α7 Integrin Mediates Cell Adhesion and Migration on Specific Laminin Isoforms

Abstract

The laminin-binding α7β1 integrin receptor is expressed at high levels by skeletal and cardiac muscles and by certain melanocytic cells. We have assessed the potential role of the α7A/B integrin isoforms in mediating cell adhesion and motility and determined the laminin isoform specificity of this integrin. When MCF-7 breast carcinoma cells, normally nonadherent to laminin 1, were stably transfected with cDNA for mouse α7, they adhered with high efficiency and migrated on laminin 1 substrates. Function-perturbing monoclonal antibodies generated to mouse α7 subunit blocked both adhesion and migration of α7 transfectants on laminin 1 substrates. Additional studies with MCF-7 transfectants revealed that α7β1 binds well to laminin 1 and to a mixture of laminin 2 and 4 but not to laminin 5. Importantly, α7β1 was capable of promoting motility on both laminin 1 and laminin 2/4 substrates. However, MCF-7 cells transfected with cDNA for either α7A or α7B showed no significant differences in cell adhesion or motility on laminin 1 substrates. Although the role for the alternatively spliced cytoplasmic variants of α7 remains unknown, the results establish that α7β1 mediates cell adhesive activities on a restricted number of laminin isoforms. The laminin-binding α7β1 integrin receptor is expressed at high levels by skeletal and cardiac muscles and by certain melanocytic cells. We have assessed the potential role of the α7A/B integrin isoforms in mediating cell adhesion and motility and determined the laminin isoform specificity of this integrin. When MCF-7 breast carcinoma cells, normally nonadherent to laminin 1, were stably transfected with cDNA for mouse α7, they adhered with high efficiency and migrated on laminin 1 substrates. Function-perturbing monoclonal antibodies generated to mouse α7 subunit blocked both adhesion and migration of α7 transfectants on laminin 1 substrates. Additional studies with MCF-7 transfectants revealed that α7β1 binds well to laminin 1 and to a mixture of laminin 2 and 4 but not to laminin 5. Importantly, α7β1 was capable of promoting motility on both laminin 1 and laminin 2/4 substrates. However, MCF-7 cells transfected with cDNA for either α7A or α7B showed no significant differences in cell adhesion or motility on laminin 1 substrates. Although the role for the alternatively spliced cytoplasmic variants of α7 remains unknown, the results establish that α7β1 mediates cell adhesive activities on a restricted number of laminin isoforms. INTRODUCTIONLaminins are adhesive glycoproteins found in basement membranes that promote diverse cellular responses. Cell adherence to laminin matrices plays an important role in maintaining normal tissue organization and in tissue renewal and repair. The interaction of cells with extracellular matrix macromolecules like laminins is mediated primarily by heterodimeric receptors from the integrin superfamily (reviewed in 1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8965) Google Scholar). Integrins provide linkage between the component elements of the extracellular matrix and the structural constituents inside the cell. Besides serving as adhesion receptors, integrins can transmit signals from the extracellular matrix to the cell interior that can activate several pathways, ultimately influencing an array of cellular properties including proliferation, differentiation, survival, and apoptosis (2Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 594-599Crossref Scopus (1460) Google Scholar).Laminin 1 was the first fully characterized isoform and is composed of α1, β1, and γ1 chains. This prototypic laminin is a large molecular weight trimer with multiple domains that are involved in cell adhesion and interactions with other basement membrane components such as nidogen, type IV collagen, and heparan sulfate proteoglycan (3Timpl R. Brown J.C. Matrix Biol. 1994; 14: 275-281Crossref PubMed Scopus (408) Google Scholar). Several other laminin isoforms have been identified and include laminin 2 (α2β1γ1, merosin), laminin 3 (α1β2γ1, S-laminin), laminin 4 (α2β2γ1, S-merosin), laminin 5 (α3β3γ2, kalinin/nicein/epiligrin), and less well characterized laminins 6-10 (reviewed in 3Timpl R. Brown J.C. Matrix Biol. 1994; 14: 275-281Crossref PubMed Scopus (408) Google Scholar, 4Delwel G.O. Sonnenberg A. Horton M.A. Adhesion Receptors as Therapeutic Targets. CRC Press, Boca Raton, Fla1995Google Scholar, 5Ziober B.L. Lin C.