Kallikrein-related Peptidase 4 (KLK4) Initiates Intracellular Signaling via Protease-activated Receptors (PARs)
2008; Elsevier BV; Volume: 283; Issue: 18 Linguagem: Inglês
10.1074/jbc.m709493200
ISSN1083-351X
AutoresA. J. Ramsay, Ying Dong, Melanie L. Hunt, MayLa Linn, Hemamali Samaratunga, Judith A. Clements, John D. Hooper,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoKallikrein-related peptidase 4 (KLK4) is one of the 15 members of the human KLK family and a trypsin-like, prostate cancer-associated serine protease. Signaling initiated by trypsin-like serine proteases are transduced across the plasma membrane primarily by members of the protease-activated receptor (PAR) family of G protein-coupled receptors. Here we show, using Ca2+ flux assays, that KLK4 signals via both PAR-1 and PAR-2 but not via PAR-4. Dose-response analysis over the enzyme concentration range 0.1–1000 nm indicated that KLK4-induced Ca2+ mobilization via PAR-1 is more potent than via PAR-2, whereas KLK4 displayed greater efficacy via the latter PAR. We confirmed the specificity of KLK4 signaling via PAR-2 using in vitro protease cleavage assays and anti-phospho-ERK1/2/total ERK1/2 Western blot analysis of PAR-2-overexpressing and small interfering RNA-mediated receptor knockdown cell lines. Consistently, confocal microscopy analyses indicated that KLK4 initiates loss of PAR-2 from the cell surface and receptor internalization. Immunohistochemical analysis indicated the co-expression of agonist and PAR-2 in primary prostate cancer and bone metastases, suggesting that KLK4 signaling via this receptor will have pathological relevance. These data provide insight into KLK4-mediated cell signaling and suggest that signals induced by this enzyme via PARs may be important in prostate cancer. Kallikrein-related peptidase 4 (KLK4) is one of the 15 members of the human KLK family and a trypsin-like, prostate cancer-associated serine protease. Signaling initiated by trypsin-like serine proteases are transduced across the plasma membrane primarily by members of the protease-activated receptor (PAR) family of G protein-coupled receptors. Here we show, using Ca2+ flux assays, that KLK4 signals via both PAR-1 and PAR-2 but not via PAR-4. Dose-response analysis over the enzyme concentration range 0.1–1000 nm indicated that KLK4-induced Ca2+ mobilization via PAR-1 is more potent than via PAR-2, whereas KLK4 displayed greater efficacy via the latter PAR. We confirmed the specificity of KLK4 signaling via PAR-2 using in vitro protease cleavage assays and anti-phospho-ERK1/2/total ERK1/2 Western blot analysis of PAR-2-overexpressing and small interfering RNA-mediated receptor knockdown cell lines. Consistently, confocal microscopy analyses indicated that KLK4 initiates loss of PAR-2 from the cell surface and receptor internalization. Immunohistochemical analysis indicated the co-expression of agonist and PAR-2 in primary prostate cancer and bone metastases, suggesting that KLK4 signaling via this receptor will have pathological relevance. These data provide insight into KLK4-mediated cell signaling and suggest that signals induced by this enzyme via PARs may be important in prostate cancer. Kallikrein-related peptidase 4 (KLK4) 2The abbreviations used are: KLK, Kallikrein-related peptidase; AP, activating peptide; BPH, benign prostatic hyperplasia; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; PAR, protease-activated receptor; PIN, prostatic intraepithelial neoplasial; siRNA, small interfering RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LMF, lung murine fibroblasts; PBS, phosphate-buffered saline; rKLK4, recombinant KLK4; BNG, benign glands; Ca, cancer. 