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B-Inducing Kinase Is Dispensable for Activation of NF-B in Inflammatory Settings but Essential for Lymphotoxin 
Receptor Activation of NF-B in Primary Human Fibroblasts1
*
Kennedy Institute of Rheumatology Division, Imperial College School of Medicine, Hammersmith, London, United Kingdom; and
Royal Free Hospital, Hampstead, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B is of major importance in the
biology of pro-inflammatory cytokines, such as TNF-
and
IL-1, and thereby is intimately involved in the process of
inflammation. Understanding the mechanisms by which NF-B is
activated in response to inflammatory stimuli has become a major goal
of inflammation research. The discovery of NF-B-inducing kinase
(NIK) as a TNFR-associated factor-interacting enzyme and a potential
activator of the IB-kinase complex appeared to have identified an
important element of the NF-B activition pathway, a view that was
supported by several subsequent studies. However, recent experiments in
the alymphoplasia (aly/aly) mouse, which has missense
point mutation (G885R) in NIK, has challenged that view. The reasons
for the discrepancy between the different studies is unclear and could
be due to multiple factors, such as cell type, species of cell, or
primary vs transformed cell lines. One system that has not been
investigated is primary human cells. Using an adenoviral vector
encoding kinase-deficient NIK, we have investigated the role of NIK in
LPS, IL-1, TNF-, and lymphotoxin (LT) 
R signaling in
primary human cells and TNF- expression from rheumatoid tissue.
These data show that, in the primary systems tested, NIK has a
restricted role in LTR signaling and is not required by the other
stimuli tested. Also, there is no apparent role for NIK in the process
of TNF- production in human rheumatoid arthritis. These data also
highlight the potential problems in extrapolating the function of
signaling pathways between primary and transfected cell
lines. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B/Rel transcription factor family plays an essential role in the
regulation of many cellular processes including inflammation, immune
surveillance and response, cell proliferation, and apoptosis
(1, 2, 3, 4, 5). Consequently, inappropriate levels of NF-B
activity have been associated with several human diseases including
atherosclerosis, diabetes, cancer, and rheumatoid arthritis
(RA),3 among other
inflammatory disorders (1, 6, 7, 8, 9). NF-B transcription
factors achieve such diverse regulatory roles by modulating the
expression of a large number of genes central to immune function and
inflammatory response. Indeed, the aberrant overproduction of TNF-
in RA is largely NF-B dependent (10, 11). Therefore, it
is not surprising that the NF-B pathway has been subject to intense
investigation over the last 10 to 15 years to facilitate the
development of novel therapeutic strategies in combating disease
(3).
NF-B is comprised of homodimers or heterodimers of a family of
related transcription factors sharing a conserved sequence known as the
Rel-homology domain: Rel A/p65, RelB, c-Rel, NF-B1 (p50/105), and
NF-B2 (p52/p100) (1). In most cell types, NF-B
exists in a latent state in the cytoplasm, bound to one of a number of
inhibitory proteins: IB, IB, IB
, p105, and p100
(7, 12, 13). The classical and best-studied form of
NF-B consists of a p50 and p65 heterodimer bound to IB. Upon
exposure to a wide variety of stimuli, including TNF- and IL-1,
IB is phosphorylated on Ser32 and
Ser36 and rapidly degraded by the
ubiquitin-proteasome pathway (14, 15, 16, 17, 18, 19, 20, 21, 22). Degradation of
IB unmasks the nuclear localization motif present on the p50-p65
NF-B heterodimer, allowing nuclear translocation of the active
transcription factor. Phosphorylation of IB is mediated by the
IB kinase (IKK) complex, which consists of two closely related
protein kinases, IKK1 (IKK) and IKK2 (IKK), and a regulatory
subunit, IKK
/NEMO/IKKAP-1 (23, 24, 25, 26, 27, 28, 29).
The immediate upstream activators of the IKK complex remain undefined.
