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• W1969132581 abstract "The IκB kinase (IKK) signaling complex is responsible for activating NF-κB-dependent gene expression programs. Even though NF-κB-responsive genes are known to orchestrate stress-like responses, critical gaps in our knowledge remain about the global effects of NF-κB activation on cellular physiology. DNA microarrays were used to compare gene expression programs in a model system of 70Z/3 murine pre-B cellsversus their IKK signaling-defective 1.3E2 variant with lipopolysaccharide (LPS), interleukin-1 (IL-1), or a combination of LPS + phorbol 12-myristate 13-acetate under brief (2 h) or long term (12 h) stimulation. 70Z/3-1.3E2 cells lack expression of NEMO/IKKγ/IKKAP-1/FIP-3, an essential positive effector of the IKK complex. Some stimulated hits were known NF-κB target genes, but remarkably, the vast majority of the up-modulated genes and an unexpected class of repressed genes were all novel targets of this signaling pathway, encoding transcription factors, receptors, extracellular ligands, and intracellular signaling factors. Thirteen stimulated (B-ATF, Pim-2, MyD118,Pea-15/MAT1, CD82, CD40L,Wnt10a, Notch 1, R-ras,Rgs-16, PAC-1, ISG15, andCD36) and five repressed (CCR2,VpreB, λ5, SLPI, andCMAP/Cystatin7) genes, respectively, were bona fide NF-κB targets by virtue of their response to a transdominant IκBαSR (super repressor). MyD118 andISG15, although directly induced by LPS stimulation, were unaffected by IL-1, revealing the existence of direct NF-κB target genes, which are not co-induced by the LPS and IL-1 Toll-like receptors. The IκB kinase (IKK) signaling complex is responsible for activating NF-κB-dependent gene expression programs. Even though NF-κB-responsive genes are known to orchestrate stress-like responses, critical gaps in our knowledge remain about the global effects of NF-κB activation on cellular physiology. DNA microarrays were used to compare gene expression programs in a model system of 70Z/3 murine pre-B cellsversus their IKK signaling-defective 1.3E2 variant with lipopolysaccharide (LPS), interleukin-1 (IL-1), or a combination of LPS + phorbol 12-myristate 13-acetate under brief (2 h) or long term (12 h) stimulation. 70Z/3-1.3E2 cells lack expression of NEMO/IKKγ/IKKAP-1/FIP-3, an essential positive effector of the IKK complex. Some stimulated hits were known NF-κB target genes, but remarkably, the vast majority of the up-modulated genes and an unexpected class of repressed genes were all novel targets of this signaling pathway, encoding transcription factors, receptors, extracellular ligands, and intracellular signaling factors. Thirteen stimulated (B-ATF, Pim-2, MyD118,Pea-15/MAT1, CD82, CD40L,Wnt10a, Notch 1, R-ras,Rgs-16, PAC-1, ISG15, andCD36) and five repressed (CCR2,VpreB, λ5, SLPI, andCMAP/Cystatin7) genes, respectively, were bona fide NF-κB targets by virtue of their response to a transdominant IκBαSR (super repressor). MyD118 andISG15, although directly induced by LPS stimulation, were unaffected by IL-1, revealing the existence of direct NF-κB target genes, which are not co-induced by the LPS and IL-1 Toll-like receptors. inhibitors of NF-κB IκB kinase NF-κB essential modulator lipopolysaccharide phorbol 12-myristate 13-acetate interleukin-1 isopropyl-1-thio-β-d-galactopyranoside reverse transcription-polymerase chain reaction tumor necrosis factor α super repressor Epstein-Barr virus cAMP-response element-binding protein mitogen-activated protein MAP kinase MAPK/ERK kinase kinase dendritic cells interferon interleukin-1 β converting enzyme B-cell lymphoma 2 epidermal