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B, NF-AT, and Early Growth Factors 2/31
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Department of Biological Sciences, Rutgers University, Newark, NJ 07102; and
Departamento Biologia Celular, Facultad de Biologia, Universidad Complutense, Madrid, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B, NF-ATp, and early growth factors (Egr)
2/3. The inhibition of NF-B binding is due to the stabilization of
I-B (inhibitory protein that dissociates from NF-B), through the
inhibition of I-B kinase 
activity. Subsequently, p65 nuclear
translocation is significantly reduced. The inhibition in NF-ATp
binding results from a calcineurin-independent reduction in NF-ATp
nuclear translocation. VIP/PACAP inhibit the expression of Egr2 and 3,
but not of Egr1. The effects on the transcriptional factors are
mediated through type 2 VIP receptor with cAMP as secondary
messenger. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Apoptosis of activated T cells represents an important mechanism for the maintenance of immune homeostasis, and is mediated primarily through Fas ligand (FasL)/Fas interactions (reviewed in Ref. 14). In contrast to Fas, which is constitutively expressed in T cells, FasL expression is induced following activation. Engagement of Fas by FasL leads to Fas oligomerization and generation of apoptotic signals in the Fas-bearing targets (reviewed in Ref. 15). The inhibition of AICD in peripheral T cells by VIP and PACAP is mediated through the down-regulation of FasL expression (13). The purpose of the present study was to investigate the molecular mechanisms by which VIP and PACAP control FasL expression in T cells.
Expression of FasL following TCR stimulation involves various
transcriptional factors. Several reports indicated that c-Myc
(16, 17, 18), NF-AT (19, 20), and NF-B
(21, 22, 23, 24) are essential for FasL expression and subsequent
apoptosis in activated T cells. In addition, it has been reported that
early growth factors (Egr) 2 and 3, two members of the early
growth-response family of transcriptional factors, are required for
optimal FasL gene transcription (25, 26).
In the present study, we determined that VIP and PACAP inhibit
TCR-stimulated FasL expression and apoptosis of 2B4.11 T cell hybridoma
through specific receptors and induction of intracellular cAMP. VIP and
PACAP down-regulate c-Myc synthesis, the NF-AT-dependent Egr2 and Egr3
expresion, and the nuclear translocation and DNA binding of NF-AT and
NF-B.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Synthetic VIP and PACAP38 were purchased from Novabiochem
(Laufelfingen, Switzerland). The PACAP receptor (PAC1)/type 2 VIP
receptor (VPAC2) antagonist PACAP6–38 was
obtained from Peninsula Laboratories (Belmont, CA). mAbs to murine FasL
(CD95L, MFL3), and CD3
(145-2C11) were purchased from PharMingen
(San Diego, CA). Calphostin C, PMA, ionomycin, MTT, cyclosporin A
(CsA), protease inhibitors, dibutyryl cAMP (dbcAMP), and forskolin were
purchased from Sigma (St. Louis, MO), and
N-[2-(p-bromocinnamyl-amino)ethyl]-5-iso-quinolinesulfonamide
(H89) from ICN Pharmaceuticals (Costa Mesa, CA). rI-B (inhibitory
protein that dissociates from NF-B) 1–317(1–317)-tagged fusion protein
and Abs against c-Myc, p65, p50, I-B, I-B-kinase (IKK),
phosphorylated I-B, NF-ATp, Egr1, Egr2, and Egr3 were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA). The VPAC1 antagonist
[Ac-His1,
D-Phe2,
K15, R16,
L27] VIP (3, 4, 5, 6, 7)-GRF (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27) and the VPAC1
agonist [K15, R16,
L27] VIP (1, 2, 3, 4, 5, 6, 7)-GRF (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27) were kindly donated
by Dr. Patrick Robberecht (Universite Libre de Bruxelles, Brussels,
Belgium). The VPAC2 agonist Ro 25-1553 Ac-[Glu8,
Lys12, Nle17,
Ala19, Asp25,
Leu26, Lys27, 28,
Gly29,30, Thr31]-VIP cyclo
(21, 22, 23, 24, 25) was a generous gift from Drs. Ann Welton and
David R. Bolin (Hoffmann-La Roche, Nutley, NJ). The synthetic PAC1
agonist maxadilan was a generous gift from Dr. Ethan A. Lerner
(Massachusetts General Hospital, Charlestown, MA). The VPAC1, VPAC2,
and PAC1 agonists and the VPAC1 antagonist have been previously
described (13).
Cell lines
The murine T cell hybridoma 2B4.11 has been described previously (27, 28). L1210 (a leukemia cell line) and L1210-Fas+ (L1210 cells transfected with fas) cells were kindly provided by Dr. P. Golstein (Center d’Immunologie Institut National de la Santé et de la Recherche Médicale-Centre National de la Recherche Scientifique, Marseille, France) (29). All cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated FCS (Life Technologies), containing 10 mM HEPES buffer, 1 mM pyruvate, 0.1 M nonessential amino acids, 2 mM glutamine, 50 mM 2-ME, 100 U/ml penicillin, and 10 µg/ml streptomycin (complete medium).
Induction of apoptosis
For induction of apoptosis, 2B4.11 cells (5 x 105/ml) were cultured in 96-well plates (Corning Glass, Corning, NY) with immobilized anti-CD3 mAb (1 µg/ml), or with PMA (10 ng/ml) and ionomycin (1 µg/ml), in the presence or absence of VIP, PACAP, VIP/PACAP receptor antagonists, and other agents, as described in text. Apoptosis was determined at 24 h, as described below.
