Membrane-Type Serine Protease-1/Matriptase Induces Interleukin-6 and -8 in Endothelial Cells by Activation of Protease-Activated Receptor-

【摘要】  Objective- The serine protease MT-SP1/matriptase plays an important role in cell migration and matrix degradation. Hepatocyte growth factor (HGF), urokinase-type plasminogen activator (uPA), and protease-activated receptor 2 (PAR-2) have been identified as in vitro substrates of MT-SP1/matriptase. Because PAR-2 is expressed in endothelial cells and contributes to inflammatory processes, we sought to investigate the effects of MT-SP1/matriptase on endothelial cytokine expression and analyzed MT-SP1/matriptase expression in vascular cells and atherosclerotic lesions.

Methods and Results- In endothelial cells, recombinant MT-SP1/matriptase dose-dependently induced interleukin (IL)-8 and IL-6 mRNA and protein expression dependent on its proteolytic activity. MT-SP1/matriptase time-dependently induced phosphorylation of p38 MAPK and p42/44 MAPK. Inhibitor experiments revealed that p38 MAPK and PKC were necessary for IL-8 induction. PAR-2 downregulation abolished and PAR-2 overexpression augmented MT-SP1/matriptase-induced IL-8 expression as evidence for PAR-2 signaling. In human atherectomies, MT-SP1/matriptase was expressed in blood cells adherent to the endothelium. Concordantly, basal MT-SP1/matriptase expression was detected in isolated monocytes. Coincubation of monocytes and endothelial cells resulted in an increased IL-8 release, which was reduced after downregulation of endothelial PAR-2 and monocytic MT-SP1/matriptase.

Conclusion- MT-SP1/matriptase induces release of proinflammatory cytokines in endothelial cells through activation of PAR-2. MT-SP1/matriptase is expressed in monocytes, thus, interaction of monocytic MT-SP1/matriptase with endothelial PAR-2 may contribute to atherosclerosis.

In endothelial cells, soluble MT-SP1/matriptase induced IL-8 and IL-6 expression. MT-SP1/matriptase proteolytic activity, activation of p38 MAPK and PKC were required for cytokine release, which was abolished by downregulation of PAR-2. In the interaction of monocytes and endothelial cells MT-SP1/matriptase and PAR-2, respectively, were crucial for IL-8 release.

【关键词】  pathophysiology growth factors endothelium

Introduction

MT-SP1/matriptase is a trypsin-like, multi-domain serine protease expressed primarily in epithelial cells. 1-4 Its importance in the biology of surface-lining epithelial cells became apparent in MT-SP1/matriptase knockout mice presenting with a severe deficient epidermal barrier function as well as abnormal hair follicle development and disturbed thymic homeostasis. 5 Moreover, MT-SP1/matriptase is upregulated in different malignant tissues 6-8 and may be expressed in microvascular endothelial cells. 9 Besides its N-terminal transmembrane signal anchor MT-SP1/matriptase contains two putative regulatory modules: 2 tandem repeats of a CUB domain ( C 1r/s, U egf, B one morphogenetic protein-1) and 4 tandem repeats of a low density lipoprotein (LDL) receptor domain. 1,4 The C-terminal serine protease domain consists of a catalytic triad comprising His-57, Asp-102, and Ser-195 according to chymotrypsin numbering. 10 In addition to the membrane-anchored form of MT-SP1/matriptase, a soluble form of the protease has been identified lacking the N-terminal 172 amino acids. 1 Shedding from the extracellular surface 11,12 or alternative splicing 3,10 may be the mechanisms leading to the truncated form of MT-SP1/matriptase isolated from human milk. 13 Cleavage within its activation motif generates the 2-chain active protease from a single-chain zymogen. The activation of MT-SP1/matriptase requires its cognate Kunitz-type inhibitor hepatocyte growth factor activator inhibitor (HAI)-1, its noncatalytic domains as well as its serine protease domain. 14 Three macromolecular substrates of MT-SP1/matriptase have been identified: urokinase-type plasminogen activator (uPA), hepatocyte growth factor (HGF), and protease-activated receptor-2 (PAR-2). 3,15,16 Both HGF and uPA have been proposed to regulate matrix degradation, cell proliferation, survival, and motility. 17,18 Because various cancer cells express MT-SP1/matriptase, tumor progression may be regulated through activation of HGF, uPA, or by extracellular matrix degradation. 15