growth factor oxidized low density lipoprotein secretory leukocyte protease inhibitor signal transducers and activators of transcription AP-1 transcription factor IL-1R-associated kinase TNF receptor-associated factor 6 evolutionarily conserved signaling intermediate in Toll pathways Fas-associated protein with death domain NF-κB transcription factors are established nuclear regulators of gene expression programs culminating in a host of cellular stress-like responses that play important roles in an organism's acquired and innate immune responses (reviewed in Refs. 1Baldwin Jr., A. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5552) Google Scholar, 2May M.M. Ghosh S. Immunol. Today. 1998; 19: 80-88Abstract Full Text Full Text PDF PubMed Scopus (1046) Google Scholar, 3Mercurio F. Manning A.M. Oncogene. 1999; 18: 6163-6171Crossref PubMed Scopus (359) Google Scholar, 4Barkett M. Gilmore T.D. Oncogene. 1999; 18: 6910-6924Crossref PubMed Scopus (1072) Google Scholar). A variety of extracellular and endogenous stimuli, including viral and bacterial infections, oxidative and DNA-damaging agents, hyperosmotic shock, chemotherapeutics, and pro-inflammatory cytokines all result in NF-κB activation (1Baldwin Jr., A. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5552) Google Scholar, 3Mercurio F. Manning A.M. Oncogene. 1999; 18: 6163-6171Crossref PubMed Scopus (359) Google Scholar, 4Barkett M. Gilmore T.D. Oncogene. 1999; 18: 6910-6924Crossref PubMed Scopus (1072) Google Scholar, 5Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4585) Google Scholar). NF-κB factors bind to DNA as heterodimers assembled from five known proteins (RelA, c-Rel, RelB, p50, and p52) with each subunit contacting one-half of a conserved 10-base pair consensus motif (GGGRNWTYCC) (1Baldwin Jr., A. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5552) Google Scholar, 5Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4585) Google Scholar). NF-κB is generally held in an inactive state, tethered in the cytoplasm to inhibitory factors termed inhibitors of NF-κB (IκBs)1 (1Baldwin Jr., A. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5552) Google Scholar, 5Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4585) Google Scholar). Activators of NF-κB cause the specific phosphorylation of pairs of amino-terminal serines in the IκBs, which mark them for ubiquitination and subsequent proteasomal destruction. NF-κB then becomes available to activate its nuclear target genes (1Baldwin Jr., A. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5552) Google Scholar, 5Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4585) Google Scholar). IκB phosphorylation is mediated by a high molecular weight signalsome complex comprising at least two direct IκB kinases (IKKα and IKKβ, also called IKK1/CHUK and IKK2) and a regulatory, docking/adapter protein (NEMO, NF-κBessential modulator, also called IKKγ/IKKAP-1/FIP-3) (reviewed in Refs. 3Mercurio F. Manning A.M. Oncogene. 1999; 18: 6163-6171Crossref PubMed Scopus (359) Google Scholar, 4Barkett M. Gilmore T.D. Oncogene. 1999; 18: 6910-6924Crossref PubMed Scopus (1072) Google Scholar, 6Karin M. 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Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1575) Google Scholar). The essential role of the IKK signalsome in NF-κB activation has been demonstrated in mice lacking IKKβ or IKKα (13Li Q. Van Antwerp D. Mercurio F. Lee K.F. Verma I.M. Science. 1999; 284: 321-325Crossref PubMed Scopus (850) Google Scholar, 14Li Z.W. Chu W. Hu Y. Delhase M. Deerinck T. Ellisman M. Johnson R. Karin M. J. Exp. Med. 1999; 189: 1839-1845Crossref PubMed Scopus (815) Google Scholar, 15Tanaka M. Fuentes M.E. Yamaguchi K. Durnin M.H. Dalrymple S.A. Hardy K.L. Goeddel D.V. Immunity. 