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Am J Physiol Heart Circ Physiol 289: H2073-H2080, 2005. First published June 24, 2005; doi:10.1152/ajpheart.01216.2004
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Fas-associated death-domain protein inhibits TNF-{alpha} mediated NF-{kappa}B activation in cardiomyocytes

Wei Chao,1,2,* Yan Shen,1,2,* Ling Li,1 Huailong Zhao,2 Steffen E. Meiler,4 Stuart A. Cook,1 and Anthony Rosenzweig1,3

1Program in Cardiovascular Gene Therapy, Cardiovascular Research Center, Massachusetts General Hospital, Charlestown; 2Department of Anesthesia and Critical Care, and 3Division of Cardiology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts; and 4Department of Anesthesia, Medical College of Georgia, Augusta, Georgia

Submitted 6 December 2004 ; accepted in final form 3 June 2005


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Fas-associated death-domain protein (FADD) is an adaptor molecule that links death receptors to caspase-8 in many cell types including cardiomyocytes (CMs). Although FADD has previously been reported to play an important role in CM apoptosis, the effect of FADD on CM NF-{kappa}B signaling, which is a proinflammatory pathway, has not been delineated. To investigate the role of FADD in CM NF-{kappa}B activation, we utilized adenoviral gene transfer of wild-type FADD and a truncation mutant that lacks the death-effector domain (FADD-DED) in rat CMs in vitro TNF-{alpha} activated NF-{kappa}B in CMs as demonstrated by phosphorylation and degradation of inhibitory-{kappa}B (I{kappa}B)-{alpha}-enhanced nuclear p65 and NF-{kappa}B DNA-binding activity as well as increased mRNA for the NF-{kappa}B-dependent adhesion molecule VCAM-1 (19 ± 4.1-fold) as measured by quantitative RT-PCR. Gene transfer of FADD inhibited TNF-{alpha}-induced I{kappa}B-{alpha} phosphorylation, decreased p65 nuclear translocation and NF-{kappa}B DNA-binding activity, and reduced VCAM-1 transcript levels by 53–65%. Interestingly, FADD-DED exhibited a similar but weaker inhibitory effect on NF-{kappa}B activation. The effects of FADD on NF-{kappa}B were cell-type specific. FADD expression also inhibited TNF-{alpha}-mediated NF-{kappa}B activation in human endothelial cells but not in rat pulmonary artery smooth muscle cells. In contrast, FADD expression actually activated NF-{kappa}B in human embryonic kidney (HEK)-293 cells. In CMs, FADD inhibited NF-{kappa}B activation as well as phosphorylation of I{kappa}B-{alpha} and I{kappa}B kinase (IKK)-{beta} in response to cytokine stimulation or expression of the upstream kinases NF-{kappa}B-inducing kinase and IKK-{beta}. These data demonstrate that FADD inhibits NF-{kappa}B activation in CMs, and this inhibition likely occurs at the level of phosphorylation and activation of IKK-{beta}.

signal transduction; cardiac; inflammation; tumor necrosis factor; nuclear factor-{kappa}B

INFLAMMATORY SIGNALING MAY contribute to a variety of cardiac conditions including injury after transient ischemia, myocarditis, and heart failure. For this reason, understanding the mechanisms that control cardiomyocyte (CM) inflammatory signaling and learning to safely modulate them could have important clinical implications.

