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*
Section on Chemical Immunology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892;
Perinatal Research Facility, Department of Obstetrics and Gynecology PHC-3, Georgetown University School of Medicine, Washington, DC 20012; and
Department of Microbiology and Immunology, Baylor College of Medicine, Houston, TX 77030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and TGF-ß. Fc
RI engagement did not further
enhance IL-2 and IL-6 mRNA levels and only slightly enhanced NF-AT
activity, but dramatically increased the mRNA levels of other tested
cytokines. To understand the signal transduction required, we focused
primarily on IL-6 induction by measuring mitogen-activated protein
kinase activity and analyzing the effects of mutant or dominant
negative forms of Vav, Rac1, and c-Jun N-terminal kinase-1 (JNK1). Vav
overexpression resulted in the constitutive activation of JNK1 with
little or no effect on p38 mitogen-activated protein kinase and ERK2.
This was dependent on Vav-mediated activation of Rac1 as a Dbl
domain-mutated Vav, inactive Rac N17, and inactive JNK1 down-regulated
the Vav-induced JNK1 or IL-6 responses. Vav expression, but not
expression of domain-mutated Vav, increased IL-6 secretion from
nonimmortalized bone marrow-derived mast cells upon FcRI engagement.
We conclude that Vav phosphorylation contributes to IL-6 induction in
mast cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tyrosine phosphorylation of Vav in response to engagement of the high
affinity receptor for IgE (FcRI) was reported (11). We
previously demonstrated that a fraction of Vav associates with the
FcRI 
-chain immunoreceptor tyrosine-based activation motif
(12). A more recent study using a chimeric Tac-FcRI
chain construct demonstrated a link between the FcRI -chain and
Vav-dependent activation of JNK1 (7). Other studies
demonstrated the interaction of the Vav SH2 domain with phosphorylated
tyrosines between the C-terminal SH2 domain and the catalytic domain of
Syk (13). However, little is known about what
FcRI-dependent mast cell response(s) may be regulated by
Vav-dependent activation of JNK1. Early studies in T cells demonstrated
that Vav overexpression resulted in increased NF-AT activity and
induced the response of an IL-2 reporter construct in T cells
(14). Later studies showed that Vav and the adapter
molecule SLP-76 were synergistically coupled in IL-2 production
(15). Thus, in the present study we explored whether Vav
could also modulate cytokine production in mast cells.
What signaling pathways regulate cytokine production in mast
cells is not clearly understood. An apparent species or developmental
difference may exist, which has led to reports of JNK activity as
important to TNF- production in MC/9 murine mast cells
(16), while ERK2 regulates this cytokine in RBL cells
(17). In addition the activation of JNK in mast cells can
be mediated by PKC-dependent and -independent pathways, with the latter
being initiated by two distinct effectors, namely SEK1 and MKK7
(18). In the present study we took advantage of the
observation that overexpression of Vav in the RBL cell line resulted in
its tyrosine phosphorylation in the absence of additional stimuli to
analyze its role in the absence of the activation of multiple signaling
pathways by FcRI engagement. We also tested the effects of FcRI
engagement on Vav-initiated signals. Finally, we examined whether the
Vav-mediated induction of IL-6 observed in RBL cells could also be
observed in nonimmortalized bone marrow-derived mast cells (BMMC).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The enzymes used in these studies were purchased from either New
England Biolabs (Beverly, MA) or Life Technologies (Gaithersburg,
MD). Deoxyadenosine
5'--[35S]thiotrisphosphate, deoxyadenosine
5'--[32P]trisphosphate,
L-[35S]cysteine, and enhanced
chemiluminescence reagents were purchased from Amersham (Arlington
Heights, IL). The PRISM Ready Reaction DyeDeoxy Terminator Cycle
Sequencing Kit was purchased from Applied Biosystems (Foster City, CA).
Piceatannol was purchased from Calbiochem (San Diego, CA). All
other chemicals were purchased from Sigma (St. Louis, MO).