Assessment of cell viability and apoptosis
Cell viability was assessed by trypan blue exclusion and loss of mitochondrial function with the MTT staining method. For MTT staining, 100 µl of culture was placed in one well of a 96-well tissue culture plate, and 10 µl of MTT solution (2.5 mg/ml) was added. After incubation at 37°C for 4 h, 100 µl of acid-isopropanol (0.04 N HCl in isopropanol) was added and mixed by gently pipetting, and the OD 560 nm was assessed with an ELISA reader.
Apoptosis was assessed by TUNEL assay, using a fluorescein in situ cell death detection kit (Boehringer Mannheim, Indianapolis, IN), according to the manufacturer’s instructions. Briefly, at the various time points, 1 x 106 T cells were harvested and fixed in 1% formaldehyde for 15 min on ice, washed with PBS, and stored in 70% ethanol at 4°C. For analysis, the cells were washed and incubated with 50 µl primary buffer (0.2 M potassium cacodylate, 25 mM Tris-HCl, pH 6.6, 2.5 mM cobalt chloride, 0.25 mg/ml BSA, 100 U/ml TdT, and 10 mM biotin-16-dUTP) for 30 min at 37°C. Washed samples were resuspended in 100 µl of secondary buffer (2.5 µg/ml FITC-streptavidin, 0.1% Triton X-100, and 5% nonfat dry milk in 4x saline sodium citrate buffer (Sigma)). After incubation at room temperature in the dark for 30 min, the apoptotic cells were identified as FITC positive by flow cytometry using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA).
DNA fragmentation was assessed by agarose gel electrophoresis. After culture for the indicated times, 5 x 105 cells were harvested and centrifuged at 750 x g for 10 min, resuspended in 100 µl hypotonic lysis buffer A (100 mM Tris-HCl, 40 mM EDTA pH 8, 0.8% sodium lauryl sarcosinate, and 0.5 mg/ml proteinase K), incubated at 50°C for 4 h, followed by the addition of RNase to a final concentration of 0.2 mg/ml. After incubation at 37°C for another 30 min, the resulting DNA fragments were precipitated with 0.5 mM NaCl and 1 vol of isopropanol at -20°C overnight. The samples were centrifuged at 14,000 x g for 20 min at 4°C, and the pellets were washed with 70% ethanol and allowed to dry at room temperature. The DNA resuspended in TE solution (10 mM Tris-HCl, 1 mM EDTA, pH 7.4) was fractionated by agarose gel electrophoresis and visualized under UV light after staining with ethidium bromide.
FACS analysis
The 2B4.11 T cells (1 x 106 cells/ml) stimulated in 96-well plates under conditions indicated in text were harvested in ice-cold RPMI complete medium and washed twice with PBS containing 0.1% sodium azide plus 2% heat-inactivated FCS (wash buffer). The cells were incubated in wash buffer containing 2.5 µg/ml normal mouse Ig for 15 min, followed by anti-FasL (MFL3) mAb (2.5 µg/ml) and incubation at 4°C for 1 h. Isotype-matched mouse Abs were used as controls, and IgG block (Sigma) was used to block the nonspecific binding of the mAb to FcR. The cells were washed and further stained with 2.5 µg/ml of FITC-conjugated goat F(ab')2 anti-hamster IgG (Sigma) for 30 min at 4°C. After extensive washing, cells were fixed in 1% buffered paraformaldehyde. Stained lymphocytes, gated according to scatter characteristics, were analyzed with a FACScan flow cytometer (Becton Dickinson). Fluorescence data are expressed as mean channel fluorescence and as percentage of positive cells after subtraction of background isotype-matched values.
Analysis of functional FasL expression
Activation-induced FasL expression on the anti-CD3-stimulated 2B4.11 T cells was assessed by determining the ability of these cells to cause lysis of Fas+ target cells, as described previously (20). Briefly, 2B4.11 T cells were activated with immobilized anti-CD3 mAbs in the presence or absence of VIP or PACAP (10-8 M) for 8 h to allow FasL expression. After washing twice, the 2B4.11 T cells were resuspended in complete medium, and added to the wells of a 96-well V-bottom microtiter plate (Corning) in graded dilutions to obtain the desired E:T ratio. The target cells, L1210 (wild type) or L1210-Fas+ cells (106 cells/ml), were labeled for 2 h at 37°C with 150 µCi of sodium [51Cr]chromate (Amersham), washed three times with PBS/5% FCS, resuspended in complete medium, and added to the microtiter plates at a concentration of 1 x 104 cells/well. In other experiments, VIP and PACAP were added directly to the cocultures to determine their effects on Fas-mediated lysis. The plates were incubated overnight at 37°C and 5% CO2, then centrifuged, and a 100-µl aliquot of the supernatant was removed for measurement in a Beckman gamma 8000 counter (Beckman, Fullerton, CA). The percent lysis was determined as follows: % lysis = (E - S)/(M - S) x 100, in which E is the release from experimental samples, S is the spontaneous release, and M is the maximum release upon lysis with 10% SDS.
RNA extraction and Northern blot analysis
Northern blot analysis was performed according to standard methods. The 2B4.11 cells were prepared and stimulated as described above. At the various time points, 1 x 107 cells were harvested and total RNA was extracted by the acid guanidinium-phenol-chloroform method, electrophoresed on 1.2% agarose-formaldehyde gels, transferred to S&S Nytran membranes (Schleicher & Schuell, Keene, NJ), and cross-linked to the nylon membrane using UV light.
The probes for murine FasL, c-myc, egr2,
egr3, and GAPDH were generated by RT-PCR, as described
previously, using the following primers: FasL,
5'-TCACCAACCAAAGCCTTAAAGTAT-3' and 5'-TCAACCTCTTCTCCTCCATTAGCA-3';
c-myc, 5'-ACAGAGGGAGTGAGCGGACG-3' and
5'-TTCACGTTGAGGGGCATCG-3'; egr2,
5'-GGTGACCATCTTTCCCAATGCC-3' and
5'-CTCCTGCACAGCCAGAATAAGGA-3'; egr3,
5'-GTCTGACCAACGAGAAGCCCAA-3' and 5'-AGGCCGG CTTGGCCGATTGGTA-3'; and
GAPDH, 5'-TCCTGCACCACCAACTGCTTAG CC-3' and
5'-GTTCAGCTCTTGGATGACCTTGCC-3'. Oligonucleotides were end
labeled with [
-32P]dATP (3000 Ci/mmol;
Amersham, Arlington, IL) by using T4 polynucleotide kinase. The
RNA-containing membranes were prehybridized for 16 h at 42°C,
and hybridized at 60°C for 16 h with the appropriate probes. The
membranes were then washed twice in 2x SSC containing 0.1% SDS at
room temperature (20 min each time), once at 37°C for 20 min, and
once in 0.1x SSC containing 0.1% SDS at 50°C (20 min). The
prehybridization and hybridization buffers were purchased from 5'
Prime
3' Prime (Boulder, CO). The membranes were exposed to x-ray
films (Kodak, Rochester, NY). Signal quantitation was performed in a
PhosphorImager SI (Molecular Dynamics, Sunnyvale, CA).
EMSA
Nuclear extracts were prepared by the mini-extraction procedure of Schreiber et al. (30) with slight modifications. The 2B4.11 cells were cultured at a density of 107 cells in six-well plates, stimulated with anti-CD3 as described above, washed twice with ice-cold PBS/0.1% BSA, and harvested. The cell pellets were homogenized with 0.4 ml of buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 1 mM NaN3). After 15 min on ice, Nonidet P-40 was added to a final 0.5% concentration, the tubes were gently vortexed for 15 s, and nuclei were sedimented and separated from cytosol by centrifugation at 12,000 x g for 40 s. Pelleted nuclei were washed once with 0.2 ml of ice-cold buffer A, and the soluble nuclear proteins were released by adding 0.1 ml of buffer C (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 25% glycerol, 1 mM DTT, 0.5 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 1 mM NaN3). After incubation for 30 min on ice, followed by centrifugation for 10 min at 14,000 rpm at 4°C, the supernatants containing the nuclear proteins were harvested, the protein concentration was determined by the Bradford method, and aliquots were stored at -80°C for later use in EMSAs.
Oligonucleotides corresponding to the proximal NF-B site
(-138/-128, 5'-GAGAAAGGTGTTTCCCTTGAC-3'), NF-AT
(-246/-229, 5'-CTGGGCGGAAACTTCCTG-3'), and FasL
regulatory element (FLRE) motif (-220/-205, 5'-AAGTGAGTGGGTGTTT-3')
of the murine FasL promoter, and to the NF-AT site (-134/-108,
5'-TAATGTTCCATTGTGAGGAGCTTCCAT-3') of the murine egr3
promoter were synthesized and annealed. Aliquots of 50 ng of the
double-stranded oligonucleotides were end labeled with
[-32P]ATP by using T4 polynucleotide kinase.
For EMSAs with hybridoma nuclear extracts, 20,000–50,000 cpm of
double-stranded oligonucleotides, corresponding to 
0.5 ng, were used
for each reaction. The binding reaction mixtures (15 µl) consisted
of: 0.5–1 ng DNA probe, 5 µg nuclear extract, 2 µg
poly(dI-dC)·poly(dI-dC), and binding buffer (50 mM NaCl, 0.2 mM EDTA,
0.5 mM DTT, 5% glycerol, and 10 mM Tris-HCl, pH 7.5). The mixtures
were incubated on ice for 15 min before adding the probe, followed by
another 20 min at room temperature. Samples were loaded onto 4%
nondenaturing polyacrylamide gels and electrophoresed in TGE buffer (50
mM Tris-HCl, pH 7.5, 0.38 M glycine, and 2 mM EDTA) at 100 V, followed
by transfer to Whatman paper, drying under vacuum at 80°C, and
autoradiography. In competition and Ab supershift experiments, the
nuclear extracts were incubated for 15 min at room temperature with the
specific Ab (1 µg) or competing cold oligonucleotide (50-fold excess)
before the addition of the labeled probe.
RT-PCR for the detection of VPAC1, VPAC2, and PAC1 mRNA
Total RNA was isolated from unstimulated and anti-CD3-stimulated 2B4.11 T cells (1 x 107 cells) using the Ultraspec RNA reagent (Biotecx, Houston, TX), as recommended by the manufacturer. Two micrograms of total RNA were reverse transcribed in the presence of 200 U Moloney murine leukemia virus reverse transcription, 40 U RNasin, 1 µg random primers, 0.5 mM dNTPs, 3 µg BSA, and Moloney murine leukemia virus reaction buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2) in a total volume of 30 µl at 37°C for 1 h.
The cDNA were amplified with specific primers. Amplification with
-actin primers was used as a control. The primers for VPAC1, VPAC2,
and PAC1 receptors have been described before (9, 13), and
have the following sequence: VPAC1 sense,
5'-CCTTCTTCTCTGAGCGGAAGTACTT-3', and antisense,
5'-CCTGCACCTCACCATTGAGGAAGCAG-3'; VPAC2 sense,
5'-GTCAAGGACAGCGTGCTCTACTCC-3', and antisense,
5'-CCTTACAATGCTGATGAAGAGGGC-3'; PAC1 sense,
5'-CAAGAAGGAGCAAGCCATGTGC-3', and antisense,
5'-CATCGAGTAATGGGGGAAGGG-3'; -actin sense,
5'-GATGGTGGGTATGGGTCAGGGG-3', and antisense,
5'-GCTCATTGCCGATAGTGATGACCT-3'. The expected sizes for the amplified
fragments are: 450 bp for VPAC1, 572 bp for VPAC2, 317 bp for PAC1, and
660 bp for -actin. Specific fragments were previously amplified with
these primers in macrophages (VPAC1, VPAC2, and PAC1) (9)
and T cells (VPAC1 and VPAC2) (13). Five microliters of
reverse-transcribed cDNA were subjected to PCR in the presence of 0.5 U
of pyrostase, 1 µM sense and antisense primers, 0.2 mM dNTPs, and
polymerase buffer (50 mM Tris-HCl, pH 9, 1.5 mM
MgCl2, 20 mM
(NH4)2SO4,
50 µg/ml BSA). The PCR conditions were: denaturation, 94°C, 45
s; annealing, 55°C, 45 s; primer extension, 72°C, 90 s
for 35 cycles. The PCR products were size separated on 2% agarose gels
and visualized by UV light.
Western blot
Lysates, cytoplasmic fraction, or nuclear extract (see above)
containing 20–30 µg of protein were subjected to reducing SDS-PAGE
(12.5%). After electrophoresis, the gel was electroblotted in
Tris-glycine buffer containing 40% methanol onto a reinforced
nitrocellulose membrane (Schleicher & Schuell). The membrane was
blocked with TBS-T buffer (10 mM Tris, pH 8, 150 mM NaCl, 0.05% Tween
20) containing 5% milk powder for 1 h at room temperature, then
incubated with primary Abs: rabbit anti-mouse IgG against I-B
(1:250), IKK (1:1000), NF-B p50 (1:1000), NF-B p65 (1:1000),
Egr1 (1:1000), Egr2 (1:500), Egr3 (1:500), NF-ATp (1:500), or c-Myc
(1.5 µg/ml), or with mouse IgG against phosphorylated I-B
(1:500), in TBS-T containing 1% milk powder for 2 h at room
temperature. The membrane was washed with TBS-T, and incubated with
secondary Ab: peroxidase-conjugated goat anti-rabbit IgG or rat
anti-mouse IgG at 1/5000 dilution for 1 h at room temperature.
After washing three times in TBS-T for 5 min each, and once in TBS for
5 min, the membrane was dried briefly and subjected to the ECL
detection system (Amersham). The x-ray films were exposed for
5–20 min.
IKK immunoprecipitation and kinase assay
Cell lysates were prepared from 2 x
106 cells in 200 µl lysis buffer (20 mM HEPES,
pH 7.4, 150 mM NaCl, 2 mM EGTA, 50 mM glycerol phosphate, 1% Triton
X-100, 10% glycerol, 1 mM DTT, 2 µg/ml leupeptin, 5 µg/ml
aprotinin, 1 mM PMSF, 5 mM NaF, 10 mM p-nitrophenyl
phosphate, and 1 mM
Na3VO4). The cell lysates
were kept on ice and vigorously vortexed every 5 min for 20 min. The
lysate was cleared by centrifugation at 13,000 x g for
3 min, and the supernatant was stored at -80°C. Endogenous IKK
was immunoprecipitated from 150–250 µg of cell lysate by incubation
with 0.5 µg of anti-IKK Ab for 2 h at 4°C. The immune
complexes were collected by incubation with protein A/G-Sepharose beads
(Sigma) for 45 min at 4°C. The beads were extensively washed with
lysis buffer, twice with LiCl buffer (500 mM LiCl, 100 mM Tris-HCl, pH
7.6, and 0.1% Triton X-100), and twice with kinase buffer (20 mM MOPS,
pH 7.6, 2 mM EGTA, 10 mM MgCl2, 1 mM DTT, 0.1%
Triton X-100, 1 mM p-nitrophenyl phosphate, and 1 mM
Na3VO4). The pelleted beads
were resuspended in 30 µl kinase buffer with 15 µM of ATP, 10 µCi
of [-32P]ATP (3000 Ci/mmol), containing 5
µg of rI-B. The kinase reaction was performed at 30°C for 30
min, and stopped by the addition of 15 µl of 2x SDS sample buffer.
Samples were boiled for 5 min and subjected to 9% SDS-PAGE. Proteins
were transferred onto a nitrocellulose membrane (Schleicher-Schuell),
followed by autoradiography. The IKK kinase activity was determined
by incorporation of 32P into its substrate
(rI-B-tagged fusion protein). The phosphorylated proteins were
quantitated by a PhosphorImager. Expression of the IKK was verified
by immunoblotting of cell lysates, as described above.
