Trypsin stimulates integrin alpha(5)beta(1)-dependent adhesion to fibronectin and proliferation of human gastric carcinoma cells through activation of proteinase-activated receptor-2.

Trypsin is widely expressed in various non-pancreatic tissues at low levels and overexpressed in some types of human cancers. In the present study, we found that trypsin stimulates integrin-dependent adhesion and growth of MKN-1 human gastric carcinoma cells. MKN-1 cells expressed both proteinase-activated receptor-1 (PAR-1) and PAR-2, which are activated by thrombin and trypsin, respectively. Both trypsin and the PAR-2 ligand SLIGKV promoted integrin alpha(5)beta(1)-mediated adhesion of MKN-1 cells to fibronectin, and less effectively integrin alpha(v)beta(3)-mediated cell adhesion to vitronectin, but not that to type IV collagen or laminin-1 at all. Thrombin and the PAR-1 ligand SFLLRN promoted the cell adhesion to vitronectin more strongly than trypsin or the PAR-2 ligand, but not the cell adhesion to fibronectin at all. The cell adhesion-stimulating effect of the PAR-2 ligand was significantly reduced by the pre-treatment of cells with trypsin, indicating that the effect of trypsin is mediated by PAR-2 activation. The trypsin-stimulated cell adhesion to vitronectin, but not to fibronectin, was effectively inhibited by the G(i) protein blocker pertussis toxin, and both cell adhesions were completely inhibited by the Src kinase inhibitor herbimycin A. Furthermore, trypsin and the PAR-2 ligand stimulated growth of MKN-1 cells more strongly than thrombin or the PAR-1 ligand. These results show that trypsin regulates cellular adhesion and proliferation by inducing PAR-2/G protein signalings, and that the integrin alpha(5)beta(1)- and integrin alpha(v)beta(3)-dependent cell adhesions are regulated by different PAR/G protein signalings.

Trypsin is an essential food-digestive enzyme. The zymogen of trypsin, trypsinogen, is synthesized in the acinar cells of pancreas, secreted into the duodenum, activated into the mature form of trypsin by enterokinase. Recent studies have shown that trypsin is widely expressed at low levels in various non-pancreatic epithelial tissues, vascular endothelial cells, leukocytes and neurons (1)(2)(3), and overexpressed in some kinds of cancer cells and tissues (3)(4)(5)(6)(7). In human ovarian and gastric cancers and in a nude mouse model, the trypsin expression is associated with the malignant potential of tumor cells (6 -8). It has been well recognized that proteinases capable of degrading extracellular matrix (ECM) 1 proteins play a critical role in tumor invasion and metastasis (9). Trypsin has potent proteolytic activity toward a wide variety of proteins including ECM proteins (4). Trypsin is also known as a potent activator for the latent forms of various matrix metalloproteinases and serine proteinases (10 -12). We recently found that overexpression of exogenous trypsinogen-1 cDNA in human gastric carcinoma cells causes their elevated growth in the abdominal cavity of nude mice and their enhanced adhesion to fibronectin and vitronectin substrates in culture (13). These results suggest that tumor-derived trypsin not only contributes to the ECM degradation but also modulates growth and adhesion properties of cancer cells in an autocrine manner. On the other hand, physiological roles of trypsin expressed by normal non-pancreatic cells remain to be clarified.
