Proteolytic Activation of Pro-Macrophage-Stimulating Protein by Hepsin

Published OnlineFirst August 29, 2011; DOI: 10.1158/1541-7786.MCR-11-0004

Molecular Cancer Angiogenesis, Metastasis, and the Cellular Microenvironment Research

Proteolytic Activation of Pro-Macrophage-Stimulating Protein by Hepsin

Rajkumar Ganesan1, Ganesh A. Kolumam2, S. Jack Lin1, Ming-Hong Xie3, Lydia Santell1, Thomas D. Wu4, Robert A. Lazarus1, Amitabha Chaudhuri3, and Daniel Kirchhofer1

Abstract Macrophage-stimulating protein (MSP) is a plasminogen-related growth factor and ligand for the receptor tyrosine kinase RON. The MSP/RON system promotes wound healing and invasive tumor growth and suppresses proinflammatory immune response. MSP binding to RON requires proteolytic conversion of the inactive single- chain form (pro-MSP) into the disulfide-linked a/b heterodimer. The pro-MSP cleavage sequence (Ser-Lys-Leu- Arg483#Val484) closely matches the substrate recognition sequences of hepsin, a type II transmembrane serine protease, that is overexpressed in several cancers. Here, we show that recombinant hepsin cleaves pro-MSP at the consensus site Arg483-Val484 with superior efficiency compared with the known activators MT-SP1 and hepatocyte growth factor activator (HGFA). At least 50% of pro-MSP was processed within 1 hour at a hepsin concentration of 2.4 nmol/L and at a molar enzyme to substrate ratio of 1:500. An uncleavable single-chain variant – of MSP weakly bound to a RON Fc fusion protein, whereas hepsin-cleaved MSP bound with a KD of 10.3 nmol/ L, suggesting that the high-affinity binding site in MSP b-chain was properly formed. LNCaP prostate cancer cells overexpressing hepsin on the cell surface efficiently activated pro-MSP, which was blocked by a specific anti-hepsin antibody. Incubation of pro-MSP with hepsin led to robust RON-mediated phosphorylation of mitogen-activated protein kinase, ribosomal S6 protein, and Akt in human A2780 ovarian carcinoma cells stably expressing RON protein. In macrophages, pro-MSP with hepsin induced chemotaxis and attenuated lipopolysaccharide-dependent production of nitric oxide. These findings suggest that the MSP/RON signaling pathway may be regulated by hepsin in tissue homeostasis and in disease pathologies, such as in cancer and immune disorders. Mol Cancer Res; 9(9); 1175–86. 2011 AACR.

Introduction (2). The a-chain of MSP consists of an N-terminal PAN module followed by 4 kringle domains and is disulfide linked Macrophage-stimulating protein (MSP, also known as to the trypsin-like b-chain (3). MSP is constitutively expressed hepatocyte growth factor–like protein, HGFL) is a plasmi- by hepatic parenchymal cells, as well as in lungs, adrenal nogen-like growth factor that mediates its biological activ- glands, placenta, kidney, and pancreas (4, 5). It is secreted as ities by activating the receptor tyrosine kinase RON an inactive single-chain precursor pro-MSP, which requires [recepteur d'origine nantais, also known as macrophage proteolytic cleavage at the Ser-Lys-Leu-Arg483#Val484 bond stimulating receptor-1, (MSTR1)], a member of the to attain functional activity (5, 6). In HGF, cleavage at the MET proto-oncogene family (1). MSP shares high sequence corresponding Arg494#Val495 bond results in distinct struc- and structural domain homology with HGF, the ligand for tural rearrangements within the HGF b-chain and the for- MET; similarly, their respective cognate receptors, RON mation of a MET-binding site that is competent for signal and MET, also share high sequence and domain homology transduction (7–9). Comparable conformational rearrangements centered at the "pseudo-active site" in the MSP b-chain are likely to occur on activation cleavage of pro-MSP (7).

Authors' Affiliations: Departments of 1Early Discovery Biochemistry, However, unlike HGF, where the high-affinity MET-binding 2Tumor Biology and Angiogenesis, 3Molecular Oncology, and 4Bioinfor- site resides in the a-chain, the high-affinity RON-binding site matics/Computational Biology, Genentech, Inc., South San Francisco, for MSP is located on the mature b-chain that forms following California pro-MSP cleavage (10). Several trypsin-like serine proteases, Note: Supplementary data for this article are available at Molecular Cancer including MT-SP1 (also known as matriptase) and HGF Research Online (http://mcr.aacrjournals.org/). activator (HGFA; refs. 11–15), are known to activate pro- Corresponding Author: Daniel Kirchhofer, Department of Early Discovery Biochemistry, Genentech, Inc., 1 DNA Way, MS #27, South San Francisco, MSP, leading to typical cellular responses mediated by the CA 94080. Phone: 650-225-2134; Fax: 650-225-3734; RON signaling pathway (11, 12). E-mail: dak@gene.com Hepsin is a cell surface–expressed trypsin-like protease doi: 10.1158/1541-7786.MCR-11-0004 and a member of the type II transmembrane serine protease 2011 American Association for Cancer Research. family (16, 17). It consists of an N-terminal cytoplasmic

