Richard B. Gaynor

Therapeutic potential of inhibition of the NF-κB pathway in the treatment of inflammation and cancer

NF-κB comprises a family of inducible transcription factors that serve as important regulators of the host immune and inflammatory response. In addition, NF-κB is also involved in protecting cells from undergoing apoptosis in response to DNA damage or cytokine treatment. Stimulation of the NF-κB pathway is mediated by diverse signal transduction cascades. These signals activate the IκB kinases, IKKα and IKKβ, which phosphorylate inhibitory proteins known as IκB to result in their ubiquitination and degradation by the proteasome. The degradation of IκB results in the translocation of NF-κB from the cytoplasm to the nucleus where it activates the expression of specific cellular genes. As we better understand the regulation of the NF-κB pathway, the potential for inhibiting this pathway has received attention. Agents that inhibit this pathway, such as glucocorticoids and aspirin, can reduce the inflammatory response, while other agents such as dominant negative IκB proteins potentiate the effects of chemotherapy and radiation therapy in the treatment of cancer. Here, we discuss cellular genes and disease states associated with activation of the NF-κB pathway and consider therapeutic strategies to prevent the prolonged activation of the NF-κB pathway.

Histone H3 phosphorylation by ikk-α is critical for cytokine-induced gene expression

Cytokine-induced activation of the IκB kinases (IKK) IKK-α and IKK-β is a key step involved in the activation of the NF-κB pathway. Gene-disruption studies of the murine IKK genes have shown that IKK-β, but not IKK-α, is critical for cytokine-induced IκB degradation. Nevertheless, mouse embryo fibroblasts deficient in IKK-α are defective in the induction of NF-κB-dependent transcription. These observations raised the question of whether IKK-α might regulate a previously undescribed step to activate the NF-κB pathway that is independent of its previously described cytoplasmic role in the phosphorylation of IκBα. Here we show that IKK-α functions in the nucleus to activate the expression of NF-κB-responsive genes after stimulation with cytokines. IKK-α interacts with CREB-binding protein and in conjunction with Rel A is recruited to NF-κB-responsive promoters and mediates the cytokine-induced phosphorylation and subsequent acetylation of specific residues in histone H3. These results define a new nuclear role of IKK-α in modifying histone function that is critical for the activation of NF-κB-directed gene expression.

Sulindac Inhibits Activation of the NF-κB Pathway

Sulindac is a non-steroidal anti-inflammatory agent that is related both structurally and pharmacologically to indomethacin. In addition to its anti-inflammatory properties, sulindac has been demonstrated to have a role in the prevention of colon cancer. Both its growth inhibitory and anti-inflammatory properties are due at least in part to its ability to decrease prostaglandin synthesis by inhibiting the activity of cyclooxygenases. Recently, we demonstrated that both aspirin and sodium salicylate, but not indomethacin, inhibited the activity of an IκB kinase β (IKKβ) that is required to activate the nuclear factor-κB (NF-κB) pathway. In this study, we show that sulindac and its metabolites sulindac sulfide and sulindac sulfone can also inhibit the NF-κB pathway in both colon cancer and other cell lines. Similar to our previous results with aspirin, this inhibition is due to sulindac-mediated decreases in IKKβ kinase activity. Concentrations of sulindac that inhibit IKKβ activity also reduce the proliferation of colon cancer cells. These results suggest that the growth inhibitory and anti-inflammatory properties of sulindac may be regulated in part by inhibition of kinases that regulate the NF-κB pathway.

TAK1 is Critical for IκB Kinase-mediated Activation of the NF-κB Pathway

Cytokine treatment stimulates the IκB kinases, IKKα and IKKβ, which phosphorylate the IκB proteins, leading to their degradation and activation of NF-κB regulated genes. A clear definition of the specific roles of IKKα and IKKβ in activating the NF-κB pathway and the upstream kinases that regulate IKK activity remain to be elucidated. Here, we utilized small interfering RNAs (siRNAs) directed against IKKα, IKKβ and the upstream regulatory kinase TAK1 in order to better define their roles in cytokine-induced activation of the NF-κB pathway. In contrast to previous results with mouse embryo fibroblasts lacking either IKKα or IKKβ, which indicated that only IKKβ is involved in cytokine-induced NF-κB activation, we found that both IKKα and IKKβ were important in activating the NF-κB pathway. Furthermore, we found that the MAP3K TAK1, which has been implicated in IL-1-induced activation of the NF-κB pathway, was also critical for TNFα-induced activation of the NF-κB pathway. TNFα activation of the NF-κB pathway is associated with the inducible binding of TAK1 to TRAF2 and both IKKα and IKKβ. This analysis further defines the distinct in vivo roles of IKKα, IKKβ and TAK1 in cytokine-induced activation of the NF-κB pathway.

Role of the NF-kB Pathway in the Pathogenesis of Human Disease States

The NF-kappaB family consists of a group of inducible transcription factors which regulate immune and inflammatory responses and protect cells from undergoing apoptosis in response to cellular stress. A number of signal transduction cascades can activate the NF-kappaB pathway to result in the translocation of the NF-kappaB proteins from the cytoplasm to the nucleus where they activate the expression of specific cellular genes. In this review, we discuss cellular genes which are regulated by NF-kappaB and disease states which are associated with constitutive activation of the NF-kappaB pathway. Strategies to prevent prolonged activation of the NF-kappaB pathway are also discussed.