1999; 10: 421-429Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar, 16Li Q. Lu Q. Hwang J.Y. Buscher D. Lee K.F. Izpisua-Belmonte J.C. Verma I.M. Genes Dev. 1999; 13: 1322-1328Crossref PubMed Scopus (416) Google Scholar, 17Hu Y. Baud V. Delhase M. Zhang P. Deerinck T. Ellisman M. Johnson R. Karin M. Science. 1999; 284: 316-320Crossref PubMed Scopus (708) Google Scholar, 18Takeda K. Takeuchi O. Tsujimura T. Itami S. Adachi O. Kawai T. Sanjo H. Yoshikawa K. Terada N. Akira S. Science. 1999; 284: 313-316Crossref PubMed Scopus (537) Google Scholar). NEMO is an essential non-catalytic, adapter/docking component of the IKK signalsome. Loss of NEMO in cultured cells resulted in a complete lack of signal-induced NF-κB activation (19Courtois G. Whiteside S.T. Sibley C.H. Israel A. Mol. Cell. Biol. 1997; 17: 1441-1449Crossref PubMed Google Scholar, 20Mercurio F. Murray B.W. Shevchenko A. Bennett B.L. Young D.B. Li J.W. Pascual G. Motiwala A. Zhu H. Mann M. Manning A.M. Mol. Cell. Biol. 1999; 19: 1526-1538Crossref PubMed Google Scholar, 21Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (845) Google Scholar, 22Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (945) Google Scholar). More importantly, murine embryos that were genetically null for NEMO, akin to IKKβ KO mice, succumbed to severe liver apoptosis due to a virtually complete block in NF-κB activation (23Rudolph D. Wen-Chen Y. Wakeham A. Rudolph B. Nallainathan D. Potter J. Elia A.J. Mak T.W. Genes Dev. 2000; 14: 854-862PubMed Google Scholar). Major issues about the IKK signaling pathway remain unexplored, including the short versus long term effects of NF-κB activation to program cellular gene expression on a genomic scale. Because NF-κB regulates a variety of ubiquitous and cell-type-specific gene products in different cellular contexts, we elaborated the signal-induced, NEMO-dependent gene expression program in the context of the 70Z/3 murine pre-B lymphoma line. 70Z/3 pre-B cells recapitulate aspects of the pre-B to immature B cell transition in response to NF-κB activation (24Paige C.J. Kincade P.W. Ralph P. J. Immunol. 1978; 121: 641-647PubMed Google Scholar, 25Sakaguchi N. Kishimoto T. Kikutani H. Watanabe T. Yoshida N. Shimizu A. Yamawaki-Kataoka Y. Honjo T. Yamamura Y. J. Immunol. 1980; 125: 2654-2659PubMed Google Scholar, 26Paige C.J. Kincade P.W. Ralph P. Nature. 1981; 292: 631-633Crossref PubMed Scopus (41) Google Scholar). In response to LPS, they uniformly differentiate into an immature B lymphocyte-like state by activating the transcription of a pre-rearranged κ-light-chain allele (24Paige C.J. Kincade P.W. Ralph P. J. Immunol. 1978; 121: 641-647PubMed Google Scholar, 25Sakaguchi N. Kishimoto T. Kikutani H. Watanabe T. Yoshida N. Shimizu A. Yamawaki-Kataoka Y. Honjo T. Yamamura Y. J. Immunol. 1980; 125: 2654-2659PubMed Google Scholar, 26Paige C.J. Kincade P.W. Ralph P. Nature. 1981; 292: 631-633Crossref PubMed Scopus (41) Google Scholar, 27Mains P.E. Sibley C.H. J. Biol. Chem. 1983; 258: 5027-5033Abstract Full Text PDF PubMed Google Scholar). By employing immunoselection against surface-bound IgM, Mains and Sibley (28Mains P.E. Sibley C.H. Somat. Cell Genet. 1983; 9: 699-720Crossref PubMed Scopus (20) Google Scholar) isolated spontaneously arising mutants of 70Z/3 that were completely unresponsive to LPS treatment, failing to express κ light chains (28Mains P.E. Sibley C.H. Somat. Cell Genet. 