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J. Biol. Chem., Vol. 280, Issue 14, 14057-14069, April 8, 2005

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Gene Expression Profiling in Conjunction with Physiological Rescues of IKK-null Cells with Wild Type or Mutant IKK Reveals Distinct Classes of IKK/NF-B-dependent Genes^*

Paul E. Massa¶, Xiang Li||, Adedayo Hanidu||, John Siamas, Milena Pariali¶, Jessica Pareja**, Anne G. Savitt**, Katrina M. Catron||, Jun Li||, and Kenneth B. Marcu, Senior scholar of the Institute of Advanced Studies of the University of Bologna¶**

From the Genetics Graduate Program, the Departments of Biochemistry and Cell Biology and ^**Microbiology, Institute for Cell and Developmental Biology, State University of New York at Stony Brook, Stony Brook, New York 11794-5215, the ^||Department of Immunology and Inflammation, Boehringer Ingelheim Pharmaceuticals, Ridgefield, Connecticut 06877-0368, and the ^¶Center for Applied Biomedical Research, San Orsola Hospital, University of Bologna, Via Massarenti 9, Bologna 40138, Italy

Received for publication, December 22, 2004

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cellular responses to stress-like stimuli require the IB kinase (IKK) signalsome (IKK, IKK, and NEMO/IKK) to activate NF-B-dependent genes. IKK and NEMO/IKK are required to release NF-B p65/p50 heterodimers from IB, resulting in their nuclear migration and sequence-specific DNA binding; but IKK was found to be dispensable for this initial phase of canonical NF-B activation. Nevertheless, IKK(-/-) mouse embryonic fibroblasts (MEFs) fail to express NF-B targets in response to proinflammatory stimuli, uncovering a nuclear role for IKK in NF-B activation. However, it remains unknown whether the global defect in NF-B-dependent gene expression of IKK(-/-) cells is caused by the absence of IKK kinase activity. We show by gene expression profiling that rescue of near physiological levels of wild type IKK in IKK(-/-) MEFs globally restores expression of their canonical NF-B target genes. To prove that the kinase activity of IKK was required on a genomic scale, the same physiological rescue was performed with a kinase-dead, ATP binding domain IKK mutant (IKK(K44M)). Remarkably, the IKK(K44M) protein rescued 28% of these genes, albeit in a largely stimulus-independent manner with the notable exception of several genes that also acquired tumor necrosis factor- responsiveness. Thus the IKK-containing signalsome unexpectedly functions in the presence and absence of extracellular signals in both kinase-dependent and -independent modes to differentially modulate the expression of five distinct classes of IKK/NF-B-dependent genes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The NF-B pathway is important for a host of cellular processes including its central role in responses to stress-like stimuli, the antiapoptotic cascade, the initiation and maintenance of immune responses, embryonic and adult tissue development, and cell cycle progression (for review, see Refs. 1-11). In mammals the NF-B family of transcription factors is comprised of five subunits characterized by the presence of a conserved Rel homology DNA binding domain (for review, see Refs. 2, 12, and 13). The p65(RelA), c-Rel, and RelB NF-B subunits are fully functional transcriptional activators, whereas the p50 and p52 subunits lack a transcriptional activation domain (for review, see Refs. 12 and 13). NF-Bs function as specific hetero- or homodimers that bind to a GGGRNWTYCC consensus DNA sequence found in the promoters or enhancers of NF-B target genes (for review, see Refs. 12 and 13). Transcriptional activating NF-B subunits are normally sequestered in the cytoplasm of unstimulated cells in a complex with one of the IB family proteins, which block their nuclear import and DNA binding activity. A large variety of extracellular activating stimuli induce the proteosomal dependent destruction of IBs thereby differentially freeing NF-Bs to bind DNA and activate the transcription of their genomic targets (for review, see Ref. 14). Stress-like inducers of NF-B (including proinflammatory cytokines such as TNF¹ and IL-1) function in a classical monophasic capacity to drive the canonical NF-B activation pathway rapidly, which largely involves the activation of p65(RelA)/p50 DNA binding activity and transcriptional competence (for review, see Refs. 1, 2, 8, and 15). More recently, a distinct class of NF-B stimuli (exemplified by LT, BAFF, and CD40 ligand), which also contribute to the implementation of differentiation programs and the adaptive phase of immune responses, have been shown to function as biphasic activators initially acting via the rapid canonical pathway and subsequently feeding into a delayed noncanonical protein synthesis-dependent route characterized by the activation of RelB/p52 heterodimers (for review, see Refs. 8, 9, 15, and 16).

