Mycobacterium bovis bacillus Calmette–Guérin (BCG)-induced CXCL8 production is mediated through PKCα-dependent activation of the IKKαβ signaling pathway in epithelial cells

Upon contact with airway epithelial cells, mycobacteria activate several signal transduction events that are required for induction of NF-κB-dependent chemokine gene expression. However, downstream signaling pathways, especially that of Ca2+-dependent protein kinase C α (PKCα), and in particular, the identity of the IKKαβ signal pathway for CXCL8 secretion in Mycobacterium bovis BCG-induced epithelial cells are still unknown. In this study, we demonstrated that the phosphoinositide-phospholipase C (PI-PLC) downstream signaling pathway is involved in M. bovis BCG-induced CXCL8 release, since A549 cells pretreated with U73122, a PI-PLC inhibitor, inhibited CXCL8 release, whereas U73343 the inactive analog had no effect. In addition, our results demonstrated that M. bovis BCG-induced CXCL8 production by A549 cells was significantly blocked by using neomycin (another well-described inhibitor of PI-PLC with a different mechanism of action), Ro-32-0432 and Ro-31-8220 (two PKCα inhibitors), PP1 and PP2 (two potent and selective inhibitors of the Src-family tyrosine kinases), and Bay 11–7082 (an IκB phosphorylation inhibitor). We also demonstrated that M. bovis BCG can rapidly induce translocation of PKCα from the cytosol to the membrane, and that treatment of cells with M. bovis BCG caused time-dependent increases in phosphorylation of c-Src at tyrosine 416. Finally, our studies revealed that M. bovis BCG induced the association of c-Src and IKKαβ during the interaction of PKCα and IKKαβ. Altogether, these results represent the first evidence to date suggesting that M. bovis BCG activates the PI-PLC/PKCα/c-Src/IKKαβ signaling pathway to induce CXCL8 release in human epithelial cells.