Kevin N. Pennington

Transcription of the RelB gene is regulated by NF-κB

RelA and RelB are two members of the NF-κB family that differ structurally and functionally. While RelA is regulated through its cytosolic localization by inhibitor proteins or IκB and not through transcriptional mechanisms, the regulation of RelB is poorly understood. In this study we demonstrate that stimuli (TNF or LPS) lead within minutes to the nuclear translocation of RelA, but require hours to result in the nuclear translocation of RelB. The delayed nuclear translocation of RelB correlates with increases in its protein synthesis which are secondary to increases in RelB gene transcription. RelA is alone sufficient to induce RelB gene transcription and to mediate the stimuli-driven increase in RelB transcription. Cloning and characterization of the RelB 5′ untranslated gene region indicates that RelB transcription is dependent on a TATA-less promoter containing two NF-κB binding sites. One of the NF-κB sites is primarily involved in the binding of p50 while the other one in the binding and transactivation by RelA and also RelB. Lastly, it is observed that p21, a protein involved in cell cycle control and oncogenesis known to be regulated by NF-κB, is upregulated at the transcriptional level by RelB. Thus, RelB is regulated at least at the level of transcription in a RelA and RelB dependent manner and may exert an important role in p21 regulation.

Protein Kinase C and Calcineurin Synergize to Activate IκB Kinase and NF-κB in T Lymphocytes

The nuclear factor of κB (NF-κB) is a ubiquitous transcription factor that is key in the regulation of the immune response and inflammation. T cell receptor (TCR) cross-linking is in part required for activation of NF-κB, which is dependent on the phosphorylation and degradation of IκBα. By using Jurkat and primary human T lymphocytes, we demonstrate that the simultaneous activation of two second messengers of the TCR-initiated signal transduction, protein kinase C (PKC) and calcineurin, results in the synergistic activation of the IκBα kinase (IKK) complex but not of another putative IκBα kinase, p90 rsk . We also demonstrate that the IKK complex, but not p90 rsk , is responsible for thein vivo phosphorylation of IκBα mediated by the co-activation of PKC and calcineurin. Each second messenger is necessary, as inhibition of either one reverses the activation of the IKK complex and IκBα phosphorylation in vivo. Overexpression of dominant negative forms of IKKα and -β demonstrates that only IKKβ is the target for PKC and calcineurin. These results indicate that within the TCR/CD3 signal transduction pathway both PKC and calcineurin are required for the effective activation of the IKK complex and NF-κB in T lymphocytes.

Protein Kinase Cα (PKCα) Acts Upstream of PKCθ To Activate IκB Kinase and NF-κB in T Lymphocytes

ABSTRACT NF-κB is an ubiquitous transcription factor that is a key in the regulation of the immune response and inflammation. T-cell receptor (TCR) cross-linking leads to NF-κB activation, an IκB kinase (IKK)-dependent process. However, the upstream kinases that regulate IKK activity following TCR activation remain to be fully characterized. Herein, we demonstrate using genetic analysis, pharmacological inhibition, and RNA interference (RNAi) that the conventional protein kinase C (PKC) isoform PKCα, but not PKCβ1, is required for the activation of the IKK complex following T-cell activation triggered by CD3/CD28 cross-linking. We find that in the presence of Ca2+ influx, the catalytically active PKCαA25E induces IKK activity and NF-κB-dependent transcription; which is abrogated following the mutations of two aspartates at positions 246 and 248, which are required for Ca2+ binding to PKCα and cell membrane recruitment. Kinetic studies reveal that an early phase (1 to 5 min) of IKK activation following TCR/CD28 cross-linking is PKCα dependent and that a later phase (5 to 25 min) of IKK activation is PKCθ dependent. Activation of IKK- and NF-κB-dependent transcription by PKCαA25E is abrogated by the PKCθ inhibitor rottlerin or the expression of the kinase-inactive form of PKCθ. Taken together, our results suggest that PKCα acts upstream of PKCθ to activate the IKK complex and NF-κB in T lymphocytes following TCR activation.

IκB Kinase-Dependent Chronic Activation of NF-κB Is Necessary for p21WAF1/Cip1 Inhibition of Differentiation-Induced Apoptosis of Monocytes

ABSTRACT The molecular mechanisms regulating monocyte differentiation to macrophages remain unknown. Although the transcription factor NF-κB participates in multiple cell functions, its role in cell differentiation is ill defined. Since differentiated macrophages, in contrast to cycling monocytes, contain significant levels of NF-κB in the nuclei, we questioned whether this transcription factor is involved in macrophage differentiation. Phorbol 12-myristate 13-acetate (PMA)-induced differentiation of the promonocytic cell line U937 leads to persistent NF-κB nuclear translocation. We demonstrate here that an increased and persistent IKK activity correlates with monocyte differentiation leading to persistent NF-κB activation secondary to increased IκBα degradation via the IκB signal response domain (SRD). Promonocytic cells stably overexpressing an IκBα transgene containing SRD mutations fail to activate NF-κB and subsequently fail to survive the PMA-induced macrophage differentiation program. The differentiation-induced apoptosis was found to be dependent on tumor necrosis factor alpha. The protective effect of NF-κB is mediated through p21WAF1/Cip1, since this protein was found to be regulated in an NF-κB-dependent manner and to confer survival features during macrophage differentiation. Therefore, NF-κB plays a key role in cell differentiation by conferring cell survival that in the case of macrophages is mediated through p21WAF1/Cip1.