Studies in transformed cell lines have suggested that atypical protein
kinase C, mitogen-activated protein kinase kinase kinase 1 (MEKK1), or
Cot/TPL2 may fulfill this role in response to diverse stimuli
(30, 31, 32, 33, 34). The MEKK-related kinase, NF-B-inducing
kinase (NIK), identified via its interaction with TNFR-associated
factor 2 (TRAF2), was demonstrated to be a key element of the NF-B
signaling pathway in response to TNF, IL-1, and CD95 (35).
Moreover, NIK was used as bait in yeast hybrid studies to identify the
first IKK (25). Overexpressed wild-type NIK interacted
with IKK1 and IKK2 and increased IKK complex kinase activity and
subsequent NF-B function, in the absence of exogenous stimuli
(26, 36). NIK was hypothesized to be the convergence point
for NF-B activation in response to TNF- family or LPS/IL-1
Toll-like receptors via TRAF-dependent pathways (35).
Furthermore, studies with a dominant-negative (NIK-kd) construct on a
variety of stimuli, including CD27, IL-17, CD3/CD28, and dsRNA
(34, 37, 38, 39, 40), suggested that NIK was a key kinase in
signaling to NF-B by numerous factors and that it might be a major
element of the NF-B pathway (26, 35, 36).
However, the in vivo physiological role of some of these intermediates
in cytokine signaling and inflammation remains controversial. Whereas
knockout mice have substantiated the role of IKK2 and IKK in
cytokine-mediated NF-B activation and apoptotic protection
(41, 42, 43, 44, 45), IKK1 has been shown to be dispensable
(46). IKK1 appears to repress basal activity of the IKK
complex and also plays an NF-B-independent role in the regulation of
keratinocyte differentiation (46, 47). The alymphoplasia
(aly) mouse phenotype has recently been described to be due
to an autosomal recessive missense point mutation (G855R) in the TRAF
binding domain of NIK (48). Alymphoplasia mice display a
systemic absence of lymph nodes and Peyer’s patches and
disorganized splenic and thymic structures with immunodeficiency
(49, 50, 51), a phenotype similar to that observed in
lymphotoxin (LT)-- and LTR-deficient mice (52, 53).
Alymphoplasia mice remain sensitive to LPS-induced endotoxic shock, and
up-regulation of VCAM-1 in response to TNF- in
aly-/- mouse embryonic fibroblasts (MEFs)
is intact (48). These data suggest that, in mice at least,
NIK may be dispensable for proinflammatory activation of NF-B, but
it may play a role in the formation of secondary lymphoid structures in
response to LT signaling. However, although not elaborated upon by the
authors, Shinkura and colleagues (48) also found that, in
human HEK 293 cells, the aly mutation does not preclude NIK
from interacting with components of the TNF- signaling pathway.
Combined with the fact that catalytically active mutant NIK protein is
present in the alymphoplasia mouse, these data do not allow
definitive conclusions to be drawn about the role of NIK in TNF-
signaling or production. Therefore, it is still relevant and essential
to determine the inflammatory role of NIK in primary human tissues to
subjugate differences induced by species or transformation.
The studies described here were undertaken to determine whether NIK
played a proinflammatory role in the induction of NF-B-dependent
cytokines in physiologically relevant primary human cells, namely
macrophages and inflammatory diseased human tissues (e.g. rheumatoid
synovium). To achieve this, adenovirus constructs encoding
NIK-kd were generated to deliver this inhibitor to primary human
cells and tissues, and the activation of NF-B in response to various
stimuli was assessed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The adherent cell lines HEK 293 (Quantum, Montreal, Canada) and
HeLa 57A (54) (gift from Prof. R. Hay, University of St.
Andrews, U.K.) were cultured in DMEM containing 10% (v/v) FBS and 100
U/ml penicillin/streptomycin at 37°C and 5%
CO2. Human monocytes were obtained from single
donor plateletphoresis residues (North London Blood Transfusion Center,
London, U.K.) and differentiated to macrophages, as described
previously (10). Monocytes/macrophages were cultured in
RPMI 1640 medium containing 5% (v/v) FBS and 100 U/ml
penicillin/streptomycin at 37°C and 5% CO2.
