Cytokine-induced activation of the IkappaB kinases (IKK) IKK-alpha and IKK-beta is a key step involved in the activation of the NF-kappaB pathway. Gene-disruption studies of the murine IKK genes have shown that IKK-beta, but not IKK-alpha, is critical for cytokine-induced IkappaB degradation. Nevertheless, mouse embryo fibroblasts deficient in IKK-alpha are defective in the induction of NF-kappaB-dependent transcription. These observations raised the question of whether IKK-alpha might regulate a previously undescribed step to activate the NF-kappaB pathway that is independent of its previously described cytoplasmic role in the phosphorylation of IkappaBalpha. Here we show that IKK-alpha functions in the nucleus to activate the expression of NF-kappaB-responsive genes after stimulation with cytokines. IKK-alpha interacts with CREB-binding protein and in conjunction with Rel A is recruited to NF-kappaB-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-alpha in modifying histone function that is critical for the activation of NF-kappaB-directed gene expression.

fibroblasts (CAFs). These data will provide a resource for future studies aimed at further characterizing and targeting specific cell populations in PDA. ResultsCellular heterogeneity during PDA progression. We sought to determine the composition of single cells during the progression of PDA GEMMs. Normal mouse pancreas; 40-day-old Kras LSL−G12D/+ Ink4a fl/fl Ptf1a Cre/+ (KIC) (5) mouse pancreas, termed "early KIC" (with the early lesion initially confirmed by ultrasound; Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci. insight.129212DS1); and 60-day-old KIC pancreas, termed "late KIC" (Figure 1A) were freshly isolated and enzymatically digested followed by single-cell cDNA library generation using the 10× Genomics platform (6). Libraries were subsequently sequenced at a depth of more than 10 5 reads per cell. We performed stringent filtering, normalization, and graph-based clustering, which identified distinct cell populations in the normal pancreas and both stages of PDA.In the normal mouse pancreas, 2354 cells were sequenced and classified into appropriate cell types based on the gene expression of known markers: acinar cells, islet and ductal cells (Supplemental Figure 2), macrophages, T cells, and B cells, as well as 3 distinct populations of fibroblasts (Figure 1, B and E) were noted. In the early KIC lesion (3524 cells sequenced), the emergence of an expanded ductal population was observed (9.9% of cells), expressing known ductal markers, such as Krt18 and Sox9 (7), and displaying early neoplastic changes (Figure 1, A, C, and F, and Supplemental Figure 3). The acinar cell population was substantially reduced, while there was a marked increase in total macrophages and fibroblasts. Of note, the same 3 populations of fibroblasts seen in the normal pancreas were identified in the early KIC lesion. Additionally, endothelial cells were observed at this stage. This indicates that the expansion of fibroblasts and macrophages is an early event during PDA development. We next characterized the late KIC pancreas (804 cells sequenced) and noted the absence of normal exocrine (acinar) and endocrine (islet) cells (Figure 1, D and G). Instead, 2 distinct populations of cancer cells were present, suggesting phenotypic cancer cell heterogeneity as a late event in the course of the disease. We also observed the presence of only 2 distinct fibroblast populations, which had a similar percentage in relation to total cells. Noticeably, macrophages became a predominant cell population in the late KIC tumor. Moreover, we observed lymphocytes at this stage. The cellular heterogeneity in cancer cells and stromal cells in the early and late KIC lesions highlighted the dynamic cellular changes that occur during PDA progression.Cancer cells enriched with mesenchymal markers emerge in advanced PDA. Gene expression analysis of epithelial markers (Cdh1, Epcam, Gjb1, and Cldn3) and mesenchymal markers (Cdh2, Cd44, Axl, Vim, and S100a4) revealed that the early KIC neoplastic cell...

