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B Activity1
* Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada;
Max Planck Institute for Experimental Endocrinology, Hannover, Germany; and
Department of Molecular Immunology, Biology III, University of Freiburg and Max Planck Institute for Immunobiology, Freiburg, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B and that IB
phosphorylation/degradation and NF-B translocation, DNA binding and
transactivation are much higher in SHIP-/- BMMCs.
Interestingly, using various pathway inhibitors, it appears that the
phosphatidylinositol 3-kinase/PKB and PKC pathways elevate IL-6 mRNA
synthesis, at least in part, by enhancing the phosphorylation of IB
and NF-B DNA binding while the Erk and p38 pathways enhance IL-6
mRNA synthesis by increasing the transactivation potential of NF-B.
Taken together, our data are consistent with a model in which SHIP
negatively regulates NF-B activity and IL-6 synthesis by reducing
IgE + Ag-induced phosphatidylinositol-3,4,5-trisphosphate levels and
thus PKB, PKC, Erk, and p38 activation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-6) (2, 4). IgE initiates these processes by
binding to mast cells via the high-affinity IgER, Fc
RI, which
belongs to the immune receptor superfamily and exists as a tetramer of
one -, one 
-, and two identical disulfide-linked 
-chains. The
subunit binds the Fc portion of a single IgE molecule. Following
crosslinking of IgE-bound FcRIs by a multivalent Ag, the - and
-chains mediate signal transduction events via their immunoreceptor
tyrosine-based activation motifs, which, upon phosphorylation by
members of the Src kinase family, attract Src homology-2-containing
signaling proteins (5, 6). One of the proteins that
becomes tyrosine phosphorylated in mast cells in response to IgE + Ag
is the hemopoietic-specific Src homology-2-containing inositol
5'-phosphatase (SHIP)3
(7, 8, 9). Although this phosphorylation event does not
appear to increase the enzymatic activity of SHIP (8), it
may be involved in the localization of SHIP at the plasma membrane
(10) where it cleaves the 5'-phosphate from the
phosphatidylinositol 3-kinase (PI-3K)-generated product
phosphatidylinositol-3,4,5-trisphosphate (PIP3)
to yield PI-3,4-P2. This in turn reduces the
ability of certain pleckstrin homology (PH)-containing proteins (e.g.,
protein kinase B (PKB)/Akt, phosphoinositide-dependent
protein kinase (PDK)-1, Bruton’s tyrosine kinase) to target to the
plasma membrane and be activated (11, 12, 13).
Using bone marrow-derived mast cells (BMMCs), we recently demonstrated
that SHIP is a key negative regulator of IgE + Ag-induced mast cell
degranulation (7). To further explore the role that SHIP
plays in regulating IgE + Ag-induced mast cell activation, we have now
compared cytokine production in murine BMMCs from
SHIP+/+ and SHIP-/-
littermates. We demonstrate in this study that SHIP negatively
regulates IgE + Ag-induced IL-6 mRNA and protein levels and requires
its phosphatase activity to exert this negative effect. Moreover, we
show that SHIP represses IL-6 mRNA levels, at least in part, by
reducing PKB, protein kinase C (PKC), p38, and extracellular
signal-related kinase (Erk) activity. This culminates in a
marked reduction in NF-B activity and, thus, IL-6 mRNA and protein
synthesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bone marrow cells aspirated from 4- to 8-wk-old
SHIP+/+ and SHIP-/- C57B6
or PKC
+/+ and
PKC-/- 129/SV mice were cultured as
described previously (7, 14). By 8 wk in culture, greater
than 98% of the cells were c-kit- and FcRI-positive, as
assessed by FITC-labeled anti-c-kit Abs (BD PharMingen,
Mississaga, Canada) and FITC-labeled IgE (anti-erythropoietin 26),
respectively (7).
Introduction of SHIP constructs into SHIP-/- BMMCs
The hemagglutinin (HA)-tagged murine SHIP cDNA in a Bluescript KS+ vector, was the starting material for the PCR-based point mutation in the D675G SHIP construct using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) (15). SHIP-/- bone marrow cells were infected with the wild-type (WT) or D675G SHIP construct and BMMCs derived as described in (15).
