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Biol. Chem., Vol. 389, pp. 653–668, June 2008 • Copyright by Walter de Gruyter • Berlin • New York. DOI 10.1515/BC.2008.078

Review

Prostatic -like -related peptidases (KLKs) and other prostate-expressed tryptic proteinases as regulators of signalling via proteinase-activated receptors (PARs)

Andrew J. Ramsay1, Janet C. Reid1, Mark N. cesses by selective cleavage of specific substrates to Adams1, Hemamali Samaratunga2, Ying Dong1, influence cell behaviour (Turk, 2006). Well known physio- Judith A. Clements1 and John D. Hooper1,* logical examples include the proteinases of the blood (e.g., ) (Davie et al., 1991), digestive 1 Institute of Health and Biomedical Innovation, (e.g., trypsin) (Yamashina, 1956) and wound healing (e.g., Queensland University of Technology, 6 Musk Avenue, ) (Castellino and Ploplis, 2005) cascades. It is Kelvin Grove, Queensland 4059, Australia also clear that dysregulated serine proteinase activity 2 Department of Anatomical Pathology, Sullivan facilitates progression of various diseases including can- Nicolaides Pathology, Taringa 4068, Australia cer (Dano et al., 2005; Turk, 2006; Szabo and Bugge, * Corresponding author 2008). The cleavage site specificity of these is e-mail: [email protected] indicated by the residue six amino acids before the cat- alytic serine. In the active conformation of the proteinase, this residue is located at the bottom of a pocket in which Abstract the side-chain of the scissile bond of the substrate is inserted. An aspartate or, rarely, a glutamate at this site The prostate is a site of high expression of serine pro- indicates that the serine proteinase will preferentially teinases including members of the kallikrein-related cleave after substrate or lysine residues (trypsin- peptidase (KLK) family, as well as other secreted and like or tryptic substrate specificity); a small hydrophobic membrane-anchored serine proteinases. It has been residue in this position specifies preferential cleavage known for some time that members of this family after large hydrophobic amino acids (-like elicit cellular responses by acting directly on cells. More substrate specificity); and larger, usually non-polar resi- recently, it has been recognised that for serine protein- dues at this site specify preferential proteolysis following ases with specificity for cleavage after arginine and lysine small hydrophobic amino acids (-like substrate residues (trypsin-like or tryptic enzymes) these cellular specificity) (Perona and Craik, 1995, 1997). responses are often mediated by cleavage of members The largest contiguous collection of S1A serine pro- of the proteinase-activated receptor (PAR) family – a four teinase encoding genes is located within the kallikrein- member sub-family of G protein-coupled receptors. related peptidase (KLK) locus at chromosome Here, we review the expression of PARs in prostate, the 19q13.3–13.4 (Gan et al., 2000; Harvey et al., 2000; You- ability of prostatic trypsin-like KLKs and other prostate- sef et al., 2000) which consists of 15 genes, 12 of which expressed tryptic enzymes to cleave PARs, as well as the encode tryptic serine proteinases: KLK1–2, KLK4–6, prostate cancer-associated consequences of PAR acti- KLK8, KLK10–15 (Borgono and Diamandis, 2004; Cle- vation. In addition, we explore the dysregulation of tryp- ments et al., 2004). A common site of expression for the sin-like serine proteinase activity through the loss of KLKs is the prostate with this tissue a predominant site normal inhibitory mechanisms and potential interactions of mRNA expression for KLK2, KLK3 (also known as between these dysregulated enzymes leading to aberrant prostate-specific antigen or PSA), KLK4, KLK11, KLK14 PAR activation, intracellular signalling and cancer-pro- and KLK15 (Clements et al., 2004; Shaw and Diamandis, moting cellular changes. 2007). In addition, mRNA of KLK6, KLK9, KLK12 and Keywords: cancer; kallikrein-related peptidase; KLK13 is highly expressed in this tissue, while low or very prostate; proteinase-activated receptor; serine low prostatic mRNA expression has been reported for proteinase. each of the remaining KLKs (Clements et al., 2004; Shaw and Diamandis, 2007). In summary, as listed in Table 1 (and described in the references cited therein), tran- Introduction scripts of each KLK are expressed in prostate and all family members, except KLK8, have been detected at the Serine proteinases of the S1A sub-family are characte- protein level in normal or cancerous prostate. rised by a triad of amino acids consisting of histidine, Over the last 15 years, it has been increasingly recog- aspartate and serine residues in highly conserved nised that many of the cellular effects elicited by trypsin- sequence motifs, which are essential for catalytic activity like serine proteinases are mediated by activation (Rawlings and Barrett, 1994). These enzymes, which of cell surface proteins known as proteinase activated encompass as many as 140 (Rawlings et al., 2006) of the receptors (PARs). These receptors, which comprise a G total 569 human degradome complement (Lo´ pez-Otı´n protein coupled receptor sub-family consisting of and Matrisian, 2007), modulate a variety of cellular pro- PAR1–PAR4, are irreversibly activated by the action of

