No abstract

Phosphorylation of inhibitor of kappa B (IkappaB) proteins is an important step in the activation of the transcription nuclear factor kappa B (NF-kappaB) and requires two IkappaB kinases, IKK1 (IKKalpha) and IKK2 (IKKbeta). Mice that are devoid of the IKK2 gene had extensive liver damage from apoptosis and died as embryos, but these mice could be rescued by the inactivation of the gene encoding tumor necrosis factor receptor 1. Mouse embryonic fibroblast cells that were isolated from IKK2-/- embryos showed a marked reduction in tumor necrosis factor-alpha (TNF-alpha)- and interleukin-1alpha-induced NF-kappaB activity and an enhanced apoptosis in response to TNF-alpha. IKK1 associated with NF-kappaB essential modulator (IKKgamma/IKKAP1), another component of the IKK complex. These results show that IKK2 is essential for mouse development and cannot be substituted with IKK1.

The potent transcriptional activities of Rel/NF-κB proteins are regulated in the cytoplasm and nucleus by the inhibitor, IκBα. The mechanism, by which IκBα can either sequester NF-κB in the cytoplasm or act as a nuclear post-induction repressor of NF-κB, is uncertain. We find that IκBα shuttles continuously between the nucleus and cytoplasm. This shuttling requires a previously unidentified CRM1-dependent nuclear export signal (NES) located within the N-terminal domain of IκBα at amino acids 45-55. Deletion or mutation of the N-terminal NES results in nuclear localization of IκBα. NF-κB (p65) association with IκBα affects steady-state localization but does not inhibit its shuttling. Endogenous complexes of IκBα-NF-κB shuttle and will accumulate in the nucleus when CRM1 export is blocked. We find TNFα can activate the nuclear IκBα-NF-κB complexes by the classical mechanism of proteasome-mediated degradation of IκBα. These studies reveal a more dynamic nucleocytoplasmic distribution for IκBα and NF-κB suggesting previously unknown strategies for regulating this ubiquitous family of transcription activators.

The possibility that bacteria may have evolved strategies to overcome host cell apoptosis was explored by using Rickettsia rickettsii, an obligate intracellular Gram-negative bacteria that is the etiologic agent of Rocky Mountain spotted fever. The vascular endothelial cell, the primary target cell during in vivo infection, exhibits no evidence of apoptosis during natural infection and is maintained for a sufficient time to allow replication and cell-to-cell spread prior to eventual death due to necrotic damage. Prior work in our laboratory demonstrated that R. rickettsii infection activates the transcription factor NF-B and alters expression of several genes under its control. However, when R. rickettsiiinduced activation of NF-B was inhibited, apoptosis of infected but not uninfected endothelial cells rapidly ensued. In addition, human embryonic fibroblasts stably transfected with a superrepressor mutant inhibitory subunit IB that rendered NF-B inactivatable also underwent apoptosis when infected, whereas infected wild-type human embryonic fibroblasts survived. R. rickettsii, therefore, appeared to inhibit host cell apoptosis via a mechanism dependent on NF-B activation. Apoptotic nuclear changes correlated with presence of intracellular organisms and thus this previously unrecognized proapoptotic signal, masked by concomitant NF-B activation, likely required intracellular infection. Our studies demonstrate that a bacterial organism can exert an antiapoptotic effect, thus modulating the host cell's apoptotic response to its own advantage by potentially allowing the host cell to remain as a site of infection.