Platelet-derived Growth Factor C (PDGF-C), a Novel Growth Factor That Binds to PDGF α and β Receptor
2001; Elsevier BV; Volume: 276; Issue: 29 Linguagem: Inglês
10.1074/jbc.m101056200
ISSN1083-351X
AutoresDebra G. Gilbertson, Meghan Duff, James W. West, James D. Kelly, Paul O. Sheppard, Philip D. Hofstrand, Zeren Gao, Kimberly Shoemaker, Thomas R. Bukowski, Margaret Moore, Andrew L. Feldhaus, Jacqueline M. Humes, Thomas E. Palmer, Charles E. Hart,
Tópico(s)Lymphatic System and Diseases
ResumoWe have characterized platelet-derived growth factor (PDGF) C, a novel growth factor belonging to the PDGF family. PDGF-C is a multidomain protein with the N-terminal region homologous to the extracellular CUB domain of neuropilin-1, and the C-terminal region consists of a growth factor domain (GFD) with homology to vascular endothelial growth factor (25%) and PDGF A-chain (23%). A serum-sensitive cleavage site between the two domains allows release of the GFD from the CUB domain. Competition binding and immunoprecipitation studies on cells bearing both PDGF α and β receptors reveal a high affinity binding of recombinant GFD (PDGF-CC) to PDGF receptor-α homodimers and PDGF receptor-α/β heterodimers. PDGF-CC exhibits greater mitogenic potency than PDGF-AA and comparable or greater mitogenic activity than PDGF-AB and PDGF-BB on several mesenchymal cell types. Analysis of PDGF-CC in vivo in a diabetic mouse model of delayed wound healing showed that PDGF-CC significantly enhanced repair of a full-thickness skin excision. Together, these studies describe a third member of the PDGF family (PDGF-C) as a potent mitogen for cells of mesenchymal origin inin vitro and in vivo systems with a binding pattern similar to PDGF-AB. We have characterized platelet-derived growth factor (PDGF) C, a novel growth factor belonging to the PDGF family. PDGF-C is a multidomain protein with the N-terminal region homologous to the extracellular CUB domain of neuropilin-1, and the C-terminal region consists of a growth factor domain (GFD) with homology to vascular endothelial growth factor (25%) and PDGF A-chain (23%). A serum-sensitive cleavage site between the two domains allows release of the GFD from the CUB domain. Competition binding and immunoprecipitation studies on cells bearing both PDGF α and β receptors reveal a high affinity binding of recombinant GFD (PDGF-CC) to PDGF receptor-α homodimers and PDGF receptor-α/β heterodimers. PDGF-CC exhibits greater mitogenic potency than PDGF-AA and comparable or greater mitogenic activity than PDGF-AB and PDGF-BB on several mesenchymal cell types. Analysis of PDGF-CC in vivo in a diabetic mouse model of delayed wound healing showed that PDGF-CC significantly enhanced repair of a full-thickness skin excision. Together, these studies describe a third member of the PDGF family (PDGF-C) as a potent mitogen for cells of mesenchymal origin inin vitro and in vivo systems with a binding pattern similar to PDGF-AB. expressed sequence tag platelet-derived growth factor PDGF receptor vascular endothelial growth factor domains first found in complement subcomponents Clr/Cls, urchin EGF-like protein and bone morphogenetic protein-1 baby hamster kidney cells cytomegalovirus growth factor domain monoclonal antibody human aortic smooth muscle cells human aortic adventitial fibroblasts human umbilical vein endothelial cells human pulmonary artery endothelial cells human aortic endothelial cells normal human lung fibroblasts normal human dermal fibroblast-neonatal 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol polyacrylamide gel electrophoresis polymerase chain reaction base pair Dulbecco's modified Eagle's medium phosphate-buffered saline 4-morpholinepropanesulfonic acid As a result of the increased bioinformatics effort to analyze the rapidly expanding expressed sequence tag (EST)1 and genomic sequence data bases, many families of growth factors have experienced a rapid increase in members, including the fibroblast growth factor (1Szebenyi G. Fallon J.F. Int. Rev. Cytol. 1999; 185: 45-106Crossref PubMed Google Scholar, 2Coulier F. Pontarotti P. Roubin R. Hartung H. Goldfarb M. Birnbaum D. J. Mol. Evol. 1997; 44: 43-56Crossref PubMed Scopus (184) Google Scholar, 3Smallwood P.M. Munoz-Sanjuan I. Tong P. Macke J.P. Hendry S.H. Gilbert D.J Copeland N.G. Jenkins N.A. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9850-9857Crossref PubMed Scopus (329) Google Scholar), VEGF (4Tischer E. Gospodarowicz D. Mitchell R. Schilling J. Lau K. Crisp T. Abraham J.A. Biochem. Biophys. Res. Commun. 1989; 165: 1198-1206Crossref PubMed Scopus (253) Google Scholar, 5Olofsson B. Korpelainen E. Pepper M.S. Mandriota S.J. Aase K. Kumar V. Gunji Y. Jeltsch M.M. Shibuya M. Alitalo K. Eriksson U. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11709-11714Crossref PubMed Scopus (443) Google Scholar, 6Joukov V. Pajusola K. Kaipainen A. Chilov D. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1143) Google Scholar, 7Achen M.G. Jeltsch M. Kukk E. Makinen T. Vitali A. Wilks A.F. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 548-553Crossref PubMed Scopus (1011) Google Scholar), and transforming growth factor-β (8Clark D.A. Coker R. Int. J. Biochem. Cell Biol. 1998; 30: 293-298Crossref PubMed Scopus (259) Google Scholar, 9Piek E. Heldin C.H. Dijke P.T. FASEB J. 1999; 13: 2105-2124Crossref PubMed Scopus (737) Google Scholar) families. During the past 15 years, however, the PDGF family has remained unchanged in size, consisting of two members, PDGF-A and -B, that are able to form three dimeric structures, AA, AB, and BB. All three forms of PDGF have been identified in natural sources and have been shown to possess biological activity (10Stroopbant P. Waterfield M.D. EMBO J. 1984; 3: 2963-2967Crossref PubMed Scopus (148) Google Scholar, 11Heldin C.H. Johnsson A. Wennergren S. Wernstedt C. Betsholtz C. Westermark B. Nature. 