(Received for publication, June 7, 1995; and in revised form, December 11, 1995)
From the
Adhesion of RBL-2H3 mucosal mast cells to fibronectin-coated surfaces has been linked to changes in secretion and tyrosine kinase activity. We now show that adhesion affects the sensitivity of RBL cells to the protein kinase C activator phorbol 12-myristate 13-acetate (PMA). In suspended cells, PMA inhibited antigen-induced calcium influx (as measured by manganese influx) and changes in intracellular free calcium and had complex effects on antigen-stimulated secretion. However, in adherent cells PMA had little effect on these responses. Suspended cells only secreted in response to thapsigargin if they were co-treated with PMA, while adherent cells secreted in response to thapsigargin alone. The thapsigargin-induced secretion in adherent cells was inhibited by protein kinase C down-regulation and by the protein kinase C inhibitor GF 109203X, but not by calphostin C. We suggest that protein kinase C is constitutively activated in adherent cells, possibly due to modification of the regulatory domain of the enzyme.
The RBL-2H3 mucosal mast cell line has been used extensively as
a model of stimulus secretion coupling(1) . Activation of these
cells by antigen leads to a complex series of events including tyrosine
phosphorylation of various proteins(2, 3) , including
the receptor for immunoglobulin E (IgE)()(4) ,
phosphoinositide breakdown (5) leading to activation of protein
kinase C(6) , emptying of intracellular calcium stores by
inositol 1,4,5-trisphosphate (IP
)(7, 8) ,
and influx of calcium across the plasma
membrane(9, 10, 11) . These events culminate
in the secretion of various mediators of the inflammatory
response(1, 8) . It is clear that both the increase in
intracellular calcium and protein kinase C activation are important
steps in the signaling pathway and that these two signals act
synergistically to promote secretion(12, 13) .
Activation of protein kinase C with the phorbol ester phorbol
12-myristate 13-acetate (PMA), alone, does not induce secretion in rat
basophilic leukemia (RBL) cells(12, 13, 14) .
Some laboratories have reported that PMA potentiates antigen-induced
secretion at concentrations below 15
nM(12, 15) , but other reports do not support
this finding(13, 14) . Nevertheless, there is general
agreement that PMA markedly potentiates secretion in response to
calcium ionophore(12, 13, 14) . A similar
synergism has been seen when protein kinase C is activated by PMA while
intracellular calcium is increased by treatment with the endoplasmic
reticulum Ca-ATPase inhibitors thapsigargin (
)or cyclopiazonic acid(16) . Additionally, the
protein kinase C inhibitors staurosporine, Ro31-7549, and
calphostin C have been shown to inhibit antigen-stimulated
secretion(17) . In general, it appears that the combination of
protein kinase C activation and increases in intracellular calcium are
sufficient to induce secretion.
In addition to promoting secretion,
activation protein kinase C by PMA has a second, inhibitory effect on
RBL cells in suspension(12, 13, 15) .
Increases in intracellular Ca are inhibited at
concentrations above 10
nM(12, 13, 15) , possibly by the
inhibition of phospholipase C-
(13, 18) , thus
preventing phosphoinositide breakdown. Some groups have also shown that
antigen-stimulated secretion is inhibited by high concentrations of PMA (12, 15) , presumably due to the inhibition of the
Ca
response.
In the past, experiments on RBL cells
have been performed interchangeably with cells in suspension or with
adherent cells. However, recent experiments have shown that adhesion
itself affects RBL cell responses. Adhesion of RBL cells results in the
tyrosine phosphorylation of several proteins including pp125(19) . In addition, antigen-stimulated secretion is
enhanced in adherent RBL cells(20) . In studying the effects of
protein kinase C activation on secretion and calcium handling, we have
discovered another effect of adhesion on RBL cell responses, namely a
loss of sensitivity to the effects of the protein kinase C activator,
PMA.
Immunoprecipitation of protein kinase C isozymes for analysis of
tyrosine phosphorylation was done as described previously(27) .
Triton X-100 (final concentration 0.5%) was added to the remaining
volume of the particulate fraction (see above), and the detergent
lysates were used for immunoprecipitation of the individual protein
kinase C isozymes. Antibodies for immunoprecipitations have been
described(28) . Proteins were resolved and transferred to
nitrocellulose as above. The tyrosine phosphorylation of protein kinase
C-, -
, and -
derived from suspended or adherent cells
was analyzed by immunoblotting with a mouse monoclonal antibody to
phosphotyrosine (4G10, Upstate Biotechnology, Inc., Lake Placid, NY).
