). If damage is beyond repair, ATMkinase employs apoptosis to remove excessivelydamaged cells and stimulates cytokine secretionto alert neighboring cells. A defect in ATM kinasehas severe consequences in repairing certaintypes of damage to DNA. All ataxia telangiectasia(AT) patients contain mutations in the ATM gene.ATM kinase is the DNA repair gene shown to beinvolved in cancer predisposition. Leukemia andlymphoma is the most frequently malignancyfound in 38% of AT homozygotes (62). ATMkinase alterations are present at diagnosis inabout 25% of individuals with chronic lymphocyticleukemia. These alterations are associated witha poor prognosis (63). The mutated ATM genespredispose to breast cancer development andadverse radiotherapy responses (64). ATMkinase augments cell survival by activating NF-kappaB (65,66).ATM kinase is also able topromote the phosphorylation of cyclin D1 Thr286(67). Phosphorylation of cyclin D1 promotes thenuclear-to-cytoplasmic redistribution of cyclin D1during S phase of the cell cycle and inhibits the

process of cell division (68). Therefore, ATMkinase appears essential for regulated celldivision. There has thus far, been one report inwhich activation of PAR2 promoted DNAsynthesis, upregulated Cyclin D1 activity, andincreased colonic cancer cell proliferation (69).The present study shows that activation of PAR2promotes ATM kinase secretion by smoothmuscle cell. This result implies possible role ofPAR2 in the regulation of cell division and mayinvolve in the manipulation of cell survivaloutcomes after cell stress.

Breast cancer type 2 susceptibility protein(BRCA2) is a human tumor suppressor gene andresponsible for repairing DNA. BRCA2 proteinhas been shown to be able to bind to RAD51,p53, and p21WAF-1/CIP1 proteins. It has been shownthat BRCA2-defective cancer cells are unable torepair the double-strand DNA breaks induced byionizing radiation (70). Deficiency of eitherRAD51 or BRCA2 protein in embryonic miceleads to extreme sensitivity to irradiation,suggesting a role in double-stranded DNA breakrepair (71). Both p21WAF-1/CIP1 and p53 protein areknown to cause cell cycle arrest, implicatingBRCA2 in the DNA repair process. BRCA2protein is associated with high risks of breastcancer.BRCA2 is a nuclear protein and is inducedin late G1/earlyS phase, before DNA synthesis(72,73). In spite of this BRCA2 protein hassequence homology and biochemical analogy tothe granin family of secreted proteins, suggestingBRCA2 is a secreted protein (74). Bernard-Gallon et al. (75) demonstrated that BRCA2proteins are localized in the cytoplasm and cellnuclei of normal mammary tissues, ofcarcinomas in situ, and of invasive carcinomasas well as in secretions inside the ducts ofmammary tissue. However, the breast is anexocrine gland, thus becoming secretory innature. Surprisingly, we identify BRCA2 proteinfrom supernatant of primary cultured smoothmuscle cell using LC-MS/MS method. Althoughthe primary role of smooth muscle cells iscontraction, they exhibit their specializedsecretory function. Our finding suggests that



216

BRCA2 protein is secreted in the secretome ofsmooth muscle cell and the secretion isdecreased in the presence of PAR2-AP. Reducedlevels of BRCA2 protein in cells may allow theaccumulation of unrepaired DNA damages andcan lead to increased mutations. Some of thesemutations can cause cells to divide in anuncontrolled way and form a tumor.To ourknowledge, this is the first report to identify thesmooth muscle cells as a source of BRCA2protein.

Apoptosis-stimulating of p53 (ASPP)protein 2 (also called ASPP2) is a pro-apoptoticregulator that belongs to a member of ASPPfamily of p53 interacting proteins. ASPP2regulates apoptosis and cell growth throughinteractions with other regulatory moleculesincluding members of the p53 family.ASPP2induces apoptosis via stimulation the apoptoticfunction of p53 but not cell cycle arrest (76). Co-expression of ASPP2 with p53 significantlyenhanced the transactivation function of p53 onpromoters of proapoptotic genes, such as Baxand PIG3 (76).The expression of ASPP2 isfrequently suppressed in many cancers (77-79).Here, we identify ASPP2 from supernatant ofprimary cultured smooth muscle cells. Theprotein level of ASPP2 is absent in cellsstimulated with PAR2-AP but increases inuntreated samples. In pathological conditions, theexpression of ASPP2 can suppress tumor growthand confer cellular sensitivity to treatments. Lackof costimulators of p53, as in case of PAR2-activation, could also account for the loss of thetumor suppressor function of p53 which maycontribute to increase cell survival. Hence, PAR2may inhibit the apoptotic function of p53 by lowerASPP2 protein level.