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654 A.J. Ramsay et al. b c ND ND ND ND Cancer a Protein expression in normal and cancerous prostate Expression pattern SF; N; B; P; CLovgren et al., 1999b;Veveris-Lowe Magklara et et al., al., 2005; 2000; Shaw Herrala and et Diamandis, al., 2007) 2001; Up Shaw and Diamandis, 2007) Tremblay et al., 1997; Veveris-Lowe et al., 2005) Shaw and Diamandis, 2007) (Shaw and Diamandis, 2007) Not detected ND Shaw and Diamandis, 2007) SF; N; B Up Lys SF; N ) Lys SF; N; B; P; C Up Lys, Phe, Tyr, Gly, AspLys, Leu, Phe, Tyr, Leu, Gly, Ser, Thr, Pro SF; N; B; C SF; N Down Lys SF; N; B; C Down Phe, Leu SF; N , Met, Phe ) ) ) ) ) (Sueiras-Diaz et al., 1994;Bourgeois Chagas et et al., al., 1997; 1995; Chen et al.,Arg 2000; Borgono et(Bourgeois al., et 2007b) al., 1997;Lovgren Mikolajczyk et et al., al., 1999a; 1998; Cloutier et al., 2002) Shaw and Diamandis, 2007) (Geiger and Clausnitzer, 1981; Fink et al., 1985; (Deperthes et al., 1995; Young et al., 1996; Darson et al., 1997, Darson et al., 1999; Tremblay et al., 1997; (Mitsui et al., 2000; Luo et al., 2006) (Diamandis et al., 2002; Nakamura et al., 2003; Arg (Akiyama et al., 1987;Denmeade Christensson et et al., al., 1997; 1990;Coombs Takayama et et al., al., 1997; 1998; Malm et al., 2000) (Darson et al., 1997, 1999; Herrala et al., 2001; Ishida et al., 2003; Magklara et al., 2000; Shaw and Diamandis, 2007; Simone et al., 2000; Arg (Takayama et al., 2001; MatsumuraDebela et et al., al., 2005; 2006a,b; Obiezu et al., 2006; Borgono et al., 2007a) Veveris-Lowe et al., 2005; Klokk et al., 2007; (Day et al., 2002; Obiezu et al., 2002, 2005; Dong et al., 2005; Arg (Brattsand et al., 2005;Oikonomopoulou Michael et et al., al., 2006; 2005; Borgono Debela et et al.,Arg al., 2007a,b) 2006b; (Shaw and Diamandis, 2007) (Little et al., 1997;Magklara Okui et et al., al., 2003; 2001;Oikonomopoulou Debela Bernett et et et al., al., al., 2006; 2006b; 2002; Borgono et al., 2007b) (Diamandis et al., 2000; Petraki et al., 2001, 2003a) (Egelrud, 1993; Skytt et al., 1995)Arg, Lys (Rajapakse et al., 2005; Kishi et al., 2006; Stefansson et al., 2008) (Kishi et al., 2004; Shaw and Diamandis, 2007) Arg (Debela et al., 2006b) (Luo et al., 2001a; Petraki et al., 2002, 2003a; Arg Summary of studies examining proteolytic substrate specificity and prostate expression of kallikrein-related peptidases. 2 Tryptic 3 Chymotryptic Leu, Tyr, Phe, Gln, His, Asn, Leu, Ser SF; N; B; P; C Down 1 Tryptic 4 Tryptic 56 Tryptic Tryptic 7 Chymotryptic Try 89 Tryptic Chymotryptic Not done SF; N Table 1 KLK Specificity P1 Preference 10 Tryptic 11 Tryptic

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Tryptic serine proteinases and PARs in prostate 655 b

c proteinases (Vu et al., 1991; Bohm et al., 1996a; Ishihara ND Up et al., 1997; Xu et al., 1998; Boire et al., 2005; Vesey et Cancer al., 2007). In fact, PAR activation is almost exclusively mediated by serine proteinases with trypsin-like sub- strate specificity for cleavage following arginine or lysine residues. As shown in Figure 1A cleavage occurs within the amino-terminal exodomain of the receptor following either an arginine or lysine residue generating a new ami- no-terminal. This neo-epitope serves as a tethered ligand which binds intramolecularly, causing allosteric changes within the PAR, followed by receptor coupling to hete- rotrimeric G proteins and signal transduction (Figure 1B, activation). Importantly, it is also possible for proteinases to cleave downstream of the activation site or within a PAR extracellular loop leading to disarming of the recep- tor (Oikonomopoulou et al., 2006) (Figure 1B, disarming). PARs are involved in a number of physiological process- es, such as cardiovascular responses, neuronal cell sur- vival, platelet aggregation, inflammation and epidermal function, and also mediate disease-associated cellular a changes including those necessary for cancer progres- Protein expression in normal and cancerous prostate sion, such as dysregulated cell proliferation and migra- tion (Macfarlane et al., 2001). Recently, several prostatic KLKs with trypsin-like sub- strate specificity have been shown to cleave PARs lead- ing either to receptor activation or disarming. It is also Expression pattern known that normal and diseased prostate is the site of expression of many other tryptic serine proteinases. This review summarises the current understanding of signal- ling via PARs in prostate initiated by KLK and other tryp- tic serine proteinases and highlights the possibility that as yet untested trypsin-like members of this enzyme family will also regulate prostatic PAR signalling.

PAR expression in prostate tissue and cell lines

The mRNA and protein expression patterns of PARs in prostate, analysed by a number of approaches, are sum- marised in Table 2. Tissue microarray analysis of a large

number of patient samples indicated elevated PAR1 expression in benign prostatic hyperplasia (BPH) com- pared to normal prostate stroma (Tantivejkul et al., 2005).

Increased PAR1 expression was also observed in high grade prostatic intraepithelial neoplasial (PIN) and pros- tate adenocarcinoma in comparison to normal prostate stroma, BPH and atrophic prostate tissues (Tantivejkul et LysLys, Tyr, Phe, Tyr, Gly, Leu SF; N SF; N; B; C Down Lys SF; N

Argal., 2005). Kaushal SF; B; C and colleagues have also recently ND ) ) ) ) reported correlation of increased PAR1 mRNA and protein Lys (Memari et al., 2007b)Arg (Sotiropoulou et al., 2003;Borgono Kapadia et et al., al., 2007a) 2004;Arg (Kapadia et al., 2003; Petraki et al., 2003a,b; (Memari et al., 2007a; Shaw and Diamandis, 2007) Shaw and Diamandis, 2007) (Takayama et al., 2001) (Shaw and Diamandis, 2007; Shaw et al., 2007) (Brattsand et al., 2005;Oikonomopoulou Felber et et al., al., 2006; 2005;Stefansson Borgono et et al., al., 2008) 2007a,b,c; Arg levels (Borgono et with al., 2007c; Shaw and Diamandis, 2007) prostate cancer stage. These workers, employing quantitative PCR analysis of early versus late stage prostate cancer tissue, showed an increase in

PAR1 mRNA in the late stage cancer samples. Increasing

PAR1 mRNA with prostate cancer stage was similarly

reflected in PAR1 protein expression, as Western blot

analysis of PAR1 protein levels showed a 2.75-fold increase in late versus early stage disease. This obser- vation was consistent with their immunohistochemical

analyses that showed low PAR1 expression in 56% (15 (Continued) of 27) of BPH samples, low to medium expression levels in 44% (14 of 32) and strong staining in 22% (7 of 32) of Up- or down-regulated in prostate cancer. ND, not done; up/down, increase/decrease in detectable levels compared with benign or normal. SF, seminal fluid; N, normal prostate tissue; B, benignDetermined prostate from disease; serum. P, prostatic intraepithelial neoplasia; C, cancer. Table 1 KLK Specificity P1 Preference 1213 Tryptic 14 Tryptic Tryptic a b c 15 T early stage prostate cancer, while strong staining was

