From the Departments of Dermatology and Cutaneous
Biology and § Biochemistry and Molecular Pharmacology,
¶ Jefferson Medical College, Jefferson Institute of Molecular
Medicine and the
Kimmel Cancer Center,
Thomas Jefferson University, Philadelphia, Pennsylvania 19107, the
** Division of Pulmonary and Critical Care Medicine, Stanford University
Medical Center, Stanford, California 94305, and ¶¶ INSERM
U532, Skin Research Institute, Hôpital
Saint-Louis, 75010 Paris, France
Received for publication, May 24, 2000, and in revised form, November 16, 2000
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ABSTRACT |
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The TNF- Tumor necrosis factor- Within the cytoplasmic tail of TNFR1 lies the death domain (6). This
region is responsible for clustering the TNF- The TNFR1 death domain, through its association with TRADD and FADD,
activates acidic sphingomyelinase (A-SMase) (13). A more upstream
segment of the TNFR1 intracellular domain binds FAN, a coupling protein
linked to neutral sphingomyelinase (N-SMase) (14-16). Acting at either
acidic or neutral pH optima, these enzymes cleave sphingomyelin from
different cellular compartments. N-SMase acts at the plasma membrane,
while A-SMase is presumed to act at endolysosomal compartments, and
both result in ceramide generation (17, 18). These aliphatic cleavage
products act as potent second messengers, activating NF- In most cell types, NF- The discrete steps linking the various upstream TNF- Cell Culture and Reagents--
Human dermal fibroblast cultures,
established by explanting tissue specimens from neonatal foreskin, were
utilized in passages 3-6. The cultures were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 50 µg/ml of
streptomycin. Cultures of the human embryonic kidney cell line, 293 (generous gift of Dr. Antonio Giordano, Thomas Jefferson University,
Philadelphia, PA) were also maintained in the same conditions. Human
epidermal keratinocyte cultures, initiated by explanting foreskin
specimens, were grown in serum-free, low calcium (0.15 mM)
keratinocyte growth medium (Clonetics Corp., San Diego, CA),
supplemented with epidermal growth factor, hydrocortisone, insulin, and
bovine pituitary extract. Keratinocyte cultures were utilized in
passages 1-2 to avoid differentiation inherent to prolonged
subculturing of these cells. Human recombinant TNF- Plasmid Constructs--
Transient transfection experiments were
performed with either the NF- Western Blot Analysis--
Whole cell lysates from fibroblasts
were prepared by first washing cells twice in 1× phosphate-buffered
saline and then scraping directly into Laemmli buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.5 mM phenylmethylsulfonyl fluoride). Protein concentration of
lysates was assayed with the Bio-Rad protein reagent, and 10 µg of
protein was denatured by heating at 95 °C for 3 min prior to
resolution by SDS-PAGE. After electrophoresis, proteins were transferred to Hybond ECL nitrocellulose filters (Amersham Pharmacia Biotech). Filters were placed in blocking solution (1×
phosphate-buffered saline, 5% nonfat milk) for 1 h, followed by
incubation with either rabbit anti-NIK (1:200 in 1× phosphate-buffered
saline, 0.5% nonfat milk) or anti-TRAF2 (1:200) polyclonal antisera
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h. After
incubation, filters were washed and incubated with horseradish
peroxidase-conjugated goat-anti-rabbit secondary antibody (Bio-Rad) for
1 h. Filters were then washed and developed according to
chemiluminescence (ECL) protocols (Amersham Pharmacia Biotech) and
exposed to x-ray film at room temperature.
Transient Transfection and CAT Assays--
Transient
transfections of human foreskin fibroblasts and 293 cells were
performed by the calcium phosphate/DNA coprecipitation method, as
described previously (25). Briefly, the cells were transfected with 2 µg of reporter DNA mixed with 2 µg of the pRSV- EMSA Analysis--
Nuclear extracts were prepared from human
dermal fibroblast cultures according to established protocols (28). Two
oligonucleotides, a 22-bp oligonucleotide containing a consensus
NF- NIK Activates NF-
Next, nuclear extracts from epidermal keratinocyte cultures transiently
transfected with NIK and TRAF2 expression vectors were prepared and
used in EMSA experiments to identify the Rel subunits involved in NIK
effect. Fig. 1C demonstrates the differential induction of
NF-
The identity of the shifted complexes shown in Fig. 1C was
determined by competition and gel supershift assays. As shown in Fig.
