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The Journal of Immunology, 2001, 166: 6839-6846.
Copyright © 2001 by The American Association of Immunologists

I{kappa}B Kinase Is Critical for TNF-{alpha}-Induced VCAM1 Gene Expression in Renal Tubular Epithelial Cells1

Zheng Tu*, Vicki Rubin Kelley{dagger}, Tucker Collins{ddagger} and Frank S. Lee2,*

* Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; and {dagger} Laboratory of Molecular Autoimmune Disease, Renal Division, Department of Medicine, and {ddagger} Vascular Research Division, Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The expression of VCAM1 is up-regulated in renal proximal tubular epithelial cells (TEC) in a variety of inflammatory renal diseases, a prominent example of which is acute renal allograft rejection. VCAM1 may play an important role in these diseases because it binds to the integrins very late Ag-4 and {alpha}4{beta}7 on lymphocytes and monocytes, thereby providing a potential mechanism to recruit these leukocytes to sites of inflammation. The molecular mechanisms underlying VCAM1 regulation in renal TEC are essentially unknown. We now report that VCAM1 mRNA is dramatically up-regulated in C1, a cell line derived from renal TEC, on exposure to TNF-{alpha}. Two NF-{kappa}B binding sites in the VCAM1 promoter are critical for the TNF-{alpha}-induced VCAM1 transcriptional up-regulation, and both sites bind to p65-p50 NF-{kappa}B complexes. TNF-{alpha} induces activation of inhibitor of NF-{kappa}B (I{kappa}B) kinase-{beta} (IKK-{beta}), a protein kinase that phosphorylates the NF-{kappa}B inhibitor I{kappa}B, and thereby targets the latter for degradation via the ubiquitin-proteasome pathway. Moreover, dominant negative versions of IKK inhibit TNF-{alpha} activation of a VCAM1 promoter reporter. We conclude that the IKK/NF-{kappa}B pathway is critical in the TNF-{alpha}-induced up-regulation of VCAM1 mRNA in renal TEC.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Renal proximal tubular epithelial cells (TEC)3 are targeted in a variety of immune and inflammatory diseases of the kidney. These include systemic lupus erythematosus, nonsteroidal anti-inflammatory drug-induced interstitial nephritis, and IgA nephropathy, among others (for reviews, see Refs. 1, 2, 3, 4). One striking example is acute renal allograft rejection; renal proximal tubules are characteristically infiltrated by mononuclear leukocytes (tubulitis). Indeed, tubulitis is one of the diagnostic features of acute cellular rejection of the kidney, and the renal TEC is a primary target (5). Episodes of acute renal allograft rejection can limit the function and lifetime of a renal allograft (5). Hence, understanding the molecular mechanisms underlying rejection form a rationale for anti-rejection therapies.

Therefore, it is relevant that VCAM1 is increased in renal TEC during episodes of acute renal allograft rejection (6, 7, 8). VCAM1 is a cell surface glycoprotein of the Ig gene superfamily, and its increase in episodes of acute renal allograft rejection is associated with a T cell and monocytic infiltrate that is not seen in normal kidney (6). Because VCAM1 binds to the integrins very late Ag-4 ({alpha}4{beta}1) and {alpha}4{beta}7 on the surfaces of lymphocytes and monocytes (9, 10, 11), VCAM1 may be responsible for recruiting immune cells to sites of active inflammation. This potential pathophysiologic mechanism is not restricted to graft rejection because TEC VCAM1 is up-regulated in other inflammatory diseases of the kidney including systematic lupus erythematosus, nonsteroidal anti-inflammatory drug-induced interstitial nephritis, IgA nephropathy, and various glomerulonephritides (1, 2, 3, 4).

Although VCAM1 was originally identified in endothelial cells (12), it is expressed in other cell types as well, including vascular smooth muscle cells (13), differentiating skeletal muscle cells (14), renal and neural epithelial cells (15, 16), dendritic cells (17, 18, 19), and bone marrow stromal cells (20). VCAM1 protein expression is increased in pathologic conditions such as cardiac allograft rejection, rheumatoid arthritis, inflammatory bowel disease, and atherosclerosis (21, 22, 23, 24, 25). Thus, increased expression of VCAM1 is a marker of a broad range of disease. Targeted disruption of the VCAM1 gene in mice by homologous recombination results in embryonic lethality at day 10–12 of gestation (26). These mice die from deficiencies of umbilical cord and placenta formation, thereby implicating VCAM1 in these developmental events as well.

VCAM1 gene regulation is tissue specific. In skeletal muscle cells, VCAM1 is constitutively expressed and is not cytokine responsive (14). In contrast, VCAM1 gene transcription in endothelial cells is regulated by the transcription factor NF-{kappa}B in a cytokine-responsive manner (27, 28, 29). Essentially nothing is known about the regulatory elements controlling the VCAM1 gene in renal TEC, nor of the signal transduction mechanisms that impinge on these elements in these parenchymal cells. Indeed, little is known of the signal transduction mechanisms involved in VCAM1 gene expression in any cell type.

In this report, we examined VCAM1 gene expression in C1 cells, a cell line derived from renal TEC (30). C1 cells were generated by transforming murine TEC with origin-defective SV40 DNA. They display alkaline phosphatase and {gamma}-glutamyl-transpeptidase enzymatic activities, properties characteristic of renal proximal TEC. In addition, they proliferate in response to epidermal growth factor and display sodium-dependent glucose uptake (30). Importantly, VCAM1 is up-regulated at both protein and mRNA levels in C1 cells on TNF-{alpha} stimulation (15). We find here that NF-{kappa}B is essential for the induction of VCAM1 gene transcription in response to TNF-{alpha} in C1 cells. Moreover, inhibitor of NF-{kappa}B (I{kappa}B) kinase-{beta} (IKK-{beta}), a protein kinase involved in the NF-{kappa}B activation pathway, plays a critical role in this induction.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and transfections

The C1 cell line was maintained in DMEM/F12 medium supplemented as described previously (30), except that the concentration of epidermal growth factor was 20 ng/ml instead of 25 ng/ml, the HEPES concentration was 15 mM instead of 25 mM, and tissue culture was performed on Primaria (Falcon, Bedford, MA) plastic ware. Transient transfections were performed with Fugene 6 (Roche, Gipf-Oberfrick, Switzerland) as described previously (31). Typically, the cells were split the day before transfection into 3.5-cm diameter wells so as to achieve 60–80% confluence. Cells then were transfected with a 2:1 Fugene/DNA ratio. Twenty-four hours after transfection, cells were incubated with or without 20 ng/ml mouse TNF-{alpha} (Roche) for 8 h.

Plasmids

The human VCAM1 cDNA plasmid and VCAM1 promoter reporter genes (Ffl, F0, F1, F2, F3, F4, F1 mAP1, F2 mGATA, F3 mA, and F3 mB) have been described previously (27, 32). The plasmid pCMV4-FlagI{kappa}B{alpha}(S32A/S36A) was a gift from Dean Ballard (Vanderbilt University, Nashville, TN) and has been described (33). The plasmid pGEM2-{gamma}actin was a gift from Tom Maniatis (Harvard University, Cambridge, MA). pRK-FlagIKK-{alpha}(K40A) and pRK-FlagIKK-{beta}(K40A) were gifts from David Goeddel (Tularik, South San Francisco, CA) and have been described (34, 35). The source of pCMV-LacZ has been described (36).

