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Blood, Vol. 95 No. 5 (March 1), 2000:
pp. 1752-1757
IMMUNOBIOLOGY
Inhibition of degranulation and interleukin-6 production in mast
cells derived from mice deficient in protein kinase C
Hovav Nechushtan,
Michael Leitges,
Cellina Cohen,
Gillian Kay, and
Ehud Razin
From the Department of Biochemistry, Hebrew University-Hadassah
Medical School; and the Max-Planck-Institut fuer Immunbiologie
Stuebeweg, Freiburg, Germany.
 |
Abstract |
The antigen-mediated activation of mast cells by means of IgE
antibodies bound to the cell surface leads to direct interactions between Fc RI receptor cytoplasmic domains and various intracellular proteins. These interactions initiate diverse signal-transduction pathways, and the activation of these pathways results in the immediate
release of proinflammatory agents. A delayed response also occurs and
includes the release of various cytokines. It is clear that the
activation of kinases is a requirement for the exocytosis observed in
mast cells. In addition to the tyrosine phosphorylation of the affected
system by soluble tyrosine kinases, activity of protein kinase C (PKC)
results in serine or threonine phosphorylation of multiple protein
substrates. In this study, we found that mast cells derived from
PKC -deficient mice produce less interleukin 6 in response to IgE-Ag.
The inhibition of exocytosis in the PKC-deficient mast cells
occurred whether the stimuli were due to the aggregation of the mast
cell surface FcRI or to the calcium ionophore, ionomycin. However,
no significant changes were observed in the proliferative response of
the mast cells to interleukin 3 (IL-3) or in their apoptotic rate after
IL-3 depletion.
(Blood. 2000;95:1752-1757)
© 2000 by The American Society of Hematology.
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Introduction |
Protein kinase C (PKC) enzymes constitute a family of
regulatory proteins that play a major role in receptor-mediated signal transduction and are involved in the regulation of a variety of physiologic processes, including differentiation, proliferation, and
apoptosis.1 The role of a specific PKC isozyme in
exocytosis was first described in studies in rat pituitary cells, in
which secretion of luteinizing hormone was reconstituted by PKC and PKC.2 It was observed that the rat mucosa-like mast cell
line, RBL-2H3, contains at least 5 species of PKC isozymes: , ,
 ,  , and  .3 Both calcium-dependent and
calcium-independent PKC isoenzymes are activated in RBL cells in
response to FcRI-receptor aggregation.3 It was also
shown that PKC and, to some extent, PKC are the isonymous
involved in the secretory response to antigen activation in
reconstituted permeabilized RBL cells.3 Moreover, PKC
was implicated as a key element in the early events of phosphorylation of the mast cell FcRI.4
Evidence for a requirement of PKC activity in gene expression has
accumulated during the past 20 years. Studies in our laboratory implicated PKCs in the regulation of mast cell growth and in the expression of transcription factors in response to the aggregation of
FcRI.5,6 These studies suggested a role for PKC activity in the regulation of the transcription factor complex activating protein 1 (AP-1). We also reported that PKC and PKC could serve as a link between the aggregation of FcRI and the subsequent expression of c-fos and c-jun.7 Moreover,
we found that PKC could serve as a link for upstream stimulating
factor 2 expression in response to aggregation of FcRI.8
Interleukin-6 (IL-6) is a multifunctional cytokine that is produced by
activated mast cells9 and regulates cellular function in
many cell lineages. A clear association between PKC activity and IL-6
synthesis has been found in studies using various culture systems.10-12 It was reported that up-regulation of PKC
in mast cells was associated with a significant increase in the
accumulation of IL-6 messenger RNA (mRNA).13 The IL-6
promoter contains the AP-1 binding site, and it was shown that a
specific JunD-JunD AP-1 complex is essential for IL-6 induction by
transforming growth factor- (TGF-).14 In the current
study, we analyzed the level of JunD mRNA in PKC-deficient cells to
provide insights into the possible role of this member of the AP-1
family in IL-6 gene regulation by PKC.
