Kallikrein …Cells were grown in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum, 100 units/ml penicil-lin,and100 g/mlstreptomycin(Invitrogen)andpropagatedin

Kallikrein-related Peptidase 4 (KLK4) Initiates IntracellularSignaling via Protease-activated Receptors (PARs)KLK4 AND PAR-2 ARE CO-EXPRESSED DURING PROSTATE CANCER PROGRESSION*

Received for publication, November 19, 2007, and in revised form, February 15, 2008 Published, JBC Papers in Press, February 28, 2008, DOI 10.1074/jbc.M709493200

Andrew J. Ramsay‡, Ying Dong‡, Melanie L. Hunt‡, MayLa Linn‡, Hemamali Samaratunga§, Judith A. Clements‡,and John D. Hooper‡1

From the ‡Institute of Health and Biomedical Innovation and School of Life Sciences, Queensland University of Technology,Corner Musk Ave. and Blamey St., Kelvin Grove, Queensland 4059, Australia and the §Department of Anatomical Pathology,Sullivan Nicolaides Pathology, Taringa, Queensland 4068, Australia

Kallikrein-related peptidase 4 (KLK4) is one of the 15 mem-bers of the human KLK family and a trypsin-like, prostate can-cer-associated serine protease. Signaling initiated by trypsin-like serine proteases are transduced across the plasmamembrane primarily by members of the protease-activatedreceptor (PAR) family of G protein-coupled receptors. Here weshow, using Ca2� flux assays, that KLK4 signals via both PAR-1and PAR-2 but not via PAR-4. Dose-response analysis over theenzyme concentration range 0.1–1000 nM indicated that KLK4-induced Ca2� mobilization via PAR-1 is more potent than viaPAR-2, whereas KLK4 displayed greater efficacy via the latterPAR.We confirmed the specificity of KLK4 signaling via PAR-2using in vitro protease cleavage assays and anti-phospho-ERK1/2/total ERK1/2 Western blot analysis of PAR-2-overexpressingand small interfering RNA-mediated receptor knockdown celllines. Consistently, confocal microscopy analyses indicated thatKLK4 initiates loss of PAR-2 from the cell surface and receptorinternalization. Immunohistochemical analysis indicated theco-expression of agonist and PAR-2 in primary prostate cancerand bone metastases, suggesting that KLK4 signaling via thisreceptor will have pathological relevance. These data provideinsight into KLK4-mediated cell signaling and suggest that sig-nals induced by this enzyme via PARsmay be important in pros-tate cancer.

Kallikrein-related peptidase 4 (KLK4)2 is a trypsin fold serineprotease from the 15-member human KLK family (1–3). Con-sistentwith its predicted substrate specificity, which is based onthe presence of an aspartate six residues before the catalytic

serine (1), KLK4 cleaves peptide substrates following arginineor lysine residues (4–6). In addition, several macromolecularsubstrates of potential pathophysiological relevance have beenidentified from in vitro studies, including another KLK familymember, pro-prostate-specific antigen (also known as KLK3)and pro-urokinase-type plasminogen activator (4, 7) as well asfibrinogen (6) and the urokinase-type plasminogen activatorreceptor (7).KLK4 is highly expressed in normal prostate (1, 3, 8) and

recently this protease has been associated with prostate cancerprogression. For example, stable overexpression of KLK4 inprostate cancer PC-3 cells resulted in an increased ability ofthese cells to migrate, accompanied by a transition from anepithelial morphology to a fibroblastic shape and, consistently,a significant decrease in E-cadherin protein levels and anincrease in vimentin expression (11). In addition, using aninducible expression system, it has been demonstrated thatoverexpression of KLK4 results in significantly increased col-ony formation, migration, and proliferation of PC-3 cells andanother prostate cancer cell line, DU145 (12). Furthermore,KLK4 protein levels are elevated in malignant prostate com-pared with normal tissue (12, 13), while prostate cancer patientserum contains antibodies that bind recombinant KLK4 (14).Most recently, using co-culture systems, it has been shown thatKLK4 is a potential mediator of cellular interactions betweenprostate cancer cells and osteoblasts (bone-forming cells) inbone metastases (15).Members of the G protein-coupled receptor (GPCR) sub-

family comprising protease-activated receptor (PAR)-1 toPAR-4, in contrast to other GPCRs, which are activated bydocking of soluble ligands, are irreversibly activated by theaction of proteases (16–19). Indeed, PAR activation is almostexclusively mediated by trypsin fold serine proteases with sub-strate specificity for cleavage following arginine or lysine resi-dues. Cleavage-inducing activation occurs at a unique sitewithin the amino-terminal exodomain of the receptor, gener-ating a new amino terminus that serves as a tethered ligand thatbinds intramolecularly, causing allosteric changes within thePAR, followed by receptor coupling to heterotrimeric G pro-teins and signal transduction (16). Importantly, cleavage down-streamof the activation site fails tomobilize Ca2� and results inunresponsiveness to protease agonists (20, 21). Recently, sev-eral members of the KLK family have been shown to initiate

* This work was supported by National Health and Medical Research Councilof Australia Fellowship 390125 (to J. A. C.) and Fellowship 339732 (toJ. D. H.), a Queensland Cancer Fund scholarship (to A. J. R.), and the Rama-ciotti Foundation. The costs of publication of this article were defrayed inpart by the payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

1 To whom correspondence should be addressed. Tel.: 61-7-3138-6197; Fax:61-7-3138-6030; E-mail: [email protected].

2 The abbreviations used are: KLK, Kallikrein-related peptidase; AP, activatingpeptide; BPH, benign prostatic hyperplasia; ERK, extracellular signal-regu-lated kinase; GFP, green fluorescent protein; GPCR, G protein-coupledreceptor; PAR, protease-activated receptor; PIN, prostatic intraepithelialneoplasial; siRNA, small interfering RNA; GAPDH, glyceraldehyde-3-phos-phate dehydrogenase; LMF, lung murine fibroblasts; PBS, phosphate-buff-ered saline; rKLK4, recombinant KLK4; BNG, benign glands; Ca, cancer.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 18, pp. 12293–12304, May 2, 2008© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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trans-plasmamembrane signal transduction via PARs.Oikono-mopoulou et al. (22) demonstrated that KLK14 activates PAR-2and PAR-4 but inactivates (or “disarms”) PAR-1. This groupalso showed that KLK5 and KLK6 activate PAR-2 (22). In addi-tion, Angelo et al. (23) have shown that KLK6 is capable ofcleaving a peptide spanning the PAR-2 activation site but notpeptides spanning the activation site of the other PARs. KLK5and KLK14 signaling via PAR-2 has been demonstrated inde-pendently in a study that also showed that KLK7 and KLK8 arenot capable of signaling through this receptor (24).Since the mechanism by which KLK4 mediates its effects on

cells is not known, we have examined the ability of this proteaseto initiate cell signaling via members of the PAR family byexamining changes in intracellular calcium ion concentration.We show that KLK4 initiates Ca2� mobilization via PAR-1 andPAR-2 but not via PAR-4. Focusing on PAR-2, we have alsoexamined the ability of KLK4 to activate extracellular signal-regulated kinase 1/2 (ERK1/2), including siRNA knockdownapproaches, to demonstrate that KLK4 signaling is specificallymediated by this receptor. The potential physiological rele-vance of KLK4 signaling via PAR-2 was explored by immuno-histochemical analysis of agonist and receptor expression inprimary prostate cancer and bone metastasis lesions. We havealso examined cellular consequences of KLK4-mediated signal-ing via PAR-2 in prostate cancer PC-3 cells.