With the exceptions of UV radiation and the effects of some DNA-damaging agents (17, 18), the release of NF-Bs from IBs is mediated by the cytoplasmic signalsome complex, which consists of two serine-threonine kinases (IKK, IKK) and NEMO/IKK, a regulatory/docking protein (for review, see Refs. 1, 7, 8, and 15). IKK is essential for the phosphorylation of IBs on a pair of amino-terminal serines (residues 32 and 36 in IB) thereby targeting IB for ubiquitination and subsequent proteosomal destruction (for review, see Refs. 1, 7, and 8). In contrast, IKK is not required for the phosphorylation of IBs via the canonical NF-B activation pathway in vivo, with the exception of receptor activator of NF-B (RANK) ligand signaling in mammary epithelial cells (19). Rather, IKK plays an essential role in epidermal keratinocyte differentiation independent of both its kinase activity and NF-B activation and has also recently been found to play NF-B-dependent and -independent roles in tooth development (20-22). With respect to its physiological role in NF-B signaling pathways, IKK is instead essential for the activation of the noncanonical NF-B activation pathway, which requires neither IKK nor NEMO/IKK (for review, see Refs. 8, 15, and 16). In this context, via NF-B-inducing kinase-dependent signaling, IKK phosphorylates multiple serines of the p100 precursor of the p52 subunit, thereby inducing its proteosome-dependent processing into mature p52 subunits that are then freed to activate NF-B target genes as RelB/p52 heterodimers (8, 9, 23, 24). We and other groups have also found that IKK is required to activate the transcription of canonical NF-B target genes (25-29). The latter dependence on IKK is independent of IB destruction and instead appears to involve one or more nuclear targets perhaps including histone H3 (27, 28) and the SMRT transcriptional corepressor (29) resulting in the de-repression of NF-B target genes.

In this report we have investigated the physiological requirement of the kinase activity of IKK for the expression of NF-B-dependent genes on a genomic scale in IKK-null MEFs. Physiological expression of Wt. IKK in IKK(-/-) MEFs by retroviral transduction resulted in the rescue of specific NF-B-dependent genes in the presence and absence of TNF stimulation. Comparative microarray screens with NF-B-compromised MEFs (p50(-/-) and Wt. + IB(S32A,S36A)) revealed that the large majority of these IKK-rescued genes are either dependent on basal or TNF-inducible NF-B, thus demonstrating that 1) IKK plays an essential role in controlling the expression of both signal-induced and basal NF-B-dependent genes, and 2) IKK does not appear to influence the expression of a large number of genes outside the NF-B pathway. Comparable physiological rescue with a kinase-dead IKK mutant protein (IKK(K44M)) showed that most of these genes are dependent on IKK kinase activity for their stimulus-dependent and -independent expression. However, the expression of up to 28% of these NF-B-dependent genes was also surprisingly rescued by the kinase-inactive IKK(K44M) mutant. Furthermore both wild type and mutant IKK are also required for the basal levels of expression of specific NF-B-dependent genes. Thus, our findings collectively reveal that the levels of expression of different downstream NF-B-dependent genes are differentially codependent on catalytically active IKK in the presence or absence of extracellular stimuli.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Tissue Culture—Growth of IKK(-/-) MEFs and their stimulation with TNF were performed as described previously. Wt. IKK/CHUK-HA or IKK(K44M)-HA (a kinase-inactive ATP binding domain mutant with lysine 44 mutated to methionine) with carboxyl-terminal HA epitope tags (30-32) was introduced into IKK(-/-) MEFs by transduction with a retroviral vector coexpressing a puromycin resistance gene followed by 6-8 days of puromycin selection (26, 33). IKK(-/-) cells harboring an empty retroviral vector (EV cells) were simultaneously generated as a matched negative control.

RNA Preparation—Total cellular RNAs were extracted from cell lysates with an RNeasy kit (Qiagen). Purified RNAs were converted to double-stranded cDNA with a Super Script Double Stranded cDNA synthesis kit (Invitrogen) and an oligo(dT) primer containing a T7 RNA polymerase promoter (GENSET). Biotin-labeled cRNAs were generated from the cDNA samples by 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 (Affymetrix Gene Chip Expression Analysis Technical Manual; (34)).