Tyrosine phosphorylation of the
isozyme was not assessed due to
the unavailability of an immunoprecipitating antibody. Detection was by
enhanced chemiluminescence, and relative quantitation of immunoblots
was performed by densitometry as described(29) .
Figure 1:
PMA shows both
enhancing and inhibitory effects on antigen-stimulated secretion in RBL
cell suspensions, but has much less effect on secretion in adherent
cells. Antigen-stimulated -hexosaminidase secretion was measured
in suspended (A) and adherent (B) RBL-2H3 cells in
the presence of the indicated concentrations of PMA. Spontaneous
secretion was subtracted from stimulated secretion at each PMA
concentration. Data are expressed as a fraction of control
(antigen-stimulated secretion without PMA) and represent the mean and
standard deviation of four experiments. Control secretion was 31.9
± 10.5% in suspended cells and 45.2 ± 12.1% in adherent
cells. Inset, a single experiment with suspended cells showing
the mean and range of two replicates. The antigen concentration was 1
µg/ml. Spontaneous secretion was 5.5 ± 1.7% in suspended
cells and 8.3 ± 2.0% in adherent cells; it was unaffected by
PMA.
Fig. 2shows that the protein
kinase C inhibitor GF 109203X (30) inhibits antigen-stimulated
secretion in both suspended and adherent RBL cells, thus confirming the
central role of protein kinase C in secretion from RBL cells. Although
high concentrations of PMA can abolish antigen-stimulated secretion
from cells in suspension (Fig. 1A), while PMA has
little effect on adherent cells (Fig. 1B), the results
in Fig. 2clearly demonstrate that protein kinase C activity is
necessary for secretion in both adherent and suspended cells. This
result supports previous studies showing that secretion can be
reconstituted in protein kinase C-depleted cells by the protein kinase
C isozymes and
(31) .
Figure 2:
The
protein kinase C inhibitor GF 109203X inhibits antigen-stimulated
secretion in suspended and adherent cells. Antigen-stimulated
-hexosaminidase secretion was measured in suspended (A)
and adherent (B) cells in the presence of the indicated
concentrations of GF 109203X. Spontaneous secretion was subtracted from
stimulated secretion at each inhibitor concentration. Data are
expressed as the fraction of control (antigen-stimulated secretion
without inhibitor) and represent the mean and standard deviation of
three experiments. Control secretion was 30.9 ± 15.7% in
suspended cells and 45.1 ± 8.1% in adherent cells. The antigen
concentration was 1 µg/ml. Spontaneous secretion was 7.6 ±
5.9% in suspended cells and 5.4 ± 2.4% in adherent cells; it was
unaffected by GF 109203X.
Figure 3:
PMA abolishes antigen-induced increases in
intracellular Ca in cell suspensions, but has little
effect on adherent cells. Suspended (A) and adherent (B) cells loaded with the Ca
indicator
fura-2 were stimulated with 1 µg/ml antigen (Ag) 1 min
after treatment with 50 nM PMA as indicated. 5 µM GF 109203X (GF) was added 2 min before antigen in the trace
indicated, and was able to reverse the effect of PMA. The quench in
fluorescence during the addition of GF 109203X was due to the strong
absorbance of the compound. Data show fluorescence traces from one of
three representative experiments. PMA had no effect on fluorescence
measurements in unstimulated cells.
Figure 4: Dose-response curves for the effect of PMA on the antigen-induced increase in intracellular calcium in suspended and adherent cells. Antigen-induced changes in fura-2 fluorescence were measured in suspended (A) and adherent (B) cells in experiments similar to those shown in Fig. 3. The maximal change in fluorescence from the pre-stimulation baseline was expressed as a percent of total fluorescence, after correcting for non-fura-2 fluorescence and for leakage of fura-2 from the cells during the experiment. The percent maximal change in fluorescence was then plotted as a fraction of control (percent maximal change in fluorescence without PMA). Data represent the mean and standard deviation of four experiments. Control maximal fluorescence changes were 26.0 ± 5.5% in suspended cells and 26.1 ± 3.3% in adherent cells. The antigen concentration was 1 µg/ml.