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The Wnt/␤-catenin/Tcf and IB/NF-B cascades are independent pathways involved in cell cycle control, cellular differentiation, and inflammation. Constitutive Wnt/␤-catenin signaling occurs in certain cancers from mutation of components of the pathway and from activating growth factor receptors, including RON and MET. The resulting accumulation of cytoplasmic and nuclear ␤-catenin interacts with the Tcf/LEF transcription factors to induce target genes. The IB kinase complex (IKK) that phosphorylates IB contains IKK␣, IKK␤, and IKK␥. Here we show that the cyclin D1 gene functions as a point of convergence between the Wnt/␤-catenin and IB pathways in mitogenic signaling. Mitogenic induction of G 1 -S phase progression and cyclin D1 expression was PI3K dependent, and cyclin D1 Ϫ/Ϫ cells showed reduced PI3K-dependent S-phase entry. PI3K-dependent induction of cyclin D1 was blocked by inhibitors of PI3K/Akt/IB/IKK␣ or ␤-catenin signaling. A single Tcf site in the cyclin D1 promoter was required for induction by PI3K or IKK␣. In IKK␣ Ϫ/Ϫ cells, mitogen-induced DNA synthesis, and expression of Tcf-responsive genes was reduced. Reintroduction of IKK␣ restored normal mitogen induction of cyclin D1 through a Tcf site. In IKK␣ Ϫ/Ϫ cells, ␤-catenin phosphorylation was decreased and purified IKK␣ was sufficient for phosphorylation of ␤-catenin through its N-terminus in vitro. Because IKK␣ but not IKK␤ induced cyclin D1 expression through Tcf activity, these studies indicate that the relative levels of IKK␣ and IKK␤ may alter their substrate and signaling specificities to regulate mitogeninduced DNA synthesis through distinct mechanisms. INTRODUCTIONThe Wingless/Wnt pathway plays a crucial role in development and cell cycle control (Cadigan and Nusse, 1997;Huelsken and Behrens, 2000). Dysregulation of the Wingless/ (Wnt)/␤-catenin/Tcf pathway has been implicated in tumorigenesis of diverse types (Polakis, 2000a). Axin/Conductin, together with APC, promote ␤-catenin degradation through serine-threonine phosphorylation of the ␤-catenin N-terminus by GSK3␤, which targets ␤-catenin for ubiquitination by a SCF ␤-TRCP (␤-transducin repeat-containing protein) ubiquitin ligase complex (Fuchs et al., 1999;Winston et al., 1999) and its degradation by the proteasome. On induction of Wnt signaling by extracellular ligands, the Frizzled receptors are activated. The activity of GSK3␤ and its effect on ␤-catenin is antagonized by Dishevelled, a downstream target of Frizzled, thus preventing the degradation of ␤-cate- nin by the proteasome. The resulting accumulation of ␤-catenin leads to its nuclear translocation and binding to Tcf/Lef transcription factors to induce target genes including cyclin D1 and c-Myc (He et al., 1998;Shtutman et al., 1999;Huelsken and Behrens, 2000). In addition to components in the Wnt signaling pathway, several other pathways can regulate ␤-catenin/Tcf signaling and gene expression and confer aberrant cellular growth. The protein encoded by Gas6, a growth factor of the vitamin K-dependent family, which binds member...

Both the ␤-catenin and the nuclear factor B (NF-B) proteins are important regulators of gene expression and cellular proliferation. Two kinases, IKK␣ and IKK␤, are critical activators of the NF-B pathway. Here we present evidence that these kinases are also important in the regulation of ␤-catenin function. IKK␣-and IKK␤-deficient mouse embryo fibroblasts exhibited different patterns of ␤-catenin cellular localization. IKK␤ decreases ␤-catenin-dependent transcriptional activation, while IKK␣ increases ␤-catenin-dependent transcriptional activity. IKK␣ and IKK␤ interact with and phosphorylate ␤-catenin using both in vitro and in vivo assays. Our results suggest that differential interactions of ␤-catenin with IKK␣ and IKK␤ may in part be responsible for regulating ␤-catenin protein levels and cellular localization and integrating signaling events between the NF-B and Wingless pathways.␤-Catenin, the mammalian homologue of the Drosophila armadillo protein, is a ubiquitously expressed protein that has at least two distinct roles in the cell. First, it participates in cell-cell adhesion by mediating the association of E-cadherin with the cytoskeleton (1, 2). Second, it is a critical downstream component of the Wnt 1 or Wingless signal transduction pathway (3-5). The Wnt family of secretory glycoproteins plays an important role in embryonic development, in the induction of cell polarity, and in the determination of cell fate. Deregulation of Wnt signaling disrupts axis formation in embryos (5-8) and is associated with multiple human malignancies (9).The current model of Wnt signaling indicates that the binding of the Wnt proteins to their receptor, frizzled, stabilizes ␤-catenin by inhibiting the activity of a serine/threonine kinase glycogen synthase kinase-3 or GSK-3␤ (9). GSK-3␤ is associated with ␤-catenin in a multiprotein complex that includes the adenomatous polyposis coli tumor suppressor protein (APC), axin or conductin, protein phosphatase 2A, and dishevelled. GSK-3␤ phosphorylation of residues in the amino terminus of ␤-catenin results in APC-mediated ␤-catenin degradation via the ubiquitin-proteosome pathway (10, 11). Increased levels of ␤-catenin are frequently found in colon cancer due to mutations in either the APC gene (12)(13)(14) or at residues in the amino terminus of ␤-catenin that are phosphorylated by . In the nucleus, ␤-catenin forms a complex with members of the T-cell factor (TCF)/lymphocyte-enhancer factor (LEF) family and activates gene expression of a variety of target genes (18 -23) including c-myc (24) and cyclin D1 (25, 26).NF-B comprises a family of transcription factors which are critical in activating the expression of genes involved in the immune and inflammatory response and in the regulation of cellular apoptosis (27, 28). NF-B is sequestered in the cytoplasm by a family of inhibitory proteins known as IB. Upon stimulation of this pathway by a variety of agents including IL-1 and TNF␣, the kinases IKK␣ and IKK␤ (29 -33) in conjunction with the scaffold protein IKK␥/NEMO (34 -36) l...