-S. Kramer R.H. Semin. Cancer Biol. 1996; 7: 119-128Crossref PubMed Scopus (54) Google Scholar). The different laminin isoforms are tissue-specific and are expressed in a developmentally regulated pattern. Laminin 1 contains multiple sites for integrin-mediated cell attachment, which is effectuated by several β1 integrins (5Ziober B.L. Lin C.-S. Kramer R.H. Semin. Cancer Biol. 1996; 7: 119-128Crossref PubMed Scopus (54) Google Scholar). Of these α3β1, α6β1, and α7β1 bind to the long arm fragment E8 of laminin, produced by elastase digestion. The α1β1 and α2β1 integrins, which both contain the I domain, bind to the cross-region of laminin represented by the short arm fragment.The biological response to laminin appears to be cell type-specific, and this may be due in part to the specific integrin receptors expressed by individual cells. α7β1, originally found in melanoma cells, is a muscle-specific integrin (6Song W.K. Wang W. Foster R.F. Bielser D.A. Kaufman S.J. J. Cell. Biol. 1992; 117: 643-657Crossref PubMed Scopus (210) Google Scholar) that binds to laminin (7Kramer R.H. Vu M.P. Cheng Y.F. Ramos D.M. Timpl R. Waleh N. Cell Regul. 1991; 2: 805-817Crossref PubMed Scopus (120) Google Scholar, 8von der Mark H. Durr J. Sonnenberg A. von der Mark K. Deutzmann R. Goodman S.L. J. Biol. Chem. 1991; 266: 23593-23601Abstract Full Text PDF PubMed Google Scholar, 9Echtermeyer F. Schober S. Poschl E. von der Mark H. von der Mark K. J. Biol. Chem. 1996; 271: 2071-2075Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Although there is only one α7 gene, complicated splicing mechanisms result in several α7 isoforms. Several studies have shown that alternative splicing generates two isoform subsets: (i) X1 and X2 and (ii) A, B, and C, which differ at extracellular and cytoplasmic regions, respectively (10Ziober B.L. Vu M.P. Waleh N. Crawford J. Lin C.-S. Kramer R.H. J. Biol. Chem. 1993; 268: 26773-26783Abstract Full Text PDF PubMed Google Scholar, 11Collo G. Starr L. Quaranta V. J. Biol. Chem. 1993; 268: 19019-19024Abstract Full Text PDF PubMed Google Scholar, 12Song W.K. Wang W. Sato H. Bielser D.A. Kaufman S.J. J. Cell Sci. 1993; 106: 1139-1152Crossref PubMed Google Scholar, 13Martin P.T. Kaufman S.J. Kramer R.H. Sanes J.R. Dev. Biol. 1996; 174: 125-139Crossref PubMed Scopus (152) Google Scholar). Upon terminal differentiation of myoblasts, isoform switching and up-regulation of α7 expression are detected. The extracellular variants have altered sequence in the ligand binding domain and may have different laminin isoform specificity or affinity. The cytoplasmic isoforms, which share the common extracellular and transmembrane domains but differ at the cytoplasmic region, may trigger different biological functions when cells interact with laminin.In the present study, we have stably transfected MCF-7 carcinoma cells, which normally do not adhere to laminin 1, with mouse α7 cDNA. We also generated function-perturbing monoclonal antibodies to mouse α7 integrin to inhibit α7-extracellular matrix interactions. Using these approaches, we demonstrated that both α7A and α7B mediate adhesion and migration of MCF-7 transfectants on laminin 1 and laminin 2/4 substrates; however, α7β1 does not bind to laminin 5.DISCUSSIONIn this study, we have demonstrated that the α7β1 integrin can mediate adhesion and migration on a restricted number of laminin isoforms. We used two approaches successfully: (i) a gain of function approach by transfecting α7 cDNA into MCF-7 cells and (ii) a loss of function approach by using function-perturbing antibodies against α7 on cells expressing