Factor Xa Induces Mitogenesis of Vascular Smooth Muscle Cells via Autocrine Production of Epiregulin ,

Factor Xa has been reported to elicit smooth muscle cell proliferation via autocrine release of platelet-derived growth factor. However, this study has shown that factor Xa-induced mitogenesis of rat aortic smooth muscle cell is independent of platelet-derived growth factor. We also could not observe any platelet-derived growth factor isoforms in the cultured medium of factor Xa-stimulated cells. Our finding that the cultured medium of factor Xa-stimulated cells strongly induces rat aortic smooth muscle cell mitogenesis in the absence of factor Xa activity led us to explore the existence of a novel autocrine pathway. The autocrine growth factor was purified from the cultured medium and was identified to be epiregulin. Recombinant epiregulin was also able to induce the mitogenesis. The secretion of epiregulin from factor Xa-stimulated rat aortic smooth muscle cell required mRNA expression and protein synthesis of the growth factor. The mitogenic effect of factor Xa on rat aortic smooth muscle cell was significantly reduced by anti-epiregulin antibody or by antisense oligodeoxynucleotide to epiregulin. Several lines of experimental evidence clearly indicate that the autocrine production of epiregulin, an epidermal growth factor-related ligand, is induced in the factor Xa-stimulated mitogenic process of rat aortic smooth muscle cell. Factor Xa has been reported to elicit smooth muscle cell proliferation via autocrine release of platelet-derived growth factor. However, this study has shown that factor Xa-induced mitogenesis of rat aortic smooth muscle cell is independent of platelet-derived growth factor. We also could not observe any platelet-derived growth factor isoforms in the cultured medium of factor Xa-stimulated cells. Our finding that the cultured medium of factor Xa-stimulated cells strongly induces rat aortic smooth muscle cell mitogenesis in the absence of factor Xa activity led us to explore the existence of a novel autocrine pathway. The autocrine growth factor was purified from the cultured medium and was identified to be epiregulin. Recombinant epiregulin was also able to induce the mitogenesis. The secretion of epiregulin from factor Xa-stimulated rat aortic smooth muscle cell required mRNA expression and protein synthesis of the growth factor. The mitogenic effect of factor Xa on rat aortic smooth muscle cell was significantly reduced by anti-epiregulin antibody or by antisense oligodeoxynucleotide to epiregulin. Several lines of experimental evidence clearly indicate that the autocrine production of epiregulin, an epidermal growth factor-related ligand, is induced in the factor Xa-stimulated mitogenic process of rat aortic smooth muscle cell. Vascular smooth muscle cell (SMC)1 migration and proliferation are necessary events that contribute to the formation of neointimal hyperplasia in atherosclerosis, restenosis, and venous bypass graft disease (1Luscher T.F. Turina M. Braunwald E. Coronary Artery Graft Disease: Mechanisms and Prevention. Springer, Heidelberg, Germany1994: 42-52Crossref Google Scholar, 2Ross R. N. Engl. J. Med. 1986; 314: 488-500Crossref PubMed Scopus (4043) Google Scholar). SMC mitogenesis is triggered by a large number of growth factors, cytokines, and extracellular matrix proteins that participate in the pathogenic process (3Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (10041) Google Scholar). The induction of various mitogenic factor expressions in mechanically injured arteries (4Majesky M.W. Lindner V. Twardzik D.R. Schwartz S.M. Reidy M.A. J. Clin. Invest. 1991; 8: 904-910Crossref Scopus (475) Google Scholar, 5Inoue S. Orimo A. Hosoi T. Matsuse T. Hashimoto M. Yamada R. Ouchi Y. Orimo H. 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Invest. 1993; 91: 94-98Crossref PubMed Scopus (466) Google Scholar). Thrombin also regulates cellular responses during inflammation (16Bar-Shavit R. Benezra M. Sabbah V. Bode W. Vlodavsky I. Am. J. Respir. Cell Mol. Biol. 1992; 6: 123-130Crossref PubMed Scopus (80) Google Scholar) and inhibits the migration and invasion of breast cancer cells (17Kamath L. Meydani A. Foss F. Kuliopulos A. Cancer Res. 2001; 61: 5933-5940PubMed Google Scholar). After stimulation by thrombin, SMCs produce and secrete autocrine growth factors, including basic fibroblast growth factor (18Weiss R.H. Maduri M. J. Biol. Chem. 1993; 268: 5724-5727Abstract Full Text PDF PubMed Google Scholar), PDGF (19Stouffer G.A. Sarembock I.J. McNamara C.A. Gimple L.W. Owens G.K. Am. J. Physiol. 