Northern blot analysis

Total RNA was isolated from C1 cells grown in 15-cm plates with a RNeasy Mini Kit (Qiagen, Chatsworth, CA). Total RNA (5 µg) was subjected to agarose gel electrophoresis and transferred to nylon membranes. Radiolabeled antisense RNA probes were prepared from the VCAM1 cDNA plasmid or pGEM2-{gamma}Actin ({gamma}-actin control) with T3 and T7 RNA polymerase, respectively, and 50 µCi of [{alpha}-32P]ATP with an Ambion Maxiscript Kit (Ambion, Austin, TX). Probes (5 x 106 cpm) then were hybridized to the membrane for 16–18 h at 65°C, washed with low- and high-stringency wash solutions (Ambion Northern Max-Gly kit), dried, and examined by autoradiography.

Chloramphenicol acetyltransferase (CAT) assays

C1 cells grown in 3.5-cm wells were washed twice with PBS/EDTA (1 mM), detached by repeated pipetting, collected by centrifugation at 13,000 x g for 1 min at 22°C, and then lysed by three freeze/thaw cycles in 200 µl of 0.25 M Tris, pH 7.8. CAT and {beta}-galactosidase activities then were measured as described previously (36, 37).

Nuclear extract preparations

C1 cells grown in two 10-cm plates were washed twice with PBS/EDTA (1 mM), detached by repeated pipetting, pooled, and then collected by centrifugation at 250 x g for 5 min at 4°C. The cell pellet was resuspended with four packed cell volumes of Buffer N (10 mM HEPES, pH 7.9, 0.2 mM EDTA, 10% glycerol, 1.5 mM MgCl2, 1 mM DTT, 1 mM PMSF, 10 µg/ml leupeptin). After a 15-min incubation on ice, cells were passed through a 25-gauge needle 30 times. The nuclei were collected by centrifugation at 3000 x g for 5 min at 4°C, washed once with Buffer N, and then resuspended in one packed cell volume of Buffer N. Potassium chloride from a 2 M stock solution then was added to a final concentration of 400 mM. Nuclei were placed on a rocker for 30 min at 4°C and then centrifuged at 10,000 x g for 5 min at 4°C. The supernatant was saved as the nuclear extract and stored at -80°C. Protein concentrations were determined by using the Bradford reagent and BSA as a standard.

EMSA

Oligonucleotide duplex probes used for EMSA were prepared as follows: the {kappa}B1 probe was prepared by first annealing the following two oligonucleotides (NF-{kappa}B binding site is underlined): 5'-GCCCTGGGTTTCCCCT-3' and 5'-TTCAAGGGGAAACCCA-3'. The {kappa}B2 probe was prepared in an analogous manner with the following two oligonucleotides: 5'-CCTTGAAGGGATTTCCCT-3' and 5'-GCGGAGGGAAATCCCTT-3'. The annealed duplexes then were radiolabeled with the Klenow fragment of Escherichia coli DNA polymerase I in the presence of 20 µCi each of [{alpha}-32P]dCTP and [{alpha}-32P]dATP. The radiolabeled probes (20,000 cpm) then were incubated with 15 µg of nuclear extract, 1 µg of poly(dI.dC), 1 µg of sheared salmon sperm DNA, and 2 µg of BSA in 20 µl of 10 mM Tris, pH 7.5, 50 mM NaCl, 5 mM MgCl2, 5% glycerol, 0.2 mM EDTA, 20 µM DTT. Incubations were performed at 22°C for 15 min and then subjected to 5% nondenaturing PAGE in 45 mM Tris-borate, 1 mM EDTA. For certain experiments, a 100-fold molar excess of unlabeled probe was included in reactions. In other experiments, either recombinant I{kappa}B{alpha} protein (5 µg) or Abs (2 µg, except for anti-p50 where 6 µg was used; Trans Cruz version; Santa Cruz Biotechnology, Santa Cruz, CA) against NF-{kappa}B p65 (sc-7151), p50 (sc-1190), p52 (sc-298), c-Rel (sc-6955), or RelB (sc-226) were preincubated with nuclear extracts for 15 min at 22°C before the addition of radiolabeled oligonucleotide probes. I{kappa}B{alpha} (polyhistidine tagged) was purified from E. coli as described previously (36).

Protein kinase assays

C1 cells grown in 3.5-cm wells were washed once with PBS/EDTA (1 mM). Cells then were lysed by the addition of 1 ml of Buffer L (20 mM Tris, pH 7.6, 150 mM NaCl, 25 mM {beta}-glycerophosphate, 2 mM EDTA, 2 mM pyrophosphate, 10% glycerol, 1% Triton X-100, 1 mM DTT, and 1 mM orthovanadate) containing 1 mM PMSF and 10 µg/ml leupeptin. After 15 min on ice, lysates were collected and clarified by centrifugation at 15,000 x g for 10 min at 4°C. Immunoprecipitations were performed by the addition of 10 µl (2 µg) of anti-IKK-{beta} polyclonal Ab (sc-7607; Santa Cruz Biotechnology) to the cell lysate, and then placement on a rocker for 4 h at 4°C. Protein A-agarose (20 µl, Santa Cruz Biotechnology) then was added to each sample and rocked for an additional 2 h at 4°C. The resins were washed three times with Buffer L, and then once with Buffer K (20 mM HEPES, pH 7.6, 20 mM {beta}-glycerophosphate, 10 mM MgCl2, 50 mM NaCl, 1 mM DTT, and 0.1 mM orthovanadate). A 10-µl aliquot of Buffer K containing 0.5 µg of GST-I{kappa}B{alpha} 5–55(5–55), 5 µCi of [{gamma}-32P]ATP, and 100 µM ATP was then added to the resin and incubated at 30°C for 1 h. Products were subjected to 12% SDS-PAGE. GST-I{kappa}B{alpha} 5–55(5–55) was purified from E. coli as described (38). Protein kinase activities were quantitated using a Molecular Dynamics Storm 860 phosphorimager (Molecular Dynamics, Sunnyvale, CA).

Western blot analysis

Aliquots of whole-cell lysates were subjected to 12% SDS-PAGE and then transferred to Immobilon-P membranes (Millipore). The membranes were then blotted with anti-I{kappa}B{alpha} (sc-371; Santa Cruz Biotechnology), anti-I{kappa}B{beta} (sc-945; Santa Cruz Biotechnology), anti-I{kappa}B{epsilon} polyclonal (sc-7156; Santa Cruz Biotechnology), anti-IKK-{beta} monoclonal (05-535; Upstate Biotechnology, Lake Placid, NY) or anti-Flag monoclonal (M2; Sigma, St. Louis, MO) Abs. Western blots were performed as described previously (31).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The VCAM1 gene in C1 cells is transcriptionally regulated in response to TNF-{alpha}

TNF-{alpha} is a potent proinflammatory cytokine implicated in diseases such as renal allograft rejection (2), and one means by which it exerts its effects is through the induction of gene expression (39). To examine the effect of TNF-{alpha} on VCAM1 mRNA levels in C1 TEC, total RNA was isolated from TNF-{alpha}-treated or control C1 cells, and a Northern blot then was performed with a radiolabeled VCAM1 probe. As shown in Fig. 1Go, the VCAM1 mRNA is increased dramatically in C1 cells on TNF-{alpha} treatment. This is consistent with a previous report using this cell line (15).