this integrin. Evidence obtained from these approaches clearly shows that exogenously expressed α7 confers on MCF-7 cells both the ability to bind and the ability to migrate on laminins. While this work was in preparation, Echtermeyer et al. (9Echtermeyer F. Schober S. Poschl E. von der Mark H. von der Mark K. J. Biol. Chem. 1996; 271: 2071-2075Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) reported that transfection of mouse α7 cDNA into the human 293 embryonic kidney cells conferred a motile phenotype on laminin 1. Interestingly, this enhancement in motility occurred even though the parental cells expressed additional laminin 1-binding receptors.We examined the ligand specificity of the α7 receptor using available laminin isoforms. In MCF-7 α7 transfectants, α7β1 mediated binding to preparations of laminin 1 and to human placental laminins (a mixture of laminin 2 and 4). In contrast, laminin 5 was a poor substrate for α7-expressing cells. Eventually, when pure preparations are available, the functions of α7 should be tested on additional members of the laminin superfamily, especially laminin 2 (merosin), which is present in the basement membrane surrounding adult skeletal myofibers where α7 normally is detected. It is interesting that another laminin-binding integrin, α6β1, sharing high amino acid sequence homology with α7, not only binds to laminin 1 and human placental merosins (laminin 2 and 4) but also binds efficiently to laminin 5 (23Delwel G.O. Hogervorst F. Kuikman I. Paulsson M. Timpl R. Sonnenberg A. J. Biol. Chem. 1993; 268: 25865-25875Abstract Full Text PDF PubMed Google Scholar). In addition, α6 can pair with β1 or β4 subunit, whereas as we show here in the MCF-7 transfectants, α7 pairs only with β1 subunit. Thus, even though α6 and α7 share strong amino acid sequence identity, there must be distinct domains that define both ligand specificity and pairing preferences. In the parental MCF-7 or transfectants, moderate levels of α2, α3, and α6 are expressed, yet these integrins are not capable of mediating adhesion or migration on laminin 1. It appears that this set of integrins, in contrast to α7, is not constitutively active for laminin 1. However, on laminin 2/4, this same set of integrins in the parental MCF-7 cells can mediate both adhesion and motility, yet in the transfectants is not fully competent for these adhesive interactions.It is interesting that α7 transfection appeared to decrease the ability of existing integrins on the MCF-7 cells to interact with laminin 2/4 and with laminin 5. One possibility for the decrease in the activity of integrins α2 and α3 is due to a decrease in their expression. Fluorescence-activated cell sorting analysis confirms that the high expression of α7 caused a modest decrease in the expression level of α3. Another contributing factor may be that the high α7 expression produces a dominant negative effect that down-regulates the activity of the other integrins. Studies have suggested that certain integrins can produce a modulating effect on the function of other integrins. For example, in lymphocytes activation of LFA-1 can down-regulate α4 activity (24van Kooyk Y. van de Wiel-van Kemenade E. Weder P. Huijbens R.J. Figdor C.G. J. Exp. Med. 1993; 177: 185-190Crossref PubMed Scopus (69) Google Scholar). A different phenomenon is observed in the α5-deficient CHO cells where full activity of αvβ3 receptor requires the presence of transfected α5 integrin (25Bauer J.S. Schreiner C.L. Giancotti F.G. Ruoslahti E. Juliano R.L. J. Cell. Biol. 1992; 116: 477-487Crossref PubMed Scopus (116) Google Scholar). Thus an integrin may induce down-regulation of another integrin's function, or alternatively two integrins may cooperate with each other to modulate function.It is conceivable that the cytoplasmic variants of α7 function differently. RNA alternative splicing events in the α cytoplasmic region have been detected in several integrin molecules, including α3, α6, and α7 (10Ziober B.L. Vu M.P. Waleh N. Crawford J. Lin C.