Mice lacking PKC were produced and used to demonstrate specific
effects on B and T lymphocytes.15 The phenotype of these mice was similar to that of Bruton tyrosine kinase (Btk)-deficient mice
in several ways. Both have a greatly reduced number of peritoneal B1B
cells, lowered IgM and IgG-3 levels, a drastic reduction in the immune
response to T-cell-independent antigen, and a markedly reduced
proliferative response of B cells to IgM and other
stimulants.15-17 One explanation for this similarity in
phenotype is that it is due to the direct binding of Btk and PKC and
the involvement of PKC in the Btk signal-transduction
pathway.17
In the present work, bone marrow-derived mast cells (BMMC) from
PKC-deficient mice were analyzed with respect to their
proliferation, survival rate, degranulation capability, and IL-6 mRNA accumulation.
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Materials and methods |
Animals and cell culture
The production of mice lacking PKC was previously
described.15 The mice had a mixed 129/Sv/129/Ola
background, with controls derived from litter mates of heterozygous
matings. In the current study, PKC-deficient mice and
control litter mates were killed on the same day. Mouse BMMC were
obtained as previously described.18 Briefly, bone marrow
cells were obtained from the femurs and tibias of control and
PKC-deficient 2-month-old mice. We used bone marrow from 1 or 2 mice
per flask. Cells from control and PKC-deficient mast cells were
grown in individual flasks according to the date the mice were killed.
The cells were cultured at a starting density of
0.1 × 106 cells/mL at 37°C in a humidified
atmosphere containing 5% carbon dioxide (CO2). The culture
medium was RPMI 1640 supplemented with 2 mmol/L of L-glutamine,
2 mmol/L of nonessential amino acids, 100 U/mL of penicillin, 100 µg/mL of streptomycin, 50 µmol/L of -2-mercaptoethanol, 10%
fetal-calf serum, and 100 U/mL of yeast-derived interleukin 3 (IL-3;
Genzyme Diagnostics, Cambridge, MA). The nonadherent cells from the
bone marrow cultures were transferred every 7 days into fresh
IL-3-containing medium after verification that there were similar cell
densities for the control and PKC-deficient cells. The cultures were
maintained for 3 to 5 weeks. Mast cells were identified by toluidine
blue staining.
Obtaining and staining peritoneal mast cells
Mice were injected with 10 mL of normal saline intraperitoneally.
After gentle abdominal massage, the peritoneal fluid was aspirated, 250 µL of the fluid was centrifuged, and the pellet was resuspended in 1 mL of normal saline. Then, 200 µL of this suspension was spun for 5 minutes in a cytospin centrifuge. The cells were stained by using a
modification of the original Enerbac protocol19 that uses
0.5% Alcian blue and 0.3% acetic acid. They were then rinsed with
water and counterstained with 0.1% safranin and 0.1% acetic acid.
IgE-Ag and ionomycin-mediated mast cell degranulation
IgE sensitization was carried out by incubating
2 × 106 cells with 30 µg of monoclonal IgE
against dinitrophenol (DNP) (provided by F.-T. Liu, La Jolla, CA) for 2 hours at 37°C in RPMI medium. The cells were washed once with 1 mL
of Tyrode buffer and resuspended in the same buffer. Fifty-microliter
aliquots of this cell suspension were put into microfuge tubes
containing increasing amounts of human serum albumin (HSA) conjugated
to DNP (DNP-HSA) or ionomycin (Sigma, St Louis, MO) for 45 minutes. The
release of -hexosaminidase was then determined in triplicate in a
96-well plate. The results were expressed as the net percentage release
of hexosaminidase, as previously described.20
For experiments investigating cytokine mRNA production and release, the
cells were preincubated for 4 days with IgE at the same concentration
described above and then incubated with 100 ng/mL of HSA-DNP in
tissue-culture medium for 8 to 10 hours. This incubation time was based
on the work by Zhu et al21 and Marshall et
al,22 in which the production of IL-6 in activated mast
cells was determined on the level of mRNA and of protein after
overnight incubation. The cells were then centrifuged and the
supernatants were collected and frozen at  70°C for later
cytokine determination. The pellets containing the activated mast cells
were used for RNA preparation. The IL-6 secreted into the medium was
measured by an enzyme-linked immunosorbent assay kit (Endogen, Woburn, MA).