1983; 9: 699-720Crossref PubMed Scopus (20) Google Scholar, 29Weeks R.S. Sibley C.H. Somat. Cell Mol. Genet. 1987; 13: 205-219Crossref PubMed Scopus (12) Google Scholar). Molecular and biochemical analyses subsequently revealed that the 70Z/3-1.3E2 variant was defective in a crucial NF-κB signaling step, making the cells refractory to all NF-κB-activating stimuli, with the exception of anti-oxidant-insensitive pathways and the HTLV-Tax-1 gene product (19Courtois G. Whiteside S.T. Sibley C.H. Israel A. Mol. Cell. Biol. 1997; 17: 1441-1449Crossref PubMed Google Scholar). More recently, Yamaoka et al. (22Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (945) Google Scholar) showed that, unlike the 70Z/3 parental line, the 70Z/3-1.3E2 variant lacked NEMO protein expression but their wild type phenotype was rescued by NEMO. With high density oligonucleotide arrays, we have performed a genomic analysis of signal-induced, NEMO-dependent NF-κB induction in 70Z/3versus 1.3E2 cells. A large number of novel induced and repressed NEMO/IKK target genes were revealed. Experiments with an IκBα(SS/AA) super repressor revealed that many of these genes are novel and direct targets of NF-κB. 70Z/3 and 70Z/3-1.3E2 cells (19Courtois G. Whiteside S.T. Sibley C.H. Israel A. Mol. Cell. Biol. 1997; 17: 1441-1449Crossref PubMed Google Scholar, 29Weeks R.S. Sibley C.H. Somat. Cell Mol. Genet. 1987; 13: 205-219Crossref PubMed Scopus (12) Google Scholar) and CH12-IκBαAA1A2 cells (30Hsing Y. Bishop G.A. J. Immunol. 1999; 162: 2804-2811PubMed Google Scholar) were routinely cultured in growth media consisting of RPMI 1640 supplemented with 50 μmβ-mercaptoethanol, 2 mm glutamine, 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Stimulations to activate the IKK signalsome pathway in 70Z/3 and 70Z/3-1.3E2 cells were performed by supplementing growth media with 15 μg/ml LPS and 10 ng/ml PMA (both from Sigma Chemical Co.) for 12 h, or LPS alone for 2 or 12 h or 20 ng/ml recombinant murine IL-1 (Life Technologies, Inc.) for 2 or 12 h prior to isolating total cellular RNAs. In some experiments, cellular protein synthesis was inhibited by co-incubation with 100 μm anisomysin (Sigma) to block translational initiation. An IκBα(S32A/S36A) super repressor gene, with serines 32 and 36 mutated to alanines (a kind gift of Dr. Dean Ballard) (31Brockman J.A. Scherer D.C. McKinsey T.A. Hall S.M. Qi X. Lee W.Y. Ballard D.W. Mol. Cell. Biol. 1995; 15: 2809-2818Crossref PubMed Google Scholar), was introduced into 70Z/3 cells by retroviral infection. Stably infected and neomycin- or puromycin-resistant populations of 70Z/3 cells (>1000 clones) were obtained after 12 days of selection in 800 μg/ml Geneticin (Life Technologies, Inc.) or 1 μg/ml puromycin following infection with recombinant murine retroviruses harboring IκBα(S32A/S36A)-IRES-Neo or IκBα(S32A/S36A)-IRES-Puro expression cassettes. CH12-IκBαAA1A2 cells, a derivative of the CH12.LX line harboring a constitutively expressed LacI repressor and an IPTG-regulated IκBα(S32A/S36A) super-repressor, was maintained in growth media supplemented with 200 + 400 μg/ml hygromycin and Geneticin, respectively (30Hsing Y. Bishop G.A. J. Immunol. 1999; 162: 2804-2811PubMed Google Scholar). After inducing their transfected IκBα(S32A/S36A) gene with 200 μm IPTG for 24 h (30Hsing Y. Bishop G.A. J. Immunol. 1999; 162: 2804-2811PubMed Google Scholar), cells were stimulated for 2 h with 2.7 μg/ml plasma membranes from Sf21 insect cells, which had been stably infected with a murine CD40L-expressing recombinant baculovirus (32Kehry M.R. Castle B.E. Semin. Immunol. 