BMMC stimulation and Western blotting
To stimulate with IgE + Ag, BMMCs were preloaded with 1–5
µg/ml anti-DNP IgE (clone SPE-7 IgE; Sigma-Aldrich, St. Louis,
MO) for 4 h or overnight in IMDM + 10% FCS + 150 µM
monothioglycerol, washed three times to remove unbound IgE, and
then resuspended in IMDM + 0.1% BSA for 5 min at 37°C before adding
20 ng/ml DNP-human serum albumin (HSA) (30–40 moles DNP/mole HSA;
Sigma-Aldrich) for the indicated times. For inhibitor studies,
inhibitors were added 15 min before the addition of DNP-HSA at the
indicated concentrations. The inhibitors used were the PI-3K inhibitor
LY294002, the mitogen-activated/extracellular signal-regulated kinase
kinase (Mek) inhibitor PD98059, the p38 inhibitor SB203580, the
NF-B inhibitor Bay11-7082 (Bay11), the PKCinhibitor compound 3
(bisindolymaleimide I), and the classical PKC (cPKC) inhibitor
GÖ6976, all obtained from Calbiochem (La Jolla, CA), and
wortmannin (Sigma-Aldrich) and cycloheximide (Boehringer Mannheim,
Mannheim, Germany). The cells were solubilized by boiling for 1 min
with SDS-sample buffer (using 1 x 106
BMMCs/sample for total cell lysates). The phospho-PKB
(Ser473), phospho-IB
(Ser32), IB, phospho-Erk 1/2, phospho-p38,
phospho-c-Jun N-terminal kinase (JNK), and JNK Abs were obtained from
Cell Signaling (Beverly, MA). Src homology and collagen (Shc) Ab
was obtained from BD Transduction Laboratories (Mississauga, Ontario,
Canada). The Erk1 and FcRI -chain Abs were generous gifts from
Drs. S. Pellech (Vancouver, British Columbia, Canada) and R. Siraganian
(Bethesda, MD), respectively. The polyclonal anti-SHIP Ab
was generated as described in Ref. 15 .
Plasma membrane preparation
SHIP+/+ and SHIP-/- BMMCs were stimulated as above and plasma membrane-enriched fractions were prepared as described in Ref. 16 . The final Nonidet P-40 solubilized membrane fraction, which was highly enriched for plasma membranes (as assessed by biotinylating the cell surface of intact BMMCs (M. R. Hughes and G. Krystal, unpublished observations), was then subjected to Western blot analysis with anti-PKC Abs (BD Transduction Laboratories).
Degranulation assays
IgE + Ag-induced degranulation was assessed as described in Ref. 7 .
RNase protection assay (RPA) and ELISA
For mRNA analysis, BMMCs were stimulated as above and RNA was
isolated using TRIzol reagent (Life Technologies, Burlington, Canada).
Cytokine mRNA levels were quantitated using a Riboquant MultiProbe
RNase protection assay (BD PharMingen) according to the manufacturer’s
instructions, using [33P]dUTP 9NEG-307H
from NEN (Boston, MA). Quantitation of cytokine mRNA levels, using a
PhosphorImager (Storm 860; Molecular Dynamics, Sunnyvale, CA),
was standardized based on levels of the housekeeping gene
GAPDH. Mouse IL-4, TNF-, IL-6 (BD PharMingen), and
IL-13 (R&D Systems, Minneapolis, MN) ELISAs were performed according to
the manufacturer’s instructions.
EMSAs
EMSAs were performed as described in (17). Nuclear
extracts (5 µg of protein, as determined by BCA Protein Assay kit
(Pierce, Rockford, IL)) were incubated with 1 pmol
[32P]NF-B consenus oligonucleotide probe
(Santa Cruz Biotechnology, Santa Cruz, CA) in binding buffer and 1 µg
of poly(dIdC) (BD PharMingen) for 15 min at 23°C and then
electrophoresed on 5% polyacrylamide gels in 0.25x Tris-borate-EDTA.
Ab supershifts were performed by preincubating with 10 µg of
anti-NF-B Abs (Rel A, Rel B, c-Rel, p50, p52; Santa Cruz
Biotechnology) 15 min before the addition of oligonucleotide probe.
These Abs were also used for Western blot analysis with nuclear
extracts.