The nuclear factor-{kappa}B (NF-{kappa}B) family of transcription factors plays a central role in coordinately regulating expression of a wide variety of inflammatory genes that have been linked to cardiac pathology (34). NF-{kappa}B factors (p65 or RelA, p50, p52, Bcl-3, c-Rel, and RelB) generally exist as dimers in the cytoplasm bound to an inhibitory subunit, inhibitory-{kappa}B (I{kappa}B; Ref. 6). The dominant mechanism of NF-{kappa}B activation involves serine phosphorylation and degradation of I{kappa}B via the ubiquitin pathway; this is followed by translocation of NF-{kappa}B to the nucleus, where it activates transcription of specific promoter targets. This serine phosphorylation is mediated by a large, multiunit complex that contains two catalytic subunits, I{kappa}B kinase (IKK)-{alpha} and -{beta}, as well as the regulatory subunit IKK-{gamma} or NEMO (6). IKK-{beta} appears to be the major kinase responsible for phosphorylation of all three I{kappa}B subunits (-{alpha}, -{beta}, and -{epsilon}; Refs. 9, 23). IKK-{beta} itself undergoes phosphorylation that appears to be mediated through trans-autophosphorylation brought about by induced proximity (13). Although not essential for IKK-{beta} activation by TNF-{alpha} (38), NF-{kappa}B-inducing kinase (NIK) can also activate IKK-{beta} and NF-{kappa}B.

Although inhibition of NF-{kappa}B may appear to be a logical therapeutic target in inflammatory diseases, NF-{kappa}B also drives expression of survival factors such as inhibitors of apoptosis (10, 25, 29, 36). In CMs, downstream inhibition of NF-{kappa}B through expression of either a transdominant I{kappa}B or dominant-negative IKK-{beta} potentiates apoptosis (8, 25), thereby raising concerns that therapeutic strategies directed at NF-{kappa}B inhibition could have adverse effects on heart (11).

The adaptor protein Fas-associated death-domain protein (FADD), which was originally described for its ability to link death receptors to caspase activation (4, 5), can also activate NF-{kappa}B in a variety of cell types including HeLa and human embryonic kidney (HEK)-293 cells and can enhance production of proinflammatory cytokines such as monocyte chemoattractant protein-1 and IL-8 in rat vascular smooth muscle cells (3, 15, 28, 35). We recently found (2) that FADD plays an important role in CM apoptosis. Expression of FADD was sufficient to induce CM apoptosis, whereas expression of a truncation mutant that lacks the death-effector domain (FADD-DED) provided remarkably effective protection against hypoxia- and serum-deprivation-induced apoptosis. Because of the potency of these effects, we wondered whether FADD-DED-mediated activation of NF-{kappa}B might contribute to these observations even though some prior studies suggest that FADD-mediated NF-{kappa}B activation requires the DED (17, 27, 32).

Surprisingly, we found that expression of both FADD and to a lesser extent FADD-DED actually inhibited NF-{kappa}B activation in rat neonatal CMs. The level at which FADD inhibits NF-{kappa}B signaling was investigated in more detail. The regulatory effect of FADD on NF-{kappa}B signaling is cell-type specific and is likely achieved by inhibition of phosphorylation and activation of IKK-{beta}.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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Materials. Neonatal CMs were isolated using the Worthington Biochemical (Lakewood, NJ) system. Rat and human recombinant TNF-{alpha} were from R&D Systems (Minneapolis, MN). All protease inhibitors were purchased from Calbiochem (La Jolla, CA). Polyclonal and monoclonal (clone 10A9B6) anti-IKK-{beta} antibodies were purchased from Cell Signaling Technology (Beverly, MA) and from Active Motif (Carlsbad, CA), respectively. Antibodies to I{kappa}B-{alpha}, phospho-I{kappa}B-{alpha}, and phospho-IKK-{beta} were from Cell Signaling. Alexa Fluor 568-labeled anti-rabbit IgG was from Molecular Probes (catalog no. A11011; Eugene, OR). Antibodies against FADD (H-181), p65 NF-{kappa}B, Oct-1, and NIK were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and {beta}-actin monoclonal antibody was from Sigma.