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  		FIGURE 1. Northern blot for the VCAM1 mRNA in C1 cells. Total RNA was isolated from control C1 cells (left) or C1 cells that had been treated with 20 ng/ml TNF-{alpha} for 4 h (right). The RNA then was subjected to agarose gel electrophoresis, transferred to a nylon membrane, hybridized with radiolabeled antisense RNA probes to either VCAM1 (top) or {gamma}-actin (bottom), and examined by autoradiography.

 
The increased VCAM1 mRNA level on TNF-{alpha} stimulation raises the possibility that the VCAM1 gene might be transcriptionally regulated. To examine this possibility, C1 cells were transfected with a chimeric reporter gene (Ffl) containing 2.2 kb of the VCAM1 5' promoter upstream of a CAT reporter gene (Fig. 2GoA, top construct). As shown in Fig. 2GoB (first two columns), this construct is TNF-{alpha} inducible, thus providing evidence that transcriptional regulation does indeed contribute to the regulation of VCAM1 mRNA level. To examine in more detail potential regulatory elements, we measured the activities of CAT reporter genes containing progressive 5' deletions of the VCAM1 promoter (to position -755 (F0), -518 (F1), -258 (F2), -98 (F3), and -44 (F4); see Fig. 2GoA; position numbers are in reference to the transcription initiation site; Ref. 27). As shown in Fig. 2GoB, Ffl, F0, F1, and F2 display similar inducibility (~3- to 5-fold) in response to TNF-{alpha}. The F3 deletion (to position -98) resulted in a diminished magnitude of reporter gene activity relative to these constructs (Fig. 2GoB). Importantly, however, F3 is still TNF-{alpha} inducible. In marked contrast, further deletion to position -44 (F4 construct) results in the abolition of TNF-{alpha} inducibility. Thus, sequences residing between position -98 and -44 are essential for TNF-{alpha} inducibility of the VCAM1 promoter in C1 cells.



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  		FIGURE 2. Reporter gene analysis of the VCAM1 promoter in C1 cells. A, Schematic diagram of VCAM1 reporter gene constructs. Names of the constructs are given to the left. AP1, GATA, and NF-{kappa}B denote positions of potential transcription factor binding sites. Arrow indicates transcription initiation site. Box denotes CAT reporter gene. B, C1 cells were cotransfected with 4 µg of the indicated constructs and 0.25 µg of pCMV-LacZ with Fugene 6. Twenty-four hours after transfection, some samples were treated with 20 ng/ml TNF-{alpha} for 8 h. Cells were harvested 32 h posttransfection, and CAT activities were measured and normalized to {beta}-galactosidase. Shown is a representative result, performed in duplicate with SDs, from three independent experiments.

 
Inspection of the VCAM1 promoter reveals the presence of potential binding sites for a number of transcription factors, including AP1, GATA, and NF-{kappa}B (27). Indeed, both AP1 and NF-{kappa}B are cytokine-inducible transcription factors (40, 41). To investigate the importance of these transcriptional elements in the cytokine-induced regulation of VCAM1, we examined reporter genes containing block mutations in these binding sites. A mutation of the potential AP1 site (F1 mAP1) had no effect on reporter gene activity (Fig. 3GoA). Consistent with this, the behaviors of F1 and F2, which differ by 260 bp of sequence containing this potential AP1 site, are indistinguishable (Fig. 2GoB). Mutation of the potential GATA site (F2 mGATA), like that of AP1, is similarly without effect (Fig. 3GoB). It might be noted that F3 showed a diminished magnitude of reporter activity in comparison with F2, which differs by 160 bp of sequence containing this potential GATA site (Fig. 2GoB). This suggests that DNA sequences in this region (between -258 and -98), but not the GATA site, are important for optimal TNF-{alpha} inducibility.



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  		FIGURE 3. Reporter gene analysis of regulatory elements in the VCAM1 promoter. A–C. C1 cells were cotransfected with 4 µg of VCAM1 constructs containing block mutations in potential (A) AP1 (F1 mAP1), (B) GATA (F2 mGATA), or (C) NF-{kappa}B (F3 mA and F3 mB) sites, or appropriate control constructs, and 0.25 µg of pCMV-LacZ. Twenty-four hours after transfection, some samples were treated with 20 ng/ml TNF-{alpha} for 8 h. Cells were harvested 32 h posttransfection, CAT activities were measured and normalized to {beta}-galactosidase. Shown is a representative result, performed in duplicate with SDs, from three independent experiments. D, Sequence of the wild-type (WT) VCAM1 promoter from position -86 to -52. Underlined are the two NF-{kappa}B binding sites, {kappa}B1 and {kappa}B2. Shown below are the block mutations of {kappa}B1 and {kappa}B2 sites present in the F3 mA and F3 mB constructs, respectively.

 
In striking contrast to the potential AP1 and GATA sites, mutation of either of two potential NF-{kappa}B binding sites (F3 mA and F3 mB; Fig. 3GoD), results in complete abolition of TNF-{alpha} responsiveness (Fig. 3GoC). Consistent with this, F4, which lacks both of the NF-{kappa}B binding sites, is also completely unresponsive to TNF-{alpha} (Fig. 2GoB). Therefore, these results identify a critical role for NF-{kappa}B binding sites in TNF-{alpha}-stimulated VCAM1 gene transcription in C1 cells. It may be noted that the three constructs lacking NF-{kappa}B binding sites, F3 mA, F3 mB, and F4, all display virtually undetectable basal levels of reporter gene activity in comparison to those containing them (Ffl, F0, F1, F2, and F3; Figs. 2GoB and 3C). This suggests that there is a significant basal level of NF-{kappa}B activity in these cells, which in turn, could account for the relatively modest inducibility of the VCAM1 reporter gene constructs analyzed (Fig. 2GoB). Consistent with this, EMSA analysis described below indicates the presence of a basal level of NF-{kappa}B binding activity in uninduced C1 cell nuclear extracts.

NF-{kappa}B binds to the VCAM1 promoter in C1 cells

To identify the nuclear proteins that bind to these sites, EMSA was conducted with C1 cell nuclear extracts. We synthesized two oligonucleotide duplexes (see Materials and Methods for sequences), each containing the sequences of one of the two potential NF-{kappa}B binding sites, {kappa}B1 and {kappa}B2 (Fig. 3GoD). In uninduced C1 cell nuclear extracts, we detected two weakly binding bands (designated complexes A and B) with the {kappa}B1 and {kappa}B2 probes (Fig. 4GoA, lanes 2 and 9, respectively). On TNF-{alpha} treatment, complexes of identical mobility are induced (compare lane 5 with lane 2 and lane 12 with lane 9). Because unlabeled competitor abolishes both of these complexes, they are specific (lanes 6 and 13).



