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B Regulation by IB Kinase-2 in Rheumatoid Arthritis Synoviocytes1
*
Division of Rheumatology, Allergy and Immunology, University of California San Diego School of Medicine, La Jolla, CA; and
Signal Research Division of Celgene, San Diego, CA
 |
Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B kinase-1 and IB kinase-2 (IKK1 and IKK2; also called
IKK
and IKK
, respectively) are part of the signal complex that
regulates NF-B activity in many cell types, including
fibroblast-like synoviocytes (FLS). We determined which of these two
kinases is responsible for cytokine-induced NF-B activation in
synoviocytes and assessed the functional consequences of IKK1 or IKK2
overexpression and inhibition. FLS were infected with adenovirus
constructs encoding either wild-type (wt) IKK1 or IKK2, the dominant
negative (dn) mutant of both kinases, or a control construct encoding
green fluorescence protein. Analysis of the NF-B pathway revealed
that cytokine-induced IKK activation, IB degradation, and NF-B
activation was prevented in cells expressing the IKK2 dn mutant,
whereas baseline NF-B activity was increased by IKK2 wt. In
addition, synthesis of IL-6 and IL-8, as well as expression of ICAM-1
and collagenase, was only increased by IKK2 wt, and their
cytokine-induced production was abrogated by IKK2 dn mutant. However,
the IKK1 dn mutant did not inhibit cytokine-mediated activation of
NF-B or any of the functional assays. These data indicate that IKK2
is the key convergence pathway for cytokine-induced NF-B activation.
Furthermore, IKK2 regulates adhesion molecule, matrix
metalloproteinase, and cytokine production in
FLS. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B plays a pivotal role in inflammation by virtue of its
ability to induce transcription of an array of proinflammatory genes
(1). In chronic inflammatory diseases, such as rheumatoid
arthritis (RA),3
NF-B is activated within the synovial tissue and contributes to
cytokine production, adhesion molecule expression, and matrix
metalloproteinase (MMP) activation (2, 3, 4, 5). In particular,
NF-B binding activity is significantly greater in RA synovium
compared with noninflammatory joint samples. NF-B-regulated genes,
such as IL-6, IL-8, cyclooxygenase 2, and inducible NO synthase, are
also abundantly expressed in rheumatoid synovitis. Animal models of
arthritis provide additional evidence for the importance of NF-B by
demonstrating that joint inflammation is preceded by synovial NF-B
activation and can be prevented by abrogating NF-B function
(6, 7). These data suggest that NF-B-targeted
therapeutics might be effective in inflammatory diseases like
RA.
NF-B is normally retained in the cytoplasm by its natural inhibitor,
IB (8). Upstream, a signaling complex consisting of two
IB kinases, IKK1 and IKK2 (also called IKK and IKK), regulates
IB activity. The IKK complex is activated by cytokines like IL-1 and
TNF-, which subsequently leads to IKK-mediated phosphorylation and
ubiquitin-mediated degradation of IB. The NF-B proteins can then
translocate to the nucleus where they bind to DNA and induce gene
transcription (9, 10, 11). We recently reported that both
kinases are part of the IKK complex in fibroblast-like synoviocytes
(FLS) and that IKK regulates NF-B activation in these cells
(12). Using adenoviral constructs encoding activated and
dominant negative forms of IKK genes, we have now further characterized
the biology of IKK in human FLS. These studies demonstrate that IKK2 is
the primary convergence site for cytokine-induced NF-B binding
activity in FLS and is a key regulator of inflammatory gene
expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FLS were isolated from RA and osteoarthritis synovial tissues
obtained at joint replacement surgery as previously described
(13). The diagnoses conformed to the 1987 revised American
College of Rheumatology criteria (14). Briefly, the
tissues were minced and incubated with 1 mg/ml collagenase in
serum-free DMEM (Life Technologies, Gaithersburg, MD) for 2 h at
37°C, filtered through a nylon mesh, extensively washed, and cultured
in DMEM supplemented with 10% FCS (Life Technologies; endotoxin
content, <0.006 ng/ml), penicillin, streptomycin, gentamicin, and
L-glutamine in a humidified 5% CO2
atmosphere. After overnight culture, nonadherent cells were removed,
and adherent cells were cultivated in DMEM plus 10% FCS. At
confluence, cells were trypsinized, split at a 1:3 ratio, and
recultured in medium. Synoviocytes were used from passages 3 through 9
where they comprised a homogeneous population of fibroblast-like
synoviocytes (<1% CD11b, <1% phagocytic, and <1% Fc
RII
receptor positive).
