Phosphorylation of NF-kB in calyculin A-induced apoptotic cells

Objectives: Calyculin A is a serine/threonine protein phosphatase inhibitor and plays important roles in cell growth, differentiation, inflammation, and transformation. Nuclear factor-κB (NF-κB) is a transcriptional factor and its pathway is a key component of cellular response to a variety of extracellular stimuli. In the present study, we investigated the effects of calyculin A on induction of apoptosis and regulation of phosphorylation of p65NF-κB in human osteosarcoma MG63 cells. Methods: MG63 cells were treated with calyculin A and apoptosis was determined by WST-8 assay, Hoechst staining, and DNA ladder formation. Expression of proapoptotic protein PTEN, FasL, and FasR mRNA was detected by RT-PCR. Phosphorylation was analyzed by Western blotting using anti-phospho-p65NF-κB antibodies. The phosphorylation status was further confirmed by using the phosphorylation site-mutated p65NF-κB gene transfected cells. To detect the localization of p65NF-κB, cell fractionation was done in MG63 cells and each fraction was analyzed by Western blotting. Degradation of IκBa was detected by immunoprecipitation and Western blotting. Localization of p65NF-κB in MG63 cells was also examined by immunocytochemistry. Results: Calyculin A induced apoptosis in MG63 cells in dose- and time-dependent manners and increased the PTEN, FasL, and FasR mRNA levels. Calyculin A enhanced the phosphorylation level of p65NF-κB on serine 536 after 2-h treatment. However, TNF-a stimulated the phosphorylation of p65NF-κB at serine 529 and 536 after 15-min treatment. Nuclear translocation of p65NF-κB occurred with TNF-a-treatment. Although IκBa was degraded within 30 min by TNF-a treatment, IκBa was not degraded by calyculin A. Conclusion: Our results indicated that calyculin A induces apoptosis in MG63 cells. Our results also demonstrated that mechanisms of calyculin A-induced phosphorylation of p65NF-κB and degradation of IκBa are different from that of the TNF-a