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656 A.J. Ramsay et al.

absent. PAR expression in prostate cancer samples was elevated in comparison to normal prostate glands for

PAR1 (45%), PAR2 (42%) and PAR4 (68%), with elevated expression most notable in cancerous epithelial cells. Additionally, higher Gleason grade cancers (G7–10) had

increased expression of PAR1 in periglandular stromal cells compared to lower grade tumours (G5–9), signifying a shift from epithelial to periglandular expression in pros- tate cancer progression (Black et al., 2007).

We have also examined the expression of PAR2 in pri- mary prostate cancer, as well as in bone metastasis. In these experiments, we used an affinity purified rabbit

anti-PAR2 polyclonal antibody generated against a pep- tide spanning amino acids 133–146 located in the first extracellular loop of the receptor (Figure 2A). As shown

in Figure 2B–D, PAR2 is expressed by cells of epithelial

origin with little evidence of stromal staining. PAR2 was weakly detected in benign glands of BPH (Figure 2B), more strongly in cells of epithelial origin in cancerous glands (Figure 2C), as well as strongly in cancer cells in bone metastases (Figure 2D). A prostate tissue section

incubated without the anti-PAR2 antibody was free of staining (Figure 2E). These data are consistent with the observations of Black and co-workers and suggest that

PAR2 expression levels increase during prostate cancer progression.

Consistent with the prostatic expression of PAR1, Figure 1 Proteinase-activated receptor cleavage. androgen regulation of the encoding gene has been (A) Activation sites of the four human PARs. The arrow indicates recently reported (Salah et al., 2005). These workers the site of proteolytic cleavage necessary for activation and intracellular signal transduction to occur. (B) Members of the showed that stimulation of LNCaP cells with dihydrotes- PAR family of seven transmembrane G protein-coupled recep- tosterone (DHT) increased the transcription of PAR1, tors are cleaved either at the receptor activation site leading to whereas PC-3 cells, which lack an androgen receptor, signal transduction across the plasma membrane, or down- were unresponsive. Consistently, PAR1 promoter activity stream of the activation site resulting in receptor inactivation was increased 3-fold in response to DHT and attributed (also known as disarming). Receptor activation involves cleav- to an androgen response element located at -1791 bp to age at an arginine or lysine to expose a tethered ligand which binds intramolecularly, inducing allosteric changes in the recep- -1777 bp. Furthermore, consistent with PAR1 androgen tor. Receptor disarming involves cleavage distal to the activation responsiveness, all nine grade- and age-matched neo- site causing removal of the tethered ligand and inhibition of plastic prostate tissue samples before and after andro- receptor activation. gen ablation therapy displayed a substantial decrease in

PAR1 mRNA levels after androgen ablation. However, as

PAR1 levels are elevated in late, androgen independent also observed in all 22 advanced prostate cancer tissues. stage disease it is apparent that other mechanisms must Interestingly, approximately 50% of the strong staining also transcriptionally regulate the encoding gene. Con- seen in early and advanced tumours was in areas sistent with this proposal, an aggressive, hormone resis- of blood vessels and a weak correlation was found tant sub-line of LNCaP cells (CL1 cells) generated between levels of PAR1 and the pro-angiogenic factor through in vitro androgen deprivation, has high levels of vascular endothelial growth factor (VEGF)-2 (Kaushal et PAR1 mRNA and protein and small amounts of AR pro- al., 2006). tein; a phenomenon that is also seen in PC-3 cell which

Similar to PAR1, other members of this receptor family lack androgen receptor (Salah et al., 2005). More recent- are also expressed in normal and cancerous prostate. Of ly, Salah and colleagues employed fluorescence in situ note, in the first paper reporting the cloning of the human chromosome hybridisation to indicate that differences in PAR cDNA, Nystedt and co-workers demonstrated high 2 PAR1 mRNA levels in cell lines of high (CL1 or PC-3) and expression of the mRNA for this receptor in normal pros- low (LNCaP) expression is not due to gene amplification. tate (Nystedt et al., 1995). Further, Black and co-workers These workers also showed that PAR1 mRNA stability is demonstrated that six matched normal and cancerous the same in each of these lines. Importantly, nuclear run- patient samples showed increased mRNA levels of PAR 1 on assays indicated markedly enhanced PAR1 mRNA (5 of 6), PAR (4 of 6) and PAR (3 of 6), while PAR levels 2 4 3 elongation rates in cell lines with high levels of PAR1 were consistently low (Black et al., 2007). In addition, mRNA in comparison to cells which have low levels of immunohistochemical analysis of 40 patient tissues this transcript (Salah et al., 2007). These observations showed little PAR (7.5%) and PAR (13%) in normal 1 2 suggest that androgen is involved in regulating PAR1 prostate glands, with PAR4 staining apparent in approx- expression in normal prostate and early stages of pros- imately 55% of samples and PAR3 expression completely tate cancer, and that an unknown mechanism may reg-

