Tumor Necrosis Factor-alpha Induces Distinctive NF-kappa B Signaling within Human Dermal Fibroblasts*

David J. KoubaDagger §||, Hajime NakanoDagger , Takafumi NishiyamaDagger , Jason Kang**, Jouni UittoDagger §Dagger Dagger §§, and Alain MauvielDagger ¶¶||||

From the Departments of Dagger  Dermatology and Cutaneous Biology and § Biochemistry and Molecular Pharmacology,  Jefferson Medical College, Jefferson Institute of Molecular Medicine and the Dagger Dagger  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



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The TNF-alpha receptor-associated factor 2 (TRAF2) and its downstream mediator, the NF-kappa B-inducing kinase (NIK), have been shown to induce NF-kappa 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-kappa B-dependent promoters linked to the CAT reporter in human dermal fibroblast cultures, while epidermal keratinocyte cultures demonstrated NIK-dependent signaling. Further, NF-kappa B activation by TNF-alpha 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-kappa B activation; however, the specific proteosome inhibitor, lactacystin, failed to do so. Furthermore, TNF-alpha receptor mutants lacking a functional death domain failed to stimulate NF-kappa B, while phosphatidylcholine-phospholipase C inhibition and alkalization of endolysosomal compartments blocked its activation by TNF-alpha . These data indicate that, while epidermal keratinocytes utilize previously defined, NIK-dependent NF-kappa B pathways, dermal fibroblasts demonstrate unique NIK/TRAF2-independent signal transduction, where both acidic sphingomyelinase and calpain activity act as surrogate mediators for NF-kappa B activation.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Tumor necrosis factor-alpha (TNF-alpha )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-alpha propeptide is proteolytically converted to its active 17-kDa form. After subsequent trimerization, TNF-alpha 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-kappa 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).

Within the cytoplasmic tail of TNFR1 lies the death domain (6). This region is responsible for clustering the TNF-alpha 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-kappa B (8, 10), while TRADD also initiates apoptosis (9). Bridging the gap between TRAF2 and Ikappa B is the NF-kappa B-inducing kinase (NIK), a MEKKK family member that phosphorylates and thereby activates the Ikappa B kinase (IKK) in response to both TNF-alpha and interleukin-1 (11). IKK completes the pathway by phosphorylating Ikappa B at two key serine residues prior to its subsequent degradation (12).

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-kappa B through an unknown mechanism.

In most cell types, NF-kappa 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 Ikappa B (19). In response to appropriate stimulation, a series of phosphorylation events occurs, terminating on Ikappa 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 Ikappa B proteolysis have been recently described that rely on calcium-dependent, calpain-mediated Ikappa B degradation (22-24).

The discrete steps linking the various upstream TNF-alpha signal transduction cascades with the multiple Ikappa B degradation mechanisms are not completely understood. Herein we have explored the function of NIK in regulating NF-kappa B activation within human skin. As has been described in other cell types, NF-kappa 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-kappa B by TNF-alpha .


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ABSTRACT
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EXPERIMENTAL PROCEDURES
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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-alpha 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).

Plasmid Constructs-- Transient transfection experiments were performed with either the NF-kappa 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-kappa B-SV2-CAT construct, containing five copies of the NF-kappa 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-kappa 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-kappa 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 pADbeta -TR55 and pADbeta -TR55Delta 394 constructs were from Dr. Dieter Adam (Universität zu Kiel, Kiel, Germany). The pADbeta parental expression vector was used as a control (CLONTECH, Palo Alto, CA). The pRSV-beta -galactosidase control vector was used as a standard for control of transfection efficiency (Promega, Madison, WI).

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-beta -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-alpha . After 40 h of incubation, cells were rinsed twice with phosphate-buffered saline, harvested by scraping, and lysed in reporter lysis buffer (Promega). The beta -galactosidase activities were measured according to standard protocols (26). Aliquots corresponding to identical beta -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 beta -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.

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-kappa B binding element (underlined) from the human immunoglobulin kappa  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 [gamma -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-kappa B1 (p50) and RelA (p65) (30), NF-kappa 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-kappa 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.


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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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NIK Activates NF-kappa 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-kappa B-dependent reporter constructs. As shown in Fig. 1A, NIK potently stimulated NF-kappa B-mediated transcription in a dose-dependent manner with maximal NF-kappa B-dependent transcription at ~250 ng of transfected expression vector. At higher doses (>1000 ng), overexpression of the NIK vector depressed NF-kappa 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/Ikappa B proteins. TRAF2, at any of the concentrations tested, did not induce either the pSV2-NF-kappa B-CAT (Fig. 1B) or pHI-CAT (not shown) reporters.