PAR-2, a heptahelical G protein-coupled receptor, has been identified in various cell types including vascular endothelial cells. 19,20 PAR-2 is activated by trypsin, mast-cell derived tryptase, 21 tissue factor/factor VIIa complex, and factor Xa. 22 Proteolytic cleavage of PAR-2 at its N terminus unmasks new amino-terminal residues serving as a tethering ligand, which irreversibly activates the maternal receptor. Subsequently, cleaved receptors undergo lysosomal degradation. PAR-2 is upregulated by cytokines, 23 vascular injury, and in atherosclerosis 24 and contributes to inflammatory responses. 25,26 Considering the potent trypsin-like activity of MT-SP1/matriptase and its potential role in the activation of PAR-2, we analyzed the role of MT-SP1/matriptase on endothelial cell release of inflammatory cytokines and the expression and distribution of MT-SP1/matriptase in atherosclerotic lesions.

Materials and Methods

Human umbilical vein endothelial cells (HUVECs) were treated with the active catalytic domain of MT-SP1/matriptase C122S or with the proteolytically inactive C122S/S195A mutant, both recombinantly expressed in E coli. Cytokine expression was analyzed by immunoassay and mRNA expression was assessed using quantitative RT-PCR. RNAi and overexpression experiments were performed using RNAiFect and Effectene transfection reagents (Qiagen), respectively. Atherectomies were obtained from carotid arteries and analyzed for mRNA expression. Coincubation of HUVECs and the monocytic cell line Mono Mac 6 (MM6) was performed under static conditions at a 1:1 ratio for 16 hours. Signaling experiments are described in detail in Materials and Methods in an online-only data supplement available at http://atvb.ahajournals.org.

Results

Expression, Renaturation, and Purification of MT-SP1/matriptase

The serine protease domain of MT-SP1/matriptase comprising residues 596 to 855 was expressed in E coli as His-tagged protein and purified from inclusion bodies. The N-terminal His-tag including the prodomain was cleaved autocatalytically during refolding as previously described 4 leading to the catalytic domain of MT-SP1/matriptase (V615-V855). MT-SP1/matriptase C122S was generated to prevent attachment of the prodomain to the catalytic domain via a disulfide bridge and displayed the same K m -value for substrate Boc-Gln-Ala-Arg-AMC as the wild-type form (25.4±2.7 µmol/L, data not shown). MT-SP1/matriptase C122S cleaved the prosequence of the active site mutant MT-SP1/matriptase C122S/S195A at a ratio of 1:100 resulting in a clear shift of the mature, processed protein in electrophoretic mobility as described. 4

MT-SP1/Matriptase Induces IL-8 and IL-6 mRNA Expression and Release Dependent on its Catalytic Activity

HUVECs showed a dose-dependent increase of IL-8 release after treatment with MT-SP1/matriptase C122S ( Figure 1 A). Because preincubation with actinomycin D resulted in abrogation of MT-SP1/matriptase C122S-induced IL-8 mRNA expression, de novo synthesis of IL-8 is required ( Figure 1 B). To assess the relevance of the MT-SP1/matriptase active site a proteolytically inactive mutant MT-SP1/matriptase C122S/S195A was compared with MT-SP1/matriptase C122S. In an enzymatic assay using the trypsin substrate Boc-Gln-Ala-Arg-AMC MT-SP1/matriptase C122S/S195A showed no catalytic activity in contrast to MT-SP1/matriptase C122S ( Figure 1 C) and did not alter cytokine release, whereas MT-SP1 C122S significantly enhanced IL-8 release ( Figure 1 D). The proinflammatory effect of MT-SP1/matriptase C122S was confirmed in human coronary arterial endothelial cells (data not shown). In the presence of serum MT-SP1/matriptase C122S induced a 1.5-fold increase in IL-8 expression as compared with an approximately 3-fold increase under serum free conditions (data not shown). Hence, secondary effects by other MT-SP1/matriptase substrates can be excluded. In addition to IL-8, the increased protein secretion and mRNA expression of IL-6 was observed after treatment of HUVECs with MT-SP1/matriptase C122S (supplemental Figure IA and IB). This was elicited only by proteolytically active MT-SP1/matriptase C122S, but not by the inactive mutant C122S/S195A (supplemental Figure IB).