1D, NIK-induced complexes were effectively competed by a 50-fold excess of unlabeled homologous NF-
Because TRAF2 was unable to activate NF- TRAF2 and NIK Fail to Activate NF-
To determine the possible reasons for the failure of TRAF2 and NIK to
induce NF-
To rule out the possibility that NIK and TRAF2 were not endogenously
expressed in skin cells or that differences in transfection efficiencies could be responsible for poor NF- NIK Is Not Required for NF-
To investigate the role played by NIK in mediating signal transduction
between TNF-
TNF- Calcium and Ceramide Dependence of TNF-
In the past, cell-permeable analogs of ceramide with abbreviated fatty
acid chains have been added to cultured cells in vitro, to
mimic the effect of endogenous ceramide. However, these less lipophilic
C-2 and C-4 ceramides differ physiologically from the native forms and
may not adequately represent the in vivo products of
sphingomyelinase. We therefore used inhibitors of various nodes in the
ceramide activation cascade to examine their contribution to
TNF-
To further investigate the possible involvement of A-SMase in NF-
Although both A-SMase and N-SMase cleave sphingomyelin to release the
second messenger ceramide, they result in very different signaling
responses. While N-SMase activates phospholipase A2, and
mitogen-activated protein kinase pathways, ceramide liberated by the
action of A-SMase does not (34). To determine the potential involvement
of these pathways in NF-
Previous studies have demonstrated that the intracellular calcium
chelator, TMB-8, could block NF-
Recently, new evidence has emerged regarding the involvement of
nonproteosome-mediated I Over the past decade, tremendous progress has been made regarding
the various signal transduction mechanisms mediating the numerous
effects of TNF- In this study, we have found striking differences in the mechanisms
involved in the regulation of NF- TRAF2 knockout experiments have demonstrated that TRAF2 is not a
stringent requirement for NF- Interestingly, in fibroblasts, neither TRAF2 nor NIK is functional in
the NF- The intracellular domain of TNFR1 required for activation of A-SMase
has been mapped to the death domain where TRADD, FADD, and
receptor-interacting protein also interact. Both TRADD and FADD have
been linked with A-SMase activation (13). These studies demonstrated
that overexpression of TRADD and FADD failed to induce A-SMase activity
over base line but rather could enhance TNF- The emergence of proteosome-independent I In conclusion, we have shown that both NIK/TRAF2-independent and
TRADD/A-SMase divergent signaling occur in dermal fibroblasts, while
epidermal keratinocyte signaling follows previously reported mechanisms. To our knowledge, these findings in dermal fibroblasts represent the first such account of divergent signal transduction pathways initiated by TNF- receptor-associated factor 2 (TRAF2)
and its downstream mediator, the NF-
B-inducing kinase (NIK), have
been shown to induce NF-
B activation in 293 cells. Investigating the
role these mediators play in human skin, we found that both NIK and TRAF2 failed to evoke transcription from NF-
B-dependent
promoters linked to the CAT reporter in human dermal fibroblast
cultures, while epidermal keratinocyte cultures demonstrated
NIK-dependent signaling. Further, NF-
B activation by
TNF-
was unaffected by overexpression of a dominant negative mutant
NIK in fibroblasts, despite detection of endogenous TRAF2 and NIK by
Western analysis. To explore alternative signaling mechanisms in dermal
fibroblasts, we found that the intracellular calcium chelator,
3,4,5-trimethoxybenzoic acid, and the calpain inhibitor,
N-acetyl-Leu-Leu-norleucinal, both blocked NF-
B
activation; however, the specific proteosome inhibitor, lactacystin,
failed to do so. Furthermore, TNF-
receptor mutants lacking a
functional death domain failed to stimulate NF-
B, while
phosphatidylcholine-phospholipase C inhibition and alkalization
of endolysosomal compartments blocked its activation by TNF-
. These
data indicate that, while epidermal keratinocytes utilize previously
defined, NIK-dependent NF-
B pathways, dermal fibroblasts
demonstrate unique NIK/TRAF2-independent signal transduction, where
both acidic sphingomyelinase and calpain activity act as surrogate
mediators for NF-
B activation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TNF-
)1 is a potent
proinflammatory cytokine elaborated predominantly by migratory immune
cells and implicated in the pathogenesis of a variety of clinical
conditions, including rheumatoid arthritis, HIV reactivation, graft
versus host disease, and shock (1). Prior to its activation,
the 26-kDa TNF-
propeptide is proteolytically converted to its
active 17-kDa form. After subsequent trimerization, TNF-
binds and
activates two distinct membrane-bound receptors, thereby initiating a
diverse intracellular signal transduction cascade (2). The 55-kDa type I receptor (TNFR1) and 75-kDa type II receptor (TNFR2) are found at the
surface of most human cell types. Although the TNFR2 has a
significantly higher ligand affinity and dissociation rate, most
biological effects are transduced through TNFR1 (3). Signaling via
TNFR2 is largely redundant to TNFR1, since both receptors are able to
activate NF-
B. However, TNFR2 is unable to initiate apoptosis or
generate the second messenger ceramide (4). Both receptors lack
intrinsic enzymatic activity and must oligomerize to initiate
intracellular signaling. Two partially overlapping pathways mediate
TNFR1 signaling: activation of sphingomyelinases and aggregation of the
TNF receptor-associated molecules (5).
receptor-associated death domain protein (TRADD), the Fas-associated death domain protein
(FADD), and the receptor-interacting protein, as well as indirectly
binding TRAF2 (7-9). Ligand-independent overexpression of either TRADD
or TRAF2 activates NF-
B (8, 10), while TRADD also initiates
apoptosis (9). Bridging the gap between TRAF2 and I
B is the
NF-
B-inducing kinase (NIK), a MEKKK family member that
phosphorylates and thereby activates the I
B kinase (IKK) in response
to both TNF-
and interleukin-1 (11). IKK completes the pathway by
phosphorylating I
B at two key serine residues prior to its
subsequent degradation (12).
B through an
unknown mechanism.
B is an inducible transcriptional modulator,
activated by a myriad of factors including cellular stress, viral
infection, inflammatory cytokines, free radicals, and UV irradiation
(12). Prior to activation, homo- and heterodimeric Rel family members
are held latent in the cytoplasm by I
B (19). In response to
appropriate stimulation, a series of phosphorylation events occurs,
terminating on I
B and leading to its eventual degradation. In
addition to polyubiquitinization (Ubn) with concomitant
degradation in the 26 S proteosome (20, 21), other mechanisms for I
B
proteolysis have been recently described that rely on
calcium-dependent, calpain-mediated I
B degradation (22-24).
signal
transduction cascades with the multiple I
B degradation mechanisms are not completely understood. Herein we have explored the function of
NIK in regulating NF-
B activation within human skin. As has been
described in other cell types, NF-
B activity in keratinocytes is an
NIK-dependent phenomenon. In fibroblasts however, we
provide evidence for an essential, yet NIK-independent, role of
sphingomyelinase action and calpain proteolysis in governing the
activation of NF-
B by TNF-
.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was purchased
from Roche Molecular Biochemicals. 3,4,5-Trimethoxybenzoic acid
(TMB-8), monesin, N-acetyl-Leu-Leu-norleucinal (ALLN), and pertussis toxin were purchased from Sigma. Lactacystin and PD98059 were
purchased from BIOMOL (Plymouth Meeting, PA). SB203580 was purchased
from SmithKline Beecham (Philadelphia, PA).