-S. Kramer R.H. J. Biol. Chem. 1993; 268: 26773-26783Abstract Full Text PDF PubMed Google Scholar, 11Collo G. Starr L. Quaranta V. J. Biol. Chem. 1993; 268: 19019-19024Abstract Full Text PDF PubMed Google Scholar, 12Song W.K. Wang W. Sato H. Bielser D.A. Kaufman S.J. J. Cell Sci. 1993; 106: 1139-1152Crossref PubMed Google Scholar, 26Hogervorst F. Admiraal L.G. Niessen C. Kuikman I. Janssen H. Daams H. Sonnenberg A. J. Cell. Biol. 1993; 121: 179-191Crossref PubMed Scopus (125) Google Scholar, 27Cooper H.M. Tamura R.N. Quaranta V. J. Cell. Biol. 1991; 115: 843-850Crossref PubMed Scopus (140) Google Scholar, 28Hogervorst F. Kuikman I. van Kessel A.G. Sonnenberg A. Eur. J. Biochem. 1991; 199: 425-433Crossref PubMed Scopus (159) Google Scholar, 29Tamura R.N. Rozzo C. Starr L. Chambers J. Reichardt L.F. Cooper H.M. Quaranta V. J. Cell. Biol. 1990; 111: 1593-1604Crossref PubMed Scopus (264) Google Scholar). However, functional significance of the α chain-cytoplasmic isoforms has not been well established. Results from recent studies searching for functional differences between α6A and α6B are still controversial (23Delwel G.O. Hogervorst F. Kuikman I. Paulsson M. Timpl R. Sonnenberg A. J. Biol. Chem. 1993; 268: 25865-25875Abstract Full Text PDF PubMed Google Scholar, 30Hogervorst F. Kuikman I. Noteboom E. Sonnenberg A. J. Biol. Chem. 1993; 268: 18427-18430Abstract Full Text PDF PubMed Google Scholar, 31Shaw L.M. Lotz M.M. Mercurio A.M. J. Biol. Chem. 1993; 268: 11401-11408Abstract Full Text PDF PubMed Google Scholar, 32Shaw L.M. Mercurio A.M. Mol. Biol. Cell. 1994; 5: 679-690Crossref PubMed Scopus (56) Google Scholar, 33O'Toole T.E. Katagiri Y. Faull R.J. Peter K. Tamura R. Quaranta V. Loftus J.C. Shattil S.J. Ginsberg M.H. J. Cell. Biol. 1994; 124: 1047-1059Crossref PubMed Scopus (578) Google Scholar). Our results indicate that α7A and α7B receptors are equally active in their adhesive or migratory activities. It is possible that α7A/B, a muscle-specific integrin, will show differential activities only in the context of a muscle-specific environment.In summary, the results presented here show that α7β1 can mediate both cell adhesion and migration on laminin 1 and laminin 2/4. Importantly, we have demonstrated that α7 can interact with these different laminin substrates but not with epithelial cell-specific laminin 5. These results strongly support the role of the α7 receptor in mediating interactions with specific laminin isoforms. The tissue-specific expression of different family members of integrins and laminins (e.g. α7β1 and merosins in skeletal muscle (13Martin P.T. Kaufman S.J. Kramer R.H. Sanes J.R. Dev. Biol. 1996; 174: 125-139Crossref PubMed Scopus (152) Google Scholar, 34Engvall E. Earwicker D. Jaaparanta T. Touslahti E. Sanes J.R. Cell Regulation. 1990; 1: 731-740Crossref PubMed Scopus (297) Google Scholar)) suggests that there is a selective interaction that may be important in both embryonic development and tissue homeostasis. INTRODUCTIONLaminins are adhesive glycoproteins found in basement membranes that promote diverse cellular responses. Cell adherence to laminin matrices plays an important role in maintaining normal tissue organization and in tissue renewal and repair. The interaction of cells with extracellular matrix macromolecules like laminins is mediated primarily by heterodimeric receptors from the integrin superfamily (reviewed in 1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8965) Google Scholar). Integrins provide linkage between the component elements of the extracellular matrix and the structural constituents inside the cell. Besides serving as adhesion receptors, integrins can transmit signals from the extracellular matrix to the cell interior that can activate several pathways, ultimately influencing an array of cellular properties including proliferation, differentiation, survival, and apoptosis (2Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 594-599Crossref Scopus (1460) Google Scholar).Laminin 1 was the first fully characterized isoform and is composed of α1, β1, and γ1 chains. This prototypic