(Received for publication, July 17, 1995; and in revised form, August 17, 1995)
From the
Thrombin initiates many physiological processes in platelets and
other megakaryocyte-lineage cells by interacting with surface receptors
and generating rises in cytoplasmic Ca; these rises
result from both Ca
release from intracellular stores
and receptor-mediated Ca
entry. Regulators that limit
Ca
entry after its initiation by thrombin have not
been identified. In this study, prevention of expression of a single
protein kinase C isoenzyme (PKC
) by antisense cDNA overexpressed
in HEL cells, a human megakaryoblastic cell line that expresses
thrombin receptors, promotes thrombin receptor-mediated Ca
entry without altering thrombin-induced intracellular release of
Ca
. The cytoplasmic Ca
rise
initiated by endoperoxide analogs was not affected by inhibiting
PKC
. Overexpression of a cDNA encoding wild-type PKC
mutated
to prevent recognition by the antisense cDNA abolished the enhancement
of Ca
influx following thrombin. Thus, PKC
appears to be a specific negative regulator of thrombin
receptor-mediated Ca
entry.
The protease thrombin is generated at sites of vascular injury
and is a central mediator of hemostasis, thrombosis, inflammation, and
vascular proliferation(1) . Thrombin stimulates several cell
types, including platelets and other cells of megakaryocytic lineage,
monocytes, endothelium, and vascular smooth muscle, by triggering
surface receptors to generate intracellular
messengers(2, 3) . The addition of thrombin to
platelets initiates a rise in cytoplasmic Ca ([Ca
]
) (
)that
results from release of Ca
from intracellular stores (4) followed by Ca
entry via a
receptor-regulated cation channel(5, 6) . Several
messengers, including an unidentified Ca
influx
factor (8) and inositol 1,3,4,5-phosphate(9) , have
been postulated to initiate receptor-mediated Ca
influx(10, 11) ; however, few regulators that
limit receptor-mediated Ca
entry, which is necessary
to prevent excessive [Ca
]
,
have been characterized. Activation of the intracellular
phospholipid-dependent protein kinase C (PKC) with phorbol esters
prevents both entry and intracellular mobilization of Ca
induced by thrombin(12, 13) . PKC can also be
activated by generation of lipid regulators (14) that are
mobilized following thrombin stimulation(2) ; some isoenzymes
of PKC (e.g. PKC
) are regulated by a rise in
[Ca
]
(15) . Since
limiting or negative mediators of Ca
influx might be
reasonably considered to be regulated by a process requiring
Ca
(16) , we asked whether selective
inhibition of a Ca
-dependent PKC isoenzyme would
modify the thrombin receptor-mediated Ca
influx. For
these experiments, we utilized human erythroleukemic (HEL)
cells(17) , a megakaryoblastic cell line that has functional
thrombin receptors and shares with platelets many components of the
thrombin signaling mechanism(12, 18) . HEL cells offer
an additional advantage for the present studies, in that they express
only one (PKC
) of the Ca
-responsive PKC
isoenzymes (12, 19) and therefore required only a
single intervention to eliminate Ca
-dependent PKC
activity from the cells. As there are few chemical PKC inhibitors that
are specific for individual isoenzymes, a strategy based on antisense
DNA was utilized to reduce selectively PKC
in HEL cells.
Low passage HEL cells (a generous gift of Thalia
Papayannopoulou, University of Washington, Seattle) were either
transfected with the antisense PKC construct or with the vector
only (control cells) by electroporation. Cells that stably expressed
anti-PKC
cDNA (anti-
cells) were selected by limiting
dilution and on the basis of cell survival in the presence of Geneticin
(1.2 mg/ml). For Northern blot analysis, total RNA was prepared from
the cells using the method described previously(12) . The cDNA
probes of PKC
, PKC
, and PKC
are the same as described
previously(19) . The cDNA probes for
,
-thromboglobulin, and the thrombin receptor were generous gifts
from Drs. Peter Newman (Blood Research Institute of Southeastern
Wisconsin), Mortimer Poncz, and Lawrence Brass (University of
Pennsylvania), respectively; the anti-thrombin receptor antibody was
kindly provided by Dr. Brass.