B-dependent
promoter/reporter gene construct, pHI-CAT, containing a 197-bp
TaqI/HindIII fragment of the HIV LTR cloned into
pUX-CAT (kindly provided by Dr. Nancy Rice, NCI-Frederick Cancer
Research and Development Center, Frederick, MD), or the NF-
B-SV2-CAT
construct, containing five copies of the NF-
B consensus cis-element cloned upstream of the SV40 enhancer (gift of
Dr. Timothy Bird (Immunex Corp., Seattle, WA). The expression vectors encoding wild-type NIK, pRK-Myc-NIK, and a dominant negative mutant, pRK-Myc-NIKmut(KK429-430AA), previously shown to interfere with the
NF-
B-inducing activity of the wild type, were gifts of Dr. Mike
Rothe (Tularik Corp., S. San Francisco, CA). The mammalian TRAF2
expression vector, pRK-TRAF2, was from Dr. David Goeddel (Tularik). The
plasmid pRKe was constructed by digesting the pRK-TRAF2 plasmid with
SalI/NotI and religating using an oligonucleotide linker composed of SalI/NotI overhangs. The
integrity of all constructs was verified by automated sequencing.
pRSV-NF-
B1 (p50) and pRSV-RelA (p65) expression vectors were
obtained from Dr. Gary Nabel and Dr. Neil Perkins (AIDS Research and
Reference Reagent Program, NIAID, National Institutes of Health). The
N-SMase expression vector, pBSM13, was kindly provided by Dr. Leena
Obeid (Duke University, Durham, NC). The pAD
-TR55 and
pAD
-TR55
394 constructs were from Dr. Dieter Adam
(Universität zu Kiel, Kiel, Germany). The pAD
parental
expression vector was used as a control (CLONTECH,
Palo Alto, CA). The pRSV-
-galactosidase control vector was used as a
standard for control of transfection efficiency (Promega, Madison, WI).
-galactosidase plasmid to monitor transfection efficiencies. After glycerol shock, cells were placed in Dulbecco's modified Eagle's medium containing 10% fetal calf serum 2 h prior to the addition of TNF-
. After 40 h of incubation, cells were rinsed twice with
phosphate-buffered saline, harvested by scraping, and lysed in reporter
lysis buffer (Promega). The
-galactosidase activities were measured
according to standard protocols (26). Aliquots corresponding to
identical
-galactosidase activity were used for each CAT assay with
[14C]chloramphenicol as substrate (27) and analyzed using
TLC. Following autoradiography, TLC plates were cut and counted by liquid scintillation to quantify the acetylated
[14C]chloramphenicol. The percentage of acetylation was
determined as the average acetylated products from the total
(unacetylated and acetylated) chloramphenicol ± S.D. Human
epidermal keratinocytes grown in keratinocyte growth medium were
transiently transfected with the liposome-based DOTAP method (Roche
Molecular Biochemicals), according to the manufacturer's protocol. Six
hours after transfection, medium was replaced, and cells were incubated
for an additional 40 h. At the end of incubation, the cells were
harvested by scraping and lysed by three cycles of freeze-thawing in
200 µl of reporter lysis buffer. Aliquots corresponding to identical
-galactosidase activity were used for each CAT assay, using
[14C]chloramphenicol as substrate, and results were
quantitated in an identical fashion to those experiments performed in
fibroblast and 293 cultures.
B binding element (underlined) from the human immunoglobulin
light chain gene, 5'-GATCGAGGGGACTTTCCCTAGC-3' (29), and a
26-bp oligonucleotide harboring the consensus AP-2 binding element
(underlined), 5'-GATCGAACTGACCGCCCGCGGCCCGT-3', were used
as either probes or unlabeled competitors. Oligonucleotides were
end-labeled with [
-32P]dATP. Following gel
purification, 4 × 104 cpm were incubated on ice for
1 h with 6 µg of nuclear extract in 20 µl of binding reaction
buffer (10 mM HEPES-KOH, pH 7.5, 4 mM Tris, pH
7.9, 40 mM KCl, 0.4 mM EDTA, 4% glycerol, and
0.4 mM dithiothreitol) in the presence of 1 µg of
poly(dI-dC) (Roche Molecular Biochemicals). DNA-nucleoprotein complex
specificity was determined by coincubation of nuclear extracts with a
50-fold molar excess of unlabeled homologous or nonhomologous
competitors. The following antibodies were used in supershift
experiments: rabbit polyclonal antibodies against NF-
B1 (p50) and
RelA (p65) (30), NF-
B2 (p52) (31), Rel (p75) (32), RelB (p68)
(raised against a synthetic 17-amino acid peptide covering the
c-terminus of human RelB:
REAAFGGGLLPGPEAT),2 and a
rabbit polyclonal anti-c-Jun (Santa Cruz Biotechnology, Inc., Santa
Cruz, CA). All NF-
B antibodies were generous gifts from Dr. Nancy
Rice. The polyclonal antisera were added to nuclear extracts (0.2 µg
of antiserum proteins per 6 µg of nuclear extract) and incubated on
ice for 2 h prior to the binding reaction. DNA-protein complexes
were separated from unbound oligonucleotide on 4% acrylamide gel in
0.5× TBE. The gels were fixed for 30 min in 30% methanol, 10% acetic
acid, vacuum-dried, and exposed to x-ray film at
70 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B Independently of TRAF2 in
Keratinocytes--
To investigate the effects of NIK and TRAF2 in
keratinocytes, we performed transient cotransfection experiments in
human epidermal keratinocyte cultures with NIK or TRAF2 expression
vectors and NF-
B-dependent reporter constructs. As shown
in Fig. 1A, NIK potently
stimulated NF-
B-mediated transcription in a
dose-dependent manner with maximal
NF-
B-dependent transcription at ~250 ng of transfected
expression vector. At higher doses (>1000 ng), overexpression of the
NIK vector depressed NF-
B-dependent transcription (not shown). This phenomenon was similar to that seen with TRADD
overexpression in the human embryonic cell line, 293 (8). That effect
was previously attributed to TRADD's apoptotic effect; however, NIK has not been shown to activate cell death. Therefore, this inhibition may be due to overproduction of this factor in nonphysiological concentrations, potentially quelching available cytoplasmic phosphate necessary for downstream kinases or possibly inhibiting the synthesis of further Rel/I
B proteins. TRAF2, at any of the concentrations tested, did not induce either the pSV2-NF-
B-CAT (Fig. 1B)
or pHI-CAT (not shown) reporters.
