Laminin-3B11, a Novel Vascular-type Laminin Capable of Inducing Prominent Lamellipodial Protrusions in Microvascular Endothelial Cells
2010; Elsevier BV; Volume: 285; Issue: 45 Linguagem: Inglês
10.1074/jbc.m110.146126
ISSN1083-351X
AutoresTaizo Mori, Kota Ono, Yoshinobu Kariya, Takashi Ogawa, Shouichi Higashi, Kaoru Miyazaki,
Tópico(s)Caveolin-1 and cellular processes
ResumoThe basement membrane (BM) proteins laminins, which consist of α, β, and γ chains, support tissue structures and cellular functions. To date only α4 and α5 types of laminins have been identified in the BMs of blood vessels. Our recent study suggested the presence of novel α3B-containing laminins in vascular BMs. Here we identified and characterized the third member of vascular laminins, laminin-3B11 (Lm3B11). RT-PCR analysis showed that microvascular endothelial (MVE) cells and umbilical vein endothelial cells expressed the messages for the α3B, β1, β2, and γ1 chains. In the culture of MVE cells, α3B was associated with β1 and γ1, producing Lm3B11. Recombinant Lm3B11 was overexpressed by introducing the cDNAs of the three chains into HEK-293 cells and purified to homogeneity. Purified Lm3B11 exhibited relatively weak cell adhesion activity through both α3β1 and α6β1 integrins. Most characteristically, Lm3B11 strongly stimulated MVE cells to extend many lamellipodial protrusions. This pseudopodial branching was blocked by an inhibitor for Src or phosphatidylinositol 3-kinase. Consistently, Lm3B11 stimulated the phosphorylation of Src and Akt more strongly than other laminins, suggesting that the integrin-derived signaling is mediated by these factors. The unique activity of Lm3B11 appears to be favorable to the branching of capillaries and venules. The basement membrane (BM) proteins laminins, which consist of α, β, and γ chains, support tissue structures and cellular functions. To date only α4 and α5 types of laminins have been identified in the BMs of blood vessels. Our recent study suggested the presence of novel α3B-containing laminins in vascular BMs. Here we identified and characterized the third member of vascular laminins, laminin-3B11 (Lm3B11). RT-PCR analysis showed that microvascular endothelial (MVE) cells and umbilical vein endothelial cells expressed the messages for the α3B, β1, β2, and γ1 chains. In the culture of MVE cells, α3B was associated with β1 and γ1, producing Lm3B11. Recombinant Lm3B11 was overexpressed by introducing the cDNAs of the three chains into HEK-293 cells and purified to homogeneity. Purified Lm3B11 exhibited relatively weak cell adhesion activity through both α3β1 and α6β1 integrins. Most characteristically, Lm3B11 strongly stimulated MVE cells to extend many lamellipodial protrusions. This pseudopodial branching was blocked by an inhibitor for Src or phosphatidylinositol 3-kinase. Consistently, Lm3B11 stimulated the phosphorylation of Src and Akt more strongly than other laminins, suggesting that the integrin-derived signaling is mediated by these factors. The unique activity of Lm3B11 appears to be favorable to the branching of capillaries and venules. IntroductionLaminin, a family of large heterotrimeric glycoproteins consisting of α, β, and γ chains, is a major cell-adhesive component present in the basement membranes (BMs) 2The abbreviations used are: BMbasement membraneLmlamininMVE cellmicrovascular endothelial cellUVEumbilical vein endothelial cellDMEDulbecco's modified Eagle's medium. of various tissues. It plays critical roles in the maintenance of tissue architectures and the regulation of cellular functions such as cell adhesion, migration, proliferation, apoptosis, and differentiation (1Colognato H. Yurchenco P.D. Dev. Dyn. 2000; 218: 213-234Crossref PubMed Scopus (1035) Google Scholar). Different combinations of five α, three β, and three γ chains give rise to more than 