Dean W. Ballard

Cross-coupling of the NF-kappa B p65 and Fos/Jun transcription factors produces potentiated biological function.

NF‐kappa B and AP‐1 represent distinct mammalian transcription factors that target unique DNA enhancer elements. The heterodimeric NF‐kappa B complex is typically composed of two DNA binding subunits, NF‐kappa B p50 and NF‐kappa B p65, which share structural homology with the c‐rel proto‐oncogene product. Similarly, the AP‐1 transcription factor complex is comprised of dimers of the c‐fos and c‐jun proto‐oncogene products or of closely related proteins. We now demonstrate that the bZIP regions of c‐Fos and c‐Jun are capable of physically interacting with NF‐kappa B p65 through the Rel homology domain. This complex of NF‐kappa B p65 and Jun or Fos exhibits enhanced DNA binding and biological function via both the kappa B and AP‐1 response elements including synergistic activation of the 5′ long terminal repeat of the human immunodeficiency virus type 1. These findings support a combinatorial mechanism of gene regulation involving the unexpected cross‐coupling of two different classes of transcription factors to form novel protein complexes exhibiting potentiated biological activity.

The v-rel oncogene encodes a κB enhancer binding protein that inhibits NF-κB function

Abstract Studies of NF-κB suggest that this enhancer binding activity corresponds to a family of at least four proteins (p50, p55, p75, and p85) differentially induced with biphasic kinetics during T cell activation. While p55 and p50 are closely related to the 50 kd DNA binding subunit of NF-κB, p75 and p85 exhibit DNA binding properties that distinguish them from this 50 kd poly-peptide and its regulatory subunits IκB and p65. All four members of this κB-specific protein family are structurally related to the v-Rel oncoprotein and one, p85, appears identical to human c-Rel. v-Rel, but not nontransforming v-Rel mutants, binds to the κB enhancer and inhibits NF-κB-activated transcription from the IL-2 receptor α promoter and HIV-1 LTR. These findings suggest a Rel-related family of κB enhancer binding proteins and raise the possibility that the transforming activity of v-Rel is Iinked to its inhibitory action on cellular genes under NF-κB control.

NF-κB regulates Fas / APO-1 / CD95- and TCR-mediated apoptosis of T lymphocytes

The maintenance of lymphocyte homeostasis by apoptosis is a critical regulatory mechanism in the normal immune system. The transcription factor NF‐κB has been shown to play a role in protecting cells against death mediated by TNF. We show here that NF‐κB also has a role in regulating Fas / APO‐1 / CD95‐mediated death, a major pathway of peripheral T cell death. Transfection of Jurkat cells with the NF‐κB subunits p50 and p65 confers resistance against Fas‐mediated apoptosis. Reciprocally, inhibition of NF‐κB activation by a soluble peptide inhibitor or a dominant form of the NF‐κB inhibitor, IκB, makes the cells more susceptible to Fas‐mediated apoptosis. Furthermore, inhibition of NF‐κB activation by a soluble peptide inhibitor rendered a T cell hybridoma more susceptible to TCR‐mediated apoptosis. Correspondingly, transfection of p50 and p65 provided considerable protection from TCR‐mediated apoptosis. These observations were corroborated by studies on Fas‐mediated death in primary T cells. Concanavalin A‐activated cycling T cell blasts from mice that are transgenic for the dominant IκB molecule have increased sensitivity to Fas‐mediated apoptosis, associated with a down‐regulation of NF‐κB complexes in the nucleus. In addition, blocking TNF, itself a positive regulator of NF‐κB, with neutralizing antibodies renders the cells more susceptible to anti‐Fas‐mediated apoptosis. In summary, our results provide compelling evidence that NF‐κB protects against Fas‐mediated death and is likely to be an important regulator of T cell homeostasis and tolerance.