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Fig. 1.
NIK drives transcription from
NF- B-dependent reporter constructs
in human epidermal keratinocytes. A, keratinocyte
cultures were cotransfected with 2 µg of NF-
B-SV2-CAT and
increasing amounts of pRK-Myc-NIK expression vector. In all
experiments, the amount of DNA transfected was held constant with the pRKe
parental vector. Cell lysates, corrected for
-galactosidase
activity, were assayed for CAT activity, and acetylated forms
(AC) were separated from unacetylated substrate
(U) by TLC. B, CAT assay examining increasing
dose of pRK-TRAF2 expression vector on the NF-
B-SV2-CAT reporter is
shown (top) with corresponding quantitative histogram
(bottom). C, NIK successfully induces NF-
B
activation in keratinocytes. EMSA was performed on keratinocytes
transfected with 250 ng of pRKe, pRK-Myc-NIK, or pRK-TRAF2 or 1 µg of pRSVe or pRSV-NF-
B (p50) plus pRSV-Rel A (p65) vectors.
Following transfection, cells received either 20 ng/ml TNF-
or
vehicle. Both specific (NF-
B) and nonspecific
(NS) complexes are noted. D, NIK activates
distinct members of the NF-
B family. Nuclear extracts of
keratinocytes transfected with 1 µg of pRKe or pRK-Myc-NIK were
preincubated with the unlabeled competitor oligonucleotides indicated
or antiserum to Rel family members. Specific
(NF-
B) complexes and supershifted bands
(asterisk) are indicated. E, NIK and TRAF2 induce
NF-
B activation in 293 cells. Similarly to the experiment outlined
in Fig. 1A, increasing doses of the pRK-Myc-NIK
(top) or pRK-TRAF2 (bottom) expression vector
were cotransfected into 293 cells with 2 µg of NF-
B-SV2-CAT
reporter construct.
B-specific binding by TNF-
. Transfection of empty pRKe
(lane 3) expression vector or pRSVe vector
(lane 4) was unable to induce binding activity,
but cotransfection with 1 µg of pRSV-p50 and pRSV-p65 expression
vectors led to substantial shifting of the NF-
B probe
(lane 5). Consistent with the data presented in
Fig. 1, A and B, NIK overexpression induced
NF-
B nuclear translocation and DNA binding (lane
6), an effect not seen with either TRAF2 (lane
7) or dominant negative mutant NIK expression vector (not shown).
B probe (lane
3) but not by excess AP-2 oligonucleotide (lane
4). Furthermore, p50 (NF-
B1) and p65 (RelA) antisera
supershifted (asterisk) the NIK-induced complex
(lanes 5 and 7), whereas antisera
raised against other
B family members did not (lanes
6, 8, and 9), indicating that NIK-induced NF-
B complexes are composed of p50 and p65.
B in keratinocytes (Fig.
1B), we examined the functionality of our TRAF2 expression system by performing transient cotransfection experiments in 293 cells,
the principal line used in the literature to test the activity of TRADD
and TRAF2 (8, 10). As expected, in 293 cells, transfection of either
NIK or TRAF2 induced a dose-dependent transcription from
the NF-
B-dependent reporter construct, pRSV-SV2-CAT
(Fig. 1E). Together, these data confirm previous
observations suggesting that ligand-independent overexpression of both
TRAF2 and NIK is sufficient to induce NF-
B activation in 293 cells
(8, 10) and establish that this effect is cell
type-dependent.
B in Fibroblast
Cultures--
We next explored the potential of both TRAF2 and NIK to
activate NF-
B in fibroblasts. To our surprise, the overexpression of
either TRAF2 or NIK had no effect on transcription of the
pSV2-NF-
B-CAT reporter (Fig. 2,
A and B) at doses that had potent effects in keratinocytes. Furthermore, at doses greater than 250 ng, both factors
elicited a transcriptional inhibition, similarly to that seen in
keratinocytes (Fig. 1, A and B). These results
may indicate the presence of unidentified inhibitor(s) of TRAF2 and NIK
signaling or alternatively a cell type-specific, TRAF2- and
NIK-independent signaling mechanism in dermal fibroblasts.
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Fig. 2.