Cell transfection and luciferase assay
Cell transfection experiments were conducted as described in
Ref. 18 . Briefly, BMMCs were incubated for 4 h in
fresh growth medium, washed, and resuspended in IMDM + 10% FCS and
aliquoted (107/250 µl) into electroporation
cuvettes (4-mm gap; Bio-Rad, Hercules, CA). Reporter gene construct (10
µg/ml, pNF-B-LUC; Stratagene) was added together with 2 µg/ml of
thymidine kinase promoter-dependent renilla luciferase construct
(pRL-TK; Promega, Madison, WI) to assess transfection efficiency and
the cells electroporated (Gene Pulser; Bio-Rad) at 280 V and 960 µF.
The cells were then incubated in tissue culture flasks at 1 x
106 cells/ml in IMDM + 10% FCS + 1 µg/ml IgE
overnight and then treated ± 20 ng/ml DNP-HSA for 4 h. Luciferase
assays were performed according to the manufacturer’s instructions
(Dual Luciferase Reporter Assay; Promega).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To explore the role that SHIP plays in regulating the IgE +
Ag-induced synthesis and release of proinflammatory cytokines from
BMMCs, we first conducted RPAs with IgE + Ag-stimulated
SHIP+/+ and SHIP-/-
BMMCs. As shown in Fig. 1
A,
these studies revealed that the mRNA levels of IL-4, IL-5, IL-6, IL-13,
and TNF- were substantially higher in
SHIP-/- than in SHIP+/+
BMMCs (Fig. 1A). This difference in cytokine mRNA levels was
observed not only in response to IgE + Ag but in response to steel
factor and IL-3 as well (data not shown). To investigate how SHIP
negatively regulates cytokine expression at the signal transduction
level, we focused our attention on IL-6 because a great deal was
already known about the regulation of this cytokine in mast cells
(19). As shown in Fig. 1B, we found that IL-6
protein levels, as assessed by ELISA, were significantly higher in the
conditioned medium from SHIP-/- BMMCs,
consistent with the elevated mRNA levels observed in Fig. 1A.
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To determine whether the observed difference in IL-6 production
between SHIP+/+ and
SHIP-/- BMMCs was a result of SHIP’s enzymatic
activity or its ability to serve as an adaptor protein (9, 20), bone marrow cells from SHIP-/- mice
were retrovirally infected with N-terminal HA- and C-terminal green
fluorescent protein-tagged versions of WT or
phosphatase-deficient (D675G) SHIP (Fig. 2A, left panel)
(15). Following 10 days in methylcellulose containing
puromycin, mast cell colonies were pooled and put in suspension culture
for 8 wk (15). At this time, the two cultures were greater
than 98% IgER and c-kit positive and Western blot analysis,
using anti-SHIP Abs, revealed that the WT and D675G SHIP constructs
expressed approximately the same amount of SHIP protein and this level
was about half that present in SHIP+/+ BMMCs
(Fig. 2A, right panel). Studies with these BMMCs
revealed that the introduction of WT SHIP, which reduced IgE +
Ag-induced PIP3 (15) and
degranulation (Fig. 2B) to levels approaching those observed
in SHIP+/+ BMMCs, reverted the IgE + Ag-induced
increase in IL-6 mRNA (as assessed by RPA; Fig. 2C) and
protein expression (as assessed by ELISA; Fig. 2D) to close
to those observed in SHIP+/+ BMMCs. The D675G
SHIP, in contrast, did not revert any of the responses examined (Ref.
15 ; Fig. 2, B–D). These results suggested that
the difference in IL-6 production was dependent on the phosphatase
activity of SHIP, and thus, likely, PIP3
levels.
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To explore which PIP3-regulated pathways
might be responsible for the elevated IL-6 mRNA and protein levels seen
in IgE + Ag-induced SHIP-/- BMMCs, we first
compared the activation states of various pathways known to be
triggered by IgE + Ag in BMMCs. Specifically, we compared the
phosphorylation state of PKB, a PH domain-containing serine/threonine
kinase that is recruited to the plasma membrane by the transient IgE +
Ag-induced increase in PIP3 and activated via
phosphorylation at Thr308, by the
PIP3 binding PH-containing serine/threonine
kinase, PDK1, and at Ser473 by either an as yet
unidentified PDK2 or by autophosphorylation in a manner similar to PKCs
(21, 22, 23, 24). As expected, because SHIP down-regulates
PIP3 levels and thus the recruitment of PKB (and
likely PDK1), we observed more intense and prolonged phosphorylation of
PKB in IgE + Ag-induced SHIP-/- BMMCs (Fig. 3A, upper panel).
Reprobing with anti-FcRI -subunit Abs demonstrated equal
loading (lower panel).
