Regulation of Bone Morphogenetic Protein-4 by Matrix GLA Protein in Vascular Endothelial Cells Involves Activin-like Kinase Receptor 1

Matrix GLA protein (MGP) has previously been shown to enhance expression of vascular endothelial growth factor (VEGF) through the activin-like kinase receptor 1 (ALK1) in bovine aortic endothelial cells. MGP has also been identified as an inhibitor of bone morphogenetic protein-2 (BMP-2). This study showed that the effect of MGP on ALK1 signaling and VEGF expression in bovine aortic endothelial cells was dose-dependent, that a progressive increase of MGP levels ceased to be stimulatory and instead turned inhibitory. We identified a new regulatory pathway involving BMP that may explain this response. BMP-2 and BMP-4 induced expression of ALK1 in a dose-dependent fashion as determined by real-time PCR and immunoblotting. Activation of ALK1 signaling induced expression of MGP in addition to that of VEGF, allowing for negative feedback regulation of BMP by MGP. MGP inhibited BMP-4 activity similarly to that of BMP-2 and interacted with BMP-4 on a protein level as determined by co-immunoprecipitation. The dose-dependent effect on ALK1 expression and the stimulation of MGP and VEGF expression were dependent on signaling by transforming growth factor-β (TGF-β) and ALK1. Inhibition of TGF-β by neutralizing antibodies abolished the inhibitory effect of high BMP-4 levels on ALK1 expression and the induction of MGP and VEGF. Depletion of ALK1 by small interfering RNA abolished the induction of MGP and VEGF. MGP promoter activity was also stimulated by BMP-4 in a TGF-β-dependent fashion. The results suggest that the effects of BMP on endothelial cells occur in part through induction of ALK1, an effect that may be limited by ALK1-induced MGP. Matrix GLA protein (MGP) has previously been shown to enhance expression of vascular endothelial growth factor (VEGF) through the activin-like kinase receptor 1 (ALK1) in bovine aortic endothelial cells. MGP has also been identified as an inhibitor of bone morphogenetic protein-2 (BMP-2). This study showed that the effect of MGP on ALK1 signaling and VEGF expression in bovine aortic endothelial cells was dose-dependent, that a progressive increase of MGP levels ceased to be stimulatory and instead turned inhibitory. We identified a new regulatory pathway involving BMP that may explain this response. BMP-2 and BMP-4 induced expression of ALK1 in a dose-dependent fashion as determined by real-time PCR and immunoblotting. Activation of ALK1 signaling induced expression of MGP in addition to that of VEGF, allowing for negative feedback regulation of BMP by MGP. MGP inhibited BMP-4 activity similarly to that of BMP-2 and interacted with BMP-4 on a protein level as determined by co-immunoprecipitation. The dose-dependent effect on ALK1 expression and the stimulation of MGP and VEGF expression were dependent on signaling by transforming growth factor-β (TGF-β) and ALK1. Inhibition of TGF-β by neutralizing antibodies abolished the inhibitory effect of high BMP-4 levels on ALK1 expression and the induction of MGP and VEGF. Depletion of ALK1 by small interfering RNA abolished the induction of MGP and VEGF. MGP promoter activity was also stimulated by BMP-4 in a TGF-β-dependent fashion. The results suggest that the effects of BMP on endothelial cells occur in part through induction of ALK1, an effect that may be limited by ALK1-induced MGP. Matrix GLA protein (MGP) 2The abbreviations used are: MGP, matrix GLA protein; N-FLAG-MGP, N-terminally FLAG-tagged MGP; VEGF, vascular endothelial growth factor; ALK1, activin-like kinase receptor 1; EC, endothelial cells; BAEC; bovine aortic EC; HAEC, human