Calcineurin assay
The calcineurin (CaN) phosphatase activity was assayed in whole cellular protein extracts by using the Biomol Green Cellular Calcineurin Assay Kit Plus (Biomol Research Laboratories, Plymouth Meeting, PA), according to the manufacturer’s instructions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As previously reported (27, 28, 31, 32), the 2B4.11 T
cell hybridoma undergoes apoptosis following cross-linking of the
TCR/CD3 complex by immobilized anti-CD3 Abs, or following
activation with PMA plus ionomycin (Fig. 1
A). The presence of VIP and
PACAP during TCR stimulation decreases apoptosis at all times assayed
(Fig. 1A). The neuropeptides inhibit the DNA fragmentation
characteristic of TCR-induced apoptosis in a dose-dependent manner
(Fig. 1B). CsA, previously shown to inhibit AICD
(33), was used as positive control (Fig. 1B).
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The immunological actions of VIP and PACAP are exerted through a
family of VIP/PACAP receptors, i.e., VPAC1 and VPAC2, which exhibit
similar affinities for the two neuropeptides, and PAC1, which exhibits
a 300- to 1000-fold higher affinity for PACAP than for VIP
(34). As reported for several T cell lines (13, 35), the 2B4.11 cells express only VPAC2 (Fig. 2A). The involvement of VPAC2
in the effect of VIP/PACAP on AICD was established by using
specific agonists and antagonists. The VPAC2 agonist, but not the VPAC1
or the PAC1 agonist, inhibited anti-CD3-induced apoptosis of 2B4.11
cells (Fig. 2B). In addition,
PACAP6–38, an antagonist specific for PAC1 and
to a lesser degree for VPAC2, but not the specific VPAC1 antagonist
(13), reversed the effects of VIP and PACAP (Fig. 2C). These results indicate that VIP and PACAP exert their
action through VPAC2.
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AICD in T cell hybridomas proceeds via expression of FasL and
subsequent Fas/FasL interaction. We have recently reported that VIP and
PACAP reduce FasL expression in activated peripheral T cells
(13). In this study, 2B4.11 T cells were stimulated with
immobilized anti-CD3 Abs in the presence or absence of VIP or
PACAP, and the expression of FasL was assayed at the protein and mRNA
level by flow cytometry and Northern blot, respectively.
Activation-induced expression of FasL was greatly reduced in a time-
and dose-dependent manner by VIP and PACAP, with an almost complete
inhibition in the concentration range of
10-8–10-6 M (Fig. 3A).
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B, unstimulated). In contrast, 2B4.11 cells treated with
anti-CD3 Abs lysed the Fas+, but not the
Fas- (L1210-wild type) targets (Fig. 3B). VIP and PACAP added at the initiation of anti-CD3
treatment (time 0) inhibit in a dose-dependent fashion the lysis of
L1210-Fas+ cells (Fig. 3B). In
contrast, the addition of VIP or PACAP to cocultures of
L1210-Fas+ target cells and activated 2B4.11
cells (8 h after activation) failed to inhibit lysis in
Fas+ targets (Fig. 3C). This suggests
that VIP and PACAP inhibit AICD by preventing FasL expression
and not by blocking the Fas signaling pathway. cAMP as a second messenger for the inhibition of AICD and FasL expression by VIP and PACAP
VPAC2 induces intracellular cAMP as a secondary messenger
(reviewed in Ref. 36). Therefore, we investigated the role
of cAMP in the inhibition of AICD. First, we determined the effects of
forskolin (a cAMP-inducing agent) and dbcAMP (a cAMP analogue) on FasL
expression and apoptosis in 2B4.11 T cells. Similar to VIP and PACAP,
forskolin and dbcAMP inhibit both FasL expression and apoptosis in
anti-CD3-stimulated 2B4.11 cells (Fig. 4A). The role of cAMP as
second messenger is supported by the fact that the protein kinase A
(PKA) inhibitor H89 reverses in a dose-dependent manner the inhibitory
effects of VIP and PACAP (Fig. 4B). In contrast, calphostin
C, a PKC inhibitor, does not reverse the inhibitory effects of the two
neuropeptides (Fig. 4B). These results suggest that
the inhibitory effects of VIP and PACAP in both AICD and FasL
expression are mediated through increases in intracellular
cAMP.
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FasL expression during AICD requires the activation of several
transcription factors. Previous studies indicated that c-myc
regulates the expression of FasL and subsequent apoptosis in some
systems, including T cells. To determine whether VIP and PACAP regulate
c-myc expression in T cell hybridomas, we analyzed the
expression of c-myc by Northern and Western blots. VIP and
PACAP inhibit anti-CD3-induced c-myc expression at both
protein and mRNA levels (Fig. 5).
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B binding to the FasL promoter
Although the FasL promoter contains a complex array of
transactivating binding sites, NF-B, NF-AT, and the FLRE sites
appear to be essentials for maximal FasL transcription in T cells
stimulated through the TCR/CD3 complex (37). We
investigated whether VIP/PACAP affect NF-B nuclear translocation by
EMSA. Treatment of 2B4.11 cells with anti-CD3 Abs led to strong
NF-B binding, and VIP and PACAP inhibited this binding (Fig. 6A, left panel).