HTLV-I Tax Protein Binds to MEKK1 to Stimulate IκB Kinase Activity and NF-κB Activation

Abstract NF-κB, a key regulator of the cellular inflammatory and immune response, is activated by the HTLV-I transforming and transactivating protein Tax. We show that Tax binds to the amino terminus of the protein kinase MEKK1, a component of an IκB kinase complex, and stimulates MEKK1 kinase activity. Tax expression increases the activity of IκB kinase β (IKKβ) to enhance phosphorylation of serine residues in IκBα that lead to its degradation. Dominant negative mutants of both IKKβ and MEKK1 prevent Tax activation of the NF-κB pathway. Furthermore, recombinant MEKK1 stimulates IKKβ phosphorylation of IκBα. Thus, Tax-mediated increases in NF-κB nuclear translocation result from direct interactions of Tax and MEKK1 leading to enhanced IKKβ phosphorylation of IκBα.

Role of the TAB2-related protein TAB3 in IL-1 and TNF signaling

The cytokines IL‐1 and TNF induce expression of a series of genes that regulate inflammation through activation of NF‐κB signal transduction pathways. TAK1, a MAPKKK, is critical for both IL‐1‐ and TNF‐induced activation of the NF‐κB pathway. TAB2, a TAK1‐binding protein, is involved in IL‐1‐induced NF‐κB activation by physically linking TAK1 to TRAF6. However, IL‐1‐induced activation of NF‐κB is not impaired in TAB2‐deficient embryonic fibroblasts. Here we report the identification and characterization of a novel protein designated TAB3, a TAB2‐like molecule that associates with TAK1 and can activate NF‐κB similar to TAB2. Endogenous TAB3 interacts with TRAF6 and TRAF2 in an IL‐1‐ and a TNF‐dependent manner, respectively. Further more, IL‐1 signaling leads to the ubiquitination of TAB2 and TAB3 through TRAF6. Cotransfection of siRNAs directed against both TAB2 and TAB3 inhibit both IL‐1‐ and TNF‐induced activation of TAK1 and NF‐κB. These results suggest that TAB2 and TAB3 function redundantly as mediators of TAK1 activation in IL‐1 and TNF signal transduction.

RNA Interference Directed against Viral and Cellular Targets Inhibits Human Immunodeficiency Virus Type 1 Replication

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) gene expression is regulated by both cellular transcription factors and Tat. The ability of Tat to stimulate transcriptional elongation is dependent on its binding to TAR RNA in conjunction with cyclin T1 and CDK9. A variety of other cellular factors that bind to the HIV-1 long terminal repeat, including NF-κB, SP1, LBP, and LEF, are also important in the control of HIV-1 gene expression. Although these factors have been demonstrated to regulate HIV-1 gene expression by both genetic and biochemical analysis, in most cases a direct in vivo demonstration of their role on HIV-1 replication has not been established. Recently, the efficacy of RNA interference in mammalian cells has been shown utilizing small interfering RNAs (siRNAs) to result in the specific degradation of host mRNAs and decreases the levels of their corresponding proteins. In this study, we addressed whether siRNAs directed against either HIV-1 tat or reverse transcriptase or the NF-κB p65 subunit could specifically decrease the levels of these proteins and thus alter HIV-1 replication. Our results demonstrate the specificity of siRNAs for decreasing the expression of these viral and cellular proteins and inhibiting HIV-1 replication. These studies suggest that RNA interference is useful in exploring the biological role of cellular and viral regulatory factors involved in the control of HIV-1 gene expression.

Methylation of SPT5 Regulates Its Interaction with RNA Polymerase II and Transcriptional Elongation Properties

SPT5 and its binding partner SPT4 function in both positively and negatively regulating transcriptional elongation. The demonstration that SPT5 and RNA polymerase II are targets for phosphorylation by CDK9/cyclin T1 indicates that posttranslational modifications of these factors are important in regulating the elongation process. In this study, we utilized a biochemical approach to demonstrate that SPT5 was specifically associated with the protein arginine methyltransferases PRMT1 and PRMT5 and that SPT5 methylation regulated its interaction with RNA polymerase II. Specific arginine residues in SPT5 that are methylated by these enzymes were identified and demonstrated to be important in regulating its promoter association and subsequent effects on transcriptional elongation. These results suggest that methylation of SPT5 is an important posttranslational modification that is involved in regulating its transcriptional elongation properties in response to viral and cellular factors.

Domains in the SPT5 Protein That Modulate Its Transcriptional Regulatory Properties

ABSTRACT SPT5 and its binding partner SPT4 regulate transcriptional elongation by RNA polymerase II. SPT4 and SPT5 are involved in both 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB)-mediated transcriptional inhibition and the activation of transcriptional elongation by the human immunodeficiency virus type 1 (HIV-1) Tat protein. Recent data suggest that P-TEFb, which is composed of CDK9 and cyclin T1, is also critical in regulating transcriptional elongation by SPT4 and SPT5. In this study, we analyze the domains of SPT5 that regulate transcriptional elongation in the presence of either DRB or the HIV-1 Tat protein. We demonstrate that SPT5 domains that bind SPT4 and RNA polymerase II, in addition to a region in the C terminus of SPT5 that contains multiple heptad repeats and is designated CTR1, are critical for in vitro transcriptional repression by DRB and activation by the Tat protein. Furthermore, the SPT5 CTR1 domain is a substrate for P-TEFb phosphorylation. These results suggest that C-terminal repeats in SPT5, like those in the RNA polymerase II C-terminal domain, are sites for P-TEFb phosphorylation and function in modulating its transcriptional elongation properties.