Mast cells play a critical role in innate immunity, allergy, and autoimmune diseases. The receptor/ligand interactions that mediate mast cell activation are poorly defined. The alpha(2)beta(1) integrin, a receptor for collagens, laminins, decorin, E-cadherin, matrix metalloproteinase-1 (MMP-1), endorepellin, and several viruses, has been implicated in normal developmental, inflammatory, and oncogenic processes. We recently reported that alpha(2) integrin subunit-deficient mice exhibited markedly diminished neutrophil and IL-6 responses during Listeria monocytogenes- and zymosan-induced peritonitis. Peritoneal mast cells require alpha(2)beta(1) integrin expression for activation in response to pathogens, yet the ligand and molecular mechanisms by which the alpha(2)beta(1) integrin induces activation and cytokine secretion remain unknown. We now report that the alpha(2)beta(1) integrin is a novel receptor for multiple collectins and the C1q complement protein. We demonstrate that the alpha(2)beta(1) integrin provides a costimulatory function required for mast cell activation and cytokine secretion. This finding suggests that the alpha(2)beta(1) integrin is not only important for innate immunity but may serve as a critical target for the regulation of autoimmune/allergic disorders.

Previous studies have established that ligation of keratinocyte alpha (2)beta (1) integrin by type I collagen induces expression of matrix metalloproteinase-1 (MMP-1) and that MMP-1 activity is required for the alpha (2)beta (1) integrin-dependent migration of primary keratinocytes across collagenous matrices. We now present evidence that MMP-1 binds the alpha (2)beta (1) integrin via the I domain of the alpha (2) integrin subunit. Using an enzyme-linked immunosorbent assay with purified human MMP-1 and recombinant alpha (2) integrin I domain, we showed that the alpha (2) integrin I domain specifically bound in a divalent cation-dependent manner to both the pro and active forms of MMP-1, but not to MMP-3 or MMP-13. Although both the I domain and MMP-1 bind divalent cations, MMP-1 bound, in a divalent cation-dependent manner, to alpha (2) integrin I domains containing metal ion-dependent adhesion sites motif mutations that prevent divalent cation binding to the I domain, demonstrating that the metal ion dependence is a function of MMP-1. Using a series of MMP-1-MMP-3 and MMP-1-MMP-13 chimeras, we determined that both the linker domain and the hemopexin-like domain of MMP-1 were required for optimal binding to the I domain. The alpha (2) integrin/MMP-1 interaction described here extends an emerging paradigm in matrix biology involving anchoring of proteinases to the cell surface to regulate their biological activities.

In injured skin, collagenase-1 (matrix metalloproteinase-1 (MMP-1)) is induced in migrating keratinocytes. This site-specific expression is regulated by binding of the alpha (2)beta (1) integrin with dermal type I collagen, and the catalytic activity of MMP-1 is required for keratinocyte migration. Because of this functional association among substrate/ligand, receptor, and proteinase, we assessed whether the integrin also directs the compartmentalization of MMP-1 to its matrix target. Indeed, pro-MMP-1 co-localized to sites of alpha (2)beta (1) contacts in migrating keratinocytes. Furthermore, pro-MMP-1 co-immunoprecipitated with alpha (2)beta (1) from keratinocytes, and alpha (2)beta (1) co-immunoprecipitated with pro-MMP-1. No other MMPs bound alpha (2)beta (1) and no other integrins interacted with MMP-1. Pro-MMP-1 also provided a substrate for alpha (2)beta (1)-dependent adhesion of platelets. Complex formation on keratinocytes was most efficient on native type I collagen and reduced or ablated on denatured or cleaved collagen. Competition studies suggested that the alpha (2) I domain interacts with the linker and hemopexin domains of pro-MMP-1, not with the pro-domain. These data indicate that the interaction of pro-MMP-1 with alpha (2)beta (1) confines this proteinase to points of cell contact with collagen and that the ternary complex of integrin, enzyme, and substrate function together to drive and regulate keratinocyte migration.