The specificity of the NF-B binding was evident by the complete
displacement with a 50-fold excess of unlabeled homologous
oligonucleotide (NF-B) (Fig. 6A, middle
panels). In contrast, a 50-fold excess of unlabeled nonhomologous
oligonucleotide (NF-AT) had no effect (Fig. 6A, middle
panels). Ab supershift experiments indicated the presence of both
p50 and p65 in the NF-B complex, whereas no supershift was observed
with an irrelevant Ab (anti-NF-ATp) (Fig. 6A,
right panel).
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B and nuclear translocation of the p65 subunit of NF-B
In unstimulated T cells, NF-B is sequestered in the cytoplasm
complexed to I-B (reviewed in Ref. 38). Stimulation
through TCR/CD3 results in the phosphorylation and proteolytic
degradation of I-B, and subsequent translocation of NF-B to the
nucleus. To determine whether VIP and PACAP act on I-B, we
examined the cytoplasmic levels of I-B by Western blot. In
anti-CD3-stimulated cells, the time-dependent I-B degradation
is apparent (Fig. 6, B and C). The decrease in
cytoplasmic I-B levels is paralleled by an increase in I-B
phosphorylation (Fig. 6C, lower panel). Treatment
with VIP or PACAP blocks the activation-mediated phosphorylation and
subsequent degradation of I-B (Fig. 6, B and
C).
Because NF-B activation involves its nuclear translocation, we
measured the levels of p65 protein in cytoplasm and nucleus. As
expected, anti-CD3 treatment decreased the level of p65 in
cytoplasm and increased the p65 levels in the nucleus (Fig. 6B). Treatment with VIP or PACAP abolished the change in
nuclear and cytoplasmic p65 levels (Fig. 6B). This indicates
that VIP and PACAP inhibit the activation-induced nuclear translocation
of p65, which is consistent with the inhibition of I-B
phosphorylation and degradation. Neither anti-CD3 treatment by
itself, nor in combination with VIP/PACAP, affected nuclear and
cytoplasmic p50 levels (Fig. 6B). The equal levels
of p50 also serve as loading controls (Fig. 6B).
Because phosphorylation of I-B by the I-B kinases (IKK) is
crucial for NF-B activation, we investigated whether VIP and PACAP
regulate IKK activity by using an in vitro kinase assay. Stimulation
of 2B4.11 cells with anti-CD3 Abs results in a time-dependent
increase in IKK activity (Fig. 6D). VIP and PACAP inhibit
activation-induced IKK activity (Fig. 6D).
VIP and PACAP inhibit activation-induced nuclear translocation of NF-ATp and its binding to the FasL promoter
NF-AT was reported to play a critical role in FasL
up-regulation following TCR stimulation (19, 22, 29, 33).
Induction of FasL mRNA is prevented by CsA, an immunosuppressive drug
that inhibits CaN activity. CaN dephosphorylates NF-AT and induces its
nuclear translocation. NF-ATp has been directly implicated in FasL
transcription by its binding to a transactivating site in the
FasL promoter (19, 22), and by the fact that
anti-TCR-induced up-regulation of FasL in vivo is impaired in
NF-ATp knockout mice (39). To determine whether VIP and
PACAP regulate NF-ATp binding to the FasL promoter, we used
electromobility shift assays with probe derived from the distal NF-AT
site in the FasL promoter. Stimulation of 2B4.11 cells led to strong
NF-AT binding, and VIP and PACAP reduced this binding, similar to CsA
(Fig. 7A, left
panel). The binding specificity was confirmed by using homologous
(NF-AT) or nonhomologous (NF-B and cAMP response element)
oligonucleotides as competitors (Fig. 7A, middle
panel). The NF-AT complexes were supershifted by an
anti-NF-ATp Ab, but not by anti-p65 Abs (Fig. 7A,
right panel).
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B). As expected, in unstimulated 2B4.11
cells NF-ATp was located preferentially in the cytoplasm (Fig. 7B). However, anti-CD3 stimulation resulted in the
reduction of cytoplasmic NF-ATp, and the increase in nuclear NF-ATp
(Fig. 7B). VIP and PACAP inhibit NF-ATp nuclear
translocation, and block the decrease in cytoplasmic NF-ATp at all
times assayed (Fig. 7B). However, the inhibition of NF-AT
nuclear translocation was not due to an inhibition of CaN, because VIP
and PACAP did not affect CaN activity (Fig. 7C). As
expected, CsA led to a dramatic decrease in CaN activity (Fig. 7C). VIP and PACAP inhibit Egr2 and Egr3 expression and their subsequent binding to the FLRE site of the FasL promoter
Mittelstadt and Ashwell (25, 26) identified a FLRE in
the FasL promoter, which confers the majority of TCR-inducible FasL
promoter activity in 2B4.11 cells and in human T cell blasts. FLRE is a
binding site for Egr1, Egr2, and Egr3, members of the early
growth-response family of transcriptional factors. The effect of VIP
and PACAP on Egr1, 2, and 3 binding to the FLRE site was assessed by
EMSA. As previously described (25, 26), nuclear extracts
from unstimulated 2B4.11 T cells exhibit only a nonspecific band (ns)
(Fig. 8A, lane 1).
In contrast, extracts from anti-CD3-stimulated cells exhibit two
specific bands, a major, lower mobility band (corresponding to Egr1 and
Egr2 binding) (25, 26), and a minor, higher mobility band
(corresponding to Egr3 binding) (25, 26) (Fig. 8A, lane 2). VIP and PACAP, as well as CsA,
prevent the appearance of the minor band, without obvious effects on
the major band (Fig. 8A, lanes 3–5).