EXPERIMENTAL PROCEDURES

Reagents—PAR-2 activating peptide (AP; SLIGKV), PAR-1AP (TFLLR), PAR-4 AP (AYPGKF) as the carboxyl amide, apeptide spanning the PAR-2 serine protease activation site(ortho-aminobenzoic acid-SKGR2SLIGK(N-(2,4-dintrophe-nyl)ethylenediamine)-Asp-OH, where the downward arrowindicates the cleaved peptide bond), and a tripeptide substratefor kinetic studies (benzyl-FVR-p-nitroanilide) were fromAus-pep (Parkville, Australia); trypsin was fromWorthington; ther-molysin was from Calbiochem; 4-methylumbelliferone,4-methylumbelliferyl p-guanidinobenzoate, and the thermoly-sin inhibitor phosphoramidon were from Sigma; and EZ-linkNHS-SS-Biotin and ImmunoPure immobilized streptavidinwere from Pierce. Antibodies were purchased from the follow-ing vendors: phospho-specific monoclonal antibody to ERK1/2and rabbit anti-ERK1/2 antibody, Cell Signaling (Beverly, MA);monoclonal anti-PAR-1 (ATAP2), anti-PAR-2 (SAM11), andanti-GFP antibodies, Santa Cruz Biotechnology, Inc. (SantaCruz, CA); rabbit anti-GAPDH antibody, Abcam (SapphireBioscience Pty. Ltd., Redfern, Australia).Cell Culture—The nontumorigenic and tumorigenic pros-

tate epithelium-derived cell lines (RWPE-1 and RWPE-2,respectively) and the prostate cancer-derived cell lines LNCaP,PC-3, and DU145 were from the American Type Culture Col-lection (Manassas, VA). Lung murine fibroblasts (LMF) fromPar-1 null mice (25) stably expressing human PAR-1, PAR-2 orPAR-4 (26) were from Johnson & Johnson PharmaceuticalResearch andDevelopment (Spring House, PA). These cells aredesignated PAR-1-LMF, PAR-2-LMF, and PAR-4-LMF,respectively. Cells were grown in Dulbecco’s modified Eagle’smedium containing 10% fetal calf serum, 100 units/ml penicil-lin, and 100�g/ml streptomycin (Invitrogen) andpropagated in

95% air, 5% CO2 at 37 °C. Cultures of PAR-1-LMF, PAR-2-LMF, and PAR-4-LMF murine fibroblasts were supplementedwith 200 �g/ml hygromycin B (Invitrogen). Insect Spodopterafrugiperda Sf9 cells were grown in SF9002 serum-free medium(Invitrogen) containing 100 units/ml penicillin and 100 �g/mlstreptomycin at 27 °C.Expression Constructs and Transfections—An insect cell

KLK4 expression construct was generated by ligating thehuman KLK4 open reading frame, including the prepro regionfrom the previously published pDNA3.1:KLK4 construct (11),into the pIB/V5-His vector (Invitrogen). This construct gener-ates the complete KLK4 amino acid sequence followed by V5(GKPIPNPLLGLDST) andHis6 tags. Insect Sf9 cellswere trans-fected using Cellfectin (Invitrogen). A mammalian expressionconstruct encoding PAR-2 with green fluorescent protein(GFP) at the COOH terminus was generated in the pEGFP-N1vector (Clontech, Mountain View, CA). A PCR employing PfuDNA polymerase (Invitrogen) was used to amplify the PAR-2coding region. Following amplification and purification, thePAR-2 PCR product was restriction-digested and then ligatedinto pEGFP-N1. Mammalian cells were transiently transfectedusing Lipofectamine (Invitrogen). The sequence of both con-structs was verified.Generation and Purification of Recombinant KLK4 (rKLK4)—

Following transfection with the KLK4-pIB/V5-His construct,stable Sf9 cells were selected with 50 �g/ml Blasticidin (Invivo-Gen, San Diego, CA). KLK4 was purified from conditionedmedia from these cells using Ni2�-nitrilotriacetic acid super-flow resin by following the instructions of the manufacturer(Qiagen, Doncaster, Australia). Eluted fractions containingKLK4, identified by analysis of a Coomassie-stained polyacryl-amide gel, were pooled and then concentrated. Following dial-ysis against phosphate-buffered saline (PBS; pH 7.4) at 4 °Covernight, KLK4 was aliquoted and stored at �80 °C.Activation of rKLK4—Recombinant zymogen KLK4 was

incubated with the metalloendopeptidase thermolysin in PBSat pH 7.4 for 1 h at 37 °C (KLK4/thermolysin ratio 80:1). Theamount of active enzyme producedwas quantified by active sitetitration using the pseudosuicide inhibitor 4-methylumbel-liferyl p-guanidinobenzoate (27). Zymogen KLK4 and thermo-lysin-treated KLK4 were examined on a Coomassie-stainedpolyacrylamide gel, protein bands were excised, and the aminoterminus of the excised treated KLK4was sequenced by Edmandegradation at the Australian Proteome Analysis Facility(North Ryde, Australia). Thermolysin activity was stopped bythe addition of phosphoramidon (10 �M).Kinetic Measurements of Activated rKLK4—Determination

of kinetic parameters was performed using 40 nM active rKLK4against the tripeptide substrate benzyl-FVR-p-nitroanilide.Assays were performed in 50 mM Tris-HCl (pH 7.4), 20 mMCaCl2, 0.01% (v/v) Tween 20 at 28 °C by following p-nitroani-lide release photometrically at 405 nm. Experiments to deter-mine the kinetics of cleavage of a fluorescence-quenched pep-tide with sequence spanning the PAR-2 serine proteaseactivation site were performed at 37 °C in 50 mM Tris-HCl (pH7.4), 20mMCaCl2, 0.01% (v/v) Tween 20 by continuouslymeas-uring fluorescence at 440 nm following excitation at 330 nm.Emissions were monitored over 15 min using a Polarstar

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Optima plate reader (BMG Labtech, Melbourne, Australia).Enzyme activity was determined from a standard curve gener-ated from the fluorescence obtained following complete cleav-age by trypsin of knownamounts of peptide using an absorptioncoefficient of 104M�1 cm�1. Toobtain kcat andKm values, initialvelocity data at each substrate concentration were fitted to theMichaelis-Menten equation by nonlinear regression analysisusing GraphPad Prism 4.0 (GraphPad Software, San Diego,CA). Experiments were performed in triplicate on three inde-pendent occasions with results displayed as means � S.E.Measurement of Changes in Intracellular Ca2�—Cells grown

to 80% confluence were washed with PBS, detached nonenzy-matically, resuspended (4 � 106 cells/ml) in extracellularmedium (121 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2�6H2O, 1.8mM CaCl2, 5.5 mM glucose, 25 mMHEPES (pH 7.4)) containing0.2% (w/v) bovine serum albumin (Sigma) and then loadedwiththe fluorescence indicator Fura-2 acetoxymethyl ester (1.0 �M;Invitrogen) at room temperature for 60 min. Cells were thenpelleted followed by resuspension in extracellular medium(without bovine serum albumin) at a concentration of 2 � 106cells/ml for fluorescence measurements. The ratio of fluores-cence at 510 nm after excitation at 340 and 380 nm was moni-tored using a Polarstar Optima fluorescent plate reader. Singleagonist treatments were KLK4 (300 nM), trypsin (10 nM),thrombin (10 nM), PAR-1 AP and PAR-2 AP (100 �M), andPAR-4AP (500�M). Displayed data are representative of exper-iments performed in triplicate and repeated on three independ-ent occasions. Dose-response experiments were performedover the KLK4 concentration range of 0.1–1000 nM in triplicateand performed on three independent occasions with resultsdisplayed as means � S.E.Knockdown of PAR-2 Expression—The mammalian siRNA