15 laminin isoforms (2Aumailley M. Bruckner-Tuderman L. Carter W.G. Deutzmann R. Edgar D. Ekblom P. Engel J. Engvall E. Hohenester E. Jones J.C. Kleinman H.K. Marinkovich M.P. Martin G.R. Mayer U. Meneguzzi G. Miner J.H. Miyazaki K. Patarroyo M. Paulsson M. Quaranta V. Sanes J.R. Sasaki T. Sekiguchi K. Sorokin L.M. Talts J.F. Tryggvason K. Uitto J. Virtanen I. von der Mark K. Wewer U.M. Yamada Y. Yurchenco P.D. Matrix Biol. 2005; 24: 326-332Crossref PubMed Scopus (660) Google Scholar). These laminins are distributed in a tissue-specific manner and are thought to play differential roles in the individual tissues. Such specific distribution and function of laminins are mainly determined by the α chains. For example, α2 laminins (laminin-211 and -221) are localized in the BMs of muscle and neurons, whereas α4 laminins (laminin-411 and -421) are localized in vascular BMs.There are two splicing variants of laminin α3 chain, the truncated form α3 (or α3A) and the full-sized form α3B (3Ryan M.C. Tizard R. VanDevanter D.R. Carter W.G. J. Biol. Chem. 1994; 269: 22779-22787Abstract Full Text PDF PubMed Google Scholar, 4Galliano M.F. Aberdam D. Aguzzi A. Ortonne J.P. Meneguzzi G. J. Biol. Chem. 1995; 270: 21820-21826Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), gene expression of which is regulated by alternative splicing and different promoters (5Ferrigno O. Virolle T. Galliano M.F. Chauvin N. Ortonne J.P. Meneguzzi G. Aberdam D. J. Biol. Chem. 1997; 272: 20502-20507Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Laminin-3A32 (Lm3A32), which is previously known as laminin-5 and consists of all truncated chains (α3A, β3, and γ2), is a major laminin isoform in the skin, esophagus, lung, breast, and other epithelial tissues. This laminin has extensively been studied because it has unique structure and biological activity as compared with other laminins (6Miyazaki K. Cancer Sci. 2006; 97: 91-98Crossref PubMed Scopus (141) Google Scholar). In the skin, Lm3A32 in the BM associates with integrin α6β4 of basal keratinocytes to form the hemidesmosome structures and contributes to their stable anchorage to the underlying connective tissues (7Kivirikko S. McGrath J.A. Baudoin C. Aberdam D. Ciatti S. Dunnill M.G. McMillan J.R. Eady R.A. Ortonne J.P. Meneguzzi G. Hum. Mol. Genet. 1995; 4: 959-962Crossref PubMed Scopus (153) Google Scholar, 8Baker S.E. DiPasquale A.P. Stock E.L. Quaranta V. Fitchmun M. Jones J.C. Exp. Cell Res. 1996; 228: 262-270Crossref PubMed Scopus (60) Google Scholar). The laminin α3A chain also associates with the full-sized β chains (β1 and β2) and the full-sized, γ1 chain, thus producing laminin-3A11 (Lm3A11) and laminin-3A21 (Lm3A21) (9Marinkovich M.P. Lunstrum G.P. Burgeson R.E. J. Biol. Chem. 1992; 267: 17900-17906Abstract Full Text PDF PubMed Google Scholar, 10Champliaud M.F. Lunstrum G.P. Rousselle P. Nishiyama T. Keene D.R. Burgeson R.E. J. Cell Biol. 1996; 132: 1189-1198Crossref PubMed Scopus (226) Google Scholar, 11Hirosaki T. Tsubota Y. Kariya Y. Moriyama K. Mizushima H. Miyazaki K. J. Biol. Chem. 2002; 277: 49287-49295Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar).Although the α3B chain is known to be more widely expressed than the α3A chain in vivo, α3B-containing laminins have been poorly investigated (12Mizushima H. Miyagi Y. Kikkawa Y. Yamanaka N. Yasumitsu H. Misugi K. Miyazaki K. J. Biochem. 