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Tryptic serine proteinases and PARs in prostate 657 b PAR expression Reference Level Localisation 128) Stain intensity 1.1 Epithelium Tantivejkul et al., 2005 6) Increased in cancer (5 of 6) n/a Black et al., 2007 6) Increased in cancer (4 of 6) n/a Black et al., 2007 6) Increased in cancer (3 of 6) n/a Black et al., 2007 s s s s 40) Increased in cancer (45%) Epithelial origin Black et al., 2007 40) Increased in cancer (42%) Epithelial origin Black et al., 2007 40)40) Absent Increased in cancer (68%) Epithelial origin n/a Black et al., 2007 Black et al., 2007 s s s s 7) PCa Increased in advanced 2.39-fold n/a Kaushal et al., 2006 6) PCa Increased in advanced 2.75-fold n/a (Western blot analysis) Kaushal et al., 2006 9) ablation (semi-quantitative) s s s a 22) Strong (22 of 22) VE and epithelial origin 32) Absent/low-medium (12/14 of 32) VE and epithelial origin s 27) Absent/low (12/15 of 27) VE 303) Stain intensity 1.4 Epithelial origin s 82) Stain intensity 2.5 Epithelial origin 82) Stain intensity 2.7 Epithelial origin s s s s 9) versus Ad (n 9) versus Ad (n s s E(n PCa pre- and post-androgen ablation (n Decreased in cancer after n/a Salah et al., 2007 BPH (n E PCa (n Ad PCa (n Normal prostate stroma (n Matched N versus C (n Matched N versus PCa (n BPH (n PCa (n PIN (n Protein E (n Protein Matched N versus C (n Protein Matched N versus C (n Summary of studies examining proteinase-activated receptor (PAR) expression in normal and cancerous prostate. n/a, not applicable; VE, vascular endothelium. N, normal; E, early; Ad, advanced; BPH, benign prostatic hyperplasia; PIN, prostatic intraepithelial neoplasia; PCa, prostate cancer. Table 2 PAR1 mRNA Prostate tissue type Matched N versus PCa (n a b 2 mRNA Normal High n/a Nystedt et al., 1995 3 Protein Matched N versus C (n 4 mRNA Matched N versus PCa (n

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658 A.J. Ramsay et al.

Figure 2 Immunohistochemical analysis of PAR2 expression in primary prostate cancer and bone metastasis.

(A) Sequence and location (residues 133–146; extracellular loop 1) of the peptide used to generate a rabbit anti-PAR2 polyclonal antibody. (B) PAR2 expression in benign glands of benign prostatic hyperplasia. (C) PAR2 expression in cancerous prostate. (D) PAR2 expression in prostate cancer cells in a bone metastasis. (E) Negative control – primary antibody omitted. ulate its expression in later, hormone resistant disease the receptor in intact cells; Figure 1). Consistent data stages. were obtained from microscopy analyses of cells stably

The expression of PARs in prostate derived cell lines expressing rat PAR2. Interestingly, these workers also is summarised in Table 3. Both PAR1 and PAR2 are widely showed that KLK5 is less efficient than KLK6 and KLK14 expressed with each analysed cell line expressing these at cleaving at the PAR2 activation site. Furthermore, receptors (Chay et al., 2002; Greenberg et al., 2003; Liu experiments analysing Ca2q mobilisation yielded impor- et al., 2003, 2006; Yin et al., 2003; Wilson et al., 2004; tant insights on the ability of KLKs to induce differential

Myatt and Hill, 2005). In contrast, PAR3 protein has not signalling in intact cells via PARs. These workers have been reported to be expressed by any prostate derived proposed that PAR2 is activated by low concentrations cell line, while PAR4 expression has only been reported of KLK6, whereas higher enzyme concentration results in in LNCaP cells (Greenberg et al., 2003). However, reports receptor inactivation. Equally as interesting is the pro- from Liu et al. (2003) and Wilson and colleagues (Wilson posal that in cells expressing both PAR1 and PAR2, the et al., 2004) have been inconsistent regarding the pres- former receptor is disarmed by KLK14, whereas the latter ence of PAR4 mRNA in this cell line. is activated by this enzyme. Adding another layer of com-

In summary, it is apparent that PARs are expressed in plexity, KLK14 was also shown to signal via PAR4, where- both normal and cancerous prostate. The presence of as KLK5 and KLK6 were unable to induce signalling these receptors and the known roles of PARs as sentinels through this receptor at concentrations that initiated of tryptic serine proteinase activity indicate the potential signalling via PAR2 (Oikonomopoulou et al., 2006). The that these cell surface receptors will be important trans- observations of Oikonomopoulou and co-workers indi- ducers of signals initiated by the large number of pro- cate that individual KLKs can function as both activator static trypsin-like serine proteinases – both KLK and and disarmer of individual PARs, as well as have the abil- non-KLK. ity to cleave multiple PARs. These findings highlight the potential complexity of regulation of PAR signalling; par- ticularly in situations of dysregulated trypsin-like serine PAR cleavage by prostatic trypsin-like proteinase activity and expression of multiple PARs, which occur in prostate cancer. The data of Oikonomopoulou and colleagues have Studies examining the ability of tryptic KLKs and other been partially confirmed by others. Stefansson and co- trypsin-like serine proteinases to regulate signalling via workers confirmed the ability of KLK5 and KLK14 to acti- PARs are summarised in Table 4. In a significant recent vate PAR2 by examining calcium mobilisation in cells report Oikonomopoulou and colleagues examined the stably expressing the human receptor (Stefansson et al., ability of three KLK family members (KLK5, KLK6 and 2008). This group also examined the ability of the related KLK14) to regulate signalling via PAR1,PAR2 and PAR4 enzymes KLK7 (chymotryptic) and KLK8 (tryptic) to sig- (Oikonomopoulou et al., 2006). Mass spectral analysis of nal via PAR2. Not surprisingly, KLK7 could not signal via peptides spanning the activation site of each receptor PAR2. Interestingly, however, KLK8 was also unable to indicated that a major cleavage site for each KLK is at signal through this receptor (Stefansson et al., 2008). In the known receptor activation site. In addition, both KLK5 contrast, whereas Oikonomopoulou and colleagues have and KLK14 also cleaved upstream of the PAR2 activation reported that KLK6 cleaves peptides spanning the acti- site. Interestingly, only KLK14 was able to cleave at a site vation sites of PAR1,PAR2 and PAR4, Angelo et al. dem- downstream of the activation site of the peptide of one onstrated that while KLK6 is able to efficiently cleave a of the receptors, PAR1 (which would induce disarming of PAR2 activation site spanning peptide, this enzyme is

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Tryptic serine proteinases and PARs in prostate 659

Table 3 Summary of studies examining proteinase-activated receptor expression in prostate-derived cell lines.