expression vector pSilencer 3.1-H1 puro (Ambion, Austin, TX)was used to reduce expression of PAR-2. Candidate PAR-2siRNA target sequences were designed as previously described(28) and then aligned against the human genome data baseusing the BLAST algorithm to eliminate those with significanthomology to other genes. Three sequences selected were 5�-GATCCAGGAAGAAGCCTTATTGGTTTCAAGAGAACC-AATAAGGCTTCTTCCTTTTTTTGGAAA-3�, 5�- GATCC-AGTAGACTTGGTGTGAAGATTCAAGAGATCTTCACA-CCAAGTCTACTTTTTTTGGAAA-3�, and 5�-GATCCGTA-GTCGTGAATCTTGTTCATTCAAGAGATGAACAAGAT-TCACGACTATTTTTTGGAAA-3�. The sequences weresynthesized (Sigma) and inserted into the pSilencer 3.1-H1puro vector (Ambion) according to the instructions of themanufacturer. Fibroblasts from Par-1 null mice stablyexpressing PAR-2 (PAR-2-LMF) were transfected with thePAR-2 pSilencer 3.1-H1 constructs or the supplied pSilencer3.1-H1 negative control using Lipofectamine. After 48 h, 2�g/ml puromycin was added to the medium to select for stabletransfected cells. PAR-2 expression levels and agonist-inducedinduction of ERK phosphorylation were examined by Westernblot analysis.PAR-1 and PAR-2 mRNA Expression Analysis—Total RNA

was extracted from cells as previously described (29) and cDNAsynthesized using Superscript II (Invitrogen). Reverse tran-scription-PCRwas performed as previously described (30) with

primers specific for PAR-1 (5�-TGTATCCCATGCAGTCCC-TCTC-3� and 5�-CACCTGGATGGTTTGCTCCTT-3�),PAR-2 (5�-AGAAGCCTTATTGGTAAGGTT-3� and 5�-AACATCATGACAGGTGGTGAT-3�), or �-actin (human5�-TGTCACCTTCACCGTTCCA-3� and 5�-CAAGATCAT-TGCTCCTCCTG-3�; mouse 5�-CGTGGGCCGCCCTAGGC-ACCA-3� and 5�-TTGGCCTTAGGGTTCAGGGGGG-3�).Cell Surface Biotinylation—PC-3 cells were washed three

times with cold PBS, and then plasmamembrane proteins werebiotinylated by incubation with 1.22 mg/ml EZ-link NHS-SS-Biotin at 4 °C for 1 h with gentle agitation. The cells were thenwashed in PBS prior to lysis in a buffer containing 20 mMHEPES, 150mMNaCl, 1mMEDTA, and 1%TritonX-100. Aftercentrifugation, bead immobilized streptavidin was added intothe supernatant and incubated for 15 min on ice to capturebiotinylated proteins. The streptavidin beads were pelleted bycentrifugation, and the supernatant was recovered for analysisof cytoplasmic (nonbiotinylated) proteins. The beadswere thenwashed thoroughly, and associated cell surface (biotinylated)proteins were eluted into Laemmli sample buffer. Cytoplasmicand cell surface fractions were examined for PAR-2 byWesternblot analysis.Western Blot Analysis—Whole cell lysates were collected in a

buffer containing Triton X-100 (1%, v/v), 50 mM Tris-HCl (pH7.4), NaCl (150 mM), and protease inhibitor mixture (RocheApplied Science). Fractions containingmembrane proteins andsoluble proteins were isolated as previously described (31). Pro-tein concentrations were determined by microbicinchoninicacid assay (Pierce). For ERK1/2 phosphorylation, cells weregrown to 50% confluence and serum-deprived for 24 h beforetreatment, and then collection of whole cell lysates was asabove, except the lysis buffer also contained 1 mM sodiumorthovanadate and 50 mM NaF (Sigma). Equal amounts oflysates (20�g) or biotinylated fractions were separated by SDS-PAGE and transferred to a nitrocellulose membrane that wasblocked with Odyssey blocking buffer (LI-COR, Lincoln, NE)containing 0.1% (v/v) Tween 20. Membranes were incubatedovernight at 4 °C with an anti-PAR-1 (1:1000), anti-PAR2(1:1000), anti-GAPDH (1:1000), anti-ERK (1:1,000), or anti-phospho-ERK (1:2,000) antibody and then washed before incu-bation with species-appropriate fluorescently conjugated sec-ondary antibodies for 1 h at room temperature. Membraneswere analyzed using an Odyssey Infrared Imaging System(LI-COR; Millennium Science, Surrey Hills, Australia) andwhere relevant signal intensity determinedusing LI-COR imag-ing software and exported toMicrosoft Excel for graphical rep-resentation as mean � S.E. Significance was examined usingStudent’s t test with a p value of �0.05 considered significant.FlowCytometry—Cells grown in serum-freemedia overnight

were detached nonenzymatically, washed in PBS, and thenresuspended at 2 � l06 cells/ml. Cells were subjected to non-permeabilizing fixation in 1% formaldehyde on ice for 5 min,washedwith PBS, and then resuspended in staining buffer (PBS,pH7.4, 0.2%bovine serumalbumin). Following incubationwithan anti-PAR-2 antibody (SAM11; 2 �g/106 cells) on ice for 60min, cells were washed and then incubated with fluorescentlytagged anti-mouse secondary antibody. Cell surface PAR-2 wasassessed using an FC500 flow cytometer (Beckman Coulter,



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Gladesville, Australia). In parallel, to assess the levels of totalPAR-2, following fixation, cells were permeabilized by incubationwith 0.01% Triton X-100 in PBS for 5min at room temperature.Confocal Microscopy—Cells plated on sterile poly-L-lysine

(Sigma)-coated glass coverslips were allowed to adhere over-night then transfected with either the PAR-2-GFP expressionconstruct or vector pEGFP-N1. For agonist treatments, cellswere incubated with either KLK4 (100 nM) or PAR-2 AP (100�M) for 10 min at 37 °C before fixation with 4% (v/v) formalde-hyde. Nuclei were stained by incubating cells for 5 min at roomtemperature with 4�,6-diamidino-2-phenylindole (1:1500) inPBS. Coverslips were mounted on slides, and cells were imagedwith a Leica SP5 confocal microscope (Leica Microsystems,Sydney, Australia). Images were processed using Adobe Photo-shop CS3 and displayed using CorelDraw. The amount ofPAR-2 on the cell surface was quantified by examining the flu-orescence of randomly selected untreated and KLK4-treatedcells (n � 15) using ImageJ software (National Institutes ofHealth, Bethesda,MD), adapting the approach of Scherrer et al.(32) for quantifyingGPCR cell surface expression. Briefly, threecellular regions of interest were defined: the whole cell, theintracellular region, and the nucleus. The signal obtained forthe whole cell and the intracellular region was corrected forbackground signal by subtracting nuclear fluorescence. Cellsurface fluorescence was then obtained by subtracting the cor-rected value for the intracellular region from the correctedvalue for the whole cell. These values were then divided by thenumber of pixels contained within each region to give fluores-cence density values for the cell surface (Di surface) and cyto-plasm (Di cytoplasm). The ratio of Di surface to Di cytoplasmwas determined to normalize data across the counted cell pop-ulation. Results are displayed graphically as mean � S.E., andsignificance was examined using Student’s t test with a p valueof �0.05 considered significant.Immunohistochemical Analysis—Archival formalin-fixed

paraffin-embedded blocks from primary prostate cancers (n �6; Gleason scores 3 � 4 to 4 � 5) and prostate cancer bonemetastases (n � 2) were obtained from Sullivan NicolaidesPathology (Taringa, Australia) and the Royal Prince AlfredHospital (Sydney, Australia), respectively, following institu-tional ethics approval. Immunohistochemistry was performedas previously described (33). Briefly, serial sections (4�m)weredeparaffinized and then rehydrated, and after antigen retrievalin urea (5% w/v) in 0.1 M Tris buffer (pH 9.5), serial sectionswere incubated in H2O2 (3%, v/v) to quench endogenous per-oxidase. Sections were then blocked in normal goat serum(10%) and incubated overnight at 4 °C with either an anti-PAR-2 monoclonal (SAM11; 1:1000 dilution) or an anti-KLK4rabbit polyclonal (1:250 dilution) antibody. As negative con-trols, mouse and rabbit immunoglobulins replaced theSAM11 and anti-KLK4 primary antibodies, respectively. TheEnVision� peroxidase polymer detection system (Dako, Bot-any, Australia) was used with 3,3�-diaminobenzidine (Sigma) asthe chromogen. The sections were counterstained with Mayer’shematoxylin, visualized by microscopy (Leitz, Laborlux S, Ger-many), and photographed using a Nikon OXM1200 digital cam-era. Images were processed using Adobe Photoshop CS3 and dis-played using CorelDraw.

