Transforming JB6 cells exhibit enhanced integrin-mediated adhesion to osteopontin

Transformation of preneoplastic epidermal IBG cells with tumor promoter 12- O-tetradecanoyl-phorbol-13-acetate (TPA) is an in vitro model of late-stage tumor promotion. Osteopontin (OPN) is a secreted, adhesive protein that is highly expressed in JB6 cells With TPA treatment, and its expression persi sts for at least 4 days, which is the rime required for subsequent expressi on of transformed phonotype. These observations suggest that OPN may play a role in promoting JB6 cell transformation. To function in transformation o f JB6 cells, OPN must bind to the surface of the JB6 cell and subsequently signal within the cell. Therefore, we investigated whether JB6 cells adhere to OPN and, ii so, to which surface receptors. TPA-treated JB6 cells had s ignificantly (P < 0.05) increased adherence to OPN compared with dimethylsu lfoxide-treated control cei Is. Enhanced attachment of JB6 cells to OPN was also observed after treatment with another tumor promoter phorbol dibutyra te but not with nontumor promoters (phorbol and 1 alpha,25-dihydroxyvitamin D-3), suggesting that tumor promoters specifically modulate attachment to OPN. The argininylglycylaspartic acid (RGD) cell-binding region of OPN medi ates attachment of TPA-treated JB6 cells because RGD, but not argininylglyc ylglutamic acid (RGE), peptides inhibited adherence of these cells to OPN i n a dose-dependent manner. Flow cytometric analyses, blocking adhesion assa y using anti-a, antibody, and co-immunoprecipitation assay all indicated th at TPA-treated cells had similar levels of alpha(v), and beta(5) but decrea sed levels of beta(1) compared with untreated cells and that cell adhesion to OPN is most likely mediated through the a,P; Furthermore, calphostin C, a specific protein kinase C (PKC) inhibitor, decreased TPA-treated JB6 cell adhesion to OPN by 50%, suggesting that TPA increased integrin affinity or avidity for OPN through a PKC-mediated pathway. Collectively, these result s indicate that transforming JB6 cells adhere to OPN through its RGD sequen ce. The most likely OPN receptor is the alpha(V)beta(5) integrin, which inc reases the affinity or avidity for OPN through a PKC-dependent pathway rath er than increasing the number of receptors. (C) 2000 Wiley-Liss, Inc.