Involvement of nuclear factor-kappa B in bcl-xL-induced interleukin 8 expression in glioblastoma.
We recently reported that bcl-xL regulates interleukin 8 (CXCL8) protein expression and promoter activity in glioblastoma cells. In this paper we demonstrate that CXCL8 induction by bcl-xL is mediated through a nuclear factor-kappa B (NF-kB)-dependent mechanism. Mutational studies on the CXCL8 promoter showed that NF-kB binding site was required for bcl-xL-induced promoter activity and an enhanced nuclear expression of NF-kB subunits p65 and p50 was observed after bcl-xL over-expression. Electrophoretic mobility shift assay showed an increased DNA-binding activity of NF-kB in bcl-xL over-expressing cells and the use of specific antibodies confirmed the involvement of p65 and p50 in NF-kB activity on CXCL8 promoter sequence. NF-kB activity regulation by bcl-xL involved IkBalpha and IKK complex signaling pathway. In fact, bcl-xL over-expression induced a decrease of cytoplasmic expression of the IkBalpha protein, paralleled by an increase in the phosphorylation of the same IkBalpha and IKKalpha/beta. Moreover, the down-regulation of the ectopic or endogenous bcl-xL expression through RNA interference confirmed the ability of bcl-xL to modulate NF-kB pathway, and the transient expression of a degradation-resistant form of the cytoplasmic NF-kB inhibitor IkBalpha in bcl-xL transfectants confirmed the involvement of that inhibitor in bcl-xL-induced CXCL8 expression and promoter activity. In conclusion, our results demonstrate the role of NF-kB as the mediator of bcl-xL-induced CXCL8 up-regulation in glioblastoma cells.
Gabellini
Chiara
C
Experimental Chemotherapy Laboratory, Regina Elena Cancer Institute, Rome, Italy.
Castellini
Laura
L
Trisciuoglio
Daniela
D
Kracht
Michael
M
Zupi
Gabriella
G
Del Bufalo
Donatella
D
D015153:Blotting, Western; D045744:Cell Line, Tumor; D024202:Electrophoretic Mobility Shift Assay; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation / Q000502:physiology*; D005909:Glioblastoma / Q000378:metabolism*; D006801:Humans; D016209:Interleukin-8 / Q000096:biosynthesis* / Q000235:genetics; D016328:NF-kappa B / Q000378:metabolism*; D009389:Neovascularization, Pathologic / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection; D015854:Up-Regulation; D051020:bcl-X Protein / Q000378:metabolism*
C494811:BCL2L1 protein, human; D016209:Interleukin-8; D016328:NF-kappa B; D051020:bcl-X Protein