Cell cultures. HEK-293 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% horse serum and 5% fetal bovine serum. CMs were prepared from 1–2-day-old rats by digestion using trypsin and collagenase as described previously (21). All CMs were incubated in RPMI 1640 that contained 5% FBS and 10% horse serum. Rat pulmonary artery smooth muscle cells (rPASMCs; generously provided by Dr. Kenneth Bloch’s laboratory) were prepared and incubated in RPMI 1640 that contained 5% FBS and 10% horse serum, penicillin, and streptomycin (30). The rPASMCs were used between passages 3 and 10. Human umbilical vein endothelial cells (HUVECs) were isolated and cultured in medium 199 with 20% fetal bovine serum, endothelial cell growth factor, porcine intestinal heparin (50 µg/ml), and antibodies (12). For gene transfer, cells were either left uninfected or were infected with the indicated adenoviral constructs overnight in regular culture media. Transgene expression was confirmed by immunoblotting. For TNF-{alpha} and LPS stimulation, cells were serum deprived for 2 h before treatments.

Preparation of nuclear and cytosolic extracts. CMs (2 to 3 x 106 cells) in 60-mm dishes were washed once with cold PBS, scraped, and transferred to 15-ml Falcon tubes. Nuclear and cytosolic fractions were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Kit from Pierce (Rockford, IL) as described in the manufacturer’s manual. CER I cytoplasmic extraction reagent (100 µl) was added to each sample, and 50 µl of ice-cold NER nuclear extraction reagent was added to each nuclear fraction.

Electrophoretic mobility shift assays. Cells were harvested, and nuclear extracts were prepared as described above. Unless stated otherwise, aliquots of the nuclear extracts (8 µg) were incubated with 0.5 ng of a radiolabeled double-stranded oligonucleotide that contained the NF-{kappa}B-binding sequence (Active Motif). In some reactions such as samples from rat CMs, 1.0 ng of mutated NF-{kappa}B oligonucleotide was included to block nonspecific binding. Nuclear proteins and oligonucleotides were then separated with native PAGE and detected by autoradiography.

Immunoblotting. Protein samples were separated by 7.5% SDS-PAGE or 4–20% gradient PAGE (Bio-Rad) and transferred to nitrocellulose membranes as described previously (2). Blocking buffer that contained 5% nonfat milk in Tris-balance solution with 0.1% Tween 20 was used to minimize nonspecific binding. The membrane was then blotted with the indicated antibodies (1:1,000–1:500 dilution) at 4°C overnight. For repeated blotting, nitrocellulose membranes were stripped with Restore Western blot stripping buffer (Pierce) at room temperature for 30 min.

Quantification of immunoblotting and electrophoretic mobility shift assay data. Films were scanned and quantified using the NIH Image program. The density of each band was calculated and normalized as the percent of all bands in each group of study. For simplicity, each data point was expressed in arbitrary densitometric units derived from the percent of density multiplied by 1/10. Data from three to five separate experiments were combined and analyzed for statistical significance.

Immunocytochemistry. For confocal microscopy of p65, rat CMs were prepared and purified using Percoll density-gradient centrifugation and were incubated in 35-mm dishes at low density. CMs were infected with Ad.GFP or Ad.FADD and treated with TNF-{alpha} as described (see Cell culture). Cells were fixed and permeabilized using a Cytostaining Kit from BD PharMingen (San Diego, CA). The p65 was stained with a polyclonal antibody (1:100 dilution; Santa Cruz) overnight at 4°C and with Alexa Fluor 546-labeled anti-rabbit IgG (1:2,000 dilution) for 1 h at room temperature.

RNA extraction and purification. Cell RNA was extracted from cultured rat CMs using TRIzol reagent (Invitrogen-Life Technologies). RNA was further purified using an RNase Mini Kit (Qiagen), eluted in 50–100 µl of H2O, and quantified using RiboGreen reagent as described in the manufacturer’s instructions (Molecular Probes). RNA (5–10 µg) was treated with DNase and stored at –80°C for quantitative (q)RT-PCR analysis.

qRT-PCR analysis. Purified RNA was quantified using RiboGreen reagent as described in the manufacturer’s instructions. qRT-PCR analysis was performed and analyzed as described previously (7, 26). RNA (90–180 ng) from each sample was utilized for qRT-PCR analysis (Stratagene). For each PCR run, a separate pair of GAPDH primers was used as an internal control to ensure equal RNA loading. RT-PCR primers were as follows: rat GAPDH: forward, 5'-ATGCCATCACTGCCACTCAG-3' and reverse, 5'-CAGGGATGATGTTCTGGGCT-3'; rat VCAM-1: forward, 5'-GAAGCCGGTCATGGTCAAGT-3' and reverse, 5'-GACGGTCACCCTTGAACAGTTC-3'.

