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FEBS 29386 FEBS Letters 579 (2005) 1945–1950

Hepsin activates pro-hepatocyte growth factor and is inhibited by hepatocyte growth factor activator inhibitor-1B (HAI-1B) and HAI-2

Daniel Kirchhofera,*, Mark Peeka, Michael T. Liparia, Karen Billecib, Bin Fana, Paul Morana a Department of Physiology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA b Assay and Automation Technology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA

Received 1 December 2004; revised 27 January 2005; accepted 31 January 2005

Available online 26 February 2005

Edited by Veli-Pekka Lehto

are known and physiologically relevant inhibitors have yet to Abstract , a type II transmembrane , is highly upregulated in prostate cancer and promotes tumor pro- be identified. A role of hepsin in blood was sug- gression and metastasis. We generated a soluble form of hepsin gested by Kazama et al. [4] demonstrating that it can activate comprising the entire extracellular domain to show that it effi- coagulation factor VII. However, hepsin-deficient mice were ciently converts single-chain hepatocyte growth factor (pro- viable and showed no blood coagulation disorders [5], casting HGF) into biologically active two-chain HGF. Hepsin activity doubt on the importance of hepsin in normal hemostasis. It is was potently inhibited by soluble forms of the bi-Kunitz domain possible that hepsin may contribute to fibrin formation in inhibitors HAI-1B (IC50 21.1 ± 2.7 nM) and HAI-2 (IC50 pathologic situations, such as renal cell carcinoma [6], where 1.3 ± 0.3 nM). Enzymatic assays with HAI-1B Kunitz domain the primary initiator of blood coagulation, tissue factor, is ab- mutants (R260A and K401A) further demonstrated that inhibi- sent. Furthermore, other studies have suggested a functional tion was due to Kunitz domain-1. The results suggest a functional link between hepsin and cellular growth. Depending on the tu- link between hepsin and the HGF/Met pathway, which may con- tribute to tumor progression. mor cell line and experimental conditions used, hepsin has Ó 2005 Federation of European Biochemical Societies. Published been reported to have growth promoting [7] or growth sup- by Elsevier B.V. All rights reserved. pressing activity [8]. Hepsin expression is increased in ovarian cancer [9] and in Keywords: Hepsin; Prostate cancer; Hepatocyte growth factor; prostate cancer, where it was identified as one of the most HGFA inhibitor-1; HGFA inhibitor-2; Kunitz domain highly upregulated [10–15]. The expression of hepsin cor- related with the neoplastic transformation [12], being highest in tumors of patients with advanced disease and lowest in be- nign hyperplasia [11,15]. In contrast, one study found that low expression of hepsin protein correlated with high Gleason 1. Introduction scores and large tumors [13]. Recently, the functional signifi- cance of hepsin overexpression in prostate cancer was eluci- Hepsin (also known as TMPRSS1) is a cell surface expressed dated in an in vivo study by Klezovitch et al. [16], -like and a member of the family demonstrating that hepsin promotes prostate cancer progres- of type II transmembrane serine (TTSP), which also sion and metastasis. Therefore, hepsin may constitute an inter- include and [1]. Hepsin is a 417 esting cancer target and more insight into the substrate/ amino acid polypeptide [2] composed of a short N-terminal inhibitor system of hepsin may provide new therapeutic cytoplasmic tail, a transmembrane region and an extracellular approaches. domain (Arg45–Leu417) composed of the scavenger receptor At the epithelial cell surface, hepsin is ideally situated to cysteine-rich and protease domains. Hepsin is acti- interact with components of the extracellular matrix. Serine vated autocatalytically by cleavage at Arg162–Ile163 [3], form- proteases, including the TTSP matriptase [17,18] which is ing a heterodimeric . structurally related to hepsin, are known to activate fibrino- The physiological function of hepsin has been elusive. Ex- lytic , matrix metalloproteases and latent forms of cept for coagulation factor VII, no macromolecular substrates growth factors, such as hepatocyte growth factor (HGF). HGF is a pleiotropic factor and activates the receptor tyro- sine Met (reviewed in [19]). HGF is secreted into the extracellular matrix as an inactive single chain precursor *Corresponding author. Fax: +1 650 225 6327. E-mail address: dak@.com (D. Kirchhofer). (pro-HGF) and requires activation cleavage at Arg494– Val495 to form the biologically competent, disulfide-linked Abbreviations: HGF, hepatocyte growth factor; HGFA, HGF activa- a/b heterodimer [19]. This step is mediated by pro-HGF con- tor; HAI-1, HAI-1B, splice variants of HGFA inhibitor-1; HAI-2, vertases, including HGF activator (HGFA) [20] and matrip- HGFA inhibitor-2; KD1 and KD2, N- and C-terminal Kunitz domain of HAI-1B; u-PA, -type ; t-PA, tissue- tase [21,22]. In turn, the activities of HGFA and matriptase type plasminogen activator; TTSP, type II transmembrane serine are regulated by cell surface-expressed Kunitz-type inhibitors, protease such as the two HGFA inhibitor splice variants HAI-1 [23,24]