Primary human fibroblasts were a gift from Prof. J. Saklatvala
(Imperial College, London, U.K.). Briefly, 1-mm tissue explants from
human foreskin were cultured in a minimal volume of DMEM containing
10% (v/v) FBS to allow outgrowth of fibroblast cells. Cells were then
expanded and maintained as low-passage stocks for experimentation. RA
dissociated synoviocytes were isolated and maintained, as described
previously (10). Recombinant
LT12 was obtained
from R&D Systems (Oxford, U.K.).
Plasmid expression vectors and transfections
Expression vectors containing FLAG epitope-tagged (DYKDDDDK) wild-type NIK, kinase-dead NIK (K429A/K430A), and the pcDNA3 vector control were kindly provided by Prof. D. Wallach (Weizman Institute of Science, Rehovot, Israel) and were described previously (35). The adenoviral transfer vector pAdTRACK.CMV and adenoviral genomic vector pAdE1 were kindly provided by Prof. B. Vogelstein (Johns Hopkins Oncology Center, Baltimore, MD) and were described previously (55). pAdKS was derived from pAdTRACK.CMV by replacing its multiple cloning site with that of pBC SK+ (Stratagene, Amsterdam, The Netherlands). Briefly, the EcoRI site of pAdTRACK.CMV was destroyed by fill-in and re-ligation, creating an XmnI site. The multiple cloning site was removed via EcoRV/BglII restriction endonuclease digestion and replaced with the BssHII-flanked multiple cloning site of pBC SK+ by blunt-end ligation. The multiple cloning site of pAdKS is orientated such that the KpnI site is closest to the CMV promoter. FLAG-tagged NIK and NIK-kd cDNAs were excised from the pcDNA3 vectors using EcoRI/BglII and were incorporated directly into pAdKS. Plasmid transfections were routinely conducted using Superfect (Qiagen, Crawley, U.K.) according to the manufacturer’s instructions.
Adenoviral vectors: production, propagation, and infection
The adenoviral vectors Adv.LacZ and Adv.IB, encoding
Escherichia coli -galactosidase and nuclear-directed
porcine IB, respectively, were as previously
described (10). The adenoviral vectors AdvIB
andAdv.IKK2-kd were kindly provided by Dr. R. de Martin (University
of Vienna, Austria). Adv.GFP, Adv.NIK, and Adv.NIK-kd adenoviral
vectors were produced after pAdE1 in vivo recombination with
AdTRACK.CMV, pAdKS.NIK, and pAdKS.NIK-kd, respectively as previously
published (55). Recombinant genomes were transfected into
the HEK 293 packaging cell line to yield mature recombinant adenoviral
particles using Lipofectamine (Life Technologies, Paisley,
U.K.) according to the manufacturer’s instructions. Adenoviral
propagation to high titers and infections was as previously
described (56). Adenoviral infections were optimized to
achieve >95% efficiency for each cell type using Adv.LacZ, as
described previously (10, 11, 57, 58).
Abs/Western blot analysis, ELISA
Immunoprecipitations directed toward FLAG-tagged NIK were
performed according to the manufacturer’s instructions using 10 µl
anti-FLAG M2 affinity gel (Sigma, Poole, U.K.). Abs used for
Western blotting included the H-4 mouse monoclonal anti-human
IB Ab (Santa Cruz Biotechnology, Santa Cruz, CA) and sheep
antisera directed against human NIK (SAK14), generated using the
synthetic peptide (VMEMAYPGAPGSAVGQQKEL) representing amino acids 3–22
(kindly provided by Prof. J. Saklatvala). For Western analysis, equal
protein extracts were resolved by SDS-PAGE and transferred to polyvinyl
difluoride membrane (Millipore, Bedford, MA). Blots were blocked for
1 h with blocking buffer (5% (w/v) fat-free milk, 0.1% (v/v)
Tween 20 in PBS) followed by a 1-h incubation with the Abs, diluted
1:1,000 in blocking buffer. Blots were then incubated in HRP-conjugated
anti-mouse IgG or anti-sheep IgG (DAKO, Cambridge, U.K.)
diluted 1:2,000 in blocking buffer. Bound Ab was detected using the ECL
kit and visualized using Hyperfilm MP (Amersham Pharmacia Biotech,
Little Chalfont, U.K.). Secreted cytokines were harvested from
cell supernatants as indicated and quantified by ELISA (BD PharMingen,
San Diego, CA) according to the manufacturer’s instructions.