Figure 1. MT-SP1/matriptase induces IL-8 mRNA and release in EC. A, HUVECs were treated with increasing concentrations of MT-SP1/matriptase C122S for 16 hours. IL-8 release in the supernatants is shown (mean±SEM, n=3); asterisks indicate a probability value <0.05. B, Before treatment with MT-SP1/matriptase C122S HUVECs were incubated with 5 µg/mL Actinomycin D (ActD). Total RNA was analyzed by quantitative RT-PCR. Values were normalized on GAPDH and are shown as x -fold untreated cells. Shown is the mean±SEM of at least 3 independent experiments. Asterisks indicate P <0.05 vs unstimulated cells. C, Proteolytic activity of MT-SP1/matriptase C122S and active site mutant MT-SP1 C122S/S195A after incubation with 25 µmol/L synthetic trypsin substrate Boc-Gln-Ala-Arg-AMC. Shown is the mean±SEM of the relative fluorescence units (RFU) of 3 independent experiments. D, HUVECs were treated with MT-SP1/matriptase C122S (20 nmol/L) or proteolytically inactive MT-SP1/matriptase C122S/S195A (20 nmol/L) and IL-8 release was measured. Each column represents the mean±SEM of 3 independent experiments. The asterisk indicates P <0.05.

MT-SP1/Matriptase Induces IL-8 Through Activation of PAR-2

Downregulation of PAR-2 using siRNA (siPAR-2) decreased PAR-2 mRNA levels whereas transfection with nonsilencing negative control siRNA (siC) did not alter PAR-2 mRNA compared with nontransfected cells (ctr) ( Figure 2 A). IL-8 mRNA expression significantly increased after MT-SP1/matriptase C122S treatment of cells transfected with siC ( Figure 2 C). In contrast, downregulation of PAR-2 abolished MT-SP1/matriptase C122S-induced IL-8 transcription ( Figure 2 C).

Figure 2. Gene silencing of PAR-2 but not PAR-1 inhibits and overexpression of PAR-2 amplifies MT-SP1/matriptase-induced IL-8 expression. A through C, HUVECs transfected with siRNA against PAR-2, PAR-1 (siPAR-2, siPAR-1), with non-silencing fluorescein-labeled siRNA (siC) and untreated cells (ctr) were stimulated with 20 nmol/L MT-SP1/matriptase C122S for 2 hours. Quantitative RT-PCR was performed to assess mRNA levels relative to control cells. A, PAR-2 mRNA levels decrease after transfection with siPAR-2 but not with siC. B, PAR-1 mRNA levels decrease after transfection with siPAR-1 but not with siC. C, The increase in IL-8 mRNA after treatment of HUVECs with MT-SP1/matriptase C122S in siC cells is abolished after transfection with siPAR-2, whereas IL-8 mRNA levels remained unchanged in siPAR-1 cells. Shown are means±SEM of at least 3 independent experiments. Asterisks indicate P <0.05 vs siC or unstimulated cells. D, After transfection with cDNA encoding human PAR-2, PAR-1, or with control vector (pcDNA3.1), HUVECs were stimulated with 20 nmol/L MT-SP1/matriptase C122S. mRNA expression was analyzed by quantitative RT-PCR. Each column represents the mean±SEM of at least 3 independent experiments. The asterisk indicates a probability value <0.05.

