Cell Adhesion to Tropoelastin Is Mediated via the C-terminal GRKRK Motif and Integrin αVβ3
2009; Elsevier BV; Volume: 284; Issue: 42 Linguagem: Inglês
10.1074/jbc.m109.017525
ISSN1083-351X
AutoresDaniel V. Bax, Ursula R. Rodgers, M.M.M. Bilek, Anthony S. Weiss,
Tópico(s)Cell Adhesion Molecules Research
ResumoElastin fibers are predominantly composed of the secreted monomer tropoelastin. This protein assembly confers elasticity to all vertebrate elastic tissues including arteries, lung, skin, vocal folds, and elastic cartilage. In this study we examined the mechanism of cell interactions with recombinant human tropoelastin. Cell adhesion to human tropoelastin was divalent cation-dependent, and the inhibitory anti-integrin αVβ3 antibody LM609 inhibited cell spreading on tropoelastin, identifying integrin αVβ3 as the major fibroblast cell surface receptor for human tropoelastin. Cell adhesion was unaffected by lactose and heparin sulfate, indicating that the elastin-binding protein and cell surface glycosaminoglycans are not involved. The C-terminal GRKRK motif of tropoelastin can bind to cells in a divalent cation-dependent manner, identifying this as an integrin binding motif required for cell adhesion. Elastin fibers are predominantly composed of the secreted monomer tropoelastin. This protein assembly confers elasticity to all vertebrate elastic tissues including arteries, lung, skin, vocal folds, and elastic cartilage. In this study we examined the mechanism of cell interactions with recombinant human tropoelastin. Cell adhesion to human tropoelastin was divalent cation-dependent, and the inhibitory anti-integrin αVβ3 antibody LM609 inhibited cell spreading on tropoelastin, identifying integrin αVβ3 as the major fibroblast cell surface receptor for human tropoelastin. Cell adhesion was unaffected by lactose and heparin sulfate, indicating that the elastin-binding protein and cell surface glycosaminoglycans are not involved. The C-terminal GRKRK motif of tropoelastin can bind to cells in a divalent cation-dependent manner, identifying this as an integrin binding motif required for cell adhesion. Cellular interactions with extracellular matrix proteins are vital for cell survival and tissue maintenance. The attachment of cells to their extracellular matrix (ECM) 3The abbreviations used are: ECMextracellular matrixEBPelastin-binding proteinHDFhuman dermal fibroblastsBSAbovine serum albuminPBSphosphate-buffered salineMES4-morpholineethanesulfonic acid. 3The abbreviations used are: ECMextracellular matrixEBPelastin-binding proteinHDFhuman dermal fibroblastsBSAbovine serum albuminPBSphosphate-buffered salineMES4-morpholineethanesulfonic acid. is often mediated by cell surface integrins. As such, integrins are involved in many biological functions such cell migration and proliferation, tissue organization, wound repair, development, and host immune responses. In addition to roles under normal physiological conditions, integrins are involved in the pathogenesis of diseases such as arthritis, cardiovascular disease, inflammation, microbial and parasitic infection, and cancer. Integrins are a family of heterodimeric transmembrane receptors containing one α subunit and one β subunit (1Green L. Humphries M. Adv. Mol. Cell Biol. 1999; 28: 3-26Crossref Scopus (4) Google Scholar). Often integrins bind to ECM proteins via short RGD motifs within the matrix protein (2Newham P. Humphries M.J. Mol. Med. Today. 