Yumi Yamamoto

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.

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.

Protein Phosphatase 2Cβ Association with the IκB Kinase Complex Is Involved in Regulating NF-κB Activity

The NF-κB pathway is important in the control of the immune and inflammatory response. One of the critical events in the activation of this pathway is the stimulation of the IκB kinases (IKKs) by cytokines such as tumor necrosis factor-α and interleukin-1. Although the mechanisms that modulate IKK activation have been studied in detail, much less is known about the processes that down-regulate its activity following cytokine treatment. In this study, we utilized biochemical fractionation and mass spectrometry to demonstrate that protein phosphatase 2Cβ (PP2Cβ) can associate with the IKK complex. PP2Cβ association with the IKK complex led to the dephosphorylation of IKKβ and decreased its kinase activity. The binding of PP2Cβ to IKKβ was decreased at early times post-tumor necrosis factor-α treatment and was restored at later times following treatment with this cytokine. Experiments utilizing siRNA directed against PP2Cβ demonstrated an in vivo role for this phosphatase in decreasing IKK activity at late times following cytokine treatment. These studies are consistent with the ability of PP2Cβ to down-regulate cytokine-induced NF-κB activation by altering IKK activity.

IκB Kinase α (IKKα) Regulation of IKKβ Kinase Activity

ABSTRACT Two related kinases, IκB kinase α (IKKα) and IKKβ, phosphorylate the IκB proteins, leading to their degradation and the subsequent activation of gene expression by NF-κB. IKKβ has a much higher level of kinase activity for the IκB proteins than does IKKα and is more critical than IKKα in modulating tumor necrosis factor alpha activation of the NF-κB pathway. These results indicate an important role for IKKβ in activating the NF-κB pathway but leave open the question of the role of IKKα in regulating this pathway. In the current study, we demonstrate that IKKα directly phosphorylates IKKβ. Moreover, IKKα either directly or indirectly enhances IKKβ kinase activity for IκBα. Finally, transfection studies to analyze NF-κB-directed gene expression suggest that IKKα is upstream of IKKβ in activating the NF-κB pathway. These results indicate that IKKα, in addition to its previously described ability to phosphorylate IκBα, can increase the ability of IKKβ to phosphorylate IκBα.

IKKα Regulates Estrogen-induced Cell Cycle Progression by Modulating E2F1 Expression

The IκB kinase (IKK) complex consists of the catalytic subunits IKKα and IKKβ and a regulatory subunit, IKKγ/NEMO. Even though IKKα and IKKβ share significant sequence similarity, they have distinct biological roles. It has been demonstrated that IKKs are involved in regulating the proliferation of both normal and tumor cells, although the mechanisms by which they function in this process remain to be better defined. In this study, we demonstrate that IKKα, but not IKKβ, is important for estrogen-induced cell cycle progression by regulating the transcription of the E2F1 gene as well as other E2F1-responsive genes, including thymidine kinase 1, proliferating cell nuclear antigen, cyclin E, and cdc25A. The role of IKKα in regulating E2F1 was not the result of reduced levels of cyclin D1, as overexpression of this gene could not overcome the effects of IKKα knock-down. Furthermore, estrogen treatment increased the association of endogenous IKKα and E2F1, and this interaction occurred on promoters bound by E2F1. IKKα also potentiated the ability of p300/CBP-associated factor to acetylate E2F1. Taken together, these data suggest a novel mechanism by which IKKα can influence estrogen-mediated cell cycle progression through its regulation of E2F1.

IKKα Regulates the Mitotic Phase of the Cell Cycle by Modulating Aurora A Phosphorylation

The IKK complex includes two catalytic components, IKKα and IKKβ, in addition to the scaffold protein IKKγ/NEMO. Even though IKKα and IKKβ share significant sequence homology, they have distinct biological roles with IKKβ regulates the classical pathway of NF-κB activation and IKKα regulates the alternative pathways. In addition, it has been shown that the IKKs regulate the proliferation of both normal and tumor cells; however, the mechanisms by which the IKKs regulate the cell cycle remain to be further defined. Here, we demonstrate that IKKα, but not IKKβ, has role in regulating the M phase of the cell cycle. IKKα siRNA knock-down resulted in increased numbers of cells in the G2/M phase of the cell cycle as compared to control and IKKβ siRNA transfected HeLa cells. This effect was associated with upregulation of cyclin B1 and Plk1 protein levels and increased histone H3 phosphorylation, consistent with a potential role of IKKα in the regulation of M phase regulatory factors. IKKα was found to be associated with Aurora A in the centrosome and regulate Aurora A phosphorylation at threonine residue 288, a site which is important in modulating its kinase activity. Taken together, these data provide the evidence that IKKα regulates the M phase of the cell cycle by modulating Aurora A phosphorylation and activation leading to the regulation of the M phase of the cell cycle.

Tools to Interfere with NF-κB Activation

The NF-κB-signaling pathway has attracted widespread interest due to its fundamental role in the regulation of immune and inflammatory responses and in the control of cellular proliferation. A number of steps in the NF-κB pathway are potential targets for drug therapy to inhibit the activity of the pathway. In fact, some of the agents that inhibit these steps in the NF-κB pathway are being tested in clinical trials for the treatment of inflammatory disorders and cancer. A better understanding of the regulation of the NF-κB-signaling pathway has also led to a re-evaluation of the function of several conventional anti-inflammatory and anticancer drugs and provided new insights into the regulation of NF-κB. In this chapter, we review recent progress on key steps involved in the control of the NF-κB pathway and agents which interfere with NF-κB activation. The therapeutic potential of these agents in inhibiting the inflammatory response and increasing the efficacy of cancer therapy are also addressed.