0014-5793/$30.00 Ó 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.01.085 1946 D. Kirchhofer et al. / FEBS Letters 579 (2005) 1945–1950

and HAI-1B [22] and by HAI-2 (also known as placental HGF generated by hepsin (HGFhepsin) and by HGFA (HGFHGFA) bikunin) [25,26]). indicated that >95% of pro-HGF was converted into two-chain HGF. In the present study, we demonstrate that hepsin cleaves 125 Pro-HGF activation assays and I-labeling of pro-HGF was car- pro-HGF with an activity comparable to the potent pro- ried out as described [22,28]. Briefly, 125I-labeled pro-HGF at HGF convertase HGFA. In addition, we identified HAI-1B 0.05 mg/ml in HEPES buffer was incubated with increasing concentra- and HAI-2 as potential physiological regulators of hepsin tions (0.16–40 nM) of hepsin or HGFA at 37 °C. After 4 h, aliquots activity. The biological implications of these findings, specifi- were removed and analyzed by SDS–PAGE (4–20% gradient gel) (Invitrogen) followed by exposure to X-ray films. For inhibition stud- cally in light of co-localization of hepsin and its inhibitors in ies, hepsin (15 nM) was incubated in HEPES buffer with 1 lMof prostate cancer, are discussed. sHAI-1B, sHAI-1B(K401A), sHAI-1B(R260A), or sHAI-2 at 37 °C. After 4 h the samples were analyzed by SDS–PAGE. Plasminogen at 0.12 mg/ml was incubated with 40 nM hepsin or 40 nM t-PA (positive control) in HEPES buffer at 37 °C. Reaction ali- 2. Materials and methods quots taken at different time points were analyzed by SDS–PAGE.

2.1. Materials 2.5. Enzyme inhibition assays The chromogenic substrate S2366 was obtained from DiaPharma Hepsin (0.4 nM) was incubated with increasing concentrations of Group, Inc. (West Chester, OH), Lys-plasminogen from Haematologic inhibitors in 30 mM Tris–HCl, pH 8.4, 30 mM imidazole, 200 mM Technologies Inc. (Essex Junction, VT) and tissue-type plasminogen NaCl for 30 min. Substrate S2366 (0.2 mM) was added and the change activator (t-PA) from Genentech, Inc. (South San Francisco, CA). in absorbance at 405 nm measured on a kinetic microplate reader Pro-HGF, expressed in Chinese hamster ovary (CHO) cells, was pro- (Molecular Devices, Sunnyvale, CA). The IC50 values were calculated vided by David Kahn (Genentech). HGFA was produced as described by fitting the data to a four parameter regression curve fitting program [22]. (Kaleidagraph).