1986; 319: 511-514Crossref PubMed Scopus (271) Google Scholar, 12Hart C.E. Forstrom J.W. Kelly J.D. Seifert R.A Smith R.A. Ross R. Murray M.J. Bowen-Pope D.F. Science. 1988; 240: 1529-1531Crossref PubMed Scopus (442) Google Scholar).Two PDGF receptors have also been identified, PDGF-α receptor (PDGFR-α) and PDGF-β receptor (PDGFR-β), that demonstrate differential binding specificity for the two PDGF chains. PDGFR-α binds both PDGF A- and B-chains, whereas the PDGFR-β binds only PDGF B-chain (12Hart C.E. Forstrom J.W. Kelly J.D. Seifert R.A Smith R.A. Ross R. Murray M.J. Bowen-Pope D.F. Science. 1988; 240: 1529-1531Crossref PubMed Scopus (442) Google Scholar, 13Seifert R.A. Hart C.E. Phillips P.E. Forstrom J.W. Ross R. Murray M.J. Bowen-Pope D.F. J. Biol. Chem. 1989; 264: 8771-8778Abstract Full Text PDF PubMed Google Scholar). Dimerization of the receptors is required for high affinity binding such that α/α homodimerization is induced by PDGF-AA, -AB, and -BB; α/β heterodimerization is induced by PDGF-AB and -BB, and β/β homodimerization is induced only by BB. Thus, the response of a given cell type to the different forms of PDGF is dependent on the relative abundance of each of the PDGF receptors (14Hart C.E. Bowen-Pope D.F. J. Invest Dermatol. 1990; 94 Suppl. 6: 53S-57SAbstract Full Text PDF Scopus (51) Google Scholar).In this report we describe the characterization of a new member of the PDGF family, PDGF-C. This protein is a multidomain protein requiring enzymatic activation to release a C-terminal domain with potent biological activity. A recent article by Li et al. (15Li X. Ponten A. Aase K. Karlsson L. Abramsson A. Uutela M. Backstrom G. Hellstrom M. Bostrom H. Li H. Soriano P. Betsholtz C. Heldin C.H. Alitalo K. Ostman A. Eriksson U. Nat. Cell Biol. 2000; 2: 302-309Crossref PubMed Scopus (499) Google Scholar) describes this same protein and reports that PDGF-CC binds only to PDGFR-α, giving a binding profile that is similar to PDGF-AA. In the studies reported here, we extend the data for this ligand, demonstrating direct binding of PDGF-CC to PDGFR-β in cells co-expressing PDGFR-α. Thus the binding specificity indicated for PDGF-CC gives it a profile similar to PDGF-AB. Distinct differences in biological activity associated with binding to α/α and α/β receptors have been reported (16Rosenkranz S. Kazlauskas A. Growth Factors. 1998; 16: 201-216Crossref Scopus (181) Google Scholar). On vascular smooth muscle cells, PDGF-AB and -BB binding to α/β receptors is associated with stimulation of chemotaxis, whereas PDGF-AA binding to α receptor is associated with inhibition of chemotaxis. In wound healing processes, PDGF-AB and PDGF-BB are significantly more potent than PDGF-AA in the enhancement of granulation tissue formation (17Lepistö P. Peltonen J. Vähä-Kreula M. Söderström K.-O. Niinikoski J. Laato M. Cell Tissue Res. 1996; 286: 449-455Crossref PubMed Scopus (25) Google Scholar).We believe the identification of the expanded receptor binding properties of PDGF-C will be critical in understanding the biology of this new member of the PDGF family as well as providing new ways of assessing PDGF biology.DISCUSSIONWe have identified a third member of the PDGF ligand family using a homology-based computational algorithm. This molecule, labeled PDGF-C (15Li X. Ponten A. Aase K. Karlsson L. Abramsson A. Uutela M. Backstrom G. Hellstrom M. Bostrom H. Li H. Soriano P. Betsholtz C. Heldin C.H. Alitalo K. Ostman A. Eriksson U. Nat. Cell Biol. 2000; 2: 302-309Crossref PubMed Scopus (499) Google Scholar), is a multidomain protein with a C-terminal domain capable of binding to and activating PDGF receptors. The data presented here demonstrate that PDGF-CC binds with high affinity to PDGFR-α/α homodimers and PDGFR-α/β heterodimers but not PDGFR-β/β homodimers. Thus, PDGF-CC has a binding phenotype similar to that of PDGF-AB, the ability to bind to α/α homodimers and α/β heterodimers. The ability to bind to the β receptor is supported by several lines of evidence including the ability of β receptor blocking antibodies to decrease the binding of PDGF-CC, the ability to immunoprecipitate PDGF-CC when complexed to PDGFR-β, and the ability of PDGF-CC to stimulate tyrosine phosphorylation of PDGFR-β. These studies were all conducted with cells that expressed both PDGFR-α and -β. For cells that expressed PDGFR-β only, there was no evidence for high affinity PDGF-CC binding to PDGFR-β.The finding that PDGF-CC binds to both α/α and α/β receptor complexes is critical to the elucidation of the biology associated with this protein. If PDGF-CC binds only to α/α receptor complexes, as suggested by Li et al. (15Li X. Ponten A. Aase K. Karlsson L. Abramsson A. Uutela M. Backstrom G. Hellstrom M. Bostrom H. Li H. Soriano P. Betsholtz C. Heldin C.H. Alitalo K. Ostman A. Eriksson U. Nat. Cell Biol. 2000; 2: 302-309Crossref PubMed Scopus (499) Google Scholar), then it would be expected that PDGF-CC would have properties similar to PDGF-AA. In contrast, the ability of PDGF-CC to bind to α/α and α/β receptor complexes gives it a binding profile similar to PDGF-AB. The extension of the receptor binding data for PDGF-CC presented in this study is important to understand the biology of this molecule as it is well documented that PDGF-AA and PDGF-AB have unique biological properties.Studying mesangial cells, Abboud et al. (36Abboud H.E. Grandaliano G. Pinzani M. Knauss T. Pierce G.F. Jaffer F. J. Cell. Physiol. 