Structural Requirements for Activation of Latent Platelet-derived Growth Factor CC by Tissue Plasminogen Activator

Abstract

Platelet-derived growth factor C (PDGF-C) is one of four members in the PDGF family of growth factors, which are known mitogens and survival factors for cells of mesenchymal origin. PDGF-C has a unique two-domain structure consisting of an N-terminal CUB and a conserved C-terminal growth factor domain that are separated by a hinge region. PDGF-C is secreted as a latent dimeric factor (PDGF-CC), which undergoes extracellular removal of the CUB domains to become a PDGF receptor α agonist. Recently, the multidomain serine protease tissue plasminogen activator (tPA), a thrombolytic agent used for treatment of acute ischemic stroke, was shown to cleave and activate PDGF-CC. In this study we determine the molecular mechanism of tPA-mediated activation of PDGF-CC. Using various PDGF-CC and tPA mutants, we were able to demonstrate that both the CUB and the growth factor domains of PDGF-C, as well as the kringle-2 domain of tPA, are required for the interaction and cleavage to occur. We also show that Arg231 in PDGF-C is essential for tPA-mediated proteolysis and that the released “free” CUB domain of PDGF-C can act as a competitive inhibitor of the cleavage reaction. Furthermore, we studied how the PDGF-C/tPA axis is regulated in primary fibroblasts and found that PDGF-C expression is down-regulated by hypoxia but induced by transforming growth factor (TGF)-β1 treatment. Elucidating the regulation and the mechanism of tPA-mediated activation of PDGF-CC will advance our knowledge of the physiological function of PDGF-CC and tPA and may provide new therapeutic opportunities for thrombolytic and cardiovascular therapies. Platelet-derived growth factor C (PDGF-C) is one of four members in the PDGF family of growth factors, which are known mitogens and survival factors for cells of mesenchymal origin. PDGF-C has a unique two-domain structure consisting of an N-terminal CUB and a conserved C-terminal growth factor domain that are separated by a hinge region. PDGF-C is secreted as a latent dimeric factor (PDGF-CC), which undergoes extracellular removal of the CUB domains to become a PDGF receptor α agonist. Recently, the multidomain serine protease tissue plasminogen activator (tPA), a thrombolytic agent used for treatment of acute ischemic stroke, was shown to cleave and activate PDGF-CC. In this study we determine the molecular mechanism of tPA-mediated activation of PDGF-CC. Using various PDGF-CC and tPA mutants, we were able to demonstrate that both the CUB and the growth factor domains of PDGF-C, as well as the kringle-2 domain of tPA, are required for the interaction and cleavage to occur. We also show that Arg231 in PDGF-C is essential for tPA-mediated proteolysis and that the released “free” CUB domain of PDGF-C can act as a competitive inhibitor of the cleavage reaction. Furthermore, we studied how the PDGF-C/tPA axis is regulated in primary fibroblasts and found that PDGF-C expression is down-regulated by hypoxia but induced by transforming growth factor (TGF)-β1 treatment. Elucidating the regulation and the mechanism of tPA-mediated activation of PDGF-CC will advance our knowledge of the physiological function of PDGF-CC and tPA and may provide new therapeutic opportunities for thrombolytic and cardiovascular therapies. Platelet-derived growth factor C (PDGF-C) 1The abbreviations used are: PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; DMEM, Dulbecco's modified Eagle's medium; PAE, porcine aortic endothelial; Ni-NTA, nickel-nitrilotriacetic acid; pNA, paranitroanilide; PAI, plasminogen activator inhibitor; FGF, fibroblast growth factor; TGF, transforming growth factor; tPA, tissue plasminogen activator. was discovered a few years ago as the third member of the well characterized PDGF family of