No abstract

In mammals, the canonical nuclear factor kappaB (NF-kappaB) signaling pathway activated in response to infections is based on degradation of IkappaB inhibitors. This pathway depends on the IkappaB kinase (IKK), which contains two catalytic subunits, IKKalpha and IKKbeta. IKKbeta is essential for inducible IkappaB phosphorylation and degradation, whereas IKKalpha is not. Here we show that IKKalpha is required for B cell maturation, formation of secondary lymphoid organs, increased expression of certain NF-kappaB target genes, and processing of the NF-kappaB2 (p100) precursor. IKKalpha preferentially phosphorylates NF-kappaB2, and this activity requires its phosphorylation by upstream kinases, one of which may be NF-kappaB-inducing kinase (NIK). IKKalpha is therefore a pivotal component of a second NF-kappaB activation pathway based on regulated NF-kappaB2 processing rather than IkappaB degradation.

Stem-like cells may be integral to the development and maintenance of human cancers. Direct proof is still lacking, mainly because of our poor understanding of the biological differences between normal and cancer stem cells (SCs). Using the ErbB2 transgenic model of breast cancer, we found that self-renewing divisions of cancer SCs are more frequent than their normal counterparts, unlimited and symmetric, thus contributing to increasing numbers of SCs in tumoral tissues. SCs with targeted mutation of the tumor suppressor p53 possess the same self-renewal properties as cancer SCs, and their number increases progressively in the p53 null premalignant mammary gland. Pharmacological reactivation of p53 correlates with restoration of asymmetric divisions in cancer SCs and tumor growth reduction, without significant effects on additional cancer cells. These data demonstrate that p53 regulates polarity of cell division in mammary SCs and suggest that loss of p53 favors symmetric divisions of cancer SCs, contributing to tumor growth.

To identify functions of the IKKalpha subunit of IkappaB kinase that require catalytic activity, we generated an Ikkalpha(AA) knockin allele containing alanines instead of serines in the activation loop. Ikkalpha(AA/AA) mice are healthy and fertile, but females display a severe lactation defect due to impaired proliferation of mammary epithelial cells. IKKalpha activity is required for NF-kappaB activation in mammary epithelial cells during pregnancy and in response to RANK ligand but not TNFalpha. IKKalpha and NF-kappaB activation are also required for optimal cyclin D1 induction. Defective RANK signaling or cyclin D1 expression results in the same phenotypic effect as the Ikkalpha(AA) mutation, which is completely suppressed by a mammary specific cyclin D1 transgene. Thus, IKKalpha is a critical intermediate in a pathway that controls mammary epithelial proliferation in response to RANK signaling via cyclin D1.

IkappaB Kinase (IKK)alpha is required for activation of an alternative NF-kappaB signaling pathway based on processing of the NF-kappaB2/p100 precursor protein, which associates with RelB in the cytoplasm. This pathway, which activates RelB:p52 dimers, is required for induction of several chemokine genes needed for organization of secondary lymphoid organs. We investigated the basis for the IKKalpha dependence of the induction of these genes in response to engagement of the lymphotoxin beta receptor (LTbetaR). Using chromatin immunoprecipitation, we found that the promoters of organogenic chemokine genes are recognized by RelB:p52 dimers and not by RelA:p50 dimers, the ubiquitous target for the classical NF-kappaB signaling pathway. We identified in the IKKalpha-dependent promoters a novel type of NF-kappaB-binding site that is preferentially recognized by RelB:p52 dimers. This site links induction of organogenic chemokines and other important regulatory molecules to activation of the alternative pathway.