Anti-dinitrophenyl-specific murine IgE was obtained and purified as
previously described (19, 20), and dinitrophenylated
(DNP)-human serum albumin (HSA) was purchased from Sigma. A rabbit
polyclonal Ab and a mouse mAb to Vav were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA) and Upstate Biotechnology (Lake Placid,
NY), respectively. Goat and rabbit polyclonal Abs to JNK1 were also
purchased from Santa Cruz Biotechnology. A mouse mAb to phosphorylated
JNK was purchased from New England Biolabs (Beverly, MA). Rabbit IgG
was purchased from Jackson ImmunoResearch Laboratories (West Point,
PA). Rac1 N17 and Rac1 V12 were gifts from Marc Symons (Onyx
Pharmaceuticals, Richmond, CA). Ras N17 and Ras V12 were provided by
Deborah Morrison (National Cancer Institute, National Institutes of
Health). The nonactivatable JNK1 (APF) and the wild-type JNK1 were
described previously (21). The previously described NF-AT
(7x) (22) luciferase reporter was provided by Dr. K.-I.
Arai (Institute of Medical Sciences, University of Tokyo, Tokyo,
Japan). Catalytically inactive Btk (K430R) was a gift from T. Kawakami
(La Jolla Institute of Allergy and Immunology, La Jolla, CA).
Library construction, probe generation, cloning, and expression of rat Vav
A cDNA plasmid library of RBL-2H3 cells was custom prepared by
Clontech (Palo Alto, CA) according to a modified Gubler and Hoffman
procedure (23). This cDNA library was generated from
methylmercuric hydroxide-denatured mRNA, which was 5'-stretched, and
oligo(dT) and random primed. The library generated was directly cloned
into the BstXI site of the plasmid vector pCDM8. The probe
specific for Vav was generated by PCR from a cDNA library of RBL-2H3
made by RT-PCR of the mRNA using oligo(dT) priming. Primers used in the
amplification reaction were of the nucleotide sequence of Vav conserved
between human and mouse. The 5'- primer was
5'-GTGAGAAGTTCGGCCTCAAGC-3', and the 3'-primer was
5'-ACCGAGGTGAAGAACAACAGAGC-3', which generated a fragment of 1160 bp
starting at bp 261 and ending at bp 1419 of the mouse sequence with
98% identity. A 600-bp fragment of the 5' end of the PCR-derived rat
Vav fragment was used as a probe for screening the cDNA library. Five
independent clones were isolated, the longest of which was 4.1 kb. All
clones were partially sequenced and were identical for the regions
sequenced in the open reading frame (ORF). One clone was sequenced to
completion and was found to be missing 28 bp at the 5' end of the ORF
compared with the mouse sequence. Analysis of the remaining clones
revealed the absence of an ATG that would result in an ORF for a 95-kDa
product of Vav. To obtain the 5' sequence, a rapid amplification of
5'-cDNA ends was performed on an RT-PCR single-stranded cDNA library
using a 3' clone-specific oligonucleotide and a 5' random priming
oligonucleotide. The product obtained was subcloned into the sequenced
clone, generating a clone of 2858 bp. The nucleotide sequence was
determined and showed a complete ORF and a 5'-untranslated sequence of
56 bp.4 The translation of the ORF (with 94% identity to
murine Vav) resulted in a protein of 843 aa with a net charge of -9, a
predicted isoelectric point of 6.51, and a predicted molecular mass of
98 kDa. Expression of the rat Vav cDNA encoding the ORF in CHO cells
resulted in expression of a protein with an apparent molecular mass of
105 kDa.
Sequencing
Sequencing was performed using the PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit and the 373A DNA Sequencing System from Perkin-Elmer/Applied Biosystems (Foster City, CA) as described by the manufacturer. Plus and minus strands were sequenced with a minimum of two sequence reactions per primer. Analysis of the acquired data was performed using the GCG Sequence Analysis Package and Sequencer (Genetics Computer Group, Madison, WI).
Cell culture and transfectants
The rat basophilic leukemia cell line (RBL-2H3) was cultured
essentially as previously described (24). The hamster
kidney (BHK) and Chinese hamster ovary (CHO) cell lines was cultured as
described by American Type Culture Collection (Manassas, VA).
Transfection of plasmids carrying the complete cDNA of Vav, the mutant
cDNA of Vav, NF-AT-luc, and the IL-6 promoter-luc were in CHO or
RBL-2H3. Stable transfectants of Vav were previously described
(25), using the MTH vector (26). For
transient protein expression we used a viral expression system based on
the Semliki Forest virus (SFV) and described briefly below and in
detail previously (27).