2The abbreviations used are: KLK, Kallikrein-related peptidase; AP, activating peptide; BPH, benign prostatic hyperplasia; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; PAR, protease-activated receptor; PIN, prostatic intraepithelial neoplasial; siRNA, small interfering RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LMF, lung murine fibroblasts; PBS, phosphate-buffered saline; rKLK4, recombinant KLK4; BNG, benign glands; Ca, cancer. is a trypsin fold serine protease from the 15-member human KLK family (1Nelson P.S. Gan L. Ferguson C. Moss P. Gelinas R. Hood L. Wang K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3114-3119Crossref PubMed Scopus (205) Google Scholar, 2Stephenson S.A. Verity K. Ashworth L.K. Clements J.A. J. Biol. Chem. 1999; 274: 23210-23214Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 3Yousef G.M. Obiezu C.V. Luo L.Y. Black M.H. Diamandis E.P. Cancer Res. 1999; 59: 4252-4256PubMed Google Scholar). Consistent with its predicted substrate specificity, which is based on the presence of an aspartate six residues before the catalytic serine (1Nelson P.S. Gan L. Ferguson C. Moss P. Gelinas R. Hood L. Wang K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3114-3119Crossref PubMed Scopus (205) Google Scholar), KLK4 cleaves peptide substrates following arginine or lysine residues (4Takayama T.K. McMullen B.A. Nelson P.S. Matsumura M. Fujikawa K. Biochemistry. 2001; 40: 15341-15348Crossref PubMed Scopus (156) Google Scholar, 5Debela M. Magdolen V. Schechter N. Valachova M. 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Chem. 2006; 387: 749-759Crossref PubMed Scopus (34) Google Scholar) and the urokinase-type plasminogen activator receptor (7Beaufort N. Debela M. Creutzburg S. Kellermann J. Bode W. Schmitt M. Pidard D. Magdolen V. Biol. Chem. 2006; 387: 217-222Crossref PubMed Scopus (50) Google Scholar). KLK4 is highly expressed in normal prostate (1Nelson P.S. Gan L. Ferguson C. Moss P. Gelinas R. Hood L. Wang K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3114-3119Crossref PubMed Scopus (205) Google Scholar, 3Yousef G.M. Obiezu C.V. Luo L.Y. Black M.H. Diamandis E.P. Cancer Res. 1999; 59: 4252-4256PubMed Google Scholar, 8Harvey T.J. Hooper J.D. Myers S.A. Stephenson S.A. Ashworth L.K. Clements J.A. J. Biol. Chem. 2000; 275: 37397-37406Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) and recently this protease has been associated with prostate cancer progression. For example, stable overexpression of KLK4 in prostate cancer PC-3 cells resulted in an increased ability of these cells to migrate, accompanied by a transition from an epithelial morphology to a fibroblastic shape and, consistently, a significant decrease in E-cadherin protein levels and an increase in vimentin expression (11Veveris-Lowe T.L. Lawrence M.G. Collard R.L. Bui L. Herington A.C. Nicol D.L. Clements J.A. Endocr. Relat. Cancer. 2005; 12: 631-643Crossref PubMed Scopus (128) Google Scholar). In addition, using an inducible expression system, it has been demonstrated that overexpression of KLK4 results in significantly increased colony formation, migration, and proliferation of PC-3 cells and another prostate cancer cell line, DU145 (12Klokk T.I. Kilander A. Xi Z. Waehre H. Risberg B. Danielsen H.E. Saatcioglu F. Cancer Res. 2007; 67: 5221-5230Crossref PubMed Scopus (75) Google Scholar). Furthermore, KLK4 protein levels are elevated in malignant prostate compared with normal tissue (12Klokk T.I. Kilander A. Xi Z. Waehre H. Risberg B. Danielsen H.E. Saatcioglu F. Cancer Res. 2007; 67: 5221-5230Crossref PubMed Scopus (75) Google Scholar, 13Dong Y. Bui L.T. Odorico D.M. Tan O.L. Myers S.A. Samaratunga H. Gardiner R.A. Clements J.A. Endocr. Relat. Cancer. 2005; 12: 875-889Crossref PubMed Scopus (50) Google Scholar), while prostate cancer patient serum contains antibodies that bind recombinant KLK4 (14Day C.H. Fanger G.R. Retter M.W. Hylander B.L. Penetrante R.B. Houghton R.L. Zhang X. McNeill P.D. Filho A.M. Nolasco M. Badaro R. Cheever M.A. Reed S.G. Dillon D.C. Watanabe Y. Oncogene. 2002; 21: 7114-7120Crossref PubMed Scopus (27) Google Scholar). Most recently, using co-culture systems, it has been shown that KLK4 is a potential mediator of cellular interactions between prostate cancer cells and osteoblasts (bone-forming cells) in bone metastases (15Gao J. Collard R.L. Bui L. Herington A.C. Nicol D.L. Clements J.A. Prostate. 