2.2. Expression and purification of hepsin 2.6. Assays for cell proliferation, migration and Met phosphorylation The cDNA of full length hepsin, obtained from the I.M.A.G.E. Proliferation assays were carried out with the human pancreatic consortium (ATCC, Manassas, VA), was digested with restriction adenocarcinoma cell line BxPC3 (European Collection of Cell Cul- endonucleases EcoRI and NotI (New England Biolabs Inc. Beverly, tures, Salisbury, UK). Cells were added (10000–15000 cells/well) to MA) and inserted in the eukaryotic expression vector pRK5E. A se- 96-well white bottom MT plates (Packard/PerkinElmer, Boston, creted His-tagged hepsin cDNA was constructed by fusion of the MA) and grown in RPMI medium–10% FCS for 24 h. The medium cDNA coding for the signal sequence of human HGF (amino acids was replaced with RPMI–0.1% BSA and after 24 h, HGFhepsin and Met1–Gly31) with the cDNA coding for the extracellular domain of HGFHGFA in RPMI–0.1% BSA were added. After 72 h, cell prolifer- human hepsin (Arg45–Leu417). The final cDNA construct was ation was quantified by use of the CellTiter-Glo Luminescent Kit inserted in the eukaryotic expression vector pCMV.PD5. Hepsin (Promega, Madison, WI). Luminescence was measured on a Tropix was expressed in a CHO cell transient expression system TR717 luminometer (Berthold, Bad Wildbad, Germany). After sub- and purified by nickel–nitrilotriacetic acid affinity chromatography traction of background values (no HGF), the activities of HGFhepsin [22]. and HGFHGFA were expressed as percent of BxPC3 proliferation by 100 ng/ml of an HGF control preparation (obtained from Dr. Ralph Schwall, Genentech). 2.3. Expression and purification of sHAI-1B, sHAI-1B mutants and Cell migration assays with the breast cancer cell line MDA-MB435 sHAI-2 and kinase receptor activation assay with lung carcinoma A549 cells A soluble form of HAI-1B (sHAI-1B) comprising the extracellular were carried out as described [27]. domain was produced in a CHO cell transient expression system and purified as described [22]. Using site-directed mutagenesis, the P1 res- idues of the N-terminal Kunitz domain (KD1) (P1 residue Arg260) and the C-terminal Kunitz domain (KD2) (P1 residue Lys401) were individually changed to Ala and the resulting proteins, sHAI- 3. Results 1B(R260A) and sHAI-1B(K401A), expressed and purified as described [22]. Full length HAI-2 was obtained from a cDNA library derived from 3.1. Proteolytic processing of pro-HGF by hepsin human fetal lung RNA (BD Biosciences Clontech, Palto Alto, CA) A soluble form of hepsin encompassing the entire extracellu- using oligo(dT)/NotI site as a primer and adapator with SalI site for lar domain was expressed in CHO cells. During the purification the second strand. The cDNA was digested with SalI and NotI; process hepsin zymogen spontaneously converted into its cDNAs greater than 2.8 kb were ligated to pRK5D and single stranded two-chain form, most likely due to auto-activation [3]. N-termi- DNA was generated. Reverse primer (50-TTTCTTGAGG- CACTCCTCCTTG-30) was annealed to the single-stranded cDNA nal sequencing of the 30 kDa protease domain 163 173 pool and extended using T7 or T4 DNA polymerase. Escherichia coli ( IVGGRDTSLGR ) confirmed cleavage at the expected were transformed with the synthesized double-stranded DNA and col- Arg162–Ile163 bond rendering the active enzyme. Hep- onies were screened using standard filter hybridization methods. The sin converted factor VII zymogen into two-chain factor VIIa insert size was analyzed by PCR and restriction endonuclease diges- tion, XbaI. HAI-2 full length clones were identified and confirmed (data not shown), consistent with experiments reported by Kaz- by DNA sequencing. A soluble form of HAI-2 (sHAI-2) was produced ama et al. [4] using cell surface expressed hepsin, indicating that by constructing a cDNA coding for the extracellular domain (Ala28– soluble hepsin recapitulates the enzymatic properties of the cel- Lys197) of HAI-2 and addition of a C-terminal His8 tag with a Gly lular, full length form. Hepsin activity towards pro-HGF was spacer. The obtained cDNA was then inserted into a baculovirus examined by measuring conversion of 125I-labeled pro-HGF. expression vector derived from pVL1393 (BD Biosciences Pharmin- gen). sHAI-2 was expressed in a baculovirus expression system and The results showed that pro-HGF was cleaved by hepsin in a purified by affinity chromatography [27]. concentration-dependent fashion (Fig. 1A) and comparable to that of HGFA (Fig. 1B), achieving complete conversion at 4– 13 nM. N-terminal sequencing of the 36 and 39 kDa HGF 2.4. Pro-HGF activation and plasminogen activation assays 495 506 Pro-HGF (0.3 mg/ml) was incubated in HEPES buffer (20 mM b-chains gave identical sequences ( VVNGIPTRTNIG ), HEPES, pH 7.5, 150 mM NaCl) with 40 nM hepsin or with 40 nM showing that hepsin processed pro-HGF at the expected HGFA for 4 h at 37 °C. Analysis by SDS–PAGE of the two-chain Arg494–Val495 peptide bond. Moreover, hepsin lacked the D. Kirchhofer et al. / FEBS Letters 579 (2005) 1945–1950 1947

