Research ArticleP-selectin activates integrin-mediated colon carcinoma cell adhesion to fibronectin
Introduction
Tumor cell metastasis is a highly regulated and dynamic process in which cancer cells separate from a primary tumor, migrate across blood vessel walls into the bloodstream, and disperse throughout the body to generate colonies [1]. Hematogenous tumor cell dissemination depends on cell-adhesive interactions that are mediated by adhesion receptors, such as selectins and integrins [2], [3], [4], [5].
Integrins are heterodimeric, transmembrane, cell surface glycoproteins that act as receptors for extracellular matrix proteins or counter-receptors on other cells. Each integrin consists of an α subunit and a β subunit. So far, at least 18 different α subunits and eight different β integrin subunits have been identified. The combination of α and β subunits in the integrin dimers determines ligand specificity. Integrins are very highly regulated receptors that are subject to multifaceted regulatory pathways [6], [7], [8], [9], [10]. “Inside-out” signaling most commonly regulates the ability of integrins to bind ligands [11]. For example, protein kinase C (PKC) mediates activation of α5β1 integrin [12] and phorbol esters activate αLβ2 integrin [13]. This increase in ligand binding capacity can be the result of either a change in integrin conformation or integrin clustering. The latter process increases apparent affinity as a result of multivalent cooperative binding [14].
Selectins are vascular cell–cell adhesion receptors that usually bind to certain sialyl Lewis A (sLeA)- and sialyl Lewis X (sLex)-containing mucin-type glycoproteins found on normal leukocytes and endothelium. Three 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. P-selectin is also expressed on thrombin-activated platelets [15]. All three members of the selectin family, E-, L-, and P-selectin, have been shown to recognize sLeA and sLeX expressed on carcinoma cell surfaces [16], [17].
Accumulation of leukocytes at sites of inflammation is initiated by selectins that mediate the capturing and rolling of leukocytes on endothelium. This step is followed by activation of leukocyte integrins, which mediate firm adhesion to the endothelial cell surface. The firmly adhering cells can then actively migrate through the endothelial cell barrier [18]. Several reports have suggested that selectins and integrins can play an important role in adhesion of tumor cells to microvascular endothelium and subsequent cell migration. Several aspects of tumor cell adhesion to the microvascular endothelial cells have been even proposed to be analogous to leukocyte recruitment [1], [5], [17], [19], [20].
E-selectin and P-selectin have been proposed as critical molecules for adhesion of some cancer cells [21], [22]. Moreover, the P-selectin ligand, CD24 (also called heat-stable antigen), has been detected on several human carcinomas [23], [24], [25], further suggesting that P-selectin could play a crucial role in metastasis. However, classical selectin ligand binding plays a role primarily in transient adhesive processes due to a relatively high on–off rate and a requirement for less stable cell adhesion mechanisms necessary for cancer cell extravasation [26], [27], [28]. It has been suggested that changes in the affinity and avidity of integrin receptors may be required for stabilized tumor cell adhesion and subsequent cell migration into the host organ [1].
We have started to investigate a possible role of P-selectin in integrin-mediated adhesion of cultured human tumor cells. We have found that P-selectin binding to human adenocarcinoma Colo 320 cells increases their adhesion specifically to fibronectin through the α5β1 integrin. We found also that P-selectin binding to Colo 320 cells activates both p38 mitogen-activated protein kinase (p38 MAPK) and phosphatidylinositol 3-kinase (PI3-K), which form a signaling complex in a P-selectin dependent manner. p38 MAPK and PI3-K are required in P-selectin-mediated cell spreading but only PI3-K is required in P-selectin-increased adhesion to fibronectin. These results suggest that P-selectin ligands could be signaling molecules important in the regulation of tumor cell adhesion.
Section snippets
Materials
P-selectin-IgG Fc fusion protein and E-selectin-IgG-Fc fusion protein were obtained from BD Biosciences (San Jose, CA) and R&D Systems (Minneapolis, MN), respectively. P-selectin-IgG Fc fusion protein was also expressed in COS cells and purified as described [29] by the NIEHS Protein Expression Core Facility. The expression vector for the P-selectin-IgG Fc fusion protein was a generous gift from Dr. John Lowe, University of Michigan Medical Center. The recombinant E- and P-selectin-IgG Fc are
P-selectin induces an increase in cell adhesion to fibronectin through activation of α5β1 integrin
In a previous study, it was shown that human colon carcinoma cells can attach to immobilized P-selectin substrates, and some tumor cells, such as Colo 320 cells, attach preferentially to P-selectin rather than to E-selectin [16], [42]. To test whether the Colo 320 cells have the ability to bind to soluble P-selectin, these cells were incubated with different concentrations of a soluble recombinant P-selectin-IgG Fc fusion protein. P-selectin-IgG Fc, but not human IgG Fc or E-selectin-IgG Fc,
Discussion
In this study, we have used an in vitro model system consisting of cultured Colo 320 human carcinoma cells incubated with soluble P-selectin-IgG chimeric protein to characterize the regulation by selectins of integrin function in cancer cells. We have shown that P-selectin binding to Colo 320 cells can specifically activate the α5β1 integrin, which results in increased cell attachment and cell spreading specifically on fibronectin in a concentration- and time-dependent manner. This
Acknowledgments
We thank our colleagues at the NIEHS for critically reviewing the manuscript and Dr. Robert Petrovich for working out the large scale expression and purification of the P-selectin-IgG Fc chimeric protein. This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences.
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