1994; 6: 287-294Crossref PubMed Scopus (79) Google Scholar). Total cellular RNAs were extracted from 70Z/3 and 1.3E2 cells with Triazol reagent (Roche Molecular Biochemicals). Poly(A)+ RNAs were isolated from total RNAs of unstimulated and LPS+PMA-stimulated cells with Oligotex (Qiagen). Purified RNAs were converted to double-stranded cDNA with a SuperScript kit (Life Technologies, Inc.) and an oligo-dT primer containing a T7 RNA polymerase promoter (Genset). Biotin-labeled cRNAs were generated from the cDNA samples by an in vitro transcription with T7 RNA polymerase (Enzo kit, Enzo Diagnostics). The labeled cRNAs were fragmented to an average size of 35–200 bases by incubation at 94 °C for 35 min. Hybridization (16 h), washing, and staining protocols have been described previously (Affymetrix Gene Chip Expression Analysis technical manual (33Mahadevappa M. Warrington J.A. Nat. Biotechnol. 1999; 17: 1134-1136Crossref PubMed Scopus (130) Google Scholar)). Affymetrix murine chips (mouse 11K set, subA and subB) were used for hybridization. Chips were stained with streptavidin-phycoerythrin (Molecular Probes) and read with a Hewlett-Packard GeneArray scanner. DNA microarray chip data analysis was performed using GENECHIP 3.2 software (Affymetrix). The quantitation of each gene expression was obtained from the hybridization intensities of 20 perfectly matched and mismatched control probe pairs (34Lockhart D.J. Dong H. Byrne M.C. Follettie M.T. Gallo M.V. Chee M.S. Mittmann M. Wang C. Kobayashi M. Horton H. Brown E.L. Nat. Biotechnol. 1996; 14: 1675-1680Crossref PubMed Scopus (2798) Google Scholar). The average of the differences (perfectly matched minus mismatched) for each gene-specific probe family was calculated. The software computes a variety of different parameters to determine if an RNA molecule is present or absent (Absolute Call) and whether each transcript's expression level has changed between the baseline and experimental samples (Difference Call). In this work, all chip files were scaled to a uniform intensity value (1500) for all probe sets. For a comparative chip file (such as stimulated Wt. versus stimulated Mut.), the experimental file (stimulated Wt.) was compared with the baseline file (stimulated Mut.). To minimize false positives, the following criteria were selected for significant changes for each primary screen: 1) the change in the average difference across all probe sets was >3-fold; 2) for induced genes, a difference call of “increase” or “marginal increase” should be present, and an absolute call of “presence” should be associated with the experimental file; 3) for suppressed genes, a difference call of “decrease” or “marginal decrease” should be present, and an absolute call of “presence” should be associated with the baseline file. Hierarchical clustering was performed with the Cluster program (available at the Stanford Web site) as described previously (35Eisen M.B. Spellman P.T. Brown P.O. Botstein D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14863-14868Crossref PubMed Scopus (13157) Google Scholar). Genes that showed >3-fold changes in at least two of the Wt.+/Mut.+ comparisons (i.e. IL-1, 2 h; IL-1, 12 h; LPS, 2 h; or LPS, 12 h) were subjected to clustering analysis for eight stimulated Wt. and Mut. samples (see Fig. 2). Genes that were detected as absent in all eight arrays were removed. The average difference values (representing the quantity of mRNA, see above) of the selected genes (360) were median-centered by subtracting the median-observed value, normalized by genes to the magnitude (sum of the squares of the values) of a row vector to 1.0. The normalized data were clustered through one cycle of