RESULTS

Examination of KLK4-initiated Changes in IntracellularCa2� Ion Concentration via PARs—For these studies, rKLK4was purified from the conditioned media of Sf9 insect cells sta-bly transfected with an expression construct containing thecomplete human KLK4 coding sequence in frame with V5 andpolyhistidine-encoding sequences. Amino-terminal sequenc-ing indicated that thermolysin-activated rKLK4 had the pre-dicted NH2 terminus of mature active KLK4 (31IINED). Thekinetic parameters obtained for thermolysin-activated rKLK4against the tripeptide substrate benzyl-FVR-p-nitroanilide(Km � 46.9 �M, kcat � 1.8 s�1, and kcat/Km � 38.3 s�1�mM�1)agreed well with the findings of Matsumura et al. (Km � 48.3�M, kcat � 1.28 s�1, and kcat/Km � 38.3 s�1�mM�1) using thesame substrate and rKLK4 generated in Drosophila melano-gaster S2 cells (34).Signaling via PARs is initiated almost exclusively by arginine/

lysine-specific serine proteases and induces transient changesin intracellular Ca2� ion concentration (17). PAR activationleading to calciummobilization requires proteolytic cleavage atdefined activation sites; cleavage downstream of this site fails tomobilize Ca2� and results in receptor inactivation (18, 20, 21).Since the ability of the arginine/lysine-specific serine proteaseKLK4 to initiate intracellular signaling has not previously beenexamined, we studied the ability of this protease to inducechanges in intracellular [Ca2�] in PAR-1-LMF, PAR-2-LMF, orPAR-4-LMF cells (generated from mouse embryonic fibro-blasts from Par-1 knock-out mice stably expressing eitherPAR-1, PAR-2, or PAR-4 (26)). Cells stably expressing PAR-3are not available, and this receptor in mice does not mediatetransmembrane signaling, leading to the proposal that PAR-3acts as a co-receptor for signaling via PAR-4 (35). As shown inFig. 1, A and B, activated rKLK4 initiated a prompt, transientCa2� mobilization in PAR-1- and PAR-2-expressing cells. Inthese experiments, the maximum calcium signal initiated byrKLK4was higher via cells expressing PAR2 than those express-ing PAR1 (compare the peak heights in Fig. 1, A and B). Thespecificity of the response via PAR-1 and PAR-2 was indicatedby the lack of Ca2� flux in cells expressing PAR-4 in response toactivated rKLK4 (Fig. 1C). Positive control agonists for PAR-1(thrombin and PAR-1 AP), PAR-2 (trypsin and PAR-2 AP), andPAR-4 (trypsin and PAR-4 AP) each induced changes in Ca2�

concentration in the respective PAR-expressing cells (Fig. 1,D–I) (36–39), confirming that the receptor expressed by eachcell linewas functional. In addition, PAR-1APdid not signal viaPAR-2 or PAR-4, PAR-2 AP did not signal via PAR-1 or PAR-4,and PAR-4 AP did not signal via PAR-1 or PAR-2 (data notshown), confirming that Ca2� mobilization in these cells wasmediated only via the respective PAR. In other controls, pro-rKLK4 andphosphoramidon-inhibited thermolysin did not ini-tiate PAR signaling, while PAR-4 cells were shown to haveintact PAR following treatment with activated rKLK4 as thePAR-4 AP, applied 3 min after protease treatment, inducedtransient Ca2� mobilization (data not shown).Concentration Dependence of KLK4-initiated Signaling via

PAR-1 and PAR-2—The concentration-effect curves for KLK4-mediated activation of PAR-1 and PAR-2 Ca2� signaling are



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shown in Fig. 2. PAR-1-LMF and PAR-2-LMF cells were incu-bated with increasing concentrations of enzyme over the range0.1 nM to 1 �M. As shown in Fig. 2, A and B, increasing concen-trations of activated rKLK4 stimulated increased Ca2� mobili-zation via both PARs. The maximum response via PAR-1 wasachieved at �300 nM activated rKLK4 with a concentration tostimulate half-maximal response (EC50) of�30 nM. In contrast,a maximum dose response via PAR-2 was not achieved at themaximum concentration of rKLK4 currently available to us.However, using the dose response achieved at 1�Mof rKLK4 asthe lower limit for the maximum response, we estimated thatthe lowest value for the EC50 via PAR-2 was �300 nM, whichsuggests that KLK4 has higher potency (inverse of EC50) forPAR-1 than PAR-2.To compare the efficacy of KLK4-initiated Ca2� mobiliza-

tion via PAR-1- and PAR-2-expressing cells, fluorescence val-ues obtained at each concentration of rKLK4 were divided bythe highest response observed for each of these PARs, whichwas via PAR-2 at 1�M rKLK4. As shown in Fig. 2C, this analysissuggests that KLK4 has greater efficacy for initiating Ca2� sig-naling via PAR-2 than PAR-1.KLK4 Rapidly Cleaves a Peptide Spanning the PAR-2 Activa-

tion Site—Since concentration-effect curves indicated thatKLK4 initiated Ca2� mobilization displayed higher efficacy via

PAR-2 than via PAR-1 (Fig. 2C), KLK4 activation of PAR-2 wasanalyzed further. A fluorescence-quenched peptide spanningthe PAR-2 activation site (ortho-aminobenzoic acid-SKGR2SLIGK-N-(2,4-dintrophenyl)ethylenediamine), whichis activated by low concentrations of trypsin, tryptase, and acro-sin but not by high concentrations of thrombin (40), was used toexamine the kinetics of direct PAR-2 cleavage by activatedrKLK4 (40 nM). The activated protease was incubated withvarying concentrations of substrate, and fluorescence wasmonitored over time with initial velocities fitted to theMichae-lis-Menten equation to determine kinetic parameters. Asshown in Fig. 3, KLK4 was able to efficiently cleave the PAR-2peptide with kinetic parameters of Km 8.0, kcat � 7.9 s�1, andkcat/Km � 1.0 s�1��M�1, which are similar to the valuesobserved for KLK6 against a similar PAR-2 peptide (ortho-ami-nobenzoic acid-SSKGR2SLIGQ-N-2,4-dintrophenyl)ethyl-enediamine) of Km � 5.6 �M, kcat � 7.9 s�1, and kcat/Km � 1.4s�1��M�1 (23).KLK4 Initiates Phosphorylation of ERK via PAR-2—The abil-

ity of KLK4 to signal via PAR-2 was further examined by ana-lyzing ERK1/2 activation, another intracellular pathway down-stream of PAR activation (17). We performed Western blotanalysis to detect phosphorylated ERK1/2 in PAR-2-LMF cellsstimulated with activated rKLK4 (10 nM) using total ERK1/2 asa control for relative expression of the kinase. Trypsin and thePAR-2 AP were used as positive controls for ERK1/2 activation