1994; 158: 140-150Crossref PubMed Scopus (32) Google Scholar) demonstrated that PDGF-AB induced increase in DNA synthesis, activation of phospholipase C, and autoinduction of PDGF A- and B-chain mRNAs, whereas PDGF-AA lacked these effects. Rupp et al. (37Rupp E. Siegbahn A. Rönnstrand L. Wernstedt C. Claesson-Welsh L. Heldin C.-H. Eur. J. Biochem. 1994; 225: 29-41Crossref PubMed Scopus (47) Google Scholar), using endothelial cells transfected with both PDGFR-α and -β at similar levels, demonstrated that PDGF-AB, but not PDGF-AA, stimulated actin reorganization and chemotaxis and that PDGF-AB was more potent than PDGF-AA at stimulating DNA synthesis. Koyama et al.(22Koyama N. Hart C.E. Clowes A.W. Circ. Res. 1994; 75: 682-691Crossref PubMed Scopus (138) Google Scholar) similarly reported that PDGF-AB stimulates chemotactic activity on vascular smooth muscle cells, whereas PDGF-AA has no stimulatory effect. Studying the in vivo effects of PDGFs, Lepistöet al. (17Lepistö P. Peltonen J. Vähä-Kreula M. Söderström K.-O. Niinikoski J. Laato M. Cell Tissue Res. 1996; 286: 449-455Crossref PubMed Scopus (25) Google Scholar) demonstrated that PDGF-AB elicited a more potent granulation response in cellulose sponges implanted into rats than PDGF-AA. The increased potency of PDGF-AB over PDGF-AA described in the above studies is consistent with the increase in mitogenic activity we observed for PDGF-CC as compared with PDGF-AA in this paper.The expression of PDGF-C in BHK-570 cells suggests that cells produce it as a full-length molecule. Whereas PDGF A- and B-chains have a dibasic cleavage site adjacent to their GFDs, similar to PDGF-C, cleavage at these sites occurs within the cell, such that only the separated GFD is secreted from the cell (reviewed in Ref. 39Heldin C.H. Ostman A. Westermark B. Growth Factors. 1993; 8: 245-252Crossref PubMed Scopus (38) Google Scholar). In contrast, expression of PDGF-C in BHK-570 cells suggests that the cell secretes the full-length molecule. In our studies the cleavage of PDGF-C appears to be sensitive to as yet unidentified serum factors. Liet al. (15Li X. Ponten A. Aase K. Karlsson L. Abramsson A. Uutela M. Backstrom G. Hellstrom M. Bostrom H. Li H. Soriano P. Betsholtz C. Heldin C.H. Alitalo K. Ostman A. Eriksson U. Nat. Cell Biol. 2000; 2: 302-309Crossref PubMed Scopus (499) Google Scholar) have reported that PDGF-C can be cleaved by plasmin to release the GFD. Our data further demonstrate that PDGF-C is produced as a homodimer in culture and that both the full-length protein and the GFD version can form homodimers when expressed as recombinant proteins. Given the presence of a large N-terminal CUB domain, it seems unlikely that PDGF-CC forms heterodimers with PDGF A- or B-chain polypeptides.The multidomain structure of PDGF-C is unique within the PDGF family. Although the GFD has potent biological activity, as demonstrated in the aortic ring assay, [3H]thymidine incorporation studies, and in vivo wound healing, the function of the CUB domain remains to be determined. It is important to note that the CUB domain has homology to neuropilin-1, a cell-surface glycoprotein initially identified in Xenopus tadpole nervous tissues (40Takagi S. Tsuji T. Amagai T. Takamatsu T. Fujisawa H. Dev. Biol. 1987; 122: 90-100Crossref PubMed Scopus (140) Google Scholar, 41Takagi S. Hirata T. Agata K. Mochii M. Eguchi G. Fujisawa H. Neuron. 1991; 7: 295-307Abstract Full Text PDF PubMed Scopus (185) Google Scholar, 42Takagi S. Kasuya Y. Shimizu M. Matsuura T. Tsuboi M. Kawakami A. Fujisawa H. Dev. Biol. 1995; 170: 207-222Crossref PubMed Scopus (175) Google Scholar), which has been demonstrated to be a receptor for various members of the semaphorin family (30Kolodkin A.L. Levengood D.V. Rowe E.G. Tai Y.T. Giger R.J. Ginty D.D. Cell. 1997; 90: 753-762Abstract Full Text Full Text PDF PubMed Scopus (994) Google Scholar, 43Chen H. Chedotal A. He Z. Goodman C.S. Tessier L.M. Neuron. 1997; 19: 547-559Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar). Neuropilin-1 has also been shown to play a role in regulating neurite outgrowth via its interactions with semaphorins (44Kitsukawa T. Shimizu M. Sanbo M. Hirata T. Taniguchi M. Bekku Y. Yagi T. Fujisawa H. Neuron. 1997; 19: 995-1005Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar). The CUB domain of PDGF-C may act as a targeting domain for the full-length protein by binding to cell-surface molecules, potentially semaphorins. PDGF-C is then locally cleaved to release the GFD by as yet unidentified serum factors. The released GFD is then available to bind and activate the PDGF receptors. This scheme provides for the accumulation of PDGF-C at localized sites without the release of the active GFD until such time as local environmental events cause the release of active protein. This model requires that full-length PDGF-C does not bind to the PDGF receptors or, if it does, is not capable of stimulating receptor activation. Results reported by Li et al. (15Li X. Ponten A. Aase K. Karlsson L. Abramsson A. Uutela M. Backstrom G. Hellstrom M. Bostrom H. Li H. Soriano P. Betsholtz C. Heldin C.H. Alitalo K. Ostman A. Eriksson U. Nat. Cell Biol. 2000; 2: 302-309Crossref PubMed Scopus (499) Google Scholar) support this conclusion. In all cases where we have expressed full-length protein, there is always some limited amount of cleaved product generated. As such, it has not been possible to determine whether the full-length protein or the cleaved portion of the molecule is interacting with the PDGF receptors.In addition to expanding the binding model of PDGF/receptor interaction, the structure of PDGF-C creates additional possibilities for in vivo PDGF function. PDGF-A and -B are made by epithelial and endothelial cells in order to recruit mesenchymal cells bearing PDGF receptors during development (38Hellström M. Kalén M. Lindahl P. Abramsson A. Betsholtz C. Development. 