2007; 67: 348-360Crossref PubMed Scopus (48) Google Scholar). Members of the G protein-coupled receptor (GPCR) subfamily comprising protease-activated receptor (PAR)-1 to PAR-4, in contrast to other GPCRs, which are activated by docking of soluble ligands, are irreversibly activated by the action of proteases (16Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11023-11027Crossref PubMed Scopus (515) Google Scholar, 17Macfarlane S.R. Seatter M.J. Kanke T. Hunter G.D. Plevin R. Pharmacol. Rev. 2001; 53: 245-282PubMed Google Scholar, 18Ossovskaya V.S. Bunnett N.W. Physiol. Rev. 2004; 84: 579-621Crossref PubMed Scopus (908) Google Scholar, 19Vesey D.A. Hooper J.D. Gobe G.C. Johnson D.W. Nephrology. 2007; 12: 36-43Crossref PubMed Scopus (31) Google Scholar). Indeed, PAR activation is almost exclusively mediated by trypsin fold serine proteases with substrate specificity for cleavage following arginine or lysine residues. Cleavage-inducing activation occurs at a unique site within the amino-terminal exodomain of the receptor, generating a new amino terminus that serves as a tethered ligand that binds intramolecularly, causing allosteric changes within the PAR, followed by receptor coupling to heterotrimeric G proteins and signal transduction (16Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11023-11027Crossref PubMed Scopus (515) Google Scholar). Importantly, cleavage downstream of the activation site fails to mobilize Ca2+ and results in unresponsiveness to protease agonists (20Dulon S. Cande C. Bunnett N.W. Hollenberg M.D. Chignard M. Pidard D. Am. J. Respir. Cell Mol. Biol. 2003; 28: 339-346Crossref PubMed Scopus (112) Google Scholar, 21Dulon S. Leduc D. Cottrell G.S. D'Alayer J. Hansen K.K. Bunnett N.W. Hollenberg M.D. Pidard D. Chignard M. Am. J. Respir. Cell Mol. Biol. 2005; 32: 411-419Crossref PubMed Scopus (101) Google Scholar). Recently, several members of the KLK family have been shown to initiate trans-plasma membrane signal transduction via PARs. Oikonomopoulou et al. (22Oikonomopoulou K. Hansen K.K. Saifeddine M. Tea I. Blaber M. Blaber S.I. Scarisbrick I. Andrade-Gordon P. Cottrell G.S. Bunnett N.W. Diamandis E.P. Hollenberg M.D. J. Biol. Chem. 2006; 281: 32095-32112Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar) demonstrated that KLK14 activates PAR-2 and PAR-4 but inactivates (or “disarms”) PAR-1. This group also showed that KLK5 and KLK6 activate PAR-2 (22Oikonomopoulou K. Hansen K.K. Saifeddine M. Tea I. Blaber M. Blaber S.I. Scarisbrick I. Andrade-Gordon P. Cottrell G.S. Bunnett N.W. Diamandis E.P. Hollenberg M.D. J. Biol. Chem. 2006; 281: 32095-32112Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). In addition, Angelo et al. (23Angelo P.F. Lima A.R. Alves F.M. Blaber S.I. Scarisbrick I.A. Blaber M. Juliano L. Juliano M.A. J. Biol. Chem. 2006; 281: 3116-3126Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) have shown that KLK6 is capable of cleaving a peptide spanning the PAR-2 activation site but not peptides spanning the activation site of the other PARs. KLK5 and KLK14 signaling via PAR-2 has been demonstrated independently in a study that also showed that KLK7 and KLK8 are not capable of signaling through this receptor (24Stefansson K. Brattsand M. Roosterman D. Kempkes C. Bocheva G. Steinhoff M. Egelrud T. J. Invest. Dermatol. 2008; 128: 18-25Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Since the mechanism by which KLK4 mediates its effects on cells is not known, we have examined the ability of this protease to initiate cell signaling via members of the PAR family by examining changes in intracellular calcium ion concentration. We show that KLK4 initiates Ca2+ mobilization via PAR-1 and PAR-2 but not via PAR-4. Focusing on PAR-2, we have also examined the ability of KLK4 to activate extracellular signal-regulated kinase 1/2 (ERK1/2), including siRNA knockdown approaches, to demonstrate that KLK4 signaling is specifically mediated by this receptor. The potential physiological relevance of KLK4 signaling via PAR-2 was explored by immunohistochemical analysis of agonist and receptor expression in