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The NF-κB transcription factor has a central role in diverse processes, including inflammation, proliferation and cell survival, and its activity is dysregulated in diseases such as autoimmunity and cancer. We recently identified the TRE17/ubiquitin-specific protease 6 (USP6) oncogene as the first de-ubiquitinating enzyme to activate NF-κB. TRE17/USP6 is translocated and overexpressed in aneurysmal bone cyst (ABC), a pediatric tumor characterized by extensive bone degradation and inflammatory recruitment. In the current study, we explore the mechanism by which TRE17 induces activation of NF-κB, and find that it activates the classical NF-κB pathway through an atypical mechanism that does not involve IκB degradation. TRE17 co-precipitates with IκB kinase (IKK), and IKK activity is augmented in stable cell lines overexpressing TRE17, in a USP-dependent manner. Optimal activation of NF-κB by TRE17 requires both catalytic subunits of IKK, distinguishing its mechanism from the classical and non-canonical pathways, which require either IKKβ or IKKα, respectively. TRE17 stimulates phosphorylation of p65 at serine 536, a modification that has been associated with enhanced transcriptional activity and nuclear retention. Induction of S536 phosphorylation by TRE17 requires both IKKα and IKKβ, as well as the IKKγ/NEMO regulatory subunit of IKK. We further demonstrate that TRE17(long) is highly tumorigenic when overexpressed in NIH3T3 fibroblasts, and that inhibition of NF-κB significantly attenuates tumor formation. In summary, these studies uncover an unexpected signaling mechanism for activation of classical NF-κB by TRE17. They further reveal a critical role for NF-κB in TRE17-mediated tumorigenesis, and suggest that NF-κB inhibitors may function as effective therapeutic agents in the treatment of ABC.

The oncogenic fusion protein RET/PTC3 (RP3) that is expressed in papillary thyroid carcinoma (PTC) and thyroid epithelia in Hashimoto's thyroiditis activates nuclear factor-kappa B (NF-κB) and induces pro-inflammatory gene expression; however, the mechanism of this activation is unknown. To address this, we expressed RP3 in murine embryonic fibroblasts (MEFs) lacking key classical and noncanonical NF-κB signaling components. In wild-type MEFs, RP3 upregulated CCL2, CXCL1, granulocyte-macrophage colony-stimulating factor and tumor necrosis factor expression and activated classical but not noncanonical NF-κB. RP3-activated NF-κB in IκB kinase (IKK)β(-/-) MEFs but not IKKα- or NF-κB essential modulator (NEMO)-deficient cells and activation was inhibited by a peptide that blocks NEMO binding to the IKKs. RP3 increased the levels of NF-κB-inducing kinase (NIK) and did not activate NF-κB in NIK-deficient MEFs. Notably, NIK stabilization was not accompanied by TRAF3 degradation demonstrating that RP3 disrupts normal basal NIK regulation. Dominant-negative NIK blocked RP3-induced NF-κB activation and an RP3 signaling mutant (RP3(Y588F)) did not stabilize NIK. Finally, examination of PTC specimens revealed strong positive staining for NIK. We therefore conclude that RP3 activates classical NF-κB via NIK, NEMO and IKKα. Importantly, our findings reveal a novel mechanism for oncogene-induced NF-κB activation via stabilization of NIK.

The nuclear factor kappa B (NF-kappaB) transcription factors are activated by a range of stimuli including pro-inflammatory cytokines. Active NF-kappaB regulates the expression of genes involved in inflammation and cell survival and aberrant NF-kappaB activity plays pathological roles in certain types of cancer and diseases characterized by chronic inflammation. NF-kappaB signaling is an attractive target for the development of novel anti-inflammatory or anti-cancer drugs and we discuss here how the method of peptide transduction has been used to specifically target NF-kappaB. Peptide transduction relies on the ability of certain small cell-penetrating peptides (CPPs) to enter cells, and a panel of CPP-linked inhibitors (CPP-Is) has been developed to directly inhibit NF-kappaB signaling. Remarkably, several of these NF-kappaB-targeting CPP-Is are effective in vivo and therefore offer exciting potential in the clinical setting.

Activation of the transcription factor nuclear factor (NF)-kappaB by proinflammatory stimuli leads to increased expression of genes involved in inflammation. Activation of NF-kappaB requires the activity of an inhibitor of kappaB (IkappaB)-kinase (IKK) complex containing two kinases (IKKalpha and IKKbeta) and the regulatory protein NEMO (NF-kappaB essential modifier). An amino-terminal alpha-helical region of NEMO associated with a carboxyl-terminal segment of IKKalpha and IKKbeta that we term the NEMO-binding domain (NBD). A cell-permeable NBD peptide blocked association of NEMO with the IKK complex and inhibited cytokine-induced NF-kappaB activation and NF-kappaB-dependent gene expression. The peptide also ameliorated inflammatory responses in two experimental mouse models of acute inflammation. The NBD provides a target for the development of drugs that would block proinflammatory activation of the IKK complex without inhibiting basal NF-kappaB activity.

The transcription factor NF-kappa B, more than a decade after its discovery, remains an exciting and active area of study. The involvement of NF-kappa B in the expression of numerous cytokines and adhesion molecules has supported its role as an evolutionarily conserved coordinating element in the organism's response to situations of infection, stress, and injury. Recently, significant advances have been made in elucidating the details of the pathways through which signals are transmitted to the NF-kappa B:I kappa B complex in the cytosol. The field now awaits the discovery and characterization of the kinase responsible for the inducible phosphorylation of I kappa B proteins. Another exciting development has been the demonstration that in certain situations NF-kappa B acts as an anti-apoptotic protein; therefore, elucidation of the mechanism by which NF-kappa B protects against cell death is an important goal. Finally, the generation of knockouts of members of the NF-kappa B/I kappa B family has allowed the study of the roles of these proteins in normal development and physiology. In this review, we discuss some of these recent findings and their implications for the study of NF-kappa B.

Rel/NF-kappa B proteins constitute a family of structurally-related transcription factors that are regulated by interaction with a family of regulatory proteins, the I kappa B proteins. In this review, we will familiarize the reader with the known Rel/NF-kappa B and I kappa B molecules, and will discuss the types of interactions that occur between these proteins to regulate their function. In addition, we will describe what is known about the structure of Rel/NF-kappa B transcription complexes on DNA. Lastly, we will discuss the wide variety of biological stimuli and conditions which activate NF-kappa B and the genes whose expression are regulated by this family of transcription factors.