Regulation of αvß3 integrin by Syndecan-1 in Mammary Carcinoma

We initially demonstrated that syndecan-1 (Sdc1) is a potent regulator of the αvß3 integrin on the highly metastatic MDA-MB-231 human mammary carcinoma cell line [1]. Cells rapidly spread when plated on mAb B-B4, an antibody specific for human Sdc1. This activity relies on Sdc1 activating the αvß3 integrin and appears coupled to ligation and clustering of the syndecan (Fig. 1). Importantly, the integrin activation (and therefore cell spreading) is blocked by addition of soluble mouse Sdc1 ectodomain (GST-mS1ED). This recombinant protein competes with the Sdc1 receptor binding to a cell surface component, possibly the integrin itself, and disrupts an interaction essential for activation of the αvß3 integrin.

This finding, made initially on Sdc1-specific antibody, holds true on extracellular matrix ligands (2, 3). The adhesion and spreading of MDA-MB-231 cells on VN is strictly dependent on the αvß3 integrin, shown by using the αvß3 integrin-blocking antibody LM609.. We next used several tests to question whether Sdc1 is required when the integrin is activated by matrix ligand. One test was to see if the soluble Sdc1 ectodomain protein (S1ED) will specifically block the activity on VN. Indeed, we find that 10 µM GST-S1ED (from either human or mouse) or S1ED cleaved to remove the GST will block the αvß3-dependent spreading. It also disrupts invasion of these cells through filters coated with VN, but has no effect on the response of the cells to FN (where they rely on the α5ß1 integrin) (2). And, it has no effect on MCF-7 cells that migrate on both ligands, but depend on the αvß1 rather than the αvß3 integrin. This suggests that the Sdc1 regulation acts on the ß3 subunit. In a second test, a rabbit polyclonal antibody directed against the ectodomain of mouse Sdc1, which recognizes both human and mouse forms, also blocks the response of the αvß3-positive MDA-MB-231 cells on VN, but not the αvß1-dependent MCF-7 cells. Thus, there is a close correlation between activation of αvß3 integrin and a cell surface binding interaction mediated by the core protein of Sdc1.

In a third approach, we find that blockade of Sdc1 expression with siRNA also blocks cell spreading and invasion on VN, but not FN (Fig. 2). We have used human Sdc1-specific siRNAs for this assay; these siRNAs do not affect mouse Sdc1 mRNA, which allows us to rescue the treated cells by expressing mouse Sdc1. We find that mouse Sdc1 will rescue the αvß3 integrin activity in cells in which the human Sdc1 expression has been silenced. This is a highly useful finding, as it has allowed us to express mouse Sdc1 mutants and learn where the activity resides in the Sdc1 ectodomain. Expression of the ectodomain alone, as a GPI-linked protein, is sufficient to activate the integrin, indicating that the active site resides in the ectodomain and explaining why soluble S1ED works as a competitor. We have tested a number of deletion mutants and find that mutants that fail to rescue are those that lack the sequence between amino acids 87-122 in the ectodomain. Thus, in the example shown in Fig. 2, the mS1Δ87-252 mutant does not rescue αvß3-mediated cell spreading nor migration on VN. The mS1TDM mutant, which lacks HS, is also unable to rescue on matrix ligand, but it does rescue in spreading assays in which the cells are bound to Sdc1 antibody. This strongly suggests that ligation of the syndecan is essential for its activity, as the antibody would ligate the syndecan even without its HS chains, whereas ligation of the syndecan by the heparin-binding domain of VN would depend on expression of the HS chains on the core protein.

We are currently examining how Sdc1 accomplishes this regulation and we are testing its role in vivo during tumorigenesis. Integrin activation is classically defined as activating the integrin to bind ligand. The integrin is present in an inactive conformation and assumes an active conformation often due signaling interactions inside the cell and due to interactions outside the cell with matrix ligands and sometimes other receptors (Fig. 3). We are interested in learning when and how Sdc1 associates with the integrin during the activation process and how it controls the integrin. We are also developing peptides that inhibit the interaction and thus inactivate the integrin. We are interested in testing these peptides using mouse models of tumorigenesis.

References:

• Beauvais, D. and Rapraeger, A.C. (2003). Syndecan-1 mediated cell spreading requires signaling by αvß3 integrins in human mammary carcinoma cells. Exp. Cell Res. 286: 219-232.
• Beauvais, D.M., Burbach, B.J. and Rapraeger, A.C. (2004). The syndecan-1 ectodomain regulates αvß3 integrin activity on human mammary carcinoma cells. J. Cell Biol., 167: 171-181.
• Beauvais, D.B. and Rapraeger, A.C. (2004). Syndecans in tumor cell adhesion and signaling. Reprod. Biol. Endocrin., 2:3 (http://www.rbej.com/content/2/1/3)

Other relevant publications:

• Rapraeger, A., Jalkanen, M., and Bernfield, M. (1986). Cell surface proteoglycan associates with the cytoskeleton at the basolateral cell surface of mouse mammary epithelial cells, J. Cell Biol., 103:2683-2696.
• Lebakken, C., and Rapraeger, A.C. (1996). Syndecan-1 mediates cell spreading in transfected human lymphoblastoid (Raji) cells. J. Cell Biol., 132: 1209-1221.
• McFall, A. and Rapraeger, A.C. (1997). Identification of an adhesion site within the syndecan-4 extracellular protein domain. J. Biol. Chem., 272: 12901-12904.
• Ott, V. and Rapraeger, A.C. (1998). Tyrosine phosphorylation of syndecan-1 and -4 cytoplasmic domains in adherent B82 fibroblasts. J. Biol. Chem., 273: 35291-35298.
• Rapraeger, A.C. and Ott, V. (1998). Molecular interactions of the syndecan core proteins. Curr. Opin. Cell Biol. 10: 620-628.
• Lebakken, C., McQuade, K. J. and Rapraeger, A.C. (2000). Syndecan-1 signals independently of ß1 integrins during Raji cell spreading. Exp. Cell Res., 259: 315-325.
• McQuade, K.J. and Rapraeger, A.C. (2003). Syndecan transmembrane and extracellular domains have unique and distinct roles in cell spreading. J. Biol. Chem., 278: 46607-46615.
• Burbach, B.J. and Rapraeger, A.C. (2004). Syndecan-1 ectodomain regulates matrix-dependent signaling on mammary carcinoma cells. Exp. Cell Res., 300: 234-237.
• McQuade, K.J., Beauvais, D.M., Burbach, B.J. and Rapraeger, A.C. (2006). Syndecan-1 core protein regulates αvß5-integrin activity in B82L fibroblasts. J. Cell Sci., 119: 2445-2456.