primary prostate cancer and bone metastasis lesions. We have also examined cellular consequences of KLK4-mediated signaling via PAR-2 in prostate cancer PC-3 cells. Reagents—PAR-2 activating peptide (AP; SLIGKV), PAR-1 AP (TFLLR), PAR-4 AP (AYPGKF) as the carboxyl amide, a peptide spanning the PAR-2 serine protease activation site (ortho-aminobenzoic acid-SKGR↓SLIGK(N-(2,4-dintrophenyl)ethylenediamine)-Asp-OH, where the downward arrow indicates the cleaved peptide bond), and a tripeptide substrate for kinetic studies (benzyl-FVR-p-nitroanilide) were from Auspep (Parkville, Australia); trypsin was from Worthington; thermolysin was from Calbiochem; 4-methylumbelliferone, 4-methylumbelliferyl p-guanidinobenzoate, and the thermolysin inhibitor phosphoramidon were from Sigma; and EZ-link NHS-SS-Biotin and ImmunoPure immobilized streptavidin were from Pierce. Antibodies were purchased from the following vendors: phospho-specific monoclonal antibody to ERK1/2 and rabbit anti-ERK1/2 antibody, Cell Signaling (Beverly, MA); monoclonal anti-PAR-1 (ATAP2), anti-PAR-2 (SAM11), and anti-GFP antibodies, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); rabbit anti-GAPDH antibody, Abcam (Sapphire Bioscience Pty. Ltd., Redfern, Australia). Cell Culture—The nontumorigenic and tumorigenic prostate 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 Collection (Manassas, VA). Lung murine fibroblasts (LMF) from Par-1 null mice (25Darrow A.L. Fung-Leung W.P. Ye R.D. Santulli R.J. Cheung W.M. Derian C.K. Burns C.L. Damiano B.P. Zhou L. Keenan C.M. Peterson P.A. Andrade-Gordon P. Thromb. Haemost. 1996; 76: 860-866Crossref PubMed Scopus (131) Google Scholar) stably expressing human PAR-1, PAR-2 or PAR-4 (26Andrade-Gordon P. Maryanoff B.E. Derian C.K. Zhang H.C. Addo M.F. Darrow A.L. Eckardt A.J. Hoekstra W.J. McComsey D.F. Oksenberg D. Reynolds E.E. Santulli R.J. Scarborough R.M. Smith C.E. White K.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12257-12262Crossref PubMed Scopus (175) Google Scholar) were from Johnson & Johnson Pharmaceutical Research and Development (Spring House, PA). These cells are designated PAR-1-LMF, PAR-2-LMF, and PAR-4-LMF, respectively. Cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen) and propagated in 95% air, 5% CO2 at 37 °C. Cultures of PAR-1-LMF, PAR-2-LMF, and PAR-4-LMF murine fibroblasts were supplemented with 200 μg/ml hygromycin B (Invitrogen). Insect Spodoptera frugiperda Sf9 cells were grown in SF9002 serum-free medium (Invitrogen) containing 100 units/ml penicillin and 100 μg/ml streptomycin at 27 °C. Expression Constructs and Transfections—An insect cell KLK4 expression construct was generated by ligating the human KLK4 open reading frame, including the prepro region from the previously published pDNA3.1:KLK4 construct (11Veveris-Lowe T.L. Lawrence M.G. Collard R.L. Bui L. Herington A.C. Nicol D.L. Clements J.A. Endocr. Relat. Cancer. 2005; 12: 631-643Crossref PubMed Scopus (128) Google Scholar), into the pIB/V5-His vector (Invitrogen). This construct generates the complete KLK4 amino acid sequence followed by V5 (GKPIPNPLLGLDST) and His6 tags. Insect Sf9 cells were transfected using Cellfectin (Invitrogen). A mammalian expression construct encoding PAR-2 with green fluorescent protein (GFP) at the COOH terminus was generated in the pEGFP-N1 vector (Clontech, Mountain View, CA). A PCR employing Pfu DNA polymerase (Invitrogen) was used to amplify the PAR-2 coding region. Following amplification and purification, the PAR-2 PCR product was restriction-digested and then ligated into pEGFP-N1. Mammalian cells were transiently transfected using Lipofectamine (Invitrogen). The sequence of both constructs 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 μg/ml Blasticidin (Invivo-Gen, San Diego, CA). KLK4 was purified from conditioned media from these cells using Ni2+-nitrilotriacetic acid superflow resin by following the instructions of the manufacturer (Qiagen, Doncaster, Australia). Eluted fractions containing KLK4, identified by analysis of a Coomassie-stained polyacrylamide