K-means clustering (K = 5) and then further clustered by average linkage clustering analysis of Y axis (genes) using an uncentered correlation similarity metric, as described in the program Cluster. Average difference values of 50 or less were set to 50 before median centering and normalization. The clustered data were visualized by the program TreeView (available at the Stanford Web site). RT-PCRs were performed as previously described (36McKenzie F.R. Connelly M.A. Balzarano D. Muller J.R. Geleziunas R. Marcu K.B. Mol. Cell. Biol. 2000; 20: 2635-2649Crossref PubMed Scopus (13) Google Scholar). To establish their relative qualities, serial dilutions of cDNAs were amplified withβ-actin and GAPDH-specific primers for internal standardization. Similarly, linear response ranges were determined for each gene to semi-quantify their levels of expression as a function of LPS stimulation in 70Z/3 and 1.3E2 mutant cells. The sizes of PCR products corresponded to those expected for each gene. PCR primer pairs were 22- to 24-mers, and their nucleotide sequences are available from the authors upon request. TaqMan Real-time quantitative PCR is based on a fluorogenic 5′-nuclease assay (37Livak K.J. Flood S.J. Marmaro J. Giusti W. Deetz K. PCR Methods Appl. 1995; 4: 357-362Crossref PubMed Scopus (1321) Google Scholar). The same total RNA samples that were used to prepare probes for microarray hybridization were treated with Dnase I followed by RNeasy Mini protocol for RNA cleanup (Qiagen). The TaqMan probe consists of an oligonucleotide with a 5′-reporter dye (FAM) and a 3′-quencher dye (TAMRA). To measure the gene copy numbers of the target transcript, cloned plasmid DNA or mouse genomic DNA was serially diluted and used to produce a standard curve as described elsewhere (38Li X. Wang X. Brain Res. Brain Res. Protoc. 2000; 5: 211-217Crossref PubMed Scopus (46) Google Scholar). Data from TaqMan PCR analyses were normalized based on mRNA copy numbers of GAPDH using the TaqMan rodentGAPDH control reagents (Applied Biosystems). The 70Z/3 pre-B line (Wt.) and its 1.3E2 NF-κB signaling-defective mutant (Mut.) were initially exposed to a combination of 15 μg/ml LPS and 100 ng/ml PMA (phorbol 12-myristate 13- acetate) for ∼12 h (equivalent to one to two cell generations) to simulate a condition of long term, constitutive NF-κB activation. Employing DNA microarrays of 11,800 known cellular genes and expressed sequence tags (Affymetrix Mu11KsubA and Mu11KsubB Arrays), 1.3% of genes displayed greater than 3-fold increases whereas 0.9% revealed greater than 3-fold decreases in expression in comparisons of 12-h stimulated 70Z/3 wild type versus 1.3E2 mutant cells. Independent microarray screenings of both Wt. and Mut. cells that were either unstimulated or stimulated by 15 μg/ml LPS or 20 ng/ml IL-1 were also performed. Genes affected 3-fold or more in the primary screening (Wt.+/Mut.+, 12-h LPS+PMA) were confirmed in a 12-h LPS screen with only occasional variations. The -fold changes of these selected hits in various comparisons between Wt. and Mut. stimulated and unstimulated cells were visualized as a two-color image (Fig.1). Most hits were also confirmed in independent microarray screens of stimulations for 12 h with IL-1 or LPS (Fig. 1), indicating that the different stimuli (LPS+PMA, LPS, and IL-1) regulate these genes by a common mechanism. Given that IKK/NF-κB activation mediated by LPS, PMA, and IL-1 are all defective in the NEMO null 1.3E2 line, genes identified in the primary Wt.+ versus Mut.