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  		FIGURE 4. EMSAs of C1 cell nuclear extracts. A, C1 cells were incubated with or without 20 ng/ml TNF-{alpha} for 60 min and then nuclear extracts prepared. Radiolabeled oligonucleotide duplex probes (20,000 cpm) containing the {kappa}B1 or {kappa}B2 site of the VCAM1 promoter then were incubated with the nuclear extract (15 µg), subjected to PAGE, and examined by autoradiography. In some samples, a 100-fold molar excess of unlabeled probe or 5 µg of recombinant I{kappa}B{alpha} protein was preincubated with the nuclear extract before the addition of radiolabeled probe. A and B1, B2 denote the positions of slower migrating species (see text). NS, nonspecific bands. B, Ab analysis of complexes binding to the {kappa}B2 (top) or {kappa}B1 (bottom) probes. Abs against p65, p50, p65 + p50, p52, c-Rel, or RelB were preincubated with the nuclear extract 15 min before the addition of radiolabeled probes. A and B1, B2 denote the position of slower migrating species (see text). Supershift indicates the position of bands induced by Ab preincubation. NS denotes nonspecific bands.

 
The reporter gene analysis suggests that NF-{kappa}B might bind to these sites. NF-{kappa}B is a dimeric transcription factor composed of subunits containing Rel homology domains (42). These subunits, which can hetero- or homodimerize, include p65, c-Rel, RelB, p50, and p52. NF-{kappa}B binds to inhibitor proteins of the I{kappa}B family, which include I{kappa}B{alpha}, I{kappa}B{beta}, and I{kappa}B{epsilon}. I{kappa}B{alpha}, for example, binds preferentially to p65 and c-Rel containing dimers and inhibits their DNA binding activities (42). As an initial test of whether NF-{kappa}B binds to the VCAM1 {kappa}B sites, nuclear extracts were preincubated with recombinant I{kappa}B{alpha} followed by the addition of the radiolabeled {kappa}B probes. As shown in Fig. 4GoA, I{kappa}B{alpha} preincubation abolished the more slowly migrating band (complex A), and partially removed complex B (compare lane 7 with lane 5, and lane 14 with lane 12). We interpret these results as providing evidence that complex A contains NF-{kappa}B subunits that bind to I{kappa}B{alpha}, whereas complex B actually consists of two distinct complexes, at least one of which contains I{kappa}B{alpha}-binding NF-{kappa}B subunits (arbitrarily designated complex B1). Notably, I{kappa}B{alpha} pretreatment of nuclear extracts from control and TNF-{alpha} treated cells reveals that complex B2 is not TNF-{alpha} inducible (compare lane 4 with lane 7, and lane 11 with lane 14). Thus, complexes A and B1 appear to completely account for the inducible complexes in response to TNF-{alpha}.

To more thoroughly characterize the NF-{kappa}B family members involved in the binding to the VCAM1 promoter, we preincubated nuclear extracts with a series of Abs against different NF-{kappa}B family members, including p65, p50, p52, c-Rel, and RelB. As shown in Fig. 4GoB, the anti-p65 and anti-p50 Abs affected the nuclear complexes. The p65 Ab almost completely eliminated the more slowly migrating band (complex A), and reminiscent of the results with I{kappa}B{alpha}, substantially removed complex B (compare lane 3 with lane 2 and lane 9 with lane 8). The p50 Ab also diminished both complex A and complex B (compare lanes 10 and 8). Higher concentrations of either Ab did not increase the amount of complex B removed (data not shown). Treatment with either Ab also resulted in supershifted bands (lanes 3, 4, 9 and 10). Preincubation of nuclear extracts with both p65 and p50 Abs simultaneously abolished all complexes (lane 11). An interpretation consistent with the data is that complex A consists of p65-p50 heterodimer, whereas complex B consists of two complexes, one a p65 homodimer and the other a p50 homodimer. The p65 homodimer is in all likelihood identical with the complex B1 identified in the experiment with I{kappa}B{alpha} preincubation, because p65 is a known target of I{kappa}B{alpha}. By inference, that containing the p50 homodimer is the same as complex B2, because p50 is not a preferred binding partner of I{kappa}B{alpha} (42). Abs to other subunits, including c-Rel, RelB, and p52 failed to abolish any of the complexes (Fig. 4GoB).

Parallel experiments performed with HeLa cells and the VCAM1 probes reveal that TNF-{alpha} induces two bands by EMSA (data not shown). The more slowly migrating of these is abolished by anti-p65 Ab, and both are completely abolished by anti-p50 Ab (data not shown). These findings suggest that the more slowly migrating complex consists of p65-p50 heterodimer, whereas the other consists of a p50 homodimer; the p65 homodimer was not observed. Thus, TNF-{alpha} induces a pattern of NF-{kappa}B complexes in C1 cells that is overlapping but not identical with that induced in HeLa cells.

TNF-{alpha} activates IKK-{beta} in C1 cells

As indicated previously, NF-{kappa}B binds to I{kappa}B. Indeed, the regulated degradation of I{kappa}B is the principal means by which NF-{kappa}B is activated (42, 43, 44, 45). In other cell types, NF-{kappa}B is ordinarily sequestered in the cytoplasm by virtue of its association with I{kappa}B. On stimulation with agents such as TNF-{alpha}, IL-1{beta}, and LPS, a high-molecular mass IKK complex is activated. This complex contains two homologous catalytic subunits, IKK-{alpha} and IKK-{beta}. This IKK complex then phosphorylates the N terminus of I{kappa}B. In the case of I{kappa}B{alpha}, the most extensively studied I{kappa}B isoform, this occurs at Ser32 and Ser36. IKK also phosphorylates homologous residues in the other main I{kappa}B isoforms, I{kappa}B{beta} and I{kappa}B{epsilon} (35, 38, 46, 47, 48, 49). This phosphorylation event targets I{kappa}B for degradation by the ubiquitin-proteasome pathway.

To examine whether TNF-{alpha} activates NF-{kappa}B through this pathway in C1 cells, we first investigated the protein levels of the three major I{kappa}B isoforms by Western blotting. As shown in Fig. 5GoA, TNF-{alpha} induces degradation of all three of these, but in an isoform-specific manner. Thus, with I{kappa}B{alpha} and I{kappa}B{epsilon}, protein levels are significantly diminished at 10 to 15 min (lanes 3 and 4, top and bottom), and in the case of I{kappa}B{alpha}, preinduction levels are reestablished by 60 min (lane 6). In the case of I{kappa}B{beta}, the kinetics of degradation is somewhat slower, and no appreciable reappearance is seen at 60 min (middle).



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  		FIGURE 5. I{kappa}B western blots and IKK-{beta} immunocomplex kinase assays in C1 cells. C1 cells were incubated with 20 ng/ml TNF-{alpha} for the indicated times and whole cell extracts then prepared. A and C. Western blots. Aliquots of the cell extract were subjected to 12% SDS-PAGE and then transferred to Immobilon-P membranes. Western blots were then performed with (A) anti-I{kappa}B{alpha} (top), anti-I{kappa}B{beta} (middle), or anti-I{kappa}B{epsilon} (lower) polyclonal or (C) anti-IKK-{beta} mAbs. Positions of the relevant proteins are indicated to the right. Those of molecular mass makers are indicated on the left. B, Immunocomplex kinase assays. Endogenous IKK-{beta} was immunoprecipitated from whole cell extracts using anti-IKK-{beta} polyclonal Abs. The kinase activities of the immunoprecipitates were measured by their phosphorylation of GST- I{kappa}B{alpha} (5–55) in the presence of [{gamma}-32P]ATP, followed by 12% SDS-PAGE and phosphorimager analysis. This is representative of three independent experiments.