DNA Microarrays and Clustering Analysis—We employed Affymetrix MG-U74Av2 chips that include 12,400 genes. Chips were stained with streptavidin-phycoerythrin (Molecular Probes) and scanned with a Hewlett-Packard Gene Array Scanner. DNA microarray chip data analysis was performed using MAS5.1 software (Affymetrix) and as described previously (26). Levels of gene expression were quantitated from the hybridization intensities of 16 pairs of perfectly matched and mismatched control probes (35) (Affymetrix, Inc.). The average of the differences (perfectly matched minus mismatched) for each gene-specific probe family was calculated and expressed as signal values. The software computes how the expression level of each transcript has changed between the base line and experimental samples (difference call/change call). A change call is a qualitative call that describes whether a transcript in an experimental array has changed compared with a base-line array. One array is designated as the experimental, and another array is designated as the base line. Wilcoxon's signed rank is used to generate a change p value. A change call is assigned based on analysis parameters. Change p values between 0.00 and 0.0025 are given an Increase call. Change p values between 0.0025 and 0.003 are given a marginal increase call. Change p values between 0.997 and 0.998 are given a marginal decrease call. Change p values between 0.998 and 1.00 are given a decrease call (Affymetrix User Manual). For a comparative chip file (such as TNF-stimulated IKK(-/-) MEF + Wt. IKK versus IKK(-/-) MEF + EV, the experimental file (Wt. IKK 2T) was compared with the base-line file (EV 2T). We employed the following stringent selection criteria to identify significant changes in gene expression: 1) a change call of increase or marginal increase in both samples, and 2) average -fold change values of 1.5 or greater (minimum of 1.3-fold each) in two independently stimulated samples of IKK(-/-) MEF + Wt. IKK 2T versus IKK(-/-) MEF + EV 2T. The following additional criteria were employed to identify the spectrum of these genes that were also dependent upon NF-B for their expression: 1) a change call of either increase or marginal increase in Wt. MEF 2T versus Wt. MEF + IBSR-Iresneomycin, or 2) a change call of either increase or marginal increase in Wt. MEF 2T versus p50(-/-) MEF 2T, and 3) a valid presence call in Wt. 2T screens. By this analysis we define NF-B dependence to represent genes whose expressions either directly (true direct targets of NF-B subunits) or indirectly (other downstream genes whose expressions are affected by the NF-B pathway) require NF-B. The TNF inducibilities of genes rescued by either Wt. IKK or IKK(K44M) were based on a combination of the following stringent criteria: 1) increase calls in duplicate 2T versus US microarray screens of IKK(-/-) MEFs rescued by Wt. IKK or IKK(K44M), and/or 2) TaqMan real time PCR analysis performed at least in duplicate and an increase call in one of two screens. By these criteria, the vast majority of IKK-rescued genes that responded to TNF did so with -fold change values of 2.0 and higher in duplicate screens with a minimum unstimulated (US) average -fold change value of 1.7.

Hierarchical clustering was performed with the Cluster program (available at rana.lbl.gov/) as described previously (36). Genes that have double increase calls and are induced >1.5-fold (average -fold values) in the duplicate primary Wt. IKK versus EV-rescued IKK(-/-) MEFs (see above description) were selected. The signal values (equivalent to the quantities of mRNAs, see above) of the selected genes were median-centered by subtracting the median observed value and 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 by average linkage clustering analysis of the y axis (genes) using an uncentered correlation similarity metric, as described in the program Cluster. Signal values of 50 or less were set to 50 before centering and normalization. The clustered data were visualized with the Treeview program (available at rana.lbl.gov/).

TaqMan Real Time Quantitative PCR—TaqMan real time quantitative PCRs were performed as described previously (26, 37). Data from TaqMan PCR analyses were normalized based on glyceraldehyde-3-phosphate dehydrogenase mRNA copy numbers using rodent glyceraldehyde-3-phosphate dehydrogenase control reagents (Applied Biosystems). TaqMan probe sets were designed for the following genes using either Primer Express 1.5 or Bio-Rad Beacon Designer 2.0 software: ATF3, A20, ISG15, MyD118/GADD45, SAA3, and VCAM1. Murine IL-6 and RANTES TaqMan reagents were obtained from ABI. TaqMan real time PCR experiments were performed in an ABI PRISM 7700 sequence detector or in a Bio-Rad iCycler. DNA sequences for each of these probe sets are available upon request.