Adenoviral vectors. Adenoviral vectors that encode dominant-negative IKK-{beta} (Ad.dnIKK{beta}), wild-type FADD (Ad.FADD) or the truncation mutant that lacks DED (Ad.FADD-DED) in addition to independent expression cassettes for enhanced green fluorescent protein (GFP) have been described previously in detail (2, 22). Ad.GFP is structurally similar to Ad.FADD but encodes {beta}-galactosidase and enhanced GFP. Ad.NIK and Ad.IKK{beta} were constructed in a similar manner to Ad.FADD and encode wild-type forms of these kinases. Viral titers were determined by plaque assay in HEK-293 cells. Stock titers were >1010 plaque-forming units (PFU) per milliliter for each vector with a 10:100 particle-to-PFU ratio. Wild-type adenovirus contamination was excluded by the absence of PCR-detectable E1 sequences.

Statistical analysis. All quantitative data are expressed as means ± SD of at least three independent experiments and were analyzed using a two-tailed, unpaired Student’s t-test. The null hypothesis was rejected for P < 0.05.


   RESULTS
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 RESULTS
DISCUSSION
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FADD inhibits NF-{kappa}B activation in TNF-{alpha}-stimulated rat neonatal CMs. To examine the effects of FADD on NF-{kappa}B activation, CMs were transduced with Ad.FADD or the control virus Ad.GFP. As seen in Fig. 1 (A-C), TNF-{alpha} induced nuclear translocation of p65 NF-{kappa}B as demonstrated by immunoblotting and confocal microscopy. Adenoviral gene transfer of FADD had no effect on the basal level of nuclear p65 compared with the GFP control. However, expression of FADD dramatically inhibited TNF-{alpha}-induced p65 nuclear translocation compared with control virus-infected CMs. Similarly, NF-{kappa}B DNA-binding activity was inhibited by both the wild type and DED mutant FADD constructs (Fig. 1, D and E). To examine the consequences of these observations on transcription, we examined mRNA for VCAM-1, which is an NF-{kappa}B-dependent proinflammatory transcript, by qRT-PCR (Fig. 1, F and G). In control virus-infected CMs, TNF-{alpha} induced a 19 ± 4.1-fold increase in VCAM-1 mRNA by 2 h after stimulation (Fig. 1G). FADD expression significantly reduced TNF-{alpha}-induced VCAM-1 expression (P < 0.01). Although FADD-DED exhibited a similar trend toward inhibition of VCAM-1, this did not achieve statistical significance (P = 0.08). Together, these data suggest that FADD expression inhibits TNF-{alpha}-induced activation of NF-{kappa}B in rat neonatal CMs without affecting baseline NF-{kappa}B activity.