It has long been known that trypsin, thrombin, and other proteinases stimulate DNA synthesis and cell division of fibroblasts in culture (14,15). Thrombin is also known to enhance adhesion of tumor cells to platelets, endothelial cells, or some ECM proteins (16 -20). A few studies have suggested that the stimulatory effect of thrombin on cell adhesion is mediated by thrombin receptor, a member of G protein-coupled, seventransmembrane domain receptor family termed proteinase-activated receptors (PARs) (20). Up to now, four members of the PAR family, PARs-1, -2, -3, and -4, have been identified (21,22). PARs are cleaved by serine proteinases within the extracellular N terminus, allowing the new N terminus to bind and activate the receptors themselves (21). PAR-1 (thrombin receptor) and PAR-3, which are expressed mainly in platelets, endothelium, and leukocytes, are effectively activated by thrombin (23,24). In contrast, PAR-2, which is expressed in a wide variety of epithelial tissues, endothelial cells, T cells, smooth muscle cells and tumor cell lines, is activated by trypsin but not by thrombin (22,(25)(26)(27). Recently found PAR-4 is highly expressed in the lung, thyroid, testis, and small intestine, and activated by both thrombin and trypsin (28). In common to G protein-coupled receptors (GPCRs), activated PARs induce G protein-mediated signal transduction, stimulating generation of inositol 1,4,5-triphosphate, intracellular calcium mobilization, arachidonic acid release, and prostaglandin secretion (22,25,27). Thus PARs are thought to be involved in various pathophysiological processes, including development, cell growth and inflammation (22). However, the natural activator of PAR-2 remains uncertain despite the wide distribution of the receptor (29).
Here we report that trypsin regulates integrin ␣ 5 ␤ 1 -dependent adhesion to fibronectin and integrin ␣ v ␤ 3 -dependent adhesion to vitronectin of human gastric carcinoma cells and their proliferation through PAR-2 activation.
Cell Culture-Human gastric carcinoma cell line MKN-1 was obtained from the Japanese Cancer Resources Bank (Tokyo, Japan). This cell line was cultured at 37°C in a humidified atmosphere of 5% CO 2 and 95% air. RPMI 1640 (Nissui, Tokyo, Japan) supplemented with 15 mM HEPES, 1.2 mg/ml NaHCO 3 , and 2 mM glutamine was used as the basal medium. Culture was maintained in the basal medium supplemented with 10% fetal calf serum (JRH Biosciences, Lenexa, KS).
Cell Adhesion Assay-Adhesion of MKN-1 cells to ECM proteins was assayed by the method reported previously (30). Each well of 96-well plates (Sumibe Medical, Tokyo, Japan) was incubated with 100 l of each ECM protein solution at indicated concentrations at 37°C overnight. The plates were blocked with 200 l of 1.2% (w/v) BSA in Ca 2ϩand Mg 2ϩ -free phosphate-buffered saline (PBS) at 37°C for 1.5 h and then washed with PBS. Cells were harvested by incubating with 6.6 mM EDTA in PBS in the presence or absence of 6.25 M (150 g/ml) trypsin at 37°C for 10 min. After inactivation of trypsin with 50 M soybean trypsin inhibitor, the cells were washed with serum-free RPMI 1640 medium and suspended into the serum-free medium containing 0.1% (w/v) BSA at a density of 5 ϫ 10 5 cells/ml. The cell suspension (100 l) was inoculated into each well of the ECM protein-coated plates and incubated at 37°C for 1 h. Non-adherent cells were removed by gentle agitation, and adherent cells were fixed with 2.5% (v/v) glutaraldehyde and stained with 100 l of 0.0005% (w/v) Hoechst 33342-0.001% (w/v) Triton X-100 for 1.5 h. The fluorescent intensity of each well of the plates was measured using a CytoFluor 2350 fluorometer (Millipore, Bedford, MA).
Reverse Transcription-PCR Analysis-Total RNA was prepared from human umbilical vein endothelial cells (HUVEC), human lung carcinoma cell line A549, and human gastric carcinoma cell line MKN-1 with Trizol reagent according to manufacturer's protocol. Twenty micrograms of RNA was reverse-transcribed with Mo-MLV reverse transcriptase, and one-fifth of the transcript was amplified by polymerase chain reaction (PCR) as previously reported (2). The PCR reaction consisted of incubation for 4 min at 94°C and subsequent 30 cycles of incubations at 94°C for 1 min, at 60°C for 1 min, and at 72°C for 1.5 min. The PCR primers used for amplifying PAR-1 were 5Ј-TGTGAACT-GATCATGTTTATG-3Ј and 5Ј-TTCGTAAGATAAGAGATATGT-3Ј (23). The PCR primers used for amplifying PAR-2 were 5Ј-GGCCAATCTG-GCCTTGGCTGAC-3Ј and 5Ј-GGGCAGGAATGAAGATGGTCTGC-3Ј (27).