1996; 120: 1196-1202Crossref PubMed Scopus (94) Google Scholar, 13Garbe J.H. Göhring W. Mann K. Timpl R. Sasaki T. Biochem. J. 2002; 362: 213-221Crossref PubMed Scopus (54) Google Scholar, 14Doliana R. Bellina I. Bucciotti F. Mongiat M. Perris R. Colombatti A. FEBS Lett. 1997; 417: 65-70Crossref PubMed Scopus (21) Google Scholar). We previously expressed laminin-3B32 (Lm3B32; α3B/β3/γ2) as a recombinant protein and revealed its biological activity (15Kariya Y. Yasuda C. Nakashima Y. Ishida K. Tsubota Y. Miyazaki K. J. Biol. Chem. 2004; 279: 24774-24784Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). In addition, our recent immunohistochemical study showed that the α3B chain is colocalized with the β3 and γ2 chains in the epithelial BMs of the skin, esophagus, breast, and lung, whereas in the BMs of blood vessels it is colocalized with the β1/2 and γ1 chains, suggesting the presence of novel vascular laminin isoforms, laminin-3B11 (Lm3B11) and laminin-3B21 (Lm3B21) in the vascular BMs (16Kariya Y. Mori T. Yasuda C. Watanabe N. Kaneko Y. Nakashima Y. Ogawa T. Miyazaki K. J. Mol. Histol. 2008; 39: 435-446Crossref PubMed Scopus (17) Google Scholar). However, no previous studies have identified the α3B-containing, full-sized Lm3B11/3B21.The network of blood vessels dynamically changes under various physiological and pathological conditions such as tumor growth and inflammation. The angiogenesis in tumor tissues is essential for tumor growth and metastasis. It is expected that vascular laminins play some important roles in the formation of blood vessels or the maintenance of vascular functions, because they regulate the proliferation, adhesion, migration, and differentiation of vascular endothelial cells (17Doi M. Thyboll J. Kortesmaa J. Jansson K. Iivanainen A. Parvardeh M. Timpl R. Hedin U. Swedenborg J. Tryggvason K. J. Biol. Chem. 2002; 277: 12741-12748Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 18Hallmann R. Horn N. Selg M. Wendler O. Pausch F. Sorokin L.M. Physiol. Rev. 2005; 85: 979-1000Crossref PubMed Scopus (410) Google Scholar). It is well known that vascular BMs contain two major laminin groups, i.e. the α4 laminin-411/421 (Lm411/421) and the α5 laminin-511/521 (Lm511/521) (18Hallmann R. Horn N. Selg M. Wendler O. Pausch F. Sorokin L.M. Physiol. Rev. 2005; 85: 979-1000Crossref PubMed Scopus (410) Google Scholar). To identify and characterize the new laminin isoform Lm3B11 (Lm3B11), we examined its expression in cultured human vascular endothelial cells and expressed it as a recombinant protein in HEK-293 cells. The present study for the first time identifies Lm3B11 protein and shows its unique activity.DISCUSSIONIn the present study we identified a novel laminin isoform consisting of the laminin α3B, β1, and γ1 chains, i.e. Lm3B11, and revealed its biological activity as a recombinant protein. Previous studies have shown that the α3B chain is expressed widely in various human organs and tissues, but unlike α3A, it is scarcely detected in cultured cells (12Mizushima H. Miyagi Y. Kikkawa Y. Yamanaka N. Yasumitsu H. Misugi K. Miyazaki K. J. Biochem. 1996; 120: 1196-1202Crossref PubMed Scopus (94) Google Scholar). The present RT-PCR analysis showed that the two types of vascular endothelial cells, MVE and UVE cells, expressed the messages for the laminin α3B, β1, β2, β3, and γ1 genes but neither α3A nor γ2. This suggested that the Lm3B11/Lm3B21 heterotrimers, but not Lm3B32, could be produced by the endothelial cells. Indeed, the α3B chain was associated with the β1 and γ1 chains in the culture of MVE cells. These results are consistent with our recent finding that the α3B chain is colocalized with the β1 and γ1 chains but neither the β3 nor γ2 chain in vascular BMs of various tissues (16Kariya Y. Mori T. Yasuda C. Watanabe N. Kaneko Y. Nakashima Y. Ogawa T. Miyazaki K. J. Mol. Histol. 2008; 39: 435-446Crossref PubMed Scopus (17) Google Scholar). However, the expression level of Lm3B11 in the cultured endothelial cells appeared to be very low.The laminin α3A and α3B chains preferentially bind with the β3 and γ2 chains, both of which have truncated short arms, producing the laminin heterotrimers Lm3A32 and Lm3B32 (15Kariya Y. Yasuda C. Nakashima Y. Ishida K. Tsubota Y. Miyazaki K. J. Biol. Chem. 2004; 279: 24774-24784Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 24Kariya Y. Ishida K. Tsubota Y. Nakashima Y. Hirosaki T. Ogawa T. Miyazaki K. J. Biochem. 