The Fatty Acid Synthase Gene is a Conserved p53 Family Target Gene from Worm to Human

The discovery that the p53 family consists of three members (p53, p63 and p73) in vertebrates and of a single homolog in invertebrates has raised the challenge of understanding the functions of the ancestor and how they have evolved and differentiated within the duplicated genes in vertebrates. Here, we report that the fatty acid synthase (FAS) gene, encoding for a key enzyme involved in the biogenesis of membrane lipids in rapidly proliferating cells, is a conserved target of the p53 family throughout the evolution. We show that CEP-1, the C. elegans p53 homolog, is able to bind the two p53 family responsive elements (REs) identified in the worm fasn-1 gene. Moreover, we demonstrate that fasn-1 expression is modulated by CEP-1 in vivo, by comparing wild-type and CEP-1 knockout worms. In human, luciferase and chromatin immunoprecipitation assays demonstrate that TAp73α and ΔNp63α, but not p53, TAp73β and TAp63α bind the two p53 REs of the human FASN gene. We show that the ectopic expression of TAp73β and ΔNp63α leads to an increase of FASN mRNA levels, while their silencing produces a decrease of FASN expression. Furthermore, we present data showing a correlation between ΔNp63α and FASN expression in cellular proliferation. Of relevant importance is that fasn-1 is the first CEP-1 direct target gene identified so far in C. elegans and our results suggest a new CEP-1 role in cellular proliferation and development, besides the one already described in apoptosis of germ cells. These data confirm the hypothesis that the ancestral functions of the single invertebrate gene may have been spread out among the three vertebrate members, each of them have acquired specific role in cell cycle regulation.     

Differential usage of NF-κB activating signals by IL-1β and TNF-α in pancreatic beta cells

The cytokines interleukin (IL)-1β and tumor necrosis factor (TNF)-α induce β-cell death in type 1 diabetes via NF-κB activation. IL-1β induces a more marked NF-κB activation than TNF-α, with higher expression of genes involved in β-cell dysfunction and death. We show here a differential usage of the IKK complex by IL-1β and TNF-α in β-cells. While TNF-α uses IKK complexes containing both IKKα and IKKβ, IL-1β induces complexes with IKKα only; this effect is achieved by induction of IKKβ degradation via the proteasome. Both IKKγ and activation of the TRAF6-TAK1-JNK pathway are involved in IL-1β-induced IKKβ degradation.     

Suppression of NF-κB signaling by KEAP1 regulation of IKKβ activity through autophagic degradation and inhibition of phosphorylation

IκB kinase β (IKKβ) plays a crucial role in biological processes, including immune response, stress response, and tumor development by mediating the activation of various signaling molecules such as NF-κB. Extensive studies on the mechanisms underlying IKK activation have led to the identification of new activators and have facilitated an understanding of the cellular responses related to NF-κB and other target molecules. However, the molecular processes that modulate IKK activity are still unknown. In this study, we show that KEAP1 is a new IKK binding partner, which is responsible for the down-regulation of TNFα-stimulated NF-κB activation. The E(T/S)GE motif, which is found only in the IKKβ subunit of the IKK complex, is essential for interaction with the C-terminal Kelch domain of KEAP1. Reduction of KEAP1 expression by small interfering RNA enhanced NF-κB activity, and up-regulated the expression of NF-κB target genes. Ectopic expression of KEAP1 decreased the expression of IKKβ, which was restored by an autophagy inhibitor. IKK phosphorylation stimulated by TNFα was blocked by KEAP1. Our data demonstrate that KEAP1 is involved in the negative regulation of NF-κB signaling through the inhibition of IKKβ phosphorylation and the mediation of autophagy-dependent IKKβ degradation.     