FLS infection by adenovirus constructs
To alter IKK function in synoviocytes, Asp-Tyr-Lys-Asp-Asp-Asp-Lys (FLAG)-tagged IKK2 and influenza hemagglutinin (HA)-tagged IKK1 cDNAs were cloned in replication-deficient adenovirus vector pAx1CA. The following constructs were prepared: 1) IKK1 wild type (wt); 2) IKK2 wt; 3) IKK1 dominant negative (dn); and 4) IKK2 dn. For both dn mutants, the ATP-binding site at amino acid 44 was altered by mutating the lysine to methionine (44K>M) (11). FLS were infected with high titer purified adenovirus (Quantum, Montreal, Canada) for 6 h at concentrations between 8.75 x 108 and 8 x 109 particles per ml. The virus supernatant was then removed, and the cells were incubated in fresh medium for 24–48 h. Adenovirus encoding for green fluorescence protein (GFP) was used as control (Quantum).
Antibodies
Affinity-purified rabbit polyclonal Ab to IKK (IKK2 CT) was
raised against a peptide encoding the carboxyl terminus of IKK2 (Signal
Pharmaceuticals, San Diego, CA). Other Abs were obtained from the
following sources: rabbit polyclonal anti-IB (Santa Cruz
Biotechnology, Santa Cruz, CA); HRP-labeled goat anti-rabbit IgG
(Biolabs, Beverly, MA); mouse anti-FLAG mAb (Sigma, St. Louis, MO);
mouse anti-HA mAb (Boehringer Mannheim, Indianapolis, IN);
HRP-labeled goat anti-mouse IgG (Biolabs); mouse anti-ICAM-1
mAb (R&D Systems, Minneapolis, MN), alkaline phosphatase-labeled goat
anti-mouse IgG (Biosource, Camarillo, CA).
Western blot analysis
Whole cell lysate (50 µg) was fractionated on
Tris-glycine-buffered 10% SDS-polyacrylamide gel (Novex, San Diego,
CA) and transferred to nitrocellulose membrane (Amersham, Cleveland,
OH). Membranes were blocked with 5% nonfat milk powder (Bio-Rad,
Hercules, CA) and probed with primary Ab to FLAG, HA, or IB and
then with HRP-conjugated goat anti-mouse Ab (1:2000) or goat
anti-rabbit IgG peroxidase-conjugated Ab (1:2000) in PBS with 0.1%
Tween 20 and 5% nonfat milk powder. Immunoreactive proteins were
detected with chemiluminescence and autoradiography (Amersham).