Proliferation assay
The proliferation assay used was described previously.17
Cells were cultured in 200 µL of medium supplemented with doses of
mouse IL-3 defined according to the requirements of each experiment. Cells from each treatment were divided into 4 replicates and were incubated for 24 hours at 37°C in 5% CO2 in
round-bottomed 96-well microtiter plates (Nunc, Denmark) at a density
of 2 × 105 cells per well. The cells were labeled
with 0.037 MBq of tritium-thymidine for the last 4 hours
at 37°C and transferred onto glass-fiber filter paper (Titerek,
United Kingdom). Thymidine incorporation was measured as described
previously.18
Measurement of apoptosis
BMMC were analyzed for apoptosis by using 2-color flow cytometry.
The cells were stained with annexin V antibody (Boehringer Mannheim,
Germany)23 and then with biotin-labeled antimouse antibody,
followed by avidin-phycoerythrin (BD-Pharmingen, San Diego, CA). The
cells were also stained with the fluorescent DNA binding agent 7-amino
actinomycin D (7AAD), which was used as a substitute for propidium
iodide to mark the cells in the last phase of apoptosis.24
The percentage of cell survival was calculated by subtracting the
number of cells stained with 7AAD, which stains DNA in dead cells, from
the total cell count.
Reverse transcriptase-polymerase chain reaction (RT-PCR) of
cytokines
Cell pellets were lysed directly with 1 mL of Tri-Reagen, and total
RNA was isolated and purified according to the manufacturer's instructions. The following primer pairs were designed from the mouse
complementary DNA sequences available in GenBank: for IL-6, 5'-CTGGTGACAACCACGGCCTCCCCT-3' and the 3' primer
5'-TGGATCACGCAATACGGATTCGTA-3', which amplify a 620 base
pair (bp); for interleukin 10 (IL-10), 5'-CCAGTTTTACCTGGTAGAAGTGATG-3' and the 3' primer
5'-TGTCTAGGTCCTGGAGTCCAGCAGACTCAA-3', which amplify a 440 bp; for -actin, 5'-TGGAATCCTGTGGCATCCATGAAAC-3' and the
3' primer 5'-GCCTGACAATGACTCGACGCAAA-3', which
amplify a 348 bp; and for JunD, 5'-ATGGAAACGCCCTTCTATGGC-3'
and the 3' primer 5'-CGTTCTTGCGTGTCCATGTCG-3', which
amplify a 783 bp.
Reverse transcription and polymerase chain reaction (PCR) were done by
using the Titan one-tube RT-PCR System (Boehringer Mannheim). The
linear range of amplification was determined by measuring gene
expression after 25 to 35 cycles (nonsaturated PCR product). After the
PCR reaction, loading buffer was added to each 5-µL reaction and
aliquots were resolved on 2% agarose gel containing 0.008% ethidium
bromide. DNA molecular-weight standards were run in parallel.
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Results |
Peritoneal mast cells
Peritoneal mast cells are different in many respects from cultured
BMMC. For instance, they contain heparin in their granules that is
stained strongly by safranin, whereas BMMC contain chondroitin sulfate
E in their granules that is stained by Alcian blue.19 In
vitro, BMMC can differentiate into heparin-containing mast cells if
coincubated with fibroblasts or c-kit ligand.25,26 Thus, primary mast cell cultures like BMMC are now considered precursors of heparin-containing mast cells. If heparin-containing mast
cells are dependent on PKC for their differentiation, one would not
expect to see peritoneal mast cells from PKC-deficient mice stained
with safranin. However, we found that the peritoneal mast cells in
cytospin preparations from mouse peritoneum from both control and
PKC-deficient mice were stained with safranin (Figure
1A and Figure 1B). Hence, PKC-deficient
mice, in contrast to mouse strains such as c-kit-deficient
mice, contain large numbers of mast cells that can differentiate into
heparin-containing mast cells.
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| Fig 1.
Light micrographs of peritoneal mast cells from control
mice (A) and mice deficient in protein kinase C(B).
Peritoneal exudates were stained with Alcian blue and counterstained
with safranin.
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Replication and survival
Our initial assessment of BMMC cell growth was by cell count after
2, 3, and 4 weeks of cell culture. At this stage, most of the cells
were mast cells (as determined by toluidine blue staining). BMMC cell
density increased in similar proportions in control and
PKC-deficient mast cells (data not shown). We then measured cell
proliferation in 3 individual experiments. BMMC derived from wild-type
and from PKC-deficient mice were incubated for 48 hours in the
presence of increasing concentrations of IL-3 and were assessed for
their rate of proliferation by tritium-thymidine incorporation into DNA
and cell number (Figure 2). A similar
pattern of IL-3 dose-dependent proliferation rate was observed in these 2 types of BMMC. Thus, the deficiency of PKC in these cells did not
affect their rate of proliferation.