HIV-1, HTLV-1 and normal T-cell growth: transcriptional strategies and surprises

In this review, Warner Greene and colleagues discuss recent studies that have revealed an intriguing molecular interplay between two pathogenic human retroviruses, HIV-1 and HTLV-1, and certain cellular genes that normally control T-cell growth. Activation of T cells during an immune response results in the induction of select transcription factors that bind specifically to kappa B enhancer elements present in both the IL-2R alpha and IL-2 genes. Normal T-cell growth is in part regulated by the transient expression of these genes. The Tax protein of HTLV-1 induces these same kappa B-specific proteins, but in contrast to immune stimulation, HTLV-1 infection of T cells leads to constitutive IL-2R alpha gene expression and immortalization. A second human retrovirus, HIV-1, can subvert the normal action of the kappa B-binding factors induced by these immune stimuli. Rather than promoting T-cell growth, these factors may augment viral replication and promote T-cell death.

NF-κB is required for H- ras oncogene induced abnormal cell proliferation and tumorigenesis

Oncogenic mutations in ras lead to constitutive activation of downstream signaling pathways that modulate the activities of transcription factors. In turn, these factors control the expression of a subset of genes responsible for neoplastic cell transformation. Recent studies suggest that transcription factor NF-κB contributes to cell transformation by inhibiting the cell death signal activated by oncogenic Ras. In this study, inhibition of NF-κB activity by forced expression of a super-repressor form of IκBα, the major inhibitor of NF-κB, markedly decreased the growth rate, saturation density and tumorigenicity of oncogenic H-Ras transformed rat embryo fibroblasts. Such clonally isolated cells overexpressing IκBα super-repressor not only were viable but also exhibited no sign of spontaneous apoptosis. Inhibition of NF-κB in these cells was functionally demonstrated by both the loss of cytokine induced DNA binding activity and a profoundly increased sensitivity to cell death in response to TNF-α treatment. In contrast, inhibition of NF-κB activity in non-transformed fibroblasts had minimal effect on growth, but rendered the cells resistant to a subsequent transformation by H-ras oncogene. Similar results were also obtained with rat intestinal epithelial cells harboring an inducible ras oncogene. Taken together, these findings suggest that NF-κB activity is essential for abnormal cell proliferation and tumorigenicity activated by the ras oncogene and highlight an alternative functional role for NF-κB in oncogenic Ras-mediated cell transformation that is distinct from its anti-apoptotic activity.

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.

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.

HIV-1, HTLV-I and the interleukin-2 receptor: insights into transcriptional control.

In this study, we present direct evidence for the binding of the inducible cellular protein, HIVEN86A, to a 12-bp element present in the IL-2R alpha promoter. This element shares significant sequence similarity with the NF-kappa B binding sites present in the HIV-1 and kappa immunoglobulin enhancers. Transient transfection studies indicate that this kappa B element is both necessary and sufficient to confer tax or mitogen inducibility to a heterologous promoter. As summarized schematically in Fig. 5, the findings suggest that the HIVEN86A protein may play a central role in the activation of cellular genes required for T-cell growth, specifically the IL-2R alpha gene. In addition, the induced HIVEN86A protein also binds to a similar sequence present in the HIV-1 LTR leading to enhanced viral gene expression and ultimately T-cell death. Thus, mitogen activation of the HIV-1 LTR appears to involve the same inducible transcription factor(s) that normally regulates IL-2R alpha gene expression and T-cell growth. These findings further underscore the importance of the state of T-cell activation in the regulation of HIV-1 replication. Our results also demonstrate that HIVEN86A is induced by the tax protein of HTLV-I. Thus, in HTLV-I infected cells, normally the tight control of the transient expression of the IL-2R alpha gene is lost. The constitutive high-level display of IL-2 receptors may play a role in leukemic transformation mediated by HTLV-I (ATL). Apparently by the same mechanism, the tax protein also activates the HIV-1 LTR through the induction of HIVEN86A.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure and Regulation of the Human IL-2 Receptor

The growth of human T lymphocytes can be divided into two discrete stages resembling competence and progression. In the first stage, the binding of antigen or mitogen to specific T cell receptors triggers the transduction of intracellular signals leading to cellular activation1,2. In turn, these activation signals induce the de novo expression of genes mediating T cell growth including interleukin-2 (IL-2) and interleukin-2 receptors (IL-2R)3–5. The subsequent binding of IL-2 to its high affinity receptor promotes proliferation and clonal expansion of the cells originally stimulated by antigen.