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domain, a transmembrane domain, and an extracellular HGFA (Val373-Ser655), the MT-SP1 protease domain portion composed of a scavenger receptor–like cysteine-rich (sMT-SP1) as well as the Kunitz domain-1 (KD1) derived domain and a C-terminal protease domain with a trypsin- from HGFA inhibitor type-1 (HAI-1) were expressed and like fold (18, 19). Hepsin was identified as one of the most purified as described (40, 41). Recombinant active human highly upregulated genes in prostate cancer (20–25) and MSP was obtained from R&D Systems and used as immunohistochemical staining revealed strong expression reference material. Antibody25 (Ab25) inhibits hepsin in late-stage tumors and metastatic bone lesions (26, 27). enzymatic activity and was generated by using antibody Studies with preclinical prostate cancer models suggested phage display. Ab25 (IgG1) and Fab25 were expressed in that hepsin may play a role in invasive cancer growth and CHO cells and Escherichia coli, respectively, and were cancer progression (28, 29). Moreover, gene expression purified according to standard procedures. analyses have also implicated hepsin in ovarian cancer (30), renal cell carcinoma (31, 32), and endometrial cancer In vitro activation of pro-MSP by sHepsin, HGFA, and (33). A putative function of hepsin in tumor progression sMT-SP1 could be related to its enzymatic activity toward the macro- Pro-MSP (100 mg/mL ¼ 1.25 mmol/L) was incubated for molecular substrates pro-HGF (19, 34), pro-uPA (35), and 1hourat37C with different concentrations (100 nmol/L– laminin-332 (36). Additional substrates that have been 97 pmol/L, 2-fold dilution series) of sHepsin, HGFA, and identified are coagulation factors (37) and pro-prostasin sMT-SP1 in a buffer containing 50 mmol/L Tris-HCl, pH (38). The hepsin cleavage sequences of these substrates are ¼ 8.0, 150 mmol/L NaCl, 0.05% Triton X-100, and 2 in good agreement with the consensus sequence obtained mmol/L CaCl2 for 1 hour at 37 C. In addition, 100 nmol/L from substrate profiling by positional scanning of a syn- of the proteases were preincubated for 15 minutes with 1 thetic combinatorial peptide library (19). mmol/L of the hepsin-specific Fab25 or 1 mmol/L of KD1 Here, we report that both recombinant soluble hepsin (an inhibitor of hepsin, MT-SP1, and HGFA) before (sHepsin) and cell surface–expressed hepsin efficiently addition of pro-MSP. For time course experiments, 12.5 cleave human pro-MSP at the physiologic activation site. nmol/L sHepsin was incubated with 1.25 mmol/L of pro- The hepsin-cleaved MSP was functionally competent for MSP and aliquots were analyzed by SDS-PAGE and pro- RON binding and in triggering cellular responses mediated teins stained with SimplyBlue Safe Stain (Invitrogen). The by the RON signaling pathway. Finally, we detected sig- experiments were repeated 3 times. Similar experiments nificant coexpression of MSP and hepsin in a number of were carried out with 250 mg/mL of the scMSP mutant and organs, suggesting a potential physiologic role of hepsin in hepsin (10 nmol/L). For the densitometric analysis of pro- regulating activity of MSP. MSP degradation, the pro-MSP band intensities were quantified using ImageJ from the NIH (http://rsb.info. Materials and Methods nih.gov/ij/index.html). The effective concentration to give 50% reduction (EC50) was determined by a 4-parameter fit Cloning, expression, and purification of recombinant (Kaleidagraph; Synergy Software). proteins sHepsin-cleaved MSP was prepared by incubating 100 The extracellular domain of the human recombinant mg/mL of pro-MSP with 15 nmol/L sHepsin for 1 hour, hepsin harboring a C-terminal His-tag (sHepsin) was resulting in complete conversion to cleaved MSP. sHepsin expressed and purified as described (35). Recombinant was removed by adding molar excess of anti-hepsin Ab25 to RON (SEMA/IPT/TIG1 domains: Glu25-Met682) was the reaction mixture followed by Protein A-Sepharose made as Fc fusion protein by expressing it in Chinese chromatography. Thus purified MSP did not contain any hamster ovary (CHO) cells. RON–Fc was purified by residual hepsin as assessed by SDS-PAGE and enzymatic affinity chromatography followed by size exclusion assays. A similar protocol was used to generate HGFA- chromatography. Recombinant pro-MSP harboring a cleaved MSP except that the anti-HGFA antibody Ab40 C-terminal His6 tag along with a C672A mutation, pre- (42) was used to remove HGFA from the reaction mixture. viously shown to yield better protein expression, was expressed in CHO cells as described (39). Secreted pro- Pro-MSP activation by cell surface–expressed hepsin on MSP was purified by Ni-NTA affinity chromatography LNCaP-34 cells followed by size exclusion chromatography on Superdex- LNCaP-34 cells stably overexpressing human full-length S200 column. Most of the MSP present in the pro-MSP hepsin were described previously (35). Confluent cultures preparation was removed by incubation with RON–Fc in 24-well plates were incubated for 15 minutes at 37C followed by a protein-A column purification and pro-MSP with 500 mL serum-free RPMI-1640 medium containing 1 was collected in the flow through (Supplementary mmol/L each of Ab25 or KD1 or Ac-KQLR chloromethyl Fig. S1A and Fig. 1A). To generate a nonactivatable form ketone (Ac-KQLR-cmk; Anaspec) or 10 nmol/L sHepsin. of pro-MSP, the P1 residue Arg483 was mutated to Glu 125I-labeled pro-MSP, prepared as described for pro-HGF (scMSP) using QuikChange mutagenesis (Stratagene). (34), was added (25 mg/mL) and incubated for 3 hours at Thus, scMSP actually contains 2 mutations: R483E 37C. Aliquots were analyzed by SDS-PAGE (4%–20% and C672A. The protein was expressed and purified as gradient gel) followed by exposure to X-ray films. The described for wild-type pro-MSP. The serum form of intensities of the bands were quantified using ImageJ