+ screens should represent direct and indirect targets of the IKK/NF-κB pathway, provided that IKK/NF-κB-independent NEMO signaling pathways do not exist. Even though NEMO/IKKγ has been clearly established to function as an essential non-catalytic component of the IKK complex in vivo, this physiological role need not constitute its only cellular raison d'être. Because the IKK/NF-κB pathway is latent in unstimulated cells, similar IKK/NF-κB-dependent gene expression changes in stimulated versus unstimulated 70Z/3 Wt. cells would be anticipated. Consistently, the majority of the up-regulated and repressed genes identified in the Wt.+/Mut.+, LPS+PMA comparison in Fig. 1 were also identified in another microarray screen comparing 12-h LPS+PMA stimulated to unstimulated 70Z/3 wild type cells (see Wt.+/Wt.−, LPS+PMA in Fig. 1). As expected, genes affected in the Wt.+ versus Wt.− screen were not observed in a Mut.+versus Mut.− screen (see Fig. 1). However, a fraction of the genes identified in the primary Wt.+ versus Mut.+ screen were inversely affected in the Mut.+ versus Mut.− screen (i.e. stimulated genes being repressed and repressed genes being stimulated) (see Fig. 1). The results indicate that these latter genes can only be effected by extracellular signals in the absence of NEMO or NF-κB but not in the presence of NEMO or NF-κB. We classified the novel target genes into eight functional categories in Tables I and II. To ensure that genes identified in the primary Wt.+versus Mut.+ screens have a higher probability of belonging to the IKK/NF-κB signaling pathway and not to an unknown, NEMO-dependent, IKK-independent pathway, we employed an additional selection criteria to assemble the relevant genes. Thus, all genes exhibiting inverse Mut.+ versus Mut.− effects of 2-fold or more in at least two independent screens were filtered out of Tables I and II. Table I displays the known genes identified by the initial screen of 70Z/3 Wt.versus 1.3E2 Mut. cells stimulated with LPS+PMA for 12 h. In addition to co-stimulating with LPS and PMA for 12 h, we also performed chip screens of cells stimulated with LPS or IL-1 alone for 2 and 12 h. Most genes that were identified by the additional screenings are the same as those identified by the initial LPS+PMA screen (Table I). Table II displays genes that were revealed by the 2-h LPS, 2-h IL-1, and 12-h IL-1 screens, which were not modulated more than 3-fold in the primary 12-h LPS+PMA screen. Genes such as Etl-1, TNF-α,Bcl-2, N-myc, PAC-1, PLA2, and 2B4 were only affected in the 2-h stimulation time (Table II). Genes presented in Tables I and II also showed minimal expression changes in a subsequent screen of Wt.− versusMut.− cells (Fig. 1 and Tables I and II), providing additional confidence that the activated IKK pathway targets them. Taken together, these results are consistent with most of the genes in Tables I and IIbeing co-dependent on NEMO and the IKKs to activate the NF-κB signaling pathway.Table IIKK/NEMO-regulated genes are listed in order of their fold changes in 12 h LPS + PMA stimulated (+) 70Z/3 Wt. versus 1.3E2 Mut. cellsGenes and descriptionsLPS + PMAWt−/Mut− unstim.LPSIL-1Wt+/Mut+ (12 h)Wt+/Wt− (12 h)Mut+/Mut− (12 h)Wt+/Mut+ (2 h)Wt+/Mut+ (12 h)Wt+/Mut+ (2 h)Wt+/Mut+ (12 h)AutoregulationU36277IκBα (2 hits)25.6101.62.79.17.14.92.8M83380RelB transcription factor6.64.81.7−1.45.22.83.73AA14443Nuclear factor NF-kappaB p100 subunit (2 hits)6.56.12.53.15.45.92.73.7Growth and developmentDifferentiation/cell fateAF01702B-ATF transcription factor (2 hits)10.85.2−1.81.26.414.55.22.9X54149MyD118 (GADD-like/myeloid differentiation primary response)9.112.52.21.812.312.41.81.8aa27119Mouse Notch 1 (embryonic cell fate