gel, were pooled and then concentrated. Following dialysis against phosphate-buffered saline (PBS; pH 7.4) at 4 °C overnight, KLK4 was aliquoted and stored at -80 °C. Activation of rKLK4—Recombinant zymogen KLK4 was incubated with the metalloendopeptidase thermolysin in PBS at pH 7.4 for 1 h at 37 °C (KLK4/thermolysin ratio 80:1). The amount of active enzyme produced was quantified by active site titration using the pseudosuicide inhibitor 4-methylumbelliferyl p-guanidinobenzoate (27Jameson G.W. Roberts D.V. Adams R.W. Kyle W.S. Elmore D.T. Biochem. J. 1973; 131: 107-117Crossref PubMed Scopus (287) Google Scholar). Zymogen KLK4 and thermolysin-treated KLK4 were examined on a Coomassie-stained polyacrylamide gel, protein bands were excised, and the amino terminus of the excised treated KLK4 was sequenced by Edman degradation at the Australian Proteome Analysis Facility (North Ryde, Australia). Thermolysin activity was stopped by the addition of phosphoramidon (10 μm). Kinetic Measurements of Activated rKLK4—Determination of kinetic parameters was performed using 40 nm active rKLK4 against the tripeptide substrate benzyl-FVR-p-nitroanilide. Assays were performed in 50 mm Tris-HCl (pH 7.4), 20 mm CaCl2, 0.01% (v/v) Tween 20 at 28 °C by following p-nitroanilide release photometrically at 405 nm. Experiments to determine the kinetics of cleavage of a fluorescence-quenched peptide with sequence spanning the PAR-2 serine protease activation site were performed at 37 °C in 50 mm Tris-HCl (pH 7.4), 20 mm CaCl2, 0.01% (v/v) Tween 20 by continuously measuring fluorescence at 440 nm following excitation at 330 nm. Emissions were monitored over 15 min using a Polarstar Optima plate reader (BMG Labtech, Melbourne, Australia). Enzyme activity was determined from a standard curve generated from the fluorescence obtained following complete cleavage by trypsin of known amounts of peptide using an absorption coefficient of 104 m-1 cm-1. To obtain kcat and Km values, initial velocity data at each substrate concentration were fitted to the Michaelis-Menten equation by nonlinear regression analysis using GraphPad Prism 4.0 (GraphPad Software, San Diego, CA). Experiments were performed in triplicate on three independent occasions with results displayed as means ± S.E. Measurement of Changes in Intracellular Ca2+—Cells grown to 80% confluence were washed with PBS, detached nonenzymatically, resuspended (4 × 106 cells/ml) in extracellular medium (121 mm NaCl, 5.4 mm KCl, 0.8 mm MgCl2·6H2O, 1.8 mm CaCl2, 5.5 mm glucose, 25 mm HEPES (pH 7.4)) containing 0.2% (w/v) bovine serum albumin (Sigma) and then loaded with the fluorescence indicator Fura-2 acetoxymethyl ester (1.0 μm; Invitrogen) at room temperature for 60 min. Cells were then pelleted followed by resuspension in extracellular medium (without bovine serum albumin) at a concentration of 2 × 106 cells/ml for fluorescence measurements. The ratio of fluorescence at 510 nm after excitation at 340 and 380 nm was monitored using a Polarstar Optima fluorescent plate reader. Single agonist treatments were KLK4 (300 nm), trypsin (10 nm), thrombin (10 nm), PAR-1 AP and PAR-2 AP (100 μm), and PAR-4 AP (500 μm). Displayed data are representative of experiments performed in triplicate and repeated on three independent occasions. Dose-response experiments were performed over the KLK4 concentration range of 0.1–1000 nm in triplicate and performed on three independent occasions with results displayed 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-2 siRNA target sequences were designed as previously described (28Pei Y. Tuschl T. Nat. Methods. 