NIK and TRAF2 fail to stimulate transcription
from NF- B-dependent reporter constructs in
dermal fibroblasts. A, 2 µg of the NF-
B-SV2-CAT
were cotransfected with increasing amounts of the pRK-Myc-NIK
expression vector. A representative autoradiogram (top) and the quantitated values (mean ± S.D.) from three independent
experiments expressed as relative CAT activity are shown
(bottom). B, in a similar experiment, an
increasing dose of pRK-TRAF2 was cotransfected with the NF-
B-SV2-CAT
reporter construct. C, fibroblast cultures were transfected
with 250 ng of either pRKe vector, pRK-Myc-NIK, pRK-TRAF2, or 1 µg of
pRSVe, and pRSV-NF-
B1 plus pRSV-Rel A vectors. Following
transfection, cells were incubated with 20 ng/ml TNF-
prior to the
preparation of nuclear extracts. D, TRAF2 and NIK are
present in native human dermal fibroblasts. Proteins were separated by
SDS-PAGE for Western blot as described under "Experimental
Procedures," and following electrotransfer, blots were incubated with
either anti-NIK (lane 1) or anti-TRAF2 (lane 2)
primary antibodies. Specific bands are indicated (asterisk),
as are molecular weights. E, TRAF2 and NIK overexpression.
Keratinocytes (K), fibroblasts (F), and 293 cells
(293) were transfected with pRKe (lanes 1,
3, 5, 7, 9, and
11), pRK-TRAF2 (lanes 2, 4, and
6), or pRK-Myc-NIK (lanes 8, 10, and
12) expression vectors, and Western blots were probed with
either anti-TRAF2 (lanes 1-6) antiserum or anti-NIK
(lanes 7-12) antiserum.
B-dependent transcription, fibroblast cultures were transiently transfected with expression vectors for NF-
B subunits, pRK-Myc-NIK, or pRK-TRAF2, and nuclear proteins were isolated
for use in EMSA, similarly to the experiments outlined in Fig.
1C. Plasmids encoding the pRKe or pRSVe parental vectors, NF-
B subunits, pRK-Myc-NIK, or the pRK-TRAF2 expression vector were
used in transient transfections. As shown in Fig. 2C, NIK (lane 6) or TRAF2 (lane 7)
overexpression in fibroblasts failed to induce NF-
B binding
activity, whereas exogenous TNF-
(lane 2) or
pRSV-p50 and pRSV-p65 overexpression (lane 5)
demonstrated strong binding. In comparison with data presented in Fig.
1C, the degree of NF-
B activation in lane
5 is clearly less than when the same experiment was
performed in keratinocytes. The reduced activation is attributable to
lower transfection efficiencies inherent to the calcium phosphate
method used in fibroblasts compared with liposome-mediated transfection
in keratinocytes. A broad range of DNA concentrations were also used in
subsequent overexpression experiments in an attempt to elicit NF-
B
activation by NIK and TRAF2 but without success (data not shown).
B activating potential in fibroblasts, we performed Western blot analyses on dermal fibroblast cultures. Data presented in Fig. 2D demonstrate that in
native human dermal fibroblasts, when 10 µg of untransfected cell
lysate was probed by either NIK or TRAF2 antiserum, NIK and TRAF2 are indeed expressed in detectable quantities (lanes
1 and 2). In addition, we harvested lysates from
keratinocytes, fibroblasts, and 293 cells that had been transfected
either with the pRKe parental vector or with equal amounts of
pRK-Myc-NIK or pRK-TRAF2 expression vector in conjunction with the
pRSV-
-galactosidase expression vector (Fig. 2E). Once
lysates were corrected for differences in transfection efficiency as
measured by
-galactosidase activity, samples of each cell type were
separated by electrophoresis, and blots were probed with either
anti-TRAF2 (lanes 1-6) or anti-NIK (lanes 7-12) antiserum. At these lower lysate
concentrations and exposure times, pRKe-transfected controls
demonstrate lower NIK and TRAF2 signal, as expected. Although after
correction for transfection efficiency, the signal intensity between
keratinocytes and fibroblasts was equivalent, there was a measurable
increased intensity of 293 proteins. These results are probably due to
a slightly higher level of basal production of these signaling proteins
in 293 cells as well as nonlinearity in the
-galactosidase assay due
to the significantly higher transfection efficiency of 293 cells
compared with fibroblasts and even keratinocytes. Therefore, these
results demonstrate that transfection techniques or efficiencies cannot explain the activity failure of transfected NIK and TRAF2 in fibroblasts.
B Activation by TNF-
in Dermal
Fibroblasts--
Cotransfected p50 and p65 NF-
B subunits, as well
as exogenously added TNF-
, were able to potently stimulate
transcription from the pSV2-NF-
B-CAT vector in both keratinocytes
and fibroblasts (Fig. 3A). The
identity of TNF-
-induced Rel family members in TNF-
-stimulated
fibroblasts (Fig. 3B, upper panel) and
keratinocytes (Fig. 3B, lower panel),
was determined by gel supershift assays. In these experiments, the
TNF-
-induced complexes (lanes 3-10) were
competed only by NF-
B oligonucleotide (lane
4). Both NF-
B1 (lane 6) and Rel A
(lane 8) antibodies selectively supershifted the
complex, while antisera directed against other Rel members (lanes 7, 9, and 10) did
not, demonstrating that the pool of latent Rel proteins was identical,
composed of p50 and p65, in both cell types tested.
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Fig. 3.
Both fibroblasts and keratinocytes activate
Rel proteins in response to TNF- .