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| Fig 2.
Dose response to interleukin 3 of mast cell DNA
synthesis.
Protein kinase C (PKC) -deficient and wild-type mast cells were
incubated for 24 hours with medium containing increasing doses of
interleukin 3 (IL-3) and then with tritium-thymidine for 4 hours. The
level of incorporation of radioactivity into DNA (mean ± SE) was
quantified for each condition in quadruplicate. One representative
experiment of 3 is shown.
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The effect of IL-3 deprivation on apoptosis of BMMC derived from the
PKC-deficient mice was also assessed in 3 separate experiments. These experiments were carried out with 2 different staining reagents: annexin V, which stains phosphatidylserine and thus determines an early
event in apoptosis23; and 7AAD, which stains DNA and so
reveals apoptotic cells at a later stage.20
PKC-deficient and wild-type mast cells were incubated with or
without IL-3 for 18 hours and then stained with annexin V and 7AAD. As
shown in Figure 3A and Figure 3B, the rates
of apoptosis after IL-3 deprivation were similar in PKC-deficient
and control BMMC.
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| Fig 3.
Fluorescence flow cytometry analysis of apoptotic mast
cells.
(A) PKC-deficient and wild-type mast cells were incubated with IL-3
(upper panels) or without IL-3 for 18 hours and stained with annexin V
and 7-amino actinomycin D (7AAD). The left-hand column shows the
forward- and side-scatter analysis of the cells. The right-hand column
shows a 2-dimensional representation of cells derived from the
left-hand column by using annexin V and 7AAD. (B) Graph based on
results obtained by fluorescence-activated cell separation analysis
done in a manner similar to the analysis shown in Figure 2A for
PKC-deficient mast cells compared with wild-type cells after growth
factor deprivation. (C) A similar graph for cells treated with
mitomycin C. Here, the cells were grown with only 20 U of IL-3.
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Mitomycin C is a chemotherapeutic agent that has been shown to induce
apoptosis in a variety of cells. Therefore, the effect of mitomycin C
on both wild-type and PKC-deficient BMMC was determined. Cells were
grown in medium containing a suboptimal concentration of IL-3 (20 U/mL)
that was found to prevent BMMC apoptosis and 30 µmol/L of mitomycin
C. No significant difference in the apoptotic rate was observed between
wild-type and PKC-deficient BMMC (Figure 3C). Thus, deficiency of
PKC in mast cells did not affect their rate of proliferation or apoptosis.
Degranulation
Wild-type and PKC-deficient BMMC were sensitized with monoclonal
mouse IgE against HSA-DNP and challenged with increasing doses of HSA-DNP-released -hexosaminidase (Figure
4). In 4 consecutive experiments,
degranulation was 52% ± 6% (mean ± SE) lower in BMMC derived
from the PKC-deficient mice than in wild-type BMMC.
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| Fig 4.
Dinitrophenol-human serum albumin antigen dose-dependent
net percentage release of -hexosaminidase from IgE-sensitized
PKC-deficient mast cells and wild-type cells.
One representative experiment of 4 is shown.
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In 3 separate experiments, cells were incubated for 20 minutes at
37°C with increasing concentrations of ionomycin. Degranulation was
75% ± 7% (mean ± SE) lower in the PKC-deficient BMMC than in the wild-type BMMC (Figure 5).
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| Fig 5.
Ionomycin dose-dependent net percentage release of
-hexosaminidase from PKC-deficient mast cells and wild-type
cells.
One representative experiment of 3 is shown.
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PKC-deficient BMMC and cytokine mRNA accumulation
Our initial experiments to assess cytokine release from BMMC
failed to detect important amounts of cytokines in the medium, probably because the preincubation period with IgE was only 2 hours.
Because several articles reported up-regulation of IgE receptors by
IgE,27-29 we preincubated the cells for 4 days with IgE before stimulating them with the antigen for 8 to 10 hours. The
culture medium was then collected and the mRNA was extracted from the cells.
Because it was demonstrated that IL-10 is induced in human mast cells
by IgE-Ag stimulation,30 we investigated IL-10 mRNA accumulation in PKC-deficient and wild-type BMMC with and without IgE-Ag activation, with -actin as the control. The level of
induction of IL-10 was similar in PKC-deficient and wild-type cells
(Figure 6A), showing that there is probably
no general defect in IgE-Ag induction in PKC-deficient BMMC.