Prostate cell linea PAR Detection methodb Reference

PNT1A PAR1 mRNA PCR Liu et al., 2003

(prostate epithelium) PAR1 protein FC

No PAR3 mRNA PCR

No PAR4 mRNA PCR

Primary prostate PAR2 protein PAR2 activation Myatt and Hill, 2005 stromal cells (BPH) assay

LNCaP PAR1 mRNA PCR and NB Liu et al., 2003; Chay et al., 2002; Wilson et al., 2004

(prostate cancer lymph PAR2 mRNA PCR Wilson et al., 2004 node metastasis) *PAR3 mRNA

*PAR4 mRNA

*No PAR3 mRNA PCR Liu et al., 2003

*No PAR4 mRNA

PAR1 protein FC and IC Greenberg et al., 2003; Liu et al., 2003; Wilson et al., 2004

PAR2 protein FC and IC Greenberg et al., 2003; Wilson et al., 2004

No PAR3 protein FC Greenberg et al., 2003

PAR4 protein FC

PC-3 PAR1 mRNA PCR and NB Chay et al., 2002; Wilson et al., 2004

(prostate cancer PAR2 mRNA PCR Wilson et al., 2004 bone metastasis) No PAR3 mRNA

No PAR4 mRNA

PAR1 protein IC Chay et al., 2002; Wilson et al., 2004

PAR2 protein IC Wilson et al., 2004

DU145 PAR1 mRNA PCR and NB Chay et al., 2002; Liu et al., 2003, 2006; Wilson et al., 2004

(prostate cancer PAR2 mRNA PCR Wilson et al., 2004 brain metastasis) No PAR3 mRNA PCR Liu et al., 2003; Wilson et al., 2004

No PAR4 mRNA PCR

PAR1 protein FC and IC Chay et al., 2002; Liu et al., 2003, 2006; Wilson et al., 2004

PAR2 protein IC Wilson et al., 2004

Primary prostate PAR2 protein PAR2 activation Myatt and Hill, 2005 stromal cells (BPH) assay

DuCaP versus VCaP PAR1 protein IC Chay et al., 2002 (soft tissue versus (increased in VCaP) bone met)

AT2.1 versus AT3.1 PAR1 mRNA PCR Yin et al., 2003 (rat prostate carcinoma) (increased in AT3.1) aCell line designation (origin); BPH, benign prostatic neoplasia. bFC, flow cytometry; NB, Northern blot; IC, immunocytochemistry. *Denotes literature conflict.

unable to cleave PAR1 or PAR4 activation site spanning secreted serine proteinases, containing modular non- peptides or a PAR3 peptide (Angelo et al., 2006). proteolytic domains, such as factor XIIa (Takeuchi et al., Interestingly, early studies by Molino and co-workers 1999), plasma kallikrein (Neth et al., 2001; Fink et al., indicated that KLK1 is unable to cleave PAR1 or PAR2 in 2007), (also known as urinary plasminogen endothelial cells (Molino et al., 1997a) or to cleave a pep- activator, uPA; Gavrilov et al., 2001; Riddick et al., 2005; tide spanning the PAR2 activation site (Molino et al., Usher et al., 2005) and protein C (He et al., 1995; Takeu- 1997b). However, more recently Houle and colleagues chi et al., 1999); the multi-serine proteinase domain con- used a rodent paw oedema model to demonstrate that taining enzymes polyserase-2 and -3 (Cal et al., 2005, this proteinase plays a role in inflammation by direct or 2006); the glycosyl-phosphatidylinositol membrane- indirect activation of PAR4 (Houle et al., 2005). anchored serine proteinase prostasin (Chen et al., 2001); as well as nine members of the type II transmembrane serine proteinase (TTSP) sub-family (Hooper et al., 2001; PAR cleavage by other prostatic trypsin-like Netzel-Arnett et al., 2003) including hepsin, enteropepti- serine proteinases dase (Cottrell et al., 2004), matriptase/MT-SP1 (Saleem et al., 2006), TMPRSS2 (Lin et al., 1999), TMPRSS3 It is of significance for signalling via PARs that normal (Scott et al., 2001), TMPRSS13 (Kim et al., 2001), human and cancerous prostate and prostate-derived cell lines airway trypsin-like proteinase (HAT) (Hahner et al., 2005), express at least 16 other trypsin-like serine proteinases. DESC1 (Lang and Schuller, 2001) and the multi-catalytic These include secreted enzymes, such as trypsin I, II domain TTSP polyserase-1 (Okumura et al., 2006). It is (Paju et al., 2000; Bjartell et al., 2005) and IV (Takeuchi also interesting to note the co-expression in prostate- et al., 1999; Cottrell et al., 2004); other more complex derived cell lines of trypsinogen I, II and IV and the in vivo

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660 A.J. Ramsay et al.

Table 4 Summary of studies examining PAR activation by tryptic KLKs and other trypsin-like serine proteinases.

Serine proteinase Structurea PAR cleavageb Reference

KLK1 Secreted Unable to cleave PAR2 activation peptide sequence Molino et al., 1997a

Unable to activate PAR1 or PAR2 in intact cells Molino et al., 1997b

Activates PAR4 in rodent paw oedema model Houle et al., 2005

KLK5 Secreted Cleaves at activation site of PAR1,PAR2 and PAR4 peptides Oikonomopoulou et al., 2006

Cleaves cell surface hPAR2 and rPAR2 Oikonomopoulou et al., 2006; Stefansson et al., 2008 2q Induces Ca mobilisation via hPAR2 and rPAR2 Oikonomopoulou et al., 2006; Stefansson et al., 2008

KLK6 Secreted Cleaves at activation site of PAR1,PAR2 and PAR4 peptides Oikonomopoulou et al., 2006

Cleaves cell surface rPAR2 2q Induces Ca mobilisation via rPAR2

2q KLK7 Secreted Unable to induce Ca mobilisation via hPAR2 Stefansson et al., 2008

2q KLK8 Secreted Unable to induce Ca mobilisation via hPAR2 Stefansson et al., 2008

KLK14 Secreted Cleaves at activation site of PAR1,PAR2 and PAR4 peptides Oikonomopoulou et al., 2006

Cleaves downstream of activation site of PAR1 peptide Oikonomopoulou et al., 2006

Cleaves cell surface hPAR2 and rPAR2 Oikonomopoulou et al., 2006; Stefansson et al., 2008 2q Induces Ca mobilisation via h/rPAR2 and h/rPAR4 Oikonomopoulou et al., 2006; Stefansson et al., 2008