growth factors (1Li X. Pontén A. Aase K. Karlsson L. Abramsson A. Uutela M. Bäckström G. Hellström M. Boström H. Li H. Soriano P. Betsholtz C. Heldin C.H. Alitalo K. Östman A. Eriksson U. Nat. Cell Biol. 2000; 2: 302-309Crossref PubMed Scopus (511) Google Scholar). The classical members of this family, PDGF-A and PDGF-B, have been intensively studied and are known to be important for connective tissue growth and maintenance, and overexpression has been observed in several pathological conditions, including malignancies and atherosclerosis (2Heldin C.H. Westermark B. Physiol. Rev. 1999; 79: 1283-1316Crossref PubMed Scopus (2019) Google Scholar). Since its discovery, PDGF-C has been shown to play a role in palate formation (3Ding H. Wu X. Boström H. Kim I. Wong N. Tsoi B. O'Rourke M. Koh G.Y. Soriano P. Betsholtz C. Hart T.C. Marazita M.L. Field L.L. Tam P.P. Nagy A. Nat. Genet. 2004; 36: 1111-1116Crossref PubMed Scopus (180) Google Scholar), fibrotic disease development (4Pontén A. Li X. Thorén P. Aase K. Sjöblom T. Östman A. Eriksson U. Am. J. Pathol. 2003; 163: 673-682Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 5Eitner F. Ostendorf T. Kretzler M. Cohen C.D. Eriksson U. Gröne H.J. Floege J. J. Am. Soc. Nephrol. 2003; 14: 1145-1153Crossref PubMed Scopus (65) Google Scholar), and angiogenesis (6Li X. Tjwa M. Moons L. Fons P. Noel A. Ny A. Zhou J.M. Lennartsson J. Li H. Luttun A. Pontén A. Devy L. Bouché A. Oh H. Manderveld A. Blacher S. Communi D. Savi P. Bono F. Dewerchin M. Foidart J.M. Autiero M. Herbert J.M. Collen D. Heldin C.H. Eriksson U. Carmeliet P. J. Clin. Investig. 2005; 115: 118-127Crossref PubMed Scopus (152) Google Scholar, 7Cao R. Bråkenhielm E. Li X. Pietras K. Widenfalk J. Östman A. Eriksson U. Cao Y. FASEB J. 2002; 16: 1575-1583Crossref PubMed Scopus (185) Google Scholar). Recently a fourth member, PDGF-D, has been added to this family of growth factors (8Bergsten E. Uutela M. Li X. Pietras K. Östman A. Heldin C.H. Alitalo K. Eriksson U. Nat. Cell Biol. 2001; 3: 512-516Crossref PubMed Scopus (473) Google Scholar, 9LaRochelle W.J. Jeffers M. McDonald W.F. Chillakuru R.A. Giese N.A. Lokker N.A. Sullivan C. Boldog F.L. Yang M. Vernet C. Burgess C.E. Fernandes E. Deegler L.L. Rittman B. Shimkets J. Shimkets R.A. Rothberg J.M. Lichenstein H.S. Nat. Cell Biol. 2001; 3: 517-521Crossref PubMed Scopus (319) Google Scholar). The four PDGF chains assemble into five dimeric isoforms, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD, that exert their effects on cells through differential signaling via two known tyrosine kinase receptors, platelet-derived growth factor receptor (PDGFR)-α and PDGFR-β (10Li X. Eriksson U. Cytokine Growth Factor Rev. 2003; 14: 91-98Crossref PubMed Scopus (163) Google Scholar). Unlike the classical members, PDGF-C and PDGF-D have a unique two-domain structure, with a so-called CUB domain N-terminal of the conserved growth factor domain (1Li X. Pontén A. Aase K. Karlsson L. Abramsson A. Uutela M. Bäckström G. Hellström M. Boström H. Li H. Soriano P. Betsholtz C. Heldin C.H. Alitalo K. Östman A. Eriksson U. Nat. Cell Biol. 2000; 2: 302-309Crossref PubMed Scopus (511) Google Scholar, 8Bergsten E. Uutela M. Li X. Pietras K. Östman A. Heldin C.H. Alitalo K. Eriksson U. Nat. Cell Biol. 2001; 3: 512-516Crossref PubMed Scopus (473) Google Scholar, 9LaRochelle W.J. Jeffers M. McDonald W.F. Chillakuru R.A. Giese N.A. Lokker N.A. Sullivan C. Boldog F.L. Yang M. Vernet C. Burgess C.E. Fernandes E. Deegler L.L. Rittman B. Shimkets J. Shimkets R.A. Rothberg J.M. Lichenstein H.S. Nat. Cell Biol. 2001; 3: 517-521Crossref PubMed Scopus (319) Google Scholar). In order for the novel PDGFs to bind and activate the PDGFRs, the N-terminal CUB domains have to be removed through limited proteolysis by extracellular proteases. The origin of the protease involved in the activation of PDGF-DD still remains elusive, whereas the extracellular fibrinolytic protease tissue plasminogen activator (tPA) has been shown to be a potent activator of PDGF-CC (11Fredriksson L. Li H. Fieber C. Li X. Eriksson U. EMBO J. 2004; 23: 3793-3802Crossref PubMed Scopus (156) Google Scholar). tPA is a highly specific serine protease that consists of five structural domains, a finger domain, an epidermal growth factor-like domain, two kringle domains, and a trypsin-like protease domain (12Collen D. Hematology (Am. Soc. Hematol. Educ. Program). 