1999; 126: 3047-3055Crossref PubMed Google Scholar, 45Ansel J.C. Tiesman J.P. Olerud J.E. Krueger J.G. Krane J.F. Tara D.C. Shipley G.D. Gilbertson D. Usui M.L. Hart C.E. J. Clin. Invest. 1993; 92: 671-678Crossref PubMed Scopus (124) Google Scholar). Human platelets release all three forms of PDGF at sites of vascular damage, recruiting smooth muscle cells and fibroblasts to the wound site. It is intriguing to speculate that mesenchymal cells are not always forced to rely on other cell types for proliferative stimulus but may also synthesize a potent form of PDGF (PDGF-C) sequestered in the tissues until specific proteolytic enzymes coupled to trauma or regenerative processes release the active GFD from the inactive pro-form. The generalized expression of PDGF-C on multiple tissue Northerns (Fig. 2 A) and in cultured fibroblasts (Fig. 2 B) is consistent with this notion. It is possible that PDGF-C is a primary source of PDGF-like wound healing activity in somatic tissues and that the PDGF is synthesized and released by the responding cell types. Inappropriate activation of the pro-form may thus also lead to diseases characterized by proliferation of connective tissues.Clearly the analysis of historical data and activities attributed to the various members of the PDGF ligand family need to be reconsidered in a new light. The identification of a new member adds to the already complex nature of this ligand/receptor family. Further elucidation of PDGF-C activities will begin to clarify its true level of involvement in regulating the multitude of activities previously attributed to the known PDGF family members. As a result of the increased bioinformatics effort to analyze the rapidly expanding expressed sequence tag (EST)1 and genomic sequence data bases, many families of growth factors have experienced a rapid increase in members, including the fibroblast growth factor (1Szebenyi G. Fallon J.F. Int. Rev. Cytol. 1999; 185: 45-106Crossref PubMed Google Scholar, 2Coulier F. Pontarotti P. Roubin R. Hartung H. Goldfarb M. Birnbaum D. J. Mol. Evol. 1997; 44: 43-56Crossref PubMed Scopus (184) Google Scholar, 3Smallwood P.M. Munoz-Sanjuan I. Tong P. Macke J.P. Hendry S.H. Gilbert D.J Copeland N.G. Jenkins N.A. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9850-9857Crossref PubMed Scopus (329) Google Scholar), VEGF (4Tischer E. Gospodarowicz D. Mitchell R. Schilling J. Lau K. Crisp T. Abraham J.A. Biochem. Biophys. Res. Commun. 1989; 165: 1198-1206Crossref PubMed Scopus (253) Google Scholar, 5Olofsson B. Korpelainen E. Pepper M.S. Mandriota S.J. Aase K. Kumar V. Gunji Y. Jeltsch M.M. Shibuya M. Alitalo K. Eriksson U. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11709-11714Crossref PubMed Scopus (443) Google Scholar, 6Joukov V. Pajusola K. Kaipainen A. Chilov D. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1143) Google Scholar, 7Achen M.G. Jeltsch M. Kukk E. Makinen T. Vitali A. Wilks A.F. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 548-553Crossref PubMed Scopus (1011) Google Scholar), and transforming growth factor-β (8Clark D.A. Coker R. Int. J. Biochem. Cell Biol. 1998; 30: 293-298Crossref PubMed Scopus (259) Google Scholar, 9Piek E. Heldin C.H. Dijke P.T. FASEB J. 1999; 13: 2105-2124Crossref PubMed Scopus (737) Google Scholar) families. During the past 15 years, however, the PDGF family has remained unchanged in size, consisting of two members, PDGF-A and -B, that are able to form three dimeric structures, AA, AB, and BB. All three forms of PDGF have been identified in natural sources and have been shown to possess biological activity (10Stroopbant P. Waterfield M.D. EMBO J. 1984; 3: 2963-2967Crossref PubMed Scopus (148) Google Scholar, 11Heldin C.H. Johnsson A. Wennergren S. Wernstedt C. Betsholtz C. Westermark B. Nature. 1986; 319: 511-514Crossref PubMed Scopus (271) Google Scholar, 12Hart C.E. Forstrom J.W. Kelly J.D. Seifert R.A Smith R.A. Ross R. Murray M.J. Bowen-Pope D.F. Science. 1988; 240: 1529-1531Crossref PubMed Scopus (442) Google Scholar). Two PDGF receptors have also been identified, PDGF-α receptor (PDGFR-α) and PDGF-β receptor (PDGFR-β), that demonstrate differential binding specificity for the two PDGF chains. PDGFR-α binds both PDGF A- and B-chains, whereas the PDGFR-β binds only PDGF B-chain (12Hart C.E. Forstrom J.W. Kelly J.D. Seifert R.A Smith R.A. Ross R. Murray M.J. Bowen-Pope D.F. Science. 1988; 240: 1529-1531Crossref PubMed Scopus (442) Google Scholar, 13Seifert R.A. Hart C.E. Phillips P.E. Forstrom J.W. Ross R. Murray M.J. Bowen-Pope D.F. J. Biol. Chem. 1989; 264: 8771-8778Abstract Full Text PDF PubMed Google Scholar). Dimerization of the receptors is required for high affinity binding such that α/α homodimerization is induced by PDGF-AA, -AB, and -BB; α/β heterodimerization is induced by PDGF-AB and -BB, and β/β homodimerization is induced only by BB. Thus, the response of a given cell type to the different forms of PDGF is dependent on the relative abundance of each of the PDGF receptors (14Hart C.E. Bowen-Pope D.F. J. Invest Dermatol. 