Disarms hPAR1 in intact cells Oikonomopoulou et al., 2006

2q Trypsinogen IV Secreted Induces Ca mobilisation via hPAR2 and hPAR4 Cottrell et al., 2004

Cleaves at activation site of PAR1,PAR2 and PAR4 peptides Knecht et al., 2007 2q Induces Ca mobilisation via hPAR1 and hPAR2 Knecht et al., 2007

Causes PAR2 dependent inflammation and hyperalgesia Knecht et al., 2007

HAT TTSP IL-8 release mediated by an anti-PAR2 antibody Iwakiri et al., 2004 inhibitable pathway

2q Matriptase/MT-SP1 TTSP Induces Ca mobilisation via hPAR2 and mPAR2 Takeuchi et al., 2000 2q Unable to induce Ca mobilisation via hPAR1,PAR3 or PAR4

2q TMPRSS2 TTSP Induces Ca mobilisation via hPAR2 Wilson et al., 2005 2q Unable to induce Ca mobilisation via hPAR1 aTTSP, type II transmembrane . bh, human; r, rat; m, mouse. activator of these enzymes, the TTSP graft model. These studies resulted in reduction of both (Light and Janska, 1989; Kitamoto et al., 1994). tumour volume and in vitro cell invasion (Galkin et al., Several of these enzymes have well documented asso- 2004). Similarly, PC-3 and DU145 cells in which matrip- ciations with cancer progression. For example, the gene tase/MT-SP1 levels had been knocked down, exhibited encoding hepsin is one of the most up-regulated genes reduced growth, invasiveness and migration in vitro and in prostate cancer (Dhanasekaran et al., 2001; Luo et al., a reduced ability to grow and develop in vivo (Sanders 2001b; Magee et al., 2001; Ernst et al., 2002; Halvorsen et al., 2006). et al., 2005), while hepsin protein expression increases TMPRSS2, an androgen regulated member of the with prostate cancer progression with expression main- TTSP family, is expressed moderately in the glandular tained in bone metastases (Xuan et al., 2006). Signifi- epithelia of normal prostate, PIN and prostate cancer cantly, hepsin overexpression in mouse prostate tissue samples (Lin et al., 1999). Consistently, in situ epithelium caused basement membrane disorganisation, hybridisation analysis of patient tissue indicated that while overexpression in a mouse model of non-metas- androgen-deprivation therapy was accompanied by sig- tasising prostate cancer was accompanied by metastasis nificantly decreased TMPRSS2 mRNA levels (Vaarala et to several organs including liver, bone and lung (Klezo- al., 2001). Interestingly, although unrelated to its proteo- vitch et al., 2004). lytic activity, fusions of the ETS transcription factor genes Similar to hepsin, expression of matriptase/MT-SP1 is ERG and ETV1 with the promoter of the TTSP gene elevated in prostate cancer tissue in comparison to nor- TMPRSS2 is a common occurrence in prostate cancer. mal prostate. Significantly, elevated matriptase/MT-SP1 These gene fusion events result in the overexpression of expression is accompanied by a decrease in the expres- ERG and ETV1 driven by androgen-responsive elements sion of hepatocyte growth factor activator inhibitor-1, the within the TMPRSS2 promoter. Importantly, TMPRSS2: specific inhibitor of this tryptic proteinase (Saleem et al., ERG fusion prostate cancers have a more aggressive 2006). A role for matriptase/MT-SP1 in prostate cancer phenotype, highlighting the potential for this fusion prod- progression has also been indicated by application of the uct as a discriminating biomarker for advanced prostate selective inhibitor, CVS-3983, in a prostate cancer xeno- cancer (Tomlins et al., 2005).

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Tryptic serine proteinases and PARs in prostate 661

A number of these trypsin-like serine proteinases have icked by PAR1 and PAR2 activating peptide (AP), but not been examined for the ability to signal via PARs (Table by PAR4 AP, signifying the importance of PAR1 and PAR2 4). These include the membrane anchored tryptic serine in prostate cancer cell migration (Greenberg et al., 2003). proteinases HAT, matriptase/MT-SP1 and TMPRSS2, as Additionally, a low concentration of thrombin stimulated well as the secreted enzyme trypsin IV. Iwakiri and col- PC-3 and DU145 cell migration towards fibronectin (Shi leagues have proposed that the TTSP HAT induces et al., 2004). Interestingly, higher concentrations of release of interleukin-8 (IL-8) via a PAR2-mediated path- thrombin resulted in unchanged cell migration of PC-3 way as inhibition of PAR2 resulted in a decreased HAT- cells on fibronectin and significantly decreased cell induced IL-8 release (Iwakiri et al., 2004). Takeuchi and migration on plastic, collagen I and IV and laminin co-workers have demonstrated that matriptase/MT-SP1 (Loberg et al., 2007), while synergistic treatment with is able to activate human PAR2 and mouse PAR2, but not PAR1 and PAR2 APs enhanced migration similar to throm- human PAR1,PAR3 or PAR4 in receptor expressing bin in these cell lines. Interestingly, PAR1 AP alone stim- oocytes (Takeuchi et al., 2000). More recently, TMPRSS2 ulated no migration, while PAR2 AP resulted in only has been shown to activate PAR2 but not PAR1 in LNCaP marginal migration. Thrombin in this study was implicat- 2q cells causing Ca mobilisation (Wilson et al., 2005). In ed as an indirect activator of PAR2, with desensitisation addition, the secreted proteinase trypsin IV is able to experiments pre-treating M24met cells with PAR2 agonist cleave peptides representing the activation sequence of abolishing the effect of thrombin induced migration. Fur-

PAR1,PAR2 and PAR4, and, in intact cells, to activate the thermore, experiments employing a PAR2 cleavage corresponding receptors inducing prompt Ca2q mobili- blocking antibody demonstrated that thrombin mediated sation (Knecht et al., 2007). Consistently, treatment of effects were cleavage independent (Shi et al., 2004). One