NEMO ensures signaling specificity of the pleiotropic IKKβ by directing its kinase activity toward IκBα

Besides activating NFκB by phosphorylating IκBs, IKKα/IKKβ kinases are also involved in regulating metabolic insulin signaling, the mTOR pathway, Wnt signaling, and autophagy. How IKKβ enzymatic activity is targeted to stimulus-specific substrates has remained unclear. We show here that NEMO, known to be essential for IKKβ activation by inflammatory stimuli, is also a specificity factor that directs IKKβ activity toward IκBα. Physical interaction and functional competition studies with mutant NEMO and IκB proteins indicate that NEMO functions as a scaffold to recruit IκBα to IKKβ. Interestingly, expression of NEMO mutants that allow for IKKβ activation by the cytokine IL-1, but fail to recruit IκBs, results in hyperphosphorylation of alternative IKKβ substrates. Furthermore IKK's function in autophagy, which is independent of NFκB, is significantly enhanced without NEMO as IκB scaffold. Our work establishes a role for scaffolds such as NEMO in determining stimulus-specific signal transduction via the pleiotropic signaling hub IKK.     

Cell migration to CXCL12 requires simultaneous IKKα and IKKβ-dependent NF-κB signaling

CXCL12 and its unique receptor CXCR4, is critical for the homing of a variety of cell lineages during both development and tissue repair. CXCL12 is particularly important for the recruitment of hemato/lymphopoietic cells to their target organs. In conjunction with the damage-associated alarmin molecule HMGB1, CXCL12 mediates immune effector and stem/progenitor cell migration towards damaged tissues for subsequent repair. Previously, we showed that cell migration to HMGB1 simultaneously requires both IKKβ and IKKα-dependent NF-κB activation. IKKβ-mediated activation maintains sufficient expression of HMGB1's receptor RAGE, while IKKα-dependent NF-κB activation ensures continuous production of CXCL12, which complexes with HMGB1 to engage CXCR4. Here using fibroblasts and primary mature macrophages, we show that IKKβ and IKKα are simultaneously essential for cell migration in response to CXCL12 alone. Non-canonical NF-κB pathway subunits RelB and p52 are also both essential for cell migration towards CXCL12, suggesting that IKKα is required to drive non-canonical NF-κB signaling. Flow cytometric analyses of CXCR4 expression show that IKKβ, but not IKKα, is required to maintain a critical threshold level of this CXCL12 receptor. Time-lapse video microscopy experiments in primary MEFs reveal that IKKα is required both for polarization of cells towards a CXCL12 gradient and to establish a basal level of velocity towards CXCL12. In addition, CXCL12 modestly up-regulates IKKα-dependent p52 nuclear translocation and IKKα-dependent expression of the CXCL12 gene. On the basis of our collective results we posit that IKKα is needed to maintain the basal expression of a critical protein co-factor required for cell migration to CXCL12.     

Discovery of potent and selective rhodanine type IKKβ inhibitors by hit-to-lead strategy

Regulation of NF-κB activation through the inhibition of IKKβ has been identified as a promising target for the treatment of inflammatory and autoimmune disease such as rheumatoid arthritis. In order to develop novel IKKβ inhibitors, we performed high throughput screening toward around 8000 library compounds, and identified a hit compound containing rhodanine moiety. We modified the structure of hit compound to obtain potent and selective IKKβ inhibitors. Throughout hit-to-lead studies, we have discovered optimized compounds which possess blocking effect toward NF-κB activation and TNFα production in cell as well as inhibition activity against IKKβ. Among them, compound 3q showed the potent inhibitory activity against IKKβ, and excellent selectivity over other kinases such as p38α, p38β, JNK1, JNK2, and JNK3 as well as IKKα.     

Beyond NF-κB activation: nuclear functions of IκB kinase α

IκB kinase (IKK) complex, the master kinase for NF-κB activation, contains two kinase subunits, IKKα and IKKβ. In addition to mediating NF-κB signaling by phosphorylating IκB proteins during inflammatory and immune responses, the activation of the IKK complex also responds to various stimuli to regulate diverse functions independently of NF-κB. Although these two kinases share structural and biochemical similarities, different sub-cellular localization and phosphorylation targets between IKKα and IKKβ account for their distinct physiological and pathological roles. While IKKβ is predominantly cytoplasmic, IKKα has been found to shuttle between the cytoplasm and the nucleus. The nuclear-specific roles of IKKα have brought increasing complexity to its biological function. This review highlights major advances in the studies of the nuclear functions of IKKα and the mechanisms of IKKα nuclear translocation. Understanding the nuclear activity is essential for targeting IKKα for therapeutics.