Abstract
Post-translational modifications of RelA play an important role in regulation of NF-κB activation. We previously demonstrated that in malignant hematopoietic cells, histone deacetylase inhibitors (HDACIs) induced RelA hyperacetylation and NF-κB activation, attenuating lethality. We now present evidence that IκB kinase (IKK) β-mediated RelA Ser-536 phosphorylation plays a significant functional role in promoting RelA acetylation, inducing NF-κB activation, and limiting HDACI lethality in human multiple myeloma (MM) cells. Immunoblot profiling revealed that although basal RelA phosphorylation varied in MM cells, Ser-536 phosphorylation correlated with IKK activity. Exposure to the pan-HDACIs vorinostat or LBH-589 induced phosphorylation of IKKα/β (Ser-180/Ser-181) and RelA (Ser-536) in MM cells, including cells expressing an IκBα "super-repressor," accompanied by increased RelA nuclear translocation, acetylation, DNA binding, and transactivation activity. These events were substantially blocked by either pan-IKK or IKKβ-selective inhibitors, resulting in marked apoptosis. Consistent with these events, inhibitory peptides targeting either the NF-κB essential modulator (NEMO) binding domain for IKK complex formation or RelA phosphorylation sites also significantly increased HDACI lethality. Moreover, IKKβ knockdown by shRNA prevented Ser-536 phosphorylation and significantly enhanced HDACI susceptibility. Finally, introduction of a nonphosphorylatable RelA mutant S536A, which failed to undergo acetylation in response to HDACIs, impaired NF-κB activation and increased cell death. These findings indicate that HDACIs induce Ser-536 phosphorylation of the NF-κB subunit RelA through an IKKβ-dependent mechanism, an action that is functionally involved in activation of the cytoprotective NF-κB signaling cascade primarily through facilitation of RelA acetylation rather than nuclear translocation.
Highlights
Given the broad spectrum of NF-B biologic functions, NF-B activity is likely to be controlled by highly regulated mechanisms
Histone deacetylase inhibitors (HDACIs) represent a novel class of anti-cancer agents [48], which, in addition to effects on gene expression mediated by inhibition of histone deacetylation, block deacetylation of various non-histone proteins, including transcription factors such as NF-B [26]
The functional role that RelA phosphorylation might play in the response of transformed cells to HDACIs has not yet been defined
Summary
Human Multiple Myeloma Cell Lines—RPMI8226, U266, and NCI-H929 cells were purchased from the ATCC (Manassas, VA). The selective IKK inhibitor IKK-2 inhibitor IV ([5-(pfluorophenyl)-2-ureido]thiophene-3-carboxamide) [37], the pan IKK inhibitor Bay 11-7082, and the PP1/PP2A inhibitor calyculin-A [38] were purchased from Calbiochem and Alexis (San Diego), respectively These agents were dissolved in DMSO and stored at Ϫ80 °C under light-protected conditions and subsequently diluted with serum-free RPMI medium prior to use. Western Blot Analysis—Samples from whole cell pellets were prepared, and 30 g/condition of proteins was subjected to Western blot analysis as described previously in detail [28]. For in vivo analysis of NF-B activity, female athymic NCrnu/nu mice (The Jackson Laboratory) were inoculated subcutaneously at the flank with 5 ϫ 106 RPMI8226 cells stably transfected with 3ϫB luciferase reporter. Statistical Analysis—For analysis of cell death/apoptosis, RelA-DNA binding, and NF-B luciferase reporter assay, values represent the means Ϯ S.D. for at least three separate experiments performed in triplicate. Analysis of synergism was performed according to median dose effect analysis using the software program CalcuSyn (Biosoft, Ferguson, MO)
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