1990; 94 Suppl. 6: 53S-57SAbstract Full Text PDF Scopus (51) Google Scholar). In this report we describe the characterization of a new member of the PDGF family, PDGF-C. This protein is a multidomain protein requiring enzymatic activation to release a C-terminal domain with potent biological activity. A recent article by Li et al. (15Li X. Ponten A. Aase K. Karlsson L. Abramsson A. Uutela M. Backstrom G. Hellstrom M. Bostrom H. Li H. Soriano P. Betsholtz C. Heldin C.H. Alitalo K. Ostman A. Eriksson U. Nat. Cell Biol. 2000; 2: 302-309Crossref PubMed Scopus (499) Google Scholar) describes this same protein and reports that PDGF-CC binds only to PDGFR-α, giving a binding profile that is similar to PDGF-AA. In the studies reported here, we extend the data for this ligand, demonstrating direct binding of PDGF-CC to PDGFR-β in cells co-expressing PDGFR-α. Thus the binding specificity indicated for PDGF-CC gives it a profile similar to PDGF-AB. Distinct differences in biological activity associated with binding to α/α and α/β receptors have been reported (16Rosenkranz S. Kazlauskas A. Growth Factors. 1998; 16: 201-216Crossref Scopus (181) Google Scholar). On vascular smooth muscle cells, PDGF-AB and -BB binding to α/β receptors is associated with stimulation of chemotaxis, whereas PDGF-AA binding to α receptor is associated with inhibition of chemotaxis. In wound healing processes, PDGF-AB and PDGF-BB are significantly more potent than PDGF-AA in the enhancement of granulation tissue formation (17Lepistö P. Peltonen J. Vähä-Kreula M. Söderström K.-O. Niinikoski J. Laato M. Cell Tissue Res. 1996; 286: 449-455Crossref PubMed Scopus (25) Google Scholar). We believe the identification of the expanded receptor binding properties of PDGF-C will be critical in understanding the biology of this new member of the PDGF family as well as providing new ways of assessing PDGF biology. DISCUSSIONWe have identified a third member of the PDGF ligand family using a homology-based computational algorithm. This molecule, labeled PDGF-C (15Li X. Ponten A. Aase K. Karlsson L. Abramsson A. Uutela M. Backstrom G. Hellstrom M. Bostrom H. Li H. Soriano P. Betsholtz C. Heldin C.H. Alitalo K. Ostman A. Eriksson U. Nat. Cell Biol. 2000; 2: 302-309Crossref PubMed Scopus (499) Google Scholar), is a multidomain protein with a C-terminal domain capable of binding to and activating PDGF receptors. The data presented here demonstrate that PDGF-CC binds with high affinity to PDGFR-α/α homodimers and PDGFR-α/β heterodimers but not PDGFR-β/β homodimers. Thus, PDGF-CC has a binding phenotype similar to that of PDGF-AB, the ability to bind to α/α homodimers and α/β heterodimers. The ability to bind to the β receptor is supported by several lines of evidence including the ability of β receptor blocking antibodies to decrease the binding of PDGF-CC, the ability to immunoprecipitate PDGF-CC when complexed to PDGFR-β, and the ability of PDGF-CC to stimulate tyrosine phosphorylation of PDGFR-β. These studies were all conducted with cells that expressed both PDGFR-α and -β. For cells that expressed PDGFR-β only, there was no evidence for high affinity PDGF-CC binding to PDGFR-β.The finding that PDGF-CC binds to both α/α and α/β receptor complexes is critical to the elucidation of the biology associated with this protein. If PDGF-CC binds only to α/α receptor complexes, as suggested by Li et al. (15Li X. Ponten A. Aase K. Karlsson L. Abramsson A. Uutela M. Backstrom G. Hellstrom M. Bostrom H. Li H. Soriano P. Betsholtz C. Heldin C.H. Alitalo K. Ostman A. Eriksson U. Nat. Cell Biol. 2000; 2: 302-309Crossref PubMed Scopus (499) Google Scholar), then it would be expected that PDGF-CC would have properties similar to PDGF-AA. In contrast, the ability of PDGF-CC to bind to α/α and α/β receptor complexes gives it a binding profile similar to PDGF-AB. The extension of the receptor binding data for PDGF-CC presented in this study is important to understand the biology of this molecule as it is well documented that PDGF-AA and PDGF-AB have unique biological properties.Studying mesangial cells, Abboud et al. (36Abboud H.E. Grandaliano G. Pinzani M. Knauss T. Pierce G.F. Jaffer F. J. Cell. Physiol. 1994; 158: 140-150Crossref PubMed Scopus (32) Google Scholar) demonstrated that PDGF-AB induced increase in DNA synthesis, activation of phospholipase C, and autoinduction of PDGF A- and B-chain mRNAs, whereas PDGF-AA lacked these effects. Rupp et al. (37Rupp E. Siegbahn A. Rönnstrand L. Wernstedt C. Claesson-Welsh L. Heldin C.-H. Eur. J. Biochem. 1994; 225: 29-41Crossref PubMed Scopus (47) Google Scholar), using endothelial cells transfected with both PDGFR-α and -β at similar levels, demonstrated that PDGF-AB, but not PDGF-AA, stimulated actin reorganization and chemotaxis and that PDGF-AB was more potent than PDGF-AA at stimulating DNA synthesis. Koyama et al.(22Koyama N. Hart C.E. Clowes A.W. Circ. Res. 1994; 75: 682-691Crossref PubMed Scopus (138) Google Scholar) similarly reported that PDGF-AB stimulates chemotactic activity on vascular smooth muscle cells, whereas PDGF-AA has no stimulatory effect. Studying the in vivo effects of PDGFs, Lepistöet al. (17Lepistö P. Peltonen J. Vähä-Kreula M. Söderström K.