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B, IgE + Ag treatment resulted in a substantially greater
recruitment of PKC , , , and to the plasma membrane of
SHIP-/- than SHIP+/+
BMMCs. Reprobing with anti-FcRI -chain Abs demonstrated equal
levels of membrane protein. Importantly, total cell lysates showed
comparable levels of these PKC isoforms in
SHIP+/+ and SHIP-/- BMMCs
(Fig. 3B, right panel).
Because PKC has been shown to phosphorylate/activate Raf-1 in
hemopoietic cells (33), we then compared the IgE +
Ag-induced phosphorylation of Erk in SHIP+/+ and
SHIP-/- BMMCs and found much more intense and
prolonged phosphorylation in SHIP-/- BMMCs
(Fig. 3C, top panel). Reprobing with
anti-FcRI -subunit Abs demonstrated equal loading (Fig. 3C, bottom panel). We also looked at the effect
of SHIP on IgE + Ag-induced activation of p38 mitogen-activated protein
kinase (MAPK), because, depending on the stimulus and cell type,
this serine/threonine kinase has been shown to be activated
(34) or inhibited (35) by the PI-3K/PKB
pathway and also activated by members of the PKC family
(36). Interestingly, we found that p38 phosphorylation was
both more intense and more prolonged in response to IgE + Ag in
SHIP-/- BMMCs (Fig. 3D, top
panel). Reprobing with anti-FcRI -subunit Abs
demonstrated equal loading (Fig. 3D, bottom
panel). Lastly, we looked at the effect of SHIP on IgE +
Ag-induced phosphorylation/activation of JNK because the activation of
this stress-activated protein kinase has been shown to enhance IL-6
production in mast cells (37). As shown in the top
panel of Fig. 3E, JNK phosphorylation was both more
intense and more prolonged in response to IgE + Ag in
SHIP-/- BMMCs. Reprobing with anti-JNK Abs
demonstrated equal loading (Fig. 3E, bottom panel).
IgE + Ag-induced IL-6 production in BMMCs is dependent on the activation of the PI-3K, PKC, Erk, and p38 pathways
To determine which of these pathways, if any, contributed to the
elevated IL-6 production observed in SHIP-/-
BMMCs, we added pathway specific inhibitors to
SHIP+/+ and SHIP-/- BMMCs
and then stimulated the cells with IgE + Ag for 3 h and performed
IL-6 ELISAs on the conditioned medium. As seen in Fig. 4A, addition of the PI-3K
inhibitor LY294002 (25 µM), the PKC inhibitor compound 3
(bisindolylmaleimide; 10 µM), the Mek inhibitor PD98059 (50 µM), or
the p38 inhibitor SB203580 (2 µM) completely abrogated IgE +
Ag-induced IL-6 production in both SHIP+/+
(left panel) and SHIP-/-
(right panel) BMMCs. The PI-3K inhibitor wortmannin (25 nM)
also totally inhibited IL-6 production (data not shown). Thus, it
appeared that all of these signaling pathways were essential for IL-6
production and, given that they were all elevated in IgE + Ag-induced
SHIP-/- BMMCs, could contribute to
the elevated IL-6 production observed in these cells. To gain some
insight into which PKC isoform(s) were involved, we also examined IL-6
production from IgE + Ag-induced SHIP+/+ and
SHIP-/- BMMCs in the presence and absence of
the cPKC inhibitor, GÖ6976. As seen in Fig. 4B
(left panel), this inhibitor blocked IL-6 production,
indicating a role for a Ca2+-dependent PKC in
IL-6 regulation. This is consistent with a recent finding showing
decreased IL-6 production from PKC-deficient BMMCs
(38). To determine whether PKC-mediated regulation of IgE
+ Ag-induced IL-6 production was limited to cPKC isoforms, we compared
IL-6 production from PKC+/+ and
PKC-/- BMMCs. As shown in Fig. 4B
(right panel), IL-6 production from
PKC-/- BMMCs was significantly reduced
compared with their PKC+/+ counterparts,
suggesting a role for a novel PKC isoform in the regulation of IL-6
production as well.
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B phosphorylation/degradation and NF-B DNA binding and
transactivation are higher in IgE + Ag-induced SHIP-/-
BMMCs
Because it had been shown recently that the transcription factor
NF-B is a key regulator of IgE + Ag-induced IL-6 mRNA in BMMCs by
binding directly to B elements within the IL-6 promoter and
activating transcription (19), we next compared the
activity of NF-B in IgE + Ag-induced
SHIP+/+ and SHIP-/-
BMMCs. NF-B activity is tightly regulated by IB (39, 40) which binds and masks the NF-B nuclear localization
signal and thus sequesters NF-B in the cytoplasm. Following IgE +
Ag-stimulation, IB kinase (IKK) is activated and phosphorylates
IB, which targets the latter for ubiquitination and degradation
by the proteasome, and frees NF-B to translocate to the nucleus to
activate target gene transcription (41, 42). As expected,
we found that the Bay11, which irreversibly inhibits the
phosphorylation of IB (43), was a potent inhibitor of
IL-6 production in both SHIP+/+ and
SHIP-/- BMMCs (Fig. 5A). Then, using
phospho-specific IB Abs, we examined IgE + Ag-induced IB
phosphorylation and found much higher phosphorylation in
SHIP-/- than SHIP+/+
BMMCs (Fig. 5B). Moreover, reintroduction of WT SHIP into
SHIP-/- cells reduced this phosphorylation
close to the levels seen in SHIP+/+ BMMCs (Fig. 5B). In keeping with this increased IB phosphorylation in
SHIP-/- BMMCs, IB degradation was
significantly greater in these cells, but only when the protein
synthesis inhibitor cycloheximide was added (Fig. 5C). In
the absence of cycloheximide, IgE + Ag-induced degradation of IB was
similar in SHIP+/+ and
SHIP-/- BMMCs (Fig. 5D), most likely
because of a compensatory increase in the transcription/translation of
the NF-B target, IB, in the
SHIP-/- cells that masks the increased
IB degradation (39, 40, 42).
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B translocation to the nucleus by carrying out
anti-NF-B p65 and p50 immunoblots with nuclear extracts and
found, interestingly, that p50, but not p65, levels were substantially
higher in IgE + Ag-induced SHIP-/- than
SHIP+/+ BMMCs (Fig. 6A, left panels).
Total cell levels of NF-B (p50), in contrast, were comparable (Fig. 6A, right panel). However, a higher concentration
of nuclear NF-B is not necessarily synonymous with more IL-6 mRNA
synthesis (44). To initiate transcription of target genes,
NF-B transcription factors must bind first as dimers to B sites
(40). Therefore, using EMSAs, we investigated whether
NF-B DNA binding was higher in IgE + Ag-induced
SHIP-/- than SHIP+/+
BMMCs and found, as shown in Fig. 6B, that it was. We
then conducted supershift studies using Abs to the members of the
NF-B family (p50, p52, p65 (Rel A), Rel B, and c-Rel; Ref.
40) and found that only anti-p50 and anti-p65
supershifted the NF-B/oligonucleotide complex (data not shown).
Thus, a p50/p65 heterodimer was likely the predominant species that
bound to B sites in activated mast cells. As expected, the addition
of Bay11 (20 µM) to SHIP+/+ and
SHIP-/- BMMCs completely abrogated the nuclear
localization of NF-B, as assessed by EMSA, on nuclear preparations
from both cell types (Fig. 6B).
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B and the
subsequent nuclear translocation and DNA binding of released NF-B is
regarded as the principle mechanism for activating NF-B-dependent
gene expression, some recent studies have suggested that the
transcriptional activity of NF-B is also highly regulated
(34). We thus conducted NF-B luciferase assays to
obtain direct evidence for increased NF-B transactivation in IgE +
Ag-induced SHIP-/- BMMCs. Specifically,
SHIP+/+ and SHIP-/- BMMCs
were electroporated with a NF-B-firefly luciferase reporter gene
construct, together with a pRL-TK to assess transfection efficiency,
and the cells were then preloaded with 1 µg/ml IgE for 18 h and
subjected to 20 ng/ml DNP for 4 h. As shown in Fig. 6C,
the transactivation potential of NF-B was significantly higher in
SHIP-/- BMMCs. Interestingly, the high NF-B
transactivation observed in the SHIP-/- BMMCs
in the absence of crosslinker is consistent with our previous data
showing that IgE alone is capable of inducing NF-B transactivation
(45) and IL-6 production (46). Both of these
processes are negatively regulated by SHIP.
PI-3K/PKB and PKC enhance IB phosphorylation/degradation and
NF-B binding to DNA while Erk and p38 stimulate NF-B
transactivation
Because we found that the PI-3K/PKB, PKC, Erk and p38 pathways
were all elevated in SHIP-/- BMMCs and were all
required for IL-6 production, we asked whether and how these pathways
elevated NF-B activity. However, a complication in delineating the
relative contributions of these upstream pathways to NF-B activation
is that they "cross-talk" with each other. To examine the
contribution of cross-talk in IgE + Ag-induced
SHIP-/- BMMCs, we first looked at the effect of
LY294002 and wortmannin on the activation of PKB, Erk, and p38 and
found that these PI-3K inhibitors reduced all three phosphorylation
events (Fig. 7A). Thus,
PIP3 levels affect all three pathways in these
cells. We next looked at the effect of compound 3 on these three
pathways and found that it not only inhibited Erk phosphorylation, as
expected (33) (Fig. 7B), but also inhibited PKB
(Fig. 7B) and, to a lesser extent, p38 phosphorylation as
well (Fig. 7C). This might be due to nonspecificity of this
PKC inhibitor at this concentration (10 µM) or, at least in the case
of PKB, a PI-3K-independent, PKC-mediated activation of PKB, as has
been reported by Kroner et al. (47). As expected, PD98059
completely inhibited Erk1/2 phosphorylation, but had no effect on PKB
(Fig. 7B) or p38 phosphorylation (data not shown). SB203580,
at a concentration (2 µM) that completely blocked p38 phosphorylation
(Fig. 7C), had no effect on Erk or PKB phosphorylation (data
not shown). Of interest, Bay11 had no effect on Erk, PKB, or p38
phosphorylation (Fig. 7, B and C). Similar
results were obtained with these inhibitors using
SHIP+/+ BMMCs (data not shown).
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B phosphorylation or NF-B DNA binding was affected by
the inhibitors of these upstream pathways. Specifically, we examined
the effects of compound 3, Bay11, PD98059, and SB203580 on IB
phosphorylation. As seen in Fig. 7D (top
panel), IB phosphorylation was markedly inhibited by Bay11, as
expected, and by compound 3, but was only slightly inhibited by PD98059
and not at all by SB203580. Reprobing with anti-FcRI -chain
Abs demonstrated equal loading (Fig. 7D, bottom
panel). Because the concentration of PD98059 used in this study
totally abrogated Erk phosphorylation while that of compound 3 only
partially inhibited Erk phosphorylation (Fig. 7B, top
two panels), this indicated that the PKC-mediated phosphorylation
of IB likely occurred independent of the Erk pathway. EMSAs of
nuclear extracts confirmed and extended these findings by showing that
compound 3 as well as LY294002, but neither PD98059 nor SB203580,
inhibited NF-B DNA binding (Fig. 7E). The numbers below
each lane represent relative band intensities, determined by
densitometry.
As mentioned earlier, NF-B activity can also be regulated at the
transactivation step and one of the players involved in this regulation
is p38 (34, 48). Therefore, we tested the effects of
SB203580, as well as PD98059, compound 3, LY294002, and Bay11 in
NF-B-luciferase assays and found that they all totally abrogated the
IgE + Ag-induced increase in luciferase activity (Fig. 7F).
These results suggested that the p38 and Erk pathways, unlike the PKC
and PI-3K pathways, enhance NF-B activity via increasing NF-B
transactivation independent of IB degradation and NF-B DNA
binding.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To address how SHIP regulates IL-6 production, we honed in on the
regulation of IL-6 mRNA synthesis because IL-6 mRNA levels correlated
nicely with secreted IL-6 protein levels. Narrowing our window of
investigation even further, we focused exclusively on NF-B activity
because this transcription factor has been shown to be a major positive
regulator of IgE + Ag-induced IL-6 mRNA synthesis in BMMCs
(19). However, we fully appreciate that SHIP may also
regulate the activity of other transcription factors involved
in IL-6 mRNA synthesis (37) and/or the secretion of the
IL-6 protein from these cells (51, 52). As expected, the
NF-B inhibitor, Bay11, blocked IL-6 production in both
SHIP+/+ and SHIP-/-
BMMCs. As a test of the specificity of this inhibitor, we checked its
effect on IgE + Ag-induced IL-4 synthesis, since the IL-4
promoter does not possess a B element (19, 53), and
found no inhibition (assessed by RPA and ELISA, data not shown). As a
point of interest, we also found that Bay11 had no effect on IL-5
production (assessed by RPA, data not shown) as expected
(19), but was capable of inhibiting IL-13 and TNF-
production (assessed by RPA and ELISA, data not shown) (53, 54).