2006; 3: 670-676Crossref PubMed Scopus (240) Google Scholar) and then aligned against the human genome data base using the BLAST algorithm to eliminate those with significant homology to other genes. Three sequences selected were 5′-GATCCAGGAAGAAGCCTTATTGGTTTCAAGAGAACCAATAAGGCTTCTTCCTTTTTTTGGAAA-3′, 5′-GATCCAGTAGACTTGGTGTGAAGATTCAAGAGATCTTCACACCAAGTCTACTTTTTTTGGAAA-3′, and 5′-GATCCGTAGTCGTGAATCTTGTTCATTCAAGAGATGAACAAGATTCACGACTATTTTTTGGAAA-3′. The sequences were synthesized (Sigma) and inserted into the pSilencer 3.1-H1 puro vector (Ambion) according to the instructions of the manufacturer. Fibroblasts from Par-1 null mice stably expressing PAR-2 (PAR-2-LMF) were transfected with the PAR-2 pSilencer 3.1-H1 constructs or the supplied pSilencer 3.1-H1 negative control using Lipofectamine. After 48 h, 2 μg/ml puromycin was added to the medium to select for stable transfected cells. PAR-2 expression levels and agonist-induced induction of ERK phosphorylation were examined by Western blot analysis. PAR-1 and PAR-2 mRNA Expression Analysis—Total RNA was extracted from cells as previously described (29Hooper J.D. Campagnolo L. Goodarzi G. Truong T.N. Stuhlmann H. Quigley J.P. Biochem. J. 2003; 373: 689-702Crossref PubMed Google Scholar) and cDNA synthesized using Superscript II (Invitrogen). Reverse transcription-PCR was performed as previously described (30Hooper J.D. Zijlstra A. Aimes R.T. Liang H. Claassen G.F. Tarin D. Testa J.E. Quigley J.P. Oncogene. 2003; 22: 1783-1794Crossref PubMed Scopus (106) Google Scholar) with primers specific for PAR-1 (5′-TGTATCCCATGCAGTCCCTCTC-3′ and 5′-CACCTGGATGGTTTGCTCCTT-3′), PAR-2 (5′-AGAAGCCTTATTGGTAAGGTT-3′ and 5′-AACATCATGACAGGTGGTGAT-3′), or β-actin (human 5′-TGTCACCTTCACCGTTCCA-3′ and 5′-CAAGATCATTGCTCCTCCTG-3′; mouse 5′-CGTGGGCCGCCCTAGGCACCA-3′ and 5′-TTGGCCTTAGGGTTCAGGGGGG-3′). Cell Surface Biotinylation—PC-3 cells were washed three times with cold PBS, and then plasma membrane proteins were biotinylated by incubation with 1.22 mg/ml EZ-link NHS-SS-Biotin at 4 °C for 1 h with gentle agitation. The cells were then washed in PBS prior to lysis in a buffer containing 20 mm HEPES, 150 mm NaCl, 1 mm EDTA, and 1% Triton X-100. After centrifugation, bead immobilized streptavidin was added into the supernatant and incubated for 15 min on ice to capture biotinylated proteins. The streptavidin beads were pelleted by centrifugation, and the supernatant was recovered for analysis of cytoplasmic (nonbiotinylated) proteins. The beads were then washed thoroughly, and associated cell surface (biotinylated) proteins were eluted into Laemmli sample buffer. Cytoplasmic and cell surface fractions were examined for PAR-2 by Western blot analysis. Western Blot Analysis—Whole cell lysates were collected in a buffer containing Triton X-100 (1%, v/v), 50 mm Tris-HCl (pH 7.4), NaCl (150 mm), and protease inhibitor mixture (Roche Applied Science). Fractions containing membrane proteins and soluble proteins were isolated as previously described (31Compton S.J. Sandhu S. Wijesuriya S.J. Hollenberg M.D. Biochem. J. 2002; 368: 495-505Crossref PubMed Scopus (106) Google Scholar). Protein concentrations were determined by microbicinchoninic acid assay (Pierce). For ERK1/2 phosphorylation, cells were grown to 50% confluence and serum-deprived for 24 h before treatment, and then collection of whole cell lysates was as above, except the lysis buffer also contained 1 mm sodium orthovanadate and 50 mm NaF (Sigma). Equal amounts of lysates (20 μg) or biotinylated fractions were separated by SDS-PAGE and transferred to a nitrocellulose membrane that was blocked with Odyssey blocking buffer (LI-COR, Lincoln, NE) containing 0.1% (v/v) Tween 20. Membranes were incubated overnight 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 incubation with species-appropriate fluorescently conjugated secondary antibodies for 1 h at room temperature. Membranes were analyzed using an Odyssey Infrared Imaging System (LI-COR; Millennium Science, Surrey Hills, Australia) and where relevant signal intensity determined using LI-COR imaging software and exported to Microsoft Excel for graphical representation as mean ± S.E. Significance was examined using Student's t test with a p value of <0.05 considered significant. Flow Cytometry—Cells grown in serum-free media overnight were detached nonenzymatically, washed in PBS, and then resuspended at 2 × l06 cells/ml. Cells were subjected to nonpermeabilizing fixation in 1% formaldehyde on ice for 5 min, washed with PBS, and