1996; 2: 304-313Abstract Full Text PDF PubMed Scopus (83) Google Scholar). In addition to an RGD motif, fibronectin also contains an upstream PHSRN synergy sequence, which is required for full integrin binding activity (3Aota S. Nomizu M. Yamada K.M. J Biol. Chem. 1994; 269: 24756-24761Abstract Full Text PDF PubMed Google Scholar).Elastin confers elasticity on all vertebrate elastic tissues including arteries, lung, skin, vocal fold, and elastic cartilage (4Mithieux S.M. Weiss A.S. Adv. Protein Chem. 2005; 70: 437-461Crossref PubMed Scopus (382) Google Scholar). Elastin comprises ∼90% of the elastic fiber and is intermingled with fibrillin-rich microfibrils (5Ramirez F. Matrix Biol. 2000; 19: 455-456Crossref PubMed Scopus (22) Google Scholar). There is a single human tropoelastin gene in which alternative splicing can result in the loss of domains 22, 23, 24, 26A, 30, 32, and 33 (4Mithieux S.M. Weiss A.S. Adv. Protein Chem. 2005; 70: 437-461Crossref PubMed Scopus (382) Google Scholar). Elastin is made from the secreted monomer tropoelastin, which is a 60–72-kDa protein containing repeating hydrophobic and cross-linking domains. Hydrophobic domains are rich in GVGVP, GGVP, and GVGVAP repeats, which can associate by coacervation (6Vrhovski B. Weiss A.S. Eur. J. Biochem. 1998; 258: 1-18Crossref PubMed Scopus (387) Google Scholar). This association results in structural changes and increased α-helical content (7Muiznieks L.D. Jensen S.A. Weiss A.S. Arch. Biochem. Biophys. 2003; 410: 317-323Crossref PubMed Scopus (51) Google Scholar). The cross-linking domains are lysine-rich. Occasionally these residues are modified to allysine through the activity of members of the family of lysyl oxidase (LOX) and four LOX-like enzymes. During coacervation the allysine and other allysines or specific lysine side chains come into close proximity, allowing nonenzymatic condensation reactions to occur, forming desmosine or isodesmosine cross-links (4Mithieux S.M. Weiss A.S. Adv. Protein Chem. 2005; 70: 437-461Crossref PubMed Scopus (382) Google Scholar). This process gives a highly stable cross-linked elastin matrix which has a half-life of ∼70 years. Members of the serine, aspartate, cysteine, and matrix metalloproteinase families of proteases can degrade elastin (8Antonicelli F. Bellon G. Debelle L. Hornebeck W. Curr. Top. Dev. Biol. 2007; 79: 99-155Crossref PubMed Scopus (167) Google Scholar). The resulting elastin peptides have effects on ECM synthesis and cell attachment, migration, and proliferation (9Duca L. Floquet N. Alix A.J. Haye B. Debelle L. Crit. Rev. Oncol. Hematol. 2004; 49: 235-244Crossref PubMed Scopus (152) Google Scholar).The consequences of mutated or hemizygous elastin in the hereditary, connective tissue disorders cutis laxa, supravalvular aortic stenosis, and Williams-Beuren syndrome highlight the elastins essential role in elastic tissue function (10Kielty C.M. Expert Rev. Mol. Med. 2006; 8: 1-23Crossref PubMed Scopus (209) Google Scholar). Elastin is the major protein in large elastic blood vessels such as the aorta, where it is likely to inhibit the proliferation of vascular smooth muscle cells and so preventing vessel occlusion (11Ito S. Ishimaru S. Wilson S.E. Angiology. 1998; 49: 289-297Crossref PubMed Scopus (47) Google Scholar), which is a major cause of death in developed countries. Previous studies have shown that human and bovine tropoelastin can bind directly to a variety of cell types directly through a number of cell surface receptors (12Mecham R.P. Hinek A. Entwistle R. Wrenn D.S. Griffin G.L. Senior R.M. Biochemistry. 1989; 28: 3716-3722Crossref PubMed Scopus (160) Google Scholar, 13Rodgers U.R. Weiss A.S. Biochimie. 2004; 86: 173-178Crossref PubMed Scopus (92) Google Scholar, 14Broekelmann T.J. Kozel B.A. Ishibashi H. Werneck C.C. Keeley F.W. Zhang L. Mecham R.P. J. Biol. Chem. 2005; 280: 40939-40947Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) and also bind indirectly to cells through ECM proteins such as fibulin-5 (15Yanagisawa H. Davis E.C. Starcher B.C. Ouchi T. Yanagisawa M. Richardson J.A. Olson E.N. Nature. 