NF-B EMSA
After stimulation, cells were scraped into ice-cold PBS and
lysed in hypotonic lysis buffer (0.125% Nonidet P-40, 5 mM HEPES, pH
7.9, 10 mM KCl, 1.5 mM MgCl2), and then the
nuclei were harvested by centrifugation (13,000 x g
for 30 s). Nuclear protein extracts were prepared by incubating
the nuclei in hypertonic extraction buffer (5 mM HEPES, pH 7.9, 25%
glycerol, 500 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA)
for 2 h with constant agitation. NF-B DNA binding activities
were determined by incubating 1 to 3 µg of each extract with
[-32P]ATP-labeled double-stranded NF-B
consensus oligonucleotide (Promega, Madison, WI), followed by
resolution on a 5% (w/v) nondenaturing polyacrylamide gel. Gels were
dried onto filter paper and retarded DNA:protein complexes were
visualized using Hyperfilm MP (Amersham Pharmacia Biotech).
NF-B luciferase reporter gene assays
After stimulation, the medium surrounding the cells was aspirated and the cells were lysed (0.65% Nonidet P-40, 10 mM Tris, pH 8, 150 mM NaCl, 0.1 mM EDTA). Lysate volumes, of equal protein content, were corrected to 170 µl by addition of luciferase assay buffer (1% (v/v) Triton X-100, 25 mM Tris-phosphate, pH 7.8, 15% (v/v) glycerol, 1 mM dithiothreitol, 1 mM EDTA, 8 mM magnesium chloride, 0.5 mM adenosine triphosphate). Relative luciferase activities were determined over a 10-s period, after injection of 30 µl of 1.5 mM Luciferin (Sigma), using a Luminoskan (Labsystems, Chicago, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation induced by TNF-, IL-1, and NIK itself in a HeLa
cell line
Recombinant adenoviruses containing NIK or kinase-deficient-
kd-NIK (KK429/430AA) were constructed by first inserting the cDNA for
both constructs into the modified pAdTRACK.CMV shuttle vector pAdKS.
These were then incorporated into an adenoviral genome by in vivo
recombination with pAdEasy1 in BJ5183 recombination-competent E.
coli (55). The insertion of NIK or NIK-kd into
the pAdKS shuttle vector had no effect on the ability of cDNA to either
promote NF-B activation or inhibit TRAF2-induced NF-B activation
when transfected into HEK 293 cells, respectively (data not shown).
Transfection of the pAdKS.NIK and pAdKS.NIK-kd into HeLa cells
demonstrated comparable expression levels to the original pcDNA3
expression vectors of Malinin et al. (35) (Fig. 1
A).
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B-dependent luciferase
reporter gene, was used. Activation of these cells by either IL-1 or
TNF- induced the activation of NF-B as judged by the induction of
luciferase activity (Fig. 1
B) or the generation of
NF-B-DNA binding activity and IB degradation (Fig. 1C). Adenoviral-mediated overexpression of NIK-kd in HeLa
57A cells was found to potently inhibit both TNF- and
IL-1-induced luciferase activity after 4 h, compared with
infection with the control Adv.GFP virus (Fig. 1B). These
studies are in agreement with previous work in which transfection of
NIK-kd into cells has been used (35). The inhibition of
NF-B by NIK-kd was comparable to that observed with
adenoviral-mediated overexpression of IKK2-kd or IB. Concomitant
with the effect on the reporter gene, NIK-kd also inhibited IB
degradation and NF-B-DNA binding activity, as also seen with the
IKK2-kd and IB constructs (Fig. 1C).