Since PMA abolished not only the initial increase but also the
prolonged elevation in intracellular Ca in suspended
cells, it should inhibit both the release of calcium from intracellular
stores and calcium influx across the plasma membrane. We therefore
examined the effects of PMA on the calcium influx component of the
calcium response using the manganese influx technique(32) . In
these experiments, decreases in fura-2 fluorescence are due to
quenching of the dye by Mn
, which has entered the
cell via a calcium influx pathway(33) . As expected,
antigen-stimulated Mn
influx in cell suspensions was
abolished by 100 nM PMA (Fig. 5A). In adherent
cells, however, PMA had no effect on manganese influx in response to
antigen (Fig. 5B).
Figure 5:
PMA inhibits antigen-induced manganese
influx in suspended RBL cells, but had little effect in adherent cells.
Suspended (A) and adherent (B) cells were treated
with 100 nM PMA 1 min before the addition of 100 µM MnCl (Mn). The immediate drop in fluorescence
is a result of manganese binding to extracellular fura-2. Two minutes
later, the cells were stimulated with 1 µg/ml antigen (Ag). Data show fura-2 fluorescence traces from one experiment
representative of three. PMA had no effect on fluorescence measurements
in unstimulated cells.
Figure 6:
Thapsigargin-induced manganese influx in
suspended cells is not inhibited by PMA. Suspended cells were treated
with 0.1% MeSO (-PMA) or 100 nM PMA
(+PMA) 1 min before the addition of 100 µM MnCl
(Mn). The immediate drop in fluorescence
is a result of manganese binding to extracellular fura-2. Two minutes
later, the cells were stimulated with 100 nM thapsigargin (Tg). Data show fura-2 fluorescence traces from one experiment
representative of three. PMA had no effect on fluorescence measurements
in unstimulated cells.
Treatment of RBL cell
suspensions with thapsigargin did not induce secretion unless the cells
were also treated with PMA (Fig. 7A). This is
consistent with work using another endoplasmic reticulum
Ca-ATPase inhibitor, cyclopiazonic acid(16) .
In contrast, thapsigargin alone was able to stimulate secretion in
adherent cells, although co-treatment with 50 nM PMA enhanced
thapsigargin-induced secretion (Fig. 7B). These data
suggest that adherent cells have a constitutive protein kinase C
activity that synergizes with thapsigargin to promote secretion.
Figure 7:
PMA was required for thapsigargin-induced
secretion from cells in suspension, but not with adherent cells.
Thapsigargin-induced -hexosaminidase secretion was measured in
suspended (A) and adherent (B) cells that had been
treated with either 0 (open circles) or 50 nM PMA (closed circles). The data are plotted as means of the
indicated data points. Similar results were obtained in at least three
other experiments on different days.
Figure 8:
Thapsigargin-induced secretion in adherent
cells was inhibited by protein kinase C down-regulation and by
GF-109203X. -Hexosaminidase secretion was measured in adherent
cells that had been treated with thapsigargin alone (open
circles), with thapsigargin and 5 µM GF 109203X (crosses), or with thapsigargin after down-regulation of
protein kinase C (closed circles). Protein kinase C was
down-regulated by treating the cells with 100 nM PMA for 6 h.
Data show the mean and range of one experiment representative of four
experiments done on different days.
Protein kinase C contains two functional domains: a regulatory domain that interacts with the physiological activator diacylglycerol and with PMA, and a catalytic domain that binds ATP and contains the kinase activity. We have shown that adhesion of RBL cells results in a marked loss of sensitivity to PMA as well as an increased activity of protein kinase C, which suggests that the regulatory domain may have been altered in some way. Since calphostin C acts on the the regulatory domain, we predicted that it would be unable to inhibit protein kinase C in adherent cells. Indeed, calphostin C failed to inhibit either antigen- or thapsigargin-induced secretion in adherent cells at concentrations that completely inhibited antigen-induced secretion in cell suspensions (Fig. 9). Since GF 109203X acts on the catalytic domain of protein kinase C(30) , this inhibitor should affect suspended and adherent cells similarly, as was shown in Fig. 2.