2002; 415: 168-171Crossref PubMed Scopus (492) Google Scholar, 16Nakamura T. Lozano P.R. Ikeda Y. Iwanaga Y. Hinek A. Minamisawa S. Cheng C.F. Kobuke K. Dalton N. Takada Y. Tashiro K. Ross Jr., J. Honjo T. Chien K.R. Nature. 2002; 415: 171-175Crossref PubMed Scopus (521) Google Scholar).A mechanism by which elastin binds to cells is via the 67-kDa elastin-binding protein (EBP), which is a peripheral membrane splice variant of β-galactosidase. The EBP forms a complex with the integral membrane proteins carboxypeptidase A and sialidase, forming a transmembrane elastin receptor (12Mecham R.P. Hinek A. Entwistle R. Wrenn D.S. Griffin G.L. Senior R.M. Biochemistry. 1989; 28: 3716-3722Crossref PubMed Scopus (160) Google Scholar). The binding site for the EBP has been mapped to the consensus sequence XGXXPG within elastin and in particular to VGVAPG within exon 24 (17Senior R.M. Griffin G.L. Mecham R.P. Wrenn D.S. Prasad K.U. Urry D.W. J. Cell Biol. 1984; 99: 870-874Crossref PubMed Scopus (314) Google Scholar). The binding of elastin to the EBP results in cell morphological changes (18Karnik S.K. Wythe J.D. Sorensen L. Brooke B.S. Urness L.D. Li D.Y. Matrix Biol. 2003; 22: 409-425Crossref PubMed Scopus (38) Google Scholar, 19Karnik S.K. Brooke B.S. Bayes-Genis A. Sorensen L. Wythe J.D. Schwartz R.S. Keating M.T. Li D.Y. Development. 2003; 130: 411-423Crossref PubMed Scopus (359) Google Scholar), chemotaxis (20Senior R.M. Griffin G.L. Mecham R.P. J. Clin. Invest. 1982; 70: 614-618Crossref PubMed Scopus (119) Google Scholar), decreased cell proliferation (21Jung S. Rutka J.T. Hinek A. J. Neuropathol. Exp. Neurol. 1998; 57: 439-448Crossref PubMed Scopus (66) Google Scholar), and angiogenesis (22Robinet A. Fahem A. Cauchard J.H. Huet E. Vincent L. Lorimier S. Antonicelli F. Soria C. Crepin M. Hornebeck W. Bellon G. J. Cell Sci. 2005; 118: 343-356Crossref PubMed Scopus (174) Google Scholar). Knockouts of β-galactosidase, which remove the EBP, display correctly deposited elastin (27Hahn C.N. del Pilar Martin M. Schröder M. Vanier M.T. Hara Y. Suzuki K. Suzuki K. d'Azzo A. Hum. Mol. Genet. 1997; 6: 205-211Crossref PubMed Scopus (131) Google Scholar). Additionally tropoelastin actively promotes cell adhesion, whereas VGVAPG does not. These observations imply that receptors other than EBP can interact with elastin.Other studies have proposed a second mechanism involving the necessity of cell surface heparan and chondroitin sulfate-containing glycosaminoglycans for bovine chondrocyte interaction with bovine tropoelastin (14Broekelmann T.J. Kozel B.A. Ishibashi H. Werneck C.C. Keeley F.W. Zhang L. Mecham R.P. J. Biol. Chem. 2005; 280: 40939-40947Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Peptide binding analysis implicated the last 17 amino acids at the C terminus of bovine tropoelastin in this cell adhesive activity, with higher binding requiring the C-terminal 25 amino acids. This region is of interest, as in humans a mutation of Gly-773 to Asp in exon 33 results in blocked elastin network assembly and modulates cell binding to a peptide corresponding to exons 33 and 36 of human tropoelastin (28Kelleher C.M. Silverman E.K. Broekelmann T. Litonjua A.A. Hernandez M. Sylvia J.S. Stoler J. Reilly J.J. Chapman H.A. Speizer F.E. Weiss S.T. Mecham R.P. Raby B.A. Am. J. Respir. Cell Mol. Biol. 2005; 33: 355-362Crossref PubMed Scopus (65) Google Scholar). Indeed Broekelmann et al. (14Broekelmann T.J. Kozel B.A. Ishibashi H. Werneck C.C. Keeley F.W. Zhang L. Mecham R.P. J. Biol. Chem. 2005; 280: 40939-40947Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) have shown that synthetic peptides containing the C-terminal 29 amino acids of bovine tropoelastin possess cell adhesive activity; however, when the G773D mutation was incorporated into the peptide, it prevented cell adhesion to that peptide.Although tropoelastin does not contain an RGD motif, other data identified a third mechanism involving direct interaction between integrin αvβ3 and human tropoelastin (13Rodgers U.R. Weiss A.S. Biochimie. 