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Fig. 1.   NIK drives transcription from NF-kappa B-dependent reporter constructs in human epidermal keratinocytes. A, keratinocyte cultures were cotransfected with 2 µg of NF-kappa 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 beta -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-kappa B-SV2-CAT reporter is shown (top) with corresponding quantitative histogram (bottom). C, NIK successfully induces NF-kappa 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-kappa B (p50) plus pRSV-Rel A (p65) vectors. Following transfection, cells received either 20 ng/ml TNF-alpha or vehicle. Both specific (NF-kappa B) and nonspecific (NS) complexes are noted. D, NIK activates distinct members of the NF-kappa 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-kappa B) complexes and supershifted bands (asterisk) are indicated. E, NIK and TRAF2 induce NF-kappa 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-kappa B-SV2-CAT reporter construct.

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-kappa B-specific binding by TNF-alpha . 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-kappa B probe (lane 5). Consistent with the data presented in Fig. 1, A and B, NIK overexpression induced NF-kappa 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).

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-kappa B probe (lane 3) but not by excess AP-2 oligonucleotide (lane 4). Furthermore, p50 (NF-kappa B1) and p65 (RelA) antisera supershifted (asterisk) the NIK-induced complex (lanes 5 and 7), whereas antisera raised against other kappa B family members did not (lanes 6, 8, and 9), indicating that NIK-induced NF-kappa B complexes are composed of p50 and p65.

Because TRAF2 was unable to activate NF-kappa 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-kappa 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-kappa B activation in 293 cells (8, 10) and establish that this effect is cell type-dependent.

TRAF2 and NIK Fail to Activate NF-kappa B in Fibroblast Cultures-- We next explored the potential of both TRAF2 and NIK to activate NF-kappa B in fibroblasts. To our surprise, the overexpression of either TRAF2 or NIK had no effect on transcription of the pSV2-NF-kappa 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-kappa B-dependent reporter constructs in dermal fibroblasts. A, 2 µg of the NF-kappa 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-kappa 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-kappa B1 plus pRSV-Rel A vectors. Following transfection, cells were incubated with 20 ng/ml TNF-alpha 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.

To determine the possible reasons for the failure of TRAF2 and NIK to induce NF-kappa B-dependent transcription, fibroblast cultures were transiently transfected with expression vectors for NF-kappa 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-kappa 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-kappa B binding activity, whereas exogenous TNF-alpha (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-kappa 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-kappa B activation by NIK and TRAF2 but without success (data not shown).

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-kappa 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-beta -galactosidase expression vector (Fig. 2E). Once lysates were corrected for differences in transfection efficiency as measured by beta -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 beta -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.

NIK Is Not Required for NF-kappa B Activation by TNF-alpha in Dermal Fibroblasts-- Cotransfected p50 and p65 NF-kappa B subunits, as well as exogenously added TNF-alpha , were able to potently stimulate transcription from the pSV2-NF-kappa B-CAT vector in both keratinocytes and fibroblasts (Fig. 3A). The identity of TNF-alpha -induced Rel family members in TNF-alpha -stimulated fibroblasts (Fig. 3B, upper panel) and keratinocytes (Fig. 3B, lower panel), was determined by gel supershift assays. In these experiments, the TNF-alpha -induced complexes (lanes 3-10) were competed only by NF-kappa B oligonucleotide (lane 4). Both NF-kappa 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-alpha . A, 2 µg of the NF-kappa 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-kappa 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-alpha 6 h after transfection, and all cells were harvested and assayed for CAT activity after an additional 36 h. B, TNF-alpha 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-alpha or vehicle for 1 h before harvesting nuclear extracts to be used in conjunction with NF- kappa B probe in EMSA supershift experiments. Unlabeled competitor oligonucleotides and antisera used in supershifts were used as described under "Experimental Procedures." Specific (NF-kappa B) complexes and supershifted bands (asterisk) are noted.

To investigate the role played by NIK in mediating signal transduction between TNF-alpha and NF-kappa 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-kappa 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-kappa 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).