 
Because IKK-induced phosphorylation of I{kappa}B triggers these degradations in other cell types, we then measured IKK activity. After TNF-{alpha} treatment, endogenous IKK-{beta} was immunoprecipitated with anti-IKK-{beta} Ab and then assayed for its capacity to phosphorylate GST-I{kappa}B{alpha} (5-55) in the presence of [{gamma}-32P]ATP. After 5 min of TNF-{alpha} treatment, IKK-{beta} activity was markedly enhanced (Fig. 5GoB). At subsequent time points, it declined, and IKK-{beta} activity returned to basal levels 30 min after TNF-{alpha} treatment. IKK-{beta} protein levels were unaltered by TNF-{alpha} (Fig. 5GoC). This is consistent with the changes in kinase activity reflecting changes in IKK-{beta}-specific activity. Notably, the peak of IKK-{beta} activity (5 min) precedes the nadir of I{kappa}B protein levels (10 min), and is consistent with a causal relationship between I{kappa}B phosphorylation and degradation.

Dominant negative I{kappa}B and IKK inhibit the TNF-{alpha} inducibility of the VCAM1 promoter

To further examine the role of IKK-I{kappa}B pathway in VCAM1 regulation, we cotransfected C1 cells with a VCAM1 promoter construct (F0) along with constructs for dominant negative IKK-{alpha}(K40A), IKK-{beta}(K40A), and I{kappa}B{alpha}(S32A/S36A). The IKK-{alpha} and IKK-{beta} mutants contain substitutions of alanine for an essential lysine in the ATP binding site, and thus produce catalytically inactive proteins. The I{kappa}B{alpha} mutant is refractory to degradation because of the substitution of its critical phosphoacceptor residues (Ser32 and Ser36) by alanines. As shown in Fig. 6GoA, dominant negative IKK-{alpha}, IKK-{beta}, and I{kappa}B{alpha} all inhibit the TNF-{alpha} inducibility of the VCAM1 promoter. Compared with dominant negative IKK-{alpha}, the IKK-{beta} mutant was a more potent inhibitor. This cannot be explained by differences in expression levels, because Western blotting reveals that IKK-{beta}, if anything, is expressed at slightly lower levels than IKK-{alpha} (Fig. 6GoB, compare lanes 2 and 3). Coexpression of dominant negative IKK-{alpha} and IKK-{beta} inhibited the reporter gene to a level similar to that of dominant negative IKK-{beta} alone (data not shown). The dominant negative I{kappa}B{alpha} was even more potent, abolishing the basal and inducible reporter gene activity. We conclude that the IKK-I{kappa}B pathway plays a critical role in TNF-{alpha}-induced expression of VCAM1 in C1 renal TEC.



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  		FIGURE 6. Reporter gene analysis of the VCAM1 promoter in the presence of dominant negative IKK or dominant negative I{kappa}B{alpha} in C1 cells. C1 cells were cotransfected with 4 µg of F0, 0.25 µg of pCMV-LacZ, and 1 µg of either pcDNA3, pRK-FlagIKK-{alpha}(K40A), pRK-FlagIKK-{beta}(K40A) or pCMV4-FlagI{kappa}B{alpha}(S32A/S36A). Twenty-four hours after transfection, some of the samples were treated with 20 ng/ml TNF-{alpha} for 8 h. Cells were harvested 32 h posttransfection. A, CAT activities were measured and normalized to that of {beta}-galactosidase. Shown is a representative result, performed in duplicate with SDs, from three independent experiments. B, C1 cell lysates were subjected to Western blotting with anti-Flag (M2) mAb. Lanes 1–4, indicate cells transfected with pcDNA3, pRK-Flag-IKK-{alpha}(K40A), pRK-Flag-IKK-{beta}(K40A), or pCMV-Flag-I{kappa}B{alpha}(S32A/S36A), respectively. Positions of the relevant proteins are indicated to the right. Those of molecular mass makers are indicated on the left. NS denotes nonspecific band.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
VCAM1 is a tissue-specific adhesion molecule (14, 29). For example, in myoblast cells, VCAM1 expression is constitutive, controlled by a yet to be identified transcription factor, and is unresponsive to cytokines. In contrast, in endothelial, COS, and HeLa cells, the VCAM1 gene is primarily regulated by NF-{kappa}B in a cytokine-responsive manner (27, 28). To examine the nature of VCAM1 transcriptional regulation in renal TEC, we initially used a combination of reporter gene and EMSAs in C1 cells, a cell line of renal TEC origin. These experiments reveal that NF-{kappa}B is critical in TNF-{alpha}-induced transcriptional regulation of VCAM1 in C1 cells. In particular, NF-{kappa}B binds in a TNF-{alpha}-inducible manner to two sites, located at positions -81 and -66. Interestingly, these are the same sites NF-{kappa}B binds in endothelial cells (14, 27).

However, it should be noted that although NF-{kappa}B plays a critical role in VCAM1 gene regulation in multiple cell types, there are cell type-specific differences. In particular, in bovine aortic endothelial cells, the full length promoter (Ffl) has a much lower TNF-{alpha} inducibility than F0, F1, or F2, suggesting the presence of a negative regulatory element(s) located between position -2167 and -755 of VCAM1 promoter (27). In contrast, in C1 cells, the inducibility of Ffl is comparable to that of F0, F1, or F2 (Fig. 2GoB). Thus, this negative regulation does not appear to be significant in C1 cells. In bovine aortic endothelial and COS cells, mutation of the GATA site (F2 mGATA) decreased TNF-{alpha}-induced VCAM1 reporter gene expression by ~50% (27), thereby providing evidence for the involvement of a GATA family member in this gene regulation. This GATA site also is involved in rhinovirus-induced up-regulation of VCAM1 in respiratory epithelial cells (50). However, in the present study, the mutation of the GATA site was without effect (Fig. 3GoB); hence, a GATA factor does not appear to be involved in VCAM1 regulation by TNF-{alpha} in C1 cells. In HeLa cells, two cytokine-inducible NF-{kappa}B complexes form on both the VCAM1 {kappa}B1 or {kappa}B2 probes. One is a p50-p65 heterodimer, as evidenced by its reactivity with both anti-p50 and anti-p65 Abs, and the other is probably a p50 homodimer, as evidenced by its reactivity solely with anti-p50 Abs (data not shown). In comparison, in C1 cells there appear to be two inducible complexes, one a p65-p50 heterodimer and the other a p65 homodimer (Fig. 4GoB). In summary, different cell types achieve cytokine-induced regulation of VCAM1 by using both common and distinct mechanisms.