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Activation of Pro-MSP by Hepsin

software. The loss of pro-MSP band intensity was used to washing with PBS, 0.05% polysorbate 20, MSP proteins determine the percentage of proteolytic activity. were added and incubated for 1 hour. Bound MSP was detected using anti-His-HRP (Qiagen) and TMB/H2O2 Binding of hepsin-activated pro-MSP to RON by substrate (KPL). The reaction was stopped with 1 mol/L surface plasmon resonance and ELISA H3PO4 and the absorbance at 450 nm was measured. For surface plasmon resonance measurements on a BIA- core-3000 instrument (GE Healthcare) rabbit anti-human Peritoneal macrophage chemotaxis and morphology IgG was immobilized (amine coupling) on CM5 biosensor change assay chips and the RON–Fc fusion protein was captured to give Murine peritoneal resident macrophages were obtained approximately 250 response units (RU). Different concen- from C57BL/6 mice by washing the peritoneal cavity with tration of either sHepsin-activated MSP or reference MSP 15 mL of serum-free RPMI-1640 medium. Cells were (R&D Systems) or scMSP were injected in HBS-P buffer washed and resuspended in medium to a concentration (10 mmol/L HEPES, pH ¼ 7.5, 150 mmol/L NaCl, of 1 106 cells/mL. The macrophage chemotaxis assay was 0.005% P20) at 25C with a flow rate of 30 mL/min. conducted using a QCM chemotaxis assay kit with a pore m Association rates (ka) and dissociation rates (kd) were size of 5 m (Millipore). To the bottom wells was added obtained by using a simple one-to-one Langmuir binding RPMI-1640 medium containing the following compo- model (BIA-Evaluation software) and the equilibrium dis- nents: (a) 80 ng/mL pro-MSP, (b) 80 ng/mL scMSP, sociation constants (KD) were calculated (kd/ka). For ELISA (c) 80 ng/mL MSP from R&D Systems, (d) 80 ng/mL experiments, maxiSorp microtiter plates (Nunc) were pro-MSP and 10 nmol/L sHepsin, and (e) 80 ng/mL pro- coated overnight at 4C with 2 mg/mL of rabbit anti-human MSP, 10 nmol/L sHepsin, and 100 nmol/L Ab25. The IgG Fc-specific antibody (Jackson ImmunoResearch reaction mixtures (a–e) were preincubated at 37C for Laboratory) in 50 mmol/L sodium carbonate buffer, 1 hour before adding to the bottom wells. Macrophage pH ¼ 9.6. After blocking with assay buffer (PBS, pH ¼ suspension (105 cells/100 mL) was added to the upper wells. 7.4, 0.5% BSA, and 0.05% Tween-20, 15 ppm Proclin), After incubation at 37C for 4 hours, the migrated cells 1 mg/mL RON-Fc was added incubated for 1 hour. After were collected using detachment buffer (Millipore) and

AB sHepsin sMT-SP1 HGFA (100 nmol/L – 97 pmol/L) (100 nmol/L – 97 pmol/L) (100 nmol/L – 97 pmol/L) Pro-MSP control Hepsin + KD1 Hepsin + Fab25 Pro-MSP control + KD1 MT-SP1 + Fab25 MT-SP1 Pro-MSP control + KD1 HGFA + Fab25 HGFA

98 kDa Pro-MSP 64 kDa α 50 kDa -Chain 36 kDa β-Chain Fab25 KD1 Pro-MSP Pro-MSP + sHepsin Pro-MSP ( t = 0 h) Pro-MSP ( t = 3 h) Pro-MSP AkKQLR-cmk Pro-MSP + KD1 Pro-MSP + Ab25 EC50 = 2.4 ± 0.3 nmol/L EC50 = 11.7 ± 1.3 nmol/L EC50 = 17.7 ± 1.9 nmol/L Pro-MSP 120 120 120 α-Chain 100 100 100 80 80 80 β-Chain 60 60 60 40 40 40 20 20 20 0 0 0 0.01 0.1 1 10 100 0.01 0.1 1 10 100 0.01 0.1 1 10 100

Residual pro-MSP intensity (%) [sHepsin] (nmol/L) Residual pro-MSP intensity (%) [sMT-SP1] (nmol/L) Residual pro-MSP intensity (%) [HGFA] (nmol/L)

Figure 1. A, activation of pro-MSP by sHepsin, sMT-SP1, and HGFA. Purified pro-MSP (100 mg/mL ¼ 1.25 mmol/L) was incubated for 1 hour at 37C with different concentrations (100 nmol/L–97 pmol/L) of recombinant soluble hepsin (sHepsin) or the known activators sMT-SP1 and HGFA. For control experiments, the proteases (100 nmol/L) were incubated with the KD1 (inhibitor of Hepsin, MT-SP1, and HGFA) or the hepsin-specific antibody Fab25. Products were separated by SDS-PAGE under reducing conditions. N-terminal sequencing identified the 60-kDa band as MSP a-chain and the 30-kDa band as MSP b-chain. Intensities of the pro-MSP band were quantified by densitometry and plotted against enzyme concentrations. The EC50 values are the average SD of 3 independent experiments. B, activation of pro-MSP by cell surface–expressed hepsin. LNCaP-34 prostate cancer cells, which stably overexpress full-length hepsin, were incubated with 125I-pro-MSP alone or in combination with different inhibitors for 3 hours. In a control experiment, no significant proteolytic cleavage of pro-MSP was observed on incubation for 3 hours (lane 1), whereas addition of sHepsin (10 nmol/L) led to nearly complete (>92%) 125I-pro-MSP conversion (lane 2). Efficient pro-MSP processing (>80%) by LNCaP-34 cells was observed (lane 4) compared with the start of the experiment (lane 1). All 3 hepsin inhibitors (Ac-KQLR-cmk, KD1, and Ab25) effectively blocked pro-MSP processing (lanes 5–7).

