Proteolytic Activation of Pro-Macrophage-Stimulating ... · Angiogenesis, Metastasis, and the...

Angiogenesis, Metastasis, and the Cellular Microenvironment

Proteolytic Activation of Pro-Macrophage-StimulatingProtein 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

AbstractMacrophage-stimulating protein (MSP) is a plasminogen-related growth factor and ligand for the receptor

tyrosine kinase RON. TheMSP/RON system promotes wound healing and invasive tumor growth and suppressesproinflammatory 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 serineprotease, that is overexpressed in several cancers. Here, we show that recombinant hepsin cleaves pro-MSP at theconsensus site Arg483-Val484 with superior efficiency compared with the known activators MT-SP1 andhepatocyte growth factor activator (HGFA). At least 50% of pro-MSP was processed within 1 hour at a hepsinconcentration of 2.4 nmol/L and at a molar enzyme to substrate ratio of 1:500. An uncleavable single-chain variantof 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 cellsoverexpressing hepsin on the cell surface efficiently activated pro-MSP, which was blocked by a specific anti-hepsinantibody. Incubation of pro-MSP with hepsin led to robust RON-mediated phosphorylation of mitogen-activatedprotein kinase, ribosomal S6 protein, and Akt in human A2780 ovarian carcinoma cells stably expressing RONprotein. In macrophages, pro-MSP with hepsin induced chemotaxis and attenuated lipopolysaccharide-dependentproduction of nitric oxide. These findings suggest that the MSP/RON signaling pathway may be regulated byhepsin in tissue homeostasis and in disease pathologies, such as in cancer and immune disorders.Mol Cancer Res;9(9); 1175–86. �2011 AACR.

Introduction

Macrophage-stimulating protein (MSP, also known ashepatocyte growth factor–like protein, HGFL) is a plasmi-nogen-like growth factor that mediates its biological activ-ities by activating the receptor tyrosine kinase RON[recepteur d'origine nantais, also known as macrophagestimulating receptor-1, (MSTR1)], a member of theMET proto-oncogene family (1). MSP shares high sequenceand structural domain homology with HGF, the ligand forMET; similarly, their respective cognate receptors, RONand MET, also share high sequence and domain homology

(2). The a-chain of MSP consists of an N-terminal PANmodule followed by 4 kringle domains and is disulfide linkedto the trypsin-likeb-chain (3).MSP is constitutively expressedby hepatic parenchymal cells, as well as in lungs, adrenalglands, placenta, kidney, and pancreas (4, 5). It is secreted asan inactive single-chain precursor pro-MSP, which requiresproteolytic cleavage at the Ser-Lys-Leu-Arg483#Val484 bondto attain functional activity (5, 6). In HGF, cleavage at thecorresponding Arg494#Val495 bond results in distinct struc-tural rearrangements within the HGF b-chain and the for-mation of a MET-binding site that is competent for signaltransduction (7–9). Comparable conformational rearrange-ments centered at the "pseudo-active site" in theMSPb-chainare likely to occur on activation cleavage of pro-MSP (7).However, unlikeHGF,where the high-affinityMET-bindingsite resides in thea-chain, the high-affinity RON-binding siteforMSP is located on thematureb-chain that forms followingpro-MSP cleavage (10). Several trypsin-like serine proteases,including MT-SP1 (also known as matriptase) and HGFactivator (HGFA; refs. 11–15), are known to activate pro-MSP, leading to typical cellular responses mediated by theRON signaling pathway (11, 12).Hepsin is a cell surface–expressed trypsin-like protease

and a member of the type II transmembrane serine proteasefamily (16, 17). It consists of an N-terminal cytoplasmic

Authors' Affiliations: Departments of 1Early Discovery Biochemistry,2Tumor Biology and Angiogenesis, 3Molecular Oncology, and 4Bioinfor-matics/Computational Biology, Genentech, Inc., South San Francisco,California

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: Daniel Kirchhofer, Department of Early DiscoveryBiochemistry, Genentech, Inc., 1 DNAWay, MS #27, South San Francisco,CA 94080. Phone: 650-225-2134; Fax: 650-225-3734;E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-11-0004

�2011 American Association for Cancer Research.

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

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domain, a transmembrane domain, and an extracellularportion composed of a scavenger receptor–like cysteine-richdomain and a C-terminal protease domain with a trypsin-like fold (18, 19). Hepsin was identified as one of the mosthighly upregulated genes in prostate cancer (20–25) andimmunohistochemical staining revealed strong expressionin late-stage tumors and metastatic bone lesions (26, 27).Studies with preclinical prostate cancer models suggestedthat hepsin may play a role in invasive cancer growth andcancer progression (28, 29). Moreover, gene expressionanalyses have also implicated hepsin in ovarian cancer(30), renal cell carcinoma (31, 32), and endometrial cancer(33). A putative function of hepsin in tumor progressioncould be related to its enzymatic activity toward the macro-molecular substrates pro-HGF (19, 34), pro-uPA (35), andlaminin-332 (36). Additional substrates that have beenidentified are coagulation factors (37) and pro-prostasin(38). The hepsin cleavage sequences of these substrates arein good agreement with the consensus sequence obtainedfrom substrate profiling by positional scanning of a syn-thetic combinatorial peptide library (19).Here, we report that both recombinant soluble hepsin

(sHepsin) and cell surface–expressed hepsin efficientlycleave human pro-MSP at the physiologic activation site.The hepsin-cleaved MSP was functionally competent forRON binding and in triggering cellular responses mediatedby the RON signaling pathway. Finally, we detected sig-nificant coexpression of MSP and hepsin in a number oforgans, suggesting a potential physiologic role of hepsin inregulating activity of MSP.

