Taxol selectively blocks microtubule dependent NF-kappa B activation by phorbol ester via inhibition of I kappa-B alpha phosphorylation and degradation

Activation of the NF-kappa B transcription factors has been shown to be dir ectly influenced by changes in the microtubule cytoskeleton network, To bet ter understand cytoskeletal regulation of NF-kappa B, experiments were perf ormed to determine whether the microtubule (MT) stabilizing agent taxol cou ld modulate NF-kappa B activation in the presence of different NF-kappa B i nducers. Pretreatment of murine NIH3T3 and human 293 cells with 5 mu M taxo l resulted in complete inhibition of phorbol, 12-myristate, 13-acetate (PMA ) mediated NF-kappa B activation, detected as the loss of DNA binding and r educed NF-kappa B dependent reporter gene activity. Furthermore, in COS-7 a nd NIH3T3 cells, PMA-induced I kappa B alpha turnover was dramatically redu ced 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 t hat TNF-alpha acts through a microtubule-independent pathway. In vitro kina se assays with PMA stimulated cell extracts demonstrated that taxol reduced protein kinase C activity by 30%, thus implicating the loss of PKC activit y 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 immunof luorescence. Both PMA and nocodazole, a MT depolymerizing agent, caused mic rotubule depolymerization, whereas TNF-alpha did not alter MT integrity; co ncomitant taxol treatment blocked both nocodazole and PMA induced depolymer ization of MTs, as well as NF-kappa B induction, thus demonstrating a link between microtubule depolymerization and NF-kappa B activation, These obser vations illustrate a novel biological activity of taxol as a selective inhi bitor of NF-kappa B activity, suggesting a link between the state of microt ubule integrity and gene regulation.