Abstract
Integrins affect the motility of multiple cell types to control cell survival, growth, or differentiation, which are mediated by cell-cell and cell-extracellular matrix interactions. We reported previously that the α9 integrin splicing variant, SFα9, promotes WT α9 integrin-dependent adhesion. In this study, we introduced a new murine α4 integrin splicing variant, α4B, which has a novel short cytoplasmic tail. In inflamed tissues, the expression of α4B, as well as WT α4 integrin, was up-regulated. Cells expressing α4B specifically bound to VCAM-1 but not other α4 integrin ligands, such as fibronectin CS1 or osteopontin. The binding of cells expressing WT α4 integrin to α4 integrin ligands is inhibited by coexpression of α4B. Knockdown of α4B in metastatic melanoma cell lines results in a significant increase in lung metastasis. Expression levels of WT α4 integrin are unaltered by α4B, with α4B acting as a regulatory subunit for WT α4 integrin by a dominant-negative effect or inhibiting α4 integrin activation.
Highlights
Integrin adhesion receptors are large heterodimeric cell surface receptors that mediate the adhesion of cells to the extracellular matrix and other cells [1]
We found that ␣4B mRNAs, as well as WT ␣4 integrin levels, in the spinal cords of EAE mice were increased (Fig. 1C). ␣4B Is Expressed on the Cell Surface with 1 Integrin—Cell lysates from NIH3T3 cells expressing HA-tagged ␣4 integrin or FLAG-tagged ␣4B were analyzed by Western blotting. 150- and 140-kDa bands of ␣4 integrin, which are the mature and precursor forms, respectively [18], were detected by anti-HA antibody
Cytoplasmic domains of integrin are highly conserved during evolution, whereas extracellular domains are not as highly conserved
Summary
Mice—Mice were kept under specific pathogen-free conditions and were provided food and water ad libitum. NIH3T3 cells expressing ␣4 integrin and/or ␣4B were identified by flow cytometry using anti-␣4 antibody. For generation of CHO cells coexpressing ␣4B and ␣4 integrin, CHO cells transfected with ␣4B-pcDNA3.1neo were cultured in DMEM containing 10% FCS and 800 g/ml G418 (Invitrogen) and analyzed for expression of ␣4B by flow cytometry with anti-␣4 antibody (R1–2). Cells expressing WT ␣4 integrin at a comparable level were selected by flow cytometry with anti-␣4 antibody (19E4). Generation of Antibodies—Anti-WT ␣4 integrin antibody (clone 19E4) or anti-␣4 integrin antibody (clone 5X2) was generated in Sprague-Dawley rats or Syrian hamsters, respectively, and immunized with cells expressing WT ␣4 integrin Their splenocytes were fused with X63-Ag8-653 mouse myelomas as described previously [17]. Flow Cytometry—For ␣4 integrin or ␣4B expression, cells were blocked with normal goat serum and incubated with a phycoerythrin-labeled anti-mouse ␣4 integrin antibody. Differences were considered to be significant when p Ͻ 0.05 (*) or 0.005 (**)
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