SFV expression of Vav, Rac, Ras, and JNK1 constructs
Vav, Rac1 N17, Rac1 V12, Ras N17, Ras V12, JNK1(APF), and JNK1 were subcloned in the pSFV1 or pSFV1-green fluorescent protein (GFP) vectors, which were modified as previously described (27). The production of recombinant SFV infectious particles and the determination of titer were performed according to the procedure previously described (28). For transient protein expression by SFV infection, adherent cells (RBL cells) were plated in six-well plates or in 100- x 20-mm tissue culture dishes for RT-PCR or in-gel kinase assays, respectively. Adherent cells were washed, infected by addition of activated virus in serum-free medium in the presence of 7.5% polyethylene glycol, and incubated at 37°C for 30 min as previously described (27). Nonadherent cells (BMMC) were recovered by centrifugation of exponential growth cultures at 1000 rpm for 10 min, washed once in PBS, and resuspended in serum-free medium containing activated virus followed by rapid addition of polyethylene glycol. Following incubation of the nonadherent cells with activated virus as described above, the cells were washed once in serum-free medium to remove polyethylene glycol and excess virus, and growth medium was added. Cells were allowed to incubate for 4 h at 37°C, and expression of the recombinant proteins was determined using a FACScan cytometer (when tagged with GFP) or by Western blot.
Detection of phosphorylated JNK1 and in-gel kinase assays
Transfected cells grown in culture dishes and sensitized with IgE were rid of unbound IgE by two washes with growth medium. Subsequently, growth medium containing DNP-HSA (100 ng/ml) was added, and cells were further incubated for 8 min. After stimulation, DNP-caproic acid (25 µM/ml) in ice-cold PBS (pH 7.4) was added to stop further receptor engagement. Cells were then harvested with a cell lifter (Costar, Cambridge, MA) and lysed with 1% Nonidet P-40 lysis buffer as previously described (12). Soluble lysates was equally divided in three for immunoprecipitations with goat anti-JNK1, rabbit anti-p38 MAPK, and rabbit anti-ERK2 Abs as previously described (12). For detection of phosphorylated JNK1 the recovered proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed with Ab specific to phosphorylated JNK1.
In-gel kinase assays were performed essentially as described by
Kameshita et al. (29) with a minor modification to the
kinase buffer. Briefly, GST-ATF2 (300 µg/ml) was used as a specific
substrate for JNK1 or p38 MAPK, and MBP (0.5 mg/ml) was used as a
substrate for ERK2. After denaturation of the gel with 6 M guanidine
HCl and a 16-h renaturation with a buffer containing 50 mM Tris-HCl (pH
8.0), 5 mM 2-ME, and 0.04% Tween 40 at 4°C, the gel was equilibrated
with 20 ml of kinase buffer containing 40 mM HEPES (pH 7.5), 2 mM DTT,
0.1 mM EDTA, and 5 mM MgCl2 for 30 min at 30°C.
The kinase reaction was conducted for 1 h at 30°C with 20 ml of
fresh kinase buffer containing, in addition to the above, 10 µM cold
ATP and 50 µCi of [-32P]ATP. The gels were
then washed extensively with washing buffer (5% TCA plus 1% sodium
pyrophosphate) to remove unincorporated
[-32P]ATP before drying and exposure to
x-ray film.
RT-PCR and cytokine mRNA analysis
A semiquantitative RT-PCR (25) was used to determine the expression of cytokines in cells expressing different constructs. Briefly, transfected cells were washed twice in PBS and solubilized with Tri-Reagent (Molecular Research Center, Cincinnati, OH), and total RNA was then isolated as described in the provided protocol. First-strand cDNA synthesis was performed using reverse transcriptase, 2.5 µg of total RNA, and poly(dT) primer. The cDNA generated was then amplified by PCR using conditions where amplification remained linear and primer sets previously described (25).