-O. Niinikoski J. Laato M. Cell Tissue Res. 1996; 286: 449-455Crossref PubMed Scopus (25) Google Scholar) demonstrated that PDGF-AB elicited a more potent granulation response in cellulose sponges implanted into rats than PDGF-AA. The increased potency of PDGF-AB over PDGF-AA described in the above studies is consistent with the increase in mitogenic activity we observed for PDGF-CC as compared with PDGF-AA in this paper.The expression of PDGF-C in BHK-570 cells suggests that cells produce it as a full-length molecule. Whereas PDGF A- and B-chains have a dibasic cleavage site adjacent to their GFDs, similar to PDGF-C, cleavage at these sites occurs within the cell, such that only the separated GFD is secreted from the cell (reviewed in Ref. 39Heldin C.H. Ostman A. Westermark B. Growth Factors. 1993; 8: 245-252Crossref PubMed Scopus (38) Google Scholar). In contrast, expression of PDGF-C in BHK-570 cells suggests that the cell secretes the full-length molecule. In our studies the cleavage of PDGF-C appears to be sensitive to as yet unidentified serum factors. Liet al. (15Li X. Ponten A. Aase K. Karlsson L. Abramsson A. Uutela M. Backstrom G. Hellstrom M. Bostrom H. Li H. Soriano P. Betsholtz C. Heldin C.H. Alitalo K. Ostman A. Eriksson U. Nat. Cell Biol. 2000; 2: 302-309Crossref PubMed Scopus (499) Google Scholar) have reported that PDGF-C can be cleaved by plasmin to release the GFD. Our data further demonstrate that PDGF-C is produced as a homodimer in culture and that both the full-length protein and the GFD version can form homodimers when expressed as recombinant proteins. Given the presence of a large N-terminal CUB domain, it seems unlikely that PDGF-CC forms heterodimers with PDGF A- or B-chain polypeptides.The multidomain structure of PDGF-C is unique within the PDGF family. Although the GFD has potent biological activity, as demonstrated in the aortic ring assay, [3H]thymidine incorporation studies, and in vivo wound healing, the function of the CUB domain remains to be determined. It is important to note that the CUB domain has homology to neuropilin-1, a cell-surface glycoprotein initially identified in Xenopus tadpole nervous tissues (40Takagi S. Tsuji T. Amagai T. Takamatsu T. Fujisawa H. Dev. Biol. 1987; 122: 90-100Crossref PubMed Scopus (140) Google Scholar, 41Takagi S. Hirata T. Agata K. Mochii M. Eguchi G. Fujisawa H. Neuron. 1991; 7: 295-307Abstract Full Text PDF PubMed Scopus (185) Google Scholar, 42Takagi S. Kasuya Y. Shimizu M. Matsuura T. Tsuboi M. Kawakami A. Fujisawa H. Dev. Biol. 1995; 170: 207-222Crossref PubMed Scopus (175) Google Scholar), which has been demonstrated to be a receptor for various members of the semaphorin family (30Kolodkin A.L. Levengood D.V. Rowe E.G. Tai Y.T. Giger R.J. Ginty D.D. Cell. 1997; 90: 753-762Abstract Full Text Full Text PDF PubMed Scopus (994) Google Scholar, 43Chen H. Chedotal A. He Z. Goodman C.S. Tessier L.M. Neuron. 1997; 19: 547-559Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar). Neuropilin-1 has also been shown to play a role in regulating neurite outgrowth via its interactions with semaphorins (44Kitsukawa T. Shimizu M. Sanbo M. Hirata T. Taniguchi M. Bekku Y. Yagi T. Fujisawa H. Neuron. 1997; 19: 995-1005Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar). The CUB domain of PDGF-C may act as a targeting domain for the full-length protein by binding to cell-surface molecules, potentially semaphorins. PDGF-C is then locally cleaved to release the GFD by as yet unidentified serum factors. The released GFD is then available to bind and activate the PDGF receptors. This scheme provides for the accumulation of PDGF-C at localized sites without the release of the active GFD until such time as local environmental events cause the release of active protein. This model requires that full-length PDGF-C does not bind to the PDGF receptors or, if it does, is not capable of stimulating receptor activation. Results reported by Li et al. (15Li X. Ponten A. Aase K. Karlsson L. Abramsson A. Uutela M. Backstrom G. Hellstrom M. Bostrom H. Li H. Soriano P. Betsholtz C. Heldin C.H. Alitalo K. Ostman A. Eriksson U. Nat. Cell Biol. 2000; 2: 302-309Crossref PubMed Scopus (499) Google Scholar) support this conclusion. In all cases where we have expressed full-length protein, there is always some limited amount of cleaved product generated. As such, it has not been possible to determine whether the full-length protein or the cleaved portion of the molecule is interacting with the PDGF receptors.In addition to expanding the binding model of PDGF/receptor interaction, the structure of PDGF-C creates additional possibilities for in vivo PDGF function. PDGF-A and -B are made by epithelial and endothelial cells in order to recruit mesenchymal cells bearing PDGF receptors during development (38Hellström M. Kalén M. Lindahl P. Abramsson A. Betsholtz C. Development. 1999; 126: 3047-3055Crossref PubMed Google Scholar, 45Ansel J.C. Tiesman J.P. Olerud J.E. Krueger J.G. Krane J.F. Tara D.C. Shipley G.D. Gilbertson D. Usui M.L. Hart C.E. J. Clin. Invest. 