aortic EC; BMP, bone morphogenetic protein; TGF, transforming growth factor; siRNA, small interfering RNA; BRE-Luc, ALK1- and BMP-responsive luciferase reporter gene; Luc, luciferase; PBS, phosphate-buffered saline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; FAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetramethylrhodamine; HA, hemagglutinin; h, human; b, bovine. is considered to be an inhibitor of cardiovascular calcification based on the extensive arterial calcification observed in MGP null mice (1Luo G. Ducy P. McKee M.D. Pinero G.J. Loyer E. Behringer R.R. Karsenty G. Nature. 1997; 386: 78-81Crossref PubMed Scopus (1764) Google Scholar, 2Murshed M. Schinke T. McKee M.D. Karsenty G. J. Cell Biol. 2004; 165: 625-630Crossref PubMed Scopus (414) Google Scholar) and the arterial and valvular calcification detected after warfarin intake (3Price P.A. Faus S.A. Williamson M.K. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 1400-1407Crossref PubMed Scopus (470) Google Scholar, 4Schurgers L.J. Aebert H. Vermeer C. Bultmann B. Janzen J. Blood. 2004; 104: 3231-3232Crossref PubMed Scopus (140) Google Scholar). However, MGP is also expressed in vascular endothelium (5Engelse M.A. Neele J.M. Bronckers A.L.K.K. Pannekoek H. de Vries C.J.M. Cardiovasc. Res. 2001; 52: 281-289Crossref PubMed Scopus (114) Google Scholar, 6Simes D.C. Williamson M.K. Ortiz-Delgado J.B. Viegas C.S.B. Price P.A. Cancela M.L. J. Bone Miner. Res. 2003; 18: 244-259Crossref PubMed Scopus (39) Google Scholar), where its role is less well understood. We have previously shown that MGP enhances VEGF expression in bovine aortic endothelial cells (BAEC) by increasing the activity of transforming growth factor-β1 (TGF-β1) signaling through the activin-like kinase receptor 1 (ALK1) and the SMAD1/5/8 pathway (7Boström K. Zebboudj A.F. Yao Y. Lin T.S. Torres A. J. Biol. Chem. 2004; 279: 52904-52913Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Other studies show that expression of MGP is increased in tubular endothelial cells (8Glienke J. Schmitt A.O. Pilarsky C. Hinzmann B. Weiβ B. Rosenthal A. Thierauch K.-H. Eur. J. Biochem. 2000; 267: 2820-2830Crossref PubMed Scopus (111) Google Scholar), suggesting that MGP might be connected to resolution of angiogenesis. ALK1 is a TGF-β type I receptor associated with angiogenesis (9Lebrin F. Deckers M. Bertolino P. Ten Dijke P. Cardiovasc. Res. 2005; 65: 599-608Crossref PubMed Scopus (425) Google Scholar). It activates the SMAD1/5/8 pathway similarly to BMP-receptors, although its only known ligands are TGF-β1 and -β3 (10Lux A. Attisano L. Marchuk D.A. J. Biol. Chem. 1999; 274: 9984-9992Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Deficiency of ALK1 results in abnormal vascular formation with defects in the smooth muscle cell recruitment and arterio-venous malformations resembling human hereditary hemorrhagic telangiectasia (11Urness L.D. Sorensen L.K. Li D.Y. Nature. 2000; 26: 328-331Google Scholar, 12Carvalho R.L.C. Jonker L. Goumans M.-J. Larsson J. Bouwman P. Karlsson S. Ten Dijke P. Arthur H.M. Mummery C.L. Development (Camb.). 2004; 131: 6237-6247Crossref PubMed Scopus (132) Google Scholar). TGF-β is known to have a biphasic effect on angiogenesis (13Pepper M.S. Vassalli J.-D. Orci L. Montesano R. Exp. Cell Res. 1993; 204: 356-363Crossref PubMed Scopus (312) Google Scholar), with low and high levels of TGF-β having a stimulatory and inhibitory effect, respectively. TGF-β is also known to induce MGP expression in endothelial and other cell types (7Boström K. Zebboudj A.F. Yao Y. Lin T.S. Torres A. J. Biol. Chem. 2004; 279: 52904-52913Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 14Zhao J. Warburton D. Am. J. Physiol. 