Blockade of T-cell activation by dithiocarbamates involves novel mechanisms of inhibition of nuclear factor of activated T cells.

Dithiocarbamates (DTCs) have recently been reported as powerful inhibitors of NF-kappaB activation in a number of cell types. Given the role of this transcription factor in the regulation of gene expression in the inflammatory response, NF-kappaB inhibitors have been suggested as potential therapeutic drugs for inflammatory diseases. We show here that DTCs inhibited both interleukin 2 (IL-2) synthesis and membrane expression of antigens which are induced during T-cell activation. This inhibition, which occurred with a parallel activation of c-Jun transactivating functions and expression, was reflected by transfection experiments at the IL-2 promoter level, and involved not only the inhibition of NF-kappaB-driven reporter activation but also that of nuclear factor of activated T cells (NFAT). Accordingly, electrophoretic mobility shift assays (EMSAs) indicated that pyrrolidine DTC (PDTC) prevented NF-kappaB, and NFAT DNA-binding activity in T cells stimulated with either phorbol myristate acetate plus ionophore or antibodies against the CD3-T-cell receptor complex and simultaneously activated the binding of AP-1. Furthermore, PDTC differentially targeted both NFATp and NFATc family members, inhibiting the transactivation functions of NFATp and mRNA induction of NFATc. Strikingly, Western blotting and immunocytochemical experiments indicated that PDTC promoted a transient and rapid shuttling of NFATp and NFATc, leading to their accelerated export from the nucleus of activated T cells. We propose that the activation of an NFAT kinase by PDTC could be responsible for the rapid shuttling of the NFAT, therefore transiently converting the sustained transactivation of this transcription factor that occurs during lymphocyte activation, and show that c-Jun NH2-terminal kinase (JNK) can act by directly phosphorylating NFATp. In addition, the combined inhibitory effects on NFAT and NF-KB support a potential use of DTCs as immunosuppressants

The NF-κB and IκB proteins: New discoveries and insights

The transcription factor NF-κB has attracted widespread attention among researchers in many fields based on the following: its unusual and rapid regulation, the wide range of genes that it controls, its central role in immunological processes, the complexity of its subunits, and its apparent involvement in several diseases. A primary level of control for NF-κB is through interactions with an inhibitor protein called IκB. Recent evidence confirms the existence of multiple forms of IκB that appear to regulate NF-κB by distinct mechanisms. NF-κB can be activated by exposure of cells to LPS or inflammatory cytokines such as TNF or IL-1, viral infection or expression of certain viral gene products, UV irradiation, B or T cell activation, and by other physiological and nonphysiological stimuli. Activation of NF-κB to move into the nucleus is controlled by the targeted phosphorylation and subsequent degradation of IκB. Exciting new research has elaborated several important and unexpected findings that explain mechanisms involved in the activation of NF-κB. In the nucleus, NF-κB dimers bind to target DNA elements and activate transcription of genes encoding proteins involved with immune or inflammation responses and with cell growth control. Recent data provide evidence that NF-κB is constitutively active in several cell types, potentially playing unexpected roles in regulation of gene expression. In addition to advances in describing the mechanisms of NF-κB activation, excitement in NF-κB research has been generated by the first report of a crystal structure for one form of NF-κB, the first gene knockout studies for different forms of NF-κB and of IκB, and the implications for therapies of diseases thought to involve the inappropriate activation of NF-κB

