NF-κB Protects HIV-1-Infected Myeloid Cells from Apoptosis

AbstractHIV-1 infection of primary monocytic cells and myeloid cell lines results in sustained NF-κB activation. Recently, NF-κB induction has been shown to play a role in protecting cells from programmed cell death. In the present study, we sought to investigate whether constitutive NF-κB activity in chronically HIV-1-infected promonocytic U937 (U9-IIIB) and myeloblastic PLB-985 (PLB-IIIB) cells affects apoptotic signaling. TNFα and cycloheximide caused infected cells to undergo apoptosis more rapidly than parental U937 and PLB-985 cells. Inhibition of TNFα-induced NF-κB activation using the antioxidantN-acetylcysteine (NAC) resulted in increased apoptosis in both U937 and U9-IIIB cells, while preactivation of NF-κB with the non-apoptotic inducer IL-1β caused a relative decrease in apoptosis. Inhibition of constitutive NF-κB activity in U9-IIIB and PLB-IIIB cells also induced apoptosis, suggesting that NF-κB protects cells from a persistent apoptotic signal. TNFα plus NAC treatment resulted in a marked decrease in Bcl-2 protein levels in HIV-1-infected cells, coupled with an increase in Bax protein compared to uninfected cells, suggesting that the difference in susceptibility to TNFα-induced apoptosis may relate to the differences in relative levels of Bcl-2 and Bax. The protective role of NF-κB in blocking TNFα- and HIV-1-induced apoptosis was supported by studies in Jurkat T cells engineered to express IκBα repressor mutants (TD-IκB) under the control of a tetracycline-responsive promoter. Cells underwent apoptosis in response to TNFα only when NF-κB activation was inhibited by TD-IκB expression. As was observed for the U9-IIIB cells, TNFα treatment also induced a marked decrease in Bcl-2 protein levels in TD-IκB expressing cells. These experiments demonstrate that apoptotic signaling is perturbed in HIV-1-infected U9-IIIB cells and indicate that NF-κB activation may play an additional protective role against HIV-1-induced apoptosis in myeloid cells

Mutants of IkBa interfere with NF-kB regulated gene expression and HIV-1 replication

Human immunodeficiency virus type 1 (HIV-1) utilizes the NF-kappaB/Rel pathway to mediate, in part, its transcriptional regulation through NF-kappaB binding sites present in the HIV-1 LTR. In unstimulated cells, DNA binding NF-kappaB sub-units are retained in the cytoplasm by inhibitory IkappaB proteins. Upon stimulation by cytokines, viruses or dsRNA, IkappaBalpha is rapidly phosphorylated and degraded, resulting in the release of NF-kappaB and the subsequent increase in NF-kappaB-regulated gene expression.The effect of transdominant mutants of IkappaBalpha (TD-IkappaBalpha) on the synergistic activation of the HIV-1 LTR by TNFalpha and the HIV-1 transactivator Tat, was examined in Jurkat T cells. Co-expression of WT IkappaBalpha and TD-IkappaBalpha inhibited Tat-TNF activation of HIV-1 LTR in a dose dependent manner. TD-IkappaBalpha also effectively inhibited HIV-1 multiplication in a single cycle infection model in COS-1 cells. To examine the effect of inducible expression of TD-IkappaBalpha on de novo HIV-1 multiplication, we generated Jurkat T cells inducibly expressing TD-IkappaBalpha by doxycycline (Dox). Dox induction of TD-IkappaBalpha dramatically reduced both NF-kappaB DNA binding activity and LTR directed gene activity. The time course of de novo HIV-1 infection was altered by Dox induction of TD-IkappaBalpha in Jurkat cells, resulting in a dramatic inhibition of HIV-1 multiplication.Interestingly, induced expression of TD-IkappaBalpha also progressively decreased the expression of endogenous IkappaBalpha to undetectable levels by 24h after induction, indicating that TD-IkappaBalpha was able to repress endogenous NF-kappaB dependent gene expression. We also demonstrated that production of TD-IkappaBalpha reduced endogenous IkappaBalpha gene transcription, due to the continued cytoplasmic sequestration of RelA(p65) by TD-IkappaBalpha. In vivo genomic footprinting revealed stimuli-responsive protein-DNA binding not only to the -63 to -53 kappaB1 site but also to the adjacent -44 to -36 Spl site of the IkappaBalpha promoter. In vivo protection of both sites was inhibited by Dox-inducible TD-IkappaBalpha expression. Prolonged NF-kappaB binding and a temporal switch in the composition of NF-kappaB complexes bound to the -63 to -53 kappaB1 site of the IkappaBalpha promoter were also observed; with time after induction, decreased levels of transcriptionally active p65-p50 and increased c-rel-p50 heterodimers were detected at the kappaB1 site. Mutation of either the kappaB1 site or the Sp1 site abolished transcription factor binding to the respective sites, as well as the inducibility of the IkappaBalpha promoter in transient transfection studies. Based on these results, specific activation of TD-IkappaBalpha can block NF-kappaB-regulated gene expression and HIV-1 infection

NF-κB/Rel Regulates Inhibitory and Excitatory Neuronal Function and Synaptic Plasticity

Changes in synaptic plasticity required for memory formation are dynamically regulated through opposing excitatory and inhibitory neurotransmissions. To explore the potential contribution of NF-κB/Rel to these processes, we generated transgenic mice conditionally expressing a potent NF-κB/Rel inhibitor termed IκBα superrepressor (IκBα-SR). Using the prion promoter-enhancer, IκBα-SR is robustly expressed in inhibitory GABAergic interneurons and, at lower levels, in excitatory neurons but not in glia. This neuronal pattern of IκBα-SR expression leads to decreased expression of glutamate decarboxylase 65 (GAD65), the enzyme required for synthesis of the major inhibitory neurotransmitter, γ-aminobutyric acid (GABA) in GABAergic interneurons. IκBα-SR expression also results in diminished basal GluR1 levels and impaired synaptic strength (input/output function), both of which are fully restored following activity-based task learning. Consistent with diminished GAD65-derived inhibitory tone and enhanced excitatory firing, IκBα-SR(+) mice exhibit increased late-phase long-term potentiation, hyperactivity, seizures, increased exploratory activity, and enhanced spatial learning and memory. IκBα-SR(+) neurons also express higher levels of the activity-regulated, cytoskeleton-associated (Arc) protein, consistent with neuronal hyperexcitability. These findings suggest that NF-κB/Rel transcription factors act as pivotal regulators of activity-dependent inhibitory and excitatory neuronal function regulating synaptic plasticity and memory