The interaction of interleukin-1␤ (IL-1␤) with its type 1 cell surface receptor initiates a cascade of intracellular reactions leading to the activation of transcription factors and the expression of target genes. One of the major transcription factors mediating IL-1␤ biological activities is NF-B (for reviews, see references 2, 3, and 22). This factor is sequestered in the cytoplasm by an inhibitor from the IB family. IL-1␤ cellular stimulation leads to a rapid phosphorylation and degradation of IB␣, the most common NF-B inhibitor. This reaction allows NF-B to translocate to the nucleus, to bind DNA, and to activate the transcription of specific genes (2, 55).Following its interaction with IL-1, the type 1 IL-1 receptor recruits the IL-1 receptor-associated kinase (IRAK) protein, which subsequently interacts with the TRAF6 adapter protein (15,16,30,61,62,65). TRAF6 is required for IL-1␤-induced NF-B activation, as demonstrated in 293 cells (16).However, the signaling pathways leading to NF-B activation from the IL-1␤ receptors are still controversial. It has been demonstrated that TRAF6 interacts with a MAP kinase kinase kinase (MAPKKK) known as NIK and that NIK is required for IL-1␤-or tumor necrosis factor alpha (TNF-␣)-dependent 56). Large (500 to 900 kDa) multimeric protein kinase complexes have been purified from HeLa cells and transmit the signal from the TNF receptor type 1 (TNFR-1) and type 1 IL-1 receptors to the NF-B/IB cytoplasmic complex (17,20,33,41). From these complexes three IB kinases, IKK-␣, IKK-␤, and IKK-␥, have been purified, and their genes were cloned (20,42,49,66). Other investigators have cloned IKK kinases by virtue of their association with the NIK protein kinase (47, 64). Moreover, inactivation of these kinases by dominant negative mutants suppresses IL-1␤ and TNF-␣ induction of NF-B. These studies indicate that the activated NIK kinase phosphorylates and activates the IKK protein kinases. IKK protein kinases can in turn phosphorylate the IB␣ protein on serines located at positions 32 and 36, a reaction which targets IB␣ for ubiquitination and rapid degradation by the proteasome (12,58,59). These reactions are extremely rapid, and the cellular IB␣ protein is completely degraded within minutes following TNF-␣ or IL-1␤ cell stimulation (4, 13).Despite this simplified linear receptor-TRAF-NIK-IKK axis for IB␣ phosphorylation and degradation, other intermediates might be involved in NF-B activation by TNF-␣ or IL-1␤. First, several components of the large signaling complex remain to be identified, as the three IKK protein kinases do not account for the molecular weight of the whole complex. Second, a large number of studies, some of them being a matter of controversy, have identified other intermediates which seem to be required for TNF-␣-or IL-1␤-mediated NF-B activation. These intermediates are Raf-1, MAP kinases, the PKC and / isoforms, Rho and Rac proteins, and ceramide or reactive oxygen intermediates (ROIs) (19,24,25,32,33,38,46,(50)(51)(52)(53)57). Such a large number of controver...

Expression of B cell-activating factor (BAFF), a critical B cell survival factor, is elevated in autoimmune and lymphoproliferative disorders. Mice overproducing BAFF develop systemic lupus erythematosus (SLE)-like disease and exhibit B cell activation of classical and alternative NF-kappaB-signaling pathways. We used a genetic approach and found that both NF-kappaB-signaling pathways contributed to disease development but act through distinct mechanisms. Whereas BAFF enhanced long-term B cell survival primarily through the alternative, but not the classical, NF-kappaB pathway, it promoted immunoglobulin class switching and generation of pathogenic antibodies through the classical pathway. Activation of the alternative NF-kappaB pathway resulted in integrin upregulation, thereby retaining autoreactive B cells in the splenic marginal zone, a compartment that contributes to their survival. Thus, both classical and alternative NF-kappaB signaling are important for development of lupus-like disease associated with BAFF overproduction. The same mechanisms may be involved in the pathogenesis of human SLE.

Lung cancer is the leading cause of cancer death worldwide. Low-dose computed tomography screening (LDCT) was recently shown to anticipate the time of diagnosis, thus reducing lung cancer mortality. However, concerns persist about the feasibility and costs of large-scale LDCT programs. Such concerns may be addressed by clearly defining the target "high-risk" population that needs to be screened by LDCT. We recently identified a serum microRNA signature (the miR-Test) that could identify the optimal target population. Here, we performed a large-scale validation study of the miR-Test in high-risk individuals (n = 1115) enrolled in the Continuous Observation of Smoking Subjects (COSMOS) lung cancer screening program. The overall accuracy, sensitivity, and specificity of the miR-Test are 74.9% (95% confidence interval [CI] = 72.2% to 77.6%), 77.8% (95% CI = 64.2% to 91.4%), and 74.8% (95% CI = 72.1% to 77.5%), respectively; the area under the curve is 0.85 (95% CI = 0.78 to 0.92). These results argue that the miR-Test might represent a useful tool for lung cancer screening in high-risk individuals.