Western Blotting—Cell lysates were prepared in an isotonic lysis buffer containing 1% Nonidet P-40 supplemented with protease inhibitors. SDS-PAGE (10% gel) transfer to polyvinylidene difluoride membranes was performed as described previously, and membranes were probed with primary antibodies to either IKK (Cell Signaling Technology) or NEMO (Santa Cruz Biotechnology) followed by an anti-rabbit-horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences). Blots were developed using a Lumi-Light Plus kit (Roche Applied Science).

Immunostaining—Prior to immunostaining cells were maintained in 10-cm tissue culture-treated plates in their regular growth medium. Cells were trypsinized, resuspended in 12 ml of growth medium, and plated at 3 ml/well in 6-well plates containing 22-mm glass microscope coverslips (VWR) precoated with poly-L-lysine (Sigma). Cells were incubated overnight at 37 °C with 5% CO₂. The following day, cells were washed and then fixed in 50% methanol and 50% acetone for 10 min. These and all subsequent washes were with phosphate-buffered saline. The fixed cells were rehydrated in phosphate-buffered saline, and the coverslips were washed, blocked with phosphate-buffered saline containing 10% heat-denatured fetal bovine serum for 1 h, and washed again. In situ expression of retrovirally transduced Wt. IKK-HA and IKK(K44M)-HA proteins in IKK(-/-) cells were specifically detected with a primary anti-HA 12CA5 antibody (38). 12CA5 antibody was diluted in blocking solution and applied to the cells followed by a 1-h incubation at room temperature. After washing, alkaline phosphatase-conjugated goat anti-mouse IgG (Jackson) secondary antibody, diluted 1:2,000 in block solution, was applied for a 1-h incubation at room temperature. The coverslips were washed and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate developing substrate applied (Roche Applied Science). Cells were visualized on a Nikon Diaphot phase-contrast microscope and photographed using a Nikon D1X digital camera.

NF-B DNA Binding—Activation of NF-B p65-dependent DNA binding activity was quantitated with an enzyme-linked immunosorbent assay-based kit (Active Motif, Inc.) (39). Nuclear extracts were prepared from unstimulated or 2-h TNF-stimulated WT. IKK-null and Wt. IKK or IKK(K44M)-rescued IKK(-/-) MEFs and applied to 96-well plates containing an immobilized NF-B DNA binding consensus oligonucleotide according to the manufacturer's instructions (Active Motif, Inc.). DNA-bound NF-B was detected with a p65-specific primary antibody followed by the addition of a secondary antibody conjugated to horseradish peroxidase and absorbance quantitated at 450 nm with a microplate spectrophotometer. The specificity of NF-B DNA binding was confirmed by competitions with wild type and mutant NF-B binding sequences. Negative controls for stimulus-dependent NF-B nuclear localization and DNA binding activity also included nuclear extracts prepared from NEMO(-/-) and p65/p50(-/-) MEFs.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Physiological Rescue of IKK(-/-) MEFs with Wt. IKK IKK(K44M) Proteins Does Not Interfere with the Induction of NF-B DNA Binding Activity—By employing DNA microarray chip technology, we previously reported that the IKK protein was as essential as the IKK and NEMO/IKK signalsome subunits for the genomic NF-B-dependent transcriptional response induced by TNF or IL-1 stimulation. Our finding of a strict requirement for IKK in the regulation of NF-B-dependent transcription in MEFs was controversial because earlier studies had shown that cells derived from IKK-null mice exhibited no significant defect in the stimulus-dependent induction of NF-B nuclear localization and DNA binding activity. However, in agreement with our observations, two other studies had shown that IKK was required for the TNF- and IL-1-dependent transcriptional induction of IL-6 gene expression (25, 40). Subsequent to these reports, chromatin immunoprecipitation experiments showed that the role of IKK in engendering DNA-bound NF-B with transcriptional competence was associated with its TNF-dependent binding to the IB gene promoter and with its ability to directly phosphorylate histone H3 on serine 10 in vitro (27, 28) and more recently also to phosphorylate and thereby facilitate the release of the SMRT corepressor from specific NF-B target gene promoters (29).