2002; 132: 607-612Crossref PubMed Scopus (42) Google Scholar). These laminins have very high activities of cell adhesion and migration as compared with other laminins (6Miyazaki K. Cancer Sci. 2006; 97: 91-98Crossref PubMed Scopus (141) Google Scholar). The α3A chain also binds with the β1/2 and γ1 chains, producing Lm3A11/3A21 (11Hirosaki T. Tsubota Y. Kariya Y. Moriyama K. Mizushima H. Miyazaki K. J. Biol. Chem. 2002; 277: 49287-49295Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). In this study we for the first time expressed Lm3B11 as a recombinant protein by introducing the cDNAs of the α3B, β1, and γ1 chains into HEK-293 cells. Lm3A11 has a truncated α chain, whereas Lm3B11, like Lm111 and Lm511, has a typical laminin structure with the three full-length chains. As compared with Lm3B32, Lm3B11 had relatively low cell adhesion activity. This is similar to the case of Lm3A11 (11Hirosaki T. Tsubota Y. Kariya Y. Moriyama K. Mizushima H. Miyazaki K. J. Biol. Chem. 2002; 277: 49287-49295Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). It seems likely that the long short arms of the β1 and γ1 chains suppress the cell adhesion activity of Lm3A11 and Lm3B11. However, Lm3B11 exhibited relatively strong cell motility activity toward vascular endothelial cells as compared with epithelial cells. This may be related to a special integrin recognition of Lm3B11 by endothelial cells. Our experiments with neutral integrin antibodies indicated that an α3 integrin, most likely integrin α3β1, was primarily responsible for the attachment of epithelial cells to Lm3B11, whereas both α3 and α6 integrins, such as integrins α3β1, α6β1, and α6β4, equally contributed to the attachment of endothelial cells. In contrast, Lm511 and Lm3B32 preferentially recognized the α3 integrin for the attachment of both epithelial and endothelial cells.Most prominent activity of Lm3B11 was the induction of a unique morphological change of MVE cells. When MVE cells were seeded on the Lm3B11 substrate, they extended multiple and branching lamellipodial protrusions. Such endothelial cell branching was less evidently seen on Lm3B32, Lm511, and Lm111 but not on fibronectin or gelatin at all. It is well known that laminins regulate cytoskeletal change through interaction with integrins (21Fujiwara H. Gu J. Sekiguchi K. Exp. Cell Res. 2004; 292: 67-77Crossref PubMed Scopus (57) Google Scholar). The Rho family of small GTPases plays critical roles in the formation of actin cytoskeleton and in the capillary assembly of endothelial cells (25Yamazaki D. Suetsugu S. Miki H. Kataoka Y. Nishikawa S. Fujiwara T. Yoshida N. Takenawa T. Nature. 2003; 424: 452-456Crossref PubMed Scopus (189) Google Scholar, 26Connolly J.O. Simpson N. Hewlett L. Hall A. Mol. Biol. Cell. 2002; 13: 2474-2485Crossref PubMed Scopus (147) Google Scholar, 27Liu Y. Senger D.R. FASEB J. 2004; 18: 457-468Crossref PubMed Scopus (92) Google Scholar). Our data suggest that Lm3B11 activates intracellular signal transduction mediated by Src, PI3K, and Akt. The PI3K signaling is known to activate small G-proteins through the downstream signaling kinase Akt (28Krasilnikov M.A. Biochemistry. 