1993; 265: C806-C811Crossref PubMed Google Scholar), heparin-binding epidermal growth factor (EGF) (20Nakano T. Raines E.W. Abraham J.A. Wenzel F.G. 4th Higashiyama S. Klagsbrun M. Ross R. J. Biol. Chem. 1993; 268: 22941-22947Abstract Full Text PDF PubMed Google Scholar), and transforming growth factor-β (21Bachhuber B.G. Sarembock I.J. Gimple L.W. Owens G.K. J. Vasc. Res. 1997; 34: 41-48Crossref PubMed Scopus (37) Google Scholar). Factor Xa acts as the major mediator of thrombus formation at the sites of vascular injury by activating prothrombin to thrombin and as a potent mitogen for endothelial cells (22Bono F. Herault J.P. Avril C. Schaeffer P. Lormeau J.C. Herbert J.M. J. Cell. Physiol. 1997; 172: 36-43Crossref PubMed Scopus (35) Google Scholar), fibroblasts (23Blanc-Brude O.P. Chambers R.C. Leoni P. Dik W.A. Laurent G.J. Am. J. Physiol. Cell Physiol. 2001; 281: C681-C689Crossref PubMed Google Scholar), and aortic SMCs (24Gasic G.P. Arenas C.P. Gasic T.B. Gasic G.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2317-2320Crossref PubMed Scopus (185) Google Scholar, 25Ko F.N. Yang Y.C. Huang S.C. Ou J.T. J. Clin. Invest. 1996; 98: 1493-1501Crossref PubMed Scopus (75) Google Scholar, 26Herbert J. Bono F. Herault J. Avril C. Dol F. Mares A. Schaeffer P. J. Clin. Invest. 1998; 101: 993-1000Crossref PubMed Scopus (70) Google Scholar). Factor Xa also induces the production of cytokines and adhesion molecules such as interleukin-6, interleukin-8, monocyte chemotactic protein-1, E-selectin, intercellular adhesion molecule 1, and vascular cell adhesion molecule-1 in human umbilical vein endothelial cells, leading to pro-inflammatory responses (27Senden N.H. Jeunhomme T.M. Heemskerk J.W. Wagenvoord R. van't Veer C. Hemker H.C. Buurman W.A. J. Immunol. 1998; 161: 4318-4324PubMed Google Scholar, 28Papapetropoulos A. Piccardoni P. Cirino G. Bucci M. Sorrentino R. Cicala C. Johnson K. Zachariou V. Sessa W.C. Altieri D.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4738-4742Crossref PubMed Scopus (61) Google Scholar). Although some aspects of the effect of factor Xa on aortic SMCs through PDGF have been investigated (25Ko F.N. Yang Y.C. Huang S.C. Ou J.T. J. Clin. Invest. 1996; 98: 1493-1501Crossref PubMed Scopus (75) Google Scholar, 26Herbert J. Bono F. Herault J. Avril C. Dol F. Mares A. Schaeffer P. J. Clin. Invest. 1998; 101: 993-1000Crossref PubMed Scopus (70) Google Scholar), the PDGF isoform that mediates factor Xa-induced SMC mitogenesis has not been identified yet. Moreover, no information is available about any particular autocrine production that is associated with factor Xa-induced SMC mitogenesis. In this work we have demonstrated that factor Xa-induced mitogenesis of rat aortic smooth muscle cells (RASMCs) is independent of PDGF. More importantly, we have identified epiregulin as the major element responsible for factor Xa-induced RASMC mitogenesis. It was also revealed that autocrine production of epiregulin is essential in the mitogenesis. Epiregulin is the newest member of the family of EGF-like ligands, and it was first identified from the cultured medium of mouse fibroblast-derived tumor cell line NIH3T3/clone T7 (29Toyoda H. Komurasaki T. Uchida D. Takayama Y. Isobe T. Okuyama T. Hanada K. J. Biol. Chem. 1995; 270: 7495-7500Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). It was subsequently shown that epiregulin acts as a mitogen for various cell types, including fibroblasts, aortic SMCs, hepatocytes (29Toyoda H. Komurasaki T. Uchida D. Takayama Y. Isobe T. Okuyama T. Hanada K. J. Biol. Chem. 1995; 270: 7495-7500Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar), keratinocytes (30Shirakata Y. Komurasaki T. Toyoda H. Hanakawa Y. Yamasaki K. Tokumaru S. Sayama K. Hashimoto K. J. Biol. Chem. 2000; 275: 5748-5753Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), and pancreatic cancer cells (31Zhu Z. Kleeff J. Friess H. Wang L. Zimmermann A. Yarden Y. Buchler M.W. Korc M. Biochem. Biophys. Res. Commun. 2000; 273: 1019-1024Crossref PubMed Scopus (80) Google Scholar). Epiregulin was also purified from the cultured medium of angiotensin II-stimulated RASMCs and was identified to be a major mitogenic constituent (32Taylor D.S. Cheng X. Pawlowski J.E. Wallace A.R. Ferrer P. Molloy C.