Adenoviral-mediated NIK overexpression was found to spontaneously
induce NF-B reporter luciferase activity in agreement with previous
data using tranfected NIK (35) (Fig. 1B).
Similarly, adenoviral overexpression of NIK potently induced IB
degradation and strong NF-B-DNA binding activity, in the absence of
exogenous stimuli, in these cells (Fig. 1C). Coinfection of
Adv.NIK with Adv.NIK-kd, Adv.IKK2-kd, or Adv.IB, but not with
Adv.GFP, ameliorated these effects (Fig. 1C), suggesting
IKK2 to be a potential distal effector of NIK.
NIK is dispensable for LPS- or TNF--induced cytokine production
in primary human macrophages
The above studies demonstrated that adenoviral delivery of NIK-kd
did not impair its inhibitory activity and confirmed data that had been
obtained by others that suggested a central role for NIK in activation
of NF-B by TNF- and IL-1 by transfection of transformed cell
lines. However, the ability of adenoviral vectors to introduce
transgenes into a variety of primary human cells and tissue (10, 11, 57, 58) allowed the role of NIK to be examined in these
systems. Indeed, by using recombinant adenoviruses expressing green
fluorescent protein (GFP), primary human macrophages are observed to be
efficiently infected (>95%) at a multiplicity of infection of 100
(Fig. 2). In contrast to the results in
transformed cell lines, adenoviral-mediated expression of NIK-kd in
primary human macrophages resulted in little effect upon LPS- or
TNF--induced IL-6, IL-8, or TNF- (LPS only) (Fig. 3, A–C).
Previously, the expression of these cytokines has been shown to be
dependent on NF-B (10, 11). Similarly, NIK-kd
overexpression failed to inhibit LPS-, TNF--, or IL-1-induced
IB degradation or NF-B-DNA binding activity in these cells
(Fig. 4). However, these data could not
be attributed to Adv.NIK-kd failing to infect the macrophages, as
>90% of cells were seen to express GFP (data not shown).
Furthermore, adenoviral-mediated overexpression of IB effectively
prevented LPS-, TNF--, and IL-1-induced IB degradation and
NF-B-DNA binding activity in the same cells (Fig. 4). Studies using
higher virus titers of Adv.NIK-kd (up to a multiplicity of infection
(moi) of 1000), still failed to inhibit LPS- or TNF--mediated
cytokines beyond that of control Adv.GFP (data not shown). To
exclude another possibility that NIK-kd could not act as a functional
dominant-negative inhibitor in primary human macrophages, coinfection
experiments were performed using wild-type NIK overexpression as the
inducing stimulus. Adenoviral-mediated overexpression of wild-type NIK
induced 
2 ng/ml TNF- 24 h postinfection compared with
control virus (Fig. 3D). Wild-type NIK-induced TNF- was
inhibited by 50% when Adv.NIK-kd was coinfected at an equal titer
(moi = 100), whereas the control virus had no effect (Fig. 3D). Coinfection with a fivefold excess of Adv.NIK-kd
(moi = 500) totally ablated NIK-induced TNF-, although at this
titer, the control virus did have a small effect (25%), suggesting
that a small component of the NIK-kd inhibition at this higher titer
may be due to a competition for virus entry into cells. NIK-induced
TNF- production was also effectively inhibited by I and
IKK2-kd and was nearly complete at virus types of moi 100. The greater
effectiveness of IKK2-kd and IB in blocking NIK-induced TNF-
is probably attributable to the increased efficiency of blocking the
signal distal to the stimulus. Similarly, studies on NF-B showed
that adenoviral-mediated overexpression of wild-type NIK strongly
induced IB degradation and NF-B-DNA binding activity in these
cells (Fig. 4). Coinfection of Adv.NIK with a threefold greater moi of
Adv.NIK-kd, Adv.IKK2-kd, or Adv.IB, but not control Adv.GFP,
inhibited these effects (Fig. 4).