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quantified by incubation for 15 minutes with lysis buffer phospho-S6 (Cell Signaling Technology), or anti-phos- and CYPRO dye followed by fluorescence measurements pho-Akt (Cell Signaling Technology) antibodies. RON (RFU, relative fluorescence units) on a microplate reader phosphorylation was analyzed by immunoprecipitating (Spectramax-M5, Molecular Devices) with excitation at RON from cell lysates using anti-RON antibody, 1A2.2 480 nm and emission at 520 nm. (Genentech, Inc.), coupled to agarose beads. Immunopre- To examine the morphologic changes, peritoneal macro- cipitates were washed 3 times in cold lysis buffer, resus- phages (1 106 cells/mL) were cultured in serum-free pended in 4 SDS-PAGE sample buffer (Invitrogen), and RPMI-1640 medium overnight. Nonadherent cells were analyzed after immunoblotting with anti-phospho-tyrosine removed and 80 ng/mL each of pro-MSP, sHepsin-cleaved pTyr-100 antibody (Cell Signaling Technology). The sec- MSP, HGFA-cleaved MSP, MSP from R&D Systems, or ondary antibodies used were IRDye800-conjugated goat scMSP were added. After 1 hour, morphologic changes were anti-mouse IgG (Rockland) and AlexaFluor 680 goat anti- captured by phase contrast microscopy. rabbit IgG (Invitrogen). Protein bands were visualized and quantified on the Odyssey Infrared Imaging System Inhibition of nitric oxide synthesis by mature MSP (LI-COR Biosciences) and the experiments were repeated Bone marrow cells were isolated from femurs of C57BL/6 3 times. mice as described (11). After washing the cell suspension with Dulbecco's Modified Eagle's Media (DMEM), red Computational gene expression analysis blood cells were lysed with erythrocyte lysis buffer. Cells Microarray data on the Affymetrix HG-U133A and HG- were resuspended in macrophage differentiation medium U133B GeneChips were obtained from the GeneLogic (DMEM with glutamine, 10% FBS, 1 Pen/Strep, and 50 database. We extracted samples that were classified as ng/mL mCSF-1) and added to 24-well plates. Medium was normal or as having noncancer disease and used the signal changed the next day and subsequently every second day. intensity values from the Microarray Analysis Suite Version After 6 days, the matured macrophages were incubated for 5 Software. We used the following microarray probes to 24 hours at 37C in 300 mL/well of serum-free medium measure gene expression: MSP, 205614_x_at; Hepsin, with or without 1 mg/mL of lipopolysaccharide (LPS; 204934_s_at; HGFA, 207027_at; MT-SP1, 202005_at; Sigma) and containing the following: (a) 10 ng/mL pro- and RON, 205455_at. For each tissue type, we generated MSP, (b) 10 ng/mL pro-MSP and 1 nmol/L sHepsin, (c) 10 expression profiles to show the mean SD of gene ng/mL pro-MSP, 1 nmol/L sHepsin, and 500 nmol/L expression values. For the 4 tissue types having the highest Ab25, and (d) 10 ng/mL MSP (R&D Systems). Nitric mean expression of MSP, liver, kidney, pancreas, and small oxide (NO) production was quantified by measuring the intestine, we generated scatter plots to show the relations of concentration of nitrite in diluted aliquots of culture med- expression of MSP against that of other genes. All plots were ium by use of the Griess Reaction Kit (Molecular Probes). generated using the R statistical package.

Phosphorylation of signaling proteins downstream of Results RON A2780 human ovarian carcinoma cells engineered to In vitro activation of pro-MSP by recombinant hepsin express human RON (A2780-RON; unpublished data) Substrate profiling of hepsin by the use of a synthetic were seeded at a density of 2 105 cells per well into a combinatorial library determined (P/K)-(K/Q)-(T/L/N)-R 12-well tissue culture plate. For siRNA experiments, RON as the P4-P1 (nomenclature according to Schechter and expression was reduced in A2780-RON cells by transfecting Berger; ref. 43) consensus sequence (19), which is in good 20 pmol of either RON-specific siRNA 50-GGGCGACA- agreement with the identified cleavage site sequences from GAAAUGAGAGUtt-30 (Ambion/Applied Biosystems) or macromolecular substrates of hepsin (refs. 34–37; Table 1). nontargeting control siRNA (Dharmacon/Thermo Scien- The consensus sequence and particularly the specific hepsin tific) using RNAi-Max (Invitrogen). Forty-eight hours fol- recognition sequences of laminin-332 (SQLR#L) and pro- lowing transfection, cells were serum starved for an HGF (KQLR#V) bear a close resemblance to the cleavage additional 24 hours and incubated with indicated concen- sequence of pro-MSP (SKLR#V). Therefore, we hypothe- trations of pro-MSP, scMSP, MSP, and pro-MSP with sized that hepsin could be a pro-MSP activator. To examine sHepsin (10 nmol/L). After 1-hour incubation, cells were this hypothesis, we expressed pro-MSP in CHO cells but washed once with cold PBS followed by incubation on ice in found it partially converted into the 2-chain form (MSP). lysis buffer (10 mmol/L Tris, pH ¼ 7.5, 150 mmol/L NaCl, Because MSP, but not pro-MSP, binds to its receptor 10% glycerol, 0.1% Triton X-100, 5 mmol/L b-glycero- RON, we further purified pro-MSP by incubating it with phosphate, 2 mmol/L NaF, 1 mmol/L sodium orthovana- RON–Fc for 16 hours at 4C followed by an affinity date, and protease and phosphatase inhibitors; Sigma Life chromatographic purification step. The resulting pro- Sciences). Lysates were clarified by centrifugation at 14,000 MSP was of high purity and contained only small amounts g, subjected to SDS-PAGE under reducing conditions, of activated MSP (Supplementary Fig. S1A). The soluble transferred onto nitrocellulose membranes, and incubated form of hepsin (sHepsin) comprising the extracellular por- overnight with anti-RON C20 (Santa Cruz Biotechnology), tion cleaved pro-MSP in a concentration- and time-depen- anti-phospho-MAPK (Cell Signaling Technology), anti- dent manner at 37C. sHepsin (12.5 nmol/L) converted