NF-{kappa}B is a transcription factor that is critical for immune and inflammatory responses. In resting cells, NF-{kappa}B is sequestered in the cytoplasm by virtue of its association with I{kappa}B, which masks the nuclear localization sequence of the former (for recent reviews, see Refs. 42, 43, 44, 45). At least three distinct pathways result in NF-{kappa}B activation. First, in a pathway responsive to most NF-{kappa}B inducing stimuli, a 700-kDa IKK complex is activated (44). This complex contains two homologous catalytic subunits, IKK-{alpha} and IKK-{beta}. By mechanisms that remain under active investigation and probably involve phosphorylation of the activation loops of IKK-{alpha} and IKK-{beta}, this IKK complex is activated. Activated IKK then phosphorylates I{kappa}B at its two critical N-terminal serines. Phosphorylated I{kappa}B then is ubiquitinated in a process dependent on a {beta}-TrCP-containing ubiquitin ligase complex, and ubiquitinated I{kappa}B then is degraded by the proteasome. Freed NF-{kappa}B translocates to the nucleus and binds to the DNA of appropriate promoters and enhancers. Second, in a pathway responsive to UV irradiation, I{kappa}B is degraded. However, this degradation is IKK-independent (51), and the nature of the signal that promotes degradation remains to be determined. Third, in a pathway responsive to hypoxia-reoxygenation and pervanadate, I{kappa}B dissociates from NF-{kappa}B but is not degraded (52, 53). In this case, tyrosine phosphorylation of I{kappa}B is the signal that induces dissociation from NF-{kappa}B. In the case of I{kappa}B{alpha}, it probably occurs at Tyr42 (52).

Our data provide evidence that the first of these pathways, the IKK pathway, is operative in TNF-{alpha} treated C1 cells. Specifically, we establish that in C1 cells: 1) I{kappa}B{alpha}, I{kappa}B{beta}, and I{kappa}B{epsilon} are all degraded in a TNF-{alpha}-inducible manner (Fig. 5GoA); 2) IKK-{beta} is activated by TNF-{alpha} with kinetics consistent with it playing a causal role in I{kappa}B degradation (Fig. 5GoB); and 3) dominant negative IKK-{beta}, as well as IKK-{alpha}, inhibit cytokine-induced activation of a VCAM1 reporter construct (Fig. 6Go). Thus, these studies not only provide evidence for NF-{kappa}B regulating VCAM1 in renal TEC, but also delineate its signaling pathway.

It might be noted that the IKK-{beta} kinase assays likely reflect the activity of an IKK-{alpha}/IKK-{beta} containing IKK complex, because IKK-{beta} is tightly associated with IKK-{alpha} (35, 46, 54). That being said, IKK-{beta} has a substantially higher specific activity toward I{kappa}B{alpha} than that of IKK-{alpha} and thus, the kinase activity measured probably reflects, in large part, that of IKK-{beta} (35, 38, 54). In addition, the I{kappa}B{alpha} protein reappears at 60 min after TNF-{alpha} treatment (Fig. 5GoA, lane 6). At least in other cell types, this is the result of a feedback loop in which NF-{kappa}B induces transcription of the I{kappa}B{alpha} gene (55). It is conceivable that the same mechanism also is operative in C1 cells. This feedback loop does not appear to operative in the case of I{kappa}B{beta} (42, 56), and consistent with this, I{kappa}B{beta} does not reappear after TNF-{alpha} treatment of C1 cells.

Our experiments on IKK have focused on the IKK-{beta} isoform. Dominant negative IKK-{beta}, for example, is more potent in inhibiting VCAM1 gene activity than dominant negative IKK-{alpha} (Fig. 6Go). Furthermore, coexpression of dominant negative IKK-{alpha} and IKK-{beta} produced inhibition levels similar to that of dominant negative IKK-{beta} alone (data not shown). Experiments on knockout mice have provided compelling evidence that IKK-{beta} may be the IKK isoform central to inflammation. In particular, embryonic fibroblast and embryonic stem cells from IKK-{beta}-/- mice are markedly defective in either IKK complex or NF-{kappa}B activation in response to TNF-{alpha} or IL-1 (57, 58, 59). In marked contrast, embryonic fibroblasts, primary keratinocytes, or liver cells from IKK-{alpha}-/- mice display normal IKK complex activity and I{kappa}B{alpha} degradation in response to these same stimuli (60, 61). Interestingly, IKK-{beta}-deficient mice die at embryonic day 12.5 from massive apoptosis in the liver (57, 58, 59), similar in phenotype to p65 knockout mice (62). In contrast, IKK-{alpha} knockout mice are defective in skin and skeletal development (60, 61).

There is evidence that VCAM1 plays an important role in acute renal allograft rejection. In a recent study, perfusion of renal allografts with synthetic oligodeoxynucleotides containing {kappa}B decoy binding sites before transplantation in rats inhibited endothelial VCAM1 expression and parenchymal infiltration of monocytes and macrophages (63). Thus, renal endothelial VCAM1 may be an attractive target in the rational treatment of transplant rejection. Our studies highlight renal TEC as another attractive target. Moreover, by virtue of their identification of IKK as a mediator of VCAM1 transcriptional regulation, the present studies now raise the possibility of pharmacological inhibitors of IKK as a means of ameliorating these as well other inflammatory diseases of the kidney.


   Acknowledgments
 
We thank Drs. Tom Maniatis, David Goeddel, and Dean Ballard for gifts of plasmids.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants DK55672 (to F.S.L.), DK36149 and DK52369 (to V.R.K.), and P50 HL56985 and P01 HL36028 (to T.C.). Back

2 Address correspondence and reprint requests to Dr. Frank S. Lee, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, 605 Stellar-Chance Laboratories, 422 Curie Boulevard, Philadelphia, PA 19104. E-mail address: franklee{at}mail.med.upenn.edu Back

3 Abbreviations used in this paper: TEC, tubular epithelial cell; I{kappa}B, inhibitor of NF-{kappa}B; IKK, I{kappa}B kinase; CAT, chloramphenicol acetyltransferase. Back

Received for publication October 11, 2000. Accepted for publication March 16, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Bruijn, J. A., E. de Heer. 1995. Adhesion molecules in renal diseases. Lab. Invest. 72:387.[Medline]
  2. Briscoe, D. M., R. S. Cotran. 1993. Role of leukocyte-endothelial cell adhesion molecules in renal inflammation: in vitro and in vivo studies. Kidney Int. Suppl. 42:S27.[Medline]
  3. Rabb, H. A.. 1994. Cell adhesion molecules and the kidney. Am. J. Kidney Dis. 23:155.[Medline]
  4. Brady, H. R.. 1994. Leukocyte adhesion molecules and kidney diseases. Kidney Int. 45:1285.[Medline]
  5. Croker, B. P., and E. L. Ramos. 1994. Pathology of the renal allograft. In Renal Pathlogy with Clinical and Functional Correlations, Vol. 2. C. C. Tisher and B. M. Brenner, eds. J. B. Lippincott Company, Philadelphia, PA, p. 1591.
  6. Briscoe, D. M., J. S. Pober, W. E. Harmon, R. S. Cotran. 1992. Expression of vascular cell adhesion molecule-1 in human renal allografts. J. Am. Soc. Nephrol. 3:1180.[Abstract]
  7. Alpers, C. E., K. L. Hudkins, C. L. Davis, C. L. Marsh, W. Riches, J. M. McCarty, C. D. Benjamin, T. M. Carlos, J. M. Harlan, R. Lobb. 1993. Expression of vascular cell adhesion molecule-1 in kidney allograft rejection. Kidney Int. 44:805.[Medline]
  8. Lin, Y., J. A. Kirby, D. A. Browell, A. R. Morley, B. K. Shenton, G. Proud, R. M. Taylor. 1993. Renal allograft rejection: expression and function of VCAM-1 on tubular epithelial cells. Clin. Exp. Immunol. 92:145.[Medline]
  9. Elices, M. J., L. Osborn, Y. Takada, C. Crouse, S. Luhowskyj, M. E. Hemler, R. R. Lobb. 1990. VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site. Cell 60:577.[Medline]
  10. Carlos, T. M., B. R. Schwartz, N. L. Kovach, E. Yee, M. Rosa, L. Osborn, G. Chi-Rosso, B. Newman, R. Lobb, M. Rosso, et al 1990. Vascular cell adhesion molecule-1 mediates lymphocyte adherence to cytokine-activated cultured human endothelial cells. Blood 76:965.[Abstract/Free Full Text]
  11. Chan, B. M., M. J. Elices, E. Murphy, M. E. Hemler. 1992. Adhesion to vascular cell adhesion molecule 1 and fibronectin: comparison of {alpha}4{beta}1 (VLA-4) and {alpha}4{beta}7 on the human B cell line JY. J. Biol. Chem. 267:8366.[Abstract/Free Full Text]
  12. Osborn, L., C. Hession, R. Tizard, C. Vassallo, S. Luhowskyj, G. Chi-Rosso, R. Lobb. 1989. Direct expression cloning of vascular cell adhesion molecule 1, a cytokine-induced endothelial protein that binds to lymphocytes. Cell 59:1203.[Medline]
  13. Li, H., M. I. Cybulsky, Jr M. A. Gimbrone, P. Libby. 1993. Inducible expression of vascular cell adhesion molecule-1 by vascular smooth muscle cells in vitro and within rabbit atheroma. Am. J. Pathol. 143:1551.[Abstract]
  14. Iademarco, M. F., J. J. McQuillan, D. C. Dean. 1993. Vascular cell adhesion molecule 1: contrasting transcriptional control mechanisms in muscle and endothelium. Proc. Natl. Acad. Sci. USA 90:3943.[Abstract/Free Full Text]
  15. Wuthrich, R. P., T. A. Jenkins, T. L. Snyder. 1993. Regulation of cytokine-stimulated vascular cell adhesion molecule-1 expression in renal tubular epithelial cells. Transplantation 55:172.[Medline]
  16. Sheppard, A. M., J. J. McQuillan, M. F. Iademarco, D. C. Dean. 1995. Control of vascular cell adhesion molecule-1 gene promoter activity during neural differentiation. J. Biol. Chem. 270:3710.[Abstract/Free Full Text]
  17. Freedman, A. S., J. M. Munro, G. E. Rice, M. P. Bevilacqua, C. Morimoto, B. W. McIntyre, K. Rhynhart, J. S. Pober, L. M. Nadler. 1990. Adhesion of human B cells to germinal centers in vitro involves VLA-4 and INCAM-110. Science 249:1030.[Abstract/Free Full Text]
  18. Burkly, L. C., A. Jakubowski, B. M. Newman, M. D. Rosa, G. Chi-Rosso, R. R. Lobb. 1991. Signaling by vascular cell adhesion molecule-1 (VCAM-1) through VLA-4 promotes CD3-dependent T cell proliferation. Eur. J. Immunol. 21:2871.[Medline]
  19. Damle, N. K., A. Aruffo. 1991. Vascular cell adhesion molecule 1 induces T-cell antigen receptor-dependent activation of CD4+ T lymphocytes. Proc. Natl. Acad. Sci. USA 88:6403.[Abstract/Free Full Text]
  20. Miyake, K., K. Medina, K. Ishihara, M. Kimoto, R. Auerbach, P. W. Kincade. 1991. A VCAM-like adhesion molecule on murine bone marrow stromal cells mediates binding of lymphocyte precursors in culture. J. Cell Biol. 114:557.[Abstract/Free Full Text]
  21. Briscoe, D. M., F. J. Schoen, G. E. Rice, M. P. Bevilacqua, P. Ganz, J. S. Pober. 1991. Induced expression of endothelial-leukocyte adhesion molecules in human cardiac allografts. Transplantation 51:537.[Medline]
  22. Postigo, A. A., R. Garcia-Vicuna, F. Diaz-Gonzalez, A. G. Arroyo, M. O. De Landazuri, G. Chi-Rosso, R. R. Lobb, A. Laffon, F. Sanchez-Madrid. 1992. Increased binding of synovial T lymphocytes from rheumatoid arthritis to endothelial-leukocyte adhesion molecule-1 (ELAM-1) and vascular cell adhesion molecule-1 (VCAM-1). J. Clin. Invest. 89:1445.
  23. Wuthrich, R. P.. 1992. Vascular cell adhesion molecule-1 (VCAM-1) expression in murine lupus nephritis. Kidney Int. 42:903.[Medline]
  24. Koizumi, M., N. King, R. Lobb, C. Benjamin, D. K. Podolsky. 1992. Expression of vascular adhesion molecules in inflammatory bowel disease. Gastroenterology 103:840.[Medline]
  25. Cybulsky, M. I., Jr M. A. Gimbrone. 1991. Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis. Science 251:788.[Abstract/Free Full Text]
  26. Gurtner, G. C., V. Davis, H. Li, M. J. McCoy, A. Sharpe, M. I. Cybulsky. 1995. Targeted disruption of the murine VCAM1 gene: essential role of VCAM-1 in chorioallantoic fusion and placentation. Genes Dev. 9:1.[Abstract/Free Full Text]
  27. Neish, A. S., A. J. Williams, H. J. Palmer, M. Z. Whitley, T. Collins. 1992. Functional analysis of the human vascular cell adhesion molecule 1 promoter. J. Exp. Med. 176:1583.[Abstract/Free Full Text]
  28. Iademarco, M. F., J. J. McQuillan, G. D. Rosen, D. C. Dean. 1992. Characterization of the promoter for vascular cell adhesion molecule-1 (VCAM-1). J. Biol. Chem. 267:16323.[Abstract/Free Full Text]
  29. Shu, H. B., A. B. Agranoff, E. G. Nabel, K. Leung, C. S. Duckett, A. S. Neish, T. Collins, G. J. Nabel. 1993. Differential regulation of vascular cell adhesion molecule 1 gene expression by specific NF-{kappa}B subunits in endothelial and epithelial cells. Mol. Cell Biol. 13:6283.[Abstract/Free Full Text]
  30. Wuthrich, R. P., L. H. Glimcher, M. A. Yui, A. M. Jevnikar, S. E. Dumas, V. E. Kelley. 1990. MHC class II, antigen presentation and tumor necrosis factor in renal tubular epithelial cells. Kidney Int. 37:783.[Medline]
  31. Zhao, Q., F. S. Lee. 1999. Mitogen-activated protein Kinase/ERK kinase kinases 2 and 3 activate nuclear factor-{kappa}B through I{kappa}B kinase-{alpha} and I{kappa}B kinase-{beta}. J. Biol. Chem. 274:8355.[Abstract/Free Full Text]
  32. Cybulsky, M. I., J. W. Fries, A. J. Williams, P. Sultan, R. Eddy, M. Byers, T. Shows, Jr M. A. Gimbrone, T. Collins. 1991. Gene structure, chromosomal location, and basis for alternative mRNA splicing of the human VCAM1 gene. Proc. Natl. Acad. Sci. USA 88:7859.[Abstract/Free Full Text]
  33. Brockman, J. A., D. C. Scherer, T. A. McKinsey, S. M. Hall, X. Qi, W. Y. Lee, D. W. Ballard. 1995. Coupling of a signal response domain in I{kappa}B {alpha} to multiple pathways for NF-{kappa}B activation. Mol. Cell Biol. 15:2809.[Abstract]
  34. Regnier, C. H., H. Y. Song, X. Gao, D. V. Goeddel, Z. Cao, M. Rothe. 1997. Identification and characterization of an I{kappa}B kinase. Cell 90:373.[Medline]
  35. Woronicz, J. D., X. Gao, Z. Cao, M. Rothe, D. V. Goeddel. 1997. I{kappa}B kinase-{beta}: NF-{kappa}B activation and complex formation with I{kappa}B kinase-{alpha} and NIK. Science 278:866.[Abstract/Free Full Text]
  36. Lee, F. S., J. Hagler, Z. J. Chen, T. Maniatis. 1997. Activation of the I{kappa}B {alpha} kinase complex by MEKK1, a kinase of the JNK pathway. Cell 88:213.[Medline]
  37. Sambrook, J., E. F. Fritsch, T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
  38. Lee, F. S., R. T. Peters, L. C. Dang, T. Maniatis. 1998. MEKK1 activates both I{kappa}B kinase {alpha} and I{kappa}B kinase {beta}. Proc. Natl. Acad. Sci. USA 95:9319.[Abstract/Free Full Text]
  39. Ashkenazi, A., V. M. Dixit. 1998. Death receptors: signaling and modulation. Science 281:1305.[Abstract/Free Full Text]
  40. Natoli, G., A. Costanzo, F. Moretti, M. Fulco, C. Balsano, M. Levrero. 1997. Tumor necrosis factor (TNF) receptor 1 signaling downstream of TNF receptor-associated factor 2: nuclear factor {kappa}B (NF{kappa}B)-inducing kinase requirement for activation of activating protein 1 and NF{kappa}B but not of c-Jun N-terminal kinase/stress-activated protein kinase. J. Biol. Chem. 272:26079.[Abstract/Free Full Text]
  41. Lenardo, M. J., D. Baltimore. 1989. NF-{kappa}B: a pleiotropic mediator of inducible and tissue-specific gene control. Cell 58:227.[Medline]
  42. Jr Baldwin, A. S.. 1996. The NF-{kappa}B and I{kappa}B proteins: new discoveries and insights. Annu. Rev. Immunol. 14:649.[Medline]
  43. Zandi, E., M. Karin. 1999. Bridging the gap: composition, regulation, and physiological function of the I{kappa}B kinase complex. Mol. Cell Biol. 19:4547.[Free Full Text]
  44. Karin, M.. 1999. The beginning of the end: I{kappa}B kinase (IKK) and NF-{kappa}B activation. J. Biol. Chem. 274:27339.[Free Full Text]
  45. Stancovski, I., D. Baltimore. 1997. NF-{kappa}B activation: the I{kappa}B kinase revealed?. Cell 91:299.[Medline]
  46. Zandi, E., D. M. Rothwarf, M. Delhase, M. Hayakawa, M. Karin. 1997. The I{kappa}B kinase complex (IKK) contains two kinase subunits, IKK{alpha} and IKK{beta}, necessary for I{kappa}B phosphorylation and NF-{kappa}B activation. Cell 91:243.[Medline]
  47. Nakano, H., M. Shindo, S. Sakon, S. Nishinaka, M. Mihara, H. Yagita, K. Okumura. 1998. Differential regulation of I{kappa}B kinase {alpha} and {beta} by two upstream kinases, NF-{kappa}B-inducing kinase and mitogen-activated protein kinase/ERK kinase kinase-1. Proc. Natl. Acad. Sci. USA 95:3537.[Abstract/Free Full Text]
  48. Simeonidis, S., S. Liang, G. Chen, D. Thanos. 1997. Cloning and functional characterization of mouse I{kappa}B{epsilon}. Proc. Natl. Acad. Sci. USA 94:14372.[Abstract/Free Full Text]
  49. Whiteside, S. T., J. C. Epinat, N. R. Rice, A. Israel. 1997. I{kappa}B{epsilon}, a novel member of the I{kappa}B family, controls RelA and cRel NF-{kappa}B activity. EMBO J. 16:1413.[Medline]
  50. Papi, A., S. L. Johnston. 1999. Respiratory epithelial cell expression of vascular cell adhesion molecule-1 and its up-regulation by rhinovirus infection via NF-{kappa}B and GATA transcription factors. J. Biol. Chem. 274:30041.[Abstract/Free Full Text]
  51. Li, N., M. Karin. 1998. Ionizing radiation and short wavelength UV activate NF-{kappa}B through two distinct mechanisms. Proc. Natl. Acad. Sci. USA 95:13012.[Abstract/Free Full Text]
  52. Imbert, V., R. A. Rupec, A. Livolsi, H. L. Pahl, E. B. Traenckner, C. Mueller-Dieckmann, D. Farahifar, B. Rossi, P. Auberger, P. A. Baeuerle, J. F. Peyron. 1996. Tyrosine phosphorylation of I{kappa}B-{alpha} activates NF-{kappa}B without proteolytic degradation of I{kappa}B-{alpha}. Cell 86:787.[Medline]
  53. Jr Canty, T. G., Jr E. M. Boyle, A. Farr, E. N. Morgan, E. D. Verrier, T. H. Pohlman. 1999. Oxidative stress induces NF-{kappa}B nuclear translocation without degradation of I{kappa}B{alpha}. Circulation 100:II361.
  54. Mercurio, F., H. Zhu, B. W. Murray, A. Shevchenko, B. L. Bennett, J. Li, D. B. Young, M. Barbosa, M. Mann, A. Manning, A. Rao. 1997. IKK-1 and IKK-2: cytokine-activated I{kappa}B kinases essential for NF- {kappa}B activation. Science 278:860.[Abstract/Free Full Text]
  55. Sun, S. C., P. A. Ganchi, D. W. Ballard, W. C. Greene. 1993. NF-{kappa} B controls expression of inhibitor I{kappa}B{alpha}: evidence for an inducible autoregulatory pathway. Science 259:1912.[Abstract/Free Full Text]
  56. Thompson, J. E., R. J. Phillips, H. Erdjument-Bromage, P. Tempst, S. Ghosh. 1995. I{kappa}B-{beta} regulates the persistent response in a biphasic activation of NF-{kappa} B. Cell 80:573.[Medline]
  57. Tanaka, M., M. E. Fuentes, K. Yamaguchi, M. H. Durnin, S. A. Dalrymple, K. L. Hardy, D. V. Goeddel. 1999. Embryonic lethality, liver degeneration, and impaired NF-{kappa}B activation in IKK-{beta}-deficient mice. Immunity