Effect of Trypsin on Adhesion of MKN-1 Cells to Various
ECM Proteins-Our previous study showed that MKN-1 human gastric carcinoma cells transfected with trypsinogen-1 cDNA grow faster and adhere to fibronectin substrate more efficiently than the parent MKN-1 cells (13). In the present study, first we examined effect of trypsin treatment on adhesion of the parent MKN-1 cells, which secrete neither trypsinogen nor trypsin, to a plastic plate precoated with various concentrations of the cell adhesion proteins fibronectin. In this experiment, MKN-1 cells were harvested by incubating with EDTA in the presence or absence of 6.25 M trypsin for 10 min. After inactivation of trypsin with soybean trypsin inhibitor, cells were incubated on the fibronectin-coated plastic plate at 37°C for 1 h. Trypsin treatment increased the cell adhesion to fibronectin substrate 2-4-fold at low fibronectin concentrations (0.6 -1.2 g/ml) (Fig. 1). The trypsin-treated cells more rapidly spread on the fibronectin substrate than the control cells ( Fig.  2). Trypsin treatment at 12.5 M showed almost the same effect as that at 6.25 M (data not shown). On the other hand, trypsin treatment at 6.25 M rather decreased the cell adhesion to vitronectin, type IV collagen, and laminin-1 (data not shown).
To investigate the mechanism of the trypsin-stimulated cell adhesion of MKN-1 cells, quantitative and qualitative changes of integrin ␣ 5 ␤ 1 , the primary receptor for fibronectin, were analyzed by immunoblotting analysis after trypsin treatment of MKN-1 cells. When MKN-1 cells were treated with 6.25 M trypsin for 10 min, the apparent amount of cell surface integrin ␣ 5 was decreased to less than 50% after the trypsin treatment, and about half the amount of the 150-kDa integrin ␤ 1 molecule was partially hydrolyzed to a 140-kDa protein (data not shown). These changes were not clearly detected when the cells were treated with 0.1 M trypsin.
Effects of PAR-1 and PAR-2 Activation on Adhesion of MKN-1 Cells-It has been reported that trypsin activates PAR-2 efficiently, stimulating signal transduction through G proteins (23,25). Therefore, the activation of PARs seemed to be a possible mechanism for the stimulatory effect of trypsin on adhesion of MKN-1 cells to fibronectin. First, we examined the expression of PAR-1 and PAR-2 mRNAs in MKN-1 cells by reverse transcription-PCR analysis (Fig. 3). The PCR product amplified with PAR-1 primers was detected at a size of 708 base pairs in MKN-1 cells as well as in HUVECs as a positive control (23). The PCR product amplified with PAR-2 primers was detected at a size of 358 base pairs in MKN-1 cells as well as in A549 cells as a positive control (27). The nucleotide sequences of the PCR products of PAR-1 and PAR-2 in MKN-1 cells were all identical to their corresponding sequences (data not shown).