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Table 1. Cleavage sequence of hepsin was treated with 10 nmol/L sHepsin in the LNCaP-34 substrates cell cultures (Fig. 1B), which resulted in more than 92% conversion of pro-MSP compared with the pro-MSP only Hepsin substrates Cleavage sequence control (lane 1). Cleavage of pro-MSP was inhibited by 3 different inhibitors—KD1, which inhibits hepsin, Pro-MSP SKLR#V MT-SP1, and HGFA (40); KQLR-cmk, an irreversible Pro-HGF KQLR#V peptide inhibitor mimicking the pro-HGF cleavage Laminin-332 SQLR#L sequence KQLR#V (19, 44); and Ab25 (Fig. 1B). Pro-uPA PRFK#I Although KD1 and KQLR-cmk are not selective inhibi- FVII PQGR#I tors, the inhibition observed for them is similar as that for Proprostasin PQAR#I the selective hepsin inhibitorAb25.Thus,anyactivation PS-SCL (consensus) (P/K)-(K/Q)-(T/L/N)-R by other proteases, in particular MT-SP1 that is present on LNCaP-34 cells (35), is minimal. NOTE: # indicates cleavage site. Abbreviations: SCL, synthetic combinatorial library; PS, Binding of sHepsin-cleaved MSP to RON profiling of substrate. Cleavage of pro-MSP at the Arg483-Val484 bond leads to the formation of a high-affinity binding site on the MSP b-chain that is absent in pro-MSP. Therefore, proper more than 50% of pro-MSP (1.25 mmol/L) within 20 processing of pro-MSP, leading to the generation of the minutes and complete conversion into the a/b heterodimer high-affinity binding site for RON can be monitored was achieved within 1 hour at a molar enzyme to substrate by measuring MSP binding to RON. In ELISA assays, ratio of 1:100 (Supplementary Fig. S1B). N-terminal sequencing identified the approximately 60-kDa band as a 19 MSP -chain ( QRSPLN) and the approximately 30-kDa A band as MSP b-chain (484VVGGHPG), indicating that 1.2 sHepsin processed pro-MSP at the consensus cleavage site 1 Arg483-Val484. We compared the enzymatic activity of sHepsin with the 0.8 2 known pro-MSP–converting proteases sMT-SP1 and 0.6 HGFA. sHepsin, sMT-SP1, and HGFA cleaved pro- 450 nm

MSP in a concentration-dependent fashion, and their OD 0.4 activities were completely inhibited by KD1, the N-terminal Kunitz domain of their physiologic inhibitor HAI-1 0.2 (Fig. 1A). The anti-hepsin antibody Fab25 specifically 0 inhibited pro-MSP processing by sHepsin but not by 4 sMT-SP1 or HGFA (Fig. 1A). The relative pro-MSP– 0.0001 0.01 110010 [Protein] (nmol/L) converting potencies of the 3 proteases were quantified B by measuring the disappearance of the pro-MSP band by 120 densitometry. The results showed that sHepsin (EC ¼ 2.4 50 100 0.3 nmol/L) was 5-fold and 7-fold more efficient than ¼ sMT-SP1 (EC50 11.7 1.3 nmol/L) and HGFA (EC50 80 ¼ 17.7 1.9 nmol/L), respectively. Prolonged incubation of pro-MSP with sHepsin over a 24-hour period did not 60 result in additional cleavage products (data not shown). 40

Consistent with this result, an uncleavable single-chain Response (RU) form of pro-MSP generated by mutating the Arg483 residue 20 483 to Glu (scMSP) was resistant to cleavage by sHepsin 0 during a 24-hour reaction period (data not shown). 0 100 200 300 400 500 Pro-MSP activation by cell surface–expressed hepsin Time (s) To determine pro-MSP processing by cell surface- expressed full-length hepsin, we used the prostate cancer Figure 2. Binding of sHepsin-cleaved MSP to RON. A, ELISA measuring cell line LNCaP-34, which was engineered to stably binding of plate-immobilized RON–Fc to sHepsin-cleaved MSP (filled ¼ overexpress hepsin on the cell surface (35). Incubating circles; EC50 0.25 nmol/L) and to uncleavable scMSP (filled triangles; 125 ¼ LNCaP-34 cells with I-labeled pro-MSP over a 3-hour EC50 125 nmol/L). B, surface plasmon resonance experiments. Anti-Fc period showed that more than 80% of pro-MSP was antibody coupled to the CM5 chip was used to capture RON-Fc on the a b biosensor. Binding curves of sHepsin-cleaved MSP were obtained by converted to the 2-chain / heterodimer form. The fitting the experimental data (gray lines) to a 1:1 binding model (black lines). effect is similar to what was observed when pro-MSP The calculated KD was 10.3 nmol/L.

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sHepsin-cleaved MSP bound to immobilized RON-Fc in a concentration-dependent manner with half-maximal A binding (EC50) of 0.25 nmol/L, whereas uncleavable ¼ scMSP showed 500-fold decreased binding (EC50

Control Pro-MSPscMSP MSP sHepsin (10 nmol/L) 125 nmol/L; Fig. 2A). Surface plasmon resonance experi- Pro-MSP(10 nmol/L) + sHepsin ments (BIAcore) with sHepsin-cleaved MSP gave a k 20 100 20 100 20 100 20 100 ng/mL a 6 1 1 2 1 α of 3.47 10 (mol/L) s , kd of 3.59 10 s -RON

and a calculated KD of10.3nmol/L(Fig.2B),whichwas α-p-MAPK almost identical to the KD of 9.1 nmol/L measured for the α commercially obtained MSP. Neither pro-MSP nor the -p-S6 Cell lysates cleavage site mutant scMSP showed any detectable bind- α-p-Akt ing to RON up to a concentration of 1 mmol/L (data not α-Actin shown). We observed a minor discrepancy in the results for the binding of pro-MSP to RON in BIAcore and α-pTyr ELISA assays, which we attribute to sample heterogeneity. IP: α-RON Nonetheless, the overall results show that pro-MSP pro- α-RON cessing by sHepsin unmasked the high-affinity receptor- binding site on the MSP b-chain. B 15 pMAPK p-S6 MSP-mediated activation of signaling The biological effects of sHepsin-cleaved MSP were first 10 assessed by monitoring phosphorylation of RON and downstream signaling proteins. Incubation of human 5 A2780 ovarian cancer cells overexpressing RON with pro-MSP, scMSP, or sHepsin alone did not result in control change over Fold 0

any appreciable phosphorylation of RON or the down- Control sHepsin MSP (20) scMSP (20) MSP (100) Pro-MSP (20) scMSP (100) stream mitogen-activated protein kinase (MAPK), the Pro-MSP (100) ribosomal S6 protein, or the protein kinase Akt Pro-MSP (20) + sHepsin (Fig.3AandB).However,coincubationofA2780- C Pro-MSP (100) + sHepsin RON cells with pro-MSP and sHepsin resulted in robust Medium Pro-MSP scMSP phosphorylation of RON, MAPK, S6, and Akt to levels (serum-free) that were comparable with treatment with active MSP (Fig.3AandB).Furthermore,siRNA-mediatedsuppres- sion of RON expression completely attenuated the phosphorylation of these downstream signaling molecules, suggesting that the effect was mediated through RON (Supplementary Fig. S2).