then resuspended in staining buffer (PBS, pH 7.4, 0.2% bovine serum albumin). Following incubation with an anti-PAR-2 antibody (SAM11; 2 μg/106 cells) on ice for 60 min, cells were washed and then incubated with fluorescently tagged anti-mouse secondary antibody. Cell surface PAR-2 was assessed using an FC500 flow cytometer (Beckman Coulter, Gladesville, Australia). In parallel, to assess the levels of total PAR-2, following fixation, cells were permeabilized by incubation with 0.01% Triton X-100 in PBS for 5 min at room temperature. Confocal Microscopy—Cells plated on sterile poly-l-lysine (Sigma)-coated glass coverslips were allowed to adhere overnight then transfected with either the PAR-2-GFP expression construct or vector pEGFP-N1. For agonist treatments, cells were incubated with either KLK4 (100 nm) or PAR-2 AP (100 μm) for 10 min at 37 °C before fixation with 4% (v/v) formaldehyde. Nuclei were stained by incubating cells for 5 min at room temperature with 4′,6-diamidino-2-phenylindole (1:1500) in PBS. Coverslips were mounted on slides, and cells were imaged with a Leica SP5 confocal microscope (Leica Microsystems, Sydney, Australia). Images were processed using Adobe Photoshop CS3 and displayed using CorelDraw. The amount of PAR-2 on the cell surface was quantified by examining the fluorescence of randomly selected untreated and KLK4-treated cells (n = 15) using ImageJ software (National Institutes of Health, Bethesda, MD), adapting the approach of Scherrer et al. (32Scherrer G. Tryoen-Toth P. Filliol D. Matifas A. Laustriat D. Cao Y.Q. Basbaum A.I. Dierich A. Vonesh J.L. Gaveriaux-Ruff C. Kieffer B.L. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 9691-9696Crossref PubMed Scopus (186) Google Scholar) for quantifying GPCR cell surface expression. Briefly, three cellular regions of interest were defined: the whole cell, the intracellular region, and the nucleus. The signal obtained for the whole cell and the intracellular region was corrected for background signal by subtracting nuclear fluorescence. Cell surface fluorescence was then obtained by subtracting the corrected value for the intracellular region from the corrected value for the whole cell. These values were then divided by the number of pixels contained within each region to give fluorescence density values for the cell surface (Di surface) and cytoplasm (Di cytoplasm). The ratio of Di surface to Di cytoplasm was determined to normalize data across the counted cell population. Results are displayed graphically as mean ± S.E., and significance was examined using Student's t test with a p value of <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 bone metastases (n = 2) were obtained from Sullivan Nicolaides Pathology (Taringa, Australia) and the Royal Prince Alfred Hospital (Sydney, Australia), respectively, following institutional ethics approval. Immunohistochemistry was performed as previously described (33Hooper J.D. Nicol D.L. Dickinson J.L. Eyre H.J. Scarman A.L. Normyle J.F. Stuttgen M.A. Douglas M.L. Loveland K.A. Sutherland G.R. Antalis T.M. Cancer Res. 1999; 59: 3199-3205PubMed Google Scholar). Briefly, serial sections (4 μm) were deparaffinized and then rehydrated, and after antigen retrieval in urea (5% w/v) in 0.1 m Tris buffer (pH 9.5), serial sections were incubated in H2O2 (3%, v/v) to quench endogenous peroxidase. 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-KLK4 rabbit polyclonal (1:250 dilution) antibody. As negative controls, mouse and rabbit immunoglobulins replaced the SAM11 and anti-KLK4 primary antibodies, respectively. The EnVision+ peroxidase polymer detection system (Dako, Botany, Australia) was used with 3,3′-diaminobenzidine (Sigma) as the chromogen. The sections were counterstained with Mayer's hematoxylin, visualized by microscopy (Leitz, Laborlux S, Germany), and photographed using a Nikon OXM1200 digital camera. Images were processed using Adobe Photoshop CS3 and displayed using CorelDraw. Examination of KLK4-initiated Changes in Intracel
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