A, 2 µg of the NF-
B-SV2-CAT reporter was transiently
transfected into either dermal fibroblasts (open bars) or
epidermal keratinocytes (solid bars) with 1 µg of pRSVe,
pRSV-NF-
B1 plus pRSV-RelA, or the reporter construct alone. Cultures
transfected with reporter only were treated with either vehicle
(control) or 20 ng/ml TNF-
6 h after transfection,
and all cells were harvested and assayed for CAT activity after an
additional 36 h. B, TNF-
activates identical Rel
family members in keratinocytes and fibroblasts. Confluent fibroblast
(upper panel) or keratinocyte cultures (lower
panel) were treated with either 20 ng/ml TNF-
or vehicle for
1 h before harvesting nuclear extracts to be used in conjunction
with NF-
B probe in EMSA supershift experiments. Unlabeled
competitor oligonucleotides and antisera used in supershifts were used
as described under "Experimental Procedures." Specific
(NF-
B) complexes and supershifted bands
(asterisk) are noted.
and NF-
B, we utilized the expression vector coding
for a kinase-inactive dominant negative mutant NIK, pRK-Myc-NIKmut, in
transient cotransfections of keratinocytes, in conjunction with either
wild type pRK-Myc-NIK expression vector or pRSV-p50 and pRSV-p65
expression vectors. As expected, the introduction of free p50 and p65
subunits stimulated transcription from the
NF-
B-dependent reporter but was unaffected by the
presence of cotransfected mutant NIK (data not shown). However,
inhibition of NIK-induced transcription from the pSV2-NF-
B-CAT
vector was observed with cotransfected mutant NIK (not shown),
confirming the dominant negative activity of the mutant NIK construct
in our cell system (11, 33).
was able to trans-activate the pSV2-NF-
B-CAT
reporter in both fibroblasts and keratinocytes. However, only in the
latter cell type did NIK activate NF-
B. Therefore, to investigate
whether NIK was necessary for the activation of NF-
B by TNF-
in
either cell type, the pSV2-NF-
B-CAT reporter was transfected in
conjunction with either equivalent amounts of pRK-Myc-NIKmut expression
vector or pRKe vector. TNF-
was added to the culture medium 12 h after transfection to allow time for adequate production of
recombinant mutant NIK. As expected, in keratinocytes (Fig.
4A) mutant NIK blocked
TNF-
-induced stimulation of the NF-
B-dependent
reporter in a dose-dependent fashion. However, in
fibroblasts, mutant NIK did not affect NF-
B-mediated transcription
induced by TNF-
(Fig. 4B), a finding consistent with
previous experiments where overexpressed NIK failed to
trans-activate NF-
B-dependent reporters (Fig.
1A). These results indicate that NIK activation is not a
requisite step in TNF-
-induced NF-
B activation in
fibroblasts.
View larger version (24K):
[in a new window]
Fig. 4.
NF- B activation by
TNF-
is NIK-independent in fibroblasts and
NIK-dependent in keratinocytes. Confluent keratinocyte
(A) or fibroblast (B) cultures were cotransfected
with 2 µg of NF-
B-SV2-CAT and either 2 µg of the pRKe vector or
increasing amounts of pRK-Myc-NIKmut. Sixteen hours after transfection,
cells were incubated with either vehicle (open bars) or 20 ng/ml TNF-
(solid bars) and harvested 36 h later. In
all experiments, the quantity of DNA transfected was held constant by the addition of pRKe parental
vector. Representative autoradiograms are shown (top). In
both A and B, the results of three independent
experiments performed in duplicate were averaged and expressed
graphically (bottom) as percentage of acetylation (mean ± S.D.)
/NF-
B Signaling in
Dermal Fibroblasts--
Previous studies have shown that ceramides,
produced by two distinct sphingomyelinases acting at either an acidic
or basic pH optima, can activate NF-
B in vitro (17).
Because both TRAF2 and NIK failed to activate NF-
B in dermal
fibroblasts, we analyzed the possibility that either N-SMase- or
A-SMase-generated ceramides could mediate TRAF-independent NF-
B
activation in response to TNF-
. Consequently, we tested the effect
of various inhibitors of ceramide activation on the ability of TNF-
to induce NF-
B.
/NF-
B signaling. As a first approach, we used D609, an inhibitor of phosphatidylcholine-phospholipase C and essential primary step in the hydrolysis of membrane sphingomyelin. As shown in
Fig. 5A, D609 reduces NF-
B
binding induced by TNF-
to base-line levels (lanes
4-6) in fibroblasts. However, D609 had no measurable effect
on NF-
B activation in keratinocytes (lanes
10-12), suggesting that the A-SMase pathway is not involved
in the latter cell type. Because D609 acts near the beginning of the
cascade and may potentially inhibit protein kinase C in addition to
A-SMase, we used monesin, an Na+/H+ antiport
able to alkalize endolysosomal compartments, to study NF-
B
activation specifically through the action of A-SMase (Fig. 5B). Previous reports have demonstrated that both selective
A-SMase inhibition with monesin and alkalization with NH4Cl
can block NF-
B (17). As shown in Fig. 5B, monesin
effectively inhibited TNF-
-induced NF-
B binding in a
dose-dependent fashion. Incubation of the same nuclear
extracts with consensus AP-2 probe demonstrated no differences in
binding (not shown), attesting to both the integrity of the extracts
used and specificity of the observed effect on NF-
B. Similar results
were obtained with NH4Cl (not shown). Less intense banding
patterns seen in Fig. 5B are attributable to differences in
probe preparation and lower exposure times.
View larger version (57K):
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Fig. 5.
A-SMase is critical for
NF- B activation by TNF-
in dermal fibroblasts. A, fibroblast cultures
(F) (lanes 1-6) or keratinocyte cultures
(K) (lanes 7-12) were preincubated with either
vehicle or increasing concentrations of D609 for 1 h prior to the
addition of 20 ng/ml TNF-
. B, in a similar experiment, fibroblasts
were preincubated for 1 h with increasing concentrations of
monesin prior to the addition of 20 ng/ml TNF-
for an additional
1 h. C and D, the death domain is critical
to NF-
B activation in fibroblasts. 500 ng of the expression vector
pAD
-TR55 (C) or the mutant pAD
-TR55
394
(D) were transiently cotransfected into confluent fibroblast
cultures with 2 µg of NF-
B-SV2-CAT. In controls, the equivalent
molar amount of pAD
parental vector was used. A representative CAT
assay autoradiogram is shown (top) with quantitated values
(bottom).