Materials and Methods

Cloning, expression, and purification of recombinantproteinsThe extracellular domain of the human recombinant

hepsin harboring a C-terminal His-tag (sHepsin) wasexpressed and purified as described (35). RecombinantRON (SEMA/IPT/TIG1 domains: Glu25-Met682) wasmade as Fc fusion protein by expressing it in Chinesehamster ovary (CHO) cells. RON–Fc was purified byaffinity chromatography followed by size exclusionchromatography. Recombinant pro-MSP harboring aC-terminal His6 tag along with a C672A mutation, pre-viously shown to yield better protein expression, wasexpressed in CHO cells as described (39). Secreted pro-MSP was purified by Ni-NTA affinity chromatographyfollowed by size exclusion chromatography on Superdex-S200 column. Most of the MSP present in the pro-MSPpreparation was removed by incubation with RON–Fcfollowed by a protein-A column purification and pro-MSPwas collected in the flow through (SupplementaryFig. S1A and Fig. 1A). To generate a nonactivatable formof pro-MSP, the P1 residue Arg483 was mutated to Glu(scMSP) using QuikChange mutagenesis (Stratagene).Thus, scMSP actually contains 2 mutations: R483Eand C672A. The protein was expressed and purified asdescribed for wild-type pro-MSP. The serum form of

HGFA (Val373-Ser655), the MT-SP1 protease domain(sMT-SP1) as well as the Kunitz domain-1 (KD1) derivedfrom HGFA inhibitor type-1 (HAI-1) were expressed andpurified as described (40, 41). Recombinant active humanMSP was obtained from R&D Systems and used asreference material. Antibody25 (Ab25) inhibits hepsinenzymatic activity and was generated by using antibodyphage display. Ab25 (IgG1) and Fab25 were expressed inCHO cells and Escherichia coli, respectively, and werepurified according to standard procedures.

In vitro activation of pro-MSP by sHepsin, HGFA, andsMT-SP1Pro-MSP (100 mg/mL¼ 1.25 mmol/L) was incubated for

1 hour at 37�C with different concentrations (100 nmol/L–97 pmol/L, 2-fold dilution series) of sHepsin, HGFA, andsMT-SP1 in a buffer containing 50 mmol/L Tris-HCl, pH¼ 8.0, 150 mmol/L NaCl, 0.05% Triton X-100, and 2mmol/L CaCl2 for 1 hour at 37�C. In addition, 100 nmol/Lof the proteases were preincubated for 15 minutes with 1mmol/L of the hepsin-specific Fab25 or 1 mmol/L of KD1(an inhibitor of hepsin, MT-SP1, and HGFA) beforeaddition of pro-MSP. For time course experiments, 12.5nmol/L sHepsin was incubated with 1.25 mmol/L of pro-MSP and aliquots were analyzed by SDS-PAGE and pro-teins stained with SimplyBlue Safe Stain (Invitrogen). Theexperiments were repeated 3 times. Similar experimentswere carried out with 250 mg/mL of the scMSP mutant andhepsin (10 nmol/L). For the densitometric analysis of pro-MSP degradation, the pro-MSP band intensities werequantified using ImageJ from the NIH (http://rsb.info.nih.gov/ij/index.html). The effective concentration to give50% reduction (EC50) was determined by a 4-parameter fit(Kaleidagraph; Synergy Software).sHepsin-cleaved MSP was prepared by incubating 100

mg/mL of pro-MSP with 15 nmol/L sHepsin for 1 hour,resulting in complete conversion to cleaved MSP. sHepsinwas removed by adding molar excess of anti-hepsin Ab25 tothe reaction mixture followed by Protein A-Sepharosechromatography. Thus purified MSP did not contain anyresidual hepsin as assessed by SDS-PAGE and enzymaticassays. A similar protocol was used to generate HGFA-cleaved MSP except that the anti-HGFA antibody Ab40(42) was used to remove HGFA from the reaction mixture.

Pro-MSP activation by cell surface–expressed hepsin onLNCaP-34 cellsLNCaP-34 cells stably overexpressing human full-length

hepsin were described previously (35). Confluent culturesin 24-well plates were incubated for 15 minutes at 37�Cwith 500 mL serum-free RPMI-1640 medium containing 1mmol/L each of Ab25 or KD1 or Ac-KQLR chloromethylketone (Ac-KQLR-cmk; Anaspec) or 10 nmol/L sHepsin.125I-labeled pro-MSP, prepared as described for pro-HGF(34), was added (25 mg/mL) and incubated for 3 hours at37�C. Aliquots were analyzed by SDS-PAGE (4%–20%gradient gel) followed by exposure to X-ray films. Theintensities of the bands were quantified using ImageJ

Ganesan et al.

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software. The loss of pro-MSP band intensity was used todetermine the percentage of proteolytic activity.

Binding of hepsin-activated pro-MSP to RON bysurface plasmon resonance and ELISAFor surface plasmon resonance measurements on a BIA-

core-3000 instrument (GE Healthcare) rabbit anti-humanIgG was immobilized (amine coupling) on CM5 biosensorchips and the RON–Fc fusion protein was captured to giveapproximately 250 response units (RU). Different concen-tration of either sHepsin-activated MSP or reference MSP(R&D Systems) or scMSP were injected in HBS-P buffer(10 mmol/L HEPES, pH ¼ 7.5, 150 mmol/L NaCl,0.005% P20) at 25�C with a flow rate of 30 mL/min.Association rates (ka) and dissociation rates (kd) wereobtained by using a simple one-to-one Langmuir bindingmodel (BIA-Evaluation software) and the equilibrium dis-sociation constants (KD) were calculated (kd/ka). For ELISAexperiments, maxiSorp microtiter plates (Nunc) werecoated overnight at 4�Cwith 2 mg/mL of rabbit anti-humanIgG Fc-specific antibody (Jackson ImmunoResearchLaboratory) in 50 mmol/L sodium carbonate buffer,pH ¼ 9.6. After blocking with assay buffer (PBS, pH ¼7.4, 0.5% BSA, and 0.05% Tween-20, 15 ppm Proclin),1 mg/mL RON-Fc was added incubated for 1 hour. After

washing with PBS, 0.05% polysorbate 20, MSP proteinswere added and incubated for 1 hour. Bound MSP wasdetected using anti-His-HRP (Qiagen) and TMB/H2O2substrate (KPL). The reaction was stopped with 1 mol/LH3PO4 and the absorbance at 450 nm was measured.

Peritoneal macrophage chemotaxis and morphologychange assayMurine peritoneal resident macrophages were obtained

from C57BL/6 mice by washing the peritoneal cavity with15 mL of serum-free RPMI-1640 medium. Cells werewashed and resuspended in medium to a concentrationof 1� 106 cells/mL. The macrophage chemotaxis assay wasconducted using a QCM chemotaxis assay kit with a poresize of 5 mm (Millipore). To the bottom wells was addedRPMI-1640 medium containing the following compo-nents: (a) 80 ng/mL pro-MSP, (b) 80 ng/mL scMSP,(c) 80 ng/mL MSP from R&D Systems, (d) 80 ng/mLpro-MSP and 10 nmol/L sHepsin, and (e) 80 ng/mL pro-MSP, 10 nmol/L sHepsin, and 100 nmol/L Ab25. Thereaction mixtures (a–e) were preincubated at 37�C for1 hour before adding to the bottom wells. Macrophagesuspension (105 cells/100 mL) was added to the upper wells.After incubation at 37�C for 4 hours, the migrated cellswere collected using detachment buffer (Millipore) and

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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 37�C withdifferent concentrations (100 nmol/L–97 pmol/L) of recombinant soluble hepsin (sHepsin) or the known activators sMT-SP1 and HGFA. For controlexperiments, 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-kDaband as MSP b-chain. Intensities of the pro-MSP band were quantified by densitometry and plotted against enzyme concentrations. The EC50 values are theaverage � SD of 3 independent experiments. B, activation of pro-MSP by cell surface–expressed hepsin. LNCaP-34 prostate cancer cells, which stablyoverexpress full-length hepsin, were incubated with 125I-pro-MSP alone or in combination with different inhibitors for 3 hours. In a control experiment, nosignificant 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 theexperiment (lane 1). All 3 hepsin inhibitors (Ac-KQLR-cmk, KD1, and Ab25) effectively blocked pro-MSP processing (lanes 5–7).

Activation of Pro-MSP by Hepsin

www.aacrjournals.org Mol Cancer Res; 9(9) September 2011 1177




quantified by incubation for 15 minutes with lysis bufferand CYPRO dye followed by fluorescence measurements(RFU, relative fluorescence units) on a microplate reader(Spectramax-M5, Molecular Devices) with excitation at480 nm and emission at 520 nm.To examine the morphologic changes, peritoneal macro-

phages (1 � 106 cells/mL) were cultured in serum-freeRPMI-1640 medium overnight. Nonadherent cells wereremoved and 80 ng/mL each of pro-MSP, sHepsin-cleavedMSP, HGFA-cleaved MSP, MSP from R&D Systems, orscMSP were added. After 1 hour, morphologic changes werecaptured by phase contrast microscopy.

Inhibition of nitric oxide synthesis by mature MSPBone marrow cells were isolated from femurs of C57BL/6

mice as described (11). After washing the cell suspensionwith Dulbecco's Modified Eagle's Media (DMEM), redblood cells were lysed with erythrocyte lysis buffer. Cellswere resuspended in macrophage differentiation medium(DMEM with glutamine, 10% FBS, 1� Pen/Strep, and 50ng/mL mCSF-1) and added to 24-well plates. Medium waschanged the next day and subsequently every second day.After 6 days, the matured macrophages were incubated for24 hours at 37�C in 300 mL/well of serum-free mediumwith or without 1 mg/mL of lipopolysaccharide (LPS;Sigma) and containing the following: (a) 10 ng/mL pro-MSP, (b) 10 ng/mL pro-MSP and 1 nmol/L sHepsin, (c) 10ng/mL pro-MSP, 1 nmol/L sHepsin, and 500 nmol/LAb25, and (d) 10 ng/mL MSP (R&D Systems). Nitricoxide (NO) production was quantified by measuring theconcentration of nitrite in diluted aliquots of culture med-ium by use of the Griess Reaction Kit (Molecular Probes).

Phosphorylation of signaling proteins downstream ofRONA2780 human ovarian carcinoma cells engineered to

express human RON (A2780-RON; unpublished data)were seeded at a density of 2 � 105 cells per well into a12-well tissue culture plate. For siRNA experiments, RONexpression was reduced in A2780-RON cells by transfecting20 pmol of either RON-specific siRNA 50-GGGCGACA-GAAAUGAGAGUtt-30 (Ambion/Applied Biosystems) ornontargeting control siRNA (Dharmacon/Thermo Scien-tific) using RNAi-Max (Invitrogen). Forty-eight hours fol-lowing transfection, cells were serum starved for anadditional 24 hours and incubated with indicated concen-trations of pro-MSP, scMSP, MSP, and pro-MSP withsHepsin (10 nmol/L). After 1-hour incubation, cells werewashed once with cold PBS followed by incubation on ice inlysis buffer (10 mmol/L Tris, pH¼ 7.5, 150 mmol/LNaCl,10% glycerol, 0.1% Triton X-100, 5 mmol/L b-glycero-phosphate, 2 mmol/L NaF, 1 mmol/L sodium orthovana-date, and protease and phosphatase inhibitors; Sigma LifeSciences). Lysates were clarified by centrifugation at 14,000� g, subjected to SDS-PAGE under reducing conditions,transferred onto nitrocellulose membranes, and incubatedovernight with anti-RON C20 (Santa Cruz Biotechnology),anti-phospho-MAPK (Cell Signaling Technology), anti-

phospho-S6 (Cell Signaling Technology), or anti-phos-pho-Akt (Cell Signaling Technology) antibodies. RONphosphorylation was analyzed by immunoprecipitatingRON from cell lysates using anti-RON antibody, 1A2.2(Genentech, Inc.), coupled to agarose beads. Immunopre-cipitates were washed 3 times in cold lysis buffer, resus-pended in 4� SDS-PAGE sample buffer (Invitrogen), andanalyzed after immunoblotting with anti-phospho-tyrosinepTyr-100 antibody (Cell Signaling Technology). The sec-ondary antibodies used were IRDye800-conjugated goatanti-mouse IgG (Rockland) and AlexaFluor 680 goat anti-rabbit IgG (Invitrogen). Protein bands were visualizedand quantified on the Odyssey Infrared Imaging System(LI-COR Biosciences) and the experiments were repeated3 times.

Computational gene expression analysisMicroarray data on the Affymetrix HG-U133A and HG-

U133B GeneChips were obtained from the GeneLogicdatabase. We extracted samples that were classified asnormal or as having noncancer disease and used the signalintensity values from the Microarray Analysis Suite Version5 Software. We used the following microarray probes tomeasure gene expression: MSP, 205614_x_at; Hepsin,204934_s_at; HGFA, 207027_at; MT-SP1, 202005_at;and RON, 205455_at. For each tissue type, we generatedexpression profiles to show the mean � SD of geneexpression values. For the 4 tissue types having the highestmean expression of MSP, liver, kidney, pancreas, and smallintestine, we generated scatter plots to show the relations ofexpression of MSP against that of other genes. All plots weregenerated using the R statistical package.

Results

In vitro activation of pro-MSP by recombinant hepsinSubstrate profiling of hepsin by the use of a synthetic

combinatorial library determined (P/K)-(K/Q)-(T/L/N)-Ras the P4-P1 (nomenclature according to Schechter andBerger; ref. 43) consensus sequence (19), which is in goodagreement with the identified cleavage site sequences frommacromolecular substrates of hepsin (refs. 34–37; Table 1).The consensus sequence and particularly the specific hepsinrecognition sequences of laminin-332 (SQLR#L) and pro-HGF (KQLR#V) bear a close resemblance to the cleavagesequence of pro-MSP (SKLR#V). Therefore, we hypothe-sized that hepsin could be a pro-MSP activator. To examinethis hypothesis, we expressed pro-MSP in CHO cells butfound it partially converted into the 2-chain form (MSP).Because MSP, but not pro-MSP, binds to its receptorRON, we further purified pro-MSP by incubating it withRON–Fc for 16 hours at 4�C followed by an affinitychromatographic purification step. The resulting pro-MSP was of high purity and contained only small amountsof activated MSP (Supplementary Fig. S1A). The solubleform of hepsin (sHepsin) comprising the extracellular por-tion cleaved pro-MSP in a concentration- and time-depen-dent manner at 37�C. sHepsin (12.5 nmol/L) converted

Ganesan et al.





more than 50% of pro-MSP (1.25 mmol/L) within 20minutes and complete conversion into the a/b heterodimerwas achieved within 1 hour at a molar enzyme to substrateratio of 1:100 (Supplementary Fig. S1B). N-terminalsequencing identified the approximately 60-kDa band asMSP a-chain (19QRSPLN) and the approximately 30-kDaband as MSP b-chain (484VVGGHPG), indicating thatsHepsin processed pro-MSP at the consensus cleavage siteArg483-Val484.We compared the enzymatic activity of sHepsin with the

2 known pro-MSP–converting proteases sMT-SP1 andHGFA. sHepsin, sMT-SP1, and HGFA cleaved pro-MSP in a concentration-dependent fashion, and theiractivities were completely inhibited by KD1, the N-term-inal Kunitz domain of their physiologic inhibitor HAI-1(Fig. 1A). The anti-hepsin antibody Fab25 specificallyinhibited pro-MSP processing by sHepsin but not bysMT-SP1 or HGFA (Fig. 1A). The relative pro-MSP–converting potencies of the 3 proteases were quantifiedby measuring the disappearance of the pro-MSP band bydensitometry. The results showed that sHepsin (EC50¼ 2.4� 0.3 nmol/L) was 5-fold and 7-fold more efficient thansMT-SP1 (EC50 ¼ 11.7 � 1.3 nmol/L) and HGFA (EC50¼ 17.7 � 1.9 nmol/L), respectively. Prolonged incubationof pro-MSP with sHepsin over a 24-hour period did notresult in additional cleavage products (data not shown).Consistent with this result, an uncleavable single-chainform of pro-MSP generated by mutating the Arg483 residueto Glu483 (scMSP) was resistant to cleavage by sHepsinduring a 24-hour reaction period (data not shown).

Pro-MSP activation by cell surface–expressed hepsinTo determine pro-MSP processing by cell surface-

expressed full-length hepsin, we used the prostate cancercell line LNCaP-34, which was engineered to stablyoverexpress hepsin on the cell surface (35). IncubatingLNCaP-34 cells with 125I-labeled pro-MSP over a 3-hourperiod showed that more than 80% of pro-MSP wasconverted to the 2-chain a/b heterodimer form. Theeffect is similar to what was observed when pro-MSP

was treated with 10 nmol/L sHepsin in the LNCaP-34cell cultures (Fig. 1B), which resulted in more than 92%conversion of pro-MSP compared with the pro-MSP onlycontrol (lane 1). Cleavage of pro-MSP was inhibitedby 3 different inhibitors—KD1, which inhibits hepsin,MT-SP1, and HGFA (40); KQLR-cmk, an irreversiblepeptide inhibitor mimicking the pro-HGF cleavagesequence KQLR#V (19, 44); and Ab25 (Fig. 1B).Although KD1 and KQLR-cmk are not selective inhibi-tors, the inhibition observed for them is similar as that forthe selective hepsin inhibitor Ab25. Thus, any activationby other proteases, in particular MT-SP1 that is presenton LNCaP-34 cells (35), is minimal.

Binding of sHepsin-cleaved MSP to RONCleavage of pro-MSP at the Arg483-Val484 bond leads to

the formation of a high-affinity binding site on the MSPb-chain that is absent in pro-MSP. Therefore, properprocessing of pro-MSP, leading to the generation of thehigh-affinity binding site for RON can be monitoredby measuring MSP binding to RON. In ELISA assays,

Table 1. Cleavage sequence of hepsinsubstrates

Hepsin substrates Cleavage sequence

Pro-MSP SKLR#VPro-HGF KQLR#VLaminin-332 SQLR#LPro-uPA PRFK#IFVII PQGR#IProprostasin PQAR#IPS-SCL (consensus) (P/K)-(K/Q)-(T/L/N)-R

NOTE: # indicates cleavage site.Abbreviations: SCL, synthetic combinatorial library; PS,profiling of substrate.

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Figure 2. Binding of sHepsin-cleaved MSP to RON. A, ELISA measuringbinding of plate-immobilized RON–Fc to sHepsin-cleaved MSP (filledcircles; EC50 ¼ 0.25 nmol/L) and to uncleavable scMSP (filled triangles;EC50 ¼ 125 nmol/L). B, surface plasmon resonance experiments. Anti-Fcantibody coupled to the CM5 chip was used to capture RON-Fc on thebiosensor. Binding curves of sHepsin-cleaved MSP were obtained byfitting the experimental data (gray lines) to a 1:1 bindingmodel (black lines).The calculated KD was 10.3 nmol/L.






sHepsin-cleaved MSP bound to immobilized RON-Fc ina concentration-dependent manner with half-maximalbinding (EC50) of 0.25 nmol/L, whereas uncleavablescMSP showed 500-fold decreased binding (EC50 ¼125 nmol/L; Fig. 2A). Surface plasmon resonance experi-ments (BIAcore) with sHepsin-cleaved MSP gave a kaof 3.47 � 106 (mol/L)�1 s�1, kd of 3.59 � 10�2 s�1

and a calculated KD of 10.3 nmol/L (Fig. 2B), which wasalmost identical to the KD of 9.1 nmol/L measured for thecommercially obtained MSP. Neither pro-MSP nor thecleavage site mutant scMSP showed any detectable bind-ing to RON up to a concentration of 1 mmol/L (data notshown). We observed a minor discrepancy in the resultsfor the binding of pro-MSP to RON in BIAcore andELISA assays, which we attribute to sample heterogeneity.Nonetheless, the overall results show that pro-MSP pro-cessing by sHepsin unmasked the high-affinity receptor-binding site on the MSP b-chain.

MSP-mediated activation of signalingThe biological effects of sHepsin-cleaved MSP were first

assessed by monitoring phosphorylation of RON anddownstream signaling proteins. Incubation of humanA2780 ovarian cancer cells overexpressing RON withpro-MSP, scMSP, or sHepsin alone did not result inany appreciable phosphorylation of RON or the down-stream mitogen-activated protein kinase (MAPK), theribosomal S6 protein, or the protein kinase Akt(Fig. 3A and B). However, coincubation of A2780-RON cells with pro-MSP and sHepsin resulted in robustphosphorylation of RON, MAPK, S6, and Akt to levelsthat were comparable with treatment with active MSP(Fig. 3A and B). Furthermore, siRNA-mediated suppres-sion of RON expression completely attenuated the phos-phorylation of these downstream signaling molecules,suggesting that the effect was mediated through RON(Supplementary Fig. S2).

Peritoneal macrophage morphology change andchemotaxis assayMSP induces mouse resident peritoneal macrophages to

assume a more flat and spread-out morphology similar towhat is seen with other chemoattractants (6, 45). Therefore,we examined whether processing of pro-MSP by sHepsinproduced an active MSP capable of eliciting these responses.sHepsin-cleaved MSP was added to primary mouse peri-toneal macrophages in culture. After 1-hour incubation,peritoneal macrophages treated with MSP/sHepsin under-went distinct changes in cell shape, assuming a flat mor-phology with elongated protrusions (Fig. 3C), unlike thecells treated with pro-MSP alone, which had a morespherical morphology. The effect of sHepsin-activatedMSP was comparable with that of the MSP from a com-mercial source. No cell shape changes were observed in themedium control or with the addition of scMSP or sHepsin(Fig. 3C). However, weak morphologic changes wereobserved in pro-MSP control wells, perhaps because ofbaseline activation of pro-MSP by some of the known

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Figure 3. A, representative immunoblot analysis of RON, phospho-MAPK, phospho-S6, phospho-Akt, and phospho-RON in humanA2780 ovarian carcinoma cells. Cells were treated with pro-MSP,scMSP, MSP (from commercial source), or pro-MSP with 10 nmol/LsHepsin at the indicated concentrations for 1 hour. Phosphorylationof MAPK, S6, and Akt were detected by immunoblotting cell lysatesusing phospho-specific antibodies. Phosphorylation of RON wasdetected by first immunoprecipitating (IP) for RON followed byimmunoblotting using phospho-tyrosine antibody. B, relative signalintensities of phospho-MAPK and phospho-S6. The numbers inparentheses represent the concentration of pro-MSP, scMSP, or MSP(from commercial source) in nanogram per milliliter. The values aremean � SD of 3 experiments. C, peritoneal macrophage morphologychange assay. On stimulation with sHepsin-cleaved MSP, peritonealmacrophages underwent distinct changes in cell shape, showedby protrusion and elongation. The effect of hepsin-activated MSPwas comparable with that of a commercially available MSP.sHepsin alone, pro-MSP alone, and uncleavable scMSP had noappreciable effect.

Ganesan et al.





pro-MSP activators like MT-SP1, which are also expressedin macrophages (11).In addition, macrophages were coincubated with pro-

MSP and sHepsin, which resulted in a significant increase(P < 0.001) in peritoneal macrophage migration (Fig. 4A)that was comparable with MSP from a commercialsource. The promigratory effect was due to the generationof active MSP by sHepsin, as neither pro-MSP nor

uncleavable scMSP by themselves increased cell migration(Fig. 4A). In accord with this, the addition of hepsin-neutralizing antibody (Ab25) completely inhibited theincreased promigratory activity found with pro-MSP andsHepsin (Fig. 4A).

Inhibition of nitric oxide synthesisIn 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 associatedincrease in the production of NO in response to stimuli,including LPS (47). The ability of sHepsin to generate MSPthat actively suppresses NO production was examined in acell culture system in which primary mouse bone marrowmacrophages were exposed to LPS. Exposure to LPSresulted in a dramatic increase in macrophage NO produc-tion as measured by nitrite concentration in medium,whereas this response remained unchanged by the additionof pro-MSP alone (Fig. 4B). However, the addition ofsHepsin to pro-MSP in the culture medium led to asignificant attenuation of NO production, comparable tothe effect by mature MSP from a commercial source(Fig. 4B) and in agreement with the approximately 50%reduction (P < 0.0002) of NO production byMSP reportedin the literature (11). Inhibition of sHepsin by Ab25reversed NO production back to control levels, suggestingthat reduction of NO synthesis was entirely mediated bysHepsin-dependent activation of pro-MSP.

Gene expression profiles of MSP and hepsin incomparison to HGFA and MT-SP1Tissue distribution of MSP expression in normal and

disease tissues (Fig. 5A) showed that MSP is expressed mosthighly in liver, followed by kidney, pancreas, and smallintestine. Comparison of this tissue expression profile withthat of hepsin, HGFA, MT-SP1, and RON (Fig. 5A)revealed a similar tissue distribution for hepsin, whichwas also highly expressed in liver, kidney, and pancreasbut not in small intestine. HGFA was also expressed athighest levels in liver, but not in other tissues, whereas MT-SP1 and RON were expressed predominantly in colorectal,small intestine, and stomach samples.To evaluate these gene expression relationships in more

detail, we generated scatter plots of MSP expression againstthat of the other genes in the liver, kidney, pancreas, and smallintestine (Fig. 5B). These scatter plots showed a strongcorrelation of expression between MSP and hepsin amongnormal and disease samples of kidney, liver, and pancreas butnot of small intestine. Coexpression was also observedbetween MSP and HGFA in liver samples and betweenMSP andbothMT-SP1 andRONin small intestinal samples.

Discussion

MSP in its latent form (pro-MSP) has no biological activityand its maturation via proteolytic processing is an importantregulatory step in the MSP/RON signaling pathway. This is

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Figure 4. A, peritoneal macrophage chemotaxis assay. Coincubation ofpro-MSP and sHepsin led to a significant increase in cell migrationcomparable with mature MSP from a commercial source (¼MSP).Pretreatment with a neutralizing anti-hepsin antibody (Ab25) inhibitedmigration. sHepsin alone, pro-MSP alone, and uncleavable scMSP did notstimulate migration. SFM, serum-free medium. After 4 hours of incubationat 37�C, the migrated cells were collected and quantified by fluorescencemeasurements on a microplate reader. The values are mean � SD of 3experiments. *, P < 0.001 versus SFM control; Student's t test. B, MSP-mediated inhibition of nitric oxide synthesis. Treatment of mouse bonemarrow–derived macrophages with LPS dramatically increases NOproduction quantified by the nitrite concentration in culture medium. Nitritelevels remained unchanged after addition of pro-MSP alone or sHepsinalone 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 nitritelevels to LPS control levels. The values are mean � SD of 3 experiments.*, P < 0.002 versus SFM þ LPS; Student's t test.






akin to the related HGF/MET system in which biologicallyactive HGF is generated by an obligatory pro-HGF cleavagereaction. Hepsin activates pro-HGF by cleavage at KQLR#V(19, 34).The scissile peptide sequence of pro-MSP, SKLR#V,has strong similarity to that of pro-HGF as well as to theSQLR#L cleavage sequence of the recently identified hepsinsubstrate laminin-332 (36), suggesting that pro-MSP couldpotentially be a hepsin substrate. Here, we provide severallines of evidence to show that hepsin is an efficient pro-MSPactivator that generates the biologically active 2-chaina/b-heterodimeric MSP signaling molecule.Using the highly purified single chain pro-MSP, both

sHepsin and cell surface–expressed full-length hepsin are

capable of processing pro-MSP at the consensus cleavage site.The proteolytic activity of sHepsin on pro-MSPwas specific,as it was completely inhibited by hepsin inhibitors, such asKD1, Ac-KQLR-cmk, and most importantly, a neutralizinganti-hepsin antibody (Ab25). Additional evidence for thespecificity of the cleavage reaction came from experimentswith the cleavage site mutant scMSP, which remainedintact on prolonged treatment with high concentrations ofsHepsin. The pro-MSP cleavage site sequence recognizedby hepsin is consistent with a preference for arginine at P1,leucine at P2, and lysine at P3 positions (19). On the basisof the published structure of hepsin with bound Ac-KQLR-cmk (pro-HGF sequence, PDB 1Z8G; ref. 19),

Figure 5. A, MSP expression innormal and diseased tissues. MSPis most highly expressed in liver,followed by kidney, pancreas, andsmall intestine. Comparison of thistissue expression profile with thatof hepsin, HGFA, MT-SP1, andRON reveals a similar tissuedistribution for hepsin, which isalso highly expressed in liver,kidney, and pancreas but not insmall intestine. HGFA is alsoexpressed at highest levels in liver,but not in other tissues, whereasMT-SP1 and RON are ubiquitouslyexpressed with highestexpression in colorectal, smallintestine, and stomach samples.

Ganesan et al.





we built a model with the pro-MSP sequence SKLR(Supplementary Fig. S3). The replacement of a P4-Lys/P3-Gln with a P4-Ser/P3-Lys should have minimal effects,as the modeled SKLR peptide also orients its side chains ina very similar position as that of the KQLR peptide. Inaddition, there are potential hydrogen bond (distance < 3A�) interactions of the SKLR residues P4-Ser and P3-Lys

with the hepsin residues Gln175b and Tyr146 (Supplemen-tary Fig. S3). These additional interactions may conferfavorable effects on the preference of substrates with aserine at P4 and lysine at P3 positions.Cleavage of pro-MSP at the Arg483-Val484 bond leads to

the formation of the receptor-binding site on the MSPb-chain that is not present on pro-MSP (3). In accordance,sHepsin-cleaved MSP was capable of binding to its receptorwith high affinity as determined by surface plasmon reso-nance and ELISA experiments, suggesting that the cleavedMSP is functionally competent. This was further establishedin a set of RON-dependent cellular activity assays, namely,the phosphorylation of downstream signaling proteins inRON-expressing A2780 cells, the change in chemotaxis andmorphology in peritoneal macrophages, and the attenuationof LPS-induced NO production in bone marrow–derivedmacrophages. Consistently, RON-mediated cellularresponses were elicited only when cells were exposed tothe combination of pro-MSP and sHepsin but not toindividual pro-MSP, scMSP, or sHepsin treatments. Theseresults strongly suggest that pro-MSP processing generates afunctional MSP signaling molecule with an activity indis-tinguishable from MSP from a commercial source. A recent

report showed that recombinant human hepsin was unableto cleave pro-MSP, yet was able to cleave the internallyquenched fluorescence peptide encompassing the pro-MSPcleavage sequence SKLR-VVGG (P4-P40; ref. 48). Althoughthere is no straightforward explanation for these discrepantresults, it may be possible that the quality and the source ofthe pro-MSP used by Beliveau and colleagues (48) could be areason for the different findings.The in vitro reaction conditions, specifically the high

substrate to enzyme ratio and the short reaction time,suggested that hepsin is a highly efficient pro-MSP acti-vator. This view was further substantiated by a comparisonwith 2 recently identified pro-MSP activators MT-SP1and HGFA, suggesting that hepsin has superior pro-MSPconverting activity. A caveat is that the assays used onlyextracellular portions of MT-SP1 and hepsin, both ofwhich are integral cell surface proteases of the TTSPfamily. Experiments with LNCaP-34 cells, which expressfull-length forms of both hepsin and MT-SP1 (35),indicated that pro-MSP processing was entirely due tohepsin, because no processing was observed in the pre-sence of the hepsin-specific inhibitor Ab25. Althoughthese results are consistent with the potent pro-MSPprocessing activity of sHepsin, they do not imply thatcell surface expressed MT-SP1 lacks pro-MSP convertaseactivity; because LNCaP-34 cells were engineered to over-express hepsin, contributions by basal levels of MT-SP1may have been masked.In addition to pro-MSP, hepsin was shown to activate

pro-HGF and pro-uPA, and to cleave laminin-332 (19,

Figure 5. (Continued ) B,expression of hepsin correlateswell with MSP in liver, kidney, andpancreas (normal, green;diseases, blue) and expression ofHGFA correlates well with MSPonly in liver. MT-SP1 and RONshows minor correlation with MSPexpression in these tissues.






34, 36). The hepsin-mediated processing of these sub-strates activates enzymatic cascades and/or initiates bio-logical pathways that pertain to tumor growth andprogression. Hepsin-activated pro-uPA activates plasmi-nogen to plasmin, which then degrades extracellularmatrix proteins directly or indirectly by activating latentforms of matrix metalloproteases (49, 50). Degradation ofstromal components and basement membranes are criticalfor tumors to invade and metastasize, and it constitutes ahallmark for increased tumor aggressiveness. Similarly,hepsin enzymatic activity toward the basement membraneconstituent laminin-332, which generates promigratorylaminin-332 fragments, as well as the hepsin-mediatedactivation of the HGF/MET and MSP/RON signalingpathways, may promote tumor invasion and progression.For instance, hepsin is highly upregulated in prostatecancer epithelial cells (20, 28), which also express RON(51, 52). RON was found to regulate the production ofangiogenic chemokines promoting tumor growth andangiogenesis (52, 53). Therefore, the increased expressionof hepsin in prostate cancer could activate RON-depen-dent signaling in cancer cells to promote cancer progres-sion. In vivo studies using mouse prostate tumor modelssupport a role of hepsin in basement membrane degrada-tion and in tumor invasion and metastasis (28, 29).The plasminogen-like growth factors MSP and HGF

share the same domain architecture and activation mechan-ism and initiate intracellular signaling pathways that lead toproliferation, migration, and differentiation (Fig. 6). Thefact that the single-chain precursors, pro-HGF and pro-MSP, have no biological activity strongly suggests that the

activating proteases are critical in regulating the MSP/RONand HGF/MET pathways. It is intriguing that both pro-MSP and pro-HGF are activated by the 3 trypsin-like serineproteases hepsin, MT-SP1, and HGFA and that all 3proteases are inhibited by the same 2 Kunitz domaininhibitors, HAI-1 and HAI-2 (refs. 40, 41, 54; Fig. 6).This could mean that they are components of the samebiological systems involving growth factor–mediated cellu-lar responses such as tissue repair and tumorigenesis. Giventhe assumption that latent growth factors are abundantlypresent in the extracellular space (55–57), this protease/inhibitor system could control the availability of biologicallyactive growth factors in the microenvironment. Except forHGFA, the proteases, inhibitors, and growth factor recep-tors (RON and MET) are integral cell surface–expressedproteins, which may allow for highly localized reactions andtheir regulation by inhibitors.Recently, gene expression profiling used to identify MT-

SP1 as a pro-MSP activator showed that both MSP andMT-SP1 expression correlated in normal and disease tissues(11). In this report, we detected a strong correlation of MSPexpression with hepsin expression in the liver, kidney, andpancreas that was superior to the corresponding correlationsobserved between MSP and HGFA or MSP and MT-SP1.Both hepsin and MSP are produced by hepatocytes in theliver (58, 59) and renal tubule cells in the kidney (58, 60),which were recently shown to increase MSP productionduring the regenerative phase in a mouse renal injury model(60). In light of the potent pro-MSP convertase activity forhepsin in vitro, the coexpression results suggest that hepsinmay regulate pro-MSP activation in tissue homeostasis orafter tissue injury. The biochemical linkage between hepsinwith the MSP/RON system presented in this study is likelyto form the basis for further investigations directed atunderstanding the biological regulation of the MSP/RON pathway by proteases under normal and pathophy-siologic conditions.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank Kaushiki Mahapatra for running the phosphorylation assay,Jose Zavala for animal husbandry, Greg Bennett and Daniel Tran for their helpwith 125I-labeling of pro-MSP, and Wendy Sandoval for N-terminal amino acidsequencing.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received January 3, 2011; revised June 2, 2011; accepted June 27, 2011;published OnlineFirst August 29, 2011.

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Figure 6. Model of protease/inhibitor system regulating growth factorsignaling. The activation of the MSP/RON and HGF/MET signalingpathways is dependent on the availability of the biologically active forms ofMSP and HGF. The 3 trypsin-like serine proteases hepsin, HGFA, andMT-SP1 can activate the biologically inactive latent growth factors (pro-MSPand pro-HGF) by enzymatic cleavage at the consensus Arg-Val bond togenerate the active a/b heterodimers MSP and HGF. The enzymaticactivities of the proteases are, in turn, regulated by the cell surface–expressed Kunitz domain inhibitors HAI-1 and HAI-2.

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2011;9:1175-1186. Published OnlineFirst August 29, 2011.Mol Cancer Res Rajkumar Ganesan, Ganesh A. Kolumam, S. Jack Lin, et al. HepsinProteolytic Activation of Pro-Macrophage-Stimulating Protein by

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