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  		Fig. 1. Adenoviral expression of Fas-associated death-domain protein (FADD) inhibits TNF-{alpha}-induced NF-{kappa}B activation in rat neonatal cardiomyocytes (CMs). CMs were infected with the indicated viral constructs overnight. Cells were then serum deprived for 2 h before stimulation with TNF-{alpha} (50 ng/ml). Cells were treated for 30 min in NF-{kappa}B nuclear translocation and gel-shift experiments or 2 h for VCAM-1 mRNA production. A: Western blot analysis of p65 NF-{kappa}B nuclear translocation. Nuclear proteins were extracted, separated by electrophoresis, transferred to nitrocellulose membrane, and blotted with polyclonal anti-p65 antibody. Same membrane was blotted for Oct-1 to verify equal loading of nuclear proteins. FADD expression was confirmed in cytosolic fraction (middle). B: nuclear p65 protein was quantified using the NIH Image program; *P < 0.001 vs. Ad.GFP control; **P < 0.01 vs. Ad.GFP-infected CMs treated with TNF-{alpha}; n = 4. C: a representative image of confocal microscopy of p65 localization (red) in cultured rat CMs from one of four independent experiments. Immunostaining of p65 NF-{kappa}B was visualized by confocal microscopy in Ad.FADD and control virus-infected CMs with or without TNF-{alpha} stimulation. Green fluorescent protein (GFP), green. Merging of GFP and p65 staining images is shown (right). Scale, 20 µm. D: electrophoretic mobility shift assay (EMSA) of p65 NF-{kappa}B binding activity. Nuclear extracts from cells infected with the indicated virus with or without TNF-{alpha} stimulation were assayed for NF-{kappa}B binding activity. E: quantitative analysis of EMSA results; *P < 0.05; **P < 0.01; n = 3. F and G: quantitative RT-PCR analysis of VCAM-1 mRNA in CMs. Cells were infected and treated as described. Representative amplification plots are shown (F). Cumulative data (G) show all eight independent experiments with each condition tested five to eight times; *P < 0.01; **P = 0.08. DED, death-effector domain; wt, wild type; IKK-{beta}, inhibitory-{kappa}B kinase-{beta}; dn, dominant-negative.

 
Effects of FADD on NF-{kappa}B are cell-type specific. We next examined the effects of FADD expression on NF-{kappa}B activation in other cell types including HUVECs, rPASMCs, and HEK-293 cells (an immortalized human kidney tumor cell line). In HUVECs, FADD inhibited NF-{kappa}B activation in response to both TNF-{alpha} and LPS (Fig. 2A). Compared with wild-type FADD, FADD-DED exhibited a more modest effect on TNF-{alpha}-induced NF-{kappa}B activation but a comparable inhibition in response to LPS. In contrast, in smooth muscle cells, FADD did not inhibit TNF-{alpha}-mediated NF-{kappa}B activation, although expression of dominant-negative IKK-{beta} did induce inhibition (Fig. 2B). Consistent with this finding, TNF-{alpha} induction of VCAM-1 mRNA was also unaffected in smooth muscle cells by expression of either wild-type FADD or FADD-DED (data not shown). Of note, in both endothelial and smooth muscle cells, neither FADD construct affected the basal level of NF-{kappa}B DNA-binding activity in unstimulated cells. Interestingly, in HEK-293 cells, viral expression of FADD alone was sufficient to increase both nuclear translocation of p65 and NF-{kappa}B DNA-binding activity in the absence of cytokine stimulation (Fig. 2, C and D).



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  		Fig. 2. Effects of FADD expression on NF-{kappa}B DNA-binding activity is cell-type specific. A: human umbilical vein endothelial cells (HUVECs) were incubated with the viral constructs overnight, and transgene expression was confirmed by Western blotting (data not shown). HUVECs expressing GFP, FADD, or wild-type FADD and a truncation mutant that lacks the death-effector domain (FADD-{Delta}DED) were left untreated or were treated with TNF-{alpha} (50 ng/ml) or LPS (100 ng/ml) for 30 min. Nuclear extracts were assayed for NF-{kappa}B binding activity using EMSA; n = 2. B: neither FADD nor FADD-{Delta}DED expression affected NF-{kappa}B activation at baseline or in response to TNF-{alpha} in rat pulmonary artery smooth muscle cells (rPASMCs). In contrast, IKK-{beta}-dn inhibited both baseline and TNF-{alpha}-induced NF-{kappa}B DNA-binding activity levels; n = 2. C and D: HEK-293 cells were left uninfected (lanes 1 and 2) or were infected with GFP or FADD constructs overnight (lanes 3 and 4). FADD expression increased nuclear p65 in a manner comparable to that seen with TNF-{alpha} stimulation (C); n = 2. Consistent with this, FADD increased nuclear NF-{kappa}B DNA-binding activity in the absence of cytokine stimulation (D). In lanes 1 and 2, 7.5 µg of nuclear proteins were used; in lanes 3 and 4, 15 µg of proteins were used.