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B). No supershift was observed with
anti-NF-ATp (an irrelevant Ab). Following VIP or PACAP treatment,
the minor band disappears, and the major band is supershifted
completely by anti-Egr1 (no supershift is evident for anti-Egr2)
(Fig. 8B). Same results were obtained for CsA treatment
(Fig. 8B). Therefore, the FLRE-binding complexes in
stimulated cells consist of Egr1, 2, and 3, and treatment with VIP or
PACAP leads to complexes consisting only of Erg1.
Next we investigated whether VIP and PACAP regulate Egr1, Egr2, and
Egr3 expression. The 2B4.11 cells were stimulated with immobilized
anti-CD3 Abs in the presence or absence of VIP, PACAP, or CsA, and
expression of the three Egr proteins was detected by Western blot. The
Egr1 protein is constitutively expressed, and neither VIP/PACAP nor CsA
affects its expression. However, Egr2 and Egr3 protein levels were
increased after anti-CD3 stimulation, and VIP, PACAP, and CsA
dramatically reduced both Egr2 and Egr3 (Fig. 8C). The
inhibitory effect on Egr2 and Egr3 was exerted at the mRNA level,
because VIP, PACAP, and CsA reduced both egr2 and
egr3 steady state mRNA levels (Fig. 8C).
Because induction of Egr2 and Egr3 was sensitive to CsA (25, 26) (Fig. 8C), the expression of the egr2
and egr3 might be dependent on NF-AT. In fact, a
transcriptionally active NF-AT site was identified in the
egr3 promoter (31). Therefore, we investigated
whether VIP and PACAP regulate NF-AT binding to the egr3
promoter by using the NF-AT site from the erg3 promoter as a
specific probe. Similar to CsA, VIP and PACAP inhibit the binding to
the egr3/NF-AT site (Fig. 8D, left
panel). The binding specificity was confirmed by using homologous
(NF-AT) or nonhomologous (NF-B) oligonucleotides as competitors
(Fig. 8D, middle panel). Supershift assays
indicate that the complex binding to the egr3/NF-AT site
consists of NF-AT. An irrelevant Ab (anti-p65) was used as control
(Fig. 8D, right panel). These results suggest
that the inhibition of Egr3, and possibly Egr2, by VIP/PACAP is
mediated, at least partially, through the inhibition of NF-AT.
Involvement of VPAC2 and cAMP/PKA in the inhibitory effects of VIP
and PACAP on c-myc, NF-B, NF-AT, and Egr2/3
Because the inhibitory effect of VIP/PACAP on FasL expression and
subsequent AICD in 2B4.11 cells is mediated through VPAC2 and cAMP, we
determined the effects of PACAP6–38, a VPAC2
antagonist, and of the PKA inhibitor H89 on the changes induced by VIP
in c-myc, NF-B, NF-AT, and Egr2/3. The receptor
antagonist and the PKA inhibitor reversed the effect of VIP on
c-myc, and forskolin, a cAMP inducer, mimicked the effect of
VIP (Fig. 9A). Same
conclusions were reached for NF-B binding, p65 and I-B
cytoplasmic levels, and IKK activity (Fig. 9B). In
addition, the receptor antagonist and H89 reversed the effect of VIP,
and forskolin mimicked the effect of VIP on NF-AT binding and NF-AT
levels in nuclear extracts (Fig. 9C). A similar conclusion
was reached for Egr2 and 3 both in supershift experiments and Western
blots (Fig. 9D). These experiments indicate that, similar to
the effects on FasL expression and apoptosis, the inhibitory effects of
VIP on the expression of c-myc, Egr2 and 3, the nuclear
translocation of p65 and NF-ATp, and the IKK kinase activity are
mediated through the VPAC2 receptor and increases in intracellular
cAMP.
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We reported previously that two neuropeptides, VIP and PACAP, protect
peripheral T cells from AICD in vitro and in vivo, through the
inhibition of FasL expression (13). In this study, we
investigated the molecular mechanisms involved in the VIP/PACAP
regulation of FasL expression in T cell hybridomas. We established that
the protective effect against AICD and the inhibition of FasL
expression are mediated through the specific VPAC2 receptor and involve
cAMP as the secondary messenger. Electromobility shift and supershift
assays indicate that VIP/PACAP reduce the specific DNA binding of
several transcriptional factors, i.e., NF-B, NF-AT, and Egr2 and 3.
VIP/PACAP block the nuclear translocation of both NF-B and NF-ATp,
and inhibit the expression of c-myc, Egr2, and Egr3. The
effects of VIP/PACAP were mimicked by forskolin, and reversed by the
PKA inhibitor H89 and by the VPAC2 receptor antagonist, confirming the
involvement of VPAC2 and of intracellular cAMP as mediators for the
regulation of FasL expression by VIP and PACAP (Fig. 10).
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Studies with the FasL promoter have established the presence of several
sites important in transcription, i.e., NF-B (21, 23, 24), NF-AT (19, 20), AP-1 (37), and
Egr2/3 specific binding sites (25, 26). The relative
functional importance of these sites might differ among species. For
example, mutations in the Erg binding site of the human FasL promoter
abolish its activity (26), whereas resulting only in
50–60% reduction for the murine promoter (37). However,
mutations in both the Egr- and the B1-binding sites silence the
murine FasL promoter (37). These results suggest that
NF-B and Egr2/3 are the most important transcriptional factors for
the expression of murine FasL.