2004; 86: 173-178Crossref PubMed Scopus (92) Google Scholar, 29Rodgers U.R. Weiss A.S. Pathol. Biol. 2005; 53: 390-398Crossref PubMed Scopus (96) Google Scholar). This interaction was also localized to the C-terminal domains of tropoelastin.More recent data has shown that human umbilical vein endothelial cells can adhere to recombinant fragments of human tropoelastin (30Kielty C.M. Stephan S. Sherratt M.J. Williamson M. Shuttleworth C.A. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2007; 362: 1293-1312Crossref PubMed Scopus (55) Google Scholar, 31Williamson M.R. Shuttleworth C.A. Canfield A.E Black R.A. Kielty C.M. Biomaterials. 2007; 28: 5307-5318Crossref PubMed Scopus (58) Google Scholar). In contrast to other data, regions encoded by the N-terminal exons (1–18), the central exons (18–27), and the C-terminal exons (18–36) all supported human umbilical vein endothelial cell attachment.Although a previous study has shown a direct interaction between purified integrin αvβ3 and human tropoelastin (13Rodgers U.R. Weiss A.S. Biochimie. 2004; 86: 173-178Crossref PubMed Scopus (92) Google Scholar), the integrin dependence of cell adhesion to tropoelastin had not been demonstrated. Here we demonstrate that human dermal fibroblasts adhere to recombinant human tropoelastin and that inhibitors of the elastin-binding protein and cell surface heparan sulfate have no effect on cell adhesion. In contrast, cell adhesion was dependent upon the presence of divalent cations, indicating integrin dependence. Inhibitory monoclonal antibodies identified integrin αVβ3 as the major receptor necessary for fibroblast adherence and spreading onto human tropoelastin. The binding motif for integrin-mediated cell adhesion is unknown; therefore, through the use of synthetic peptides, the adhesive activity was localized to the extreme C-terminal GRKRK motif of tropoelastin. This data present a novel mechanism for cell adhesion to human tropoelastin and identify a novel integrin binding motif within tropoelastin.DISCUSSIONCellular interactions with extracellular matrix proteins give vital cues to the cell for cell survival and tissue maintenance. Although integrin αVβ3 has been shown to interact directly with tropoelastin by solid phase analysis, the utilization of this integrin by cells adhering to human tropoelastin has not been studied. As tropoelastin is the major constituent of elastic tissue and in vivo elastic fibers are associated with cells (30Kielty C.M. Stephan S. Sherratt M.J. Williamson M. Shuttleworth C.A. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2007; 362: 1293-1312Crossref PubMed Scopus (55) Google Scholar, 34Davis E.C. Cell Tissue Res. 1993; 272: 211-219Crossref PubMed Scopus (33) Google Scholar), we have investigated the cell binding properties of human tropoelastin.In these studies human skin fibroblasts were shown to be capable of attaching and spreading on recombinant human tropoelastin. The level of cell adhesion to human tropoelastin (68%) is consistent with the level of cell attachment observed on other elastic fiber proteins such as fibulin-5 (45% adhesion (35Lomas A.C. Mellody K.T. Freeman L.J. Bax D.V. Shuttleworth C.A. Kielty C.M. Biochem. J. 2007; 405: 417-428Crossref PubMed Scopus (67) Google Scholar)) and fibrillin-rich microfibrils (35% adhesion (36Bax D.V. Bernard S.E. Lomas A. Morgan A. Humphries J. Shuttleworth C.A. Humphries M.J. Kielty C.M. J. Biol. Chem. 