 
FADD inhibits TNF-{alpha}-induced phosphorylation of I{kappa}B-{alpha}. To explore the mechanisms by which FADD inhibits TNF-{alpha}-mediated NF-{kappa}B activation in CMs, we examined the effects of FADD on I{kappa}B-{alpha} phosphorylation as a critical step in NF-{kappa}B activation. As shown in Fig. 3, TNF-{alpha} induced phosphorylation and slight degradation of I{kappa}B-{alpha} in rat neonatal CMs within 5 min. Adenoviral expression of FADD had no effect on the level of phosopho-I{kappa}B-{alpha} but led to a dramatic decrease in the phospho-I{kappa}B-{alpha} but not total I{kappa}B-{alpha} levels compared with TNF-{alpha}-stimulated GFP-expressing cells (Fig. 3).



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  		Fig. 3. FADD inhibits TNF-{alpha}-induced phosphorylation of I{kappa}B-{alpha} in rat neonatal CMs. Cells were infected with Ad.GFP or Ad.FADD (wild type or DED mutant) overnight, which was followed by serum deprivation for 2 h. TNF-{alpha} induced I{kappa}B-{alpha} phosphorylation that was evident as early as 5 min after stimulation in Ad.GFP-infected CMs. Neither GFP nor FADD expression induced detectable phosphorylation of I{kappa}B-{alpha} in the absence of TNF-{alpha} stimulation. However, FADD expression inhibited TNF-{alpha}-induced I{kappa}B-{alpha} phosphorylation. A and B: representative blots of phospho-I{kappa}B-{alpha}. C: cumulative data from four independent experiments; *P < 0.01 vs. GFP control; **P < 0.01 vs. GFP control treated with TNF-{alpha}.

 
FADD inhibits NIK- and IKK-{beta}-induced NF-{kappa}B activation. To identify the step at which FADD modulates NF-{kappa}B activation, two kinases known to activate the NF-{kappa}B signaling pathway, NIK and IKK-{beta}, were overexpressed in CMs. As expected, expression of either NIK or IKK-{beta} was sufficient to induce phosphorylation of I{kappa}B-{alpha} (Fig. 4, A and B). Coexpression of FADD inhibited I{kappa}B-{alpha} phosphorylation in response to both NIK and IKK-{beta} expression. Although the antibodies to total and phospho-IKK-{beta} did not recognize the endogenous rat IKK-{beta}, they did detect the expressed human IKK-{beta} transgene and demonstrated that FADD inhibited IKK-{beta} phosphorylation in response to IKK-{beta} expression (Fig. 4A). Furthermore, FADD expression significantly reduced NF-{kappa}B binding activity stimulated by expression of NIK and IKK-{beta} (Fig. 4, C and D). FADD-DED also inhibited the stimulatory effect of the kinases but to a much lesser degree.