Figure 9:
Calphostin C did not inhibit thapsigargin-
or antigen-induced secretion from adherent cells at concentrations that
inhibited secretion from suspended cells. -Hexosaminidase
secretion was measured in (A) unstimulated (open
circles) or antigen-stimulated (closed circles) cells in
suspension, and in (B) unstimulated (open circles),
antigen-stimulated (closed circles), or
thapsigargin-stimulated (crosses) adherent cells. Data show
the mean and range of one experiment representative of four experiments
done on different days. The antigen concentration was 1 µg/ml, and
the thapsigargin concentration was 1
µM.
Activation of suspended and adherent
cells by antigen also revealed a difference in the ability of the
calcium-dependent protein kinase C- and -
isozymes to
translocate (Table 1). There was a 5-fold increase in
membrane-associated protein kinase C-
in both adherent and
suspended cells in response to antigen, but again the extent of
translocation was 3-fold higher in adherent cells. Antigen stimulation
caused a 2-3-fold increase in membrane-associated protein kinase
C-
in suspended cells, whereas a 6-fold increase was seen with
adherent cells. In contrast, no difference between suspended and
adherent cells was observed for translocation of protein kinase C-
and -
in response to antigen.
Treatment of cells for 3 min with
50 nM PMA (a concentration that effectively inhibited 75% of
the secretory response of suspended cells) resulted in translocation to
the membrane of all isozymes except . Although the extent of this
translocation varied between isoforms (see Table 1), no
statistically significant differences were seen between adherent and
suspended cells. However, the differential distribution of protein
kinase C-
and -
in adherent and suspended cells that was seen
in response to antigen appeared to be maintained in PMA-treated cells (Table 1). No large differences were observed for protein kinase
C-
and -
in response to antigen, since both of these isozymes
were already localized to the membrane by PMA treatment (Table 1). An additional experiment using 100 nM PMA
showed a similar trend, although the PMA alone induced a more
substantial translocation of isozymes and so additional translocation
in response to antigen was not as great (data not shown).
Thapsigargin-induced elevation of intracellular calcium in adherent and
suspended cells did not affect membrane association of any of the
isozymes except for protein kinase C-
, which increased from 7.6
± 4.6% to 21.3 ± 6.1% in adherent cells (n = 3).
Tyrosine phosphorylation of the individual protein
kinase C isozymes was assessed by immunoprecipitation of the individual
isozymes and immunoblotting of the resolved proteins with antibody to
phosphotyrosine. Only protein kinase C-, which was previously
shown to be tyrosine-phosphorylated(27) , was
tyrosine-phosphorylated in response to antigen or PMA. In resting cells
a trace amount of tyrosine phosphorylation of protein kinase C-
was also noted. However, in all cases the state of tyrosine
phosphorylation of protein kinase C-
from adherent and suspended
cells was similar (data not shown).
In the past, there have been discrepancies in the literature
describing the effects of PMA on antigen-stimulated secretion in RBL
cells. Pecht and colleagues (12, 15) found that PMA
potentiates secretion at low concentrations (<15 nM) and
inhibits secretion at higher concentrations. However, Beaven's
laboratory (13, 14) has shown that PMA has no effect
on antigen-induced secretion. One difference between these two sets of
experiments is that Pecht's group worked with cell suspensions
while Beaven's group worked with adherent cells. Our results
clearly demonstrate that adherent RBL cells are markedly resistant to
PMA. Only in suspended cells did PMA inhibit antigen-induced increases
in intracellular [Ca]
( Fig. 3and Fig. 4) and calcium influx (Fig. 5), and
have complex effects on secretion (Fig. 1). Thus, we propose
that the earlier discrepancies were due to differences between adherent
and suspended cells. In the past, results obtained using cells in
suspension have often been compared with other data obtained using
adherent cells. Our findings highlight the importance of making all
measurements under the same experimental conditions.
The importance of the adhesion process in the modification of cellular activities such as differentiation and proliferation has been recognized in many cell types(38, 39) . It is thus not very surprising that other aspects of the cellular response should also be affected by cell adhesion. One of the events following RBL cell adhesion is tyrosine kinase activation(2, 3, 19) . Although similar results have not yet been reported for serine/threonine kinases, we suggest that protein kinase C itself might be activated, either directly or indirectly, during or after adhesion. Since thapsigargin only induced secretion from RBL cell suspensions if PMA was also present (Fig. 7A), we used thapsigargin to test whether protein kinase C is constitutively active in adherent cells. If the enzyme is activated with cell adhesion, thapsigargin should induce secretion in adherent cells without the need for PMA co-treatment and this was indeed the case (Fig. 7B). Similar results were obtained with the calcium ionophore A23187 (data not shown). This secretion was inhibited by the protein kinase C inhibitor GF 109203X and by down-regulation of protein kinase C (Fig. 8), suggesting that the thapsigargin-induced secretion in adherent cells is indeed dependent upon a constitutive protein kinase C activity.