Proteases activate PARs by irreversiblecleavage, so that the exposed tethered ligand isalways available to interact with the cleavedreceptor. Thus activation would result insustained signaling unless there were efficientmechanisms to terminate the signal.Nonetheless, during normal physiologicalcondition, signaling by PAR2 is efficiently

terminated by receptor phosphorylation andassociation with β-arrestins (80). In addition, theaction of protease can be terminated by itsremoval from the vicinity of the receptor. Alteredendothelial or epithelial integrity is thought toexpose the underlying smooth muscle cells toproteases that derive from the circulation,inflammatory cells, and from intestinal lumen.Thus proteases are always available for receptoractivation. Prolonged activation of PAR2 altersprotein secretion patterns in gastrointestinalsmooth muscle cells. Here we reported that manyof the altered proteins were mainly related toregulation of small G-protein signaling,developmental process, cell cycle, apoptosis, andresponse to stress. Interference with the normalsignaling by prolonged activation of PAR2 canalter the balance among the different pathwaysresponding to that signal, thus generatingfunctional heterogeneity that may affectproliferation, quiescence, survival, apoptosis, orinflammation.Many proteases that activate PARsare produced during inflammation. Theseproteases derive from inflammatory cells, thecoagulation cascade and other sources. Besideexpression of PARs themselves are also elevatedduring inflammation (31). PAR2 agonists activatethe NF-kappa B pathway in keratinocytes (81)and endothelial cells (82). NF-kappa B is knownto regulate the expression of many genesincluding immune-mediating genes andinflammatory genes, anti-apoptotic genes, cellproliferation regulation genes, and genesencoding negative regulators of NF-kappa B(83).These observations indicate that NF-kappa Bmay play a critical role in the pathogenesis ofchronic inflammation. Recent evidences havesuggested that NF-kappa B is a key regulator ofinflammation-induced tumor development (84).NF-kappa B stimulates cell proliferation andactivates the expression of growth factor genes,proto-oncogene c-MyC (85), and cyclin D1 (86),suggesting that NF-kappa B contributes to cancerdevelopment. PAR2 antagonists can also reduceinflammation, emphasizing the importance ofPAR2 in inflammation. Growing evidencesuggests that PAR2 play important roles in



217

pathogenesis of chronic inflammation, such asinflammatory bowel disease (29). In addition,PAR2 may also contribute to tumor formation andmetastasis since PAR2-AP stimulatesproliferation of colon tumor cell lines (69,87).PAR2 is expressed by a wide range of tumor cells(1, 87, 88). These observations emphasize theroles in chronic inflammation and tumor growth.

ConclusionsIn summary, the study successfully

identified proteins that are differentially secretedby smooth muscle cells upon activation by PAR2activating peptide. The results of this studyprovide the first overview of alterations in thesecretome of culture smooth muscle cells uponactivation with PAR2-AP. Such secreted proteinsmay be of pathological significance in the role ofPAR2 in regulation the inflammation, proliferation,and apoptotic process. Combining candidatesecreted proteins with the actual abundance andfunction of these proteinsprovides insights intotheir biological importance in physiological andpathological states. Secreted proteins derivedfrom smooth muscle cells during pathologicalstates may contribute to chronic inflammation orcancer prevention. However, more studies arerequired to elucidate the mechanisms by whichPAR2 mediate its secretory effects on smoothmuscle cell in inflammation orcancer. Additionalevaluation of diseased tissues or tumorsamples with LC-MS/MS may also help identifycellular proteins biologically important for thedevelopment and progression of human disease.

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Identification of Secreted Proteins during Protease- Activated …abap.co.in/sites/default/files/Paper-1-Corrected.pdf · The aim of the present study was to determine the secretory