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  		Fig. 4. FADD inhibits NF-{kappa}B-inducing kinase (NIK)- or IKK-{beta}-mediated NF-{kappa}B activation. Rat neonatal CMs were infected with NIK or IKK-{beta} viruses in combination with either Ad.GFP or Ad.FADD. Cells were serum deprived for 2 h before assays. Phosphorylation of IKK-{beta} and I{kappa}B-{alpha} was detected by immunoblotting and NF-{kappa}B binding activity was assayed using EMSA. A: phosphorylation of I{kappa}B-{alpha}. Note the endogenous rat IKK-{beta} is not recognized by the available antibodies, and thus IKK-{beta} phosphorylation cannot be assessed in NIK-transduced CMs. B: quantification of phospho-I{kappa}B-{alpha}; *P < 0.01 vs. respective GFP control; n = 3–5. C: EMSA of p65 NF-{kappa}B binding activity. D: quantification of EMSA results; *P < 0.01 vs. respective GFP control; n = 3–4.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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The function of FADD as an adaptor molecule in death receptor-initiated, proapoptotic signaling has been well documented (4). Increasing evidence has indicated that FADD may also play an important role in regulating the proinflammatory NF-{kappa}B pathway. FADD has been implicated in both pro- and anti-inflammatory effects in various cells including tumor cells, rat smooth muscle cells, and human vascular endothelial cells (1, 15, 28, 35). Recently it has been demonstrated that Fas-L and FADD play an important role in CM cell death and ischemia-reperfusion-induced myocardia injury (2, 20). The present study was designed to examine the effects of FADD, both the wild type and a mutant that lacks the DED, on TNF-{alpha}-induced NF-{kappa}B activation and NF-{kappa}B-dependent inflammatory gene expression in primary rat CMs and to further explore the mechanisms of this regulatory effect. We found that overexpression of FADD significantly inhibited TNF-{alpha}-NF-{kappa}B signaling as demonstrated by decreased I{kappa}B-{alpha} phosphorylation, decreased NF-{kappa}B nuclear translocation and DNA-binding activity, and downregulation of VCAM-1 mRNA levels. FADD expression also inhibited NF-{kappa}B activation initiated by two signaling molecules, IKK-{beta} and NIK. FADD exhibited a similar inhibitory effect on NF-{kappa}B activity in HUVECs but had no effect in rPASMCs.

Previous reports have demonstrated that overexpression of FADD mediates death receptor-induced activation of NF-{kappa}B in a variety of cell types including Jurkat cells and HEK-293 cells. In rat smooth muscle cells, stable expression of FADD induces production of the NF-{kappa}B-dependent chemokines monocyte chemoattractant protein-1 and IL-8 but only after 4 days of expression (28). Although NF-{kappa}B was not directly examined in this prior study, our results are consistent with these observations in that we found no early activation of NF-{kappa}B after adenoviral expression of FADD in rPASMCs for ≤16–24 h. Later time points were not examined in the present study. We also found that FADD activated NF-{kappa}B in the HEK-293 cell line.

One previous study also noted, as we have, an inhibitory effect of FADD on NF-{kappa}B activation in a dermal microvascular endothelial cell line (1). Importantly, this study also documented that NF-{kappa}B activation was enhanced in FADD-null embryonic fibroblasts, thus suggesting the physiological relevance of FADD-mediated NF-{kappa}B inhibition. However, we noted potentially important differences in our studies of primary CMs. In the microvascular endothelial cell line, Bannerman et al. (1) found that FADD inhibited the response to IL-1 and LPS but not TNF-{alpha} and, in fact, the authors use this observation to hypothesize at which level in the signaling cascade (downstream of IL-1 but not TNF) the inhibition could occur. In contrast, in primary CMs, we found that FADD inhibited NF-{kappa}B activation in response to TNF-{alpha}. We are not aware of any previous documentation that FADD could inhibit TNF-induced NF-{kappa}B activation in any cell type. This finding has important implications for efforts to identify the molecular mechanism of the FADD-NF-{kappa}B interaction, since it suggests, in contrast with the study of Bannerman et al. (1), that signaling molecules downstream of the TNF receptor should not be excluded from consideration. Moreover, TNF-{alpha} has been specifically linked to cardiac pathology in a wide range of conditions from ischemic injury (14) to myocarditis (19) and heart failure (18, 33). Thus the observation that FADD (and FADD-DED) inhibit TNF-induced NF-{kappa}B in CMs may have significant practical implications in addition to the clue it provides regarding potential mechanisms.