We have attempted to identify how this increased level of protein kinase C activity might be achieved, and which isozymes are involved. An attractive hypothesis is that adhesion causes an alteration in protein kinase C that affects the function of the regulatory domain of the enzyme. Since the binding site for PMA is on the regulatory domain, this could also explain the loss of sensitivity of adherent cells to PMA as well as the apparent increase in protein kinase C activity in adherent cells. If the regulatory domain is altered, protein kinase C would still be sensitive to GF 109203X, because this inhibitor acts on the catalytic site of protein kinase C(30) . However, the protein kinase C inhibitor calphostin C should not inhibit the altered protein kinase C, because it acts on the regulatory domain(40) . Indeed, calphostin C was unable to inhibit thapsigargin-induced secretion in adherent cells at concentrations that inhibited antigen-stimulated secretion in cell suspensions (Fig. 9).
One mechanism by which this alteration
might be accomplished is by the proteolytic cleavage of the regulatory
domain of protein kinase C from the catalytic domain, leaving a
constitutively active protein kinase M
fragment(41, 42) . However, this would not explain the
potentiation of thapsigargin-induced secretion by PMA that is still
seen in adherent cells (Fig. 7B). Another possibility
is that the function of the regulatory domain is altered by
phosphorylation. This is supported by data suggesting that several of
the protein kinase C isozymes can become phosphorylated (27, 31) and that tyrosine phosphorylation of protein
kinase C- occurs on the regulatory domain(43) . However,
we failed to detect any differences between adherent and suspended
cells in the tyrosine phosphorylation of any of the protein kinase C
isozymes. One mechanism suggested by the ability of thapsigargin to
stimulate secretion in adherent cells without PMA treatment is that
protein kinase C is activated in adherent cells when intracellular
Ca
is increased, even without diacylglycerol
stimulation. However, in response to stimulation with thapsigargin, we
did not detect membrane translocation of any of the protein kinase C
isozymes except for protein kinase C-
in adherent cells. The
ability of thapsigargin to induce membrane translocation of protein
kinase C-
has been described previously in GH
C
rat pituitary cells(44) ; since protein kinase C-
is
not calcium-dependent, this effect may be an indirect consequence of
the thapsigargin-induced increase in intracellular
Ca
(44) .
Another possibility is that cell
adhesion may activate the kinase by inducing translocation of protein
kinase C to the plasma membrane in a manner similar to activation by
PMA or antigen. Our experiments suggest that protein kinase C- and
-
are indeed differentially distributed in adherent versus suspended cells, with greater membrane translocation of protein
kinase C-
in adherent cells, and of protein kinase C-
in
suspended cells (Table 1). We also observed that with antigen
stimulation the calcium-dependent protein kinase C-
and -
isozymes were translocated to the membrane to a greater extent in
adherent than in suspended cells (Table 1). Protein kinase
C-
is able to reconstitute antigen-induced secretion in
permeabilized cells(31) , while protein kinase C-
and
-
have been shown to inhibit of phospholipase C-
, thus
preventing IP
production and the release of calcium from
stores(18) . Thus, both potentiating and inhibitory protein
kinase C isozymes show differential distribution in adherent and
suspended cells.
In conclusion, we have shown that following
adhesion, RBL cells lose sensitivity to PMA and display a constitutive
activity of protein kinase C, perhaps because the regulatory domain of
protein kinase C has been altered in some way. Since mature mucosal
mast cells reside in tissues, adherent cells should be more
representative of mast cells in vivo. It is possible that the
increase in protein kinase C activity represents a regulatory mechanism
which allows mature, adherent mast cells to achieve greater sensitivity
to intracellular Ca, thus leading to full
physiological activation. Activation of protein kinase C when mast
cells adhere may therefore be an important link between physiological
stimulus and cell response in mast cells and perhaps in other cell
types as well.