B
activation in fibroblasts, we examined the necessity of the TNFR1 death
domain, which is associated with both A-SMase and TRADD activation, for
proper TNF-
/NF-
B signaling. For this purpose, cotransfection
experiments were performed, using expression vectors coding for either
wild type TNFR1 (pAD
-TR55), or a death domain-truncated mutant
(pAD
-TR55
394) together with the pSV2-NF-
B-CAT reporter
construct (Fig. 5, C and D). Interestingly,
although TRADD and the indirectly TNFR1-associated factor, TRAF2,
failed to activate NF-
B in this cell type, the intact receptor
readily activated the NF-
B-dependent reporter (Fig.
5C), while loss of the death domain blocked the ability of
the TNFR1 to activate NF-
B (Fig. 5D). Together, these
results indicate a TRADD/TRAF2-independent NF-
B activation that
still requires an intact death domain. Furthermore, expression of
N-SMase using the expression vector pBSM13 did not alter the base-line
level of pSV2-NF-
B-CAT activity when tested in both epidermal
keratinocytes or dermal fibroblasts (data not shown). Coupled with the
information gained from Fig. 5, A and B, these
data suggest the existence of an N-SMase- and TRADD-independent, yet
A-SMase-dependent, signaling process in fibroblasts.
B activation by TNF-
, we examined the
effects of the mitogen-activated protein kinase inhibitors PD98059 (50 µM) and SB203580 (15 µM) as well as the
phospholipase A2 inhibitor, pertussis toxin (up to 500 ng/ml), on TNF-
-induced NF-
B activation in fibroblasts. Within
nontoxic experimental concentrations, none of these inhibitors affected
NF-
B activation by TNF-
in fibroblasts (data not shown).
B activation by endoplasmic reticulum stress but not by TNF-
(35, 36). To explore the role of
free calcium in NF-
B activation in fibroblasts, EMSAs were performed
on nuclear extracts from cultures pretreated with TMB-8 for various
time periods prior to TNF-
treatment (Fig. 6A). After exposure, liquid
scintillation was performed on portions of the gel corresponding to
NF-
B and nonspecific (NS) complexes. When
NF-
B-specific counts were corrected to nonspecific binding, a 68%
reduction in NF-
B binding was seen between 1 and 12 h of TMB-8
treatment, demonstrating that in our experimental system, TMB-8 can
block TNF-
-induced NF-
B activation in vitro, an
apparent cell type-specific phenomenon.
View larger version (46K):
[in a new window]
Fig. 6.
Dermal fibroblasts utilize a calcium- and
calpain-dependent pathway for NF- B
activation by TNF-
. A,
confluent fibroblast cultures were pretreated with either vehicle alone
or with 50 µM of the intracellular calcium chelator,
TMB-8, for the times indicated prior to the addition of 20 ng/ml TNF-
. Cells were then incubated for an
additional 1 h before harvesting nucleoproteins used in EMSA with
the NF-
B probe. B, calpain activity is required for
NF-
B activation in fibroblasts. Either vehicle alone or increasing
concentrations of the calpain inhibitor, ALLN, were added to fibroblast
cultures prior to the addition of TNF-
. C, proteosome
inhibition does not affect NF-
B activation in fibroblasts. Either
vehicle alone or increasing concentrations of the proteosome-inhibitor,
lactacystin, were added to fibroblast cultures prior to the addition of
TNF-
. Nuclear extracts were harvested 1 h after the addition of
TNF-
and used in EMSA. Both specific (NF-
B)
and nonspecific (NS) complexes are noted.
B degradation, requiring the
calcium-dependent protease, calpain I (22). Because Rel
activation by TNF-
in fibroblasts was
Ca2+-dependent, we studied the role of calpain
I on this pathway. For this purpose, fibroblast cultures were
preincubated with the peptide aldehyde calpain inhibitor, ALLN, prior
to the addition of TNF-
and subsequent harvesting of nucleoproteins
for EMSA. Again, a dose-dependent inhibition of NF-
B
activation by ALLN was seen (Fig. 6B, lanes
4-6), but no modification of AP-2 binding was observed (not
shown). These findings suggest a significant role played by calpain
proteolysis in NF-
B activation; however, since the inhibitory
activity of ALLN for proteolytic enzymes is of limited specificity,
they do not rule out the possibility of a parallel, proteosome-mediated
pathway acting in dermal fibroblasts. Therefore, we next utilized the
highly specific proteosome inhibitor, lactacystin, in similar EMSA
experiments. At doses 2-fold greater than its IC50,
lactacystin preincubation had no effect on NF-
B activation by
TNF-
(Fig. 6C, lanes 4-6).
Collectively, these data strongly suggest a predominant role for
calpain proteolysis as opposed to I
B degradation in the proteosome.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. Through two hybrid screening techniques, the family
of TNFR-associated proteins was discovered. Separately, sphingomyelinases were found to play a role in TNF-
/NF-
B signal transduction. With the discovery of the NIK/IKK/I
B/proteosome pathway (37, 38) and the coupling associations between TRADD/A-SMase (13) and FAN/N-SMase (15), the TNF-
/NF-
B signaling pathway has
been extensively explored. However, recent reports have revealed a
novel diversity to the NF-
B activation cascades, including coexistent calpain and proteosome I
B proteolysis (22), temporally biphasic NF-
B activation that utilizes both calpain and proteosome hydrolysis of I
B (39), calpain-specific I
B degradation discovered in an immature B cell line (23), lysosomal I
B proteolysis (40), and
redox-dependent, proteosome-mediated I
B-
proteolysis
that does not require I
B phosphorylation (41).