Because of its trypsin-like activity, MT-SP1/matriptase may not only activate PAR-2 but also PAR-1. Hence, we analyzed the role of PAR-1 using specific siRNA (siPAR-1). PAR-1 expression was downregulated as compared with nontreated control (ctr) ( Figure 2 B). Gene silencing of PAR-1, however, did not affect MT-SP1/matriptase C122S-induced IL-8 transcription ( Figure 2 C). Similarly, the use of inhibitory antibodies against PAR-1 did not abolish the proinflammatory MT-SP1/matriptase C122S effect (supplemental Figure II). Moreover, flow cytometry analysis using activation-dependent (SPAN12) and independent (WEDE15) antibodies revealed that MT-SP1 C122S did not activate PAR-1 (supplemental Figure III). Thus, PAR-2 exclusively mediates MT-SP1/matriptase-induced IL-8 upregulation in HUVECs.

Overexpression of human PAR-2 and PAR-1 in HUVECs confirmed the importance of PAR-2-mediated IL-8 induction. ( Figure 2 D).

MT-SP1/Matriptase Activates p38 MAPK, p42/44 MAPK, and NF- B

To determine whether MT-SP1/matriptase-induced signaling pathways via PAR-2 involve the activation of mitogen-activated protein kinases (MAPK), we analyzed p38 MAPK and p42/44 MAPK phosphorylation. MT-SP1/matriptase C122S time-dependently induced activation of p38 MAPK ( Figure 3 A) and p42/44 MAPK ( Figure 3 B) with a maximum at 5 minutes. Luciferase reporter gene assays revealed that MT-SP1/matriptase C122S activates NF- B, which was dependent on p38 MAPK and p42/44 MAPK, because specific pharmacological inhibitors against both MAPK suppressed NF- B activation ( Figure 3 C). Transfection with pTAL-Luc control did not elicit reporter gene expression (data not shown). However, only p38 MAPK was essential for induction of IL-8 expression after MT-SP1/matriptase C122S-treatment of HUVECs ( Figure 3 D). Similar results were found for the induction of IL-6 (data not shown). NF- B activation inhibitor was included in the luciferase reporter gene assay before to assure efficacy ( Figure 3 C).

Figure 3. MT-SP1/matriptase-induced activation of p38 MAPK, p42/44 MAPK, and NF- B in HUVECs. Requirement of p38 MAPK for enhancement of IL-8 expression. A and B, HUVECs were incubated with 20 nmol/L MT-SP1/matriptase C122S for the indicated time periods (0 to 30 minutes). Cell extracts were subjected to Western blot analysis for p38 MAPK (A) and p42/44 MAPK (B) phosphorylation and for total p38 MAPK (A) and p42/44 MAPK (B) protein. Shown is 1 representative Western blot of 3. C, HUVECs were transfected with pNF- B-Luc and subsequently treated with inhibitors of p38 MAPK (SB 220025, 20 µmol/L), p42/44 MAPK (PD 98059, 20 µmol/L), or NF- B activation (1 µmol/L) and stimulated with 20 nmol/L MT-SP1/matriptase overnight. Lysates were subjected to luciferase assays. Each column represents the mean±SEM of 3 experiments, displayed as x -fold untreated. Asterisk indicates a probability value <0.05. D, HUVECs were treated with inhibitors of p38 MAPK (SB 220025, 20 µmol/L), p42/44 MAPK (PD 98059, 20 µmol/L), or NF- B activation (1 µmol/L) and stimulated with 20 nmol/L MT-SP1/matriptase C122S for 2 hours. Total RNA was analyzed by quantitative RT-PCR. Values are displayed as x -fold untreated cells. Shown are means±SEM of 3 independent experiments. Asterisks indicate P <0.05 vs MT-SP1/matriptase C122S-treated control cells.