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B by the LTR, but
not the TNFR in primary human skin fibroblasts (HSFs)
The above data appeared to contradict a generic role for NIK in
NF-B activation that had been ascribed to the molecule by previous
studies in transformed cell lines. However, it was possible that our
results were particular to monocytic cells. Studies in another primary
human cell system, HUVECs, also failed to show any role for NIK in
LPS-, TNF-, or IL-1-induced NF-B activation (M. Conron, E.
Andreakos, P. Pantelidis, C. Smith, H. Beynon, R. duBois, and B.
Foxwell, manuscript in preparation). The observation that
alymphoplasia mice displayed similar lymphoid organogenesis defects to
LT- and LTR-deficient mice (48) led us to
investigate whether NIK played a role in LTR activation of NF-B.
For this study, we used primary HSFs that respond to LT as well as to
TNF-. A recombinant form of LT12, which is normally membrane
bound and demonstrates specificity for the LTR, was used. Both
TNF- and LT12 were found to induce IB degradation and
NF-B-DNA binding activity in HSFs, and this was not affected by
infection with control Adv.GFP (Fig. 5A). Adenoviral infection of
NIK-kd into primary human fibroblasts blocked LT12, but not
TNF--induced IB degradation and subsequent NF-B-DNA binding
activity (Fig. 5A). In contrast, adenoviral-mediated
overexpression of IKK2-kd or IB was found to ablate IB
degradation and NF-B-DNA binding activity in response to both
LT12 and TNF-. Similarly, induction of IL-6 and IL-8
expression was also NIK-dependent for LT12, but NIK-independent
for TNF- (Fig. 5B). The effect of NIK was specific for
NF-B, as the activation of p42 mitogen-activated protein kinase or
p54/c-Jun N-terminal kinase by LT12 as for TNF was unaffected by
NIK-kd (data not shown).
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secretion from RA
inflammatory diseased tissues
The previous data indicate that NIK may play a restricted role in
the signaling to NF-B, in that it is required for LTR but
not by LPS, TNF, or IL-1. Therefore, we were interested in examining
what role NIK may have in the constitutive expression of TNF- in
synovial cells taken from rheumatoid arthritic joints, where the nature
of the stimulus perpetuating TNF synthesis is unknown. As
shown in Fig. 6, data from a
representative donor showed that infection of rheumatoid-dissociated
synoviocytes with Adv.NIK-kd was found not to cause any significant
reduction (expression was 96% of control) in the spontaneous
production by TNF- by these cells and was comparable to Adv.GFP
control (Fig. 5). Studies in two other donors resulted in TNF-
production of 144 and 82% of control (a mean of 108 ± 31%,
n = 3). Compared with adenoviral introduction of
IB into these cells, which reduces spontaneous TNF- production
by >75% (Fig. 6) (10), these data would suggest that NIK
plays no role in the production of this cytokine in rheumatoid
joints.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation in
primary human cells. The importance of NF-B in the processes
associated with inflammation and infection has made elucidation of the
components controlling the activation of this transcription factor the
goal of much research. The identification of NIK as a TRAF-interacting
kinase and its subsequent use in identifying IKK appeared to place
this enzyme central to the NF-B activation pathway
(25). This view was apparently confirmed by many
subsequent studies that associated NIK with NF-B activation by
multiple and diverse stimuli. However, the majority, if not all, of
these studies were conducted in transformed cell lines. The development
of adenoviral vectors as a means of introducing transgenes into primary
human monocytic cells and inflammatory disease tissue (10, 11, 59) allowed the role of NIK in NF-B activation to be assessed
in more physiologically relevant systems. The data presented here
indicate that, rather than being a generic factor in the activation of
NF-B by many stimuli, NIK is selectively used in signaling by LTR
and does not appear to be used by TNF-, IL-1, or LPS. Moreover,
there appears to be no role for NIK in the process involved
in TNF- production in the rheumatoid arthritic joint. These data
highlight the problems of extrapolating observations from transformed
cell lines to primary human models.
The discrepancies between these data and those originally found in
transformed cell lines have been reproduced recently in studies using
mutant mice (48, 60, 61). The G855R missense point
mutation in the IKK/TRAF binding domain of NIK has been described as
being the molecular basis of the alymphoplasia (aly) mouse.