Thrombin and trypsin cleave the N-terminal, extracellular tethered sequences of PAR-1 and PAR-2, respectively, at a specific site, allowing the new terminal sequences of 6 amino acids to bind to the respective PARs (21). The synthetic peptides for the new N-terminal sequences, so-called tethered ligands, can be used as the active ligands for PARs to investigate PAR-mediated signal transduction (21,31). We examined the effects of trypsin, thrombin, and the PAR-1-and the PAR-2activating peptides as the PAR ligands, on adhesion of MKN-1 cells to fibronectin, vitronectin, type IV collagen, and laminin-1 (Fig. 4). In this experiment, MKN-1 cells harvested with EDTA were incubated with each of the proteinases and the PARactivating peptides for 30 min and then inoculated on fibronectin-, vitronectin-, type IV collagen-, and laminin-1-coated plates. Trypsin at a concentration of 0.1 M, but not thrombin, enhanced the cell adhesion to fibronectin (Fig. 4A). Although the PAR-1-activating peptide (SFLLRN) did not have significant effect, the PAR-2-activating peptide (SLIGKV) enhanced the cell adhesion over 2-fold as compared with the control peptide (LSIGKV), which had the same amino acid composition as the PAR-2 peptide. In contrast, all of trypsin, thrombin, and PAR-1-and PAR-2-activating peptides dose-dependently enhanced the cell adhesion to vitronectin (Fig. 4B). The cell ad- hesion stimulating activity on vitronectin was significantly higher in thrombin than trypsin and in the PAR-1-activating peptide than the PAR-2-activating peptide at any concentrations tested. When the trypsin concentration was increased to 6.25 M, the cell adhesion was rather inhibited (data not shown). Any of the proteinases and the PAR-activating peptides did not enhance the adhesion of MKN-1 cells to type IV collagen or laminin-1 (Fig. 4, C and D). Both trypsin and thrombin at 0.1 M rather inhibited the cell adhesion to laminin-1, presumably due to the degradation of related integrins.
We also examined cell surface integrins responsible for the adhesion of MKN-1 cells to fibronectin or vitronectin enhanced by treatments with the proteinases and the PAR-activating peptides. For this experiment, the proteinase-or peptidetreated MKN-1 cells were incubated with anti-integrin ␣ 5 (P1D6), anti-integrin ␤ 1 (P4C10), or anti-integrin ␣ v ␤ 3 (LM609) monoclonal antibody, all of which are neutralizing antibodies, and then inoculated to fibronectin-or vitronectin-coated plates. The adhesion to fibronectin of MKN-1 cells stimulated by trypsin or the PAR-2-activating peptide, as well as that of nonstimulated MKN-1 cells, was almost completely blocked by any of the anti-integrin ␣ 5 and anti-integrin ␤ 1 monoclonal antibodies, indicating that integrin ␣ 5 ␤ 1 was responsible for the enhanced cell adhesion (Fig. 5A). When the cell adhesion of MKN-1 cells to vitronectin was similarly tested, it was partially blocked by anti-integrin ␣ v ␤ 3 monoclonal antibody but not by anti-integrin ␣ 5 or anti-integrin ␤ 1 monoclonal antibody, regardless of the type of treatment. This indicated that integrin ␣ v ␤ 3 is mainly responsible for the cell adhesion to vitronectin (Fig. 5B).
Activation of PAR-2 by Trypsin-The results shown above suggest that trypsin stimulates integrin ␣ 5 ␤ 1 -mediated adhesion of MKN-1 cells to fibronectin through PAR-2 activation. To confirm this possibility, we examined if trypsin treatment of MKN-1 cells desensitizes PAR-2 for the second challenge by the PAR-2-activating peptide. Although proteolytic activation of PARs is irreversible, their rapid resensitization occurs due to de novo synthesis and the trafficking of pre-formed receptors from intracellular pools (22). The rapid resensitization of PAR-2 after trypsin treatment is effectively suppressed by the protein-trafficking inhibitor brefeldin A or the translation inhibitor cycloheximide (32). In this experiment, therefore, MKN-1 cells were initially treated with trypsin for 30 min, left in the presence or absence of brefeldin A or cycloheximide for 3 h, and then treated with the PAR-2-activating peptide SLIGKV. In the absence of the inhibitors, the treatment with the PAR-2-activating peptide SLIGKV increased the cell adhesion to fibronectin of non-stimulated cells by about 110% and that of trypsin-stimulated cells by about 75%, indicating that trypsin-treated cells had been well resensitized (Fig. 6). Even in non-stimulated cells, both brefeldin A and cycloheximide significantly decreased the cell adhesion. When non-stimulated cells and trypsin-stimulated cells were compared in the presence of brefeldin A, the treatment with the PAR-2-activating peptide increased the cell adhesion of non-treated cells by 159% but increased that of trypsin-treated cells by only 27%. Essentially the same result was obtained when cycloheximide was used in stead of brefeldin A. These results indicated that the PAR-2-activating peptide could not restimulate the adhesion of the trypsin-treated cells when intracellular protein trafficking or protein synthesis was blocked. This strongly suggests that the trypsin-stimulated cell adhesion is mediated by the activation of PAR-2.