Luciferase reporter and cytokine ELISAs
Luciferase reporter assays were previously described (22, 30) with minor modification. Briefly, 36 h posttransfected
cells were IgE sensitized and serum deprived overnight before
engagement of FcRI. Luciferase activity was measured with the dual
luciferase detection kit from Promega (Madison, WI). The response of
2 x 106 cells was assayed in each sample
and was normalized to either the response observed for a control
luciferase vector or determined protein concentrations. For IL-6
secretion assays, IgE-sensitized and GFP-, Vav-GFP-, or Vav
(DH-)-GFP-expressing BMMC cells (1.0 x 107
cells) were grown for 4 h post-SFV infection, and expressing cells
were recovered by FACSorter. The observed efficiency of viral infection
for BMMC ranged from 15 to 40%. The highest infection efficiency was
observed for cultures 4–5 wk of age. The sorted cells were recovered,
washed once in growth medium, resuspended in the same containing
DNP-HSA (100 ng/ml), and further incubated for 1 h at 37°C. The
IL-6 secreted in the medium was measured by an ELISA kit purchased from
Endogen (Woburn, MA) as previously described (31).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Overexpression of the Vav cDNA in RBL-2H3 stable transfectants
gave 4- to 8-fold greater levels of Vav in these cells (data not
shown). Analysis of seven experiments of the tyrosine phosphorylation
profile of total proteins in the Vav-stable transfectants revealed no
significant change compared with control cells, except that Vav was
heavily tyrosine phosphorylated (Fig. 1
A). The level of tyrosine
phosphorylation of Vav was similar to that observed by FcRI
engagement of the vector control transfectant (Fig. 1B). The
ratio of phosphorylated to unphosphorylated Vav was the same in control
and Vav-transfected cells, with 5% of the total Vav phosphorylated
in either transfectant. SFV transient overexpression of Vav showed a
similar increase in the amount of tyrosine-phosphorylated Vav, and the
levels of Vav expression were up to 10-fold greater than the levels of
endogenous Vav (27). To determine whether the basal
activity of Syk might lead to Vav phosphorylation, we
immunoprecipitated Vav from 2.5 x 107
vector- or Vav-transfected cells either untreated or treated with
genistein or piceatannol. The phosphorylation of Vav was inhibited by
the Syk-selective inhibitor, piceatannol (32), but not by
the Src-selective inhibitor, genistein (Fig. 1C), thus
demonstrating a basal Syk activity sufficient for Vav phosphorylation.
This was further confirmed in other studies where Vav phosphorylation
was not seen in Syk-deficient cells
(33).5 In
addition, we explored whether Btk activity could contribute to Vav
phosphorylation in control and Vav-overexpressing cells. Expression of
a catalytically inactive Btk (K430R) had no effect on Vav
phosphorylation in the absence or the presence of FcRI engagement
(data not shown). Because we previously demonstrated that low levels of
Syk activity are found in resting RBL-2H3 cells (34), we
conclude that the increased fraction of phosphorylated Vav results from
the presence of increased substrate for Syk.
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The tyrosine phosphorylation of Vav has been demonstrated to be
critical for its Rac1-directed GEF activity (5). Thus, the
observation of constitutive phosphorylation of Vav by overexpression in
the RBL cells in the absence of FcRI engagement provided a suitable
system to investigate the consequences of Vav activation in mast cell
responses. Because Vav has been described to promote IL-2 production in
T cells (14, 15) we investigated, by RT-PCR, the profile
of cytokine mRNA. We found increased IL-2 and IL-6 mRNA levels in
Vav-overexpressing cells, but little (GM-CSF) or no effect on IL-3,
IL-4, TNF-, and TGF-ß mRNA levels (Fig. 2). In most experiments the increased
levels of IL-2 and IL-6 mRNA were similar to the levels observed in
response to FcRI engagement, although a slight enhancement was
observed upon FcRI engagement (Fig. 2). Because the levels of IL-2
mRNA in RBL cells were not easily detectable, further efforts were
focused on investigating the molecular mechanisms regulating the IL-6
response in these cells.
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Tyrosine-phosphorylated Vav was shown to activate JNK1 in response
to an external stimulus (7). Furthermore, JNK activity has
been implicated in mast cell cytokine production (16).