2001; : 1-9Crossref PubMed Scopus (81) Google Scholar). It is best known for its role in vascular fibrinolysis where it converts the zymogen plasminogen into plasmin, which in turn degrades the fibrin network in blood clots. The that tPA to fibrin via its finger and kringle-2 domains EMBO J. PubMed Scopus Google Scholar, H. H. U. S. A. PubMed Scopus Google Scholar), a of plasmin, has on the of tPA as a thrombolytic In tPA is used to acute and is also for treatment of acute ischemic N. J. PubMed Scopus Google Scholar). of tPA, the with and regulation of D. PubMed Scopus Google Scholar, S. PubMed Google Scholar, M. M. E. Wu D. J. Clin. Investig. 2002; PubMed Scopus Google Scholar). of the to be by plasmin, whereas show the to be of plasminogen activation in M. 2004; 14: PubMed Scopus Google Scholar). are two for tPA, PDGF-CC and the of the receptor (11Fredriksson L. Li H. Fieber C. Li X. Eriksson U. EMBO J. 2004; 23: 3793-3802Crossref PubMed Scopus (156) Google Scholar, F. C. I. Carmeliet P. D. A. Nat. 2001; PubMed Scopus Google Scholar). has been to the growth of primary which have in the and growth of fibroblasts into and in (11Fredriksson L. Li H. Fieber C. Li X. Eriksson U. EMBO J. 2004; 23: 3793-3802Crossref PubMed Scopus (156) Google Scholar). It has also been shown that PDGF-CC can in K. M. J.M. Hart C.E. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google and of ischemic (6Li X. Tjwa M. Moons L. Fons P. Noel A. Ny A. Zhou J.M. Lennartsson J. Li H. Luttun A. Pontén A. Devy L. Bouché A. Oh H. Manderveld A. Blacher S. Communi D. Savi P. Bono F. Dewerchin M. Foidart J.M. Autiero M. Herbert J.M. Collen D. Heldin C.H. Eriksson U. Carmeliet P. J. Clin. Investig. 2005; 115: 118-127Crossref PubMed Scopus (152) Google Scholar), the therapeutic of PDGF-CC. it is of to determine the structural and of PDGF-CC we the molecular mechanism of tPA-mediated cleavage of PDGF-CC. We demonstrate that both the CUB and the growth factor domains of PDGF-C and the kringle-2 domain of tPA are for interaction of the two and that Arg231 in the hinge of PDGF-CC is for cleavage by Cell cells and primary fibroblasts were in Dulbecco's modified Eagle's with and and porcine aortic cells were in The cells were in a primary fibroblast were as and were on cells (11Fredriksson L. Li H. Fieber C. Li X. Eriksson U. 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Cell Biol. 2000; 2: 302-309Crossref PubMed Scopus (511) Google Scholar), and a for were in the The were and used were and of the were by and of used in a new and cells were with the various expression in with as the was by and by The was and used in receptor the were as (1Li X. Pontén A. Aase K. Karlsson L. Abramsson A. Uutela M. Bäckström G. Hellström M. Boström H. Li H. Soriano P. Betsholtz C. Heldin C.H. Alitalo K. Östman A. Eriksson U. Nat. Cell Biol. 2000; 2: 302-309Crossref PubMed Scopus (511) Google Scholar). were to conditions, and by PDGF-C and were by PDGF-C (1Li X. Pontén A. Aase K. Karlsson L. Abramsson A. Uutela M. Bäckström G. Hellström M. Boström H. Li H. Soriano P. Betsholtz C. Heldin C.H. Alitalo K. Östman A. Eriksson U. Nat. Cell Biol. 2000; 2: 302-309Crossref PubMed Scopus (511) Google and PDGF-D (8Bergsten E. Uutela M. Li X. Pietras K. Östman A. Heldin C.H. Alitalo K. Eriksson U. Nat. Cell Biol. 2001; 3: 512-516Crossref PubMed Scopus (473) Google Scholar), tPA