FADD-DED had a more modest effect on NF-{kappa}B activation, although its consistent tendency was also to inhibit NF-{kappa}B activation. This suggests that the DED domain of FADD is not essential for its inhibitory effect on NF-{kappa}B. Thus we hypothesize that FADD induces CM apoptosis and FADD-DED inhibits it, through modulation of caspase-8 (which directly interacts with the DED), but the observed effects on NF-{kappa}B are independent of this domain and caspase-8. Consistent with this model, the caspase-8 inhibitor IETD.fmk [50 µM, which is sufficient to block caspase-8 activation and CM apoptosis (2)] failed to reverse the effect of FADD on NF-{kappa}B (data not shown). The finding that FADD-DED blocks both apoptosis and NF-{kappa}B activation in CMs is unique. Previous studies suggested that NF-{kappa}B was necessary for CM survival (25). More recently, we found that overexpression of the endogenous NF-{kappa}B inhibitor A20 could inhibit CM NF-{kappa}B activation without potentiating apoptosis but did not promote survival (8). The ability of FADD-DED to simultaneously inhibit apoptosis and NF-{kappa}B suggests it may prove useful in cardiac conditions characterized by coexistent apoptosis and inflammation as are often seen in ischemic injury or heart failure. However, additional studies utilizing expression of FADD-DED are necessary to determine whether these theoretical advantages translate into effective treatment in vivo.

The molecular mechanisms of the effect of FADD on TNF-{alpha}-NF-{kappa}B signaling have not been fully defined, although some useful clues are provided by our data. First, in HUVECs, FADD inhibited NF-{kappa}B activation in response to either TNF-{alpha} or LPS (see Fig. 2A). Although TNF-{alpha} and LPS signaling differ proximately, they both activate and converge at the level of IKK-{beta} (16, 31). Second, FADD expression inhibited IKK-{beta} phosphorylation in response to IKK-{beta} expression, which is thought to occur through autophosphorylation (16). Both of these lines of evidence suggest that FADD regulation of NF-{kappa}B activation may occur at the level of IKK-{beta} activation. However, repeated attempts have thus far failed to identify a direct physical interaction between IKK-{beta} and FADD (data not shown). It is possible, therefore, that FADD acts by interfering with assembly of the IKK-signaling complex or through an intermediary. Both of these possibilities are being actively pursued.

Some limitations of the present study should be noted. First, our findings are based on overexpression of wild-type and mutant FADD constructs and thus may be less informative regarding the physiological role of the endogenous FADD molecule. As noted above, FADD-null embryonic fibroblasts exhibit enhanced NF-{kappa}B activation consistent with a physiological regulatory role (1), but given the cell type-specific signaling observed in our study, these results cannot be directly extrapolated to CMs. Unfortunately, the embryonic lethality of FADD-null mice (37) precludes a comparable experiment in CMs without the development of FADD-conditional knockout mice. Second, these studies were performed in isolated CMs in vitro and therefore cannot address the in vivo role of FADD-NF-{kappa}B interactions.

Nevertheless, these studies present evidence for potentially important cross-talk between FADD signaling and NF-{kappa}B in CMs. Additional delineation of these interactions may help elucidate mechanisms simultaneously modulating CM survival and inflammatory signaling while laying foundation for novel approaches to intervention in a variety of cardiac conditions.


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This work was supported in part by grants from the Foundation for Anesthesia Education and Research (FAER) and the National Institutes of Health Grants HL-04336 (to W. Chao) and HL-59521 (to A. Rosenzweig).


   ACKNOWLEDGMENTS
 
The authors thank Dr. Kenneth Bloch (Massachusetts General Hospital) for providing rat pulmonary artery smooth muscle cells, and also thank Dr. Michael A. Gimbrone, Jr. and Ms. Kay Case (Vascular Research Division, Brigham and Women’s Hospital) for providing human umbilical vein endothelial cells.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Rosenzweig, MGH-EAST, 114 16th St., Rm. 2600, Charlestown, MA 02129 (E-mail: arosenzweig{at}partners.org)

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.

* W. Chao and Y. Shen contributed equally to this study. Back


   REFERENCES
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  1. Bannerman DD, Tupper JC, Kelly JD, Winn RK, and Harlan JM. The Fas-associated death domain protein suppresses activation of NF-kappa B by LPS and IL-1 beta. J Clin Invest 109: 419–425, 2002.[CrossRef][ISI][Medline]
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