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1633-1638Crossref PubMed Scopus (69) Google Scholar). Materials—Dulbecco's modified Eagle's medium (DMEM) was purchased from Invitrogen, and [3H]thymidine was from PerkinElmer Life Sciences. Bovine factor Xa, bovine thrombin, and pGEX-2T vector were purchased from Novagen, Inc., and Pefabloc®TH (Pefa-3204) was from Pentapharm Ltd. (Switzerland). Recombinant tick anticoagulant protein (rTAP) was kindly provided by Dr. Y. Jang (Cardiovascular Research Institute, Yonsei University College of Medicine, Seoul, Korea). NuPAGE™ 4–12% Bis-Tris gel was purchased from Invitrogen, and DC protein assay reagent from Bio-Rad. An antibody to phospho-p44/42 mitogen-activated protein kinase (ERK-1/2) and an antibody that recognizes both phosphorylated and nonphosphorylated forms of p44/42 mitogen-activated protein kinase were obtained from Cell Signaling Technology. ECL reagent, benzamidine-Sepharose, and were purchased from and from was purchased from and were purchased from human and human and epiregulin antibody from was purchased from Inc., and vector were obtained from and were from were of the Cell were by a of the of G.R. Ross R. Physiol. PubMed Scopus Google Scholar). The from were and in The was in of the with and for at the was with and the were a The was a of the and for at The was at for and the was in with RASMCs were cultured in with in RASMCs were with synthesis in cells, the of [3H]thymidine was RASMCs were a and for in After the cells were in for were with or After an the cells were with of [3H]thymidine for at and with The cells were in at for and with The was in at by with Cell were and at for After the cells were in for were for with or The cells were with factor Xa for The medium was and the cell was a the RASMCs were to to in and in for After stimulation with for in the or absence of the cells were in and with on Cell were and at for was in the DC protein assay reagent to the and for PDGF and epiregulin RASMCs were to to and for After stimulation with factor Xa, the cultured were a with a were on NuPAGE™ 4–12% Bis-Tris gel and to The were with by with ECL was for were to to in and for After and with the cells were with factor After the cultured medium was and at for to The of and in the medium were of Epiregulin from the of Factor Xa-stimulated was purified from the medium that was with factor Xa in for The cultured medium was and to with high cells an with a The medium was to a with and proteins were with the activity in was by [3H]thymidine in were and a with The proteins were with a of in A. were and in a gel with at a of mitogenic activity were and to at a of proteins were from the with a of was subsequently on NuPAGE™ 4–12% Bis-Tris gel for The gel was by The purified protein was for with expression of epiregulin mRNA was the the RASMCs were for in and with factor Xa for the was by and was from the of of of and of was at for After at for the was to were for at for for and for with and on the of the rat epiregulin and The was as the and The was and its was the for rat epiregulin was modified by for pGEX-2T The a The a and an The was with of at for at for and at for The modified was and its were the expression for rat epiregulin, the epiregulin vector was with and and the expression pGEX-2T forms a of to the of rat epiregulin. and of Recombinant expression vector was The were in medium to an of at induced with and for an at The expressed of epiregulin were the were in The protein was with a at for The protein was to and with thrombin for at The epiregulin was and to a at a of proteins were from the with a of to to and antisense to with were to the sites of rat epiregulin. rat epiregulin, the was and was The of the for rat epiregulin was by the of was an was to be of Factor Xa-stimulated Xa-induced mitogenesis of RASMCs was by [3H]thymidine and the cell number in RASMCs by the factor Xa [3H]thymidine the cell and the cell number with mitogenic effect was also with RASMCs the mitogenic effect of factor Xa on RASMCs is on its a factor Xa was shown in 1, significantly reduced the mitogenesis of RASMCs that was induced by factor Xa, that the induction of RASMC mitogenesis is associated with the activity of factor on its not the [3H]thymidine and proliferation of cells not the that the mitogenic of factor Xa be by the production of thrombin, the factor Xa-induced mitogenesis was in the of a thrombin clearly indicate that Pefabloc®TH not factor Xa-induced the mitogenic effect was by the Pefabloc®TH not the [3H]thymidine and proliferation of RASMCs not PDGF of Factor Xa-induced and the factor Xa-induced mitogenesis is associated with the secretion of RASMCs were with factor Xa in the