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B Kinase Is Critical for TNF-
-Induced VCAM1 Gene Expression in Renal Tubular Epithelial Cells1
*
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; and
Laboratory of Molecular Autoimmune Disease, Renal Division, Department of Medicine, and
Vascular Research Division, Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115
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Abstract |
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4
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-. Two
NF-
B binding sites in the VCAM1 promoter are critical for the
TNF--induced VCAM1 transcriptional up-regulation, and both sites
bind to p65-p50 NF-B complexes. TNF- induces activation of
inhibitor of NF-B (IB) kinase- (IKK-), a protein kinase
that phosphorylates the NF-B inhibitor IB, and thereby targets
the latter for degradation via the ubiquitin-proteasome pathway.
Moreover, dominant negative versions of IKK inhibit TNF- activation
of a VCAM1 promoter reporter. We conclude that the IKK/NF-B pathway
is critical in the TNF--induced up-regulation of VCAM1 mRNA in renal
TEC. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 (41) and
47 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-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 
-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- stimulation (15). We find here that NF-B is
essential for the induction of VCAM1 gene transcription in response to
TNF- in C1 cells. Moreover, inhibitor of NF-B (IB) kinase-
(IKK-), a protein kinase involved in the NF-B activation pathway,
plays a critical role in this induction.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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- (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-FlagIB(S32A/S36A) was a gift from Dean Ballard (Vanderbilt
University, Nashville, TN) and has been described (33).
The plasmid pGEM2-actin was a gift from Tom Maniatis (Harvard
University, Cambridge, MA). pRK-FlagIKK-(K40A) and
pRK-FlagIKK-(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-Actin (-actin control) with T3 and T7
RNA polymerase, respectively, and 50 µCi of
[-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
-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 B1 probe was prepared by first annealing the following
two oligonucleotides (NF-B binding site is underlined):
5'-GCCCTGGGTTTCCCCT-3' and
5'-TTCAAGGGGAAACCCA-3'. The 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
[-32P]dCTP and
[-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 IB 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-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.
IB (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 -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-
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 -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-IB 5–55(5–55), 5 µCi of
[-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-IB 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-IB (sc-371; Santa Cruz
Biotechnology), anti-IB (sc-945; Santa Cruz Biotechnology),
anti-IB
polyclonal (sc-7156; Santa Cruz Biotechnology),
anti-IKK- 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).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TNF- 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- on VCAM1
mRNA levels in C1 TEC, total RNA was isolated from TNF--treated or
control C1 cells, and a Northern blot then was performed with a
radiolabeled VCAM1 probe. As shown in Fig. 1
, the VCAM1 mRNA is increased
dramatically in C1 cells on TNF- treatment. This is consistent with
a previous report using this cell line (15).
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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. 2A, top construct). As shown
in Fig. 2B (first two columns), this construct is TNF-
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. 2A; position numbers are in
reference to the transcription initiation site; Ref. 27).
As shown in Fig. 2B, Ffl, F0, F1, and F2 display similar
inducibility (
3- to 5-fold) in response to TNF-. The F3 deletion
(to position -98) resulted in a diminished magnitude of reporter gene
activity relative to these constructs (Fig. 2B).
Importantly, however, F3 is still TNF- inducible. In marked
contrast, further deletion to position -44 (F4 construct) results in
the abolition of TNF- inducibility. Thus, sequences residing between
position -98 and -44 are essential for TNF- inducibility of the
VCAM1 promoter in C1 cells.
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B (27). Indeed, both AP1 and
NF-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. 3A).
Consistent with this, the behaviors of F1 and F2, which differ by 260
bp of sequence containing this potential AP1 site, are
indistinguishable (Fig. 2
B). Mutation of the potential GATA
site (F2 mGATA), like that of AP1, is similarly without effect (Fig. 3B). 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. 2B). This
suggests that DNA sequences in this region (between -258 and -98),
but not the GATA site, are important for optimal TNF-
inducibility.
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B binding sites (F3 mA and F3 mB; Fig. 3D), results in complete abolition of TNF- responsiveness
(Fig. 3C). Consistent with this, F4, which lacks both of the
NF-B binding sites, is also completely unresponsive to TNF- (Fig. 2B). Therefore, these results identify a critical role for
NF-B binding sites in TNF--stimulated VCAM1 gene transcription in
C1 cells. It may be noted that the three constructs lacking NF-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. 2B and
3C). This suggests that there is a significant basal level
of NF-B activity in these cells, which in turn, could account for
the relatively modest inducibility of the VCAM1 reporter gene
constructs analyzed (Fig. 2B). Consistent with this, EMSA
analysis described below indicates the presence of a basal level of
NF-B binding activity in uninduced C1 cell nuclear extracts.