Cytokine-induced IB kinase activity
IKK activity was detected by adding radiolabeled ATP and
recombinant IB to immunoprecipitated IKK as previously described
(11). Briefly, cells (1 x 106)
were rotated for 1 h at 4°C in lysis buffer (20 mM HEPES (pH
7.9), 0.5 M NaCl, 0.25% Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mM DTT
with phosphatase and protease inhibitors). Phosphatase and protease
inhibitors consisted of: 20 mM -glycerophosphate, 10 mM NaF, 0.3 mM
Na3VO4, 1 mM benzamidine,
10 mM p-nitrophenyl phosphate and complete protease
inhibitor cocktail (Boehringer Mannheim). Anti-IKK Ab was added to the
lysis buffer and mixed at 4°C for 2 h. Washed protein A-agarose
(40 µl; Calbiochem, San Diego, CA) was then added for an additional
hour. Immunoprecipitated material was washed four times in wash buffer
(40 mM Tris (pH 8.0), 0.5 M NaCl, 0.1% Nonidet P-40, 6 mM EDTA, 6 mM
EGTA, 1 mM DTT with phosphatase and protease inhibitors) and once with
kinase buffer (20 mM HEPES (pH 7.9), 1 mM MgCl2,
1 mM MnCl2, 1 mM DTT with phosphatase and
protease inhibitors). Kinase activity was assayed in 40 µl kinase
buffer containing 10 µM [-32P]ATP and 3
µg GST-IB1–54(1–54) for 30 min at 30°C. The reaction was stopped by
the addition of SDS gel sample buffer and analyzed by
SDS-polyacrylamide gel electrophoresis and autoradiography.
EMSA
Nuclear protein was extracted from FLS (1 x
106 cells) and assayed for DNA binding of
NF-B. After the cells were washed in ice cold PBS, the cell pellet
was resuspended in 1 ml buffer A (10 mM HEPES (pH 7.9), 1.5 mM
MgCl2, 10 mM KCl, 1 mM DTT) containing 0.1%
Triton X-100. After incubation for 10 min on ice, the lysate was
centrifuged and the nuclei were resuspended in 20 µl buffer C (20 mM
HEPES (pH 7.9), 25% (v/v) glycerol, 420 mM NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 1 mM DTT). This suspension
was incubated for 30 min on ice followed by centrifugation at
10,000 x g for 20 min. The supernatant was stored at
-80°C as nuclear extract after protein concentrations were
determined by the Bradford method using BSA as standard.
Double-stranded oligonucleotides containing a consensus NF-B
recognition sequence (Promega, Madison, WI) were end-labeled with T4
polynucleotide kinase in the presence of
[-32P]dATP. The DNA binding reaction was
performed at room temperature for 30 min in a final volume of 15 µl,
which contained 5 µg nuclear extract, oligonucleotide probe (40
fmol), and binding buffer (10 mM Tris-HCl (pH 7.5), 4% (v/v) glycerol,
50 mM NaCl, 1 mM MgCl, 0.5 mM EDTA, 0.5 mM DTT, 100 µg/ml
poly(dI-dC)). Reactions were subjected to electrophoresis on
nondenaturing 5% polyacrylamide gels in 0.5x Tris-buffered EDTA at
125 mA for 6 h at 4°C. The gels were exposed to Hyperfilm MP
(Amersham) with an intensifying screen at -70°C.
Solid phase ICAM-1 ELISA
RA FLS were seeded at 1 x 104/well
in 96-well microtiter plates (Costar, Cambridge, MA) and cultured at
37°C under 5% CO2. The next day, cells were
infected as described, and on day 3 they were synchronized with 0.5%
FCS-DMEM for 24 h. TNF- (100 ng/ml; R&D Systems) or IL-1 (10
ng/ml; R&D Systems) were added on day 4. After 24 h, the
supernatant was removed, and cells were washed with PBS and fixed with
4% paraformaldehyde/PBS for 30 min. FLS were then washed and incubated
for 2 h with ICAM-1 Ab in 0.1% BSA-0.25% sodium azide-PBS. Three
additional PBS wash steps were performed, followed by incubation with
alkaline phosphatase-labeled goat anti-mouse Ab (1:500 in 0.1%
BSA-0.25% sodium azide-PBS) for 2 h. Cells were washed three
times with PBS and then incubated with chromagenic solution (1 mg/ml
p-nitrophenyl phosphate in 1 M diethanolamine, 0.5 M MgCl,
pH 9.8) for 30 min at 37°C. Absorbance was measured at 405 nm. The
values determined for uninfected FLS cultured in control medium were
used as reference point (100%) for ICAM-1 expression.
