(Received for publication, October 9, 1995; and in revised form, November 7, 1995)
From the
The ability of c-Fos to dimerize with various proteins creates
transcription complexes which can exert their regulatory function on a
variety of genes. One of the transcription factors that binds to c-Fos
is the newly discovered Fos-interacting protein (FIP). In this report
we present evidence for the regulation of the synthesis of FIP by a
physiological stimulus. We found that the aggregation of the mast cell
high affinity receptor for IgE (FcRI) induced the synthesis of FIP
and increased its DNA binding activity. Moreover, down-regulation of
the isoenzyme protein kinase C-
(PKC-
) by a specific
antisense phosphorothioate oligonucleotide resulted in profound
inhibition of FIP-Fos DNA binding activity. Thus, aggregation of the
Fc
RI on mast cells elicits a PKC-
dependent signaling pathway
which regulates FIP-Fos DNA binding activity.
IgE-antigen (IgE-Ag)()-mediated activation of mast
cells results in the phosphorylation of the cell surface IgE Fc
receptor (Fc
RI), priming the receptor for direct interactions with
effector molecules that initiate diverse signal transduction pathways (1) . These signaling pathways ultimately lead to the release
of mast cell granule contents and the activation of genes encoding
inflammatory products.
Some of these genes are known to be regulated
by AP-1 activity(2, 3) . The AP-1 is a complex of
transcription factor proteins which play an important role in mitosis
and, in particular, the entry of cells into S
phase(4, 5, 6) . The AP-1 is comprised of a
Jun homodimer or a Fos-Jun heterodimer. The Fos protein was found to be
incapable of forming a homodimer but rapidly forms a highly stable
complex via a leucine zipper interaction with the Jun protein or with
selected members of the family of activating transcription
factor/cAMP-responsive element-binding proteins(7) . As a
heterodimer, Fos and Jun activate transcription through the AP-1
binding site found in the promoter region of a variety of
genes(2, 3) . The binding of this heterodimer to its
DNA binding site may in part be regulated by the phosphorylation of
Jun. The phosphorylation of Jun requires both mitogen-activated protein
kinase and protein kinase C (PKC) activities(8) . Recently we
found that the aggregation of the FcRI, on the
interleukin-3-dependent murine fetal liver derived mast cells (MC-9),
increased the AP-1 DNA binding activity in these cells, a process found
to be dependent on PKC activity. We also found that in IgE
antigen-activated MC-9 cells most of the newly synthesized c-Fos was
bound to proteins other than Jun.
The USF family represents one class of proteins which can interact with Fos(9) . The protein product of USF-2 is generally referred to as Fos-interacting protein (FIP)(10, 11) . Members of this family recognize the DNA sequence of CACGTG(10) . They are also characterized by the leucine zipper (Zip) and the helix-loop-helix (HLH) dimerization motifs that are found in various eukaryotic transcription factors and makes these proteins members of the basic region Zip (bZip) and/or basic region HLH (bHLH) families. Family members include TFE3, USF-1, USF-2, TFEB, AP-4, Myc, and Max(11) . The presence of the bHLH/Zip functional domains permits the formation of homodimers and heterodimers among the family members. The formation of these complexes may be regulated by protein kinase activity(8) .
Protein kinase C
regulatory enzymes have been implicated in physiological processes such
as membrane receptor function, cell differentiation, and
proliferation(12) . The functional diversity of PKC may in part
be mediated by distinct isoenzymes that play an important role in both
the activation and regulation of signaling pathways in the cell. Recent
studies in rat basophilic leukemia cells, a rat analog of mucosal mast
cells, begin to explore the role of these isoenzymes in mast cell
function. Ozawa et al. (13) found that a full
secretory response to antigen could be reconstituted by either
PKC- or -
, but not by PKC-
or -
. Our own
experiments, in these cells, showed that the increased activity of
PKC-
and -
, activated by the aggregation of the Fc
RI,
resulted in the induction of c-Fos and c-Jun mRNA
synthesis(14) . Recently we also observed a PKC-dependent
increase in the binding of the FIP-Fos complex to its target DNA in
response to the aggregation of the Fc
RI(9) . In the
present study we assess the relationship between the aggregation of the
Fc
RI, activation of PKC isoenzyme activity, synthesis of FIP
protein, and the FIP-Fos DNA binding activity.