TNF-alpha was able to trans-activate the pSV2-NF-kappa B-CAT reporter in both fibroblasts and keratinocytes. However, only in the latter cell type did NIK activate NF-kappa B. Therefore, to investigate whether NIK was necessary for the activation of NF-kappa B by TNF-alpha in either cell type, the pSV2-NF-kappa B-CAT reporter was transfected in conjunction with either equivalent amounts of pRK-Myc-NIKmut expression vector or pRKe vector. TNF-alpha 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-alpha -induced stimulation of the NF-kappa B-dependent reporter in a dose-dependent fashion. However, in fibroblasts, mutant NIK did not affect NF-kappa B-mediated transcription induced by TNF-alpha (Fig. 4B), a finding consistent with previous experiments where overexpressed NIK failed to trans-activate NF-kappa B-dependent reporters (Fig. 1A). These results indicate that NIK activation is not a requisite step in TNF-alpha -induced NF-kappa B activation in fibroblasts.



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Fig. 4.   NF-kappa B activation by TNF-alpha is NIK-independent in fibroblasts and NIK-dependent in keratinocytes. Confluent keratinocyte (A) or fibroblast (B) cultures were cotransfected with 2 µg of NF-kappa 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-alpha (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.)

Calcium and Ceramide Dependence of TNF-alpha /NF-kappa 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-kappa B in vitro (17). Because both TRAF2 and NIK failed to activate NF-kappa B in dermal fibroblasts, we analyzed the possibility that either N-SMase- or A-SMase-generated ceramides could mediate TRAF-independent NF-kappa B activation in response to TNF-alpha . Consequently, we tested the effect of various inhibitors of ceramide activation on the ability of TNF-alpha to induce NF-kappa B.

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-alpha /NF-kappa 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-kappa B binding induced by TNF-alpha to base-line levels (lanes 4-6) in fibroblasts. However, D609 had no measurable effect on NF-kappa 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-kappa 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-kappa B (17). As shown in Fig. 5B, monesin effectively inhibited TNF-alpha -induced NF-kappa 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-kappa 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.



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Fig. 5.   A-SMase is critical for NF-kappa B activation by TNF-alpha 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-alpha . 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-alpha for an additional 1 h. C and D, the death domain is critical to NF-kappa B activation in fibroblasts. 500 ng of the expression vector pADbeta -TR55 (C) or the mutant pADbeta -TR55Delta 394 (D) were transiently cotransfected into confluent fibroblast cultures with 2 µg of NF-kappa B-SV2-CAT. In controls, the equivalent molar amount of pADbeta parental vector was used. A representative CAT assay autoradiogram is shown (top) with quantitated values (bottom).

To further investigate the possible involvement of A-SMase in NF-kappa 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-alpha /NF-kappa B signaling. For this purpose, cotransfection experiments were performed, using expression vectors coding for either wild type TNFR1 (pADbeta -TR55), or a death domain-truncated mutant (pADbeta -TR55Delta 394) together with the pSV2-NF-kappa B-CAT reporter construct (Fig. 5, C and D). Interestingly, although TRADD and the indirectly TNFR1-associated factor, TRAF2, failed to activate NF-kappa B in this cell type, the intact receptor readily activated the NF-kappa B-dependent reporter (Fig. 5C), while loss of the death domain blocked the ability of the TNFR1 to activate NF-kappa B (Fig. 5D). Together, these results indicate a TRADD/TRAF2-independent NF-kappa 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-kappa 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.

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-kappa B activation by TNF-alpha , 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-alpha -induced NF-kappa B activation in fibroblasts. Within nontoxic experimental concentrations, none of these inhibitors affected NF-kappa B activation by TNF-alpha in fibroblasts (data not shown).

Previous studies have demonstrated that the intracellular calcium chelator, TMB-8, could block NF-kappa B activation by endoplasmic reticulum stress but not by TNF-alpha (35, 36). To explore the role of free calcium in NF-kappa B activation in fibroblasts, EMSAs were performed on nuclear extracts from cultures pretreated with TMB-8 for various time periods prior to TNF-alpha treatment (Fig. 6A). After exposure, liquid scintillation was performed on portions of the gel corresponding to NF-kappa B and nonspecific (NS) complexes. When NF-kappa B-specific counts were corrected to nonspecific binding, a 68% reduction in NF-kappa B binding was seen between 1 and 12 h of TMB-8 treatment, demonstrating that in our experimental system, TMB-8 can block TNF-alpha -induced NF-kappa B activation in vitro, an apparent cell type-specific phenomenon.