Expression of both RNA and protein of PKC was
significantly reduced in anti-
cells compared with that in either
wild-type HEL cells or the control cells (Fig. 1, a and b). In addition to PKC
, the predominant isoenzymes
expressed by HEL cells are PKC
and PKC
, which are also
expressed in platelets(12, 19) ; neither PKC
nor
PKC
was inhibited by anti-PKC
cDNA (Fig. 1a). Inhibition of PKC
provoked no obvious
alteration of differentiation, as shown by the similar expression of
integrin
,
-thromboglobulin, and thrombin
receptor (Fig. 1c).
Figure 1:
Inhibition of PKC expression in
megakaryocyte-lineage cells. a, Northern transfer analysis of
RNAs extracted from wild-type (HEL), control, and anti-
HEL cells.
The blot was hybridized with cDNA probes complementary to PKC
,
PKC
, PKC
, or glyceraldehyde-phosphate dehydrogenase (GAP-DH) as a control for RNA loading, as indicated.
Approximate mRNA sizes are: 2.6 kb for PKC
; 2.2 kb for PKC
;
and 2.7 kb for PKC
. b, immunoblot of total protein
extracted from wild-type, control, and anti-
HEL cells with a
PKC
-specific monoclonal antibody (Seikagaku America, Inc.). c, Northern transfer analysis of RNA extracted from wild-type
(HEL), control, and anti-
cells. The blot was hybridized with cDNA
probes encoding portions of
,
-thromboglobulin,
and the thrombin receptor, as indicated. Approximate mRNA sizes are:
3.5 kb for the thrombin receptor; 2.5 kb for
; and
1.6 kb for
-thromboglobulin
(
TG).
Because of the effect that
overall PKC activation exerts on [Ca]
homeostasis, we asked whether this sharp reduction in PKC
would
alter [Ca
]
following thrombin.
The thrombin-induced [Ca
]
in
clonal populations of anti-
HEL cells loaded with the
Ca
-sensitive fluorophore Fura-2 was significantly
enhanced when compared with that of the control cells (Fig. 2, a and b). This result suggests that thrombin-induced
elevation of [Ca
]
in HEL cells
is normally inhibited by PKC
; this effect appeared to be specific
for thrombin, because [Ca
]
induced by the endoperoxide analog U46619, which activates the
thromboxane A
receptor, is not affected in anti-
cells (Fig. 2, c and d). Enhanced
[Ca
]
does not result from
altering the expression of thrombin receptors on anti-
cells, as
assessed by flow cytometry utilizing a fluorescently labeled
anti-thrombin receptor antibody (data not shown) or by expression of
mRNA encoding the thrombin receptor (Fig. 1c).
Figure 2:
Intracellular Ca
concentration measured by Fura-2 fluorescence in both control and
anti-
HEL cells. a, representative tracing showing
changes in [Ca
]
following
addition of thrombin (0.5 unit/ml at time shown by arrow) in
both control and anti-
cells. b, bar graph showing mean
± S.E. of the differences between peak and basal
[Ca
]
in both control (n = 13) and anti-
cells (n = 23); p < 0.01, two-tail Student's t test. c,
changes in [Ca
]
following
addition of U46619 (1 µM) in control and anti-
cells. d, bar graph showing mean ± S.E. of the concentration
difference between peak values and basal level of
[Ca
]
in control (n = 8) and anti-
cells (n =
5).
To
determine whether enhanced [Ca]
resulted specifically from the reduction of PKC
expression
by the antisense construct, we restored the expression of PKC
by
stably transfecting cDNA encoding the full-length PKC
into clonal
populations of anti-
cells. To prevent inhibition of expression of
the transfected PKC
by the constitutively expressed anti-
cDNA, the degeneracy of the genetic code was exploited to generate a
mutant PKC
cDNA (mut-PKC
) that had minimal complementarity
with the anti-
cDNA but still encoded the same amino acid sequence
as native PKC
(Fig. 3a). Expression of the
mut-PKC
was not inhibited by the anti-
construct, as verified
by cotransfection of the two constructs in COS7 cells, which do not
normally express PKC
, followed by Northern transfer analysis (data
not shown). Transfection of mut-PKC
into the anti-
cells
restored the expression of PKC
, as assessed by immunoblotting (Fig. 3b), and abolished the enhancement of
thrombin-induced [Ca
]
in the
anti-
cells (Fig. 3, c and d). Thus, the
reversal of the changes induced by antisense restoration of PKC
in
the same clone of cells strengthens the argument that PKC
specifically inhibits the thrombin-induced increase in
[Ca
]
.
Figure 3:
Restoration of PKC expression with
mutant PKC
. a, wild-type human PKC
sequence against
which the antisense construct was targeted (top) and the
sequence of the corresponding portion of the mutated full-length rat
PKC
that was overexpressed in the anti-
cells. b,
immunoblot of PKC
expression in control (leftlane) and two clones of anti-
HEL cells transfected
with a mutant PKC
cDNA (mut-PKC
). Immunoblotting was
performed with a polyclonal anti-PKC
antibody (Santa Cruz). c, changes in [Ca
]
upon adding thrombin (0.2 unit/ml) to control and mutant PKC
cells. d, bar graph shows the mean ± S.E. of the
differences between peak and basal levels of
[Ca
]
in control (n = 3) and mut-PKC
(n = 10)
cells.
This inhibition of
the thrombin-induced increase in
[Ca]
by PKC
might result
from an effect on Ca
entry, on Ca
release from intracellular stores, or on both. To distinguish
among these possibilities, the contribution of Ca
entry from extracellular medium to
[Ca
]
was eliminated by either
briefly chelating extracellular Ca
with EGTA or
adding Ni
, which blocks the Ca
entry via receptor-operated cation
channels(5, 7) . Following these interventions,
[Ca
]
was not significantly
different between anti-
and control cells (Fig. 4, a and b). Furthermore, the divalent cation entry, assessed
by measuring the Mn
quench of intracellular Fura-2
fluorescence (6) after addition of thrombin, was less in the
control cells than in the anti-
cells (Fig. 4c).
Thus, PKC
reduces the magnitude of thrombin-induced Ca
entry but has little effect on release of Ca
from intracellular stores. These results also demonstrate that
some aspects of thrombin receptor function were not affected by
inhibition of PKC
; additionally, PKC-mediated inhibition of
thrombin-induced mobilization of Ca
from
intracellular storage sites (12, 13) does not require
the presence of known Ca
-regulated PKC isoenzymes in
HEL cells.
Figure 4:
Determination of the component of
[Ca]
regulated by PKC
. a, bar graphs of the mean ± S.E. of the difference
between peak thrombin-induced and basal levels of
[Ca
]
in control and anti-
cells suspended in buffer to which 2 mM EGTA has been added (n = 3). b, bar graphs of the mean ±
S.E. of the differences between peak thrombin-induced and basal levels
of [Ca
]
in control and
anti-
cells suspended in buffer to which 2 mM NiCl
has been added (n = 3). c,
representative tracing of Fura-2 fluorescence changes (360 nM excitation wavelength) of control and anti-
cells suspended
in medium containing Mn
(0.1 mM) shortly
after stimulation with thrombin (0.5
unit/ml).
After addition of thrombin (but not ADP) to
platelets(6, 7) , a measurable delay precedes
Ca entry, suggesting that the Ca
channel is not directly linked to the receptor but instead is
activated by intracellular mediators, generated perhaps by depletion of
intracellular Ca
stores (10, 11) or
by phospholipid hydrolysis(21) . Thus, PKC
might interfere
with a mediator that initiates or potentiates Ca
influx; such a mediator would presumably be generated by thrombin
but not endoperoxide analogs such as U46619. An alternative possibility
is that PKC
, once activated, might phosphorylate a
receptor-mediated Ca
channel specifically associated
with the thrombin receptor. Either model has implications for the
specificity of agonist effect. Thus, although other mechanisms for
limiting Ca
entry may exist for other agonists, this
reduction in thrombin receptor-mediated Ca
influx by
PKC
, a Ca
-regulated PKC, represents a novel and
selective cross-regulatory mechanism that could prevent excessive
accumulation of cytoplasmic Ca
following thrombin
stimulation.