was tPA and tPA growth tyrosine of and cells the PDGFRs, were for on with cells with cells with with the growth factor domain of PDGF-CC were used as treatment the cells were in and protease inhibitor and the were specific A. A. Westermark B. Heldin C.H. L. EMBO J. PubMed Scopus Google Scholar). were separated by were by an were as determine which of tPA and PDGF-CC are involved in the interaction the two PDGF-CC the expression as (1Li X. Pontén A. Aase K. Karlsson L. Abramsson A. Uutela M. Bäckström G. Hellström M. Boström H. Li H. Soriano P. Betsholtz C. Heldin C.H. Alitalo K. Östman A. Eriksson U. Nat. Cell Biol. 2000; 2: 302-309Crossref PubMed Scopus (511) Google Scholar), were to nickel-nitrilotriacetic and for with cells with the tPA were used as The were and the PDGF-CC were with were by and with to tPA The were and with to of and PDGF-C an to of CUB were as protease the tPA mutants, a was cells with the tPA were to to to The protease was in with tPA as a for The formation of the of was protease cells were used as tPA was used to of PDGF-C, tPA, and determine how PDGF-C, tPA, and its inhibitor are regulated by various growth factors and conditions, primary fibroblasts were in the was for in the of fibroblast growth factor the cells were in the were and the were and to by PDGF-C and tPA were specific and was a tPA-mediated on of the structural for of latent PDGF-CC as a for tPA of PDGF-CC in a The of PDGF-CC of PDGF-C, one the CUB domain of PDGF-D and the hinge and growth factor domain of PDGF-C and the one the CUB domain and the hinge of PDGF-C and the growth factor domain of PDGF-D in In a the CUB domain of PDGF-C was also were in and were secreted in the with tPA, the of a was as with PDGF-CC whereas cleavage was in with with were in receptor where cells were cells with expression of PDGFR-β of the receptors, was as of receptor tyrosine shown cells tPA and PDGF-CC induced activation with (11Fredriksson L. Li H. Fieber C. Li X. Eriksson U. EMBO J. 2004; 23: 3793-3802Crossref PubMed Scopus (156) Google Scholar), whereas cells tPA with induced with the PDGF-CC tPA with the of cleavage of the growth factor domain of PDGF-DD, a PDGFR-β the were also to PDGFR-β but induced PDGFR-β that both the CUB and the growth factor domains are for cleavage of latent PDGF-CC by The of the in that the to activation that the CUB domain but also of the hinge have to be removed for receptor the structural of PDGF-CC for receptor and a of of PDGF-C, the CUB domain and of the hinge was in and were secreted in the by cells The of the in the are to and of the in the growth factor domain of PDGF-C (1Li X. Pontén A. Aase K. Karlsson L. Abramsson A. Uutela M. Bäckström G. Hellström M. Boström H. Li H. Soriano P. Betsholtz C. Heldin C.H. Alitalo K. Östman A. Eriksson U. Nat. 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EMBO J. 2004; 23: 3793-3802Crossref PubMed Scopus (156) Google Scholar). which of the and is important for cleavage to the were to The expression PDGF-C were with The of PDGF-CC cleavage was by as the of the and of PDGF-C and a to tPA-mediated cleavage with the by were used as demonstrate that the cleavage for tPA is to the in PDGF-CC and that Arg231 is essential for cleavage to whereas the in the are The CUB of PDGF-C as a of tPA-mediated on our that the CUB domain of PDGF-C, but that of PDGF-D, with tPA (11Fredriksson L. Li H. Fieber C. Li X. Eriksson U. EMBO J. 2004; 23: 3793-3802Crossref PubMed Scopus (156) Google and the that the CUB domain is for specific we that the released CUB domain of PDGF-C act as a competitive inhibitor of tPA-mediated activation of PDGF-CC. this we cells with PDGF-CC and tPA in the of an expression the CUB domain of PDGF-C We were able to show that the CUB domain of PDGF-C for the of latent PDGF-CC by tPA, as by and as by of that the CUB domain may act as a competitive inhibitor of tPA-mediated