As to how SHIP negatively regulates NF-B activity, we examined four
major levels of NF-B regulation: IB phosphorylation/degradation,
NF-B translocation to the nucleus, DNA binding, and transactivation.
Our finding that IB is phosphorylated to a greater extent in
SHIP-/- BMMCs is consistent with several recent
reports showing that PKB acts as a positive regulator of NF-B
activity by transiently binding and phosphorylating/activating IKK
(34, 54, 55, 56, 57, 58, 59, 60). Relevant to our finding that IB
degradation is increased in SHIP-/- BMMCs in
the presence of cycloheximide, Koul et al. (41) recently
reported that the tumor suppressor phosphatase and tensin homolog
deleted on chromosome 10 (PTEN), which also hydrolyses
PIP3, inhibits NF-B DNA binding without
affecting IB degradation. However, they did not examine IB
degradation in the presence of cycloheximide. Gustin et al.
(61), in contrast, found, in support of our findings, that
PTEN inhibits the activation of IKK and phosphorylation of IB in
response to TNF, but they did not examine IB degradation.
Interestingly, we observe an increased translocation of the p50, but
not the p65, subunit of NF-B into the nucleus of IgE + Ag-stimulated
SHIP-/- BMMCS, as assessed by Western blot
analysis of nuclear extracts. Using EMSAs, we show that SHIP negatively
regulates NF-B DNA binding, though this may be explained entirely by
the reduced nuclear translocation of p50 in the presence of SHIP. As
well, our finding that NF-B DNA binding is inhibited by LY294002
(confirming the work of Gustin et al. (61)) strengthens
the hypothesis that SHIP represses IL-6 production via its
hydrolysis of PIP3. Related to this, the
3'-phosphatase, PTEN, has also been shown to negatively regulate
NF-B DNA binding (41, 61).
To gain some insight into the SHIP-regulated upstream pathways that
modulate IL-6 mRNA levels in BMMCs and to delineate the contribution of
each of these pathways to NF-B activation, we used specific
inhibitors to pathways that were elevated in the absence of SHIP. As
mentioned earlier, the elevated PKB activity in
SHIP-/- BMMCs likely contributes to the
increased NF-B activity in these cells by directly
phosphorylating/activating IKK. Complicating the picture, the
PIP3-dependent PDK1, besides playing a critical
role in activating PKB, has also been shown to phosphorylate/activate
various PKC isoforms (27, 28, 29). Related to this, many PKC
isoforms, such as , , 
, and 
, have been shown to positively
regulate NF-B activity in a variety of cell types (36, 62, 63) and the classical PKC isoform, PKC, is known to be a
positive regulator of IgE + Ag-induced mast cell degranulation and IL-6
production (38). In this study, we show that the novel PKC
isoform, PKC, also acts as a positive regulator of IL-6 production.
As to how PKC isoforms regulate IL-6 production, we show that PKC
isoforms regulate IB phosphorylation/degradation, and thus
subsequent NF-B DNA binding and transactivation, a finding supported
by Lallena et al. (62) who showed that PKC and atypical
PKC isoforms bind to IKKs in vitro and in vivo.
NF-B activity can also be regulated at the transactivation step and
several recent reports support our finding that p38 is capable of
up-regulating NF-B transactivation without affecting IB
degradation, NF-B nuclear translocation, or NF-B DNA binding
(34, 36, 48, 64). As to how p38 is activated, Madrid et
al. (34) found that PKB mediates IL-1-induced activation
of NF-B by activating p38 in an IKK-dependent manner. However, we
found that Bay11, which irreversibly inhibits IKK and the subsequent
phosphorylation of IB, had no effect on p38 phosphorylation in
response to IgE + Ag. We thus propose that IKK and p38 enhance NF-B
activity by separate pathways; this is supported by studies showing
that PKC regulates parallel IKK and p38 pathways to enhance NF-B
activity in response to thrombin (36). Because we found
that PKC inhibitors partially block IgE + Ag-induced p38
phosphorylation in BMMCs, it is conceivable that p38 activity is
regulated in these cells via a PI-3K/PDK1/PKC pathway in response to
IgE + Ag. Related to this, the phosphorylation of p38 and Erk are
significantly reduced in PKC-/- BMMCs (data
not shown) and this may contribute to the decreased IL-6 production
observed in these cells.