Serum antibodies to the synthetic
peptide were assayed by enzyme-linked immunosorbent assay using 20 mg
of peptide and 2 mg of BSA as control per well. Both preimmune and
immune sera were diluted by serial 10-fold dilutions to a final of
10. The end point titer was defined as the immune
serum dilution that gave values of absorbance of at least 0.2, these
values were 10 times higher than the mean background absorbance for
BSA. Detection of bound antibody was by using anti-rabbit IgG coupled
with alkaline phosphatase and 1 mg/ml p-nitrophenyl phosphate
as substrate for colorimetric detection at 405 nm. Depletion of
antibody reactivity was achieved by preincubating the serum abs at
10
dilution with MC-9 cell extracts prior to the
assay. This resulted in an 80% reduction in antibody reactivity to
peptide antigen.
Immunoprecipitation was essentially as described
previously (21) with the following modification: 2.5
10
cells were lysed by the addition of 500 µl of cold
lysis buffer (0.01 M Tris-HCl, pH 7.4, 1% deoxycholate, 1%
Triton X-100, 0.1% SDS, 0.15 M NaCl, and 0.25 mM phenylmethylsulfonyl fluoride). Cells were subsequently
homogenized, and supernatants were collected after 30 min of
microcentrifugation at 4 °C. Antibody to mouse FIP, which had been
preincubated in the absence or presence of the FIP peptide, was added
to the supernatants. After an overnight incubation at 4 °C, 10 mg
of protein A-Sepharose (Sigma) was added, and the mixture was incubated
for 3 h at 4 °C with mixing. The recovered immunoprecipitate was
washed three times with lysis buffer and once with Tris-EDTA. Proteins
were solubilized in boiling Laemmli sample buffer containing 0.5% SDS.
The proteins were resolved by 10% SDS-PAGE. Gels were dried and exposed
to Kodak X-Omat AR film at -70 °C for varying times.
Quantitation of the autoradiography was by densitometric analysis.
Figure 1:
Identification of the FIP protein by
antibody to mouse FIP in [S]methionine-labeled
MC-9 cells. A, [
S]methionine-labeled
IgE-sensitized MC-9 cells were either exposed for 60 min to DNP-BSA or
not (control). FIP protein was identified by immunoprecipitation of
cell lysates with antibody to mouse FIP which had been preincubated in
the absence or presence of FIP immunogen peptide prior to the addition
of the antibody to the cell lysates. Immunoprecipitates were analyzed
by SDS-PAGE. B, [
S]methionine-labeled
cell lysates from either MC-9 or KU812 cells were immunoprecipitated
with rabbit antibody to mouse FIP and analyzed by SDS-PAGE. Each sample
was run in duplicate. One representative experiment out of three is
shown.
Figure 2:
Kinetics of the expression of FIP in
IgE-Ag-stimulated MC-9 cells. Immunoprecipitates of the
[S]methionine-labeled FIP. Cells were sampled at
the indicated times, lysed, soluble proteins recovered, and incubated
with antibody to mouse FIP. Proteins were resolved by SDS-PAGE. One
representative experiment of three is
shown.
Figure 3: Identification of FIP in Fos-containing complex bound to the FBS. Cell lysates were prepared from MC-9 cells. Twenty µg of each lysate was incubated with radiolabeled FBS sequence in the absence (control) or presence of a 100-fold excess of unlabeled FBS (compet. FBS) or in the presence of polyclonal antibodies to c-Fos (anti-Fos) or antibody to mouse FIP (anti-FIP) or to normal rabbit serum (NRS), prior to analysis by nondenaturing PAGE and autoradiography. One representative experiment of three is shown.
To study which of the PKC isoenzymes is involved in
FIP-Fos and AP-1 DNA binding activities, the PKC isoenzymes were
down-regulated by 6 h of treatment with PMA, followed by incubation
with an antisense oligonucleotide, directed to the C2 domain of
c-PKC(12) , for an additional 48 h. Immunoblots revealed a
decrease of 65% ± 16% (n = 3) in the levels of
PKC- (Fig. 4). Only a slight decrease in the levels of
PKC-
was observed, while PKC-
and -
levels were
unaffected. Controls treated with sense oligonucleotides were minimally
affected (Fig. 4). To determine the role of PKC-
in the DNA
binding activities of FIP-Fos and AP-1, gel shift assays were performed
using either a synthetic double-stranded TRE or FBS. We initially
confirmed our previous results that aggregation of the Fc
RI on
MC-9 cells could cause an increase in the DNA binding activities of
both FIP-Fos and AP-1 complexes(9) . Kinetic analysis of the
DNA binding activity revealed that both FIP-Fos (Fig. 5) and
AP-1 (data not shown) binding reached a maximum at 15-30 min
after antigen stimulation. When PKC-
was down-regulated, FIP-Fos
DNA binding activity decreased by an average of 50% (n = 6) in IgE-Ag-stimulated MC-9 cells (Fig. 6A). In contrast, no effect was observed on the
binding activity of AP-1 in the PKC-
-depleted cells (Fig. 6B). These results suggest that the aggregation
of the Fc
RI in mast cells induces a PKC-
-dependent signaling
pathway for FIP-Fos DNA binding activity.