Peritoneal macrophage morphology change and chemotaxis assay sHepsin MSP-sHepsin MSP MSP induces mouse resident peritoneal macrophages to assume a more flat and spread-out morphology similar to what is seen with other chemoattractants (6, 45). Therefore, Figure 3. A, representative immunoblot analysis of RON, phospho- MAPK, phospho-S6, phospho-Akt, and phospho-RON in human we examined whether processing of pro-MSP by sHepsin A2780 ovarian carcinoma cells. Cells were treated with pro-MSP, produced an active MSP capable of eliciting these responses. scMSP, MSP (from commercial source), or pro-MSP with 10 nmol/L sHepsin-cleaved MSP was added to primary mouse peri- sHepsin at the indicated concentrations for 1 hour. Phosphorylation toneal macrophages in culture. After 1-hour incubation, of MAPK, S6, and Akt were detected by immunoblotting cell lysates using phospho-specific antibodies. Phosphorylation of RON was peritoneal macrophages treated with MSP/sHepsin under- detected by first immunoprecipitating (IP) for RON followed by went distinct changes in cell shape, assuming a flat mor- immunoblotting using phospho-tyrosine antibody. B, relative signal phology with elongated protrusions (Fig. 3C), unlike the intensities of phospho-MAPK and phospho-S6. The numbers in cells treated with pro-MSP alone, which had a more parentheses represent the concentration of pro-MSP, scMSP, or MSP spherical morphology. The effect of sHepsin-activated (from commercial source) in nanogram per milliliter. The values are mean SD of 3 experiments. C, peritoneal macrophage morphology MSP was comparable with that of the MSP from a com- change assay. On stimulation with sHepsin-cleaved MSP, peritoneal mercial source. No cell shape changes were observed in the macrophages underwent distinct changes in cell shape, showed medium control or with the addition of scMSP or sHepsin by protrusion and elongation. The effect of hepsin-activated MSP (Fig. 3C). However, weak morphologic changes were was comparable with that of a commercially available MSP. sHepsin alone, pro-MSP alone, and uncleavable scMSP had no observed in pro-MSP control wells, perhaps because of appreciable effect. baseline activation of pro-MSP by some of the known

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pro-MSP activators like MT-SP1, which are also expressed uncleavable scMSP by themselves increased cell migration in macrophages (11). (Fig.4A).Inaccordwiththis,theadditionofhepsin- In addition, macrophages were coincubated with pro- neutralizing antibody (Ab25) completely inhibited the MSP and sHepsin, which resulted in a significant increase increased promigratory activity found with pro-MSP and (P < 0.001) in peritoneal macrophage migration (Fig. 4A) sHepsin (Fig. 4A). that was comparable with MSP from a commercial source. The promigratory effect was due to the generation Inhibition of nitric oxide synthesis of active MSP by sHepsin, as neither pro-MSP nor In epithelial cells and macrophages, MSP/RON signaling can function as a negative regulator of NO production (46). MSP is capable of blocking the increase in macrophage- inducible nitric oxide synthase mRNA and its associated increase in the production of NO in response to stimuli, A 160 including LPS (47). The ability of sHepsin to generate MSP * that actively suppresses NO production was examined in a 120 * cell culture system in which primary mouse bone marrow macrophages were exposed to LPS. Exposure to LPS 80 resulted in a dramatic increase in macrophage NO produc- RFU tion as measured by nitrite concentration in medium, whereas this response remained unchanged by the addition 40 of pro-MSP alone (Fig. 4B). However, the addition of sHepsin to pro-MSP in the culture medium led to a 0 significant attenuation of NO production, comparable to the effect by mature MSP from a commercial source SFM MSP

scMSP (Fig. 4B) and in agreement with the approximately 50% sHepsin sHepsin pro-MSP reduction (P < 0.0002) of NO production by MSP reported pro-MSP + pro-MSP + pro-MSP in the literature (11). Inhibition of sHepsin by Ab25

sHepsin + Ab25 reversed NO production back to control levels, suggesting that reduction of NO synthesis was entirely mediated by B 40 (–) LPS (+) LPS sHepsin-dependent activation of pro-MSP.

30 Gene expression profiles of MSP and hepsin in comparison to HGFA and MT-SP1 20 * * Tissue distribution of MSP expression in normal and disease tissues (Fig. 5A) showed that MSP is expressed most 10 highly in liver, followed by kidney, pancreas, and small [Nitrite] (µmol/L) intestine. Comparison of this tissue expression profile with 0 that of hepsin, HGFA, MT-SP1, and RON (Fig. 5A) SFM MSP revealed a similar tissue distribution for hepsin, which was also highly expressed in liver, kidney, and pancreas sHepsin sHepsin pro-MSP