2003; 278: 34605-34616Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar)). Therefore, human tropoelastin is a truly cell adhesive protein.Soluble bovine elastin was not capable of supporting adhesion or spreading. Others have shown that the C terminus of tropoelastin may be lost in mature elastic fibers, which would account for the lack of cell interaction to soluble bovine elastin (37Broekelmann T.J. Ciliberto C.H. Shifren A. Mecham R.P. Matrix Biol. 2008; 27: 631-639Crossref PubMed Scopus (18) Google Scholar).A prior study using solid phase binding assays showed a direct interaction between purified integrin αvβ3 and the C-terminal exons of human tropoelastin (13Rodgers U.R. Weiss A.S. Biochimie. 2004; 86: 173-178Crossref PubMed Scopus (92) Google Scholar); however, the importance of this integrin in cell-human tropoelastin adhesion has not been studied. The attachment of cells to the extracellular matrix is often mediated by cell surface integrins in a cation-dependent manner (38Mould A.P. J. Cell Sci. 1996; 109: 2613-2618Crossref PubMed Google Scholar). We found that the presence of EDTA blocked fibroblast adhesion to human tropoelastin. In the absence of cations Mn2+ could restore cell binding, whereas Mg2+ and Ca2+ had lower efficacy. This pattern of cation dependence is indicative of integrin-mediated adhesion (38Mould A.P. J. Cell Sci. 1996; 109: 2613-2618Crossref PubMed Google Scholar). Moreover, antibody LM609 and 17E6 inhibition of cell adherence identified integrin αvβ3 as the predominant receptor for human tropoelastin on human fibroblasts. These results show that integrin αvβ3 on cells can bind to tropoelastin. In contrast, others have shown no inhibition of chondrocyte adherence to tropoelastin using either EDTA or LM609. This may be because of cell lineage: i.e. bovine chondrocytes and Chinese hamster ovary cells compared with fibroblasts utilized.The EBP, a previously identified tropoelastin receptor, binds to the sequence VGVAPG in exon 24 of tropoelastin and is inhibited by lactose (12Mecham R.P. Hinek A. Entwistle R. Wrenn D.S. Griffin G.L. Senior R.M. Biochemistry. 1989; 28: 3716-3722Crossref PubMed Scopus (160) Google Scholar, 17Senior R.M. Griffin G.L. Mecham R.P. Wrenn D.S. Prasad K.U. Urry D.W. J. Cell Biol. 1984; 99: 870-874Crossref PubMed Scopus (314) Google Scholar). In this study lactose did not inhibit cell binding, discounting the elastin-binding protein as the elastin receptor responsible for fibroblast adherence to human tropoelastin. This is consistent with reports of a lack of chondrocyte adherence observed on a fragment of tropoelastin encoded by exons 15–29, which contains VGVAPG (14Broekelmann T.J. Kozel B.A. Ishibashi H. Werneck C.C. Keeley F.W. Zhang L. Mecham R.P. J. Biol. Chem. 2005; 280: 40939-40947Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Furthermore, β-galactosidase (which is alternately spliced to give a component of the EBP) knockout in mice, which would knock out the EBP, does not affect elastin deposition, indicating that another receptor is capable of compensating for the EBP (27Hahn C.N. del Pilar Martin M. Schröder M. Vanier M.T. Hara Y. Suzuki K. Suzuki K. d'Azzo A. Hum. Mol. Genet. 