mediator)5.9−1.1−2.42.617.15.913.112.1U61969Wnt10a42.71.21.84.34.45.22.2L38971Integral membrane protein 23.92−1.11.512.39.611.710.458636Lef-1 (lymphoid enhancer binding factor) (2 hits)−3.9−12−1.41.9−2−3.2−1.4−1.1Growth arrest and apoptosisD14883KAI1/CD82 (tetraspan transmembrane protein, tumor suppressor)3.941.51.56.59.94.94.1M21828Gas2 (growth-arrest-protein)−3.4−2.31.61.3−1.6−3.3−1.3−1.4Proliferation and survivalL41495Pim-2 (serine/threonine kinase) (2 hits)46.238.6−1.7−1.626.235.220.111.4AA108330Pea-15/Mat-1 (mammary transforming gene)5.94.81.62.411.312.97.512x63027Mammary tumor virus locus 434.13.2−1.6−1.32.37.621.9AA050733GILZ/TSC22 (glucocorticoid-induced leucine zipper)−5.2−2.13.6−1.3−5.1−10.5−2.4−5.6Signal transduction/cell cycleU06924STAT1 (signal transducer and activator of transcription)8.15.8−1.11.8−1.48.94.7−2.9AA011731Pip5k2α (phosphatidylinositol-4-phosphate 5-kinase IIα)6.94.1−1.6−1.66.310.6−2.55.2Z14249MAPK/ERK-1 (mitogen-activated protein kinase)5.93.7−1.21.73.132.11.8W41501R-ras52.9−1.91.55.94.85.98.2u94828Rgs16 (regulator of G-protein signaling)4.31.11.42.98.52.43.84.2I38444T-cell-specific GTPase4.23.81.5−1.82.84.21.82.5m83749Cyclin D24−1.5−1.52.89.36.39.416.9AA013648ler5 (Immediate early response 5)3.18.31.4−1.63.82.11.12.3M90388Ptpn8/70zpep (non-receptor protein tyrosine phosphatase) (2 hits)−5.6−2.81.7−1.2−2.6−4.3−2.6−2W10739Rgs2 (regulator of G-protein signaling)−3.5−2.521.5−2.1−6.11−1.7aa184871Mkp-3 (dual specificity protein phosphatase)−3.2−2.6−1.7−2.2−4.9−5−2.7−1.8C78795Ccnd3 (mouse cyclin D3) (2 hits)−3.1−1.6−1.1−2.1−4.3−3−2.1−2.4Environmental stress/immune responsesx56602UCRP/ISG15 (interferon-induced 15-kDa protein)36.636.1−1.6−121.815.13.3−1.6ET61599Igκ (immunoglobulin kappa light chain) (19 hits)25.114.4−1.5−1.532.821.512.633w12941Mouse analogue of rat interferon-inducible protein147.9−2.1−1.25.9152.41.1AF028725Mirf5 (mouse interferon regulatory factor 5)11.46.5−1.4−1.38.28.24.44.9C77421OxyR/Y-box protein 3 (redox-dependent transcription factor)8.39.21−1.21.73.31.61.2M83312CD40 ligand85.3−1.2−2.529.111.511.18.8ET62172IgH (immunoglobulin heavy chain) (5 hits)7.66.7−1.3−1.8516.76.74.4M17122C4b complement binding protein7.43.8−1.31.16.86.85.84.5L23108CD36 antigen (2 hits)7.34.81.2−1.134.72725.511.3Y08026Immunity associated protein 384.32.2−1.11.46.14.23.94.3x96639Exostoses (multiple) 13.91.5−1.41.73.22.45.12.3AA051446Cerebellar degeneration-associated antigen3.72.42.53.51.43.11.83.1U72519Evl (Ena-vasodilator stimulated phosphoprotein)3.7−1.11.62.821.4412.416.4AA118715CD97/leukocyte antigen/DAF interactor−7.8−1.63.2−1.4−1.2−4.3−8.4−3.4x53825CD24a (heat-stable antigen)−6.1−8.5−1.9−1.8−3.5−4.3−3.4−2.3U69488G7e (viral envelope-like protein)−5.5−2.41.8−1−5.7−11.1−4.9−6Kinesis/secretion/inflammatory responsesu02298Scya5 (RANTES chemokine)41.121.81.4−1.221.950.7−1.62.5u16985Lymphotoxin-beta28.919.91.83.533.619.212.113.4X12531MIP-1/Scya3 (macrophage inflammatory protein)28105.9−1.2−3.88.28.24.12.4L31580CCR7/EBI-1 (chemokine receptor 7)27.27−1.62.11118.84.93.3AF013114EBI-3 (cytokine receptor-like molecule) (2 hits)11.46.91.7−1.328.511.319.315L06039pecam1 (platelet endothelial c" @default.
• W1969132581 created "2016-06-24" @default.
• W1969132581 creator A5003150512 @default.
• W1969132581 creator A5003635947 @default.
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• W1969132581 creator A5084448524 @default.
• W1969132581 date "2001-05-01" @default.
• W1969132581 modified "2023-10-07" @default.
• W1969132581 title "Novel NEMO/IκB Kinase and NF-κB Target Genes at the Pre-B to Immature B Cell Transition" @default.
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