of isoforms of and demonstrated in the antibody to the in [3H]thymidine and in factor Xa-stimulated cells. the of autocrine PDGF for this particular mitogenic RASMCs were by factor Xa in the of a kinase of PDGF M. A. A. C. L. J. A. Cancer Res. Google Scholar). indicate that the factor Xa-induced [3H]thymidine was by the reagent In it was demonstrated that the antibody or the mitogenic induced by The of the antibody or the itself in the cells was on such mitogenic not of PDGF in the factor Xa the secretion of PDGF from was this the cultured from or factor RASMCs was and by or The obtained indicate that the by the or antibody was not in the not the of and in the cultured the of growth were The stimulation of RASMCs with factor Xa for to not in any secretion of and in the cultured medium not The of assay in the were and Cell the factor Xa-induced mitogenesis is the cultured was from the factor Xa-stimulated RASMCs at various to and to the RASMCs to the effect of factor The cells were for [3H]thymidine and cell that the factor medium was in significantly synthesis and cell number in RASMCs the medium of cells to such mitogenic not antibody or not the [3H]thymidine induced by the cultured medium that is no for autocrine PDGF in the particular mitogenesis induced by factor Xa The mitogenic activity factor Xa stimulation and its in and of the is responsible for the factor Xa-stimulated mitogenesis of was purified to from of the medium in After the cultured medium was by affinity on a of activity was in the of the with factor Xa was in the the were to a the mitogenic activity was by the with The were and in a gel the mitogenic activity was purified to in a phase The protein in a of as a with an of the of the of the purified protein was through it was revealed to be is to that of epiregulin. of Epiregulin mRNA in Factor Xa-stimulated was to the mRNA of epiregulin in factor Xa-stimulated were on the of the of the rat epiregulin After stimulation of the RASMCs with factor Xa, it was to observe a and induction of epiregulin of the mRNA its at and to the in and of Recombinant rat epiregulin was and expressed in E. as a of The expressed protein was to the and at a to the Recombinant epiregulin was purified to phase The purified epiregulin as a to epiregulin, on The of the epiregulin with the protein was by and by we the epiregulin for its to RASMC mitogenesis the epiregulin was able to the RASMC mitogenesis in a Epiregulin of Factor Xa-induced RASMC the of epiregulin in factor Xa-stimulated we the effect of anti-epiregulin antibody on factor Xa-induced [3H]thymidine RASMCs were by factor Xa in the of anti-epiregulin antibody or The anti-epiregulin antibody significantly reduced [3H]thymidine the the not the mitogenic effect induced by factor Xa In the anti-epiregulin antibody the mitogenic effect of the epiregulin. The anti-epiregulin antibody effect on [3H]thymidine the cells not the of growth in factor Xa-induced RASMCs were with factor Xa for and the cultured medium was for RASMC mitogenesis the addition of and anti-epiregulin antibody to the of factor Xa and epiregulin, were in the cultured The medium that was the effect on RASMC that epiregulin is the major autocrine factor in factor Xa-stimulated RASMCs not the of epiregulin in factor Xa-induced RASMC we an to the protein expression of epiregulin. RASMCs were with epiregulin to the expression of its a of epiregulin production was by the cultured medium with anti-epiregulin antibody The also significantly reduced [3H]thymidine the factor Xa-stimulated RASMCs the the was not in the mitogenic of factor The itself was in RASMC mitogenesis in work that factor Xa the mitogenesis of RASMCs via autocrine release of epiregulin. It has been that factor Xa the mitogenesis of aortic SMCs (24Gasic G.P. Arenas C.P. Gasic T.B. Gasic G.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2317-2320Crossref PubMed Scopus (185) Google Scholar, 25Ko F.N. Yang Y.C. Huang S.C. Ou J.T. J. Clin. Invest. 1996; 98: 1493-1501Crossref PubMed Scopus (75) Google Scholar, 26Herbert J. Bono F. Herault J. Avril C. Dol F. Mares A. Schaeffer P. J. Clin. Invest. 1998; 101: 993-1000Crossref PubMed Scopus (70) Google Scholar) and vein SMCs E. M. A. M. E. K. Thromb. 2000; PubMed Scopus (35) Google Scholar). the study demonstrated that factor Xa both synthesis and the proliferation of RASMCs with cells. Factor Xa-induced mitogenesis of RASMCs was by its not by the thrombin that thrombin is not associated with the mitogenic pathway. factor Xa has been to elicit SMC proliferation via autocrine stimulation a isoform of PDGF (25Ko F.N. Yang Y.C. Huang S.C. Ou J.T. J. Clin. Invest. 