1993; 92: 671-678Crossref PubMed Scopus (124) Google Scholar). Human platelets release all three forms of PDGF at sites of vascular damage, recruiting smooth muscle cells and fibroblasts to the wound site. It is intriguing to speculate that mesenchymal cells are not always forced to rely on other cell types for proliferative stimulus but may also synthesize a potent form of PDGF (PDGF-C) sequestered in the tissues until specific proteolytic enzymes coupled to trauma or regenerative processes release the active GFD from the inactive pro-form. The generalized expression of PDGF-C on multiple tissue Northerns (Fig. 2 A) and in cultured fibroblasts (Fig. 2 B) is consistent with this notion. It is possible that PDGF-C is a primary source of PDGF-like wound healing activity in somatic tissues and that the PDGF is synthesized and released by the responding cell types. Inappropriate activation of the pro-form may thus also lead to diseases characterized by proliferation of connective tissues.Clearly the analysis of historical data and activities attributed to the various members of the PDGF ligand family need to be reconsidered in a new light. The identification of a new member adds to the already complex nature of this ligand/receptor family. Further elucidation of PDGF-C activities will begin to clarify its true level of involvement in regulating the multitude of activities previously attributed to the known PDGF family members. We have identified a third member of the PDGF ligand family using a homology-based computational algorithm. This molecule, labeled PDGF-C (15Li X. Ponten A. Aase K. Karlsson L. Abramsson A. Uutela M. Backstrom G. Hellstrom M. Bostrom H. Li H. Soriano P. Betsholtz C. Heldin C.H. Alitalo K. Ostman A. Eriksson U. Nat. Cell Biol. 2000; 2: 302-309Crossref PubMed Scopus (499) Google Scholar), is a multidomain protein with a C-terminal domain capable of binding to and activating PDGF receptors. The data presented here demonstrate that PDGF-CC binds with high affinity to PDGFR-α/α homodimers and PDGFR-α/β heterodimers but not PDGFR-β/β homodimers. Thus, PDGF-CC has a binding phenotype similar to that of PDGF-AB, the ability to bind to α/α homodimers and α/β heterodimers. The ability to bind to the β receptor is supported by several lines of evidence including the ability of β receptor blocking antibodies to decrease the binding of PDGF-CC, the ability to immunoprecipitate PDGF-CC when complexed to PDGFR-β, and the ability of PDGF-CC to stimulate tyrosine phosphorylation of PDGFR-β. These studies were all conducted with cells that expressed both PDGFR-α and -β. For cells that expressed PDGFR-β only, there was no evidence for high affinity PDGF-CC binding to PDGFR-β. The finding that PDGF-CC binds to both α/α and α/β receptor complexes is critical to the elucidation of the biology associated with this protein. If PDGF-CC binds only to α/α receptor complexes, as suggested by Li et al. (15Li X. Ponten A. Aase K. Karlsson L. Abramsson A. Uutela M. Backstrom G. Hellstrom M. Bostrom H. Li H. Soriano P. Betsholtz C. Heldin C.H. Alitalo K. Ostman A. Eriksson U. Nat. Cell Biol. 2000; 2: 302-309Crossref PubMed Scopus (499) Google Scholar), then it would be expected that PDGF-CC would have properties similar to PDGF-AA. In contrast, the ability of PDGF-CC to bind to α/α and α/β receptor complexes gives it a binding profile similar to PDGF-AB. The extension of the receptor binding data for PDGF-CC presented in this study is important to understand the biology of this molecule as it is well documented that PDGF-AA and PDGF-AB have unique biological properties. Studying mesangial cells, Abboud et al. (36Abboud H.E. Grandaliano G. Pinzani M. Knauss T. Pierce G.F. Jaffer F. J. Cell. Physiol. 1994; 158: 140-150Crossref PubMed Scopus (32) Google Scholar) demonstrated that PDGF-AB induced increase in DNA synthesis, activation of phospholipase C, and autoinduction of PDGF A- and B-chain mRNAs, whereas PDGF-AA lacked these effects. Rupp et al. (37Rupp E. Siegbahn A. Rönnstrand L. Wernstedt C. Claesson-Welsh L. Heldin C.-H. Eur. J. Biochem. 1994; 225: 29-41Crossref PubMed Scopus (47) Google Scholar), using endothelial cells transfected with both PDGFR-α and -β at similar levels, demonstrated that PDGF-AB, but not PDGF-AA, stimulated actin reorganization and chemotaxis and that PDGF-AB was more potent than PDGF-AA at stimulating DNA synthesis. Koyama et al.(22Koyama N. Hart C.E. Clowes A.W. Circ. Res. 1994; 75: 682-691Crossref PubMed Scopus (138) Google Scholar) similarly reported that PDGF-AB stimulates chemotactic activity on vascular smooth muscle cells, whereas PDGF-AA has no stimulatory effect. Studying the in vivo effects of PDGFs, Lepistöet al. (17Lepistö P. Peltonen J. Vähä-Kreula M. Söderström K.-O. Niinikoski J. Laato M. Cell Tissue Res. 1996; 286: 449-455Crossref PubMed Scopus (25) Google Scholar) demonstrated that PDGF-AB elicited a more potent granulation response in cellulose sponges implanted into rats than PDGF-AA. The increased potency of PDGF-AB over PDGF-AA described in the above studies is consistent with the increase in mitogenic activity we observed for PDGF-CC as compared with PDGF-AA in this paper. The expression of PDGF-C in BHK-570 cells suggests that cells produce it as a full-length molecule. Whereas PDGF A- and B-chains have a dibasic cleavage site adjacent to their GFDs, similar to PDGF-C, cleavage at these sites occurs within the cell, such that only the separated GFD is secreted from the cell (reviewed in Ref. 39Heldin C.H. Ostman A. Westermark B. Growth Factors. 