(48). The similarities between the aly/aly
phenotype and that of LT- and LTR-deficient mice suggested that
NIK was essential for LTR-dependent signaling. Indeed, while this
manuscript was in preparation, LTR-induced IB degradation and
subsequent NF-B-DNA binding activity has been shown to be deficient
in aly/aly MEFs (60, 61). In contrast,
aly/aly MEFs up-regulate NF-B-dependent VCAM in response
to TNF-, and aly/aly mice demonstrate normal
TNF--mediated endotoxemia in response to LPS administration
(48). These data suggest that NIK use is stimulus
specific. However, the presence of catalytically active, albeit
mutated, NIK protein in these mice prevents decisive conclusions from
being drawn. Especially as Shinkura et al. (48) found that
the aly mutation did not preclude NIK from interacting with
the TNF- signaling pathway. This problem has recently been overcome
by the generation of NIK-/- deficient mice
(62). These mice generally recapitulate the phenotype
observed in aly/aly mice, and NIK-/-
MEFs demonstrate normal TNF-- and IL-1-induced IKK activity
(62). NIK-/- MEFs also show a
specific impairment of LTR function in agreement with our data and
those of the aly/aly mouse. However, unlike the data
presented here and those of the aly/aly mouse,
LTR-induced IB degradation and NF-B DNA binding in
NIK-/- MEFs remain intact. Instead, the lesion
appears to fall at the level of NF-B transactivation, a previously
unknown but hypothesized role of NIK (62).
Although these mechanistic differences appear at first to conflict,
they could possibly be reconciled and explained by developmental
substitution. The absence of NIK protein in
NIK-/- deficient mice could permit substitution
by a hypothetical IKK kinase closely related to NIK. Such a kinase
could complement IKK kinase function in NIK-/-
cells but be incapable of supporting transactivation, resulting in the
effects observed. However, in the presence of aly NIK or
NIK-kd, such a kinase would not be able to gain access to the IKK
complex, i.e., be cross-dominated, thus resulting in the continued
inhibition of IB degradation and NF-B-DNA binding. The
existence of such a kinase, of course, does not explain the lack of
impairment in TNF- or IL-1 signaling, etc., where NIK-kd and
aly NIK are equally ineffective.
The observation that NIK-kd failed to block TNF-, IL-1, and LPS
signaling in a variety of primary cell systems, i.e., macrophages,
fibroblasts, and HUVECs (data not shown), would suggest that there is
not a selective use of NIK by these stimuli in different tissue
systems, although this would have to be confirmed by direct
experimentation. The ability of NIK-kd to block
LT12, but not TNF-
signaling, in the same primary human fibroblast cells shows that, in
the appropriate setting, NIK has a role and that the failure of NIK-kd
to inhibit other pathways is not due to the adenoviral vector approach.
The selective use of NIK by LTR, however, does raise the question of
what role the kinase may have in pathological inflammatory conditions,
such as RA, where a role for NF-B has been identified (10, 11), although the primary stimulus for the transcription factor
is unknown. There is preliminary evidence that a soluble LTR
Fc construct can inhibit the development of murine collagen-induced
arthritis (63). The ineffectiveness of NIK-kd on TNF
production by RA joint cell cultures, however, suggests that there is
no role for NIK toward the activation of NF-B in this inflammatory
condition. Moreover, we have obtained similar data with another
autoinflammatory disease, fibrosing alveolitis (M. Conron, E.
Andreakos, P. Pantelidis, C. Smith, H. Beynon, R. duBois, and B.
Foxwell, manuscript in preparation). However, it would be
imprudent to preclude a role for NIK for other inflammatory conditions
without direct study.