IL-6 and IL-8 ELISA
Human IL-6 and IL-8 ELISA kit (Endogen, Woburn, MA) were used
according to the manufacturer’s instructions. Briefly, RA FLS were
seeded at 1 x 104/well in 96-well
microtiter plates (Costar) and cultured at 37°C under 5%
CO2. The next day, cells were infected as
described, and on day 3 they were synchronized with 0.5% FCS-DMEM for
24 h. TNF- (100 ng/ml; R&D Systems) or IL-1 (10 ng/ml; R&D
Systems) were added on day 4. After cytokine stimulation for 24 h,
the supernatants were transferred to precoated microtiter plates and
assayed for IL-6 or IL-8 as per the instructions.
Northern blot analysis
Total RNA was isolated using RNA STAT-60 (Tel-Test, Friendswood,
TX) and fractionated in a 1.2% agarose gel containing 5.5%
formaldehyde. The RNA was transferred to a nylon membrane using the
turbo blotter (Schleicher and Schuell, Keene, NH) and cross-linked at
80°C for 45 min. The blots were prehybridized in 50% formamide, 5x
saline-sodium phosphate-EDTA, 1x Denhardt’s solution, 1% sodium
dodecyl sulfate (SDS), 200µg/ml ssDNA and 50µg/ml tRNA. cDNA probe
for collagenase was denatured and labeled by random primed
incorporation of [-32P]dATP (Ambion, Austin,
TX). The labeled probe was then denatured at 100°C and the blot
hybridized overnight at 42°C. The membrane was washed in 2x 5x
saline-sodium phosphate-EDTA-0.1% SDS at 37°C, and autoradiography
was performed with Kodak X-OMAT AR film (Kodak, Rochester, NY) and an
intensifying screen for 12–24 h at -80°C.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preliminary studies were performed to confirm the ability of
adenoviral constructs expressing IKK1 wt, IKK1 dn, IKK2 wt, and IKK2 dn
to infect FLS. Three FLS cell lines were infected with adenovirus
encoding these genes or a control gene (GFP). Western blot analysis was
used to detect expression of the FLAG-tagged IKK2 and HA-labeled IKK1
constructs. Fig. 1
shows that transgene
expression was abundant in each of the infected lines. In other
experiments, IKK1 wt transgene expression was documented by Western
blot analysis and GFP transgene expression was readily demonstrated
using fluorescence microscopy (data not shown). Approximately 80% of
the infected cells expressed GFP when FLS were infected with the
adenovirus GFP construct.
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To determine the relative contributions of IKK1 and IKK2 to
cytokine-induced FLS kinase activity, synoviocyte lines were prepared
under 5 conditions: 1) parental uninfected cells; 2) control cells
infected with adenovirus expressing GFP; and 3–5) cells infected with
adenovirus encoding IKK1 dn, IKK2 wt, or IKK2 dn. The wt constructs
function as constitutively activated forms of IKK, and the dn forms
selectively block the action of the respective IKK isoform. The
infected and control FLS were stimulated with either medium or TNF-
for 10 min, lysed, and then assayed for kinase activity in the presence
of recombinant substrate (GST-IB) and
[-32P]dATP. As shown in Fig. 2, the IKK2 dn blocked cytokine-induced
kinase activity, whereas overexpression of IKK2 wt resulted in
spontaneous kinase activity. In contrast, overexpression of the IKK1 dn
gene had no effect on basal or TNF--induced kinase activity.
Therefore, IKK2 is the primary kinase responsible for functional IKK
activity in cultured FLS.
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B degradation and NF-B activation
We then evaluated the effect of IKK1 and IKK2 on IB degradation
and NF-B DNA binding activity in FLS. Infection with IKK1 dn
construct had no effect on either basal or TNF--stimulated IB
expression and NF-B binding (Fig. 3).
In contrast, IKK2 dn prevented IB degradation and subsequent NF-B
binding after cytokine exposure. Furthermore, the IKK2 wt construct
induced constitutive NF-B activation (Fig. 3). In a separate
experiment, IKK1 wt induced a small amount of NF-B binding in FLS
and had no effect on TNF--mediated NF-B activation (data not
shown). These data indicate that IKK2 is required for TNF--induced
IB degradation and NF-B activation in FLS.