Mechanism of PP2A-mediated IKKβ dephosphorylation: a systems biological approach

BACKGROUND: Biological effects of nuclear factor-kappaB (NF kappaB) can differ tremendously depending on the cellular context. For example, NF kappaB induced by interleukin-1 (IL-1) is converted from an inhibitor of death receptor induced apoptosis into a promoter of ultraviolet-B radiation (UVB)-induced apoptosis. This conversion requires prolonged NF kappaB activation and is facilitated by IL-1 + UVB-induced abrogation of the negative feedback loop for NF kappaB, involving a lack of inhibitor of kappaB (I kappaB alpha) protein reappearance. Permanent activation of the upstream kinase IKK beta results from UVB-induced inhibition of the catalytic subunit of Ser-Thr phosphatase PP2A (PP2Ac), leading to immediate phosphorylation and degradation of newly synthesized I kappaB alpha. RESULTS: To investigate the mechanism underlying the general PP2A-mediated tuning of IKK beta phosphorylation upon IL-1 stimulation, we have developed a strictly reduced mathematical model based on ordinary differential equations which includes the essential processes concerning the IL-1 receptor, IKK beta and PP2A. Combining experimental and modelling approaches we demonstrate that constitutively active, but not post-stimulation activated PP2A, tunes out IKK beta phosphorylation thus allowing for I kappaB alpha resynthesis in response to IL-1. Identifiability analysis and determination of confidence intervals reveal that the model allows reliable predictions regarding the dynamics of PP2A deactivation and IKK beta phosphorylation. Additionally, scenario analysis is used to scrutinize several hypotheses regarding the mode of UVB-induced PP2Ac inhibition. The model suggests that down regulation of PP2Ac activity, which results in prevention of I kappaB alpha reappearance, is not a direct UVB action but requires instrumentality. CONCLUSION: The model developed here can be used as a reliable building block of larger NF kappa B models and offers comprehensive simplification potential for future modeling of NF kappa B signaling. It gives more insight into the newly discovered mechanisms for IKK deactivation and allows for substantiated predictions and investigation of different hypotheses. The evidence of constitutive activity of PP2Ac at the IKK complex provides new insights into the feedback regulation of NF kappa B, which is crucial for the development of new anti-cancer strategies

Regions of IκBα that are critical for its inhibition of NF-κB·DNA interaction fold upon binding to NF-κB

Nuclear factor κB (NF-κB) transcription factors regulate genes responsible for critical cellular processes. IκBα, -β, and -ε bind to NF-κBs and inhibit their transcriptional activity. The NF-κB-binding domains of IκBs contain six ankyrin repeats (ARs), which adopt a β-hairpin/α-helix/loop/α-helix/loop architecture. IκBα appears compactly folded in the IκBα·NF-κB crystal structure, but biophysical studies suggested that IκBα might be flexible even when bound to NF-κB. Amide H/(2)H exchange in free IκBα suggests that ARs 2–4 are compact, but ARs 1, 5, and 6 are conformationally flexible. Amide H/(2)H exchange is one of few techniques able to experimentally identify regions that fold upon binding. Comparison of amide H/(2)H exchange in free and NF-κB-bound IκBα reveals that the β-hairpins in ARs 5 and 6 fold upon binding to NF-κB, but AR 1 remains highly solvent accessible. These regions are implicated in various aspects of NF-κB regulation, such as controlling degradation of IκBα, enabling high-affinity interaction with different NF-κB dimers, and preventing NF-κB from binding to its target DNA. Thus, IκBα conformational flexibility and regions of IκBα folding upon binding to NF-κB are important attributes for its regulation of NF-κB transcriptional activity

The interleukin-1 signalling pathway in astrocytes: a key contributor to inflammation in the brain

A dysregulated inflammatory response in the central nervous system (CNS) lies at the heart of many neuropathological conditions such as multiple sclerosis and Alzheimer's disease. A key component of these inflammatory conditions is the accumulation of leukocytes in the CNS. The infiltration of leukocytes into the brain is dependent on the induction of leukocyte adhesion molecules and chemoattractant chemokines. Recent studies have suggested the astrocyte to be a key cell in mediating the inflammatory process in the brain and in expressing adhesion molecules and chemokines. Here I overview work in my laboratory and others that demonstrates interleukin-1 (IL-1) to be a key inducer of the expression of these molecules in astrocytes. The temporal expression is sustained in nature and this is due to prolonged activation of the transcription factor NFκB. The molecular basis to the sustained activation of NFκB is also discussed. The IL-1 signalling pathway thus emerges as a valuable therapeutic target in the treatment of presently incurable neuropathological conditions