NF-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-B binding sites, B1 and B2 (Fig. 3D). In
uninduced C1 cell nuclear extracts, we detected two weakly binding
bands (designated complexes A and B) with the B1 and B2 probes
(Fig. 4A, lanes 2
and 9, respectively). On TNF- 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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B might bind to these
sites. NF-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-B binds to inhibitor proteins of the IB family, which
include IB, IB, and IB. IB, 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-B binds to the VCAM1 B sites, nuclear extracts were
preincubated with recombinant IB followed by the addition of the
radiolabeled B probes. As shown in Fig. 4A, IB
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-B
subunits that bind to IB, whereas complex B actually consists of
two distinct complexes, at least one of which contains IB-binding
NF-B subunits (arbitrarily designated complex B1). Notably, IB
pretreatment of nuclear extracts from control and TNF- treated cells
reveals that complex B2 is not TNF- 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-.
To more thoroughly characterize the NF-B family members involved in
the binding to the VCAM1 promoter, we preincubated nuclear extracts
with a series of Abs against different NF-B family members,
including p65, p50, p52, c-Rel, and RelB. As shown in Fig. 4B, 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
IB, 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 IB preincubation, because p65 is a known target
of IB. By inference, that containing the p50 homodimer is the
same as complex B2, because p50 is not a preferred binding partner of
IB (42). Abs to other subunits, including c-Rel,
RelB, and p52 failed to abolish any of the complexes (Fig. 4B).
Parallel experiments performed with HeLa cells and the VCAM1 probes
reveal that TNF- 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- induces a pattern of
NF-B complexes in C1 cells that is overlapping but not identical
with that induced in HeLa cells.
TNF- activates IKK- in C1 cells
As indicated previously, NF-B binds to IB. Indeed, the
regulated degradation of IB is the principal means by which NF-B
is activated (42, 43, 44, 45). In other cell types, NF-B is
ordinarily sequestered in the cytoplasm by virtue of its association
with IB. On stimulation with agents such as TNF-, IL-1, and
LPS, a high-molecular mass IKK complex is activated. This complex
contains two homologous catalytic subunits, IKK- and IKK-. This
IKK complex then phosphorylates the N terminus of IB. In the case of
IB, the most extensively studied IB isoform, this occurs at
Ser32 and Ser36. IKK also
phosphorylates homologous residues in the other main IB isoforms,
IB and IB (35, 38, 46, 47, 48, 49). This
phosphorylation event targets IB for degradation by the
ubiquitin-proteasome pathway.
To examine whether TNF- activates NF-B through this pathway in C1
cells, we first investigated the protein levels of the three major
IB isoforms by Western blotting. As shown in Fig. 5A, TNF- induces
degradation of all three of these, but in an isoform-specific manner.
Thus, with IB and IB, protein levels are significantly
diminished at 10 to 15 min (lanes 3 and 4,
top and bottom), and in the case of IB,
preinduction levels are reestablished by 60 min (lane
6). In the case of IB, the kinetics of degradation is
somewhat slower, and no appreciable reappearance is seen at 60 min
(middle).
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B triggers these
degradations in other cell types, we then measured IKK activity. After
TNF- treatment, endogenous IKK- was immunoprecipitated with
anti-IKK- Ab and then assayed for its capacity to phosphorylate
GST-IB (5-55) in the presence of
[-32P]ATP. After 5 min of TNF- treatment,
IKK- activity was markedly enhanced (Fig. 5B). At
subsequent time points, it declined, and IKK- activity returned to
basal levels 30 min after TNF- treatment. IKK- protein levels
were unaltered by TNF- (Fig. 5C). This is consistent with
the changes in kinase activity reflecting changes in IKK--specific
activity. Notably, the peak of IKK- activity (5 min) precedes the
nadir of IB protein levels (10 min), and is consistent with a causal
relationship between IB phosphorylation and degradation.
Dominant negative IB and IKK inhibit the TNF- inducibility of
the VCAM1 promoter
To further examine the role of IKK-IB pathway in VCAM1
regulation, we cotransfected C1 cells with a VCAM1 promoter construct
(F0) along with constructs for dominant negative IKK-(K40A),
IKK-(K40A), and IB(S32A/S36A). The IKK- and IKK- mutants
contain substitutions of alanine for an essential lysine in the ATP
binding site, and thus produce catalytically inactive proteins. The
IB mutant is refractory to degradation because of the
substitution of its critical phosphoacceptor residues
(Ser32 and Ser36) by
alanines. As shown in Fig. 6A,
dominant negative IKK-, IKK-, and IB all inhibit the
TNF- inducibility of the VCAM1 promoter. Compared with dominant
negative IKK-, the IKK- mutant was a more potent inhibitor. This
cannot be explained by differences in expression levels, because
Western blotting reveals that IKK-, if anything, is expressed at
slightly lower levels than IKK- (Fig. 6B, compare
lanes 2 and 3). Coexpression of dominant negative
IKK- and IKK- inhibited the reporter gene to a level similar to
that of dominant negative IKK- alone (data not shown). The dominant
negative IB was even more potent, abolishing the basal and
inducible reporter gene activity. We conclude that the IKK-IB
pathway plays a critical role in TNF--induced expression of VCAM1 in
C1 renal TEC.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-B is critical
in TNF--induced transcriptional regulation of VCAM1 in C1 cells. In
particular, NF-B binds in a TNF--inducible manner to two sites,
located at positions -81 and -66. Interestingly, these are the same
sites NF-B binds in endothelial cells (14, 27).