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After demonstrating the dominant role of IKK2 in the regulation of
NF-B activation, we evaluated its ability to regulate expression of
genes involved in inflammation. NF-B is a key transcriptional
regulator of IL-6 and IL-8 synthesis. To identify the responsible
kinase, we evaluated the effects of the IKK constructs on IL-6 and IL-8
production. After stimulation with IL-1 (10 ng/ml), the synthesis of
IL-6 and IL-8 was markedly increased with no significant difference
between parental cells, GFP-expressing cells, or cells transduced with
the IKK1 dn construct (Figs. 5B and 6B). However, when FLS expressing
the IKK2 dn mutant were stimulated with IL-1, no increase in IL-6 and
IL-8 synthesis was observed (Figs. 5A and 6A). In
addition, overexpression of functional IKK2 by wt IKK2 infection
substantially increased baseline IL-6 and IL-8 production
(p < 0.001). Similar results
(n = 3, p < 0.001) were observed for
IL-6 and IL-8 production in response to TNF- (Figs. 5, C
and D, and 6, C and D). Therefore,
cytokine-induced IL-6 as well as IL-8 synthesis in FLS is mediated by
NF-B activation, which in turn is regulated by IKK2.
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, FLS infected with the adeno IKK1 wt
construct did not significantly alter basal IL-6 and IL-8 expression.
TNF- stimulation of cells infected with IKK1 wt responded
appropriately and produced large amounts of both cytokines.
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-induced ICAM-1 expression
Studies were then performed to assess the participation of the IKK
genes on ICAM-1 expression in FLS. As with the studies described above,
FLS were infected with each construct and then stimulated with medium,
IL-1, or TNF-. As shown in Fig. 4, B and D, cytokine stimulation increased ICAM-1
expression in parental cells and GFP FLS as well as cells infected with
IKK1 dn. IKK2 wt significantly (p < 0.001)
increased basal ICAM-1 levels, whereas the dn construct prevented
cytokine-induced ICAM-1 expression (Fig. 4, A and
C). These findings indicate that IKK2 plays a central role
in synoviocyte cytokine-induced and NF-B-mediated ICAM-1
expression.
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Recent data suggest that NF-B can directly regulate MMP
expression, perhaps through binding to NF-B-like sites in the
promoter regions. To determine which IKK regulates NF-B binding
activity at the MMP1 promoter, mRNA expression for collagenase was
evaluated by Northern blot analysis in FLS that had been infected with
the adenoviral constructs. Stimulation with IL-1 (10 ng/ml) induced
collagenase gene expression in parental FLS as well as cells infected
with the GFP and IKK1 constructs (Fig. 7). As with the other
NF-B-regulated genes, IKK2 wt overexpression increased collagenase
expression, whereas IKK2 dn blocked IL-1-induced gene expression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B has been recognized as one of the most important
regulators of proinflammatory gene expression. In RA, the synthesis of
cytokines, like IL-1 and TNF-, is mediated by NF-B, as is the
expression of cycloxygenase 2 (16). NF-B activation
increases expression of adhesion molecules E-selectin, VCAM-1, and
ICAM-1, whereas NF-B inhibition reduces leukocyte adhesion and
transmigration (17, 18). Moreover, studies in
collagen-induced arthritis in mice revealed that NF-B activation
precedes MMP expression and joint inflammation in vivo
(6). By inhibiting apoptosis, NF-B provides an
additional mechanism for cell hyperplasia found in the synovial intimal
lining (7).
NF-B activity is regulated by a multiarray complex, which consists
of a binding protein and at least two kinases, IKK1 and IKK2, also
named IKK and IKK. Activated IKK phosphorylates IB, the
natural inhibitor of NF-B, at two N-terminal serine residues.