1997; 6: 205-211Crossref PubMed Scopus (131) Google Scholar). In further support, peptides that contain VGVAPG are poorly adhesive but have potent chemotactic and signaling properties. As the EBP binding site possesses signaling rather than adhesive properties, these data indicate that adhesion and signaling activities of tropoelastin could be mediated by distinct receptors. Consistent with previous data (14Broekelmann T.J. Kozel B.A. Ishibashi H. Werneck C.C. Keeley F.W. Zhang L. Mecham R.P. J. Biol. Chem. 2005; 280: 40939-40947Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), we observed no actin stress fiber assembly in cells adherent to human tropoelastin compared with cells adherent to plasma fibronectin (data not shown). Therefore, integrin engagement is necessary for cell adhesion to human tropoelastin but may not result in signaling into the cell necessary for cytoskeletal assembly.Cell surface glycosaminoglycans and, in particular, heparan sulfate have also been shown to mediate bovine chondrocyte-bovine tropoelastin interactions. In this study when heparan sulfate was added to fibroblasts no inhibition of cell adhesion was observed. Furthermore, EDTA lifting of cells had no effect on the mechanism of cell-tropoelastin interaction compared with trypsin lifting of cells (supplemental figures). This excludes the possibility that during lifting trypsin might have removed a heparan sulfate-mediated tropoelastin receptor from the cell surface required for heparan sulfate-mediated cell adhesion to tropoelastin. Interestingly, although heparan sulfate-mediated cell adhesion to bovine tropoelastin was assigned to the C terminus of tropoelastin, this was not contained in exon 36, as a peptide corresponding to the last 14 amino acids of bovine tropoelastin was not capable of supporting cell adhesion. The minimum sequence for bovine tropoelastin adhesion required the last 3 amino acids of exon 35 in conjunction with exon 36 (i.e. C-terminal 17 amino acids), with higher binding requiring the last 11 amino acids of exon 35 in conjunction with exon 36 (i.e. C-terminal 25 amino acids) (14Broekelmann T.J. Kozel B.A. Ishibashi H. Werneck C.C. Keeley F.W. Zhang L. Mecham R.P. J. Biol. Chem. 2005; 280: 40939-40947Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Human tropoelastin does not contain the exons 34 and 35 that are involved in heparan sulfate-mediated adhesion; however, human exon 33 has been proposed to substitute for bovine exon 35 (28Kelleher C.M. Silverman E.K. Broekelmann T. Litonjua A.A. Hernandez M. Sylvia J.S. Stoler J. Reilly J.J. Chapman H.A. Speizer F.E. Weiss S.T. Mecham R.P. Raby B.A. Am. J. Respir. Cell Mol. Biol. 2005; 33: 355-362Crossref PubMed Scopus (65) Google Scholar). This implies that the different mechanisms of human tropoelastin or bovine tropoelastin adhesion to cells may simply reflect differing cell lines utilized.It has been postulated cell surface heparan sulfate-mediated cell adhesion may be through cell surface syndecans (14Broekelmann T.J. Kozel B.A. Ishibashi H. Werneck C.C. Keeley F.W. Zhang L. Mecham R.P. J. Biol. Chem. 2005; 280: 40939-40947Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Syndecans have been shown to cooperatively signal with integrins to assemble the cellular cytoskeleton (39Morgan M.R. Humphries M.J. Bass M.D. Nat. Rev. Mol. Cell Biol. 2007; 8: 957-969Crossref PubMed Scopus (442) Google Scholar). Interestingly, chondrocytes, which adhere to tropoelastin through cell surface glycosaminoglycans, but not integrins, have a predominance of filopodial extensions which are similar to those seen for syndecan-2 or -3 overexpressing cells. As we observed no heparan sulfate dependence on cell adhesion and attachment and also no actin cytoskeletal assembly, it may be possible that in our system we did not recruit syndecans. It is possible that individual sites on tropoelastin are capable of mediating cell-tropoelastin interactions by binding to either integrin αvβ3 or cell surface glycosaminoglycans, and usage of both sites may be required for full cytoskeletal assembly.Others have previously reported cell adhesion to elastin through the bridging protein fibulin-5 (15Yanagisawa H. Davis E.C. Starcher B.C. Ouchi T. Yanagisawa M. Richardson J.A. Olson E.N. Nature. 