B activation between dermal
fibroblasts and previously published reports in other cell lines. Prior
studies of the TNFR-associated proteins have predominantly involved the
human embryonic kidney cell line, 293. In this cell type, TRADD was
found to link the death domain of the TNFR1 to TRAF2 (8, 9). NIK
further coupled TRAF2 to IKK, completing a TRADD/TRAF2/NIK/IKK pathway
directly to NF-
B activation (11, 12). Although we were able to
duplicate the ligand-independent effects of TRAF2 and NIK in 293 cells,
normal human dermal fibroblasts in culture demonstrated distinct
variations in their NF-
B signaling mechanism. 293 cells were
originally chosen for their lack of TNFR1 gene expression. Although
appropriate for the study of the directly receptor-associated factors,
TRADD, FADD, and receptor-interacting protein, this particular feature
of 293 cells is not essential for the study of NIK. This is
demonstrated in our experiments showing that NIK overexpression in
keratinocyte cultures induced NF-
B in a fashion identical to that in
293 cells (Fig. 1, A and E).
B activation (42). Therefore, it was
not surprising that TRAF2 is not required for NF-
B activation in
both human skin fibroblasts and keratinocytes, despite its presence in
these cells. In keratinocytes, NIK acts independently of TRAF2 (Fig. 1,
A and B), implying that other associations
coupling NIK to the TNFR1 may exist, a likely scenario since NIK can
interact with TRAF1, -2, -3, -5, and -6, despite the fact that TRAF1
and -3 are unable to stimulate NF-
B (2).
B activation pathway. It is possible that NIK itself may be
regulated by an as yet unknown cell-specific inhibitor, resulting in a
block of upstream signals emanating from TNF receptor-associated molecules. This idea is supported by recent evidence demonstrating autoregulation of NIK by its own N-terminal negative regulatory domain,
which, when overexpressed, blocks the interaction between NIK and the
I
B-kinase (43), inhibiting NF-
B activation. These data raise the
question of the ultimate necessity of NIK in NF-
B signaling. This
has recently been partially addressed in mice with the discovery of a
naturally occurring NIK mutation that leads to the immunodeficiency,
alymphoplasia, which affects B-cell development in otherwise viable
mice and results in absent lymph nodes and Peyer's patches (44).
-induced A-SMase
activation. Further, dominant negative FADD mutants decreased TNF-
-induced A-SMase activity, suggesting that TRADD and FADD can
augment, but are not necessarily required for, A-SMase activation by
TNF-
. Caspase-like protease(s) were postulated mediators of this
TRADD/FADD-associated A-SMase activation (13). We found that although
TRADD, TRAF2, and NIK were unable to initiate NF-
B activation, an
intact death domain was required for TNFR1 activation of NF-
B in
fibroblasts. Furthermore, alkalization of endolysosomal compartments
and inhibition of phosphatidylcholine-phospholipase C, two
approaches previously shown to block A-SMase-induced NF-
B activation
(17) were effective in abrogating TNF-
-induced NF-
B in
fibroblasts. These findings suggest that TRADD-independent A-SMase
activation occurs in fibroblasts and is at least partially responsible
for NF-
B activation in response to TNF-
.
B signaling as demonstrated
in WEHI231 immature B cells (23) and human HepG2 cells, as well as
NF-
B activation in ubiquitin-defective ts20b cells (22) has enlarged
the scope of TNF-
/NF-
B signaling. Additionally, previously
conflicting reports of calcium dependence or independence (35, 45) in
TNF-
/NF-
B signaling are now resolved with the discovery of
parallel I
B degradation pathways, since a
calcium/calpain-dependent system coexists with the earlier
described, proteosome-mediated pathway (21). Furthermore, the addition
of ceramides to permeablized cells has been shown to stimulate
intracellular calpain activity (46). Our results bolster these
findings, since dermal fibroblasts were found to be dependent on
intracellular free calcium for NF-
B activation by TNF-
in
addition to being blocked by various inhibitors of ceramide activation
and loss of the A-SMase-associated death domain region of the TNFR1.
and help to explain differences in NF-
B signaling between cell lines previously reported.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Ying-Jee Song and Linda Lin for expert technical assistance.
![]() |
FOOTNOTES |
---|
* This research was supported in part by National Institutes of Health Grants R29-AR43751 (to A. M.) and R01-AR41439 (to J. U.) and a Research Career Development Award from the Dermatology Foundation (to A. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This paper was submitted in partial fulfillment of the Ph.D.
degree at Thomas Jefferson University.
§§ To whom correspondence may be addressed: Thomas Jefferson University, Philadelphia, PA 19107. Tel.: 215-503-5787; Fax: 215-503-5788; E-mail: Jouni.Uitto@mail.tju.edu.
To whom correspondence may be addressed: INSERM U532,
Pavillon Bazin, Hôpital Saint-Louis, 75010 Paris, France. E-mail:
mauviel@chu-stlouis.fr.
Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.M004511200
2 N. R. Rice, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
TNF-, tumor
necrosis factor-
;
NIK, NF-
B-inducing kinase;
TRAF, TNF-
receptor-associated factor;
FADD, Fas-associated death domain protein;
TRADD, TNF-
receptor-associated death domain protein;
CAT, chloramphenicol acetyltransferase;
EMSA, electrophoretic mobility shift
assay;
TNFR, tumor necrosis factor receptor;
IKK, I
B
kinase;
A-SMase, acidic sphingomyelinase;
N-SMase, neutral
sphingomyelinase;
TMB-8, 3,4,5-Trimethoxybenzoic acid;
ALLN, N-acetyl-Leu-Leu-norleucinal.
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