Fig. 1. Activation of pro-HGF by hepsin. 125I-labeled pro-HGF was incubated with hepsin or HGFA: 3-fold dilution steps of enzymes, from 40 nM in lane 2 down to 0.16 nM in lane 7. (A) Hepsin and (B) HGFA. Lane 1, aliquot at t = 0. (C) Activation of plasminogen by t-PA and hepsin. 1, Buffer at 0.5 h; 2, t-PA at 0.5 h; 3, hepsin at 0.5 h; 4, buffer at 5 h; 5, t-PA at 5 h; 6, hepsin at 5 h. The positions of plasminogen (Plg) and the heavy chain (h.c.) are indicated. ability to activate plasminogen during a 5 h reaction (Fig. 1C). In comparison, t-PA efficiently processed plasminogen having about 50% of zymogen already cleaved after 0.5 h (Fig. 1C).

3.2. Biological activity of HGF generated by hepsin Unlabeled pro-HGF was completely converted to HGF with hepsin or HGFA to give HGFhepsin and HGFHGFA, respectively (Fig. 2A, inset). Both HGF preparations in- duced similar concentration-dependent increases in Met phosphorylation, reaching maximal activity at 250 ng/ml (Fig. 2A). As shown previously [27], an uncleavable single chain form of HGF had no activity (Fig. 2A). Furthermore, HGFhepsin efficiently promoted proliferation of BxPC3 pan- creatic cancer cells. The activity was comparable to that of HGFHGFA in the examined range of 5–100 ng/ml (Fig. 2B). Similar results were obtained in a cell migration assay with MDA-MB435 cells using a transwell migration system. The pro-migratory effects of HGFhepsin were concentration- dependent and indistinguishable from that of HGFHGFA (Fig. 2C).

3.3. Inhibition of hepsin enzymatic activity by sHAI-1B and sHAI-2 In an amidolytic assay with the synthetic substrate S2366, the Kunitz inhibitors sHAI-1B and sHAI-2 potently inhibited Fig. 2. Biological activity of pro-HGF cleaved by hepsin. (A, inset) hepsin activity with IC50 values of 21.1 ± 2.7 and 1.3 ± 0.3 nM, Unlabeled pro-HGF was cleaved with hepsin or HGFA and the respectively (Fig. 3A). Moreover, mutant sHAI-1B(K401A) products (HGFhepsin and HGFHGFA) analyzed by SDS–PAGE. 1, no enzyme; 2, HGF ; 3, HGF . (A) Phosphorylation of Met containing a non-functional KD2 was equally potent as wild- hepsin HGFA receptor with increasing concentrations of HGFhepsin (circles), type sHAI-1B (IC50 18.2 ± 3.7 nM), whereas sHAI- HGFHGFA (squares), or to single-chain HGF (diamonds), an uncleav- 1B(R260A) had <5% of wild-type activity (IC50 > 500 nM) able single-chain form of HGF. (B) BxPC3 proliferation with (Fig. 3A). increasing concentrations of HGFhepsin (filled bars) and HGFHGFA (open bars). Average of two independent experiments. (C) MDA- In agreement, sHAI-2, wild-type sHAI-1B and sHAI- MB435 migration with increasing concentrations of HGF (filled 1B(K401A) at 1 lM completely inhibited pro-HGF cleavage hepsin bars) and HGFHGFA (open bars). Except for 5 ng/ml HGF (n = 2), the (Fig. 3B). In contrast, sHAI-1B(R260A) showed no inhibition results are the average ± S.D. of three experiments. 1948 D. Kirchhofer et al. / FEBS Letters 579 (2005) 1945–1950