2002; 415: 168-171Crossref PubMed Scopus (492) Google Scholar, 16Nakamura T. Lozano P.R. Ikeda Y. Iwanaga Y. Hinek A. Minamisawa S. Cheng C.F. Kobuke K. Dalton N. Takada Y. Tashiro K. Ross Jr., J. Honjo T. Chien K.R. Nature. 2002; 415: 171-175Crossref PubMed Scopus (521) Google Scholar). In our adhesion assays the cells are incubated with the tropoelastin surface for 40 min, reducing the possibility of cell-derived proteins functioning as a bridging molecule between tropoelastin and cellular receptors. Furthermore, previous data (13Rodgers U.R. Weiss A.S. Biochimie. 2004; 86: 173-178Crossref PubMed Scopus (92) Google Scholar) have shown a direct interaction between integrin αVβ3 and human tropoelastin. This was performed in the absence of cells on recombinantly expressed tropoelastin. Therefore, there were no other proteins in this assay system that could reasonably bridge between tropoelastin and integrin αVβ3. Interestingly the whole of the tropoelastin molecule has been shown to be necessary for fibulin-5-tropoelastin association (40Wachi H. Nonaka R. Sato F. Shibata-Sato K. Ishida M. Iketani S. Maeda I. Okamoto K. Urban Z. Onoue S. Seyama Y. J. Biochem. 2008; 143: 633-639Crossref PubMed Scopus (28) Google Scholar), and so fibulin-5 should not associate with our cell adhesive peptide 36 and GRKRK. Therefore, it is unlikely that fibulin-5 will bind to this small section of tropoelastin, making it unlikely that fibulin-5 is acting as a bridging molecule for cell adhesion. In addition, potential bridging proteins associated with elastic fibers, namely fibrillin-1 (41Rock M.J. Cain S.A. Freeman L.J. Morgan A. Mellody K. Marson A. Shuttleworth C.A. Weiss A.S. Kielty C.M. J. Biol. Chem. 2004; 279: 23748-23758Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), fibulin-5 (40Wachi H. Nonaka R. Sato F. Shibata-Sato K. Ishida M. Iketani S. Maeda I. Okamoto K. Urban Z. Onoue S. Seyama Y. J. Biochem. 2008; 143: 633-639Crossref PubMed Scopus (28) Google Scholar), and MAGP-2 (42Lemaire R. Bayle J. Mecham R.P. Lafyatis R. J. Biol. Chem. 2007; 282: 800-808Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), bind to cells via RGD motifs (36Bax D.V. Bernard S.E. Lomas A. Morgan A. Humphries J. Shuttleworth C.A. Humphries M.J. Kielty C.M. J. Biol. Chem. 2003; 278: 34605-34616Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 35Lomas A.C. Mellody K.T. Freeman L.J. Bax D.V. Shuttleworth C.A. Kielty C.M. Biochem. J. 2007; 405: 417-428Crossref PubMed Scopus (67) Google Scholar, 43Gibson M.A. Leavesley D.I. Ashman L.K. J. Biol. Chem. 1999; 274: 13060-13065Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). As we observed no inhibition of cell binding to human tropoelastin by RGD peptides, it is unlikely that these proteins act as bridging proteins between cells and human tropoelastin. Additionally, fibrillin-1 possesses dependence upon different integrin receptors to mediate cell binding than a predominance of integrin αVβ3 (36Bax D.V. Bernard S.E. Lomas A. Morgan A. Humphries J. Shuttleworth C.A. Humphries M.J. Kielty C.M. J. Biol. Chem. 2003; 278: 34605-34616Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). The protein synthesis blocker, actinomycin D, had no inhibitory effect on cell adhesion to tropoelastin (supplemental figures), giving additional evidence that cells can interact directly with tropoelastin rather than through a cell-derived bridging protein. Therefore, we have shown a direct binding interaction between integrin αVβ3 and human tropoelastin (13Rodgers U.R. Weiss A.S. Biochimie. 