MT-SP1/Matriptase-Induced Cytokine Expression Requires PKC

Because PAR-2 has not only been associated with p38 MAPK and p42/44 MAPK activation 27,28 but also with activation of PKC, 29,30 the implication of PKC in activation of p38 MAPK, p42/44 MAPK, and NF- B and in cytokine induction in response to MT-SP1/matriptase was assessed. A decrease in MT-SP1/matriptase C122S-induced IL-8 ( Figure 4 D) and IL-6 (data not shown) mRNA synthesis was observed after inhibition of PKC. Experiments analyzing the involvement of PKC in p38 MAPK and p42/44 MAPK activation demonstrated that inhibition of PKC did not alter MAPK phosphorylation ( Figure 4 A and B). Furthermore, NF- B activation was not impaired by inhibition of PKC ( Figure 4 C). Therefore, MT-SP1/matriptase C122S-induced IL-8 gene expression via PAR-2 requires 2 independent pathways, involving p38 MAPK and PKC.

Figure 4. Requirement of PKC for induction of IL-8 expression. A and B, HUVECs were treated with an inhibitor of PKC (Safingol, 20 µmol/L) and stimulated with 20 nmol/L MT-SP1/matriptase C122S. Cell extracts were subjected to Western blot analysis for p38 MAPK (A) and p42/44 MAPK phosphorylation (B) and for total p38 MAPK (A) and p42/44 MAPK protein (B). Shown is 1 representative Western blot of 6. C, HUVECs were transfected with pNF- B-Luc, treated with an inhibitor of PKC (Safingol, 20 µmol/L), and stimulated with 20 nmol/L MT-SP1/matriptase C122S overnight. Cell lysates were subjected to the luciferase assay. Each column represents the mean±SEM of 3 experiments, displayed as x -fold untreated. D, HUVECs were treated with a PKC -inhibitor (Safingol, 20 µmol/L) and stimulated with 20 nmol/L MT-SP1/matriptase C122S for 2 hours. Total RNA was analyzed by qRT-PCR. Values are displayed as x -fold untreated cells. Shown are means±SEM of 5 independent experiments. Asterisks indicate P <0.05 vs MT-SP1/matriptase C122S-treated control cells.

MT-SP1/Matriptase Expression in Human Atherectomies

By the use of an external standard curve absolute MT-SP1/matriptase mRNA copies in vascular cells and atherosclerotic lesions were determined and subsequently normalized on GAPDH. MT-SP1/matriptase mRNA levels were significantly increased in atherectomy samples as compared with control vessels ( Figure 5 A, P =0.002). A significant correlation of MT-SP1/matriptase with IL-8 mRNA expression ( R 2 =0.76, P =0.0002) provides further evidence for a role of MT-SP1/matriptase in proinflammatory changes during atherosclerosis. The high variance of MT-SP1/matriptase expression in atherectomy samples is likely attributable to varying compositions of the collected specimens. Immunohistochemical analysis revealed that MT-SP1/matriptase was highly expressed in blood cells attached to the endothelium ( Figure 5 B; supplemental Figures I and II), whereas healthy vessels mainly expressed MT-SP1/matriptase only in the adventitia (data not shown). The hypothesis that monocytic MT-SP1/matriptase may induce IL-8 in HUVECs via PAR-2 was confirmed by coincubation of HUVECs and Mono-Mac-6 (MM6) cells. Gene silencing of PAR-2 in HUVECs and of MT-SP1/matriptase in MM6 decreased IL-8 release after 8 hours coincubation in contrast to negative control siRNA (siC)-transfected cells ( Figure 5 C).

