Ectopic Expression of Human and Feline CD9 in a Human B Cell Line Confers β1 Integrin-dependent Motility on Fibronectin and Laminin Substrates and Enhanced Tyrosine Phosphorylation

Abstract

Few molecules have been shown to confer cell motility. Although the motility-arresting properties of anti-CD9 monoclonal antibody (mAb) suggest the transmembrane 4 superfamily (TM4SF) member CD9 can induce a motorgenic signal, gene transfection studies have failed to confirm this hypothesis. We report here that ectopic expression of human CD9 (CD9h) and feline CD9 (CD9f) in the CD9-negative, poorly motile, human B cell line Raji dramatically enhances migration across fibronectin-and laminin-coated polycarbonate filters. Migration of Raji/CD9h and Raji/CD9f on either substrate was inhibited by the anti-CD9 mAb 50H.19 and by the anti-␤1 integrin mAb AP-138. Migration of Raji/CD9h on laminin was potently inhibited by the anti-VLA-6 integrin mAb GoH3 and by the anti-VLA-4 integrin mAb 44H6, whereas migration of Raji/CD9h on fibronectin was inhibited only by mAb 44H6. Since CD9h-transfected Raji cells adhered to fibronectin as effectively as mock transfectants, expression of CD9 enhanced motility, but not adhesion. CD9-enhanced migration was inhibited by the protein tyrosine kinase inhibitor herbimycin A suggesting that tyrosine phosphorylation played a role in the generation of a motorgenic signal. Raji/CD9h transfectants adherent to fibronectin expressed 6-fold higher levels of phosphotyrosine than Raji. Raji/CD9f transfectants also phosphorylated proteins on tyrosine more effectively than Raji including a protein of 110 kDa which was phosphorylated on the motility-inducing substrates laminin and fibronectin, but not on bovine serum albumin. Our results support a role for CD9 in the amplification of a motorgenic signal in B cells involving ␤1 integrins and the activation of protein tyrosine kinases.

Expression of tetraspanins in human lung cancer cells: frequent downregulation of CD9 and its contribution to cell motility in small cell lung cancer

“…In this respect, it is of note that reconstitution of the Rb gene into a Rb-defective NSCLC cell inhibited tumor cell invasion in vitro (Li et al, 1996). Although a previous study showed that overexpression of CD9 into an NSCLC line suppressed tumor cell motility (Ikeyama et al, 1993), another study presented contradictory evidence that CD9 transfection into a Bcell line upregulated cell motility on FN and LM (Shaw et al, 1995). In addition, other studies reported that transfection of tetraspanins, CO-029 (Claas et al, 1998) and CD151 (Testa et al, 1999), into a pancreatic cancer cell line and a cervical cancer cell line, respectively, resulted in enhanced cell migration or metastasis.…”

The role of membrane microdomains in transmembrane signaling through the epithelial glycoprotein Gp140/CDCP1

“…Anti-CD9 antibody treatment enhanced cell spreading of MDA-MB-435 breast carcinoma cells in β4 dependent signaling as well [22]. Further, when CD9 was transfected into a poorly motile human B cell cell line, motility was enhanced on substrates that utilized β1 for motility [26]. CD9 siRNA also increased lymphocyte detachment in shear flow assays [27].…”

A Functionally Relevant Conformational Epitope on the CD9 Tetraspanin Depends on the Association with Activated β1Integrin

“…Several tetraspanins, including CD9, have been proposed to mediate some of the functional activities of associated cell surface molecules, including regulation of cell migration and adhesion-dependent signaling through ␤ 1 integrins (23,24,48,49,53). We have addressed in this study the functional consequences of CD9 engagement with mAb PAINS-13 in four different types of functional assays in which the implication of ␤ 1 integrins is well established: (i) in vitro wound healing assays in monolayers of endothelial cells, (ii) formation of angiotubular endothelial cell structures on Matrigel, (iii) cell adhesion to and spreading on ␤ 1 integrin-specific ligands laminin-1 and fibronectin, and (iv) signal transduction and activation of PI3K.…”