Trypsin/PAR-2-dependent Signaling Pathway-G proteins are heterotrimers consisting of ␣, ␤, and ␥ subunits, and the ␣ subunit is grouped into four classes on the basis of sequence homology: G s , G i , G q , and G 12 (33). PAR-1-dependent or other GPCR signalings are often inhibited by pertussis toxin, which inactivates by ADP-ribosylation the ␣ subunits of the G i class

FIG. 5. Effects of anti-integrin ␣ 5 , anti-integrin ␤ 1 , and antiintegrin ␣ V ␤ 3 monoclonal antibodies on adhesion of MKN-1 cells treated with proteinases or PAR-activating peptides to fibronectin and vitronectin.
MKN-1 cells harvested with EDTA were incubated with 0.1 M trypsin or thrombin, or 1 mM PAR-1-activating peptide (SFLLRN), the PAR-2-activating peptide (SLIGKV), or a control peptide (LSIGKV) at room temperature for 30 min. The proteinaseor peptide-treated MKN-1 cells were incubated without or with mouse IgG, anti-integrin ␣ 5 monoclonal antibody, anti-integrin ␤ 1 monoclonal antibody, or anti-integrin ␣ v ␤ 3 monoclonal antibody at room temperature for 15 min. Adhesion of these MKN-1 cells to plastic plates precoated with 0.625 g/ml fibronectin (A) or 0.625 g/ml vitronectin (B) were determined as described in Fig. 1. Other experimental conditions are described under "Experimental Procedures." Each value represents the mean Ϯ S.D. (bar) for triplicate assays. such as three forms of ␣ i (␣ i1 , ␣ i2 , and ␣ i3 ) and two forms of ␣ 0 (␣ 01 and ␣ 02 ) (34 -36). To examine the involvement of G i proteins in the trypsin-stimulated cell adhesion, effect of pertussis toxin on the stimulation of the MKN-1 cell adhesion by trypsin or the PAR-2-activating peptide was investigated in this study. The stimulatory effects of trypsin and the PAR-2 ligand on adhesion of MKN-1 cells to fibronectin were hardly blocked by pertussis toxin (Fig. 7A), whereas their stimulatory effects on the cell adhesion to vitronectin were almost completely blocked by pertussis toxin (Fig. 7B). This differential sensitivity to pertussis toxin indicates that G i protein mediates the activation of integrin ␣ v ␤ 3 -dependent cell adhesion by the PAR-2 signal, whereas a different G protein might be involved in the activation of integrin ␣ 5 ␤ 1 -dependent cell adhesion.
It is well known that the Src family nonreceptor tyrosine kinases play important roles in PAR-1-dependent and other GPCR signalings (36 -38). Using the Src kinase inhibitor herbimycin A, we examined the contribution of Src kinase in the stimulatory effects of trypsin and the PAR-2 ligand on adhesion of MKN-1 cells. Herbimycin A completely inhibited the stimulatory effects of trypsin and the PAR-2 ligand on both cell adhesions to fibronectin and to vitronectin (Fig. 7, A and B). These results showed that Src kinase is involved in the stimulation of both integrin ␣ 5 ␤ 1 -dependent and integrin ␣ v ␤ 3 -dependent cell adhesions by PAR-2/G protein signaling.
Effects of PAR Activation on Growth of MKN-1 Cells-Effects of trypsin and the PAR-activating peptides on growth of MKN-1 cells were examined in culture medium containing 1% FCS (Fig. 8). Both trypsin and the PAR-2-activating peptide dose-dependently stimulated the growth of MKN-1 cells. On the other hand, thrombin and the PAR-1-activating peptide less effectively stimulated the cell growth. The control peptide for PAR-2 showed no stimulatory effect. These results showed that the PAR-2 signal induced by trypsin or the PAR-2-activating peptide more efficiently stimulates the growth of MKN-1 cells than the PAR-1 signal induced by thrombin or the PAR-1-activating peptide.

DISCUSSION
The present study demonstrated that trypsin stimulated the integrin ␣ 5 ␤ 1 -dependent adhesion of human gastric carcinoma cells to fibronectin and less effectively integrin ␣ v ␤ 3 -dependent cell adhesion to vitronectin. Although MKN-1 cells expressed both PAR-1 and PAR-2, only the PAR-2-activating peptide SLIGKV as the PAR-2 ligand promoted the cell adhesion to fibronectin. Trypsin and the PAR-2 ligand also promoted the integrin ␣ v ␤ 3 -dependent cell adhesion to vitronectin, but less effectively than thrombin and the PAR-1-activating peptide, respectively. Together with the similar effects of trypsin and the PAR-2 ligand, the results of the desensitization experiment shown in Fig. 6 strongly suggest that the effect of trypsin depends on activation of PAR-2. Although the treatment of MKN-1 cells with a high concentration (6.25 M) of trypsin hydrolyzed integrin ␣ 5 and ␤ 1 subunits in an unlimited or limited manner, 0.1 M trypsin, which still significantly stimulated the cell adhesion to fibronectin (Fig. 4A), caused no proteolytic degradation of the integrins. Therefore, it is concluded that the trypsin-stimulated cell adhesion to fibronectin is not due either to the induction of integrin expression on cell surface or the direct proteolytic effect of trypsin on the integrins.
The binding of integrins to extracellular matrix proteins such as fibronectin and vitronectin activates focal adhesion kinases (FAKs), and the activated FAKs interact with a variety of cytoskeletal or intracellular signaling molecules, such as paxillin, tensin, talin, c-Src, Grb2, phosphatidylinositol 3-kinase, and p130 Cas , transducing the downstream cytoskeletal and mitogenic signalings (39,40). Such activation of focal adhesion complex is essential for tight cell adhesion to matrix proteins. It is also known that the binding affinity of integrins to various matrix proteins is regulated by internal mitogenic signaling, so-called "inside-out signaling" (39,41). For example, integrin ␣ IIb ␤ 3 on resting circulating platelets does not bind to soluble fibrinogen, but it is activated to bind the soluble ligand when platelets were stimulated by thrombin, epinephrine, or ADP (39,42). This integrin activation is supposed to depend on the conformational change of the integrin molecule induced by the interaction of its cytoplasmic domain with some GPCR signaling molecules (42). In the present study, therefore, it is very likely that the trypsin/PAR-2-stimulated cell adhesion to fibronectin and to vitronectin was caused by PAR-2-dependent activation of the focal adhesion complex. Many GPCRs induce Ras-dependent mitogenic signals, leading to the activation of the mitogen-activated protein kinase cascade (38). Recent studies have shown that some GPCR mitogenic signalings transactivate two types of tyrosine kinases, receptor tyrosine kinases such as epidermal growth factor receptor and platelet-derived growth factor receptor, and integrin-associated FAKs such as p125 FAK and Pyk2 (37,38). These tyrosine kinases contribute to the formation of membrane-associated scaffolds to assemble many signaling molecules such as Shc, c-Src, Grb2, and Sos, which lead to the activation of Ras and then the mitogenactivated protein kinase cascade. The stimulation of cell adhesion and cell growth by the trypsin/PAR-2 signaling found in this study are well consistent with the above model of GPCR signaling.