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| Fig 6.
IgE-Ag-mediated interleukin 10 (A), interleukin 6 (B),
and JunD (C) mRNA accumulation in bone marrow mast cells from
PKC-deficient and wild-type mice.
RNA isolated from IgE-Ag-stimulated mast cells was reverse transcribed
with specific primers and then amplified. Polymerase chain reaction
products were resolved on 2% agarose gel containing 0.008% ethidium
bromide.
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Overexpression of PKC in RBL cells induces the accumulation of IL-6
mRNA.13 Therefore, we measured the accumulation of IL-6
mRNA in IgE-Ag-activated PKC-deficient BMMC. Figure 6B shows that
the mRNA level of IL-6 was significantly reduced in the
PKC-deficient BMMC compared with the wild-type cells.
The homodimer of JunD/JunD has been suggested to be a regulator of IL-6
gene expression.14 We showed that, in mast cells, the
expression of JunD on the level of mRNA is dependent on
PKC.5 We therefore decided to determine whether the level
of JunD mRNA accumulation is different in BMMC derived from
PKC-deficient mice compared with wild-type BMMC. As shown in Figure
6C, compared with the findings in wild-type BMMC, there was indication
of a reduction in the level of JunD mRNA in PKC-deficient BMMC after activation.
To determine whether the PKC deficiency in BMMC also inhibited IL-6
production, we measured the FcRI-dependent synthesis of IL-6 in
these BMMC (Figure 7). No IL-6
protein was detected in the resting BMMC. However, the average results
from 3 separate experiments showed that IL-6 was significantly reduced
in the PKC-deficient BMMC (381 ± 66 pg/mL per 106
cells) compared with the wild-type BMMC (769 ± 154 pg/mL per 106 cells). Thus, the PKC deficiency in these cells
significantly affected their capacity to produce IL-6.
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| Fig 7.
IgE-Ag mediated interleukin 6 synthesis in bone marrow
mast cells from PKC-deficient and wild-type mice.
Cells were incubated with IgE for 4 days and with antigen for 8 to 10 hours. Supernatants were frozen at 70°C until assayed for
interleukin 6 by enzyme-linked immunosorbent assay. Results (mean ± SE) are shown for 3 separate experiments.
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Discussion |
The finding of peritoneal mast cells stained with safranin in
peritoneal exudates from PKC-deficient mast cells shows that PKC-deficient mice contain mast cells that can differentiate into
heparin-containing cells. Because no previous studies indicated that
PKC is essential for mast cell proliferation or differentiation, this result was not unexpected, and we therefore focused on the analysis of the specific functions of mast cells by using BMMC.
Our hypotheses regarding specific differences between control and
PKC-deficient mast cells were based on 2 kinds of evidence that obtained by using mice lacking in Btk, which display an immunophenotype very similar to PKC-deficient mice15; and that obtained
with in vitro cellular studies. The similarity between deficiency of Btk and deficiency of PKC has been shown in studies in knockout mice. One explanation for the similarity in phenotype could be an
association between Btk and PKC. However, since signal-transduction pathways can vary substantially between different cell types, finding a
difference in the phenotype of these 2 mutant cell types was not
surprising. Our observation that PKC-deficient mast cells undergo
apoptosis at the same rate as control cells in response to
growth-factor deprivation is in contrast to the situation in Btk-deficient mast cells. Btk-deficient mast cells undergo apoptosis at
a lower rate than control mast cells in response to growth-factor deprivation.16 Several explanations for the difference in
phenotype between Btk-deficient and PKC-deficient mast cells could
be postulated, such as the use of a different kind of PKC for
pathways involving Btk for the induction of apoptosis in
PKC-deficient mast cells. Additional work is needed to explain the
differences in the phenotypes.
Most chemotherapeutic agents cause cell death by inducing apoptosis.
Interestingly, safingol, a PKC inhibitor, was shown to greatly enhance
mitomycin C-induced apoptosis of gastric tumor cells.31
Others reported that PKC inhibition reduced the apoptotic rate of B
lymphocytes.32 We therefore tried to assess the
chemotherapy-induced apoptotic response of PKC-deficient mast cells.