Therefore, we analyzed whether the constitutive phosphorylation of Vav
induced by its overexpression in RBL cells could directly result in
increased JNK1 activity or whether FcRI engagement is a
prerequisite. Fig. 3A shows
that transient Vav overexpression led to activation of JNK1 with little
effect on p38 MAPK and ERK2 activities. In the control transfectant
(where only GFP is expressed) no stimulation of JNK1 activity was
observed. Engagement of the FcRI in the control (GFP) transfectant
resulted in a greater than 4-fold stimulation of JNK1 activity, while
in Vav-transfected cells <2-fold enhancement of the JNK1 activity was
observed, suggesting that almost complete activation of the kinase is
achieved by Vav overexpression alone (Fig. 3A). Engagement
of the FcRI potently activated both p38 MAPK and ERK2 activities in
the GFP control and Vav-transfected cells (Fig. 3A).
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RI engagement, since we found, by kinetic
analysis, that the level of JNK1 phosphorylation was a direct
reflection of its activity (J. Song and J. Rivera, unpublished
observation). As shown in Fig. 3
B, wortmannin inhibited
(40–50%) the activation of JNK1 in both the control and
Vav-transfected cells. Treatment of the transfected cells with 10–300
nM wortmannin gave similar results, although inhibition ranged from 25
to 50%. These results are consistent with prior studies demonstrating
a 60% inhibition of JNK activation in RBL cells after treatment with
100 nM wortmannin (38). Wortmannin had little effect on
the basal JNK1 activity of the GFP control transfectant before FcRI
engagement. However, the extent of inhibition for GFP control and
Vav-transfected cells was the same following FcRI engagement.
Therefore, basal and stimulated PI-3 kinase activities are required for
the complete activation of JNK1 by Vav, but Vav overexpression does not
overcome the inhibitory effect of wortmannin, demonstrating that PI-3
kinase is upstream of Vav. Role of Rac and Ras in Vav-mediated JNK1 activation
Because Vav overexpression activated JNK1 we investigated the role
of Rac and Ras in this constitutive activation by coexpression of the
inactive forms of Rac1 (N17) and Ras (N17) with Vav. Coexpression of
Ras N17 with Vav or with the control GFP-transfected cells had no
effect on JNK1 activity (Fig. 4,
lanes 1 and 2 vs 3 and 4).
In fact, a slight enhancement of JNK1 activity was observed in several
experiments, including the representative experiment shown. In
contrast, coexpression of Rac1 N17 with Vav or with the GFP control led
to the marked inhibition of JNK1 activity, with almost complete
inhibition of Vav-induced JNK1 activity in some experiments (Fig. 4, lanes 1 and 2 vs 5 and 6).
This suggested the possibility that the active form of Rac1 (Rac1 V12)
would mimic the Vav-mediated induction of JNK1. Expression of Rac1 V12
with GFP resulted in the activation of JNK1, and expression of Rac1 V12
in the presence of Vav enhanced the Vav-induced JNK activity (Fig. 4, lanes 1 and 2 vs 7 and 8).
These results demonstrated that Vav-mediated activation of JNK1 is
Rac1-dependent.
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To assess whether the Vav-dependent activation of JNK1 was a
direct result of Vav activation of Rac1, we tested whether a mutant Vav
could activate JNK1 in the RBL-2H3 cells. The Dbl homology (DH) domain
of Vav is a guanine nucleotide exchange domain with specificity to
Rac1. Deletion of this domain should inhibit JNK1 activation, if Rac1
activity is required for JNK1 activation (8) in these
cells. As shown in Fig. 5A,
the DH-Vav did not show a dominant-negative phenotype, as it did not
inhibit JNK1 activation below the level of the GFP control
(lanes 1 and 3 vs 7 and
9). However, overexpression of DH-Vav did not result in
constitutive activation of JNK1 (lane 7) to the
levels that result from overexpression of wild-type Vav
(lane 4). This suggested that Vav guanine nucleotide
exchange activity is important for JNK1 activation. Thus, one might
expect that coexpression of wild-type Vav with the inactive JNK1 (APF),
expressing a dominant-negative phenotype, should inhibit the
Vav-dependent activation of JNK1 as demonstrated in Fig. 5A
(lane 5). Furthermore, expression of wild-type JNK
enhanced the JNK1 activity of all transfectants (Fig. 5A,
lanes 3, 6, and 9). These results showed that the
guanine nucleotide exchange activity of Vav mediates the activation of
JNK1 in these cells.
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B, overexpression of the DH-Vav led to minimal
increases in the IL-6 mRNA (lane 7) above that seen
in the GFP control transfectant (lane 1). Thus, as
noted above, the inability of the DH-Vav to effectively activate JNK1
also led to a poor IL-6 mRNA response. In contrast, coexpression of
wild-type JNK with either the control GFP or DH-Vav gave a 2-fold IL-6
mRNA response (Fig. 5B, lane 3 vs 9).
Coexpression of wild-type Vav with wild-type JNK caused a 3-fold
enhancement of the IL-6 mRNA response (Fig. 5B, lane
6). Collectively, the findings demonstrated a link of JNK1
activity to IL-6 mRNA accumulation. Thus, as might be expected,
coexpression of GFP, wild-type Vav, and DH-Vav with the mutant JNK1
(APF) effectively inhibited the Vav-induced IL-6 mRNA accumulation
(Fig. 5B, lanes 2, 5, and 8). These
results demonstrated the requirement of JNK1 activity in the Vav
induction of IL-6. Overexpression of Vav-induced NF-AT activity
Vav-induced NF-AT activity was reported in T cells and correlated
with the induction of IL-2 in these cells (14). NF-AT
activity in response to FcRI engagement of mast cells has also been
demonstrated (39, 40). We explored whether Vav
overexpression induced the constitutive activation of NF-AT using an
NF-AT-luciferase reporter construct (22). We used a
suboptimal dose of Ag (1 ng) to assess whether any additional
FcRI-mediated effect of Vav on NF-AT activity might be mediated by
Vav. As shown in Fig. 6, we observed a
significant basal NF-AT activity in the control transfected cells.
Serum deprivation had no significant effect on the basal NF-AT activity
in these cells and had only a minor effect on the stimulated response
(data not shown). FcRI engagement caused a 2-fold enhancement of
NF-AT activity in the control transfectant (Fig. 6, Vector/+Agn). Vav
overexpression caused a constitutive activation of NF-AT activity that
ranged from 4- to 8-fold enhancement of the basal activity (Fig. 6, Vav/-Agn). In addition, FcRI engagement caused a small, but
consistent, increase in the Vav-induced NF-AT response (Fig. 6, Vav/+Agn). A comparison of the FcRI-stimulated control (vector/+Agn)
and Vav-transfected (Vav/+Agn) cell NF-AT response showed a 2- to
4-fold increase in NF-AT activity. Collectively, our results
demonstrated the Vav-mediated constitutive activation of NF-AT activity
(Fig. 6) and suggest that Vav may contribute to NF-AT activation by
more than one signaling pathway, because FcRI stimulation enhanced
Vav-mediated NF-AT activation.
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RI-dependent IL-6 secretion from BMMC
To determine whether Vav enhanced IL-6 production in a
nonimmortalized mast cell, we studied the FcRI-dependent secretion
of IL-6 in BMMC-transfected with wild-type and DH-Vav. For reasons that
are unclear, RBL cells did not secrete detectable levels of IL-6 (above
the background of the available assay), although the protein was
present in these cells (data not shown). Because transient transfection
or viral infection of BMMC is inefficient, cells were sorted using the
GFP tag of the expressed protein to isolate the expressing cell
population. Before stimulation both GFP- and Vav-transfected cells
secreted very low levels of IL-6 in the medium, with the latter
secreting 30–40% more IL-6 under resting conditions. Kinetic analysis
of IL-6 secretion from nontransfected BMMC showed that maximum
secretion occurred at 4 h post-FcRI engagement. Thus, we chose
to work at 1 h post-FcRI engagement because the IL-6 levels
were easily detected, and secretion was at a maximal rate. Using these
conditions we found that the overexpression of Vav resulted in an
FcRI-stimulated increase of as much as 90% in secreted IL-6
compared with the overexpression of GFP alone (Fig. 7). The GFP control transfectants
secreted 550–750 pg/ml/106 cells, while Vav
transfectants secreted 1.0–1.4 ng/ml/106 cells.
Furthermore, the increase in secreted IL-6 required the GEF activity of
Vav, as DH-Vav failed to enhance the levels of secreted IL-6 (Fig. 7).
Thus, we conclude that Vav induction of IL-6 mRNA results in increased
protein production that is secreted upon FcRI engagement.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI
engagement. This model demonstrated a direct link between Vav
activation and IL-6 induction in mast cells (see Fig. 8). Of particular importance is the
finding that Vav phosphorylation is both necessary and sufficient for
increased IL-6 mRNA in the absence of ERK2 and p38 MAPK activation. In
addition Ras V12, which enhanced ERK2 activity in RBL cells (data not
shown), failed to induce an IL-6 response, while Rac1 V12, which
activated JNK1, caused an increase in IL-6 transcripts. This
demonstrates that Vav has the potential to induce IL-6 expression in a
Rac1/JNK1-dependent and Ras/ERK2-independent manner. Nevertheless,
following FcRI engagement other signaling pathways appear to
synergize to activate JNK1, because only a partial inhibition of JNK1
activity by Rac1 N17 or JNK1 (APF) was observed under these conditions.
It is possible that this synergy is mediated by the Ras pathway,
because we previously found that components of the Ras pathway
coimmunoprecipitated with Vav (12), and in kinetic studies
we presently found that Vav overexpression enhances the rate of ERK2
activation after FcRI engagement by Agn (data not shown). This
enhancement was most pronounced when a suboptimal concentration (1 ng)
of Agn was used. These results suggest that Vav GEF activity may not
directly activate the Ras pathway, as previously suggested
(41), but at suboptimal concentrations of Agn Vav may
increase the rate of ERK2 activation by facilitating the formation of a
Vav-containing plasma membrane macromolecular complex (see Footnote 5)
that contributes to Ras and Rac signaling (12, 42, 43).
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, or TGF-ß and
only a slight effect on GM-CSF. This specificity is rather remarkable,
because the Vav-dependent increase in NF-AT activity that we found in
RBL cells should also affect other cytokine promoters
(44). To the best of our knowledge an NF-AT binding site
in the IL-6 promoter has not been described, although it is present in
the IL-2 (22) and IL-4 (44) promoters. Vav
induction of NF-
B activity has been reported (45), and
an NF-B binding site is present in the IL-6 promoter
(30). In preliminary experiments we could not detect
induction of NF-B-luciferase reporter activity in response to Vav
overexpression in RBL cells. However, since a previous study
demonstrated an NF-B-like activity (not comprised of the p50 or Rel
proteins) in RBL cells that is important for TNF- gene expression
(46), it is possible that our reporter construct may not
be activated by this NF-B-like activity, or the promoter insert did
not contain the appropriate binding site. Nevertheless, because we
observed no significant Vav-mediated induction of IL-4, GM-CSF, or
TNF- (genes known to be regulated by NF-AT), this would suggest that
the increased NF-AT, c-Jun (47), or NF-B/NF-B-like
(45, 46) activities cannot solely explain the specific
accumulation of IL-2 and IL-6. Thus, one possible explanation would be
that other unidentified transcription factors are induced by Vav that
provide the required complex for specific gene activation. The recent
findings (48) of two distinct NF-ATc isoforms ( and
ß) in mast cells whose individual expression is either constitutive
() or inducible (ß; by FcRI engagement) would support the
aforementioned hypothesis if Vav activity is also capable of inducing
early response genes.
Recent studies by Y. Kawakami et al. (49) showed that mast
cells from btk-null mice failed to activate JNK to the
levels of normal mast cells. Interestingly, these mice were also
defective in the production of cytokines, and the cytokine responses
could be reconstituted by transfection with Btk (31).
Given that a similar phenotype was observed with overexpression of Vav,
DH-Vav, and downstream effectors, this suggested a possible connection
between Btk and Vav. However, we did not find evidence for a direct
link of Btk activity to Vav function under overexpression conditions
that led to activated JNK1. In fact, when FcRI was engaged on RBL
cells expressing the inactive Btk (K430R), only a minimal
effect on Vav phosphorylation was observed. Neverthe-less, Btk and Vav
may communicate. For example, Btk and Vav activities are regulated by
binding, to their pleckstrin homology domains, of components of the
phosphoinositide pathway (35, 50, 51, 52, 53). Our finding that
wortmannin, at concentrations that effectively inhibit PI-3 kinase
(36), partially inhibited Vav-induced JNK1 activity is
consistent with prior results (38) and suggests that Vav
activity is at least partly responsible for JNK1 activation. A more
complete JNK1 inhibition by the same concentration of wortmannin was
observed in prior studies on MC/9 cells, suggesting that the primary
pathway of JNK1 activation in these cells is PI-3 kinase dependent
(16, 37). It is possible that the sustained activation of
Vav may be dependent on Btk activation and its regulation of other
effectors, such as phospholipase C (52), because in
SLP-76 (an adapter molecule for Vav)-deficient T cells, phosphorylation
of phospholipase C and the calcium response is inhibited
(54), and the latter is also seen in Vav-null T cells
(55, 56). Alternatively, Btk and Vav may be part of a
molecular signaling complex, where, in the absence of one protein, the
function of the others may be affected (54, 57, 58).