Figure 4:
Down-regulation of PKC- by antisense
oligonucleotide. MC-9 cells were incubated with PMA for 6 h, washed
extensively with growth medium, and further incubated with (ANTISENSE) or without (NOT-TREATED) 10 µM antisense or sense oligonucleotides (SENSE) for an
additional 48 h as described under ``Materials and Methods.''
An additional dose of oligonucleotides was added to the culture medium
once during the incubation period. One representative experiment of
three is shown.
Figure 5: Kinetics of the DNA binding activity of the FIP-Fos complex. Cell lysates were prepared from IgE-sensitized MC-9 cells which were activated (DNP-BSA) or not (control) for the indicated time. Twenty micrograms of each cell lysate was incubated with radiolabeled FBS in the presence (compet. FBS) or absence (control) of 100-fold excess of unlabeled FBS oligonucleotide prior to analysis by nondenaturing PAGE and autoradiography. One representative experiment of three is shown.
Figure 6:
The
effect of PKC- activity on the DNA binding activity of the FIP-Fos
complex and of AP-1. A, cell lysates were prepared from
IgE-sensitized MC-9 cells which were activated (DNP-BSA) or
not after pretreatment with PMA for 6 h and the addition of sense or
antisense oligonucleotides as described under ``Materials and
Methods.'' Twenty micrograms of each lysate was incubated with
radiolabeled FBS in the presence (compet. FBS) or absence of
100-fold excess of unlabeled FBS oligonucleotide. In addition cell
lysates were incubated with radiolabeled FBS in the presence of
100-fold excess TRE consensus sequence oligonucleotide (compet.
TRE) as an additional control for specificity of FBS binding.
Analysis was by nondenaturing PAGE followed by autoradiography. One
representative experiment of two is shown. B, cell lysates
were prepared from IgE-sensitized MC-9 cells which had been activated (DNP-BSA) or not after treatment as in A. Twenty
micrograms of each lysate was incubated with radiolabeled TRE consensus
binding sequence in the presence (compet. TRE) or absence of
100-fold excess of unlabeled TRE oligonucleotide, prior to analysis by
nondenaturing PAGE and autoradiography. One representative experiment
of two is shown.
Figure 7:
The effect of down-regulation and recovery
of PKC- activity on the expression of FIP. A, MC-9 cells
were incubated with or without antisense or sense oligonucleotides as
described under ``Materials and Methods.'' The
[
S]methionine-labeled cells were then activated
with IgE-Ag. Cells were lysed and soluble proteins immunoprecipitated
with antibody to mouse FIP. Proteins were resolved by SDS-PAGE. One
representative experiment of three is shown. B, cells were
treated or not (control) with PMA for 6 h followed by
treatment with PKC-
sense or antisense for 48 h as described under
``Materials and Methods.'' Following the 48-h incubation with
oligonucleotides, the cells were washed and incubated for an additional
24 h without the oligonucleotides to allow recovery of PKC-
. This
was followed by [
S]methionine labeling of the
cells and activation by IgE-Ag. Cell lysates were prepared and FIP was
immunoprecipitated by incubation with antibody to mouse FIP. Proteins
were resolved by SDS-PAGE. One representative experiment of three is
shown.
Our previous studies have described the possible involvement
of FIP in regulating AP-1 activity in MC-9 cells when these cells are
activated by IgE-Ag(9) . The analysis of the role of FIP was
limited in these studies by the unavailability of antibody to FIP.
Nevertheless, we defined the presence of FIP complexed to Fos by
analyzing the ability of a Fos-containing complex to bind to the
FIP-DNA binding site (9) , since Fos alone does not bind to
this DNA sequence. In the present study we show that FIP is present in
the MC-9 cells by first identifying a FIP clone with 100% homology to
mouse USF-2 (10) from a cDNA library derived from
IgE-Ag-stimulated MC-9 cells. Second, we identify the presence of FIP
protein with antibody generated to a FIP-specific peptide sequence (Fig. 1). We further demonstrate that IgE-Ag stimulation of MC-9
cells regulates the synthesis of FIP protein and that PKC-
regulates both the synthesis of FIP and its DNA binding activity.