Our data, in conjunction with the other studies discussed above, raise
two other intriguing points. The discrepancy among the data obtained
primarily from HeLa, HEK293 cells, and the human monocyte cell line
U937 (40) compared with those acquired from human and
murine primary cells could suggest that transformation, which promotes
cell cycle progression and is associated with significant chromosomal
abnormalities, implying gene dysregulation, can have a profound effect
on signaling mechanisms and the components used. The fact that
adenoviral introduction of NIK-kd into HeLa cells confirmed data
previously obtained by transfection excludes the means of delivery of
the transgene as an explanation for the different results. That
transformation could have an effect on the role of NIK is supported by
the observation that, unlike in primary cells, LTR activates NF-B
normally in transformed aly/aly MEFs (61).
However, it is difficult to understand the mechanism by which NIK has
become involved in NF-B activation in HeLa cells without further
study. A simple explanation would be that transformation increases
expression of NIK such that it dominates the signaling mechanisms used
by TNF-, LPS, and IL-1. However, we have not been able, nor to our
knowledge have others been able, to detect endogenous NIK protein in
any cell system. This may suggest that expression of this kinase is
extremely low.
The second disparate point raised by these data is that, whereas NIK
appears to be inessential for TNF- and IL-1 activation of NF-B,
TRAFs and IKKs, which are potential NIK interacting proteins, are
required (64, 65). However, the ability of NIK to interact
with these proteins has only been described in studies involving the
overexpression of NIK (26, 35). Furthermore, these studies
had been conducted in the same cell lines where NIK-kd was a successful
inhibitor of TNF- and IL-1 signaling. There is no evidence that
TRAFs or IKK interact with NIK in primary cells or at normal levels of
expression. For this reason, the data presented here cannot preclude
the involvement of TRAF2, IKK1, and IKK2 in TNF-, IL-1, or LPS
signaling in the primary systems investigated. It is likely that TRAFs
and IKKs associate with other signaling molecules in these
circumstances. The natural question to ask, therefore, is the identity
of the IKK kinase in LPS, IL-1, and TNF- signaling. One
possibility is that no kinase is required, and recent data by Zhang et
al. (66), showing that the IKK complex can directly
associate with the TNF- receptor complex, might suggest that this is
the case (66). However, the fact that NIK is required for
NF-B activation by TNF-, IL-1, and LPS in the cell line
studies and that signaling by LTR in primary cells, a member of the
TNFR family, may suggest that there is a role for an IKK kinase.
A potential candidate could have been MEKK1, which has also been
associated with NF-B activation (32, 67). However, in
our preliminary studies, infection of murine and human macrophages with
a virus encoding MEKK1-dn failed to inhibit TNF- and LPS signaling
(data not shown). These data are in agreement with those shown
previously in MEKK-1 deficient mice (68).
In summary, these data have demonstrated for the first time, in
human primary cells and inflamed RA tissue, that NIK has a selective
role in signaling by the LTR and is not a generic IKK kinase.
Moreover, these studies have helped to resolve major discrepancies that
have arisen in previous studies on NIK. The data also highlight the
potential complexity of the pathways controlling NF-B activation and
the potential problems associated with extrapolating studies between
primary and transformed cell lines.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Prof. Brian M. J. Foxwell, Kennedy Institute of Rheumatology Division, Imperial College School of Medicine, 1 Aspenlea Road, Hammersmith, London, W6 8LH, U.K. E-mail address: b.foxwell{at}ic.ac.uk
3 Abbreviations used in this paper: RA, rheumatoid arthritis; IKK, IB kinase; MEKK, mitogen-activated protein kinase kinase kinase; NIK, NF-B-inducing kinase; TRAF, TNFR-associated factor; LT, lymphotoxin; MEF, mouse embryonic fibroblast; moi, multiplicity of infection; HSF, human skin fibroblast; GFP, green fluorescent protein.
Received for publication June 14, 2001. Accepted for publication September 4, 2001.
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References |
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), 25 ng/ml TNF-
), or 10 ng/ml IL-1
). Cell lysates
were then prepared and luciferase activities assessed
(n = 3, ±SEM). C, left
panels, HeLa 57A cells were infected with Adv.NIK, Adv.GFP,
Adv.NIK-kDa, Adv.IKK2-kDa, or Adv.I
). Cell supernatants were collected and secreted IL-6, IL-8, and
TNF-