1997; 273: L282-L287PubMed Google Scholar). Bone morphogenetic proteins (BMP), also members of the TGF-β superfamily of growth factors, have been shown to stimulate vasculogenesis and angiogenesis (15Moser M. Patterson C. Thromb. Haemostasis. 2005; 94: 713-718PubMed Google Scholar). BMP-4 is necessary for induction of mesoderm and endothelial progenitor cell differentiation (16Winnier G. Blessing M. Labosky P.A. Hogan B.L. Genes Dev. 1995; 9: 2105-2126Crossref PubMed Scopus (1473) Google Scholar). BMP-2 induces tumor angiogenesis (17Langenfeld E.M. Langenfeld J. Mol. Cancer Res. 2004; 2: 141-149PubMed Google Scholar, 18Raida M. Clement J.H. Leek R.F. Ameri K. Bicknell R. Niederwieser D. Harris A.L. J. Cancer Res. Clin. Oncol. 2005; 131: 741-750Crossref PubMed Scopus (133) Google Scholar), and BMP-4 enhances VEGF secretion by human retinal pigment epithelial cells (19Vogt R.R. Unda R. Yeh L.C. Vidro E.K. Lee J.C. Tsin A.T. J. Cell. Biochem. 2006; 98: 1196-1202Crossref PubMed Scopus (39) Google Scholar). Furthermore, BMP-2, -4, and -6 stimulate VEGF secretion in preosteoblasts, and BMP-2 and -6 modulate capillary tube formation (20Deckers M.M. van Bezooijen R.L. van der Horst G. Hoogendam J. van Der Bentqq C. Papapoulos S.E. Lowik C.W. Endocrinology. 2002; 143: 1545-1553Crossref PubMed Scopus (457) Google Scholar, 21Valdimarsdottir G. Goumans M.-J. Rosendahl A. Brugman M. Itoh S. Lebrin F. Sideras P. ten Dijke P. Circulation. 2002; 106: 2263-2270Crossref PubMed Scopus (263) Google Scholar). Conversely, vascular formation is also affected by BMP inhibitors, including chordin, noggin, and the BMP-binding endothelial cell precursor-derived regulator (BMPER) (15Moser M. Patterson C. Thromb. Haemostasis. 2005; 94: 713-718PubMed Google Scholar, 22Moser M. Binder O. Wu Y. Aitsebaomo J. Ren R. Bode C. Bautch V.L. Conlon F.L. Patterson C. Mol. Cell. Biol. 2003; 23: 5664-5679Crossref PubMed Scopus (184) Google Scholar). We and others have shown that MGP binds and inhibits BMP-2 (23Wallin R. Cain D. Hutson S.M. Sane D.C. Loeser R. Thromb. Haemostasis. 2000; 84: 1039-1044Crossref PubMed Scopus (138) Google Scholar, 24Zebboudj A.F. Imura M. Boström K. J. Biol. Chem. 2002; 277: 4388-4394Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar), and we hypothesized that MGP may affect endothelial cells through BMP inhibition. Our study shows that the effect of MGP on TGF-β1/ALK1 signaling and VEGF expression is dose-dependent in BAEC and that a progressive increase of MGP levels ceases to be stimulatory and turns inhibitory. We have identified a new regulatory pathway that may contribute to explain this response. Our results show that BMP-2 and BMP-4 induce the expression of ALK1, induces expression of MGP and VEGF. Inhibition of TGF-β ALK1 expression of MGP and VEGF. MGP enhances ALK1 However, MGP also binds and inhibits BMP-2 and negative feedback for BMP to a in ALK1 expression and this may to a effect on ALK1 Our suggest that the effects of BMP on endothelial cells and angiogenesis occur in part through induction of ALK1, an effect that may be limited by ALK1-induced MGP. Cell and were and as previously (7Boström K. Zebboudj A.F. Yao Y. Lin T.S. Torres A. J. Biol. Chem. 2004; 279: 52904-52913Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The BAEC were used and for were used and of BAEC were with and of as previously for (7Boström K. Zebboudj A.F. Yao Y. Lin T.S. Torres A. J. Biol. Chem. 2004; 279: 52904-52913Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) and as for The of used for was with expression human BMP-2, and neutralizing antibodies to TGF-β were the of were as previously (7Boström K. Zebboudj A.F. Yao Y. Lin