1993; 8: 245-252Crossref PubMed Scopus (38) Google Scholar). In contrast, expression of PDGF-C in BHK-570 cells suggests that the cell secretes the full-length molecule. In our studies the cleavage of PDGF-C appears to be sensitive to as yet unidentified serum factors. Liet al. (15Li X. Ponten A. Aase K. Karlsson L. Abramsson A. Uutela M. Backstrom G. Hellstrom M. Bostrom H. Li H. Soriano P. Betsholtz C. Heldin C.H. Alitalo K. Ostman A. Eriksson U. Nat. Cell Biol. 2000; 2: 302-309Crossref PubMed Scopus (499) Google Scholar) have reported that PDGF-C can be cleaved by plasmin to release the GFD. Our data further demonstrate that PDGF-C is produced as a homodimer in culture and that both the full-length protein and the GFD version can form homodimers when expressed as recombinant proteins. Given the presence of a large N-terminal CUB domain, it seems unlikely that PDGF-CC forms heterodimers with PDGF A- or B-chain polypeptides. The multidomain structure of PDGF-C is unique within the PDGF family. Although the GFD has potent biological activity, as demonstrated in the aortic ring assay, [3H]thymidine incorporation studies, and in vivo wound healing, the function of the CUB domain remains to be determined. It is important to note that the CUB domain has homology to neuropilin-1, a cell-surface glycoprotein initially identified in Xenopus tadpole nervous tissues (40Takagi S. Tsuji T. Amagai T. Takamatsu T. Fujisawa H. Dev. Biol. 1987; 122: 90-100Crossref PubMed Scopus (140) Google Scholar, 41Takagi S. Hirata T. Agata K. Mochii M. Eguchi G. Fujisawa H. Neuron. 1991; 7: 295-307Abstract Full Text PDF PubMed Scopus (185) Google Scholar, 42Takagi S. Kasuya Y. Shimizu M. Matsuura T. Tsuboi M. Kawakami A. Fujisawa H. Dev. Biol. 1995; 170: 207-222Crossref PubMed Scopus (175) Google Scholar), which has been demonstrated to be a receptor for various members of the semaphorin family (30Kolodkin A.L. Levengood D.V. Rowe E.G. Tai Y.T. Giger R.J. Ginty D.D. Cell. 1997; 90: 753-762Abstract Full Text Full Text PDF PubMed Scopus (994) Google Scholar, 43Chen H. Chedotal A. He Z. Goodman C.S. Tessier L.M. Neuron. 1997; 19: 547-559Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar). Neuropilin-1 has also been shown to play a role in regulating neurite outgrowth via its interactions with semaphorins (44Kitsukawa T. Shimizu M. Sanbo M. Hirata T. Taniguchi M. Bekku Y. Yagi T. Fujisawa H. Neuron. 1997; 19: 995-1005Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar). The CUB domain of PDGF-C may act as a targeting domain for the full-length protein by binding to cell-surface molecules, potentially semaphorins. PDGF-C is then locally cleaved to release the GFD by as yet unidentified serum factors. The released GFD is then available to bind and activate the PDGF receptors. This scheme provides for the accumulation of PDGF-C at localized sites without the release of the active GFD until such time as local environmental events cause the release of active protein. This model requires that full-length PDGF-C does not bind to the PDGF receptors or, if it does, is not capable of stimulating receptor activation. Results reported by Li et al. (15Li X. Ponten A. Aase K. Karlsson L. Abramsson A. Uutela M. Backstrom G. Hellstrom M. Bostrom H. Li H. Soriano P. Betsholtz C. Heldin C.H. Alitalo K. Ostman A. Eriksson U. Nat. Cell Biol. 2000; 2: 302-309Crossref PubMed Scopus (499) Google Scholar) support this conclusion. In all cases where we have expressed full-length protein, there is always some limited amount of cleaved product generated. As such, it has not been possible to determine whether the full-length protein or the cleaved portion of the molecule is interacting with the PDGF receptors. In addition to expanding the binding model of PDGF/receptor interaction, the structure of PDGF-C creates additional possibilities for in vivo PDGF function. PDGF-A and -B are made by epithelial and endothelial cells in order to recruit mesenchymal cells bearing PDGF receptors during development (38Hellström M. Kalén M. Lindahl P. Abramsson A. Betsholtz C. Development. 1999; 126: 3047-3055Crossref PubMed Google Scholar, 45Ansel J.C. Tiesman J.P. Olerud J.E. Krueger J.G. Krane J.F. Tara D.C. Shipley G.D. Gilbertson D. Usui M.L. Hart C.E. J. Clin. Invest. 1993; 92: 671-678Crossref PubMed Scopus (124) Google Scholar). Human platelets release all three forms of PDGF at sites of vascular damage, recruiting smooth muscle cells and fibroblasts to the wound site. It is intriguing to speculate that mesenchymal cells are not always forced to rely on other cell types for proliferative stimulus but may also synthesize a potent form of PDGF (PDGF-C) sequestered in the tissues until specific proteolytic enzymes coupled to trauma or regenerative processes release the active GFD from the inactive pro-form. The generalized expression of PDGF-C on multiple tissue Northerns (Fig. 2 A) and in cultured fibroblasts (Fig. 2 B) is consistent with this notion. It is possible that PDGF-C is a primary source of PDGF-like wound healing activity in somatic tissues and that the PDGF is synthesized and released by the responding cell types. Inappropriate activation of the pro-form may thus also lead to diseases characterized by proliferation of connective tissues. Clearly the analysis of historical data and activities attributed to the various members of the PDGF ligand family need to be reconsidered in a new light. The identification of a new member adds to the already complex nature of this ligand/receptor family. Further elucidation of PDGF-C activities will begin to clarify its true level of involvement in regulating the multitude of activities previously attributed to the known PDGF family members. We thank B. Miller and B. Gutierrez for generation of adenovirus; J. Lehner, J. Volpone, J. Rosser, and J. Yi for generation of antibodies; C. Ostrander, J. Muilenburg, E. Giste, and H. Pelto for protein expression; M. Stamm and N. Fox for protein analysis; C. Jones for mitogenesis assays; S. Topouzis for migration assays; K. Mink for histologic tissue preparation; and M. Rogers for help in preparing this manuscript.
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