Although p38 is a well-established regulator of NF-B transactivation
(34, 48, 65), the role of Erk in NF-B regulation is
more controversial. Our inhibitor studies suggest that IgE + Ag-induced
Erk activation is highly dependent on PI-3K and PKC activation. As
well, using the Mek inhibitor, PD98059, we show that Erk activity is
required for NF-B transactivation independent of IB
phosphorylation and NF-B DNA binding. In support of our finding,
Vanden Berghe et al. (65) found that PD98059 inhibited
NF-B transactivation in response to TNF-. However, Madrid et al.
(34) did not find any significant decrease in NF-B
luciferase in 3T3 cells in the presence of PD98059, albeit using very
low (2 µM) inhibitor levels. As to how PD98059 inhibits the
transactivation of NF-B, Chen et al. (66) recently
showed that NF-B can be acetylated and that acetylated NF-B p65
interacts weakly, if at all, with IB. Related to this, Vanden
Berghe et al. (67) found that both p38 and Erk promoted
the acetylation capacity of the enhanceosome and thus stimulated
TNF-induced, NF-B-mediated, IL-6 gene expression.
Although we also found that SHIP acts as a negative regulator of
IgE + Ag-induced JNK phosphorylation, we were unable to study the role
of this MAPK family member in the regulation of NF-B activity and
IL-6 production due to the lack of JNK-specific inhibitors. However, it
has been reported that the PIP3-dependent
tyrosine phosphorylation of the PH-containing Rac guanine nucleotide
exchange factor, Vav, stimulates IL-6 production in mast
cells by a Rac/JNK-dependent pathway, and we observe
increased membrane recruitment and phosphorylation of Vav in
SHIP-/- BMMCs (M. Ware andG. Krystal,
unpublished observations).
Taken together our results suggest a model, shown in Fig. 8, in which SHIP represses IL-6
production in BMMCs, at least in part, by reducing PI-3K-generated
PIP3 levels induced by IgE + Ag. This in turn
inhibits PKB- and PKC-mediated IB phosphorylation/degradation and
the nuclear translocation and DNA binding of released NF-B, as well
as p38- and Erk-mediated NF-B transactivation. Thus, these upstream
pathways which synergize to stimulate IL-6 mRNA synthesis, are all
negatively regulated by SHIP.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Gerald Krystal, Terry Fox Laboratory, British Columbia Cancer Agency, 601 West 10th Avenue, Vancouver, British Columbia, Canada V5Z 1L3. E-mail address: gerryk{at}terryfox.ubc.ca
3 Abbreviations used in this paper: SHIP, Src homology-2 containing inositol 5'-phosphatase; PI-3K, phosphatidylinositol 3-kinase; PIP3, phosphatidylinositol-3,4,5-trisphosphate; PH, pleckstrin homology; PKB, protein kinase B; PKC, protein kinase C; PDK, phosphoinositide-dependent protein kinase; cPKC, classical PKC; BMMC, bone marrow-derived mast cells; Erk, extracellular signal-related kinase; HA, hemagglutinin; WT, wild type; RPA, RNase protection assay; HSA, human serum albumin; Mek, mitogen-activated/extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; pRL-TK, thymidine kinase promoter-dependent renilla luciferase construct; IKK, IB kinase; Bay11, NF-B inhibitor Bay11-7082; Shc, Src homology and collagen; PTEN, phosphatase and tensin homolog deleted on chromosome 10.
Received for publication December 3, 2001. Accepted for publication March 1, 2002.
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) and SHIP-/-
(
) BMMCs were preloaded with 5 µg/ml IgE for 4 h, then
stimulated ± 20 ng/ml DNP-HSA for 30 min and subjected to RPAs. The
relative band intensities of IL-4, IL-5, IL-6, IL-13, and TNF-
) and green fluorescent protein-tagged
at the C terminus (
). Total cell lysates from 1 x
106 SHIP+/+, SHIP-/-, WT, and
D675G BMMCs were subjected to Western blot analysis using anti-SHIP
Abs (right panel). B,
SHIP+/+, SHIP-/-, WT, and D675G BMMCs were
preloaded with 10 µg/ml IgE for 1 h, then stimulated for 15 min
± 20 ng/ml DNP-HSA; the percentage of degranulation was determined by
assaying supernatants and cell pellets for 