pro-MSP + pro-MSP + pro-MSP but not in small intestine. HGFA was also expressed at highest levels in liver, but not in other tissues, whereas MT- sHepsin + Ab25 SP1 and RON were expressed predominantly in colorectal, small intestine, and stomach samples. Figure 4. A, peritoneal macrophage chemotaxis assay. Coincubation of To evaluate these gene expression relationships in more pro-MSP and sHepsin led to a significant increase in cell migration detail, we generated scatter plots of MSP expression against comparable with mature MSP from a commercial source (¼MSP). that of the other genes in the liver, kidney, pancreas, and small Pretreatment with a neutralizing anti-hepsin antibody (Ab25) inhibited migration. sHepsin alone, pro-MSP alone, and uncleavable scMSP did not intestine (Fig. 5B). These scatter plots showed a strong stimulate migration. SFM, serum-free medium. After 4 hours of incubation correlation of expression between MSP and hepsin among at 37C, the migrated cells were collected and quantified by fluorescence normal and disease samples of kidney, liver, and pancreas but measurements on a microplate reader. The values are mean SD of 3 not of small intestine. Coexpression was also observed experiments. *, P < 0.001 versus SFM control; Student's t test. B, MSP- mediated inhibition of nitric oxide synthesis. Treatment of mouse bone between MSP and HGFA in liver samples and between marrow–derived macrophages with LPS dramatically increases NO MSP and both MT-SP1 and RON in small intestinal samples. production quantified by the nitrite concentration in culture medium. Nitrite levels remained unchanged after addition of pro-MSP alone or sHepsin Discussion alone but were reduced by about 50% when sHepsin was added to pro- MSP or with MSP from a commercial source (¼MSP). Addition of anti- hepsin antibody Ab25 to the pro-MSP/sHepsin mixture restored nitrite MSP in its latent form (pro-MSP) has no biological activity levels to LPS control levels. The values are mean SD of 3 experiments. and its maturation via proteolytic processing is an important *, P < 0.002 versus SFM þ LPS; Student's t test. regulatory step in the MSP/RON signaling pathway. This is

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Figure 5. A, MSP expression in normal and diseased tissues. MSP is most highly expressed in liver, followed by kidney, pancreas, and small intestine. Comparison of this tissue expression profile with that of hepsin, HGFA, MT-SP1, and RON reveals a similar tissue distribution for hepsin, which is also highly expressed in liver, kidney, and pancreas but not in small intestine. HGFA is also expressed at highest levels in liver, but not in other tissues, whereas MT-SP1 and RON are ubiquitously expressed with highest expression in colorectal, small intestine, and stomach samples.

akin to the related HGF/MET system in which biologically capable of processing pro-MSP at the consensus cleavage site. active HGF is generated by an obligatory pro-HGF cleavage The proteolytic activity of sHepsin on pro-MSP was specific, reaction. Hepsin activates pro-HGF by cleavage at KQLR#V as it was completely inhibited by hepsin inhibitors, such as (19, 34). The scissile peptide sequence of pro-MSP, SKLR#V, KD1, Ac-KQLR-cmk, and most importantly, a neutralizing has strong similarity to that of pro-HGF as well as to the anti-hepsin antibody (Ab25). Additional evidence for the SQLR#L cleavage sequence of the recently identified hepsin specificity of the cleavage reaction came from experiments substrate laminin-332 (36), suggesting that pro-MSP could withthecleavagesitemutantscMSP,whichremained potentially be a hepsin substrate. Here, we provide several intact on prolonged treatment with high concentrations of lines of evidence to show that hepsin is an efficient pro-MSP sHepsin. The pro-MSP cleavage site sequence recognized activator that generates the biologically active 2-chain by hepsin is consistent with a preference for arginine at P1, a/b-heterodimeric MSP signaling molecule. leucine at P2, and lysine at P3 positions (19). On the basis Using the highly purified single chain pro-MSP, both of the published structure of hepsin with bound Ac- sHepsin and cell surface–expressed full-length hepsin are KQLR-cmk (pro-HGF sequence,PDB1Z8G;ref.19),

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Figure 5. (Continued )B, expression of hepsin correlates well with MSP in liver, kidney, and pancreas (normal, green; diseases, blue) and expression of HGFA correlates well with MSP only in liver. MT-SP1 and RON shows minor correlation with MSP expression in these tissues.

we built a model with the pro-MSP sequence SKLR report showed that recombinant human hepsin was unable (Supplementary Fig. S3). The replacement of a P4-Lys/ to cleave pro-MSP, yet was able to cleave the internally P3-Gln with a P4-Ser/P3-Lys should have minimal effects, quenched fluorescence peptide encompassing the pro-MSP as the modeled SKLR peptide also orients its side chains in cleavage sequence SKLR-VVGG (P4-P40; ref. 48). Although a very similar position as that of the KQLR peptide. In there is no straightforward explanation for these discrepant addition, there are potential hydrogen bond (distance < 3 results, it may be possible that the quality and the source of A) interactions of the SKLR residues P4-Ser and P3-Lys the pro-MSP used by Beliveau and colleagues (48) could be a with the hepsin residues Gln175b and Tyr146 (Supplemen- reason for the different findings. tary Fig. S3). These additional interactions may confer The in vitro reaction conditions, specifically the high favorable effects on the preference of substrates with a substrate to enzyme ratio and the short reaction time, serine at P4 and lysine at P3 positions. suggested that hepsin is a highly efficient pro-MSP acti- Cleavage of pro-MSP at the Arg483-Val484 bond leads to vator. This view was further substantiated by a comparison the formation of the receptor-binding site on the MSP with 2 recently identified pro-MSP activators MT-SP1 b-chain that is not present on pro-MSP (3). In accordance, and HGFA, suggesting that hepsin has superior pro-MSP sHepsin-cleaved MSP was capable of binding to its receptor converting activity. A caveat is that the assays used only with high affinity as determined by surface plasmon reso- extracellular portions of MT-SP1 and hepsin, both of nance and ELISA experiments, suggesting that the cleaved which are integral cell surface proteases of the TTSP MSP is functionally competent. This was further established family. Experiments with LNCaP-34 cells, which express in a set of RON-dependent cellular activity assays, namely, full-length forms of both hepsin and MT-SP1 (35), the phosphorylation of downstream signaling proteins in indicated that pro-MSP processing was entirely due to RON-expressing A2780 cells, the change in chemotaxis and hepsin, because no processing was observed in the pre- morphology in peritoneal macrophages, and the attenuation sence of the hepsin-specific inhibitor Ab25. Although of LPS-induced NO production in bone marrow–derived these results are consistent with the potent pro-MSP macrophages. Consistently, RON-mediated cellular processing activity of sHepsin, they do not imply that responses were elicited only when cells were exposed to cell surface expressed MT-SP1 lacks pro-MSP convertase the combination of pro-MSP and sHepsin but not to activity; because LNCaP-34 cells were engineered to over- individual pro-MSP, scMSP, or sHepsin treatments. These express hepsin, contributions by basal levels of MT-SP1 results strongly suggest that pro-MSP processing generates a may have been masked. functional MSP signaling molecule with an activity indis- In addition to pro-MSP, hepsin was shown to activate tinguishable from MSP from a commercial source. A recent pro-HGF and pro-uPA, and to cleave laminin-332 (19,