Subsequently, IB is degraded by the 26S proteasome complex, and
NF-B is then translocated to the nucleus where it binds its target
genes to initiate transcription. Upstream of IKK, kinases such as
NF-B-inducing kinase and mitogen-activated protein
kinase/extracellular signal-related protein kinase kinase can activate
IKK in response to IL-1 or TNF- receptor ligation. IKK therefore
represents the key convergence site for NF-B activation by serving
as a conduit between multiple activation signals and nuclear
translocation of NF-B.
To understand the regulation of NF-B in synoviocytes, we recently
characterized the expression and activation of IKK1 and IKK2
(12). Both are constitutively expressed by FLS and
activation of the IKK complex resulted in sequential degradation of
IB and increased NF-B nuclear binding. Preliminary studies using
cell transfection with naked DNA indicated that both IKK1 and IKK2 can
induce NF-B nuclear translocation after FLS are stimulated with
cytokines, although IKK2 was significantly more effective. However,
because of the very low transfection efficiency of this technique
(generally <1–2%) additional studies to evaluate the biology of dn
IKK constructs could not be performed.
Because of the availability of high titer adenoviral constructs encoding these genes, we are now able to investigate the function of IKK1 and IKK2 in FLS. Overexpressing functional IKK2 by wt infection creates an environment with constitutively activated IKK (12). A dn mutant with a single amino acid change from lysine to methionine (K>M) at the kinase ATP-binding site was used to inhibit either IKK1 or IKK2 function. FLS with these quantitative and qualitative changes in IKK activity were then compared with uninfected cells and FLS infected with adenovirus encoding only GFP as an additional control.
Successful infection and transgene expression with IKK2 and IKK1
constructs were confirmed by Western blot analysis, whereas GFP
expression was confirmed by immunofluorescence. Assessment of kinase
function in FLS demonstrated that IKK activity was mainly due to IKK2
because wt infection with this construct increased kinase activity and
cytokine-induced phosphorylation of recombinant IB was blocked by
IKK2 dn mutant. In contrast, the IKK1 dn construct did not inhibit
IB degradation. Investigation of cytoplasmic IB levels by
Western blot analysis confirmed these results. Stimulation of FLS with
TNF- normally increases IKK activity and consequently IB levels
decrease in the cytoplasm. Because IKK2 dn mutant (but not IKK1 dn)
prevented IKK activation, IB was still detectable in FLS. Of
interest, NF-B DNA binding activity was further up-regulated by IKK2
wt plus TNF- as shown by EMSA.
NF-B controls the promoter region responsible for ICAM-1 gene
expression and mediates its synthesis in FLS (4, 19). We
therefore investigated which kinase regulates NF-B mediated
expression of this adhesion molecule. As with NF-B DNA-binding
activity, IKK2 was sufficient to increase ICAM-1 synthesis as
determined by ELISA. Moreover, only inhibiting IKK2 function could
prevent IL-1 or TNF--induced expression of this adhesion molecule
identifying IKK2 as the key convergence site for cytokine-induced,
NF-B-mediated synthesis of ICAM-1.
Cytokines like IL-8 and IL-6 are abundant in RA synovial fluid
(20, 21). Although IL-8 serves as a neutrophil
chemoattractant and angiogenic factor (22), IL-6 mediates
cell differentiation, proliferation, as well as acute phase protein
production (23). Both cytokines are transcriptionally
regulated by NF-B (24, 25) and synthesized by FLS
(26). Our studies demonstrated that, like ICAM-1, IKK2
controls NF-B-mediated IL-6 and IL-8 synthesis in synoviocytes
because 1) IKK2 wt overexpression substantially increased the
production of both cytokines and 2) IKK2 dn (but not IKK1 dn) blocked
IL-6 and IL-8 induction. Infection of cells with the equivalent dose of
IKK1 wt construct did not increase cytokine production and FLS
responded appropriately to TNF- stimulation. Our previously reported
studies demonstrated that IKK1 wt transfection using naked DNA vectors
was able to weakly induce nuclear translocation of NF-B in FLS
(12). These data, along with the observation that only
IKK2 dn blocks cytokine production, indicates that IKK2 is the primary
pathway used by FLS for cytokine-mediated NF-B activation.