T.S. Torres A. J. Biol. Chem. 2004; 279: 52904-52913Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), with to Cell was determined by the as previously S. K. T. Itoh H. K. H. Thromb. Vasc. Biol. 1995; PubMed Scopus Google Scholar) after with BMP-2, and N-terminally FLAG-tagged MGP in the were in the and the was as the of cells. Inhibition of ALK1 by aortic endothelial cells were used for the bovine ALK1 was for The were as previously H. P. S. G. S. C. L. Res. 2000; PubMed Scopus Google Scholar). of were with the of been as the ALK1 siRNA, and with the were with and siRNA, the ALK1 in a in ALK1 and protein levels as determined by real-time PCR and respectively. The that the was used for ALK1- and BMP-responsive luciferase reporter was ten The Cancer The M.-J. G. Itoh S. Lebrin F. Larsson J. Mummery C. Karlsson S. ten Dijke P. Mol. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The for ALK1 was the N-terminally FLAG-tagged human MGP a the for was by The was in the of the protein by of a the for the and the The was by PCR and the and of in the The human MGP luciferase reporter J. M. Cancela Price P.A. R. S. A. 1997; 94: PubMed Scopus Google Scholar) were of RNA RNA was cells the PCR were as previously (7Boström K. Zebboudj A.F. Yao Y. Lin T.S. Torres A. J. Biol. Chem. 2004; 279: 52904-52913Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The and were human MGP bovine MGP bovine VEGF human ALK1 bovine and was as previously (7Boström K. Zebboudj A.F. Yao Y. Lin T.S. Torres A. J. Biol. Chem. 2004; 279: 52904-52913Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). of protein of were were with antibodies to Cell ALK1 BMP-4 VEGF were in for and was as previously A.F. Imura M. Boström K. J. Biol. Chem. 2002; 277: 4388-4394Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). antibodies to MGP were by were used to of BMP-4 and was as previously A.F. Imura M. Boström K. J. Biol. Chem. 2002; 277: 4388-4394Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). BMP-4 was to 1 of BAEC after of the was antibodies and was antibodies as and were and respectively. of of in BAEC with the was determined an protein was used as a the of BAEC protein in was with of and used for of for 1 were with of with bovine and for 1 with PBS, the were for of in was to well and for 1 with the to the was by a with in with was well and for 1 with the were with and were for by of with The were were a of MGP on ALK1 and VEGF have previously shown that an increase in MGP levels enhances ALK1 and VEGF expression (7Boström K. Zebboudj A.F. Yao Y. Lin T.S. Torres A. J. Biol. Chem. 2004; 279: 52904-52913Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). However, MGP expression is high in (8Glienke J. Schmitt A.O. Pilarsky C. Hinzmann B. Weiβ B. Rosenthal A. Thierauch K.-H. Eur. J. Biochem. 2000; 267: 2820-2830Crossref PubMed Scopus (111) Google Scholar), may also be connected with resolution of angiogenesis. the effect of MGP on ALK1 signaling is dose-dependent, we BAEC with a of the reporter and increasing of an expression The cells were with TGF-β1 for h, after the level of was determined in the by and the luciferase activity was determined and to The results showed that low levels of MGP stimulated ALK1 signaling to similarly to results (7Boström K. Zebboudj A.F. Yao Y. Lin T.S. Torres A. J. Biol. Chem. 2004; 279: 52904-52913Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), high levels of MGP inhibited This that the effect of MGP is dose-dependent in a biphasic fashion. we that VEGF expression induced by TGF-β1 (7Boström K. Zebboudj A.F. Yao Y. Lin T.S. Torres A. J. Biol. Chem. 2004; 279: 52904-52913Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). and