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Activated Protein C Directly Activates Human Endothelial Gelatinase A

2000; Elsevier BV; Volume: 275; Issue: 13 Linguagem: Inglês

10.1074/jbc.275.13.9095

1083-351X

Minh Nguyen, Jacky Arkell, Chris Jackson,

Hemostasis and retained surgical items

Angiogenesis (formation of new blood vessels) occurs in a number of diseases such as cancer and arthritis. The matrix metalloproteinase (MMP), gelatinase A, is secreted by endothelial cells and plays a vital role during angiogenesis. It is secreted as a latent enzyme and requires extracellular activation. We investigated whether activated protein C (APC), a pivotal molecule involved in the body's natural anti-coagulant system, could activate latent gelatinase A secreted by human umbilical vein endothelial cells (HUVEC). APC induced the fully active form of gelatinase A in a dose (100–300 nm)- and time (4–24 h)-responsive manner. The inactive zymogen, protein C, did not activate gelatinase A when used at similar concentrations. APC did not up-regulate membrane type 1 MMP (MT1-MMP) mRNA in HUVEC. In addition, the MMP inhibitor, 1,10-phenanthroline (10 nm), was unable to inhibit APC-induced activation. These results suggested that MT1-MMP was not involved in the activation process. APC activation of gelatinase A occurred in the absence of cells, indicating that it acts directly. APC may contribute to the physiological/pathological mechanism of gelatinase A activation, especially during angiogenesis. Angiogenesis (formation of new blood vessels) occurs in a number of diseases such as cancer and arthritis. The matrix metalloproteinase (MMP), gelatinase A, is secreted by endothelial cells and plays a vital role during angiogenesis. It is secreted as a latent enzyme and requires extracellular activation. We investigated whether activated protein C (APC), a pivotal molecule involved in the body's natural anti-coagulant system, could activate latent gelatinase A secreted by human umbilical vein endothelial cells (HUVEC). APC induced the fully active form of gelatinase A in a dose (100–300 nm)- and time (4–24 h)-responsive manner. The inactive zymogen, protein C, did not activate gelatinase A when used at similar concentrations. APC did not up-regulate membrane type 1 MMP (MT1-MMP) mRNA in HUVEC. In addition, the MMP inhibitor, 1,10-phenanthroline (10 nm), was unable to inhibit APC-induced activation. These results suggested that MT1-MMP was not involved in the activation process. APC activation of gelatinase A occurred in the absence of cells, indicating that it acts directly. APC may contribute to the physiological/pathological mechanism of gelatinase A activation, especially during angiogenesis. matrix metalloproteinase membrane-type MMP activated protein C human umbilical vein endothelial cells tissue inhibitor of metalloproteinase-2 Angiogenesis is a prominent feature of cancer and arthritis. The matrix metalloproteinase (MMP),1 gelatinase A, plays a vital role during angiogenesis by degrading the collagens present in the basement membrane (1.Basbaum C.B. Werb Z. Curr. Opin. Cell Biol. 1996; 8: 731-738Crossref PubMed Scopus (293) Google Scholar) and allowing the endothelial cells to invade the stroma. The enzyme is constitutively expressed by human endothelial cells in a latent form and can be activated by membrane-type MMPs (MT-MMPs) on the cell surface (2.Sato H. Seiki M. J. Biochem. (Tokyo). 1996; 119: 209-215Crossref PubMed Scopus (195) Google Scholar). Activation can be induced in endothelial cells by non-physiological agents, such as phorbol myristate acetate, resulting in the generation of the intermediate active 62-kDa and the fully active 59-kDa species (3.Foda H.D. George S. Conner C. Drews M. Tompkins D.C. Zucker S. Lab. Invest. 1996; 74: 538-545PubMed Google Scholar). Recently, two physiological agents, thrombin and type I collagen, have been shown to activate gelatinase A in human and rat endothelial cells, respectively (4.Nguyen M. Arkell J. Jackson C.J. Lab. Invest. 1999; 79: 467-476PubMed Google Scholar, 5.Hass T.L. Davis S.J. Madri J.A. J. Biol. Chem. 1998; 273: 3604-3610Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). Activated protein C (APC) is a serine protease that plays a central role in physiological anticoagulation. The inactive precursor, protein C, is a vitamin K-dependent glycoprotein synthesized by the liver and found in the plasma. Activation of protein C occurs on the endothelial cell surface and is triggered by a complex formed between thrombin and thrombomodulin (6.Esmon C.T. FASEB J. 1999; 9: 946-955Crossref Scopus (361) Google Scholar). APC functions as an anticoagulant by binding to the co-factor, protein S, which inactivates the clotting factors Factor VIIIa and Factor Va. The importance of APC as an anticoagulant is reflected by the findings that deficiencies in this molecule result in familial disorders of thrombosis (7.Baker W.F. Bick R.L. Semin. Thromb. Hemostasis. 1999; 25: 387-405Crossref PubMed Scopus (46) Google Scholar). In addition to its anti-coagulant activity, APC has been reported to have an anti-inflammatory effect (8.Cicala C. Cirino G. Life Sci. 1998; 62: 1817-1824Crossref PubMed Scopus (154) Google Scholar). In the current report we describe a new role for APC, demonstrating that it can activate gelatinase A in human endothelial cells. Human APC and human protein C were obtained from ICN Biomedicals (Aurora, OH). TIMP2 was purchased from Oncogene Science (Uniondale, NY). 1,10-Phenanthroline was obtained from Sigma. Human umbilical vein endothelial cells (HUVEC) were isolated and maintained as described previously (9.Jaffe E.A. Nachman R.L. Becker C.G. Minick C.R. J. Clin. Invest. 1973; 52: 2745-2756Crossref PubMed Scopus (6019) Google Scholar). HUVEC were grown in Biorich containing 20% fetal calf serum plus 50 μg/ml endothelial cell growth supplement (Sigma) and 50 μg/ml heparin (Sigma). Cells were used at passage four. Cells were plated down at 30,000 cells/well in 96-well plates in growth medium for 5 days. They were washed twice with Hanks' balanced salt solution and preincubated for 6 h in basal medium (Biorich plus 1% normal pooled human serum, which was stripped of gelatinases by running through a gelatin-Sepharose column) (Amersham Pharmacia Biotech). The culture medium was then replaced with fresh basal medium, and test agents were added for 24 h. The conditioned media were collected for analysis. To ensure that the results were standardized between wells, the cell numbers were quantitated using the CellTiter One Solution cell proliferation assay (Promega, Madison, WI). The cell numbers did not differ between any of the treatments used in the experiments (data not shown). Gelatinase A was detected using gelatin zymography under non-reducing conditions as described previously (10.Herron G.S. Banda M.J. Clark E.J. Gavrilovic J. Werb Z. J. Biol. Chem. 1986; 261: 2814-2818Abstract Full Text PDF PubMed Google Scholar). The gels were scanned into an IBM PC, and the intensity of the bands was semi-quantitated using Scion Image (Meyer Instruments, Houston, TX). Latent and active gelatinase A were detected by Western analysis after SDS-polyacrylamide gel electrophoresis. A monoclonal antibody to gelatinase A (Oncogene Science) was used at 2 μg/ml. The extraction of total RNA was performed using the acid guanidine thiocyanate/phenol/chloroform method of Chomczynski and Sacchi (11.Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). Ten μg of total RNA was run on a 1% agarose gel containing 1.25 m formaldehyde. The RNA was transferred to a Hybond-N+ nylon membrane (Sigma) and cross-linked by ultraviolet irradiation. Northern analysis for MT1-MMP was performed as described previously (4.Nguyen M. Arkell J. Jackson C.J. Lab. Invest. 1999; 79: 467-476PubMed Google Scholar). The MT1-MMP probe was generously provided by Prof. Paul Basset (Illkirch, France). HUVEC were treated with human APC or no test agent for 24 h, and the conditioned media were analyzed for gelatinase A by zymography. Results are shown in Fig. 1 a. Consistent with our previous report (12.Jackson C.J. Nguyen M. Int. J. Biochem. Cell Biol. 1997; 29: 1167-1177Crossref PubMed Scopus (116) Google Scholar), under basal conditions, HUVEC expressed a prominent latent form, a 62-kDa intermediate form (∼3% total gelatinase A activity, as determined by scanning densitometry) and a barely detectable level of the 59-kDa fully active form of gelatinase A (<1% total protein). Treatment of cells with 100 nm APC substantially enhanced the amount of the 59-kDa fully active form to ∼8% of total gelatinase A activity. Western analysis confirmed the activation of gelatinase A by APC (Fig.1 b). APC was dose-responsive in its activation of gelatinase A (Fig.1 a). When used at 200 nm, APC increased the amount of the 59-kDa fully active enzyme by approximately 2.1-fold, compared with 100 nm. Interestingly, there was a concomitant decrease in the amount of the intermediate species generated as the concentration of APC increased. At 300 nm, APC converted almost all the intermediate form to the fully active form. In contrast, the inactive zymogen, protein C, did not activate gelatinase A when used at similar concentrations to APC (Fig.1 c). Time course experiments revealed that APC induced the fully active form as early as 4 h, the levels of which progressively increased after 12 and 24 h of exposure to APC (Fig.2). Previous workers have shown that plasmin (13.Mazzieri R. Masiero L. Zanetta L. Monea S. Onisto M. Garbisa S. Mignatti P. EMBO J. 1997; 16: 2319-2332Crossref PubMed Scopus (373) Google Scholar, 14.Baramova E.N. Bajou K. Remacle A. Lhoir C. Krell H.W. Weidle U.H. Noel A. Foidart J.M. FEBS Lett. 1997; 405: 157-162Crossref PubMed Scopus (241) Google Scholar) can activate gelatinase A to the fully active form in HT1080 cells. To determine whether contaminating plasmin was contributing toward endothelial gelatinase A activation by APC, we tested whether the serine protease inhibitor, aprotinin, was able to inhibit activation. Aprotinin inhibits plasmin, trypsin, and kallikrein but not APC (15.Gebhard N. Tschesche H. Fritz H. Barrett A.J. Salvesen G. Biochemistry of Aprotinin and Aprotinin-like Inhibitors in Protease Inhibitors. Elsevier Science Publishers B.V., Amsterdam1986: 375-388Google Scholar). The inhibitor was added to HUVEC in the presence of 100 nm APC for 16–20 h at concentrations of 10 or 25 μm. Aprotinin did not inhibit APC-induced gelatinase A activation at either concentration (Fig. 3). This suggests that activation of endothelial gelatinase A is attributable to APC and is not due to plasmin. We examined whether the activation by APC was mediated through the well described MT1-MMP pathway. First, we measured the levels of mRNA for MT1-MMP by Northern analysis. HUVEC did not up-regulate MT1-MMP mRNA after stimulation with 100 nmAPC for 24 h (Fig. 4). To confirm that MT1-MMP was not involved, we tested the effects of the MMP inhibitor, 1,10-phenanthroline, at 10 μg/ml. Previous workers have shown that MT1-MMP-mediated activation of gelatinase A is blocked by 1,10-phenanthroline at this concentration (4.Nguyen M. Arkell J. Jackson C.J. Lab. Invest. 1999; 79: 467-476PubMed Google Scholar). HUVEC were stimulated with APC (100 nm) for 24 h in the presence of 1,10-phenanthroline, and the conditioned medium was analyzed using zymography. Results are shown in Fig. 3. As expected, phenanthroline (shown as Phenan in Fig. 3) inhibited the production of the 62-kDa intermediate form, which has previously been shown to be generated by constitutively expressed MT1-MMP (12.Jackson C.J. Nguyen M. Int. J. Biochem. Cell Biol. 1997; 29: 1167-1177Crossref PubMed Scopus (116) Google Scholar). In contrast, the generation of the 59-kDa fully active form by APC was not affected by phenanthroline. Together, these results suggested that activation of gelatinase A by APC does not require active MT1-MMP. To determine whether APC was dependent upon MT1-MMP or another endothelial membrane protein(s), we examined its effect on gelatinase A in the absence of cells. HUVEC-conditioned medium, which contains latent gelatinase A, was incubated in the presence or absence of 100 nm APC for 24 h at 37 °C. The samples were then measured for gelatinase A activity using zymography (Fig. 5). In the absence of APC, conditioned medium contained an intermediate band and barely detectable fully active band of gelatinase A. In response to APC, the levels of the fully active band were markedly elevated, indicating that APC directly activated gelatinase A and did not require the presence of cells. TIMP2 has several functions, which are independent of its inhibition of MMPs. For example, TIMP2 binds to the C-terminal domain of gelatinase A and at low concentrations promotes activation via MT1-MMP (16.Zucker S. Drews M. Conner C. Foda H.D. DeClerck Y.A. Langley K.E. Bahou W.F. Docherty A.P. Cao J. J. Biol. Chem. 1998; 273: 1216-1222Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 17.Cao J. Drews M. Lee H.M. Conner C. Bahou W.F. Zucker S. J. Biol. Chem. 1998; 273: 34745-34752Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). We tested the effect of TIMP2 on APC-induced activation of gelatinase A. TIMP2 (10 or 25 nm) was added to HUVEC in the presence of 100 nm APC for 24 h. The results of zymographic analysis of the conditioned medium are shown in Fig. 6. When used at 10 and 25 nm, TIMP2 completely inhibited the generation of the intermediate form. This observation is in agreement with previous workers who reported that excess TIMP2 inhibits the formation of the 62-kDa intermediate species generated by MT1-MMP (18.Kinoshita T. Sato H. Okada A. Ohuchi E. Imai K. Okada Y. Seiki M. J. Biol. Chem. 1998; 273: 16098-16103Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 19.Will H. Atkinson S.J. Butler G.S. Smith B. Murphy G. J. Biol. Chem. 1996; 271: 17119-17123Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar). Interestingly, at both concentrations, TIMP2 partially prevented the formation of the 59-kDa fully active species generated by APC. At 25 nm, TIMP2 inhibited the fully active form by 83 ± 1.7% as determined by scanning densitometry (mean of 3 cell lines). Since we have shown that MT1-MMP is not involved in this process (Figs.3 and 4), it appears that TIMP2 is playing an independent role during APC-induced gelatinase A activation. It is feasible that excess TIMP2 may interfere with an interaction between APC and gelatinase A (and possibly other molecules) and thus partially reduces activation. The mechanism of TIMP2 inhibition needs to be further explored. Our report is the first to show that the serine protease, APC, activates gelatinase A. Two other serine proteases, plasmin and thrombin, have previously been shown to activate gelatinase A. In contrast to our results for APC, activation by plasmin is fully inhibited by TIMP2 in HT1080 cells (14.Baramova E.N. Bajou K. Remacle A. Lhoir C. Krell H.W. Weidle U.H. Noel A. Foidart J.M. FEBS Lett. 1997; 405: 157-162Crossref PubMed Scopus (241) Google Scholar). In addition, in the absence of cell membranes, APC activates gelatinase A, whereas plasmin does not activate but rapidly generates degradation products that lack catalytic activity. Thus, it appears that APC works via a different mechanism to plasmin. Zucker et al. (20.Zucker S. Conner C. Dimassmo B.I. Ende H. Drews M. Seiki M. Bahou W.F. J. Biol. Chem. 1995; 270: 23730-23738Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) first reported that thrombin can induce gelatinase A activation in human endothelial cells. We have recently shown that activation of gelatinase A by thrombin is rapid, efficient, and independent of MT1-MMP (4.Nguyen M. Arkell J. Jackson C.J. Lab. Invest. 1999; 79: 467-476PubMed Google Scholar). Similarly, we have shown here that APC generates the fully active form within 4 h and does not require MT1-MMP. Thrombin, through its interaction with thrombomodulin on the endothelial cell surface, is a physiological activator of protein C (21.Esmon C.T. Fukudome K. Mather T. Bode W. Regan L.M. Stearns-Kurosawa D.J. Kurosawa S. Haematologica. 1999; 84: 254-259PubMed Google Scholar). It is feasible that thrombin-induced activation is mediated through APC. This is currently under investigation. MT1-MMP has recently been implicated as the key participant in physiological activation of gelatinase A in most cell types, including human endothelial cells (3.Foda H.D. George S. Conner C. Drews M. Tompkins D.C. Zucker S. Lab. Invest. 1996; 74: 538-545PubMed Google Scholar). HUVEC express MT1-MMP under basal conditions, which can be up-regulated by the potent tumor-promoting chemical, phorbol myristate acetate (3.Foda H.D. George S. Conner C. Drews M. Tompkins D.C. Zucker S. Lab. Invest. 1996; 74: 538-545PubMed Google Scholar). Surprisingly, there have been no reported physiological agents that activate gelatinase A via MT1-MMP in human endothelial cells. Prior to the current report, thrombin, which acts via a mechanism independent of MT1-MMP (4.Nguyen M. Arkell J. Jackson C.J. Lab. Invest. 1999; 79: 467-476PubMed Google Scholar), was the only known physiological substance that can activate gelatinase A in these cells. The contribution of MT1-MMP, thrombin, or APC in physiological/pathological activation of endothelial gelatinase A is unknown. It is possible that these molecules act synergistically to generate active gelatinase A. Whereas the biological actions of plasmin and thrombin are multifactorial, APC is thought to be a relatively selective enzyme. In the presence of its cofactor, Protein S, it inactivates Factors Va and VIIIa, which leads to anti-coagulation (6.Esmon C.T. FASEB J. 1999; 9: 946-955Crossref Scopus (361) Google Scholar). The reason(s) that this pivotal molecule involved in physiological anti-coagulation activates gelatinase A is unclear. It is unlikely that the active gelatinase A directly contributes to fibrinolysis, as Bini et al. (22.Bini A. Itoh Y. Kudryk B.J. Nagase H. Biochemistry. 1996; 35: 13056-13063Crossref PubMed Scopus (90) Google Scholar) have shown that gelatinase A does not cleave fibrin. However, there is ample evidence to show that gelatinase A plays a vital role during angiogenesis. It induces vascular network formation when added to endothelial cells cultured on Matrigel (23.Schnaper H.W. Grant D.S. Stetlerstevenson W.G. Fridman R. Dorazi G. Murphy A.N. Bird R.E. Hoythya M. Fuerst T.R. French D.L. Quigley J.P. Kleinman H.K. J. Cell. Physiol. 1993; 156: 235-246Crossref PubMed Scopus (281) Google Scholar). Brooks et al.(24.Brooks P.C. Silletti S. Schalscha T. Friedlander M. Cheresh D.A. Cell. 1998; 92: 391-400Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar) demonstrated that a fragment of the hemopexin-like domain of gelatinase A, termed PEX, significantly disrupted angiogenesis in the CAM system. Itoh et al. (25.Itoh T. Tanioka M. Yoshida H. Yoshioka T. Nishimoto H. Itohara S. Cancer Res. 1998; 58: 1048-1051PubMed Google Scholar) have recently reported a substantial reduction in both angiogenic activity and tumor progression in gelatinase A-deficient mice. Our finding that APC activates gelatinase A suggests that a link exists between anticoagulation and angiogenesis. This is supported by the recent findings of O'Reillyet al. (26.O'Reilly M.S. Pirie-Shepherd S. Lane W.S. Folkman J. Science. 1999; 285: 1926-1928Crossref PubMed Scopus (424) Google Scholar) who demonstrated that a cleaved conformation of antithrombin III has potent anti-angiogenic activity. They concluded that the clotting and fibrinolytic pathways are directly involved in the regulation of angiogenesis. It is tempting to speculate that APC activates gelatinase A in angiogenic diseases such as cancer and arthritis, where clotting abnormalities are present. The inhibition of APC-induced gelatinase A activation may prove useful as a potential therapeutic target in angiogenic diseases. We thank Dr. Paul Basset for providing the cDNA for MT1-MMP, Professor Phillip Sambrook, Dr. Ross Davey, and Dr. Jim Melrose for helpful comments, Amanda Burke for expert technical assistance, and Eddie Jozefiak and Paula Ellis for photography.