To determine whether the intrinsic defect of IKK(-/-) MEFs to express NF-B-dependent genes on a genomic scale was solely caused by the absence of a functional IKK kinase, we employed retroviral transduction to rescue physiological levels of Wt. IKK expression in a large population of IKK(-/-) MEFs. To determine whether IKK kinase activity was required to rescue the expression of their NF-B-dependent genes, we also derived a similar population of IKK(-/-) MEFs expressing near physiological levels of a kinase-dead IKK mutant (IKK(K44M)) (31, 32). To rule out any effects of stable retroviral transduction, we also generated a matched negative control population of IKK(-/-) MEFs harboring the empty retroviral vector (IKK(-/-)-EV cells). Western blotting revealed that the levels of Wt. IKK and IKK(K44M) expression in IKK(-/-)-rescued MEFs were similar to the expression of endogenous IKK in wild type NF-B competent MEFs (see Fig. 1). Importantly, as shown in Fig. 2, in situ immunostaining also revealed uniform expression of either the Wt. IKK-HA or mutant IKK(K44M)-HA proteins in their respective stably transduced cell populations. These stable populations of IKK(-/-) cells expressing physiologically comparable levels of either Wt. IKK-HA or IKK(K44M)-HA were used in all subsequent experiments.

View larger version (45K): [in this window] [in a new window]   FIG. 1. Physiological levels of expression of Wt. IKK and IKK(K44M) in rescued populations of IKK(-/-) MEFs. Populations of IKK(-/-) MEFs were retrovirally transduced to express stably either murine Wt. IKK-HA or an IKK(K44M)-HA mutant protein. SDS-PAGE was performed to determine the levels of IKK expression obtained in the infected populations (top) (see "Experimental Procedures"). The membrane was stripped and reprobed with a NEMO-specific antibody as a reference control (bottom), which showed comparable expression in each cell background.

View larger version (89K): [in this window] [in a new window]   FIG. 2. Immunostaining of Wt. IKK-HA and IKK(K44M)-HA proteins in retrovirally transduced populations of IKK(-/-) cells. Wt. IKK-HA and IKK(K44M)-HA proteins, stably expressed by populations of retrovirally transduced IKK(-/-) MEFs, were visualized by in situ immunostaining. Cells were plated on coverslips coated with poly-L-lysine, fixed, and immunostained using 12CA5 anti-HA monoclonal antibody or no primary antibody as a negative control. Stained cells were viewed by a phase-contrast Nikon Diaphot microscope and photographed using a Nikon D1X digital camera, as described under "Experimental Procedures." In the absence of primary antibody (top panels) or in IKK(-/-) parental cells, no immunostaining is seen, whereas populations of IKK(-/-) cells stably transduced by either Wt. IKK-HA- or IKK(K44M)-HA-expressing retroviruses (bottom panels) show uniform cytoplasmic staining.

View larger version (26K): [in this window] [in a new window]   FIG. 3. Physiological expression of Wt. IKK or IKK(K44M) in IKK(-/-) MEFs does not interfere with stimulus-dependent NF-B DNA binding. RelA/p65 DNA binding was assayed using the TransAM NF-B p65 Transcription factor assay kit (Active Motif), following the manufacturer's instructions for the preparation of nuclear extracts. All samples are presented as (-) unstimulated or (+) stimulated for 2 h with 20 ng/ml TNF prior to lysis and nuclear extract preparation. Data shown represent each data point done in quadruplicate, with standard deviations presented as error bars. Nuclear p65 was measured as the absorbance at 450 nm, with a reference wavelength of 650 nm using a fluorescent plate reader. Specificity of p65-DNA binding within the Wt. IKK- and IKK(K44M)-infected populations was determined by competition for binding using an excess of either Wt. (W)or mutant (M) NF-B synthetic oligonucleotide.

View larger version (40K): [in this window] [in a new window]   FIG. 4. Hierarchical cluster image of the NF-B dependences of genes rescued by Wt. IKK in IKK(-/-) MEFs. Signal values of 118 IKK-dependent genes derived from two independent microarray screens of IKK(-/-) cells expressing Wt. IKK (first two columns) were subjected to hierarchical clustering compared with the following samples: third column, IKK(-/-) MEFs + EV 2T, fourth column, IKK(-/-) MEFs 2T; fifth column, p50(-/-) 2T; and sixth column, Wt. MEFs+IB(S32A,S36A)2T.The118IKK-dependent genes employed in these comparisons were selected on the basis of average -fold change values of 1.5 or greater (minimum of 1.3-fold each) and difference calls of increase or marginal increase in two independent samples of 2-h TNF (2T)-stimulated IKK(-/-) MEF + Wt.IKK 2T versus IKK(-/-) MEF + EV 2T MEFs. The locations of a number of genes are indicated. Signal values were all derived from MAS 5.1 calculation and normalized as described under "Experimental Procedures." Gene expression values are shown in color according to the indicated expression scale bar.