PC-3 cells with trypsin IV caused calcium ion mobilisa- explanation is that PAR1 and PAR2 are forming hetero- tion, presumably through PAR1 and PAR2, as PC-3 cells dimers at the cell surface with thrombin mediated chang- lack PAR4 (Cottrell et al., 2004). Potentially, these data es via PAR1 requiring the interaction of these receptors indicate that, as trypsinogen I, II and IV and the endo- (Shi et al., 2004). genous activator enteropeptidase are expressed by pros- The study by Greenberg and co-workers also indicated tate cancer derived cell lines and tissues, an enzymatic the ability of thrombin and trypsin to induce changes in cascade that mirrors that seen for pancreatic tryptic pro- the morphology of LNCaP cells. Proteolytic treatment teinases in the gastro-intestinal tract may also regulate induced cytoskeletal reorganisation after 8 h, and, after PAR signalling in normal and diseased prostate. Such a 48 h, the projection of filapodia containing actin filaments cascade may induce activation of an individual PAR or and the development of punctate focal adhesions con- synergistic activation of multiple members of this recep- nected with stress fibres consisting of thick actin bundles tor family. It is also possible that other membrane bound (Greenberg et al., 2003). Consistently, PAR1 and PAR2 and secreted trypsin-like proteinases (and other protein- APs were able to initiate activation of the cytoskeleton ases) will interact similarly to mediate regulation of PAR reorganising GTPases RhoA, Rac1 and Cdc42 (Hall, signalling. 2005) in a manner similar to thrombin and trypsin in LNCaP cells (Greenberg et al., 2003). Loberg and col- leagues have also shown that thrombin treatment of Cancer-associated consequences of PAR PC-3 cells causes retraction of the actin cytoskeleton activation in prostate-derived cell lines and numerous microspike extensions from the cell body (Loberg et al., 2007). This treatment was accompanied Activation of PARs in prostate cancer cell lines initiates a by rapid activation of RhoA and Cdc42 with no change variety of cellular processes that are associated with in the level of Rac-1 activation (Loberg et al., 2007). cancer progression including proliferation, secretion of Thrombin has also been reported to promote adhesion tumour promoting factors, changes in morphology and of prostate cancer cells to extracellular matrix proteins. increased migration (Arora et al., 2007). As indicated For example, Liu and colleagues have shown that throm- below most of these studies have employed peptides bin increases DU145 adhesion to fibronectin and laminin mimicking PAR tethered ligand sequences to induce sig- via a mechanism involving PAR1 (Liu et al., 2004). In con- nalling via PARs. In addition, several studies, recognising trast to DU145 cells, treatment of PC-3 cells with a sim- the role of thrombin as the endogenous activator of PAR 1 ilar concentration of thrombin and PAR1 AP decreased and PAR4 in the vasculature (Vu et al., 1991; Xu et al., cell adhesion to collagen I and IV and laminin and result- 1998), the correlation between hyperactivation of the ed in no change in adhesion to fibronectin (Loberg et al., coagulation system and tumour progression (Rickles et 2007). In addition to cell migration and adhesion, exam- al., 1992) and the access of thrombin to cancer cells dur- ples of the co-opting of other normal cellular processes ing cancer progression (Trikha and Nakada, 2002), have essential for cancer progression, such as tissue remod- also employed this proteinase in studies of the conse- elling, cell growth, angiogenesis and mechanisms regu- quences of PAR activation in prostate-derived cells. lating apoptosis, have recently been shown to be A critical component of metastasis is the ability of cells mediated via PARs. For example, Wilson and colleagues to migrate/invade, undergo morphological changes and have demonstrated that activation of PAR2, but not PAR1, adhere (Hanahan and Weinberg, 2000). In this respect, in LNCaP cells induced increased activity of the tissue thrombin as well as trypsin stimulated LNCaP migration remodelling enzyme matrix metalloproteinase (MMP)-2. towards foetal calf serum in a dose-dependent manner. In addition, these workers demonstrated that activation The observed proteinase induced migration was mim- of both PAR1 and PAR2 induced increased MMP-2 activ-

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662 A.J. Ramsay et al.