TGF-β1 MGP expression and in BAEC (7Boström K. Zebboudj A.F. Yao Y. Lin T.S. Torres A. J. Biol. Chem. 2004; 279: 52904-52913Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), we that MGP with TGF-β1 stimulate VEGF we BAEC with increasing of and the cells with TGF-β1 RNA was after h, and VEGF expression was determined by real-time PCR and to VEGF was by immunoblotting. The results showed that VEGF expression was only stimulated by MGP the of TGF-β1 that the effects of TGF-β1 and MGP are of human and bovine MGP, was determined by real-time PCR to the BMP-2 and BMP-4 of ALK1, of MGP and MGP is a BMP inhibitor that to ALK1, we the effect of BMP-2 and BMP-4 on ALK1 BAEC were for with BMP-2 BMP-4 and ALK1 expression was determined by real-time PCR and to The results showed that BMP-2 and BMP-4 increased ALK1 expression in a dose-dependent with stimulation by high BMP-4 levels and BMP-4 the ALK1 induction and was used in that ALK1 protein also increased after BMP the cells were with BMP-4 for Cell were and by for ALK1. that the ALK1 antibodies the we the ALK1 with cells with ALK1, been with antibodies antibodies were to the by the antibodies in cells. BMP-4 in a dose-dependent increase in ALK1 protein as was for ALK1 We hypothesized that ALK1 induces MGP, be to enhance ALK1 activity (7Boström K. Zebboudj A.F. Yao Y. Lin T.S. Torres A. J. Biol. Chem. 2004; 279: 52904-52913Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) and also negative feedback for ALK1 induces MGP expression in addition to that of VEGF, we BAEC with an expression for ALK1 in the of neutralizing antibodies to TGF-β ALK1 for activity and is to MGP. The was with real-time PCR to and for ALK1 of MGP was determined by real-time PCR with to and with VEGF The results showed that expression of MGP increased and that of VEGF increased VEGF was by with the increased MGP protein was by also with the increased The results that ALK1 MGP and VEGF Cell was determined to for the effects of BMP-2 BMP-4 TGF-β1 and the in cell was detected for of the the The of cells was for for BMP-2, for and for MGP and BMP-4 is to BMP-2, has been shown that MGP inhibits BMP-4 the effect of MGP on BMP-4 activity in with that of BMP-2, BAEC were with increasing of expression and a of the reporter and with BMP-2 BMP-4 in the of neutralizing antibodies to the effects of TGF-β on MGP The BMP were the that signaling activity in h, was in the by with and the luciferase activity was determined and to The results showed that increased MGP levels inhibited luciferase activity induced by BMP-2 and BMP-4 MGP with we used BAEC that and We with antibodies to BMP-4 to in the FLAG-tagged MGP. The BMP-4 was by for the and the was by the The results showed that BMP-4 and in suggesting that the proteins the antibodies high and low of MGP detected in the Other have shown that the in of MGP is to of Williamson M.K. Price P.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). that the MGP we the a of the The results that BMP-4 and the MGP the with BMP-4 on of MGP and VEGF on TGF-β and that of ALK1 signaling and MGP induction BMP-4 stimulation of ALK1 expression high BMP-4 we used neutralizing antibodies to the only known ALK1 BAEC expressed TGF-β We BAEC with BMP-4 in the of antibodies for and with BMP-4 only The of antibodies the dose-dependent ALK1 response. expression of ALK1 to increase high BMP-4 levels as determined by real-time PCR with to and ALK1 Furthermore, we expression of MGP and VEGF to ALK1 signaling was This was with real-time PCR with to The results showed that expression of MGP and VEGF was abolished that the BMP-4 effects on MGP and VEGF are The of ALK1 expression that of MGP in This may be