The -fold change values of a representative set of 40 genes rescued by restoring Wt. IKK expression in IKK-null MEFs are shown in Table I. The relative NF-B dependences of these genes are presented as positive -fold change values in one or both of the two microarray screens (Wt. MEF versus Wt. MEF + IBSR or Wt. MEF versus p50(-/-)). Importantly, many of these genes were also rescued to levels of expression observed in Wt. MEFs. Comparisons of the relative mRNA expression levels of genes in duplicate TNF-stimulated and unstimulated samples revealed that the genes rescued by Wt. IKK protein in IKK-null cells fell into two distinct stimulus-dependent and -independent classes (Table I, Fig. 5, and data not shown). The signal values (equivalent to mRNA quantities) of three representative examples of the stimulus-dependent (Fas ligand, C/EBP-, and CXCL10) and independent (Clast1, NOV, and C1r) classes of these Wt. IKK-rescued genes are presented in bar graph format in Fig. 5. Collectively, these results reveal that the expression of a large number of NF-B-dependent genes was restored in IKK-null MEFs by retroviral transduction of a Wt. IKK protein in a TNF-responsive manner, including, IL-6, GADD45, RANTES, ScyB5/LIX, A20, IB, IFITR-1, C/EBP-, ATF3, Fas ligand, caspase-11, M/CSF-1, serum amyloid A3, MIP2, VCAM, JunB, ScyD1, ISG15, Gro1, MMP3, MMP13, and Met 1 (see Table I and selected signal value comparisons in Fig. 5A). Among these 40 representative genes rescued by the Wt. IKK protein, examples of signal-independent rescues by Wt. IKK include ClastI/LR8, C1r, IFP35, Plf2, Plf3, Itm2b, NOV/CNN3, Decorin, Snx10, and IFITR2 (see genes highlighted in gray in Table I and also selected signal value comparisons in Fig. 5B). In agreement with these results, the expression of this same class of genes without extracellular stimulation was also found to be similarly reduced in Wt. MEFs harboring a constitutively expressed IB super-repressor or in p50-null MEFs compared with their wild type counterparts (data not shown). Thus, these observations show that the Wt. IKK-containing signalsome is required for both the stimulus-dependent and basal levels of expression of NF-B-dependent genes. Because this class of NF-B-dependent genes required IKK without TNF stimulation, they define a novel class of IKK-dependent genes that are downstream of basally activated NF-B.

View this table: [in this window] [in a new window]   TABLE I A Wt. kinase-competent IKK protein rescues the expression of two classes of NF-B-dependent genes in IKK-null cells 40 representative IKK-dependent genes, which met the stringent selection criteria outlined-under "Experimental Procedures," are shown. The first data column shows the -fold change values of each gene obtained in two independent microarray screens of IKK(-/-) + Wt. IKK 2T versus IKK(-/-) + EV 2T. The second column displays their relative dependences on IKK in the context of wt. MEFs (i.e. Wt. MEF 2T versus IKK(-/-) 2T as described previously) (26) (and data not shown). The third and fourth columns show the NF-B dependences of each gene by comparing their induced expressions in Wt. MEF 2T compared with either p50-null MEF 2T (third column) or Wt. MEF + IBSR(super-repressor) 2T (26) (fourth column). The criteria for assigning the TNF responsiveness of each rescued gene was determined on the basis of duplicate S (2T) versus US microarray screens of Wt. IKK-rescued IKK(-/-) MEFs (see description of criteria under "Experimental Procedures"). Examples of signal values of three genes are shown in Fig. 5, and TaqMan real time PCR analyses of selected genes are shown in Fig. 9. Genes induced by TNF are assigned a (+) sign, and genes whose expressions were not significantly stimulated by TNF were given a (-) sign. The expressions of 9 of this representative group of 40 genes were rescued independently of TNF stimulation and are highlighted in gray.