2004; 86: 173-178Crossref PubMed Scopus (92) Google Scholar), which we have now shown results in cell adhesion to tropoelastin.In this study we have used synthetic peptides to map cell binding to the C-terminal 5 amino acids of exon 36. Furthermore, we have shown integrin-mediated cell adhesion to this motif, identifying GRKRK as a novel integrin binding motif. Indeed there is no classical RGD integrin recognition site in tropoelastin; however, not all integrin-receptor ligation is via RGD motifs. For example, Cyr61, fibrinogen, tumstatin, and the collagen α3(IV) NC1 domain do not bind via RGD motifs. Furthermore, integrin α1β1 recognizes the motif RKKH, which is very similar to the RKRK motif that we have identified as being necessary for cell interaction (44Nymalm Y. Puranen J.S. Nyholm T.K. Käpylä J. Kidron H. Pentikäinen O.T. Airenne T.T. Heino J. Slotte J.P. Johnson M.S. Salminen T.A. J. Biol. Chem. 2004; 279: 7962-7970Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Interestingly the RKRK motif is highly conserved between divergent species, indicating that it possesses a vital function.Although we have identified integrin αVβ3 as a cellular receptor for human tropoelastin, the in vivo importance of this interaction is unknown. Integrins have been implicated in many biological functions such as cell migration and proliferation, tissue organization, and roles in the pathogenesis of disease.Integrin αVβ3 could be involved in tropoelastin deposition onto maturing elastic fibers. This is consistent with the identified interaction with tropoelastin, i.e. the non-deposited, progenitor of elastin. Integrins are implicated in ECM assembly, including αVβ3 participation in fibronectin network deposition (45Leiss M. Beckmann K. Girós A. Costell M. Fässler R. Curr. Opin. Cell Biol. 2008; 20: 502-507Crossref PubMed Scopus (210) Google Scholar). However, the αVβ3 knock-out mouse has apparently functional elastic lamellae in blood vessels (46Choi E.T. Khan M.F. Leidenfrost J.E. Collins E.T. Boc K.P. Villa B.R. Novack D.V. Parks W.C. Abendschein D.R. Circulation. 2004; 109: 1564-1569Crossref PubMed Scopus (52) Google Scholar). Therefore, this receptor may not be solely responsible for elastic fiber deposition and assembly and may point to involvement of multiple tropoelastin receptors in elastogenesis.In this study we have shown that cell adhesion is predominantly mediated through integrin αVβ3 and the C terminus of tropoelastin, which could implicate this integrin in elastin fiber assembly. Mutation G773D, which is present in exon 33, N-terminal to exon 36 in human tropoelastin, blocks cell binding but also prevents cellular deposition of elastin to form fiber-like structures, implicating cell adhesion in the assembly of tropoelastin fibers (28Kelleher C.M. Silverman E.K. Broekelmann T. Litonjua A.A. Hernandez M. Sylvia J.S. Stoler J. Reilly J.J. Chapman H.A. Speizer F.E. Weiss S.T. Mecham R.P. Raby B.A. Am. J. Respir. Cell Mol. Biol. 2005; 33: 355-362Crossref PubMed Scopus (65) Google Scholar). Additionally, cutis laxa (the hyper-extensible elastic tissue disorder) mutations tend to cluster in exons 30, 32, and 33 and generally result in disruption or removal of exon 36 (24Zhang M.C. He L. Giro M. Yong S.L. Tiller G.E. Davidson J.M. J. Biol. Chem. 1999; 274: 981-986Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 25Tassabehji M. Metcalfe K. Hurst J. Ashcroft G.S. Kielty C. Wilmot C. Donnai D. Read A.P. Jones C.J. Hum. Mol. Genet. 1998; 7: 1021-1028Crossref PubMed Scopus (141) Google Scholar, 26Rodriguez-Revenga L. Iranzo P. Badenas C. Puig S. Carrió A. Milà M. Arch. Dermatol. 2004; 140: 1135-1139Crossref PubMed Scopus (63) Google Scholar). These examples suggest that the C terminus is important in human elastic fiber assembly.The migration of dermal fibroblasts in a wound environment model relies on integrin interactions that include αVβ3 (23Greiling D. Clark R.A. J. Cell Sci. 1997
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