FIGURE 1. KLK4 initiated changes in intracellular Ca2� ion concentrationvia PARs. PAR-1-LMF, PAR-2-LMF, or PAR-4-LMF cells were incubated witheither activated rKLK4 (300 nM), trypsin (10 nM), thrombin (10 nM), PAR-1 APand PAR-2 AP (100 �M), or PAR-4 AP (500 �M) as indicated, and fluorescence at510 nm was measured following alternating excitation at 340 and 380 nm(Em510(340/380)) using a fluorescent plate reader. The ratio of Em510(340/380) isproportional to intracellular Ca2� ion concentration. The arrow indicates timeof treatment. Data are representative of experiments performed in triplicate,repeated three times. A–C, PAR-1-, PAR-2-, and PAR-4-expressing cells treatedwith KLK4. D–F, PAR-1-, PAR-2-, and PAR-4-expressing cells treated with serineprotease agonist (D, thrombin; E and F, trypsin). G–I, PAR-1-, PAR-2-, and PAR-4-expressing cells treated with AP (G, PAR-1 AP; H, PAR-2 AP; I, PAR-4 AP).

FIGURE 2. Concentration dependence of KLK4 signaling via PAR-1 andPAR-2. PAR-1-LMF and PAR-2-LMF cells were incubated with activated rKLK4(0.1 nM to 1 �M), and fluorescence at 510 nm was measured following alter-nating excitation at 340 and 380 nm (Em510(340/380)) using a fluorescent platereader. Intracellular Ca2� ion mobilization is displayed graphically as a per-centage of the highest recorded response. Experiments were performed intriplicate and repeated three times with data displayed as mean � S.E.A, response of PAR-1-LMF cells to KLK4. B, response of PAR-2-LMF cells toKLK4. C, graphical comparison of responses of PAR-1-LMF and PAR-2-LMFcells to activated rKLK4. To normalize values obtained via PAR-1 and PAR-2,fluorescence values obtained at each concentration of rKLK4 were divided bythe highest response observed for each of these two PARs, which was viaPAR-2 at 1 �M rKLK4. The maximum response initiated via PAR-1 (300 nM) andPAR-2 (1 �M) is indicated by an arrow and arrowhead, respectively.



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via PAR-2. As shown in Fig. 4A, KLK4 induced ERK1/2 phos-phorylation within 5 min, with reduced levels of activationapparent at 15 min. PAR-2 AP and trypsin induced ERK1/2phosphorylation in a similar time-dependent manner (Fig. 4, Band C, respectively). The dose response of KLK4-initiated sig-naling via PAR-2was examined by incubating PAR-2-LMF cellswith increasing amounts of enzyme (0.1–300 nM) and examin-ing changes in ERK1/2 activation. Consistent with Ca2� mobi-lization assays, as shown in Fig. 4D, increasing concentrationsof activated rKLK4 stimulated increased ERK1/2 activation.

We also examined the specificity of KLK4 signaling viaPAR-2 by using siRNA to reduce the expression levels of thisreceptor in PAR-2-LMF cells. We generated three PAR-2siRNAconstructs and stably transfected these intomouse PAR-2-LMF cells. As shown in Fig. 5A (left) byWestern blot analysis,it was apparent that construct 2 effectively reduced PAR-2 lev-els. Densitometric analysis indicated that this constructreduced PAR-2 levels by up to �80% (mean �60%), whereassiRNA constructs 1 and 3 failed to consistently reduce PAR-2expression levels (Fig. 5A, right). The effect of PAR-2 knock-down on KLK4-initiated intracellular signaling was examinedby assessing levels of activated and total ERK1/2 in lysates fromcontrols and cells stably transfected with siRNA construct 2.Densitometric analysis was used to determine a value for phos-phorylated ERK at each time point relative to untreated cellsand a value for total ERKat each timepoint relative to untreatedcells; the ratio of these values is the -fold increase of ERK acti-vation over basal levels (41). When treated with KLK4, cellstransfected with construct 2 showed �70% reduction inERK1/2 phosphorylation after 5min relative to cells transfected

FIGURE 3. KLK4 cleavage of a fluorescent quenched peptide substratespanning the PAR-2 activation site. Activated rKLK4 was incubated withvarying concentrations of the PAR-2 peptide SKGR2SLIGK (the downwardarrow indicates the activation site), and the inverse of initial velocity (deter-mined using a fluorescent plate reader) was plotted against the inverse ofsubstrate concentration to determine the displayed kinetic parameters.Experiments were performed in triplicate on three separate occasions, andthe data are means � S.E.

FIGURE 4. KLK4 activation of PAR-2 induces transient phosphorylation ofERK. PAR-2-LMF cells were incubated with either activated rKLK4 (10 nM) (A),PAR-2 activating peptide (100 �M) (B), or trypsin (10 nM) (C), and cell lysatescollected at the indicated times were analyzed by Western blot analysis usinganti-ERK and anti-phospho-ERK (p-ERK) antibodies. Each agonist experimentwas performed three times with similar results. D, concentration dependenceof KLK4 on ERK1/2 activation. Lysates from PAR-2-LMF cells incubated withactivated rKLK4 (0.1–300 nM) were examined by anti-phospho-ERK and anti-ERK Western blot analysis. The ratio of activated to total ERK was determinedby densitometry and is displayed graphically. The data are the mean � S.E. ofthree individual experiments.

FIGURE 5. Changes in levels of phospho-ERK (p-ERK) induced by KLK4 aremediated by PAR-2. A, siRNA-mediated knock-down of PAR-2. PAR-2-LMFcells were stably transfected with one of three PAR-2 siRNA constructs or ascrambled sequence siRNA control construct (NS, nonspecific siRNA). Celllysates were analyzed by Western blot analysis probing with either an anti-PAR-2 (SAM-11) or anti-GAPDH antibody. The level of PAR-2 expression rela-tive to GAPDH was determined by densitometry and is displayed graphicallyas the mean � S.E. of three individual experiments. B, cells stably transfectedwith either PAR-2 siRNA construct 2 or the nonspecific siRNA construct wereincubated with activated rKLK4. Lysates were collected at the indicated timepoints and analyzed by anti-ERK and anti-phospho-ERK (p-ERK) Western blotanalysis. Each Western blot analysis is representative of three experiments.The ratio of phospho-ERK to total ERK is also represented graphically as themean � S.E. of three individual experiments. C, as for B, except cells wereincubated with PAR-2 AP. *, p � 0.05.