Our results indicate that VIP and PACAP reduce NF-B binding to the
B1 site, and that the inhibitory effect is mediated through VPAC2
and intracellular cAMP. The inhibition of NF-B binding is associated
with a reduction in p65 nuclear translocation. p65 is retained in the
cytoplasm due to the stabilization of I-B. As previously
demonstrated for macrophages (44, 45), VIP and PACAP
inhibit I-B phosphorylation. This is accomplished through an
inhibitory effect on the IKK, a kinase specific for I-B. Although
the link between cAMP and IKK is not elucidated, one possibility is
that mitogen-activated protein kinase kinase 1 (MEKK1), which was shown
to synergize with NF-B-inducing kinase in the activation of IKK
and (46, 47), is inhibited by increased levels of
intracellular cAMP. We reported recently that VIP and PACAP inhibit
MEKK1 in macrophages through a cAMP-dependent mechanism
(48). In addition, increases in cAMP lead to cAMP
regulatory element binding protein (CREB) phosphorylation and nuclear
translocation. CREB has a high avidity for the CREB-binding protein
(CBP), a nuclear cofactor required for NF-B transcriptional activity
(49). Because CBP is present in limiting amounts, its
sequestration by CREB leads to a reduction in the transcriptional
activity of NF-B.
Our results indicate that VIP and PACAP also inhibit NF-AT binding, and
NF-ATp translocation to the nucleus, and that the effects on NF-AT are
mediated through VPAC2 and intracellular cAMP. FasL expression was
reported to be sensitive to CsA, and NF-AT sites were indeed identified
in the FasL promoter (19, 20). NF-ATp nuclear
translocation and transcriptional activity depends on its
dephosphorylation by CaN (50). Therefore, CaN represents
one of the potential targets for the inhibitory effect of VIP/PACAP on
NF-ATp translocation. However, in contrast to CsA, VIP and PACAP did
not affect CaN activity. The cAMP-dependent pathway initiated through
VPAC2 by VIP and PACAP could affect NF-AT activity in several ways. A
direct PKA-mediated phosphorylation of NF-AT, which results in a
decrease in transcriptional activity, was reported recently
(51). In addition, previous reports indicated the
generation of cAMP-dependent transcriptional inhibitors for NF-AT
(52). Finally, because both NF-AT and NF-B require the
coactivator CBP for transcriptional activity (53),
sequestration of CBP by CREB following cAMP induction will interfere
with both these transcriptional factors.
Our results are in agreement with Hsu et al. (54), who
indicated that cAMP inhibits FasL expression by suppressing NF-B,
but contradict a previous report by Lee et al. (55), who
reported no changes in NF-AT and NF-B binding by cAMP analogues.
This might be due to the difference in the oligonucleotides used
as probes, i.e., Lee et al. used a human IL-2 enhancer NF-AT
site and an Ig -chain NF-B site, whereas we used the specific
proximal NF-B and NF-AT sites from the murine FasL promoter
(28, 33).
Finally, our results indicate that VIP and PACAP inhibit Erg2 and 3 binding to the FLRE site, and inhibit the expression of the inducible Egr2 and 3 factors at protein and mRNA level. In contrast, Egr1, which is constitutively expressed in the 2B4.11 cells, is not affected by VIP or PACAP. Egr2 and 3 emerged recently as probably the most important transcriptional factors for FasL expression (25, 26). Induction of both Erg2 and 3 is CsA sensitive, suggesting that NF-AT participates in the expression of the erg2/3 genes (26). Therefore, the effect of VIP/PACAP on Egr2/3 synthesis is probably mediated, at least partially, through the reduction in NF-AT translocation. However, at the present time, a direct connection between cAMP-dependent events and Erg2/3 expression cannot be eliminated.
Although neuropeptides such as VIP and PACAP were initially characterized as antiinflammatory agents, a more accurate functional description is as endogenous modulators of the immune response. The inhibition of activation-induced T cell apoptosis through mechanisms described in this study is in agreement with the proposed immunomodulatory role. During an immune response, mechanisms must be in place to allow the survival of a small number of activated T cells and their differentiation into memory cells. Naive T cells, although Fas+, are apoptosis resistant, and upon activation switch to an apoptosis-sensitive phenotype during the proliferative stage (14). Through the down-regulation of FasL expression, VIP and PACAP might favor the local generation of apoptosis-resistant memory T cells. Another physiological relevance for the inhibition of FasL expression by VIP and PACAP is the effect on Fas/FasL-dependent cell-mediated cytotoxicity, especially in organ-specific autoimmune diseases in which Fas/FasL-dependent cytotoxicity against bystander targets contributes to tissue destruction.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Doina Ganea, Rutgers University, Department Biological Sciences, 101 Warren Street, Newark, NJ 07102.
3 Abbreviations used in this paper: VIP, vasoactive intestinal peptide; AICD, activation-induced cell death; CaN, calcineurin; CBP, CREB-binding protein; CREB, cAMP regulatory element binding protein; CsA, cyclosporin A; dbcAMP, dibutyryl cAMP; Egr, early growth factor; FasL, Fas ligand; FLRE, FasL regulatory element; I-B, inhibitory protein that dissociates from NF-B; IKK, I-B kinase; MEKK, mitogen-activated protein kinase kinase; NF-ATp, ??; PAC1, PACAP receptor; PACAP, pituitary adenylate cyclase-activating polypeptide; PKA, protein kinase A; VPAC, VIP receptor.
Received for publication August 7, 2000. Accepted for publication October 23, 2000.
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