Whereas the IL-6 promoter can be targeted by p50 as well as p65, the
initiation of IL-8 transcription is usually achieved by the p65
homodimer (27). The increased IL-8 production in IKK2
wt-infected cells might, at least in part, be due to p65 homodimers,
which have been identified in FLS (28). Alternatively,
activation of c-Rel-p65 heterodimers might also be involved because
IL-1-stimulated IL-8 production in synoviocytes can be reduced by
antisense to both p65 and c-Rel (29). Effects downstream
of NF-B might also be involved. For instance, NF-B might
interfere with the mitogen-activated kinase pathway, as was recently
suggested in chondrocytes (30). The ability of specific
IKK activation pathways to alter NF-B composition is suggested by
studies such as the EMSA in Fig. 3
. In this case, the relative density
of the high and low mobility bands varies with the stimulation. For
instance, wt IKK2 induces an increase in the low mobility band, whereas
TNF- increases both bands. Therefore, TNF- activation of NF-B
probably involves other pathways besides IKK with regard to selection
of specific dimers. Depending on the specific proteins present in
NF-B dimers, the pattern of gene expression and suppression
could vary.
Production of matrix metalloproteinases by the synovium is a major
feature of inflammatory joint destruction (31).
Collagenase and stromelysin are thought to participate in these
processes, and recently a NF-B binding site was identified within
the collagenase promoter of synoviocytes. Notably, NF-B binding to
this site by p50 homodimers enhances collagenase gene transcription
(32). Our data suggest that this process is also under the
control of IKK because wt IKK2 increased MMP1 mRNA synthesis. In
addition, abrogating functional IKK2 with dn mutant blocked
IL-1-induced collagenase gene expression. As with the other studies,
IKK1 appeared to have no effect.
These results are consistent with other reports suggesting that IKK2,
but not IKK1, regulates cytokine-induced NF-B activation in tumor
cells and hepatocytes (33, 34). In monocytes, LPS-mediated
TNF- promoter activity is also under the control of IKK2
(35). Thus, IKK2 appears to be the primary pathway of
inflammatory stimuli in a variety of cells, including T cells
(36), whereas IKK1 seems to have different functions.
Studies in IKK1 knockout mice revealed that loss of IKK1 interfered
with multiple morphogenetic events, including limb and skeletal
patterning as well as proliferation and differentiation of epidermal
keratinocytes (37). However, its role as a regulator of
NF-B in adult tissues is uncertain.
In conclusion, our studies indicate that IKK2, but not IKK1, is the key
convergence site for cytokine-induced NF-B activation in primary
synoviocytes and also regulates cytokine, adhesion molecule, and MMP
expression. The in vivo relevance of this observation is suggested by
recent studies with intraarticular gene therapy in rats using the same
constructs (38). For instance, activation of IKK2 in the
synovium with the IKK2 wt construct induced arthritis in normal rats,
whereas suppression of IKK activity with IKK2 dn suppressed clinical
arthritis in the rat adjuvant arthritis model. These data provide
evidence that IKK2 represents a potential therapeutic target for
inflammatory arthritis.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Gary S. Firestein, Division of Rheumatology, Allergy and Immunology, Mail Code 0656, University of California San Diego School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093-0656.
3 Abbreviations used in this paper: RA, rheumatoid arthritis; IKK, IB kinase; FLS, fibroblast-like synoviocytes, wt, wild type; dn, dominant negative; GFP, green fluorescence protein; MMP, matrix metalloproteinase; FLAG, Asp-Tyr-Lys-Asp-Asp-Asp-Lys; HA, influenza hemagglutinin.
Received for publication February 1, 2000. Accepted for publication December 8, 2000.
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