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with a nonspecific control (Fig. 5B). A similar reduction inERK1/2 phosphorylation after 5 min was also apparent in cellswith reduced PAR-2 levels that had been treated with PAR-2AP (Fig. 5C). Consistent with results of Ca2� flux assays, thesedata indicate that KLK4 initiates intracellular signaling viaPAR-2.PAR-1 and PAR-2 in Prostate-derived Cell Lines—We exam-

ined PAR-1 and PAR-2 expression in five prostate cell linesderived from a range of pathologies, by reverse transcription-PCR and Western blot analysis using PAR-1-LMF and PAR-2-LMF cells as positive controls for PAR-1 and PAR-2mRNAandprotein. The cell lines were derived from the following sourc-es: RWPE1, virus-transformed normal epithelium (42);RWPE2, Ki-ras-transformed RWPE1 cells (42); LNCaP,lymph node metastasis (43); PC-3, bone metastasis (44); andDU145, brain metastasis (45). As shown in Fig. 6A, PAR-1 andPAR-2 mRNA was detected in each of these cell lines, as indi-cated by the presence of a specific 125- and 583-bp product,respectively. Similarly, PAR-1 and PAR-2 protein was detectedat high levels in each of the analyzed cell lines (Fig. 6, B and C).Consistent with a previous report (46), PAR-1 was predomi-nantly expressed as an�65-kDa protein in each cell line, whichreduced to�40 kDa upon treatmentwithN-glycosidase F (datanot shown). Higher PAR-1 molecular weight bands present in

lysates from RWPE-1, RWPE-2, PC-3, and D145 cells arethought to bemultimeric forms of the protein. In each prostatecell line, PAR-2 was apparent as bands at �55, �65, �85, and�105 kDa. Consistently, PAR-2 bands at �65, �85, and �105kDa reduced to the�55 kDa band on tunicamycin treatment ofbreast cancer-derived cell lines, whereas the �85 kDa was alsophosphorylated in these cells (47). Interestingly, for PAR-2 onlythe 55 kDa band was apparent in PAR-2-LMF cells, potentiallyindicating differential post-translational modification (e.g. gly-cosylation) of PAR-2 as previously proposed (31).KLK4 Initiated Signaling in a Prostate-derived Cell Line—

PC-3 cells were selected for analysis of KLK4-initiated signalingin a prostate-derived cell line. For serine protease-mediatedPAR activation to occur, the receptor must be located on thecell surface (36). PC-3 cell surface expression of PAR-1 has beenpreviously reported (48).We assessed plasmamembrane local-ization of PAR-2 in these cells by cell surface biotinylation andflow cytometry approaches.In biotinylation experiments, plasma membrane proteins

were isolated by treating intact PC-3 cells with a biotinylatingagent, and biotinylated (cell surface) proteins were purifiedusing streptavidin-conjugated beads. As shown in Fig. 7A byWestern blot analysis using a PAR-2-specific monoclonal anti-body, PAR-2 was detected in both the cytoplasm and plasmamembrane fractions. Reprobing this membrane with an anti-GAPDH antibody indicated that the plasma membrane frac-tion purified by biotinylation was largely uncontaminated byintracellular proteins. Consistent data were obtained from ourflow cytometry analyses. In these experiments, cell surface-lo-calized PAR-2was assessed by examining PC-3 cells, which hadbeen subjected to nonpermeabilizing fixation. In parallel, totalPAR-2 was determined by examining PC-3 cells permeabilizedwith the detergent Triton X-100. As shown in Fig. 7B, the anti-PAR-2 antibody detected both plasma membrane-localizedPAR-2 (left) and total PAR-2 (right) well above background lev-els of fluorescence obtained when the cells were stained onlywith the secondary antibody.The ability of KLK4 to initiate signaling in these cells was

assessed by examining transient changes in Ca2� ion mobiliza-tion. PAR-1 AP and PAR-2 AP were used as positive controlsfor signaling via the respective PARs. KLK4 initiated a prompt,transient Ca2� mobilization in these cells as did PAR-1 AP andPAR-2 AP (Fig. 7C). The dose response of KLK4-initiated Ca2�

mobilization was examined by incubating PC-3 cells withincreasing concentrations of enzyme (0.1–1000 nM). As shownin Fig. 7D, increasing concentrations of KLK4 stimulatedincreased Ca2� mobilization consistent with levels observedfrom PAR-2-LMF cells (Fig. 2B). These data indicate thatPAR-2 is expressed in a range of prostate cell lines, that PC-3cells express functional PAR-1 and PAR-2, and that KLK4 ini-tiates Ca2�mobilization in these cells, consistentwith signalingvia PAR-1 and PAR-2.KLK4 Induces Loss of PAR-2 from the Surface of Prostate Can-

cer-derived PC-3 Cells—It is known that following activation,PAR-2 is processed by receptor uncoupling from G proteinsand endocytosis followed by ubiquitination-mediated lysoso-mal degradation (49–52). We employed confocal microscopyto quantitatively examine internalization of PAR-2 from the cell

FIGURE 6. PAR-1 and PAR-2 in prostate-derived cell lines. A, RT-PCR analy-sis of PAR-1 and PAR-2 mRNA expression (30 cycles) in PAR-1-LMF, PAR-2-LMF, PAR-4-LMF, and prostate-derived cell lines. B, Western blot analysis ofPAR-1 protein expression in prostate-derived cell lines and mouse-derivedPAR-1-LMF cells using an anti-PAR-1 antibody (ATAP2). C, Western blot anal-ysis of PAR-2 protein expression in prostate-derived cell lines and mouse-derived PAR-2-LMF cells using an anti-PAR-2 antibody (SAM11).



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surface of KLK4-stimulated PC-3 cells. Cells transientlyexpressing PAR-2-GFP were used to facilitate localization ofthe receptor. As shown in Fig. 8A, in unstimulated PC-3 cells,PAR-2was detected at the plasmamembrane (arrowheads) andin cytoplasmic vesicles, including within structures previouslyidentified as the Golgi apparatus (50, 53). Stimulation of PAR-2-GFP-expressing cells with activated rKLK4 (100 nM) for 10min resulted in internalization of PAR-2 from the cell surfaceand endocytosis (Fig. 8A, arrow) (54). In controls, PAR-2 APtreatment also caused PAR-2 internalization and redistributionthroughout PC-3 cells in a manner similar to KLK4 (data notshown). As described under “Experimental Procedures,” quan-titative analysis of these data employing a modification of apreviously described approach (32) was used to determine the

amount of PAR-2 on the cell surface before and after KLK4stimulation. Cellular regions of interest were defined for thewhole cell, the intracellular region, and the nucleus, (marked onthe example unstimulated cell shown in Fig. 8B (top) as red,yellow, and blue, respectively (middle)). As shown in Fig. 8B(bottom), graphical representation of the values obtained fromthis analysis indicated that PAR-2 cell surface levels decreasedby�80% following stimulation with KLK4. These data indicatethat KLK4 induces loss of PAR-2 from the cell surface andreceptor internalization.KLK4andPAR-2AreCo-expressed in Primary andBoneMet-

astatic Prostate Cancer—Since we demonstrated that the pros-tate cancer-associated protease KLK4 initiates intracellular sig-naling via PAR-2, efficiently cleaves a peptide spanning thePAR-2 activation site, and induces PAR-2 cellular processing,we next examined the potential pathological relevance of theseobservations by performing immunohistochemical analysis ofsix primary prostate cancer tissue specimens. The expressionpattern of receptor and agonist in consecutive tissue sectionswas examined using a PAR-2-specificmonoclonal antibody andour previously described anti-KLK4 antibody (55). Represent-ative images are shown in Fig. 9. PAR-2 and KLK4 had similarexpression patterns, with both expressed by glandular epithelialcells with little evidence of stromal staining (Fig. 9, panels A andB and panels D and E, respectively). PAR-2 was detected inbenign glands (BNG) of benign prostatic hyperplasia (BPH),prostatic intraepithelial neoplasia (PIN), and cancer (Ca).Expression levels were higher in regions of PIN and Ca than inBNG regions of BPH (comparing BNG and PIN in Fig. 9A andBNGandCa in Fig. 9B). A similar staining patternwas apparentfor KLK4. As published previously (13), prostate cancer tissuesamples showed little KLK4 staining in benign glands, withstronger staining in regions of PIN (comparing BNGand PIN inFig. 9D) and Ca (comparing BNG and Ca in Fig. 9E). Negativecontrols were free of staining (Fig. 9, D and H).Bone metastasis is the cause of significant morbidity and

mortality in prostate cancer patients (56). Recently, we haveshown that KLK4 is expressed by both prostate cancer cells andosteoblasts in the in vivometastatic bone environment and thatosteoblast-like SaOs2 cells induce KLK4 expression in co-cul-ture systems with prostate cancer-derived LNCaP and PC-3cells (15). Accordingly, we performed immunohistochemistryto examine the expression pattern of KLK4 and PAR-2 in con-secutive sections from prostate cancer bone metastases fromtwo patients. As shown in Fig. 10, receptor and agonist hadsignificant overlap in expression in prostate cancer bonemetas-tasis. In regions of prostate cancer lesions, PAR2 and KLK4were highly expressed by prostate cancer cells (Fig. 10,A andC,respectively). In regions containing prostate cancer lesions andbone, strong staining of osteoblasts lining the bone surface wasalso apparent for both PAR2 andKLK4 (arrows in Fig. 10,B andD, respectively). Negative controls were free of staining (datanot shown).