a of the of MGP to enhance ALK1 activity low to levels (7Boström K. Zebboudj A.F. Yao Y. Lin T.S. Torres A. J. Biol. Chem. 2004; 279: 52904-52913Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), enhance MGP expression ALK1 MGP expression also to However, we signaling ALK1 expression and increasing MGP expression high BMP-4 We that of the ALK1 receptor expression of MGP and VEGF. the bovine ALK1 is we used human aortic endothelial cells ALK1. The were with ALK1 and with of ALK1 in in ALK1 levels after as determined by real-time PCR and the affect ALK1 expression with ALK1 were with BMP-4 after of expression of MGP and VEGF was determined by real-time PCR and to The results showed that the ALK1 BMP-4 induction of ALK1 expression and abolished the stimulatory effect of BMP-4 on expression of MGP and VEGF the results suggest that TGF-β and ALK1 the effect of BMP-4 on MGP and VEGF The MGP by BMP-4 in a TGF-β-dependent BMP-4 the MGP promoter and stimulation is we used luciferase reporter of the MGP promoter J. M. Cancela Price P.A. R. S. A. 1997; 94: PubMed Scopus Google Scholar). promoter in TGF-β signaling are and by the and only be in the promoter BAEC were with the luciferase reporter and with BMP-4 only in the of TGF-β1 antibodies h, luciferase activity was determined and to The results for the MGP promoter showed that BMP-4 only increased promoter activity the addition of TGF-β to the BMP-4 increased promoter activity on the other promoter activity by The results for the MGP promoter showed that BMP-4 a effect on promoter activity the addition of TGF-β to the BMP-4 only increased promoter activity activity by a The results suggest that BMP-4 of the MGP promoter is in a to MGP also the of a in the promoter We that BMP-4 also the MGP promoter through other signaling Our studies suggest that ALK1, and MGP are part of a regulatory in vascular endothelium and that BMP-4 on signaling to affect expression of MGP and VEGF. The regulatory may in part explain the dose-dependent effect of MGP on signaling and VEGF to levels of MGP enhances ALK1 signaling and VEGF MGP also a progressive negative feedback for BMP that induction of ALK1 and VEGF expression This regulatory may also explain the dose-dependent effect of TGF-β on angiogenesis (13Pepper M.S. Vassalli J.-D. Orci L. Montesano R. Exp. Cell Res. 1993; 204: 356-363Crossref PubMed Scopus (312) Google Scholar) TGF-β induces MGP expression in cell types including The have been for to stimulate vasculogenesis in the and the (15Moser M. Patterson C. Thromb. Haemostasis. 2005; 94: 713-718PubMed Google Scholar). BMP-4 is necessary for endothelial progenitor cell differentiation (16Winnier G. Blessing M. Labosky P.A. Hogan B.L. Genes Dev. 1995; 9: 2105-2126Crossref PubMed Scopus (1473) Google Scholar). BMP-2 induces tumor angiogenesis (17Langenfeld E.M. Langenfeld J. Mol. Cancer Res. 2004; 2: 141-149PubMed Google Scholar, 18Raida M. Clement J.H. Leek R.F. Ameri K. Bicknell R. Niederwieser D. Harris A.L. J. Cancer Res. Clin. Oncol. 2005; 131: 741-750Crossref PubMed Scopus (133) Google Scholar), and BMP-2 and -6 modulate capillary tube formation (20Deckers M.M. van Bezooijen R.L. van der Horst G. Hoogendam J. van Der Bentqq C. Papapoulos S.E. Lowik C.W. Endocrinology. 2002; 143: 1545-1553Crossref PubMed Scopus (457) Google Scholar, 21Valdimarsdottir G. Goumans M.-J. Rosendahl A. Brugman M. Itoh S. Lebrin F. Sideras P. ten Dijke P. Circulation. 2002; 106: 2263-2270Crossref PubMed Scopus (263) Google Scholar). of ALK1 expression a for BMP to affect angiogenesis and enhance VEGF secretion as in cells with BMP-2, -4, and -6 (19Vogt R.R. Unda R. Yeh L.C. Vidro E.K. Lee J.C. Tsin A.T. J. Cell. Biochem. 