View larger version (36K): [in this window] [in a new window]   FIG. 5. Relative mRNA expression levels of examples of stimulus-dependent and -independent classes of genes only rescued by a Wt. IKK protein. A, comparisons of signal values of three NF-B-dependent genes, which were rescued only by a kinase-competent Wt. IKK protein, in a TNF-responsive manner. B, signal value comparisons of three additional genes, whose basal levels of NF-B-dependent expression were rescued only by Wt. IKK and were unaffected by TNF stimulation.

A Kinase-inactive IKK Mutant Rescues a Portion of the Genes Dependent on Wt. IKK in IKK(-/-) MEFs—Because the IKK mechanism of action to activate NF-B-dependent transcription in the canonical pathway remains poorly understood, we next investigated whether the kinase activity of IKK was essential for the expression of the 118 genes rescued by the Wt. IKK protein in IKK(-/-) MEFs. Lysine 44 in the IKK kinase domain is essential for its binding of ATP, and its mutation to methionine prevents ATP binding, thereby completely destroying IKK kinase activity (32, 44). To determine the contribution of IKK kinase function for the rescue of NF-B-dependent genes, we used duplicate microarray analysis to determine the ability of a kinase-dead IKK(K44M) mutant to rescue IKK/NF-B-dependent targets when expressed at near physiological levels in the IKK(-/-) cells (see hierarchical Treeview comparisons of Wt. IKK and IKK(K44M) expressing IKK(-/-) cells in the first and second columns and third and fourth columns, respectively, in Fig. 6). These results demonstrate that 72% of the genes rescued by Wt. IKK in IKK(-/-) cells were unaffected by the presence of comparable levels of an IKK(K44M) mutant protein. Surprisingly, 28% (33 of the 118 IKK-dependent genes) were reproducibly rescued by the IKK(K44M) mutant. Most of these 33 IKK(K44M)-rescued genes can be visualized as two coclustered groups in the upper and lower portions of the hierarchical Treeview image presented in Fig. 6.

View larger version (41K): [in this window] [in a new window]   FIG. 6. Hierarchical cluster image of genes rescued by Wt. IKK compared with an IKK(K44M) mutant. Signal values of the 118 genes rescued by Wt. IKK in duplicate screens (first two columns) were evaluated by hierarchical clustering (as described in Fig. 3) compared with their signal values in duplicate screens of 2-h TNF (2T)-stimulated IKK(-/-) MEFs expressing a kinase-inactive IKK(K44M) mutant (third and fourth columns). As in Fig. 4, the IKK specificities of the rescues can be visualized in the fifth and sixth columns, which display the signal values of the 118 genes in IKK(-/-)-null MEFs or the same cells expressing EV.

View this table: [in this window] [in a new window]   TABLE II NF-B-dependent genes rescued by IKK independently of its kinase activity 20 representative genes meeting the criteria of being rescued by both the Wt. IKK and IKK(K44M) proteins are shown. -Fold change values of these 20 genes obtained from duplicate microarray comparisons using independent samples of IKK(-/-) + IKK(K44M) 2T versus IKK(-/-) + EV 2T are indicated in the first column. The second column shows the results obtained for the same genes in duplicate microarray comparisons of IKK(-/-) + Wt. IKK 2T versus IKK(-/-) + EV 2T. The third column displays their relative dependences on IKK in the context of Wt. MEFs (i.e. Wt. MEF 2T versus IKK(-/-) 2T as described previously (26 and data not shown). The fourth and fifth columns show their NF-B dependences by comparing their expressions in Wt. MEF 2T with either p50-null MEF 2T (fourth column) or Wt. MEF + IBSR(super-repressor) 2T (26) (fifth column). The TNF dependences for the IKK-rescued expression of each gene are shown in the context of their independent rescues by physiological levels of either IKK(K44M) (sixth column 6) or Wt. IKK (seventh column). The criteria for assigning the TNF responsiveness of each rescued gene was determined on the basis of duplicate S (2T) versus US microarray screens (for Wt. IKK or IKK(K44M)-rescued IKK(-/-) MEFs as indicated) (see description of criteria under "Experimental Procedures"). Examples of signal values of three genes are shown in Fig. 7, and TaqMan real time PCR analyses of selected genes are in Fig. 9. Genes induced by TNF are assigned a (+) sign, and genes whose expression was not significantly stimulated by TNF were given a (-). The IKK(K44M) mutant in a TNF-responsive manner comparable to that achieved by Wt. IKK rescued three genes highlighted in gray.