that soluble hepsin recapitulates substrate and inhibitor speci- ficity of the surface-expressed form. This notion is supported by our finding that soluble hepsin activated zymogen factor VII similar to cell-surface expressed hepsin [4] and by studies using different recombinant or purified forms of the related TTSP matriptase [21,22,24,31,32]. A recent in vivo study demonstrated that overexpression of hepsin in a mouse model of non-metastasizing prostate cancer results in cancer progression and metastasis [16]. The observed disruption of basement membrane architecture and the disor- ganization of epithelial structure could, at least in part, be due to generation of active HGF by hepsin. HGF is known to activate proteolytic pathways [e.g., urokinase-type plasmin- ogen activator (u-PA), u-PA receptor, and matrilysin] leading to basement membrane degradation and cell–cell dissociation [33–35]. For instance, it was shown that HGF-mediated matri- lysin activation resulted in E-cadherin cleavage and disruption of cell–cell adhesion leading to increased invasive potential of LNCap prostate cancer cells [35]. It remains to be seen whether hepsin itself is capable of degrading basement membrane com- ponents, such as laminin 5 or collagen IV. HAI-1B, HAI-1 and HAI-2 are epithelial cell surface inhib- itors and, as such, are ideally located to regulate the enzymatic activity of epithelial cell-expressed TTSPs and other cell sur- face associated serine proteases. Here, we demonstrate that both sHAI-1B and sHAI-2 are potent inhibitors of hepsin. Fig. 3. Inhibition of hepsin enzymatic activity by sHAI-1B and Moreover, P1 residue-directed mutagenesis experiments dem- sHAI-2. (A) Amidolytic assay. Hepsin was incubated with inhibitors onstrated that inhibition is mediated by KD1 of sHAI-1B. and enzymatic activity towards S2366 was measured. sHAI-1B (squares), sHAI-1B(R260A) (inverted triangles), sHAI-1B(K401A) Thus, hepsin, matriptase and HGFA not only have compara- (filled circles), sHAI-2 (open circles). (B) Pro-HGF activation assay. ble pro-HGF converting activities but are also inhibited by 125I-labeled pro-HGF was incubated with hepsin and 1 lMof sHAI-1B with equal potencies (16–30 nM) in a KD1-specific inhibitors for 4 h at 37 °C. 1, aliquot at t = 0; 2, no inhibitor; 3, fashion [22]. The role of HAI-2 Kunitz domains was not spe- sHAI-1B; 4, sHAI-1B(R260A); 5, sHAI-1B(K401A); 6, sHAI-2. cifically addressed in our study. For most of its target enzymes, HAI-2 utilizes both the N-and C-terminal Kunitz domains [26]. Whether or not this also applies to the inhibition of hep- and pro-HGF activation proceeded to complete conversion sin remains to be seen. (Fig. 3B). Both HAI-1 splice variants and HAI-2 are expressed in nor- mal prostate [22,36] and HAI-1 antigen is localized to the epi- thelial cell type in normal prostate [36]. In prostate cancer, 4. Discussion hepsin expression is strongly upregulated (>7-fold) [10–15], whereas expression of HAI-1 and HAI-2 increases only slightly This study demonstrates that hepsin, a serine protease highly (about 1.5-fold) according to gene expression results reported upregulated in prostate and ovarian cancer, is a potent activa- by Welsh et al. [10]. As a consequence, hepsin enzymatic activ- tor of pro-HGF. Therefore, hepsin may contribute to tumor ity in prostate cancer may be inadequately controlled and progression by activating the HGF/Met signalling pathway, could lead to enhanced pro-HGF processing and tumor pro- which has been implicated in both prostate [29] and ovarian gression. Similar imbalances of pro-HGF convertase/inhibitor cancer [30]. systems have been described for matriptase/HAI-1 in ovarian Hepsin proteolytically cleaved pro-HGF at the Arg494– cancer [37] and HGFA/HAI-1 and HGFA/HAI-2 in renal cell Val495 peptide bond without any additional cleavage at carcinoma [38]. The upregulated expression of hepsin in some Arg424–His425 in Kringle domain 4, a site recognized by fac- of these cancers suggests that certain convertase/inhibitor tor XIa and plasma [28]. The two-chain HGF gener- systems comprise multiple enzymes and therefore increased ated by hepsin was fully functional with biological activities enzyme/inhibitor ratios could magnify the consequences for comparable to HGF generated by HGFA. The proteolytic malignancy. activity of hepsin towards pro-HGF appears specific, since it In conclusion, the results suggest a functional link between did not cleave plasminogen, a close structural homolog of hepsin and the HGF/Met pathway, which may contribute to pro-HGF. In support, hepsin has no proteolytic activity to- tumor progression. The finding that HAI-1B and HAI-2 are wards other serine protease substrates, such as prothrombin, potent inhibitors of hepsin may provide new treatment ap- , and factor IX [4]. The soluble hepsin used proaches. For example, the functional Kunitz domains of in our experiments consisted of the entire extracellular non- HAI-1B or HAI-2 could serve as scaffolds for generating more catalytic and the protease domains and, thus, contained the specific and/or more potent enzyme inhibitors by use of phage essential structural elements which determine substrate speci- display technology, which has been successfully applied to ficity ( region and exosites). Therefore, we presume other Kunitz domain scaffolds. D. Kirchhofer et al. / FEBS Letters 579 (2005) 1945–1950 1949

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