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Fig. 6.   Dermal fibroblasts utilize a calcium- and calpain-dependent pathway for NF-kappa B activation by TNF-alpha . 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-alpha . Cells were then incubated for an additional 1 h before harvesting nucleoproteins used in EMSA with the NF-kappa B probe. B, calpain activity is required for NF-kappa 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-alpha . C, proteosome inhibition does not affect NF-kappa 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-alpha . Nuclear extracts were harvested 1 h after the addition of TNF-alpha and used in EMSA. Both specific (NF-kappa B) and nonspecific (NS) complexes are noted.

Recently, new evidence has emerged regarding the involvement of nonproteosome-mediated Ikappa B degradation, requiring the calcium-dependent protease, calpain I (22). Because Rel activation by TNF-alpha 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-alpha and subsequent harvesting of nucleoproteins for EMSA. Again, a dose-dependent inhibition of NF-kappa 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-kappa 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-kappa B activation by TNF-alpha (Fig. 6C, lanes 4-6). Collectively, these data strongly suggest a predominant role for calpain proteolysis as opposed to Ikappa B degradation in the proteosome.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Over the past decade, tremendous progress has been made regarding the various signal transduction mechanisms mediating the numerous effects of TNF-alpha . Through two hybrid screening techniques, the family of TNFR-associated proteins was discovered. Separately, sphingomyelinases were found to play a role in TNF-alpha /NF-kappa B signal transduction. With the discovery of the NIK/IKK/Ikappa B/proteosome pathway (37, 38) and the coupling associations between TRADD/A-SMase (13) and FAN/N-SMase (15), the TNF-alpha /NF-kappa B signaling pathway has been extensively explored. However, recent reports have revealed a novel diversity to the NF-kappa B activation cascades, including coexistent calpain and proteosome Ikappa B proteolysis (22), temporally biphasic NF-kappa B activation that utilizes both calpain and proteosome hydrolysis of Ikappa B (39), calpain-specific Ikappa B degradation discovered in an immature B cell line (23), lysosomal Ikappa B proteolysis (40), and redox-dependent, proteosome-mediated Ikappa B-alpha proteolysis that does not require Ikappa B phosphorylation (41).

In this study, we have found striking differences in the mechanisms involved in the regulation of NF-kappa 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-kappa 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-kappa 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-kappa B in a fashion identical to that in 293 cells (Fig. 1, A and E).

TRAF2 knockout experiments have demonstrated that TRAF2 is not a stringent requirement for NF-kappa B activation (42). Therefore, it was not surprising that TRAF2 is not required for NF-kappa 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-kappa B (2).

Interestingly, in fibroblasts, neither TRAF2 nor NIK is functional in the NF-kappa 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 Ikappa B-kinase (43), inhibiting NF-kappa B activation. These data raise the question of the ultimate necessity of NIK in NF-kappa 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).

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-alpha -induced A-SMase activation. Further, dominant negative FADD mutants decreased TNF-alpha -induced A-SMase activity, suggesting that TRADD and FADD can augment, but are not necessarily required for, A-SMase activation by TNF-alpha . 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-kappa B activation, an intact death domain was required for TNFR1 activation of NF-kappa B in fibroblasts. Furthermore, alkalization of endolysosomal compartments and inhibition of phosphatidylcholine-phospholipase C, two approaches previously shown to block A-SMase-induced NF-kappa B activation (17) were effective in abrogating TNF-alpha -induced NF-kappa B in fibroblasts. These findings suggest that TRADD-independent A-SMase activation occurs in fibroblasts and is at least partially responsible for NF-kappa B activation in response to TNF-alpha .

The emergence of proteosome-independent Ikappa B signaling as demonstrated in WEHI231 immature B cells (23) and human HepG2 cells, as well as NF-kappa B activation in ubiquitin-defective ts20b cells (22) has enlarged the scope of TNF-alpha /NF-kappa B signaling. Additionally, previously conflicting reports of calcium dependence or independence (35, 45) in TNF-alpha /NF-kappa B signaling are now resolved with the discovery of parallel Ikappa 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-kappa B activation by TNF-alpha in addition to being blocked by various inhibitors of ceramide activation and loss of the A-SMase-associated death domain region of the TNFR1.

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-alpha and help to explain differences in NF-kappa 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-alpha , tumor necrosis factor-alpha ; NIK, NF-kappa B-inducing kinase; TRAF, TNF-alpha receptor-associated factor; FADD, Fas-associated death domain protein; TRADD, TNF-alpha receptor-associated death domain protein; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; TNFR, tumor necrosis factor receptor; IKK, Ikappa 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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