Regardless, the present study is most consistent with prior studies
supporting the critical role for Syk activity in Vav phosphorylation
and activation (33, 59). In addition, we recently found
that the presence of Syk is critical not only to Vav phosphorylation
but also to Vav compartmentation and function (see Footnote
5).
The overexpression of the inactive JNK1 (APF), which competes with
wild-type JNK1 (TPY) as a nonphosphorylatable substrate, also
demonstrated the inhibition of Vav-induced JNK1 activity and of the
IL-6 mRNA response, thus establishing the link of
(pY)Vav
Rac1JNK1IL-6 (Fig. 8). Given the specificity observed
for IL-2 and IL-6 mRNA responses, our findings underscore published
studies that suggest that no single signaling pathway is capable of
inducing a general mast cell cytokine response (16, 17, 25, 31, 48). It is clear, however, that Vav-dependent activation of JNK1
is sufficient for the induction of an IL-6 response in RBL and BMMC.
Thus, one might speculate that because Vav-dependent induction of IL-6
may be relatively direct, IL-6 production could occur in circumstances
where multiple and converging signaling pathways might not be active
(47, 60, 61, 62).
Recent studies clearly demonstrate that Vav regulates cytoskeletal
reorganization by the TCR (55, 56). In these studies,
vav-/- T cells were found to be defective
in TCR-mediated actin cap formation and in IL-2 production.
Surprisingly, these studies also demonstrated that JNK/SAPK activity is
normal in vav-/- T cells, a finding that
would not be predicted from previous (7, 8, 9, 10) and present
studies. These differences may be explained by the presence of
redundant pathways or the up-regulation of compensatory mechanisms,
such as the possible increased expression or activation of another GEF
activity such as Vav2 (63), which in the absence of Vav
may target an alternate pathway leading to JNK activation
(18). Regardless, the present study demonstrates the
potential of Vav to activate JNK1 activity via the activation of Rac1
(Fig. 8). We also found that IL-6 mRNA accumulation required the GEF
activity of Vav, could be induced by Rac, and required activation of
JNK1. Finally, because Vav, but not DH-Vav, expression in BMMC promoted
increased FcRI-dependent IL-6 secretion, we conclude that Vav GEF
activity contributes to the production of IL-6 in mast cells.
Collectively, these findings support the idea that activation of a
particular signaling pathway can have specific consequences. However,
the cross-talk of signaling pathways in response to receptor engagement
is likely to modulate the kinetics, extent, and profile of cellular
responses.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Juan Rivera, National Institutes of Health, Building 10, Room 9N228, 10 Center Drive, MSC 1820, Bethesda, MD 20892-1820. E-mail address:
3 Abbreviations used in this paper: GEF, guanine nucleotide exchange factor; JNK, c-Jun N-terminal kinase; FcRI, high affinity receptor for IgE; BMMC, bone marrow-derived mast cells; Agn, Ag (refers to DNP-HSA); DH-Vav, Dbl homology domain-deleted Vav; DNP-HSA, dinitrophenylated human serum albumin; ORF, open reading frame; SFV, Semliki Forest virus; GFP, green fluorescent protein; MAPK, mitogen-activated protein kinase; PI, phosphoinositide; DH, Dbl homology; DH-, domain mutated.
4 Reported cDNA sequence of rat vav: GenBank accession no. U39476.
5 Arudchandran, R., M. J. Brown, M. J. Peirce, J. S. Song, J. Zhang, R. P. Siraganian, U. Blank, and J. Rivera. Compartmentation of Vav in plasma membrane glycolipid-enriched microdomains is required for Fc receptor-mediated activation of JNK1. Submitted for publication.
Received for publication March 10, 1999. Accepted for publication April 29, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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