However, even in this context, we observed no significant difference
between PKC-deficient BMMC and wild-type BMMC in the mitomycin
C-induced death rate.
Our observations that the total number of mast cells grown with IL-3
and the proliferation rates were not different in control and
PKC-deficient mast cells (Figure 2) indicate that PKC has a
nonessential role (if any) in mast cell growth. This is in disagreement with the evidence from studies in a rat mast cell line that
overexpressed PKC and had an increase in cellular proliferation
rate.13 Unless these results are due to dissimilarities of
the mast cell subtypes assessed in the different studies, it seems that
although an abnormal signal from PKC can increase cellular
proliferation rate, this enzyme does not have an essential role in this
process. In contrast to this discrepancy, our finding of a substantial
decrease in mast cell degranulation in PKC-deficient mast cells
compared with control cells (Figure 4) is in agreement with results
from reconstitution and overexpression experiments. Although
PKC could also allow degranulation in reconstitution experiments,
it could not totally replace the absence of PKC in cells
lacking this enzyme. Hence, PKC has an essential role in degranulation.
Because ionomycin was unable to rescue the degranulation defect in the
PKC-deficient mice, it appears that PKC might function further
downstream of the FcRI receptor itself. It has been shown that
calcium ionophore-induced secretion from mast cells correlates with
myosin light-chain phosphorylation by PKC33; thus, myosin light-chain might present a target for PKC. Alternatively, it has
been reported that PKC is involved in the regulation of coat protein
assembly in mast cells and therefore in the modulation of
secretion.34 Additional experiments are needed to elucidate the physiologic target of PKC in mast cells.
IgE-Ag induction of 2 cytokine mRNAs, IL-6 and IL-2, was increased in
mast cells that overexpressed PKC.13 We detected a
decrease in IL-6 released by activated PKC-deficient mast cells compared with control cells. An even greater decrease in the amount of
IL-6 mRNA was observed in activated PKC-deficient mast cells compared with control cells. It is almost impossible to determine whether the decrease in the level of IL-6 protein in mast cells activated overnight was due to a decay in the level of IL-6 mRNA. Additional work must be performed to find out whether the control of
IL-6 synthesis is on the transcriptional, translation, or mRNA accumulation level. Experiments to investigate calcium flux in these
cells would be of great interest because degranulation is dependent on
calcium, and IL-6 production is probably dependent on calcium by means
of the activation of an NF-AT or NF-AT-like factor, although a
classical NF-AT site is not present in the IL-6 promoter.35
Interestingly, there was no significant change in the amount of mRNA
for IL-10 between the different cell types, which suggests that there
is no general defect in IgE-receptor signal transduction in these
cells. In contrast, mRNA levels of JunD, which has been shown to be a
regulator of the IL-6 gene,14 were lower in activated
PKC-deficient mast cells than in wild-type cells. This suggests that
JunD induction by PKC is important for IgE-Ag induction of the IL-6
gene. In previous work, we demonstrated that JunD mRNA accumulation is
dependent on PKC.5 We also showed that the PKC isozyme
is the predominant PKC isozyme in BMMC.8 Therefore, it is
not surprising that in this study we found indication that
PKC-deficient BMMC had lower levels of IgE-induced JunD mRNA.
Thus, our study not only supports the main conclusions from
reconstitution and overexpression experiments regarding the role of
PKC in signal transduction leading to degranulation and cytokine induction, but it also shows that under normal conditions the role of
this enzyme is not redundant and cannot be totally replaced by other
PKC isozymes. Specific PKC isozyme inhibitors are currently being
formulated. Studies using different PKC knockout mice should prove to
be invaluable not only for assessing the possible effects of such
inhibitors but also for increasing our understanding of the complex
role of different PKC isozymes in signal transduction in different cell types.
| |
Acknowledgments |
We thank the members of the A. Tarakhovsky's laboratory, especially I. Mecklenbrauker.
| |
Footnotes |
Submitted August 3, 1999; accepted November 10, 1999.
Supported by grants from the Israel Academy for Science (to E.R.) and
the Israeli Ministry of Health (to E.R.). H.N. was supported by a
short-term EMBO fellowship.
Reprints: Ehud Razin, Department of Biochemistry, Hebrew
University-Hadassah Medical School, PO Box 12272, Jerusalem, 91120, Israel; e-mail: ehudr{at}cc.huji.ac.il.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction