Identification of membrane-bound serine proteinase matriptase as processing enzyme of insulin-like growth factor binding protein-related protein-1 (IGFBP-rP1/angiomodulin/mac25) Sanjida Ahmed1,2, Xinlian Jin1, Motoki Yagi1,2, Chie Yasuda1, Yuichiro Sato1,2, Shouichi Higashi1, Chen-Yong Lin3, Robert B Dickson3 and Kaoru Miyazaki1,2 Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, Japan Graduate School of Integrated Sciences, Yokohama City University, Japan Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA Keywords angiomodulin; insulin-like growth factor; insulin-like growth factor binding proteinrelated protein-1; matriptase; proteolytic processing Correspondence K Miyazaki, Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, 641–12 Maioka-cho, Totsuka-ku, Yokohama 244–0813, Japan Fax: +81 458201901 Tel: +81 458201905 E-mail: miyazaki@yokohama-cu.ac.jp (Received 25 August 2005, revised 15 November 2005, accepted December 2005) doi:10.1111/j.1742-4658.2005.05094.x Insulin-like growth factor (IGF) binding protein-related protein-1 (IGFBP-rP1) modulates cellular adhesion and growth in an IGF ⁄ insulindependent or independent manner It also shows tumor-suppressive activity in vivo We recently found that a single-chain IGFB-rP1 is proteolytically cleaved to a two-chain form by a trypsin-like, endogenous serine proteinase, changing its biological activities In this study, we attempted to identify the IGFBP-rP1-processing enzyme Of nine human cell lines tested, seven cell lines secreted IGFBP-rP1 at high levels, and two of them, ovarian clear cell adenocarcinoma (OVISE) and gastric carcinoma (MKN-45), highly produced the cleaved IGFBP-rP1 Serine proteinase inhibitors effectively blocked the IGFBP-rP1 cleavage in the OVISE cell culture The conditioned medium of OVISE cells did not cleave purified IGFBP-rP1, but their membrane fraction had an IGFBP-rP1-cleaving activity The membrane fraction contained an 80-kDa gelatinolytic enzyme, which was identified as the membrane-type serine proteinase matriptase (MT-SP1) by immunoblotting When the membrane fraction was separated by SDS ⁄ PAGE, the IGFBP-rP1-cleaving activity comigrated with matriptase A soluble form of matriptase purified in an inhibitor-free form efficiently cleaved IGFBP-rP1 at the same site as that found in a naturally cleaved IGFBP-rP1 Furthermore, small interfering RNAs for matriptase efficiently blocked both the matriptase expression and the cleavage of IGBP-rP1 in OVISE cells These results demonstrate that IGFBP-rP1 is processed to the two-chain form by matriptase on the cell surface Insulin-like growth factor (IGF) binding proteins (IGFBPs) regulate cellular proliferation by modulating the actions of insulin and IGFs [1,2] Recent studies have revealed a group of IGFBP-related proteins (IGFBP-rPs), which have low affinity for IGFs ⁄ insulin and low structural homology to IGFBPs [3] Abbreviations AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride; AGM, angiomodulin; FBS, fetal bovine serum; HAI-1, hepatocyte growth factor activator inhibitor-1; HGF, hepatocyte growth factor; HLE, hepatocellular carcinoma; IGF, insulin-like growth factor; IGFBP, IGF-binding protein; IGFPB-rP1, IGFBP-related protein-1; MMP, matrix metalloproteinase; MT-SP1, membrane-type serine proteinase matriptase; PSF, prostacyclin-stimulating factor; TAF, tumor-derived cell adhesion factor; siRNA, small interfering RNA; tPA, tissue plasminogen activator uPA, urokinase-type plasminogen activator FEBS Journal 273 (2006) 615–627 ª 2006 The Authors Journal compilation ª 2006 FEBS 615 Processing of IGFBP-rP1 by matriptase S Ahmed et al Angiomodulin (AGM) was initially purified as a tumor-derived cell adhesion factor (TAF) from human bladder carcinoma cells [4,5] Its cDNA was cloned from human leptomeningial cells as mac25 [6] and from human fibroblasts as prostacyclin-stimulating factor (PSF) [7] Since AGM has a relatively low structural homology to IGFBPs, the name of IGFBP-related protein-1 (IGFBP-rP1) has recently been proposed for AGM ⁄ mac25 ⁄ PSF AGM ⁄ IGFBP-rP1 exerts a weak cell adhesion activity through heparan sulfate proteoglycans on the cell surface [4,5,8] and stimulates cell growth in culture medium containing insulin or IGFs [9,10] The IGFBP-rP1 mRNA is expressed in a wide range of normal tissues including the heart, spleen, ovary, small intestine and colon [11] Immunohistochemical analysis has shown that IGFBP-rP1 is highly expressed in the blood vessels of various human cancer tissues [5] and in invading tumor cells [12] On the other hand, other studies have shown that IGFBP-rP1 exhibits a tumor-suppressive activity when overexpressed in cancer cells [13–15] Thus, exact biological functions of IGFBP-rP1 remain to be clarified Various extracellular proteinases regulate cellular functions by degrading or processing protein substrates including extracellular matrix proteins, growth factors and cell surface proteins For example, matrix metalloproteinases (MMPs), such as membrane type-1 matrix metalloproteinase (MT1-MMP), matrilysin (MMP-7) and gelatinases A ⁄ B (MMP-2 ⁄ 9), are known to play important roles in the process of tumor invasion and metastasis [16–18] Serine proteinases such as plasminogen activators, plasmin and trypsin also contribute to expression of malignant phenotypes in tumor cells [19,20] Recently, considerable attention has been focused on the physiological and pathological functions of a membrane-bound serine proteinase, matriptase (MT-SP1) [21–23] It is well known that IGFBPs often undergo proteolytic processing in various kinds of biological fluids such as blood, synovial fluid and interstitial fluid, as well as culture media [24,25] Several types of proteinases, such as pregnancy-associated plasma proteins [26,27], prostate specific antigen [28] and MMP-3 [29], have been reported to cleave IGFBPs The proteolysis of IGFBPs is thought to modulate the actions of IGFs towards cells [29] We recently found that IGFBP-rP1 is converted from a single-chain form to a two-chain form by the action of a trypsin-like serine proteinase [10] The proteolytic processing of IGFBP-rP1 greatly reduced its insulin ⁄ IGF-dependent growth promoting activity but enhanced its syndecan-1-mediated cell adhesion activity [10] We report here that the membrane-bound serine 616 proteinase matriptase is responsible for the processing of IGFBP-rP1 Results Expression and processing of IGFBP-rP1 in various cell lines We have reported that IGFBP-rP1 is proteolytically converted to a two-chain form during purification [10] The cleavage of IGFBP-rP1 leads to complete loss of insulin ⁄ IGF-1-dependent cell growth-stimulatory activity due to its loss of insulin ⁄ IGF-binding ability To examine whether the specific cleavage of IGFBPrP1 also occurs in cultured cell systems, we tested expression and processing of IGFBP-rP1 in eight human cancer cell lines and one immortalized epithelial cell line (HEK293) When the conditioned media were analyzed by immunoblotting after reducing SDS ⁄ PAGE, seven of the nine cell lines tested were found to secrete IGFBP-rP1 protein (Fig 1) Among them, OVISE ovarian adenocarcinoma cells and MKN-45 gastric adenocarcinoma cells secreted significant levels of the 25-kDa, cleaved form of IGFBP-rP1 On nonreducing SDS ⁄ PAGE, IGFBP-rP1 in the twoconditioned media was separated to a single band of 33 kDa (data not shown), indicating that the cleaved IGFBP-rP1 was a two-chain form consisting of a 25 kDa chain and an kDa chain [10] These results suggested that OVISE and MKN-45 cells expressed a high level of proteinase(s) responsible for the processing of IGFBP-rP1 To determine the class of the IGFBP-rP1-cleaving proteinase, OVISE cells were cultured in the presence of various proteinase inhibitors and the processing of IGFBP-rP1 was analyzed As shown in Fig 2, serine proteinase inhibitors, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) and aprotinin, significantly inhibited the production of the cleaved form of IGFBP-rP1 (25 kDa), whereas a cysteine proteinase inhibitor (leupeptin), an aspartic proteinase inhibitor (pepstatin) and a metalloproteinase inhibitor (N-(R)(2-(hydroxyaminocarbonyl)methyl)-4-methylpentanoyl-l3-(2¢-naphthyl)alaninyl-l-alanine 2-aminoethyl amide; TAPI-1) did not affect the processing A commercially available proteinase inhibitor mixture containing AEBSF, aprotinin and some other inhibitors inhibited the processing to the same extent as AEBSF alone As trypsin-type serine proteinases, but neither chymotrypsin-type nor elastase-type, are susceptible to the AEBSF inhibition, a trypsin-type serine proteinase was thought to be responsible for the IGFBP-rP1 cleavage FEBS Journal 273 (2006) 615–627 ª 2006 The Authors Journal compilation ª 2006 FEBS S Ahmed et al Processing of IGFBP-rP1 by matriptase Fig Analysis of noncleaved and cleaved forms of IGFBP-rP1 in conditioned media of eight cancer cell lines and one immortalized cell line (HEK293) Concentrated conditioned media were prepared from the cultures of the nine indicated human cell lines, and each sample containing the same amount of protein (5 lg) was subjected to SDS ⁄ PAGE under reducing conditions on a 14% gel, followed by immunoblotting with the antiTAF ⁄ IGFBP-rP1 antibody (upper panel) Bars indicate the noncleaved form (33 kDa) and the cleaved form (25 kDa) The cleaved form is detected highly in OVISE and MKN-45 cell lines and slightly in HLE and HEK293 cell lines As a loading control, the same volumes of conditioned media as those for the immunoblotting were subjected to SDS ⁄ PAGE followed by protein staining with Coomassie Brilliant Blue R-250 (lower panel) Bars indicate the molecular size in kDa Other experimental conditions and the types of the cell lines used are described in ‘Experimental procedures’ Fig Effects of proteinase inhibitors on processing of IGFBP-rP1 in culture of OVISE cells OVISE ovarian carcinoma cells were incubated in a serum-free culture medium supplemented without (None) or with one of the following proteinase inhibitors: AEBSF (100 lM), aprotinin (75 nM), leupeptin (10 lM), pepstatin A (1 lM), TAPI-1 (2 lM), and a mixture (100 lM AEBSF, 75 nM aprotinin, lM bestatin, 1.5 lM E-64, lM leupeptin, and lM pepstatin A) After incubation for days, IGFBP-rP1 in each conditioned medium was analyzed by immunoblotting, as described in Fig The upper panel shows immunoblots for matriptase Bars indicate the noncleaved form (33 kDa) and the cleaved form (25 kDa) of IGFBP-rP1 The lower panel shows protein staining patterns of conditioned media Bars indicate the molecular size in kDa Analysis of serine proteinases secreted by various cell lines Trypsin-type serine proteinases present in the conditioned media of the nine cell lines were analyzed by gelatin zymography (Fig 3A) To eliminate the activities of metalloproteinases, the renatured gel was first treated with mm EDTA and then incubated in the presence of 10 mm CaCl2, as described in Experimental procedures Of the nine cell lines tested, OVISE, FEBS Journal 273 (2006) 615–627 ª 2006 The Authors Journal compilation ª 2006 FEBS 617 Processing of IGFBP-rP1 by matriptase EJ -1 EC V3 HT 10 80 OV IS E M KN -4 HL E HS C4 DL D1 HE K2 93 A S Ahmed et al 110 95 75 93 K2 HE D- CHS E HL DL -45 KN M E OV IS 80 10 HT V30 EC EJ -1 B 110 95 75 MKN-45 and DLD-1 cells commonly expressed a major gelatinolytic activity at approximately 75 kDa and additional weak activities at 95 kDa, 105 kDa and lower molecular weight positions The 75 kDa activity was faintly detected in HSC-4 and HEK293 cells To date, several families of type II transmembrane serine proteinases have been identified [30] Among them, MT-SP1 is known to be expressed in many types of epithelial cell lines and carcinoma cell lines, and to be proteolytically released from the cell membranes [21,31,32] The molecular size of a major form of soluble matriptase is approximately 75 kDa To determine whether or not the 75 kDa proteinase in Fig 3A was matriptase, the conditioned media of the nine cell lines were subjected to immunoblotting with an antimatriptase antibody (M32) As shown in Fig 3B, the conditioned media of OVISE, MKN-45 and DLD-1 cells clearly showed doublet bands at approximately 75 kDa and two minor bands at 95 and 110 kDa The conditioned media of HSC-4 and HEK293 cells also showed weak immunoreactive bands at the same posi618 Fig Analysis of serine proteinases secreted by nine human cell lines Conditioned media were prepared from the cultures of the indicated, nine human cell lines The concentrated conditioned media containing the same amount of protein (5 lg) were subjected to gelatin zymography (A) and immunoblotting with the antimatriptase antibody M32 (B) as described in ‘Experimental procedures’ In the gelatin zymography, metalloproteinase activities were eliminated by incubating the renatured gel with mM EDTA for 30 (A) Gelatin zymograms of the conditioned media from the nine cell lines The conditioned media of OVISE and MKN-45, which showed high IGFBP-rP1 cleavage in Fig 1, showed a major activity at 75 kDa and minor activities at 95 and 110 kDa, which were indicated by bars (B) Immunoblots for matriptase The 75 kDa major band seems to correspond mainly to a single-chain, latent matriptase, while the 95 and 110 kDa bands seem to correspond to two-chain, active forms complexed with HAI-1 [18,32] tions These results attributed the gelatinolytic activities at 75, 95 and 105 kDa in Fig 3A to matriptase The 75 kDa doublet has been reported to result from glycosylation [33] The glycosylation is thought to increase the stability of this protein against trypsin and other proteinases [34] To further characterize the gelatinolytic activities in the OVISE cell conditioned medium, we examined effect of a serine proteinase inhibitor, AEBSF, on the proteinase activities (Fig 4) The sample was treated with mm AEBSF before and ⁄ or after SDS ⁄ PAGE and subsequent renaturation of separated proteins The pretreatment of the sample with the inhibitor partially blocked only the 75 kDa activity, whereas the inhibitor treatment after the protein renaturation strongly blocked the activities at 75, 95 and 110 kDa This indicated that only a part of the 75 kDa enzyme existed in an inhibitor-free active form in the concentrated conditioned medium A weak activity of 40 kDa was not inhibited by the inhibitor, suggesting that it might be a metalloproteinase It has been reported that FEBS Journal 273 (2006) 615–627 ª 2006 The Authors Journal compilation ª 2006 FEBS S Ahmed et al Processing of IGFBP-rP1 by matriptase The correlation between the IGFBP-rP1-cleaving activity (Fig 1) and the expression of matriptase in MKN-45 and OVISE cell lines suggested matriptase as a candidate for the IGFBP-rP1-processing enzyme Moreover, it has been reported that matriptase prefers basic P1, P3 and P4 residues to cleave substrates [33] This consensus sequence is found in the cleavage site sequence of IGFBP-rP1, where Lys97, Lys95 and Arg94 residues are located at P4, P3 and P1 sites, respectively [10] These facts prompted us to examine whether or not matriptase is the IGFBP-rP1-processing enzyme However, when purified IGFBP-rP1 was incubated with the conditioned medium of OVISE or DLD-1 cells, the latter of which contained the highest amount of matriptase, its processing to a two-chain form was not observed (data not shown) Fig Effect of serine proteinase inhibitor AEBSF on gelatinolytic activities of OVISE cell conditioned medium AEBSF was added to make a final concentration of mM into the OVISE cell conditioned medium before SDS ⁄ PAGE (lanes and 4) and ⁄ or into the reaction buffer after the SDS ⁄ PAGE and subsequent renaturation (lanes and 4) –, No addition, +, addition Ordinate indicates the molecular size in kDa Arrowheads show the matriptase bands Other experimental conditions are described in ‘Experimental procedures’ and in the legends to Fig 3A membrane-bound matriptase is shed into culture medium in some different forms [21] The soluble matriptase includes a 70 kDa single-chain, latent form as a major component, a two-chain, active enzyme complexed with two different sizes of hepatocyte growth factor (HGF) activator inhibitor (HAI-1) (total molecular sizes of 95 and 110 kDa), and a trace of an inhibitor-free active enzyme [21,31,32] The 95 and 110 kDa matriptase ⁄ HAI-1 complexes are expected not to react with AEBSF, because their active serine residues are bound to HAI-1 Based on these facts, the 95- and 110-kDa bands in zymography (Figs 3A and 4) and immunoblotting (Fig 3B) are most likely to correspond to the matriptase ⁄ HAI-1 complexes The 95- and 110-kDa gelatinolytic activities seemed to result from partial dissociation of an active matriptase from its inhibitor HAI-1 after SDS ⁄ PAGE Similarly, the 75-kDa activity that was not inhibited by the pretreatment with AEBSF was thought to be an active matriptase, which had been dissociated from HAI-1 by the SDS treatment In addition, immunoblotting under reducing conditions suggested that the majority of the 75-kDa immunoreactive band was a single-chain latent matriptase (data not shown) [32] IGFBP-rP1-cleaving activity of membrane fraction from OVISE cells As IGFBP-rP1 is cleaved in cultured OVISE cells, we next examined the possibility that cell-associated serine proteinase(s) might cleave IGFBP-rP1 A membrane fraction of OVISE cells was prepared as described under ‘Experimental procedures’ and incubated with purified IGFBP-rP1 Figure 5A (lanes and 2) shows that the cleaved form of IGFBP-rP1 significantly increased during the incubation This cleavage was inhibited effectively by aprotinin, suggesting that a serine proteinase(s) catalyzed this processing (Fig 5A, lane 3) We further analyzed serine proteinase activities in the membrane fraction of OVISE cells by gelatin zymography This analysis revealed the presence of a gelatinolytic enzyme, with an approximate molecular size of 80 kDa, in the membrane fraction (Fig 5B, lane 1) When analyzed on the same gel, the apparent molecular size of the gelatinolytic activity in the membrane was slightly higher than the 75 kDa activity found in the conditioned medium (data not shown) Immunoblotting with the antimatriptase antibody showed an immunoreactive band at the same position as the gelatinolytic activity (Fig 5B, lane 2), suggesting that the 80-kDa serine proteinase was a membrane-bound matriptase To verify that the 80 kDa proteinase has the IGFBP-rP1-cleaving activity, we separated the proteins in the membrane fraction of OVISE cells by SDS ⁄ PAGE After electrophoresis, the proteins on the gel were renatured and the gel was horizontally divided into eight equal parts Each piece of the gel was then incubated with purified IGFBP-rP1 and the cleavage of the protein was analyzed as described under Experimental procedures As shown in Fig 6A, gel fractions FEBS Journal 273 (2006) 615–627 ª 2006 The Authors Journal compilation ª 2006 FEBS 619 Processing of IGFBP-rP1 by matriptase A S Ahmed et al 33 25 B 80 80 Fig Analysis of IGFBP-rP1-cleaving enzyme present in membrane fraction of OVISE cells A membrane fraction was prepared from OVISE cells as described in ‘Experimental procedures’ and used for the following assays (A) IGFBP-rP1-cleaving activity Purified IGFBP-rP1 (500 ng) was incubated with the membrane fraction (20 lg protein) in the presence (lane 3) or absence (lane 2) of 75 nM aprotinin (serine proteinase inhibitor) The incubated samples were subjected to immunoblotting under reducing conditions with the anti-TAF ⁄ IGFBP-rP1 antibody Lane 1, no incubation Bars indicate the noncleaved form (33 kDa) and the cleaved form (25 kDa) (B) Gelatin zymography (lane 1) and immunoblotting with the antimatriptase antibody M32 The membrane fraction containing 15 lg protein was run on a gelatin-containing gel under nonreducing conditions for the zymography (lane 1), and the same sample containing 10 lg protein was subjected to immunoblotting with the antimatriptase antibody M32 The bar indicates a gelatinolytic band at approximately 80 kDa in lane and a matriptase band at almost the same position in lane Other experimental conditions are described in ‘Experimental procedures’ 4–6 had the IGFBP-rP1-cleaving activity and fraction showed the highest activity When the proteins in the gel pieces were analyzed by immunoblotting, an 80 kDa matriptase was also detected in fraction 620 Fig Fractionation of IGFBP-rP1-cleaving enzyme and matriptase present in OVISE cell membrane by SDS ⁄ PAGE The membrane fraction (20 lg proteinỈlane)1) of OVISE cells was separated by nonreducing SDS ⁄ PAGE on two lanes of a 7.5% gel After the gel was washed with 1% Triton X-100 for protein renaturation, each of the two lanes was divided into eight fractions and used for one of the two following assays (A) IGFBP-rP1-cleaving activity Each gel fraction was incubated with IGFBP-rP1 (500 ng) at °C for 24 h followed by 37 °C for 24 h The incubated samples were subjected to immunoblotting under reducing conditions with the anti-TAF ⁄ IGFBP-rP1 antibody (None), IGFBP-rP1 incubated without gel fraction Bars indicate the noncleaved form (33 kDa) and the cleaved form (25 kDa) Fraction shows the highest activity (B) Fractionation of matriptase Gel fractions from another lane were used to detect matriptase Each gel fraction was extracted with the SDS sample buffer and applied to SDS ⁄ PAGE under nonreducing conditions on a 10% gel and immunoblotted with the antimatriptase antibody M32 An arrow indicates an immunoreactive band of matriptase MF, the membrane fraction before separation Other experimental conditions are described in ‘Experimental procedures’ (Fig 6B), indicating that this membrane-bound serine proteinase is the prime candidate for the IGFBP-rP1cleaving proteinase in OVISE cells IGFBP-rP1-cleaving activity of purified matriptase The results of the SDS ⁄ PAGE separation of the membrane-bound enzyme suggested that the soluble form of matriptase might cleave IGFBP-rP1 if separated from inhibitors To test this possibility, the conditioned medium of DLD-1 cells, which contained the highest activity of matriptase, was separated by SDS ⁄ PAGE and assayed for the IGFBP-rP1-cleaving FEBS Journal 273 (2006) 615–627 ª 2006 The Authors Journal compilation ª 2006 FEBS S Ahmed et al Processing of IGFBP-rP1 by matriptase activity The IGFBP-rP1-cleaving activity was detected at fractions corresponding to approximately 75 and 95 kDa, which also showed immunoreactive bands to the antimatriptase antibody in immunoblotting (data not shown) A similar result was also obtained when the conditioned medium of OVISE cells was separated by SDS ⁄ PAGE (data not shown) These results strongly suggested that soluble forms of matriptase are able to cleave IGFBP-rP1 in the absence of HAI-1 or other inhibitors To further confirm this possibility, we purified an inhibitor-free, soluble form of matriptase from the conditioned medium of DLD-1 cells, as described in ‘Experimental procedures’ The purified matriptase preparation contained the 75-kDa matriptase and a few minor proteins, as analyzed by SDS ⁄ PAGE (Fig 7A) Gelatin zymography under nonreducing conditions confirmed that the 75 kDa matriptase had a proteolytic activity (Fig 7B) Immunoblotting analysis under nonreducing conditions showed a single immunoreactive band for matriptase at 75 kDa, but under reducing conditions it was split into a major 75-kDa A B C band of the single-chain, latent enzyme and a minor 50 kDa band of the two-chain, active enzyme (Fig 7C) [10] Based on the relative band intensity, the percentage of the active enzyme to the total matriptase was estimated to be approximately 30% We previously reported that trypsin cleaves IGFBPrP1 at the same site as an endogenous processing enzyme [10] The IGFBP-rP1-cleaving activities of matriptase and trypsin were compared at varied concentrations (Fig 8) Fifty nanograms of the 33 kDa IGFBP-rP1 was almost completely cleaved to the 25 kDa form by ng of the total matriptase, which contained the active enzyme as a minor component On the other hand, 10 ng of trypsin converted a major part of the 33 kDa IGFBP-rP1 to the 25 kDa form, but an increased amount (50 ng) of trypsin nonspecifically degraded both forms of IGFBP-rP1 These results indicated that the two-chain, active matriptase has a much higher IGFBP-rP1-cleaving activity than trypsin Furthermore, we tried to identify the cleavage site of IGFBP-rP1 by the purified matriptase The 25 kDa form of IGFBP-rP1 obtained by the treatment with matriptase was applied to an automated protein sequencer The N-terminal amino acid sequence of the 25 kDa band of IGFBP-rP1 was determined to be A98GAAAGGPG106, suggesting that this protein had been cleaved between K(Lys)97 and A(Ala)98 This cleavage site was identical to that previously determined for a naturally cleaved IGFBP-rP1 All these results demonstrate that the soluble form of active matriptase cleaves IGFBP-rP1 but HAI-1 or some other inhibitors block its activity in culture medium Effects of matriptase siRNAs on IGFBP-rP1 cleavage in OVISE cells Fig Electrophoretic analyses of purified matriptase Soluble matriptase was purified from the conditioned medium of DLD-1 cells as described in the text The purified matriptase (approximately 200 ng of total proteins) was subjected to the following analyses (A) SDS ⁄ PAGE under nonreducing conditions followed by silver staining Arrowhead indicates the matriptase band Bars indicate the molecular size in kDa (B) Gelatin zymography Arrowhead indicates the gelatinolytic activity by matriptase (C) Immunoblotting with the antimatriptase antibody M32 under nonreducing conditions (lane 1) and reducing conditions (lane 2) In lane 2, the 75-kDa band corresponds to the single-chain, latent enzyme, while the 50 kDa band corresponds to the heavy chain of the two-chain, active enzyme Other experimental conditions are described in ‘Experimental procedures’ To show that matriptase is an endogenous IGFBPrP1-processing enzyme in OVISE cells, we designed three siRNAs for matriptase and examined their effects on the IGFBP-rP1 cleavage As negative controls, OVISE cells were treated with a scrambled RNA or with the lipofectamine reagent alone Although the scrambled RNA had some cytotoxic effect, there was no significant difference in the profiles of secreted proteins among the control and siRNA-treated cultures (Fig 9A) Immunoblotting with the antimatriptase antibody showed that all of the three siRNAs decreased the amount of soluble matriptase to less than 20% of the control levels (Fig 9B) When the IGFBP-rP1 secreted into culture medium by OVISE cells was analyzed by immunoblotting, contrasting patterns of the secreted IGFBP-rP1 were obtained FEBS Journal 273 (2006) 615–627 ª 2006 The Authors Journal compilation ª 2006 FEBS 621 Processing of IGFBP-rP1 by matriptase S Ahmed et al Fig Cleavage of IGFBP-rP1 by purified matriptase and trypsin (A) Matriptase IGFBP-rP1 (50 ng) was incubated with the indicated amounts of purified matriptase at 37 °C for h in 10 lL of a reaction mixture containing 20 mM Tris ⁄ HCl (pH 7.5), 0.1 M NaCl and 10 mM CaCl2 The original concentration of matriptase was determined for the total 75-kDa protein based on the band intensity relative to that of bovine serum albumin as standard (Fig 7A) (B) Trypsin IGFBP-rP1 was incubated with the indicated amounts of TPCK-trypsin under the same conditions except for the absence of CaCl2 in the reaction mixture The proteolytic cleavage of IGFBP-rP1 in (A) and (B) was analyzed by immunoblotting as described in Fig Bars indicate the 33 kDa, uncleaved form and the 25 kDa, cleaved form of IGFBP-rP1 A B C Fig Effects of matriptase siRNAs on IGFBP-rP1 processing in culture of OVISE cells OVISE cells at 50–60% confluence in 60 mm culture dishes were transfected with 200 pmol of each of three siRNAs (si973, si1513 and si2578) using Lipofectamine 2000 reagent As negative controls, the cells were treated with a scrambled RNA (sc) or with the lipofectamine reagent alone (Cont.) These cultures were incubated in serum-containing medium overnight, and then in serum-free medium for days The resultant conditioned media were collected and concentrated The concentrated samples containing 10 lg protein were subjected to reducing SDS ⁄ PAGE followed by the Coomassie Brilliant Blue staining (A), nonreducing immunoblotting to detect matriptase (B), and reducing immunoblotting to detect IGFBP-rP1 (C) Bars indicate the molecular sizes of marker proteins in (A), the 110-, 95- and 75-kDa bands of matriptase in (B), and the 33-kDa, uncleaved form and the 25-kDa, cleaved form of IGFBP-rP1 in (C) The scrambled RNA-treated culture (2nd lane) was low in the total band intensity of matriptase (B) and IGFBP-rP1 (C) as compared with the control or siRNA-treated cultures This seemed due to the cytotoxic effect of the scrambled RNA Other experimental conditions are described in Experimental procedures between the control and siRNA-treated cultures (Fig 9C) In the two control cultures, the 33-kDa, uncleaved IGFBP-rP1 was faintly detected compared with the 25-kDa, cleaved form, whereas in the siRNAtreated cultures, the uncleaved IGFBP-rP1 was a major component This indicated that the cleavage of IGFBP-rP1 was effectively reduced by the siRNA treatment These results confirmed that matriptase acts 622 as an IGFBP-rP1-processing enzyme in the culture of OVISE cells Discussion In this study, we first identified the type II membranebound serine proteinase matriptase as a processing enzyme of IGFBP-rP1 ⁄ AGM Matriptase was initially FEBS Journal 273 (2006) 615–627 ª 2006 The Authors Journal compilation ª 2006 FEBS S Ahmed et al found as a trypsin-like serine proteinase secreted by human breast cancer cells [35] and later purified as a complex with its natural inhibitor HAI-1 from human milk [21] Matriptase is expressed in normal epithelial tissues such as the skin, stomach, colon, kidney, breast, ovary and pancreas, but not in mesenchyma [36,37] A recent study with matriptase-deficient mice has shown that matriptase plays critical roles in the epidermal barrier function, hair follicle development and thymic homeostasis [38] Matriptase is also expressed in cancer tissues of the breast, ovary, uterus and colon and also by some mammary and ovarian carcinoma cell lines in vitro [36] Since matriptase is able to degrade extracellular matrix proteins and to activate urokinasetype plasminogen activator (uPA) [33,39], hepatocyte growth factor (HGF) [39] and protease-activated receptor-2 (PAR-2) [33], it is expected to play some roles in the growth, invasion and metastasis of human carcinoma cells We previously reported that IGFBP-rP1 is cleaved to a two-chain form by a trypsin-like serine proteinase [10] In the present study, we found that OVISE ovarian carcinoma cells cleaved endogenous IGFBP-rP1, and their membrane fraction cleaved exogenous IGFBP-rP1 in a cell-free solution The IGFBP-rP1cleaving activity in the OVISE cell membrane comigrated with membrane-bound matriptase on SDS ⁄ PAGE Furthermore, the treatment of OVISE cells with matriptase siRNAs efficiently blocked both matriptase expression and the cleavage of IGFBP-rP1 It is also noted that the cleavage sequence of IGFBP-rP1 is consistent with the most preferable sequence in substrate proteins of matriptase [33] Indeed, a soluble form of matriptase purified in an inhibitor-free form efficiently cleaved IGFBP-rP1 at the same site as that found in a naturally cleaved IGFBP-rP1 All these facts indicate that the membrane-bound matriptase is a natural processing enzyme of IGFBP-rP1 On the other hand, soluble forms of matriptase, which are released from cell membranes by proteolysis, were detected in the conditioned media of at least cell lines out of cell lines tested The processing of endogenous IGFBP-rP1 was correlated with the amount of soluble matriptase in the conditioned media Although the purified soluble matriptase could cleave IGFBPrP1, the conditioned media of OVISE and DLD-1 cells did not show the IGFBP-rP1-processing activity unless they were separated by SDS ⁄ PAGE Our recent analysis has detected soluble matriptase in 19 of 24 human carcinoma cell lines tested, which included carcinomas of the breast, lung, stomach and colon [32] The soluble matriptase mostly existed in a single-chain, latent form as a major component and two-chain forms Processing of IGFBP-rP1 by matriptase complexed with its inhibitor HAI-1, in agreement with the past reports [18,30] Therefore, soluble matriptase released from cell membrane is expected to have a very low, if any, proteolytic activity The IGFBP-rP1-cleaving activities of the SDS ⁄ PAGE fractions and the matriptase purified from DLD-1 conditioned medium indicate that the soluble form of activated matriptase can cleave IGFBP-rP1, but its activity is masked by HAI-1 in culture medium It was recently reported that the matriptase zymogen might be auto-activated by interacting with HAI-1 on the cell surface [40] The proteolytic action of matriptase, including the processing of IGFBP-rP1, seems to be restricted to the cell surface and its close vicinity Although the present study demonstrates that matriptase cleaves IGFBP-rP1, our data not exclude the possibility that other proteinases, especially serine proteinases, also cleave IGFBP-rP1 We previously reported that many human cancer cell lines secrete an active or latent form of trypsin and a 75-kDa serine proteinase [20], the latter of which was identified as matriptase in a recent study [32] We have also reported that OVISE cells secrete uPA, but neither trypsin nor tissue plasminogen activator (tPA) [41] In addition, it was previously found that trypsin cleaves IGFBP-rP1 to the same two-chain form as that found in conditioned media [10] In the present study, we examined whether or not uPA and tPA cleave IGFBPrP1 in test tubes, but the IGFBP-rP1 cleavage was seen with neither uPA nor tPA (data not shown) Therefore, matriptase seems to be a major IGFBP-rP1-processing enzyme, at least in OVISE cells, and possibly in MKN-45 cells Recent studies have revealed the presence of four families of type II transmembrane serine proteinase [30] The matriptase subfamily constitutes three members (matriptase, matriptase and matriptase 3) It is conceivable that some of these membrane-bound serine proteinases or their soluble forms are also involved in the processing of IGFBP-rP1 It is well known that some IGFBPs undergo proteolytic cleavage [24,25] In biological fluids, IGFBPs bind IGFs with high affinity, protecting the growth factors from proteolytic degradation The proteolytic cleavage of IGFBPs is thought to contribute to the release of IGFs from IGF ⁄ IGFBP complexes to interact with IGF receptors on the cell surface This mechanism may not be directly applicable to the case of IGFBPrP1, because IGFBP-rP1 has a far lower affinity for IGFs than IGFBPs The high affinity binding of IGFs to IGFBPs limits the interaction of the growth factors with the cell surface receptors Therefore, IGFBPs generally inhibit IGF-stimulated cell growth in vitro [25] In contrast, IGFBP-rP1 stimulates cell growth in the FEBS Journal 273 (2006) 615–627 ª 2006 The Authors Journal compilation ª 2006 FEBS 623 Processing of IGFBP-rP1 by matriptase S Ahmed et al presence of insulin or IGF-1 in vitro, presumably due to its low affinity for the factors [9,10] On the other hand, some studies have shown that IGFBP-rP1 exhibits a tumor-suppressive activity when overexpressed in cancer cells [13–15] The apparent discrepancy between the in vitro and in vivo studies is not clearly explained IGFBP-rP1 also shows IGF ⁄ insulin-independent activities It has affinity for heparin, type IV collagen and syndecan-1 [5,8,10] These activities seem to be responsible for the cell adhesion activity of IGFBP-rP1 in vitro and its dense deposition on the basement membrane of blood vessels in tumor tissues [5] These IGF ⁄ insulin-dependent and independent activities of IGFBP-rP1 are notably altered by its proteolytic cleavage [10] For example, IGFBP-rP1 loses its IGF ⁄ insulin-binding activity and IGF ⁄ insulin-dependent growth-stimulating activity but acquires high cell adhesion activity by proteolytic cleavage It seems conceivable that matriptase regulates tumor growth by modulating biological activities of IGFBP-rP1, as well as other growth-regulating proteins, in vivo In this study, we identified a new substrate of matriptase Matriptase may exert a broad range of functions in regulating cellular growth, apoptosis and differentiation by degrading or processing a variety of extracellular proteins Experimental procedures Materials The sources of materials used are as follows: aprotinin, leupeptin, pepstatin A, AEBSF and proteinase inhibitor mixture that contains AEBSF, aprotinin, bestatin, E-64, leupeptin and pepstatin A from Wako Pure Chemical Industries (Osaka, Japan); gelatin from Difco (Detroit, MI, USA); N-(R)-(2-(hydroxyaminocarbonyl)methyl)-4-methylpentanoyll-3-(2¢-naphthyl)alaninyl-l-alanine 2-aminoethyl amide (TAPI-1) from Peptide Institute (Osaka, Japan) IGFBP-rP1 was purified from the conditioned medium of the human bladder carcinoma cell line EJ-1, as described previously [10] An anti-TAF ⁄ IGFBP-rP1 monoclonal antibody (#88) [5] and an antimatriptase monoclonal antibody (M32) [21] were raised against purified IGFBP-rP1 and purified matriptase, respectively All other chemicals were of analytical grade or the highest quality commercially available Cell cultures and preparation of conditioned medium Types of human cancer cell lines used are as follows: HSC4, tongue squamous cell carcinoma; HT1080, fibrosarcoma; EJ-1 and ECV-304, bladder carcinomas; DLD-1, colon 624 adenocarcinoma; OVISE, ovarian clear cell adenocarcinoma; MKN-45, adenosquamous carcinoma of the stomach; HLE, hepatocellular carcinoma The source and properties of OVISE cells were described before [41] The human embryonic kidney cell line HEK293 (ATCC CRL-1573) was purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) The other cell lines were obtained from Japanese Cancer Resources Bank (JCRB) in National Institute of Biomedical Innovation (Osaka, Japan) To prepare conditioned medium, each cell line was grown to semiconfluence in 90-mm culture dishes containing a : mixture of Dulbecco’s modified Eagles medium and Ham’s F12 medium (Gibco; Grand Island, NY, USA), DME ⁄ F12, supplemented with 10% fetal calf serum (FCS) The cells were rinsed three times with serum-free DME ⁄ F12, and the culture was further continued in the presence or absence of various proteinase inhibitors in serum-free DME ⁄ F12 After incubation for days, the resultant conditioned medium was collected, clarified by centrifugation and dialyzed against distilled water at °C The dialyzed sample was then lyophilized and dissolved in a 100th volume of 10 mm Tris ⁄ HCl (pH 7.5) to the original conditioned medium Preparation of membrane fractions OVISE cells were grown to confluence in the serum-containing medium, rinsed three times with ice cold NaCl ⁄ Pi and then scraped in the presence of 20 mm Hepes (pH 7.5) containing 250 mm sucrose at °C The cell suspension was then homogenized with a Dounce homogenizer The homogenate was centrifuged at 1500 g for to remove the nuclei The postnuclear supernatant was further centrifuged at 50 000 g for 30 The resultant pellet was then dissolved in 20 mm Tris ⁄ HCl (pH 7.5) containing 0.5 m KCl, 0.15 m NaCl and 1% Triton X-100, and the insoluble substances were removed by centrifugation at 14 000 g for 20 The supernatant was used as a crude membrane fraction SDS–polyacrylamide gel electrophoresis (SDS/PAGE) and immunoblotting SDS ⁄ PAGE was performed on 7.5, 10, or 14% polyacrylamide gel slabs (85 mm wide, mm thick, and 70 mm long) under reducing or nonreducing conditions Separated proteins were stained with silver Immunoblotting was performed as described previously [42] Briefly, proteins on gels were transferred onto nitrocellulose membranes (Schleicher & Schuell, Keene, NH, USA) The membrane was blocked with skimmed milk and successively treated with an anti-TAF ⁄ IGFBP-rP1 monoclonal antibody (#88) or an antimatriptase monoclonal antibody (M32) as the first antibody, the second antibody (biotinylated antimouse IgG, Vector Laboratory, Burlingham, CA, USA), and alkaline FEBS Journal 273 (2006) 615–627 ª 2006 The Authors Journal compilation ª 2006 FEBS S Ahmed et al phosphatase-coupled avidin (Vector Laboratory) Immunoreactive signals were visualized by the enhanced chemiluminescence (ECL) detection method (Amersham Biosciences, Piscataway, NJ, USA) Gelatin zymography Zymography was carried out on 10% polyacrylamide gels containing mgỈmL)1 of gelatin, as described previously [20] Briefly, after SDS ⁄ PAGE, the gel was incubated in 50 mm Tris ⁄ HCl (pH 7.5) buffer containing 100 mm NaCl and 2.5% Triton X-100 at room temperature for 1.5 h to renature the proteins on the gel After the incubation, the gel was treated with 50 mm Tris ⁄ HCl (pH 7.5) containing mm EDTA at 37 °C for 30 to inactivate metalloproteinases After the treatment, the gel was further incubated in 50 mm Tris ⁄ HCl (pH 7.5) containing 10 mm CaCl2 at 37 °C for 18 h Gelatinolytic bands were visualized by staining the gel with Coomassie Brilliant Blue R-250 Assay of IGFBP-rP1-cleaving activity of membrane fraction and conditioned medium Purified IGFBP-rP1 (500 ng) was incubated with a membrane fraction (20 lg protein) or concentrated conditioned medium (20 lg protein) of OVISE or DLD-1 cells in 40 lL of 50 mm Tris ⁄ HCl (pH 7.5) containing 0.15 m NaCl and 10 mm CaCl2 first at °C for 24 h and then at 37 °C for 24 h IGFBP-rP1 in the reaction mixture was precipitated with cold 10% trichloroacetic acid, washed with cold ethanol, and then applied to SDS ⁄ PAGE on a 14% gel, followed by immunoblotting with the anti-TAF ⁄ IGFBP-rP1 antibody #88 Assay of IGFBP-rP1-cleaving activity after SDS/PAGE separation of proteinases IGFBP-rP1-cleaving proteinases present in the membrane fraction of OVISE cells and conditioned medium of DLD-1 cells were separated by SDS ⁄ PAGE on 7.5% gels and assayed as follows Each sample containing 20 lg protein was applied to two lanes on a gel After electrophoresis, the gel was incubated in the renaturation buffer described above for 1.5 h and then washed with pure water for 15 Each lane (5 mm wide, mm thick, and 70 mm long) of the gel was divided horizontally into eight or 16 equal parts For the assay of IGFBP-rP1 cleavage, each gel piece from one lane was incubated with purified IGFBPrP1 (500 ng protein) in 40 lL of 50 mm Tris ⁄ HCl (pH 7.5) containing 0.15 m NaCl and 10 mm CaCl2 first at °C for 24 h, and then at 37 °C for 24 h The resultant IGFBP-rP1 fragments were precipitated by 10% trichloroacetic acid and analyzed by immunoblotting with the anti-TAF ⁄ IGFBP-rP1 antibody #88 as described above To detect Processing of IGFBP-rP1 by matriptase matriptase, gel pieces from another lane were individually subjected to repeated freeze-thawing in 20 lL of three-fold concentrated SDS sample buffer and then incubated at room temperature for h Each extract was subjected to nonreducing SDS ⁄ PAGE on a 10% gel followed by immunoblotting with the antimatriptase antibody M32 Purification of soluble matriptase Serum-free conditioned medium of DLD-1 cells was collected, concentrated by ammonium sulfate precipitation, and subjected to molecular-sieve chromatography on a Cellullofine GCL-2000 m column (Seikagaku Kogyo, Tokyo, Japan) pre-equilibrated with 20 mm Tris ⁄ HCl (pH 7.5) buffer containing 0.5 m NaCl, 0.1% CHAPS and 0.01% Brij35 Fractions containing matriptase were pooled, dialyzed against 20 mm Tris ⁄ HCl (pH 7.5) containing 0.01% Brij35 and applied to a Reactive Red agarose column (Sigma-Aldrich, St Louis, MO, USA) Proteins bound to the column were sequentially eluted with 0.2 m and 0.4 m NaCl in the buffer The 0.4 m NaCl fraction was dialyzed against 20 mm Tris ⁄ HCl (pH 8.0) and applied to a Mono Q HR5 ⁄ column (Amersham Biosciences) pre-equibrated with the same buffer Proteins bound to the column were eluted with a linear gradient of 0–0.5 m NaCl Matriptase eluted from the column was finally applied to reverse-phase HPLC on a TSKgel TMS-250 column (Tosoh, Tokyo, Japan) in the presence of 0.1% trifluoroacetic acid (TFA) and eluted with a linear gradient of 0–80% acetonitrile in 0.1% trifluoroacetic acid Matriptase was dissociated and separated from HAI-1 in this HPLC The matriptase-containing fractions were concentrated by centrifugation under vacuum and then freeze-dried The dried materials were dissolved in 20 mm Tris ⁄ HCl (pH 7.5) and used for the activity assay The main fraction contained a 75 kDa matriptase as a major component and a few contaminating proteins as analyzed by SDS ⁄ PAGE RNAi experiments with OVISE cells Matriptase siRNAs and a scrambled RNA as a control were designed and synthesized at iGENE (Tsukuba, Japan) The forward sequences of the siRNAs were: #973, sense 5¢-UCAUCACACUGAUAACCAACACUGA-AG-3¢; #2578, sense 5¢-GGAUCAAAGAGAACACUGGGGUAUA-AG-3¢; and #1513 sense, 5¢-AGUUCACGUGCAAGAACAAG UUCUG-AG-3¢ The forward sequence of the scrambled RNA was 5¢-GAUCCAAGUAAUACAGAGAUGGGAG AG-3¢ OVISE cells were plated the day before transfection at a cell density of 50–60% saturation in 60 mm culture dishes The cells were transfected with 200 pmol siRNA using Lipofectamine 2000 reagent according to the manufacturer protocol (Invitrogen, Carlsbad, CA, USA) The FEBS Journal 273 (2006) 615–627 ª 2006 The Authors Journal compilation ª 2006 FEBS 625 Processing of IGFBP-rP1 by matriptase S Ahmed et al transfected cells were incubated in serum containing medium overnight, and further incubated in serum-free medium for days The resultant serum-free conditioned medium was collected from each culture as described above, and matriptase and IGFBP-rP1 in the 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matriptase was purified from the conditioned medium of DLD-1 cells as described in the text The purified matriptase (approximately 200 ng of total proteins) was... this study, we first identified the type II membranebound serine proteinase matriptase as a processing enzyme of IGFBP-rP1 ⁄ AGM Matriptase was initially FEBS Journal 273 (2006) 615–627 ª 2006 The