View larger version (39K): [in this window] [in a new window]   FIG. 7. Relative mRNA expression levels of examples of stimulus-dependent and -independent classes of genes rescued by the IKK protein regardless of its kinase activity. A, comparisons of signal values of three NF-B-dependent genes, which were comparably rescued by both Wt. IKK and the IKK(K44M) mutant, in a TNF-responsive manner. B, signal value comparisons of three additional NF-B target genes, whose basal levels of NF-B-dependent expression were rescued by either a wild type or a kinase-inactive IKK mutant but were unresponsive to TNF stimulation.

View larger version (31K): [in this window] [in a new window]   FIG. 8. TaqMan real time PCR analysis of selected genes differentially rescued by Wt. IKK and IKK(K44M). Independent samples of Wt. MEFs and IKK(-/-) MEFs expressing Wt. IKK, EV, or IKK(K44M) were stimulated with 20 ng/ml TNF for 2 h., and RNAs were prepared and subjected to TaqMan real time PCR analysis. The levels of expression of eight representative genes (IL-6, ISG15, SAA3, RANTES, VCAM1, A20, ATF3, and GADD45) were quantitatively compared. Each bar represents data obtained at least in duplicate with the indicated standard deviations. All samples were normalized to a glyceraldehyde-3-phosphate dehydrogenase probe set, and mRNA copy numbers were determined compared with a genomic DNA standard for each probe set.

View larger version (30K): [in this window] [in a new window]   FIG. 9. TaqMan real time PCR analysis of the TNF dependences of selected genes rescued by Wt. IKK or IKK(K44M). The relative levels of expression in TNF-stimulated and unstimulated cells of the eight selected genes in Fig. 8 (IL-6, ISG15, SAA3, RANTES, VCAM1, A20, ATF3, and GADD45) are shown. TaqMan PCRs were performed and quantitated as described under "Experimental Procedures" and in the legend of Fig. 8.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Modifications of NF-B subunits and other post-translational nuclear processes are also necessary for the induction of NF-B target genes, and a number of reports have implicated IKK in these events. Phosphorylation of serines 276 (in the Rel Homology domain/RHD) and serines 529 and 536 in the transcriptional activation domain (TAD) of the NF-B p65/ RelA subunit have been suggested to play activating roles, and a number of kinases have been directly implicated in this step including the IKKs (45-52). Transactivation by p65/RelA in response to TNF was localized to serine 529 within the p65/RelA TAD and found to mediate its transcriptional activation independent of the ability of NF-B to bind DNA (89). Phosphorylation of serine 276 was also found to be involved in the activation of p65/RelA at least in part by controlling its association with the p300/CBP coactivator or the histone deacetylase-1 (53). IKK and IKK were both implicated as downstream effectors of Akt-dependent signaling targeted to serines 529 and 536 in the p65/RelA TAD, which in part appeared to involve the engagement of the CBP/p300 transcriptional coactivator (90, 91). PTEN, a negative upstream effector of Akt, was also reported to inhibit TNF-induced NF-B activation (92, 93), which was subsequently shown to occur solely at the level of p65 transactivation (94). Additionally, IL-1- and Akt-mediated NF-B activation was found to involve p65 TAD phosphorylations with a codependence on IKK and IKK (25). TNF-induced phosphorylation of p65/RelA serine 536 was recently shown to be dependent on both IKK and IKK and mediated by TRAF2, TRAF5, and TAK1 signaling (54), and a requirement for IKK in p65 serine 529 phosphorylation and NF-B-dependent transcriptional activation in response to LT stimulation has also recently been reported (55). In addition, the activation of NF-B by the HTLV-Tax1 protein involves the specific phosphorylation of p65 serines 529 and 536, requiring IKK, but not IKK (56). The IKK mechanism of action in the canonical NF-B pathway has also been proposed to be purely nuclear in nature. In this context, IKK has been shown to migrate into the nucleus (57) and associate with the promoters of NF-B-dependent genes upon TNF stimulation (27, 28). More recently mitogenic activation of the c-fos gene by epidermal growth factor-dependent signaling was found to require the constitutive and induced recruitment of p65/RelA and IKK, respectively, to the c-fos promoter (58). Because IKK was also found to phosphorylate serine 10 of histone H3 in vitro, and the phosphorylation status of histone H3 in IKK(-/-) MEFs was enhanced by introduction of Wt. IKK