IκB Kinase Subunits α and γ Are Required for Activation of NF-κB and Induction of Apoptosis by Mammalian Reovirus

ABSTRACT Reoviruses induce apoptosis both in cultured cells and in vivo. Apoptosis plays a major role in the pathogenesis of reovirus encephalitis and myocarditis in infected mice. Reovirus-induced apoptosis is dependent on the activation of transcription factor NF-κB and downstream cellular genes. To better understand the mechanism of NF-κB activation by reovirus, NF-κB signaling intermediates under reovirus control were investigated at the level of Rel, IκB, and IκB kinase (IKK) proteins. We found that reovirus infection leads initially to nuclear translocation of p50 and RelA, followed by delayed mobilization of c-Rel and p52. This biphasic pattern of Rel protein activation is associated with the degradation of the NF-κB inhibitor IκBα but not the structurally related inhibitors IκBβ or IκBε. Using IKK subunit-specific small interfering RNAs and cells deficient in individual IKK subunits, we demonstrate that IKKα but not IKKβ is required for reovirus-induced NF-κB activation and apoptosis. Despite the preferential usage of IKKα, both NF-κB activation and apoptosis were attenuated in cells lacking IKKγ/Nemo, an essential regulatory subunit of IKKβ. Moreover, deletion of the gene encoding NF-κB-inducing kinase, which is known to modulate IKKα function, had no inhibitory effect on either response in reovirus-infected cells. Collectively, these findings indicate a novel pathway of NF-κB/Rel activation involving IKKα and IKKγ/Nemo, which together mediate the expression of downstream proapoptotic genes in reovirus-infected cells.

Human Immunodeficiency Virus Reactivation by Phorbol Esters or T-Cell Receptor Ligation Requires both PKCα and PKCθ

ABSTRACT Latently human immunodeficiency virus (HIV)-infected memory CD4+ T cells represent the major obstacle to eradicating HIV from infected patients. Antigens, T-cell receptor (TCR) ligation, and phorbol esters can reactivate HIV from latency in a protein kinase C (PKC)-dependent manner; however, it is unknown which specific PKC isoforms are required for this effect. We demonstrate that constitutively active (CA) forms of both PKCθ, PKCθA148E, and PKCα, PKCαA25E, induce HIV long terminal repeat (LTR)-dependent transcription in Jurkat and primary human CD4+ T cells and that both PKCθA148E and PKCαA25E cause HIV reactivation in J1.1 T cells. Suppression of both PKCα and PKCθ with short hairpinned (sh) RNA inhibited CD3/CD28-induced HIV LTR-dependent transcription and HIV reactivation in J1.1 T cells. Both prostratin and phorbol myristate 13-acetate induced HIV LTR-dependent transcription and HIV reactivation in J1.1 T cells that was blocked by shRNA against either PKCα or PKCθ. Since suppression of PKCα and PKCθ together has no greater inhibitory effect on HIV reactivation than inhibition of PKCα alone, our data confirm that PKCα and PKCθ act in sequence. The requirement for PKCα and PKCθ for prostratin-induced HIV reactivation and the ability of selective PKCα or PKCθ agonists to induce HIV transcription indicate that these PKC isoforms are important targets for therapeutic drug design.

Site-specific Monoubiquitination of IκB Kinase IKKβ Regulates Its Phosphorylation and Persistent Activation

Transcription factor NF-κB governs the expression of multiple genes involved in cell growth, immunity, and inflammation. Nuclear translocation of NF-κB is regulated from the cytoplasm by IκB kinase-β (IKKβ), which earmarks inhibitors of NF-κB for polyubiquination and proteasome-mediated degradation. Activation of IKKβ is contingent upon signal-induced phosphorylation of its T loop at Ser-177/Ser-181. T loop phosphorylation also renders IKKβ a substrate for monoubiquitination in cells exposed to chronic activating cues, such as the Tax oncoprotein or sustained signaling through proinflammatory cytokine receptors. Here we provide evidence that the T loop-proximal residue Lys-163 in IKKβ serves as a major site for signal-induced monoubiquitination with significant regulatory potential. Conservative replacement of Lys-163 with Arg yielded a monoubiquitination-defective mutant of IKKβ that retains kinase activity in Tax-expressing cells but is impaired for activation mediated by chronic signaling from the type 1 receptor for tumor necrosis factor-α. Phosphopeptide mapping experiments revealed that the Lys-163 → Arg mutation also interferes with proper in vivo but not in vitro phosphorylation of cytokine-responsive serine residues located in the distal C-terminal region of IKKβ. Taken together, these data indicate that chronic phosphorylation of IKKβ at Ser-177/Ser-181 leads to monoubiquitin attachment at nearby Lys-163, which in turn modulates the phosphorylation status of IKKβ at select C-terminal serines. This mechanism for post-translational cross-talk may play an important role in the control of IKKβ signaling during chronic inflammation.