Contribution of GADD45 family members to cell death suppression by cellular Bcl-xL and cytomegalovirus vMIA

Mammalian cells and viruses encode inhibitors of programmed cell death that localize to mitochondria and suppress apoptosis initiated by a wide variety of inducers. Mutagenesis was used to probe the role of a predicted alpha-helical region within the hydrophobic antiapoptotic domain (AAD) of cytomegalovirus vMIA, the UL37x1 gene product. This region was found to be essential for cell death suppression activity. A screen for proteins that interacted with the AAD of functional vMIA but that failed to interact with mutants identified growth arrest and DNA damage 45 (GADD45alpha), a cell cycle regulatory protein activated by genotoxic stress, as a candidate cellular binding partner. GADD45alpha interaction required the AAD alpha-helical character that also dictated GADD45alpha-mediated enhancement of death suppression. vMIA mutants that failed to interact with GADD45alpha were completely nonfunctional in cell death suppression, and any of the three GADD45 family members (GADD45alpha, GADD45beta/MyD118, or GADD45gamma/OIG37/CR6/GRP17) was able to cooperate with vMIA; however, none influenced cell death when introduced into cells alone. GADD45alpha was found to increase vMIA protein levels comparably to treatment with protease inhibitors MG132 and ALLN. Targeted short interfering RNA knockdown of all three GADD45 family members maximally reduced vMIA activity, and this reduction was abrogated by additional GADD45alpha. Interestingly, GADD45 family members were also able to bind and enhance cell death suppression by Bcl-xL, a member of the Bcl-2 family of cell death suppressors, suggesting a direct cooperative link between apoptosis and the proteins that regulate the DNA damage response.     

NAMPT pathway is involved in the FOXO3a-mediated regulation of GADD45A expression

Nicotinamide-phosphoribosyltransferase (NAMPT), induced under stress, converts nicotinamide (NA) to nicotinamide mononucleotide (NMN), which then reacts with ATP to regenerate NAD(+). Despite the pivotal role of NAD(+) in metabolic reactions, the molecular pathways triggered by the intracellular changes in NAD(+) level in cancer cells are largely unknown. Growth Arrest and DNA Damage-inducible Gene (GADD45A) is regulated by multiple cellular factors which play an important role in the control of cell-cycle checkpoint, DNA repair process and signal transduction. The present study was designed to assess the significance of intracellular NAD(+) levels on the regulation of GADD45A expression. The results of this study demonstrate an inverse relationship between NAMPT expression and the regulation of GADD45A gene. Thus, an overexpression of NAMPT led to a decreased expression of GADD45A, whereas, the inhibition of NAMPT by the known chemical inhibitor FK866 increased the expression of GADD45A in cells. Inhibition of SIRT1, an NAD(+)-dependent deacetylase, using shRNA also led to an increased expression of GADD45A gene. In further experiments we could show that the increased expression of GADD45A under the above experimental conditions, NAMPT inhibition by FK866, involves acetylation of FOXO3a, a member of the important family of forkhead (FOXO) proteins. This knowledge should contribute to our understanding of the role played by NAMPT and SIRT1 in the regulation of GADD45A expression by FOXO3a.