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activating proteases are critical in regulating the MSP/RON Pro-HGF HGF HGF/MET and HGF/MET pathways. It is intriguing that both pro- MSP and pro-HGF are activated by the 3 trypsin-like serine proteases hepsin, MT-SP1, and HGFA and that all 3 Hepsin Cell proliferation, proteases are inhibited by the same 2 Kunitz domain HGFA HAI-1 migration and inhibitors, HAI-1 and HAI-2 (refs. 40, 41, 54; Fig. 6). HAI-2 differentiation This could mean that they are components of the same MT-SP1 biological systems involving growth factor–mediated cellular responses such as tissue repair and tumorigenesis. Given pro-MSP MSP MSP/RON the assumption that latent growth factors are abundantly present in the extracellular space (55–57), this protease/ inhibitor system could control the availability of biologically Figure 6. Model of protease/inhibitor system regulating growth factor active growth factors in the microenvironment. Except for signaling. The activation of the MSP/RON and HGF/MET signaling HGFA, the proteases, inhibitors, and growth factor recep- pathways is dependent on the availability of the biologically active forms of tors (RON and MET) are integral cell surface–expressed MSP and HGF. The 3 trypsin-like serine proteases hepsin, HGFA, and MT- SP1 can activate the biologically inactive latent growth factors (pro-MSP proteins, which may allow for highly localized reactions and and pro-HGF) by enzymatic cleavage at the consensus Arg-Val bond to their regulation by inhibitors. generate the active a/b heterodimers MSP and HGF. The enzymatic Recently, gene expression profiling used to identify MT- activities of the proteases are, in turn, regulated by the cell surface– SP1 as a pro-MSP activator showed that both MSP and expressed Kunitz domain inhibitors HAI-1 and HAI-2. MT-SP1 expression correlated in normal and disease tissues (11). In this report, we detected a strong correlation of MSP 34, 36). The hepsin-mediated processing of these sub- expression with hepsin expression in the liver, kidney, and strates activates enzymatic cascades and/or initiates bio- pancreas that was superior to the corresponding correlations logical pathways that pertain to tumor growth and observed between MSP and HGFA or MSP and MT-SP1. progression. Hepsin-activated pro-uPA activates plasmi- Both hepsin and MSP are produced by hepatocytes in the nogen to plasmin, which then degrades extracellular liver (58, 59) and renal tubule cells in the kidney (58, 60), matrix proteins directly or indirectly by activating latent which were recently shown to increase MSP production forms of matrix metalloproteases (49, 50). Degradation of during the regenerative phase in a mouse renal injury model stromal components and basement membranes are critical (60). In light of the potent pro-MSP convertase activity for for tumors to invade and metastasize, and it constitutes a hepsin in vitro, the coexpression results suggest that hepsin hallmark for increased tumor aggressiveness. Similarly, may regulate pro-MSP activation in tissue homeostasis or hepsin enzymatic activity toward the basement membrane after tissue injury. The biochemical linkage between hepsin constituent laminin-332, which generates promigratory with the MSP/RON system presented in this study is likely laminin-332 fragments, as well as the hepsin-mediated to form the basis for further investigations directed at activation of the HGF/MET and MSP/RON signaling understanding the biological regulation of the MSP/ pathways, may promote tumor invasion and progression. RON pathway by proteases under normal and pathophy- For instance, hepsin is highly upregulated in prostate siologic conditions. cancer epithelial cells (20, 28), which also express RON (51, 52). RON was found to regulate the production of Disclosure of Potential Conflicts of Interest angiogenic chemokines promoting tumor growth and angiogenesis (52, 53). Therefore, the increased expression No potential conflicts of interest were disclosed. of hepsin in prostate cancer could activate RON-dependent signaling in cancer cells to promote cancer progres- Acknowledgments sion. In vivo studies using mouse prostate tumor models support a role of hepsin in basement membrane degrada- The authors thank Kaushiki Mahapatra for running the phosphorylation assay, tion and in tumor invasion and metastasis (28, 29). Jose Zavala for animal husbandry, Greg Bennett and Daniel Tran for their help with 125I-labeling of pro-MSP, and Wendy Sandoval for N-terminal amino acid The plasminogen-like growth factors MSP and HGF sequencing. share the same domain architecture and activation mechan- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance ism and initiate intracellular signaling pathways that lead to with 18 U.S.C. Section 1734 solely to indicate this fact. proliferation, migration, and differentiation (Fig. 6). The fact that the single-chain precursors, pro-HGF and pro- Received January 3, 2011; revised June 2, 2011; accepted June 27, 2011; MSP, have no biological activity strongly suggests that the published OnlineFirst August 29, 2011.

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1186 Mol Cancer Res; 9(9) September 2011 Molecular Cancer Research

Proteolytic Activation of Pro-Macrophage-Stimulating Protein by Hepsin

Rajkumar Ganesan, Ganesh A. Kolumam, S. Jack Lin, et al.

Mol Cancer Res 2011;9:1175-1186. Published OnlineFirst August 29, 2011.

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