2006; 98: 1196-1202Crossref PubMed Scopus (39) Google Scholar, M.M. van Bezooijen R.L. van der Horst G. Hoogendam J. van Der Bentqq C. Papapoulos S.E. Lowik C.W. Endocrinology. 2002; 143: 1545-1553Crossref PubMed Scopus (457) Google Scholar), that the for ALK1 is only TGF-β1 and -β3 have been shown to stimulate ALK1 signaling (10Lux A. Attisano L. Marchuk D.A. J. Biol. Chem. 1999; 274: 9984-9992Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), ALK1 may also on identified BMP-4 has been identified as an induced by in endothelial cells M. D. D. A. J. H. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, H. J. S. D.A. B. H. Res. 2004; PubMed Scopus Google Scholar), and is that BMP-4 and ALK1 induction are in of angiogenesis and of the vascular in as and and arterial and valvular J.C. J. M.A. J. 2006; PubMed Scopus (39) Google Scholar, J.H. E. S. F. C. B. O. S. P. G. J. Am. 2004; Scopus Google Scholar). of MGP and VEGF expression might contribute to the of hereditary hemorrhagic telangiectasia in by arterio-venous malformations and recruitment of smooth muscle cell cells (11Urness L.D. Sorensen L.K. Li D.Y. Nature. 2000; 26: 328-331Google Scholar, 12Carvalho R.L.C. Jonker L. Goumans M.-J. Larsson J. Bouwman P. Karlsson S. Ten Dijke P. Arthur H.M. Mummery C.L. Development (Camb.). 2004; 131: 6237-6247Crossref PubMed Scopus (132) Google Scholar). in MGP levels have been associated with tumor MGP was in that were detected in that were F. J. G. R. M. C. K. F. Clin. Cancer Res. 2004; PubMed Scopus Google Scholar). levels of MGP were also detected in with C. D. H. K. C. E. Cancer 2001; 165: PubMed Scopus Google Scholar). high levels of MGP have been associated with tubular (8Glienke J. Schmitt A.O. Pilarsky C. Hinzmann B. Weiβ B. Rosenthal A. Thierauch K.-H. Eur. J. Biochem. 2000; 267: 2820-2830Crossref PubMed Scopus (111) Google Scholar) and may ALK1 is that a of MGP results in a of angiogenesis. The of ALK1 expression might be in involving BMP 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). A. Y. D. Boström K. S. A. 2004; PubMed Scopus Google Scholar) have also shown that BMP-2 and MGP are to as an that formation in vascular cells as by a ALK1 expression has been in vascular and Am. J. Physiol. Google Scholar, Mummery C.L. Dev. 1997; PubMed Scopus (111) Google Scholar) and might be in the BMP and MGP. Our studies that the inhibitory effect of MGP is limited to BMP-2 also MGP and BMP-4 similarly to MGP and BMP-2 A.F. Imura M. Boström K. J. Biol. Chem. 2002; 277: 4388-4394Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). Furthermore, BMP-4 also with a of MGP that by of MGP J. M. Cancela Price P.A. R. S. A. 1997; 94: PubMed Scopus Google Scholar). It that this of MGP However, may affect matrix of BMP the of MGP has been in to M. Matrix Biol. 2005; PubMed Scopus Google Scholar). Our studies showed that MGP abolished of BMP-2 to cell A.F. Imura M. Boström K. J. Biol. Chem. 2002; 277: 4388-4394Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar), MGP might affect the receptor by interfering with other proteins that are to the as Our results explain the stimulatory effect of low to levels of MGP on ALK1-induced VEGF expression (7Boström K. Zebboudj A.F. Yao Y. Lin T.S. Torres A. J. Biol. Chem. 2004; 279: 52904-52913Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) It is that MGP of type receptor for ALK1. However, we have in TGF-β expression and the type BMP be after an increase in BMP has been connected with ALK1 R. R. M.A. Circulation. 2005; PubMed Scopus Google Scholar). studies have identified a new regulatory pathway involving ALK1, and MGP, allowing for a regulation of BMP and MGP through ALK1 in a TGF-β-dependent fashion.