Bacillus Calmette-Guerin initiates intracellular signaling in a transitional carcinoma cell line by crosslinking α5β1 integrin

Purpose: The adherence of bacillus Calmette-Guerin (BCG) to the surface of transitional carcinoma tumor cells initiates nuclear factor (NF)-kappaB signal transduction pathways that modulate the expression of proteins important in the antitumor response to BCG. We tested the hypothesis that BCG initiates NF-kappaB signaling as a consequence of cross-linking alpha5beta1 integrin receptors present on the tumor cell surface. Materials and Methods: The effect of alpha5beta1 antibody mediated cross-linking on interleukin (IL)-6 mRNA expression, IL-6 promoter activation and activation of a specific NF-kappaB reporter construct was determined. A series of reporter constructs containing nonfunctional mutations in the AP-1, NF-IL-6 and NF-kappaB sites were used to determine the relative importance of these response elements in alpha6beta1 cross-linking mediated activation of the IL-6 promoter. A final series of experiments assessed the role of alpha5beta1 receptor occupancy by fibronectin (FN) in initiating antibody or BCG mediated signaling. Results: Antialpha5 and antibeta1 mediated cross-linking of alpha5beta1 integrin initiated NF-kappaB signaling, IL-6 promoter activation and IL-6 mRNA expression. Deletion mutants demonstrated that alpha5beta1 cross-link initiated, IL-6 promoter transactivation required intact NF-kappaB and AP-1 response elements. Receptor occupancy by FN was required for BCG but not for antibody initiated signaling. Conclusions: Cross-linking the alpha5beta1 receptor present on the surface of human transitional carcinoma cells lines initiates signal transduction in a manner identical to that observed for BCG. We propose a model in which multiple FN binding sites present on BCG interact with alpha5beta1 receptor bound FN molecules to cross-link alpha5beta1 receptors and initiate intracellular signaling.