However, it should be noted that although NF-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-
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. 2B). 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--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. 3B); hence, a GATA factor
does not appear to be involved in VCAM1 regulation by TNF- in C1
cells. In HeLa cells, two cytokine-inducible NF-B complexes form on
both the VCAM1 B1 or 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. 4B). In summary, different cell types achieve
cytokine-induced regulation of VCAM1 by using both common and distinct
mechanisms.
NF-B is a transcription factor that is critical for immune and
inflammatory responses. In resting cells, NF-B is sequestered in the
cytoplasm by virtue of its association with IB, 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-B activation. First, in a pathway responsive to most NF-B
inducing stimuli, a 700-kDa IKK complex is activated (44).
This complex contains two homologous catalytic subunits, IKK- and
IKK-. By mechanisms that remain under active investigation and
probably involve phosphorylation of the activation loops of IKK- and
IKK-, this IKK complex is activated. Activated IKK then
phosphorylates IB at its two critical N-terminal serines.
Phosphorylated IB then is ubiquitinated in a process dependent on a
-TrCP-containing ubiquitin ligase complex, and ubiquitinated IB
then is degraded by the proteasome. Freed NF-B translocates to the
nucleus and binds to the DNA of appropriate promoters and enhancers.
Second, in a pathway responsive to UV irradiation, IB 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, IB dissociates from NF-B but is not degraded
(52, 53). In this case, tyrosine phosphorylation of IB
is the signal that induces dissociation from NF-B. In the case of
IB, it probably occurs at Tyr42
(52).
Our data provide evidence that the first of these pathways, the IKK
pathway, is operative in TNF- treated C1 cells. Specifically, we
establish that in C1 cells: 1) IB, IB, and IB are all
degraded in a TNF--inducible manner (Fig. 5A); 2) IKK-
is activated by TNF- with kinetics consistent with it playing a
causal role in IB degradation (Fig. 5B); and 3) dominant
negative IKK-, as well as IKK-, inhibit cytokine-induced
activation of a VCAM1 reporter construct (Fig. 6). Thus, these studies
not only provide evidence for NF-B regulating VCAM1 in renal TEC,
but also delineate its signaling pathway.
It might be noted that the IKK- kinase assays likely reflect the
activity of an IKK-/IKK- containing IKK complex, because IKK-
is tightly associated with IKK- (35, 46, 54). That
being said, IKK- has a substantially higher specific activity toward
IB than that of IKK- and thus, the kinase activity measured
probably reflects, in large part, that of IKK- (35, 38, 54). In addition, the IB protein reappears at 60 min after
TNF- treatment (Fig. 5A, lane 6). At least in
other cell types, this is the result of a feedback loop in which
NF-B induces transcription of the IB 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
IB (42, 56), and consistent with this, IB does
not reappear after TNF- treatment of C1 cells.
Our experiments on IKK have focused on the IKK- isoform. Dominant
negative IKK-, for example, is more potent in inhibiting VCAM1 gene
activity than dominant negative IKK- (Fig. 6). Furthermore,
coexpression of dominant negative IKK- and IKK- produced
inhibition levels similar to that of dominant negative IKK- alone
(data not shown). Experiments on knockout mice have provided compelling
evidence that IKK- may be the IKK isoform central to inflammation.
In particular, embryonic fibroblast and embryonic stem cells from
IKK--/- mice are markedly defective in
either IKK complex or NF-B activation in response to TNF- or IL-1
(57, 58, 59). In marked contrast, embryonic fibroblasts,
primary keratinocytes, or liver cells from
IKK--/- mice display normal IKK complex
activity and IB degradation in response to these same stimuli
(60, 61). Interestingly, IKK--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- 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 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.
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Acknowledgments |
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Footnotes |
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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
3 Abbreviations used in this paper: TEC, tubular epithelial cell; IB, inhibitor of NF-B; IKK, IB kinase; CAT, chloramphenicol acetyltransferase.
Received for publication October 11, 2000. Accepted for publication March 16, 2001.
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