laminin is a large molecular weight trimer with multiple domains that are involved in cell adhesion and interactions with other basement membrane components such as nidogen, type IV collagen, and heparan sulfate proteoglycan (3Timpl R. Brown J.C. Matrix Biol. 1994; 14: 275-281Crossref PubMed Scopus (408) Google Scholar). Several other laminin isoforms have been identified and include laminin 2 (α2β1γ1, merosin), laminin 3 (α1β2γ1, S-laminin), laminin 4 (α2β2γ1, S-merosin), laminin 5 (α3β3γ2, kalinin/nicein/epiligrin), and less well characterized laminins 6-10 (reviewed in 3Timpl R. Brown J.C. Matrix Biol. 1994; 14: 275-281Crossref PubMed Scopus (408) Google Scholar, 4Delwel G.O. Sonnenberg A. Horton M.A. Adhesion Receptors as Therapeutic Targets. CRC Press, Boca Raton, Fla1995Google Scholar, 5Ziober B.L. Lin C.-S. Kramer R.H. Semin. Cancer Biol. 1996; 7: 119-128Crossref PubMed Scopus (54) Google Scholar). The different laminin isoforms are tissue-specific and are expressed in a developmentally regulated pattern. Laminin 1 contains multiple sites for integrin-mediated cell attachment, which is effectuated by several β1 integrins (5Ziober B.L. Lin C.-S. Kramer R.H. Semin. Cancer Biol. 1996; 7: 119-128Crossref PubMed Scopus (54) Google Scholar). Of these α3β1, α6β1, and α7β1 bind to the long arm fragment E8 of laminin, produced by elastase digestion. The α1β1 and α2β1 integrins, which both contain the I domain, bind to the cross-region of laminin represented by the short arm fragment.The biological response to laminin appears to be cell type-specific, and this may be due in part to the specific integrin receptors expressed by individual cells. α7β1, originally found in melanoma cells, is a muscle-specific integrin (6Song W.K. Wang W. Foster R.F. Bielser D.A. Kaufman S.J. J. Cell. Biol. 1992; 117: 643-657Crossref PubMed Scopus (210) Google Scholar) that binds to laminin (7Kramer R.H. Vu M.P. Cheng Y.F. Ramos D.M. Timpl R. Waleh N. Cell Regul. 1991; 2: 805-817Crossref PubMed Scopus (120) Google Scholar, 8von der Mark H. Durr J. Sonnenberg A. von der Mark K. Deutzmann R. Goodman S.L. J. Biol. Chem. 1991; 266: 23593-23601Abstract Full Text PDF PubMed Google Scholar, 9Echtermeyer F. Schober S. Poschl E. von der Mark H. von der Mark K. J. Biol. Chem. 1996; 271: 2071-2075Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Although there is only one α7 gene, complicated splicing mechanisms result in several α7 isoforms. Several studies have shown that alternative splicing generates two isoform subsets: (i) X1 and X2 and (ii) A, B, and C, which differ at extracellular and cytoplasmic regions, respectively (10Ziober B.L. Vu M.P. Waleh N. Crawford J. Lin C.-S. Kramer R.H. J. Biol. Chem. 1993; 268: 26773-26783Abstract Full Text PDF PubMed Google Scholar, 11Collo G. Starr L. Quaranta V. J. Biol. Chem. 1993; 268: 19019-19024Abstract Full Text PDF PubMed Google Scholar, 12Song W.K. Wang W. Sato H. Bielser D.A. Kaufman S.J. J. Cell Sci. 1993; 106: 1139-1152Crossref PubMed Google Scholar, 13Martin P.T. Kaufman S.J. Kramer R.H. Sanes J.R. Dev. Biol. 1996; 174: 125-139Crossref PubMed Scopus (152) Google Scholar). Upon terminal differentiation of myoblasts, isoform switching and up-regulation of α7 expression are detected. The extracellular variants have altered sequence in the ligand binding domain and may have different laminin isoform specificity or affinity. The cytoplasmic isoforms, which share the common extracellular and transmembrane domains but differ at the cytoplasmic region, may trigger different biological functions when cells interact with laminin.In the present study, we have stably transfected MCF-7 carcinoma cells, which normally do not adhere to laminin 1, with mouse α7 cDNA. We also generated function-perturbing monoclonal antibodies to mouse α7 integrin to inhibit α7-extracellular matrix interactions. Using these approaches, we demonstrated that both α7A and α7B mediate adhesion and migration of MCF-7 transfectants on laminin 1 and laminin 2/4 substrates; however, α7β1 does not bind to laminin 5.