ity in PC-3 and DU145 cells, while PAR2 activation protein C inhibitor, another serpin which complexes with stimulated the activity of another matrix remodelling pro- KLK1 (Ecke et al., 1992; Espana et al., 1995), KLK2 teinase, MMP9, in PC-3 cells (Wilson et al., 2004). The (Deperthes et al., 1995; Heeb and Espana, 1998; Lovgren importance of PAR1 for prostate tumour growth in vivo et al., 1999a; Mikolajczyk et al., 1999), KLK5 (Luo and has also recently been reported. Yin and co-workers Jiang, 2006), KLK8 (Luo and Jiang, 2006), KLK12 (Luo demonstrated that rat Dunning prostate carcinoma cells and Jiang, 2006; Memari et al., 2007b) and KLK14 (Luo expressing human PAR1 displayed a 3.7-fold increase in and Jiang, 2006), is down-regulated in prostate cancer tumour mass above controls when grown subcutaneous- (Cao et al., 2003). Further, levels of a-2 macroglobulin, ly in rats (Yin et al., 2003). In addition, low concentrations which forms complexes with KLK2 (Frenette et al., 1997; of thrombin have been reported to modulate proliferation Grauer et al., 1998; Heeb and Espana, 1998; Mikolajczyk of DU145, LNCaP and PNT1A cells (Liu et al., 2003). In et al., 1998), KLK4 (Obiezu et al., 2006), KLK5 (Michael another study, high concentrations of thrombin, acting et al., 2005) and KLK13 (Kapadia et al., 2004), also through PAR1, impaired the proliferation of DU145 cells decrease during prostate cancer progression (Kanoh et by a mechanism proposed to involve the induction of al., 2001). In addition, as noted above, increased expres- apoptosis (Zain et al., 2000). In contrast, pre-treatment sion of the TTSP matriptase/MT-SP1 in prostate cancer of DU145 and PC-3 cells with thrombin or PAR1 AP pro- is accompanied by decreased expression of the specific vided a protective effect against docetaxel-induced inhibitor of this enzyme (Saleem et al., 2006). Interesting- apoptosis by mediating increased expression of the anti- ly, in addition to reduced levels of expression of protein- apoptotic protein Bcl-xL (Tantivejkul et al., 2005). Also of ase inhibitors during prostate cancer progression there is interest, thrombin treatment of DU145 cells has been evidence that certain trypsin-like serine proteinases also shown to induce, via PAR1 activation, elevated secretion modulate inhibitor levels by inactivation. For example, of two cytokines that are implicated in tumour cell KLK2 has been reported to inactivate the serpin plas- growth, IL-8 and VEGF (Liu et al., 2006). This study is minogen activator inhibitor-1 by direct cleavage of the consistent with a previous report showing that thrombin inhibitor (Mikolajczyk et al., 1999). Trypsin IV has also stimulation mediated increased VEGF mRNA levels in been reported to deactivate proteinase inhibitors includ- PC-3 cells (Huang et al., 2001). ing kunitz and kazal type inhibitors and also is generally resistant to inhibition mediated by proteinaceous inhibi- Dysregulated tryptic proteolytic activity tors (Rinderknecht et al., 1984; Nyaruhucha et al., 1997; Szmola et al., 2003; Salameh et al., 2008). An essential step in aberrant signalling initiated by tryp- sin-like serine proteinases is dysregulation of proteolytic activity. This dysregulation requires, quite separate from Conclusion increased proteinase expression, the loss of mechanisms regulating normal activation and cessation of proteolytic Many serine proteinases with trypsin-like substrate spec- activity. In prostate cancer, as in other diseases, this dys- ificity are expressed in normal and diseased prostate and regulation is mediated by changes in levels of various several of these have been shown to regulate intracellular factors including metal ions (e.g., Zn2q) and protein- signalling via PARs. Studies on prostate-derived cell aceous inhibitors. For example, in prostate and other tis- lines, largely employing activating peptides, the blood sues, Zn2q ions provide an effective means of regulating coagulation enzyme, thrombin, and the food digestion the action of several KLK and other trypsin-like enzymes proteinase trypsin, have indicated cancer associated including KLK2 (Lovgren et al., 1999a), KLK4 (Debela et consequences of PAR activation. In addition, several al., 2006a), KLK5 (Michael et al., 2006; Debela et al., reports indicate that cascades involving KLKs and other 2007), KLK8 (Kishi et al., 2006), KLK12 (Memari et al., serine proteinases will be important for prostatic regula- 2007a) and KLK14 (Borgono et al., 2007c), as well as tion of PARs. For example, recently Yoon and colleagues, prostasin (Shipway et al., 2004) and uPA (Ishii et al., exploring the ability of 12 members of the KLK family to 2001). Of relevance to normal prostate physiology as well process the pro-peptide sequences of all 15 members of as prostate cancer, Zn2q levels are up to 10-fold higher this enzyme family, have demonstrated a number of sig- in normal prostate than in other tissues (Kavanagh, nificant cross-activation and autolytic relationships (Yoon 1985), while there is a 10- to 20-fold decrease in these et al., 2007). In addition, the ability of the membrane levels in patients with advanced prostate cancer (Zai- anchored serine proteinase enteropeptidase to activate chick et al., 1996, 1997). Based on in vitro studies this trypsinogen to trypsin (Yamashina, 1956), the well known reduction in Zn2q levels would be expected to be accom- PAR signalling proteinase (Bohm et al., 1996b), may be panied by a corresponding increase in the activity of tryp- a model for the interaction of other secreted and tic proteinases. membrane bound serine proteinases, localising proteo- Progression of prostate cancer is also often accom- lytic activity at the cell surface in close proximity to PARs. panied by loss of expression of proteinaceous inhibitors Furthermore, the observation that MMP1 can promote of trypsin-like enzymes. For example, the serpin anti- cell migration and invasion through PAR1 (Boire et al., thrombin III which complexes with KLK2 (Cao et al., 2005) indicates that proteinases from multiple enzyme 2002) KLK4 (Obiezu et al., 2006), KLK6 (Magklara et al., families can signal via PARs raising the possibility that 2003), KLK12 (Memari et al., 2007b) and KLK14 (Borgono interactions between these proteinase families will be et al., 2007c) is down-regulated during prostate cancer important in PAR-mediated signalling in physiology and progression (Frenette et al., 1997; Cao et al., 2002). Also, disease. Also, it is clear that loss of the normal inhibitory

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Tryptic serine proteinases and PARs in prostate 663 mechanisms regulating the action of activated trypsin- Borgono, C.A., Michael, I.P., Shaw, J.L., Luo, L.Y., Ghosh, M.C., like proteinases, such as reduced levels of Zn2q ions and Soosaipillai, A., Grass, L., Katsaros, D., and Diamandis, E.P. serpins, have the capacity to contribute significantly to (2007c). Expression and functional characterization of the cancer-related serine protease, human tissue kallikrein 14. J. progression of prostate cancer and other cancers. Finally, Biol. Chem. 282, 2405–2422. as tryptic proteinases and PARs are widely expressed it Bourgeois, L., Brillard-Bourdet, M., Deperthes, D., Juliano, M.A., is also likely that interactions between these proteins will Juliano, L., Tremblay, R.R., Dube, J.Y., and Gauthier, F.(1997). be important in many diseases in addition to prostate Serpin-derived peptide substrates for investigating the sub- cancer. strate specificity of human tissue kallikreins hK1 and hK2. J. Biol. Chem. 272, 29590–29595. Brattsand, M., Stefansson, K., Lundh, C., Haasum, Y., and Egelrud, T. (2005). A proteolytic cascade of kallikreins in the Acknowledgements stratum corneum. J. Invest. Dermatol. 124, 198–203. Cal, S., Quesada, V., Llamazares, M., Dı´az-Perales, A., Gara- This work was supported by the Queensland Cancer Fund baya, C., and Lo´ pez-Otı´n, C. (2005). Human polyserase-2, (Scholarship to A.J.R.) and the National Health and Medical a novel enzyme with three tandem serine protease domains Research Council of Australia (R.D. Wright Fellowship number in a single polypeptide chain. J. Biol. Chem. 280, 1953– 339732 to J.D.H. and grant numbers 101202 and 242220 to 1961. J.A.C.). Cal, S., Peinado, J., Llamazares, M., Quesada, V., Moncada- Pazos, A., Garabaya, C., and Lo´ pez-Otı´n, C. (2006). Identi- fication and characterization of human polyserase-3, a novel References protein with tandem serine-protease domains in the same polypeptide chain. BMC Biochem. 7,9. 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