Figure 5. Increased MT-SP1/matriptase expression in human atherectomies: interaction of monocytic MT-SP1/matriptase and endothelial PAR-2 contributes to IL-8 release. A, Total RNA was prepared from human atherectomies (n=26) and from undiseased vessels (n=8). Quantitative RT-PCR for MT-SP1/matriptase was performed, and mRNA copies were determined using a standard curve of pre-quantified PCR products specific for the target mRNA. MT-SP1/matriptase copies are shown relative to the GAPDH copies in the sample. Each dot represents one analyzed sample. B, Immunohistochemistry of human atherectomies using anti-MT-SP1/matriptase monoclonal antibody M32 (10 µg/mL). Nuclei were stained with Mayer hemalaun. Panels I and II (magnification 40 x ) show selective MT-SP1/matriptase staining in atherosclerotic vessels. Arrows mark cells adhering to the endothelial layer. Panel IgG (40 x ) shows the isotype control. C, Coincubation of HUVECs and monocytic Mono-Mac-6 (MM6) cells under static conditions at a 1:1 ratio. Cells were transfected with siPAR-2 (HUVECs) or siMT-SP1 (MM6) and 32 hours later coincubated for 16 hours. Supernatants were analyzed for IL-8 by immunoassay. Data are shown as means±SEM of 3 independent experiments, asterisks indicate P <0.05 vs HUVECs.

Expression of MT-SP1/Matriptase in Peripheral Blood Monocytic Cells

Mononuclear cells (MNCs) expressed MT-SP1/matriptase mRNA, whereas endothelial cells (HUVECs) and smooth muscle cells (SMCs) showed no basal expression under culture conditions (supplemental Figure IVA). In mononuclear cells MT-SP1/matriptase was exclusively expressed in CD14+ monocytes (supplemental Figure IVB I-III). Isotype controls mouse IgG and mouse 1-PE did not show fluorescence (data not shown).

Discussion

Major findings of this study are: (1) Dependent on its proteolytic activity, MT-SP1/matriptase stimulates de novo synthesis of the proinflammatory cytokines IL-8 and IL-6 through activation of PAR-2 in endothelial cells. MT-SP1/matriptase-induced IL-8 expression is dependent on activation of p38 MAPK and PKC. (2) In atherosclerotic lesions, enhanced mRNA and protein expression of MT-SP1/matriptase was found as compared with nondiseased vessels. Immunohistochemistry showed a strong staining for MT-SP1/matriptase in blood cells attached to the endothelium. (3) Only monocytes, but not lymphocytes, endothelial or smooth muscle cells, expressed MT-SP1/matriptase under culture conditions. Concordantly, monocytic MT-SP1/matriptase and endothelial PAR-2 were crucial for increased IL-8 release in a coincubation model.

MT-SP1/matriptase may play a role in tumor invasion and metastasis by altering migratory responses and by matrix degradation. 31 In this study, we identify a proinflammatory role of MT-SP1/matriptase and provide evidence for potential implications in atherosclerosis. MT-SP1/matriptase dose-dependently induced IL-8 and IL-6 release in human endothelial cells, dependent on its catalytic activity. The increased IL-8 secretion was attributable to upregulation of IL-8 gene transcription. Using RNA interference, we specifically knocked down PAR-2, a known in vitro substrate for MT-SP1/matriptase, to analyze its role as mediator of MT-SP1/matriptase-induced cytokine release. Applying this technique, we identified PAR-2 as the mediator of MT-SP1/matriptase-induced IL-8 expression and established an efficient tool to analyze PAR-2-mediated signaling pathways.

Phosphorylation of p38 MAPK and p42/44 MAPK occurred after stimulation with MT-SP1/matriptase, even though only p38 MAPK activation was necessary for IL-8 induction. These results are in line with a previous study in human peripheral blood eosinophils that identified activation of p38 MAPK as mechanism for an increased IL-8 release after activation of PAR-2 by tryptase. 32 In contrast, we did not find dependence on p42/44 MAPK activation. Furthermore, activation of PKC was required for MT-SP1/matriptase-induced IL-8 expression, independent of p38 MAPK. Although MT-SP1/matriptase activated NF- B as shown by reporter gene assays, NF- B activation was not necessary for IL-8 gene expression. NF- B activation after PAR-2 activation was demonstrated in several cell lines including microvascular endothelial cells, coronary artery smooth muscle cells, and keratinocytes. However, there was no direct evidence for NF- B-mediated cytokine expression, even though intercellular adhesion molecule-1 (ICAM-1) upregulation was shown to depend on NF- B activation. 33,34 Optimal IL-8 expression may require the activation of several transcription factors (NF- B, AP-1, and nuclear factor for IL-6/NF-IL-6), which may explain the lack of effect of NF- B inhibition on MT-SP1/matriptase-induced IL-8 expression. 35

Recent studies identified proinflammatory effects of PAR-2 activation in neutrophils. 36 Moreover, in vivo studies in PAR-2-deficient mice highlight the critical roles of PAR-2 in skin progression and joint inflammation as well as sepsis. 37,38 In vascular disease, PAR-2-derived peptide agonists reduce vascular tone and increase blood flow via nitric oxide-dependent and -independent actions of the endothelium. 39 In addition, enhanced expression of PAR-2 was found in human coronary atherosclerosis. 24 Yet, the role of PAR-2 in atherosclerosis and the underlying mechanisms have not been elucidated. This study reveals a novel physiological PAR-2 agonist abundantly expressed in monocytes, which may enhance local inflammatory responses in the endothelial inner layer of the vessel wall. Compared with IL-1ß, which induces IL-8 mRNA in endothelial cells up to 70-fold (data not shown), MT-SP1/matriptase may not be the main proinflammatory stimulus but rather contribute to amplification and perpetuation of the inflammatory reaction. Investigation of MT-SP1/matriptase expression in different vascular cell types revealed that MT-SP1/matriptase is expressed in mononuclear leukocytes but not in smooth muscle and endothelial cells. Immunohistochemistry confirmed expression of MT-SP1/matriptase mainly in blood cells adhering to the endothelium of the atherosclerotic vessel wall. The biological importance of monocytic MT-SP1/matriptase for the induction of inflammatory mediators in the endothelium via PAR-2 was demonstrated in a coincubation experiment. Interaction of monocytes with the endothelium plays a major role in the progression of atherosclerosis. Considering the fact that PAR-2 agonists stimulate P-selectin expression in endothelial cells and increase neutrophil adherence to the endothelium, one might assume that PAR-2 contributes to both adherence and inflammation in progression of atherosclerosis. 40 Furthermore, previous studies have shown increased PAR-2 expression in atherosclerotic coronary arteries. 24 Thus, PAR-2 may represent a possible therapeutic target to modify responses to vascular injury.

Because we were interested in the biological effects of MT-SP1/matriptase, we expressed soluble human MT-SP1/matriptase to exclusively analyze the effects of the serine protease domain in endothelial cells. Experiments were performed in a serum-free environment to exclude interference with the other known substrates uPA and HGF. 3,15 Under these conditions, we identified PAR-2 as the mediator of proinflammatory cytokine release in response to MT-SP1/matriptase. Secondary effects of MT-SP1/matriptase, eg, via uPA with subsequent PAR-1 activation by plasmin, 41 are unlikely because of the reduced induction of IL-8 in the presence of serum providing additional MT-SP1/matriptase substrates. After adhesion of monocytes to the endothelium in atherosclerosis, activation of PAR-2 by MT-SP1/matriptase may contribute to local inflammatory changes. This mechanism of cytokine induction will presumably not constitute an initial event in inflammation but may perpetuate the inflammatory process within the atherosclerotic vessel wall. In summary, in addition to its role in cancer invasion, metastasis, and oncogenic transformation or as activator of uPA and HGF, we identified a novel proinflammatory role of MT-SP1/matriptase via activation of PAR-2 in endothelial cells.

Acknowledgments

We thank Mrs B. Campbell, A. Stobbe, and C. Bauer for invaluable technical assistance.

Sources of Funding

The study was supported by grants from the Sander Stiftung and Deutsche Herzstiftung.

Disclosures

None.

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作者单位：Isabell Seitz; Sibylle Hess; Henk Schulz; Robert Eckl; Gabriele Busch; Hans Peter Montens; Richard Brandl; Stefan Seidl; Albert Schömig; Ilka OttFrom the Deutsches Herzzentrum und 1. Medizinische Klinik (I.S., G.B., A.S., I.O.), Technische Universität München, Germany; Morphochem AG M