of tPA for of determine which of the structural domains of tPA is for cleavage of latent PDGF-CC we of tPA in and in cells that the tPA were a tPA was added to and the formation of pNA, of the of tPA was tPA induced formation in a and protease tPA was used to and induced formation of cells with was used as the structural of tPA for cleavage of PDGF-CC, the tPA were in cells with PDGF-CC, and cleavage was by the formation of the of show that of the tPA the trypsin-like protease domain, with PDGF-CC the of the as with of the tPA is and the kringle-2 domain is required for tPA to cleave PDGF-CC. tPA and PDGF-CC by kringle-2 in the that the kringle-2 domain of tPA the interaction tPA and PDGF-CC (11Fredriksson L. 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The of the CUB domain of PDGF-C to with tPA and act as a competitive inhibitor of tPA-mediated proteolysis may the of the activation by tPA in the The of the activation is that of of PDGF-C will two of the CUB this mechanism is used in is but it may provide a potent mechanism the activation of PDGF-CC. limited of the PDGF-C can also the CUB domain of the have to provide of PDGF-C CUB domains The of CUB on tPA may have in of the tPA in thrombolytic treatment. The domains of tPA have been to interaction tPA and various the finger domain fibrin H. H. U. S. A. PubMed Scopus Google and PubMed Google Scholar, J. J. Biol. Full Text PDF PubMed Google Scholar), and the kringle domains, in the kringle domain, also bind fibrin EMBO J. PubMed Scopus Google Scholar, H. H. U. S. A. 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Biol. 2004; Full Text Full Text PDF PubMed Scopus Google it is that of the of the kringle-2 domain is through and of PDGF-CC Furthermore, we that the interaction of PDGF-CC with the domains of tPA with as the known to both with tPA G. S. U. S. A. PubMed Scopus Google and signaling P. M. Li J. 2003; PubMed Scopus Google and a of PDGF-CC. tPA via interaction with the and proteolysis of an as M. M. J. Clin. Investig. 2003; PubMed Scopus Google Scholar). the PDGF-CC and the novel PDGFR-β PDGF-DD, tPA to and cleavage of this factor (11Fredriksson L. Li H. Fieber C. Li X. Eriksson U. EMBO J. 2004; 23: 3793-3802Crossref PubMed Scopus (156) Google Scholar). is known the activation of We have that the for the classical and novel PDGFs separated and that the novel PDGFs a L. Li H. Eriksson U. Cytokine Growth Factor Rev. 2004; PubMed Scopus Google Scholar). It is that the protease involved in PDGF-DD activation has a structural as Using a we several serine with domain as tPA, including the plasminogen of can cleave and activate PDGF-DD remains to be in are a and by fibroblast K. G. A. K. J. 16: Full Text PDF PubMed Scopus Google Scholar). therapeutic it has become a to the mechanism of the and the fibroblast in that the growth of primary fibroblasts in on a we that of PDGF-CC signaling be involved in the function of fibroblasts in demonstrate that expression of the PDGF-C/tPA axis in primary fibroblasts but that known to play an important role in S. 2001; PubMed Scopus Google Scholar), the expression of PDGF-C and a for It is that is used to the of of M. G. N. F. K. J. 2002; 16: PubMed Scopus Google Scholar). Furthermore, we show that treatment with induced the expression of PDGF-C in the primary which is that overexpression of PDGF-C (4Pontén A. Li X. Thorén P. Aase K. Sjöblom T. Östman A. Eriksson U. Am. J. Pathol. 2003; 163: 673-682Abstract Full Text Full Text PDF PubMed Scopus (123) Google U. C. R. S. T. K. M. J. Clin. Investig. 2002; PubMed Scopus Google in in and it is known that PDGF-CC can in K. M. J.M. Hart C.E. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google it still remains to be PDGF-C expression is in In the molecular mechanism by which PDGF-CC a through tPA-mediated proteolysis will the of PDGF signaling in and pathological We for the of