As compared with the PAR-1-dependent signaling, the PAR-2-dependent signaling has poorly been understood. Like activated PAR-1, activated PAR-2 stimulates generation of inositol triphosphate and mobilization of intracellular Ca 2ϩ (25,27), activity of mitogen-activated protein kinases ERK-1/-2 (43), and cell growth (44) in some cell lines. However, little is known about PAR-2-dependent regulation of cell adhesion. In the present study, the stimulatory effects of trypsin and the PAR-2 ligand on adhesion of MKN-1 cells to vitronectin were almost completely blocked by the G i inhibitor pertussis toxin, whereas the cell adhesion to fibronectin was not blocked at all. This implies that the activations of integrin ␣ 5 ␤ 1 and integrin ␣ v ␤ 3 by the PAR-2-stimulated signaling are mediated by different G proteins. In this regard, there are reports showing that at least two types of G proteins mediate similar PAR-1-derived mitogenic signalings in a single cell type: G q and G i2 in mouse Balb/c3T3 cells (35) and G q and G o in Chinese hamster CCL39 cells (45). Our results first show a specific linkage between integrins and G proteins in the trypsin/PAR-2-dependent regulation of cell adhesion in a single cell type. We also found that trypsin-PAR-2 and thrombin-PAR-1 signalings differently regulate integrin functions in a single cell type. The important role of the Src family tyrosine kinases in GPCR signalings has been reported in many cell types (34, 36 -38). In accordance with these studies, the present study showed that Src kinase plays an essential role in the regulation of integrins ␣ 5 ␤ 1 and ␣ v ␤ 3 by the trypsin/PAR-2 signaling. Thus, we propose the model that trypsin activates PAR-2, and the activated PAR-2 stimulates the functions of integrins ␣ 5 ␤ 1 and ␣ v ␤ 3 using different G proteins in an as yet unknown mechanism, leading to the efficient adhesion to fibronectin and vitronectin, respectively. More extensive studies are needed to clarify the detailed molecular mechanisms of the specific regulation of the two integrin functions by the trypsin/PAR-2 and thrombin/PAR-1 signalings.
The present study showed that low concentrations of exogenous trypsin stimulated cell growth and cell adhesiveness through PAR-2 activation. In our previous study, MKN-1 cell transfectants overexpressing trypsinogen grew faster and adhered to fibronectin more efficiently than the control, trypsinogen-non-producing MKN-1 cells only when the trypsinogen activator enterokinase was added into the culture (13). This indicates that trypsinogen secreted by tumor cells, if activated to trypsin, can stimulate the growth and adhesiveness of the producer cells in an autocrine manner. In addition, the MKN-1 transfectants overexpressing trypsinogen showed high tumor-igenicity in the abdominal cavity of nude mice compared with the parent MKN-1 cells (13). From the present results, it may be concluded that these characteristics of the trypsinogen-1overexpressing cells depend on the PAR-2 activation by the self-produced trypsin. Increased adhesiveness of tumor cells is thought to be important for their metastasis. Many types of human cancer cell lines secrete trypsin in an active or inactive form in culture (7), and in vivo expression of trypsin is associated with malignant potential of tumor cells in gastric and ovarian cancers (6 -8). Taken together, tumor-derived trypsin seems to contribute to growth, invasion, and metastasis of human cancer cells not only by proteolysis of surrounding ECM proteins but also by activation of PAR-2. Furthermore, recent studies have shown that trypsin is a ubiquitous enzyme. It is expressed in various epithelial tissues including the gastrointestinal tract, kidney, liver, airway and skin, endothelial cells, leukocytes, and neuronal cells (1)(2)(3). Most of these tissues and cells, as well as cancer cell lines, also express PAR-2 (25)(26)(27). PAR-2 is activated by acrosin from sperm and tryptase from mast cells, besides trypsin (29). Of these enzymes, trypsin has been suggested to act as the natural PAR-2 activator in bronchial epithelial cells (32) and enterocytes in the small intestine (46). The present study strongly suggests that the tissue trypsin might regulate various cellular functions as a natural activator of PAR-2 under physiological and pathological conditions.