Taxol selectively blocks microtubule dependent NF-κB activation by phorbol ester via inhibition of Iκ-Bα phosphorylation and degradation

Activation of the NF-kappa B transcription factors has been shown to be directly influenced by changes in the microtubule cytoskeleton network, To better understand cytoskeletal regulation of NF-kappa B, experiments were performed to determine whether the microtubule (MT) stabilizing agent taxol could modulate NF-kappa B activation in the presence of different NF-kappa B inducers. Pretreatment of murine NIH3T3 and human 293 cells with 5 mu M taxol resulted in complete inhibition of phorbol, 12-myristate, 13-acetate (PMA) mediated NF-kappa B activation, detected as the loss of DNA binding and reduced NF-kappa B dependent reporter gene activity. Furthermore, in COS-7 and NIH3T3 cells, PMA-induced I kappa B alpha turnover was dramatically reduced in taxol treated cells, mediated via the inhibition of I kappa-B alpha phosphorylation, However, taxol did not prevent TNF-alpha induced I kappa B alpha phosphorylation, degradation, or NF-kappa B activation, indicating that TNF-alpha acts through a microtubule-independent pathway. In vitro kinase assays with PMA stimulated cell extracts demonstrated that taxol reduced protein kinase C activity by 30%, thus implicating the loss of PKC activity as a possible regulatory target of taxol-mediated suppression of NF-kappa B, Since PMA causes modulation of cytoarchitecture through PKC activation, microtubule integrity and cell morphology was analysed by indirect immunofluorescence. Both PMA and nocodazole, a MT depolymerizing agent, caused microtubule depolymerization, whereas TNF-alpha did not alter MT integrity; concomitant taxol treatment blocked both nocodazole and PMA induced depolymerization of MTs, as well as NF-kappa B induction, thus demonstrating a link between microtubule depolymerization and NF-kappa B activation, These observations illustrate a novel biological activity of taxol as a selective inhibitor of NF-kappa B activity, suggesting a link between the state of microtubule integrity and gene regulation.