DISCUSSION

Intracellular signaling, initiated via activation of members ofthe protease-activated receptor family of GPCRs, by the actionof trypsin-like serine proteases is important in physiology and

FIGURE 7. PAR-2 in PC-3 cells. A, Western blot analysis of protein fractionscollected from PC-3 cells partitioned between cytoplasmic and plasma mem-brane fractions by cell surface biotinylation. Fractions were analyzed forPAR-2 using antibody SAM11 and for purity using an antibody against a cyto-plasmic marker (GAPDH). B, flow cytometry analysis for PAR-2, cell surface(fixed, nonpermeabilized cells; left) and total (fixed, permeabilized cells; right)PAR-2. Cells were incubated with either an anti-PAR-2 SAM11 antibody fol-lowed by a fluorescently tagged secondary antibody (black line) or a fluores-cently tagged secondary antibody only (gray line). C, KLK4 (left), PAR-1 AP(middle), and PAR-2 AP (right) initiated changes in intracellular Ca2� ion con-centration in the prostate cancer-derived PC-3 cell line. Cells were incubatedwith either KLK4 (300 nM) or activating peptide (100 �M) as indicated, andfluorescence at 510 nm was measured following alternating excitation at 340and 380 nm (Em510(340/380)) using a fluorescent plate reader. The ratio ofEm510(340/380) is proportional to intracellular Ca2� ion concentration. Experi-ments were performed in triplicate and repeated three times. D, concentra-tion dependence of KLK4-induced changes in intracellular Ca2� ion concen-tration in prostate-derived PC-3 cells. Cells were incubated with KLK4 (0.1 nM

to 1 �M), and fluorescence was measured as described in D. Experiments wereperformed in triplicate and repeated three times.



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disease (17, 18, 57). The serine protease agonists responsible forPAR activation in these settings, where many proteolyticenzymes can be present, are not well defined. Here we haveanalyzed the ability of the arginine/lysine-specific (4, 6, 58),prostate cancer-associated (11–15) serine protease KLK4 toinitiate intracellular signaling via members of the PAR family.Our data indicate that KLK4 induces Ca2� mobilization via

PAR-1 and PAR-2 but not via PAR-4. This ability to signal viamore than one PAR is known for other serine proteases. Theseinclude thrombin, which signals via PAR-1 and PAR-4 and alsocleaves PAR-3 (16), trypsin, which activates PAR-2 (37, 53) andPAR-4 (39), activated Factor X, which, in complex with acti-vated Factor VII and tissue factor, signals via PAR-1 and PAR-2(59, 60), and KLK14, which activates both PAR-2 and PAR-4and inactivates PAR-1 (22). Interestingly, our analysis of Ca2�

mobilization in response to KLK4 suggests that although sig-naling via PAR-1 is more potent than via PAR-2, KLK4 dis-played greater efficacy for signaling via the latter PAR. Thiscontrasts with trypsin IV, which had similar potency as well asefficacy for inducing Ca2� mobilization via these PARs inreceptor-overexpressing cells (61). It is possible that the maxi-mum signaling via PAR-1 observed by us occurred at lower

KLK4 concentration either becauseof lower levels of expression of thisreceptor or because at higherenzyme concentration, KLK4cleaves PAR-1 other than at the acti-vation site, thereby inactivating or“disarming” the receptor. Althoughthese proposals remain to be tested,the latter has been suggested toexplain the observations that Ca2�

mobilization via PAR-2 in responseto KLK6 reached a maximum at amuch lower concentration thanKLK14, and this maximumresponse to KLK6 was much lowerthan observed for KLK14 (22). Itwas proposed that the responses toKLK6 were due to a receptor acti-vating ability at lower enzyme con-centrations via cleavage of PAR-2 atthe consensus activation site and toa “disarming” ability at higher con-centration via cleavage downstreamof the activation site. Thus, it is pos-sible that KLK4 regulates cellularactivity by differential activation ofPAR-1 and PAR-2, at lower enzymeconcentration signaling via bothPARs and as KLK4 concentrationincreases via PAR-2 as PAR-1 is dis-armed. Alternatively, it is possiblethat efficacy was lower via PAR-1,because this receptor is expressed atlevels lower than PAR-2 on the sur-face of the cells used in this study.Due to the ability of KLK4 to sig-

nal via PAR-2 over a broader enzyme concentration range, weanalyzed signaling via this receptor-agonist system in greaterdetail and also examined the expression of these proteins dur-ing prostate cancer progression. Our enzymatic studies, using afluorescence-quenched peptide indicated that KLK4 efficientlycleaves a peptide spanning the PAR-2 activation site, suggestingthat intracellular signaling mediated by KLK4 occurs via directproteolytic activation of this receptor. The specificity of KLK4signaling via PAR-2 was assessed by Western blot analysis ofERK1/2 activation. We observed that KLK4-induced phospho-rylation of ERK1/2 could be substantially reduced by siRNA-mediated knock-down of PAR-2 levels. Our confocal micros-copy analyses indicated that KLK4-induced intracellularsignaling via PAR-2 is accompanied by loss of this GPCR fromthe cell surface and receptor internalization.Our observation of increased PAR-2 expression during pros-

tate cancer progression is consistent with a recent report show-ing elevated PAR-2 in regions of prostate cancer comparedwithadjacent normal glandular epithelial cells in 42%of patient sam-ples (n� 40) (62). These workers also showed that PAR-2 levelsincreased with increasing Gleason score (62). In addition, ourfinding of PAR-2 expression by osteoblasts in prostate cancer

FIGURE 8. KLK4 induces loss of PAR-2 from the surface of prostate cancer PC-3 cells. A, confocal micros-copy analysis of PC-3 cells transiently transfected with a PAR-2-GFP expression construct. Cells, either unstimu-lated (left panels) or stimulated with KLK4 (100 nM) for 10 min (right panels), were stained with 4�,6-diamidino-2-phenylindole (DAPI) to identify nuclei, fixed, and then imaged on a Leica SP5 confocal microscope. Cellsurface PAR-2 is indicated by arrowheads in an unstimulated cell (left overlay panel). Endocytosed PAR-2 isindicated by an arrow in a KLK4-stimulated cell (right overlay panel). B, quantification of cell surface PAR-2 wasperformed by examining the fluorescence of randomly selected unstimulated and KLK4 stimulated cells (n �15) using ImageJ software. An unstimulated cell is shown in the upper panel. Cellular regions of interest weredefined for this cell (middle panel), including the whole cell, the intracellular region, and the nucleus (red, yellow,and blue, respectively). The signals obtained for the whole cell and the intracellular region were corrected forbackground signal by subtracting nuclear fluorescence. Cell surface fluorescence was then obtained by sub-tracting the corrected value for the intracellular region from the corrected value for the whole cell. These valueswere then divided by the number of pixels contained within each region to give fluorescence density values forthe cell surface (Di surface) and cytoplasm (Di cytoplasm). The ratio of Di surface to Di cytoplasm was deter-mined to normalize data across the counted cell population. Results are displayed graphically as mean � S.E.(bottom).