2000; 65: 59-67PubMed Google Scholar). The signaling pathway of Src induces capillary assembly through activation of Rac (27Liu Y. Senger D.R. FASEB J. 2004; 18: 457-468Crossref PubMed Scopus (92) Google Scholar). Taken together, it is expected that the interaction of Lm3B11 with α3 and α6 integrins induces the Src and then PI3K signaling pathways, which may activate Rac rather than Rho, leading to the unique cytoskeletal change that allows the cells to produce many thin and long lamellipodial protrusions. The α3Β chain contains the second integrin-binding site in its N-terminal region in addition to the LG1–3 domain (15Kariya Y. Yasuda C. Nakashima Y. Ishida K. Tsubota Y. Miyazaki K. J. Biol. Chem. 2004; 279: 24774-24784Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 29Hirosaki T. Mizushima H. Tsubota Y. Moriyama K. Miyazaki K. J. Biol. Chem. 2000; 275: 22495-22502Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). In this study the N-terminal fragment of the α3Bnt weakly stimulated the protrusion formation in MVE cells, suggesting that the N-terminal integrin binding site contributes to the unique activity of Lm3B11 toward MVE cells.It has long been believed that there are only two groups of laminin isoforms, Lm411/421 and Lm511/521, in vascular BMs. Their distinctive roles are still unclear. Lm411 is expressed in the BMs of all types of blood vessels including developing embryonic endothelium (E8.5) (30Frieser M. Nöckel H. Pausch F. Röder C. Hahn A. Deutzmann R. Sorokin L.M. Eur. J. Biochem. 1997; 246: 727-735Crossref PubMed Scopus (111) Google Scholar), whereas Lm511 occurs in mature vessels at later stages, predominantly in capillaries (31Sorokin L.M. Pausch F. Frieser M. Kröger S. Ohage E. Deutzmann R. Dev. Biol. 1997; 189: 285-300Crossref PubMed Scopus (215) Google Scholar). A study with laminin α4 null mice indicates that this laminin is not essential for blood vessel formation, but it plays roles in maturation of vessels (32Thyboll J. Kortesmaa J. Cao R. Soininen R. Wang L. Iivanainen A. Sorokin L. Risling M. Cao Y. Tryggvason K. Mol. Cell. Biol. 2002; 22: 1194-1202Crossref PubMed Scopus (253) Google Scholar). Because the laminin α5 chain is expressed in not only the endothelium but also various kinds of epithelial tissues, laminin α5 null mice die during embryogenesis before it appears in vascular BMs. An in vitro study with mouse embryoid bodies also showed that although both α4 and α5 laminins are deposited on the surface of capillary-like tubes, the laminin deposition is dispensable for vasculogenesis (33Jakobsson L. Domogatskaya A. Tryggvason K. Edgar D. Claesson-Welsh L. FASEB J. 2008; 22: 1530-1539Crossref PubMed Scopus (49) Google Scholar). This study also showed that laminin-deficient embryoid bodies produced vascular structures with large lumens, but the addition of purified laminins increased capillary branches and produced more slender vessels. Taken together, it is considered that vascular laminins play roles in organizing and stabilizing blood vessels. In addition, these laminins may influence the barrier function of blood vessels and the transmigration of leukocytes and tumor cells. T-cell transmigration through the endothelial cell BM occurs exclusively at sites having the α4 but not α5 laminins (34Spessotto P. Yin Z. Magro G. Deutzmann R. Chiu A. Colombatti A. Perris R. Cancer Res. 2001; 61: 339-347PubMed Google Scholar).Lm3B11 found in this study is the third member of vascular laminins. The laminin α3B chain has the highest homology to the α5 chain (14Doliana R. Bellina I. Bucciotti F. Mongiat M. Perris R. Colombatti A. FEBS Lett. 1997; 417: 65-70Crossref PubMed Scopus (21) Google Scholar). Therefore, Lm3B11 is most close to Lm511 in structure. Like the laminin α5 chain, the α3B chain is expressed in both endothelial and epithelial cells. However, the α3B chain seems to exist as Lm3B32 in epithelial BMs because of its higher affinity to the β3 and γ2 chains than the β1 and γ1 chains. The present study demonstrates that Lm3B11 is distinguished from Lm511 and Lm3B32 with respect to the biological activity and integrin requirement. At present it is unknown what vascular function is attributed to the prominent activity of Lm3B11 in promoting the formation of multiple pseudopodial protrusions of MVE cells. Endothelial cell branching is required for the angiogenic process (23Fischer R.S. Gardel M. Ma X. Adelstein R.S. Waterman C.M. Curr. Biol. 2009; 19: 260-265Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). The unique activity of Lm3B11 appears to be favorable to the branching of capillaries during angiogenesis. However, we previously found that the α3B laminin chain was less frequently detected in human skin cancer tissues as compared with normal skin tissues (16Kariya Y. Mori T. Yasuda C. Watanabe N. Kaneko Y. Nakashima Y. Ogawa T. Miyazaki K. J. Mol. Histol. 