Cell adhesion and migration contribute to normal processes such as differentiation, embryonic development, and wound healing as well as to the progression of diseases and pathological conditions that can result from either acute or chronic exposure to environmental toxicants, such as cancer and inflammatory responses. Key mechanistic steps in these processes involve the interactions of extracellular glycoproteins--such as fibronectin, laminin, and collagens--with specific adhesive receptors, the best characterized of which are the integrins, a family of heterodimeric complexes consisting of an alpha subunit and a beta subunit. Integrins are highly regulated receptors that can exist in either an active or inactive state. Selectins are vascular cell-cell adhesion molecules involved in leukocyte trafficking, inflammation, thrombosis, autoimmunity and cancer. Accumulation of leukocytes at sites of inflammation is initiated by selectins that mediate the capturing and rolling of leukocytes on endothelium. Three major members of the selectin family have been identified: L-selectin, E-selectin and P-selectin. L-Selectin is constitutively expressed on leukocytes. P-and E-selectins are expressed on activated endothelial cells in response to microenvirnomental stimuli. P-selectin is also expressed on thrombin-activated platelets. All three members of the selectin family, E-, L-, and P-selectin can bind to human tumor cells and cancer-derived cell line. Our research has focused recently on the possible role of cues from the tumor microenvironment that can regulate integrin-mediated tumor cell migration and invasion. As a model system, we are examining the ability of one selectin, P-selectin, to trigger integrin-mediated adhesion and migration of cultured human tumor cells. We focus on two closely related aspects of this project: to characterize the mechanisms of P-selectin-induced activation of integrin-mediated cell adhesion and cell migration. We have previously shown that binding of soluble, recombinant P-selectin-IgG Fc chimeric protein to Colo-320 cells stimulates cell adhesion to fibronectin through the specific activation of the alpha5-beta1 integrin by means of the p38 MAP kinase and PI-3 kinase (PI3-k) signaling pathways. We have now identified nucleolin as a novel cell surface P-selectin receptor on Colo-320 cells using affinity chromatography and a proteomic approach. We have also found that P-selectin binding to Colo-320 cells induces tyrosine phosphorylation specifically of cell-surface nucleolin and formation of a signaling complex containing cell surface nucleolin, PI 3-K, and p38 MAPK. Using siRNA approaches, we showed that both P-selectin binding to Colo-320 cells and formation of the P-selectin-mediated p38 MAPK/ PI 3-K signaling complex require nucleolin expression. Thus, we have characterized nucleolin (or a nucleolin-like protein) as a novel, signaling cell-surface receptor for P-selectin on Colo-320 cells and suggest a mechanism for linkage of nucleolin to P-selectin-induced signal transduction pathways that regulate the adhesion of Colo-320 on fibronectin substrates. We have recently been characterizing the stimulation of integrin-mediated tumor cell migration by the binding of soluble, recombinant P-selectin-IgG Fc chimeric protein. We have found that P-selectin stimulates the secretion of matrix metalloproteinase-9 (MMP-9) by A375 human melanoma cells and that direct addition of exogenous MMP-9 to A-375 cells can stimulate the migratory phenotype. The current paradigm in the field states that MMP-9 stimulates migration by digesting basement membrane proteins, allowing tumor cells to clear a migratory pathway. However, we have preliminary data that show that stimulation of cell migration by MMP-9 can occur even in the absence of catalytic activity.