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bone metastases is consistent with studies in mice demonstrat-ing that murine osteoblasts also express this receptor in vitroand in vivo (63). The key observation from our immunohisto-chemical analysis of PAR-2 andKLK4 co-expression in primaryprostate cancer as well as bone metastasis lesions suggests that

KLK4will contribute to PAR-2 activation in these settings. Thisis potentially significant, since recent reports have demon-strated cancer-associated functional consequences of PAR-2activation in prostate cancer-derived cell lines. For example, inLNCaP cells, PAR-2 AP induced activation of members of theRho GTPase family (62, 64), proteins that are of critical impor-tance in cytoskeletal reorganization and cancer (65). In addi-tion, AP mediated activation of PAR-2-induced activity of thecollagenases matrix metalloprotease 2 and 9 by the prostatecancer cell lines LNCaP, PC-3, and DU145 (66). Further, it hasbeen suggested that activation of PAR-2 in bone mediates inhi-bition of osteoclast differentiation and bone lytic activity, andthis effect is probably mediated by osteoblasts (67). This ispotentially significant, since prostate adenocarcinomas haveprimarily an osteoblastic phenotype in bone metastasis lesions(68). Similarly, cancer-associated functional consequences ofKLK4 expression have been noted in prostate cancer-derivedcell lines. For example, KLK4 overexpression induced anincreased ability of PC-3 cells to migrate, accompanied by atransition from an epithelial morphology to a fibroblastic shapeand, consistently, a significant decrease in E-cadherin proteinlevels and an increase in vimentin (11). In addition, inducibleoverexpression of KLK4 resulted in significantly increased col-ony formation, migration, and proliferation of both PC-3 andDU145 prostate cancer-derived cells (12). Recently, we haveshown that KLK4 levels increased in LNCaP and PC-3 cellsco-cultured with osteoblast-like SaOs2 cells, whereas KLK4-overexpressing PC-3 cells had increased migration towardSaOs2 cell conditionedmedia (15). These reports are suggestivethat KLK4 activation of PAR-2 will be important in prostatecancer progression in both primary and bone lesions.It is possible that KLK4-initiated signaling via PAR-1will also

be relevant in prostate cancer. Recently, two reports have con-sistently identified an increase in expression atmRNA and pro-tein levels of this receptor during prostate cancer progression,although the expression pattern reported by these groups dif-fered (62, 69). Kaushal et al. (69) noted predominant expressionin endothelial cells in cancerous regions with cancer cells alsoshowing PAR-1 expression in some early and advanced stagespecimens. In contrast, using a different antibody, Black et al.(62) reported PAR-1 expression in cancer cells in 45% of pros-tate cancer samples and in periglandular stromal cells in 55% ofhigher grade cancers. Thus, although the identity of the celltypes expressing PAR-1 in cancerous prostate is contentious,loss of prostate organ structure during cancer progression willexpose PAR-1 to KLK4. In addition, of relevance, PAR-1expression has also been reported in human osteoblast-likecells (70) and mouse osteoblasts (71), and this GPCR mediatesthrombin-induced proliferation of primary rat osteoblasts (72).The action of KLK4 on PAR-1 and PAR-2 may be particularlyrelevant in more advanced primary prostate cancer and bonemetastatic lesions, since the level of Zn2�, an efficient inhibitorof KLK4 proteolytic activity (IC50 � 16 �M (58)) in normalprostate, is known to drop 10–20-fold in prostatic tissue andfluid from prostate cancer patients (73, 74).Of importance to the regulation of PAR-2 activation in nor-

mal and diseased prostate, in addition to KLK4, three otherprostate-expressed serine proteases can initiate intracellular

FIGURE 9. Immunohistochemical analysis of PAR-2 and KLK4 in prostate.Consecutive sections were stained with either an anti-PAR-2 monoclonalantibody (A and B), an anti-KLK4 polyclonal antibody (D and E), or controlanti-mouse (C) or anti-rabbit (F) secondary antibodies. BNG regions, PIN, andCa are indicated.

FIGURE 10. Immunohistochemical analysis of PAR-2 and KLK4 in prostatecancer bone metastasis. Consecutive sections were stained with either ananti-PAR2 or an anti-KLK4 antibody. A, PAR-2 staining in prostate cancer cellsin a prostate cancer bone metastasis lesion. B, PAR-2 staining in a prostatecancer bone metastasis lesion showing a region of bone. C, KLK4 staining inprostate cancer cells in a prostate cancer bone metastasis lesion. D, KLK4staining in a prostate cancer bone metastasis lesion showing a region ofbone. Bone, Ca cells, and osteoblasts lining the bone surface (arrows) areindicated.



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signaling via PAR-2 in in vitro systems. These include the type IItransmembrane serine proteases (75) TMPRSS2 (76), andmatriptase/MT-SP1 (77) and another KLK serine protease,KLK14 (24). The ability of four prostatic serine proteases tosignal via PAR-2 indicates that activation of this receptor inprostate will require both tight regulation of the activating pro-teases (for example by the presence of activators and inhibitors)and spatial and temporal expression of receptor and agonist. Inthis respect, although protein or mRNA expression has beenreported for TMPRSS2, matriptase/MT-SP1 and KLK14 bynormal and cancerous prostate epithelial cells (13, 78–80), co-expression of these enzymes with PAR-2 in normal or diseasedprostate, or bone metastases has not yet been addressed.Another aspect of regulation of PAR-2 signaling in prostate isthe possibility that these serine proteases will most efficientlyactivate PAR-2 via a proteolytic cascade reminiscent of theblood coagulation (81) and wound healing (82) serine proteasecascades.PAR-1 and PAR-2 constitute a growing number of KLK4

substrates, including pro-prostate-specific antigen (4), fibrino-gen (6), pro-urokinase-type plasminogen activator (4), and theurokinase-type plasminogen activator receptor (7) as well astype I and type IV collagen at lower efficiency (6). In this study,we observed that PAR-2-mediated activation of ERK1/2 wasinitiated at a lower KLK4 concentration than Ca2� mobiliza-tion (compare Figs. 2B and 4D). Of relevance, ERK1/2 activa-tion and Ca2� mobilization can both be initiated via disparateevents at the cell surface, includingGPCR and receptor tyrosinekinase ligand docking (83–85). Thus, it is possible that KLK4 isable to cleave/activate other cell surface proteins in addition toPAR-1 and PAR-2 to initiate the differential induction of Ca2�

ion flux and ERK1/2 phosphorylation observed by us.This study has shown for the first time that the receptors

PAR-1 and PAR-2 are activated by the serine protease KLK4and that PAR-2 and KLK4 are co-expressed during prostatecancer progression. In addition to a putative role in this disease,KLK4 is thought to be critical in dental enamel formation (86).Also, cell culture approaches (9) and immunohistochemicalanalysis (10) indicate potential involvement in ovarian cancer.Thus, KLK4-mediated cell signaling via PARs may be impor-tant in prostate cancer and other pathophysiological processes.

Acknowledgments—We thank Dr. Patricia Andrade-Gordon andDr. Claudia Derian (Johnson & Johnson Pharmaceutical Researchand Development) for PAR cell lines and Nigel Bennett, MarkAdams, Andreas Wortmann, and Carson Stephens for technicalassistance.

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Samaratunga, Judith A. Clements and John D. HooperAndrew J. Ramsay, Ying Dong, Melanie L. Hunt, MayLa Linn, Hemamali

DURING PROSTATE CANCER PROGRESSIONProtease-activated Receptors (PARs): KLK4 AND PAR-2 ARE CO-EXPRESSED

Kallikrein-related Peptidase 4 (KLK4) Initiates Intracellular Signaling via

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