2008; 39: 435-446Crossref PubMed Scopus (17) Google Scholar). It seems possible that Lm3B11 contributes to the formation or maintenance of complex vascular network in normal tissues. To clarify the biological function of Lm3B11, it is important to investigate its precise distribution in comparison with Lm411 and Lm511 in blood vessels of various tissues including cancers. IntroductionLaminin, a family of large heterotrimeric glycoproteins consisting of α, β, and γ chains, is a major cell-adhesive component present in the basement membranes (BMs) 2The abbreviations used are: BMbasement membraneLmlamininMVE cellmicrovascular endothelial cellUVEumbilical vein endothelial cellDMEDulbecco's modified Eagle's medium. of various tissues. It plays critical roles in the maintenance of tissue architectures and the regulation of cellular functions such as cell adhesion, migration, proliferation, apoptosis, and differentiation (1Colognato H. Yurchenco P.D. Dev. Dyn. 2000; 218: 213-234Crossref PubMed Scopus (1035) Google Scholar). Different combinations of five α, three β, and three γ chains give rise to more than 15 laminin isoforms (2Aumailley M. Bruckner-Tuderman L. Carter W.G. Deutzmann R. Edgar D. Ekblom P. Engel J. Engvall E. Hohenester E. Jones J.C. Kleinman H.K. Marinkovich M.P. Martin G.R. Mayer U. Meneguzzi G. Miner J.H. Miyazaki K. Patarroyo M. Paulsson M. Quaranta V. Sanes J.R. Sasaki T. Sekiguchi K. Sorokin L.M. Talts J.F. Tryggvason K. Uitto J. Virtanen I. von der Mark K. Wewer U.M. Yamada Y. Yurchenco P.D. Matrix Biol. 2005; 24: 326-332Crossref PubMed Scopus (660) Google Scholar). These laminins are distributed in a tissue-specific manner and are thought to play differential roles in the individual tissues. Such specific distribution and function of laminins are mainly determined by the α chains. For example, α2 laminins (laminin-211 and -221) are localized in the BMs of muscle and neurons, whereas α4 laminins (laminin-411 and -421) are localized in vascular BMs.There are two splicing variants of laminin α3 chain, the truncated form α3 (or α3A) and the full-sized form α3B (3Ryan M.C. Tizard R. VanDevanter D.R. Carter W.G. J. Biol. Chem. 1994; 269: 22779-22787Abstract Full Text PDF PubMed Google Scholar, 4Galliano M.F. Aberdam D. Aguzzi A. Ortonne J.P. Meneguzzi G. J. Biol. Chem. 1995; 270: 21820-21826Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), gene expression of which is regulated by alternative splicing and different promoters (5Ferrigno O. Virolle T. Galliano M.F. Chauvin N. Ortonne J.P. Meneguzzi G. Aberdam D. J. Biol. Chem. 1997; 272: 20502-20507Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Laminin-3A32 (Lm3A32), which is previously known as laminin-5 and consists of all truncated chains (α3A, β3, and γ2), is a major laminin isoform in the skin, esophagus, lung, breast, and other epithelial tissues. This laminin has extensively been studied because it has unique structure and biological activity as compared with other laminins (6Miyazaki K. Cancer Sci. 2006; 97: 91-98Crossref PubMed Scopus (141) Google Scholar). In the skin, Lm3A32 in the BM associates with integrin α6β4 of basal keratinocytes to form the hemidesmosome structures and contributes to their stable anchorage to the underlying connective tissues (7Kivirikko S. McGrath J.A. Baudoin C. Aberdam D. Ciatti S. Dunnill M.G. McMillan J.R. Eady R.A. Ortonne J.P. Meneguzzi G. Hum. Mol. Genet. 1995; 4: 959-962Crossref PubMed Scopus (153) Google Scholar, 8Baker S.E. DiPasquale A.P. Stock E.L. Quaranta V. Fitchmun M. Jones J.C. Exp. Cell Res. 1996; 228: 262-270Crossref PubMed Scopus (60) Google Scholar). The laminin α3A chain also associates with the full-sized β chains (β1 and β2) and the full-sized, γ1 chain, thus producing laminin-3A11 (Lm3A11) and laminin-3A21 (Lm3A21) (9Marinkovich M.P. Lunstrum G.P. Burgeson R.E. J. Biol. Chem. 1992; 267: 17900-17906Abstract Full Text PDF PubMed Google Scholar, 10Champliaud M.F. Lunstrum G.P. Rousselle P. Nishiyama T. Keene D.R. Burgeson R.E. J. Cell Biol. 1996; 132: 1189-1198Crossref PubMed Scopus (226) Google Scholar, 11Hirosaki T. Tsubota Y. Kariya Y. Moriyama K. Mizushima H. Miyazaki K. J. Biol. Chem. 2002; 277: 49287-49295Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar).Although the α3B chain is known to be more widely expressed than the α3A chain in vivo, α3B-containing laminins have been poorly investigated (12Mizushima H. Miyagi Y. Kikkawa Y. Yamanaka N. Yasumitsu H. Misugi K. Miyazaki K. J. Biochem. 1996; 120: 1196-1202Crossref PubMed Scopus (94) Google Scholar, 13Garbe J.H. Göhring W. Mann K. Timpl R. Sasaki T. Biochem. J. 2002; 362: 213-221Crossref PubMed Scopus (54) Google Scholar, 14Doliana R. Bellina I. Bucciotti F. Mongiat M. Perris R. Colombatti A. FEBS Lett. 1997; 417: 65-70Crossref PubMed Scopus (21) Google Scholar). We previously expressed laminin-3B32 (Lm3B32; α3B/β3/γ2) as a recombinant protein and revealed its biological activity (15Kariya Y. Yasuda C. Nakashima Y. Ishida K. Tsubota Y. Miyazaki K. J. Biol. Chem. 2004; 279: 24774-24784Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). In addition, our recent immunohistochemical study showed that the α3B chain is colocalized with the β3 and γ2 chains in the epithelial BMs of the skin, esophagus, breast, and lung, whereas in the BMs of blood vessels it is colocalized with the β1/2 and γ1 chains, suggesting the presence of novel vascular laminin isoforms, laminin-3B11 (Lm3B11) and laminin-3B21 (Lm3B21) in the vascular BMs (16Kariya Y. Mori T. Yasuda C. Watanabe N. Kaneko Y. Nakashima Y. Ogawa T. Miyazaki K. J. Mol. Histol. 2008; 39: 435-446Crossref PubMed Scopus (17) Google Scholar). However, no previous studies have identified the α3B-containing, full-sized Lm3B11/3B21.The network of blood vessels dynamically changes under various physiological and pathological conditions such as tumor growth and inflammation. The angiogenesis in tumor tissues is essential for tumor growth and metastasis. It is expected that vascular laminins play some important roles in the formation of blood vessels or the maintenance of vascular functions, because they regulate the proliferation, adhesion, migration, and differentiation of vascular endothelial cells (17Doi M. Thyboll J. Kortesmaa J. Jansson K. Iivanainen A. Parvardeh M. Timpl R. Hedin U. Swedenborg J. Tryggvason K. J. Biol. Chem. 2002; 277: 12741-12748Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 18Hallmann R. Horn N. Selg M. Wendler O. Pausch F. Sorokin L.M. Physiol. Rev. 2005; 85: 979-1000Crossref PubMed Scopus (410) Google Scholar). It is well known that vascular BMs contain two major laminin groups, i.e. the α4 laminin-411/421 (Lm411/421) and the α5 laminin-511/521 (Lm511/521) (18Hallmann R. Horn N. Selg M. Wendler O. Pausch F. Sorokin L.M. Physiol. Rev. 2005; 85: 979-1000Crossref PubMed Scopus (410) Google Scholar). To identify and characterize the new laminin isoform Lm3B11 (Lm3B11), we examined its expression in cultured human vascular endothelial cells and expressed it as a recombinant protein in HEK-293 cells. The present study for the first time identifies Lm3B11 protein and shows its unique activity.
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