Although PKC- was found to be the predominant isoenzyme in MC-9
cells, differential localization of the various PKC isoenzymes may lead
to their involvement in the regulation of FIP expression and DNA
binding activity. Moreover, the relative enzymatic activity of the
individual PKC isoenzymes does not necessarily correlate with their
intracellular concentrations. Thus, we cannot exclude the possibility
that other isoenzymes of PKC may also participate in regulating the
expression and DNA binding activity of FIP. However, our results are
conclusive for a role of PKC-
activity in either directly or
indirectly regulating the synthesis of FIP and of FIP DNA binding
activity in response to the aggregation of the Fc
RI.
Protein
kinase C- plays a major role in the secretory response of mast
cells (13) and also links the Fc
RI to the induction of
AP-1 component expression (14) in the rat basophilic leukemia
cell line. However in rat basophilic leukemia cells, PKC-
is the
predominant isoenzyme(14) , while in the murine MC-9 cells,
PKC-
is the most highly expressed. These differences may be
species-specific (rat versus mouse), influenced by the state
of differentiation, state of retroviral/chemical transformation, or
growth conditions, yet PKC-
regulates transcription factor
synthesis in both cell lines. Nevertheless it still remains unclear as
to how PKC-
is capable of regulating the secretory response,
expression of FIP, and expression of AP-1 components in mast cells. It
could be argued that the calcium influx in response to the aggregation
of the Fc
RI leads to the preferential activation of the
calcium-dependent PKC-
. In contrast, activation of the
calcium-independent PKC-
, which also is capable of stimulating
c-Fos and c-Jun expression but inhibits phosphatidylinositol
hydrolysis(23) , might be attenuated in vivo by
negative regulatory influences in response to aggregation of the
Fc
RI. Furthermore, differences in the amino acid sequence of these
isoenzymes may determine their interactions with other proteins in
vivo which may play a role in the regulation of their activity and
thereby of cellular responses.
Based on the results of this study we
conclude that the induction of FIP synthesis and increased FIP-Fos DNA
binding activity, by aggregation of the FcRI, requires the
activity of PKC-
. According to our results the decrease in FIP-Fos
DNA binding activity in the antisense oligonucleotide-treated cells
could at minimum be partially attributed to the reduction in the level
of FIP in these cells. The ability of Fos to bind to other nuclear
proteins such as FIP may generate novel complexes that regulate
transcription in a receptor-specific manner. Furthermore, formation of
receptor-specific complexes may serve to regulate gene transcription in
a direct and indirect manner. For the latter, it is well known that the
affinity of binding of Fos-Jun to the TRE is greater than that of
Jun-Jun(2) . Thus, the formation of FIP-Fos complexes may
attenuate the activity of the AP-1 by decreasing the amount of Fos that
might be available for association with Jun. Thus, the
receptor-specific synthesis of FIP would indirectly control genes whose
expression was regulated by AP-1. Protein kinase C-
would also
influence this transcriptional regulation by increasing the formation
of FIP-Fos complexes and increasing the DNA binding activity of this
complex.
The specificity of the antisense oligonucleotide approach
was determined in our system by the specific inhibition of the
expression of only PKC- and not other isoenzymes and by the
inability of sense oligonucleotides (24) to cause a similar
effect. However, we cannot exclude the possibility that the effect
observed in this study was due to an indirect effect of the antisense
oligonucleotides that ultimately resulted in the down-regulation of
PKC-
. As previously mentioned the antisense oligonucleotide was
designed to the C2 domain responsible for the calcium dependence of
c-PKC(12) . The amino acid sequence of this site is 90%
homologous between the murine PKC-
and
-
(17, 25) . Despite the high homology there was
only a minimal decrease in the level of PKC-
, while the expression
of PKC-
was decreased by greater than 60%. This preferential
effect of the antisense might be explained by the difference in the
concentrations of PKC-
and -
in MC-9 cells. Thus, since
PKC-
is present in these cells at a concentration greater than
2-fold that of PKC-
, it is possible that the oligonucleotide which
entered the cells could be preferentially bound by the PKC-
mRNA.
Alternatively, the preferential binding of the oligonucleotide to
PKC-
might be explained by differences in the variable regions
adjacent to the C2 domains of these isoenzymes that may influence the
conformation and availability of these domains.
Examples for the cross-talk between signaling pathways in biological systems are growing in number. The ability of c-Fos to bind to various proteins enables the cross-talk between the enzymes that are activated by receptor stimulation and genes that are activated or inactivated as a consequence of a particular stimulus. It has to be determined whether other stimuli, like growth factors, might initiate an association of c-Fos with c-Jun rather than with FIP. Furthermore, it is possible that the PKC isoenzymes involved in increasing the DNA binding activity of the AP-1 complex differ from that which initiate binding at the FBS. This complexity in regulation of gene expression might be expected in light of the diverse responses initiated by diverse stimuli.