1996; 98: 1493-1501Crossref PubMed Scopus (75) Google Scholar), it was investigated to the PDGF isoform that mediates factor Xa-induced SMC mitogenesis. isoforms of PDGF are able to SMC proliferation with However, of the PDGF isoforms cellular on vascular SMCs B. S. L. 1996; Full Text PDF PubMed Scopus (67) Google Scholar). and are from in the of The isoform can also with an that to its or is able to of and the PDGF we the factor Xa-induced mitogenesis is associated with the release of PDGF in on the of experimental obtained with antibody and with PDGF we have that the factor Xa-induced RASMC mitogenesis is independent of the autocrine release of PDGF. obtained from provided evidence that no PDGF isoform is from the factor Xa-stimulated finding is in to the of (25Ko F.N. Yang Y.C. Huang S.C. Ou J.T. J. Clin. Invest. 1996; 98: 1493-1501Crossref PubMed Scopus (75) Google Scholar) and Herbert J. Bono F. Herault J. Avril C. Dol F. Mares A. Schaeffer P. J. Clin. Invest. 1998; 101: 993-1000Crossref PubMed Scopus (70) Google Scholar), that factor Xa-induced mitogenesis on PDGF release in human and rat aortic E. M. A. M. E. K. Thromb. 2000; PubMed Scopus (35) Google Scholar), that factor Xa-induced and mitogenic activity in human venous SMCs are independent of PDGF. It was for us to on the of a novel autocrine pathway that is associated with the factor Xa-induced mitogenesis of In the cultured medium of factor Xa-stimulated we an autocrine that was identified to be epiregulin, an growth factor (29Toyoda H. Komurasaki T. Uchida D. Takayama Y. Isobe T. Okuyama T. Hanada K. J. Biol. Chem. 1995; 270: 7495-7500Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, H. Komurasaki T. Y. M. S. FEBS Lett. 1995; PubMed Scopus (75) Google Scholar). Epiregulin was purified to from the cultured medium of factor Xa-stimulated RASMCs through a gel and phase affinity has been to PDGF and a number of growth that to However, it was to that epiregulin, a growth not to in the absence of In this of epiregulin from the cultured any mitogenic in the were not the of heparin-binding growth that are to the mitogenic response to factor It has been that epiregulin to both and not to and T. Toyoda H. Uchida D. S. 1997; PubMed Scopus Google Scholar). Epiregulin not the of both and also M. R. H. Wang L. M. A. Yarden Y. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). is not expressed in as or as with or to as the mitogenic for epiregulin (32Taylor D.S. Cheng X. Pawlowski J.E. Wallace A.R. Ferrer P. Molloy C.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1633-1638Crossref PubMed Scopus (69) Google Scholar). Although epiregulin affinity for is epiregulin potent mitogenic M. R. H. Wang L. M. A. Yarden Y. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). affinity and is to a that and in a M. R. H. Wang L. M. A. Yarden Y. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). factor Xa can its mitogenic in RASMCs through the autocrine production of epiregulin, acts as an and potent demonstrated that epiregulin is potent and or transforming growth in Komurasaki T. J. Cell. Biochem. 89: PubMed Scopus Google Scholar). In the process of the blood coagulation pathway is leading to the formation of factor Xa and After blood in the injured the coagulation be in by the production of potent including epiregulin, in cells. The mitogenic effect of factor Xa on RASMCs was not by an antibody epiregulin or by to epiregulin. The mitogenic by anti-epiregulin antibody or by in factor Xa-induced RASMCs be to the of growth that are during the cell However, this not to be the the cultured medium and anti-epiregulin antibody was not able to RASMC mitogenesis. are that factor Xa can both and in vascular cells D.S. FEBS Lett. PubMed Scopus Google Scholar, F. Schaeffer P. Herault J.P. C. J.C. Herbert J.M. Arterioscler. Thromb. Vasc. Biol. 2000; PubMed Google Scholar, E. Huang W. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar, M. W. 2001; PubMed Scopus Google Scholar). are that elicit responses to the for is that factor Xa its mitogenic effect on RASMCs via The study on the of mitogenic effect of factor Xa on We have shown that the autocrine production of epiregulin, an ligand, is induced in the factor Xa-stimulated mitogenic process of on the factor Xa-induced leading to the epiregulin expression in work information for the cellular of factor Xa that is associated with RASMC mitogenesis.