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Inhibition of early platelet-derived growth factor responses in BALBc-3T3 cells by interferon
BIOCHEMICAL
Vol. 150, No. 3, 1988 February 15; 1988
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1207-1212
INHIBITION OF EARLYPLATELET-DERIVEDGROWTH FACTORRESPONSES IN BALB/C-3T3 CELLSBY INTERFERON M. Zagari",
J.R. Hepler@, C. Harris/l,
and B. Herman*+
*Laboratories for Cell Biology, Department of Cell Biology and Anatomy, @Programin Neurobiology and @Pharmacology, +Lineberger Cancer Research Center, University of North Carolina at Chapel Hill, CB d 7090, Chapel Hill, NC 27599 Received January 6, 1988 SUMMARY:Stimulation of total inositol phosphate production, alteration of cytosolic free calcium ([Ca++]f), vinculin disruption from adhesion plaques, and DNA synthesis caused by PDGFwere examined in normal and INF pretreated density arrested BALB/c-3T3 fibroblasts. In normal cells, PDGFcaused an increase in total inositol phosphates, a rapid, transient increase in [Ca++]i, disappearance of vinculin from adhesion plaques, and stimulation of DNA synthesis. Pretreatment of cells with INF inhibited PDGF-stimulated increases in [Ca++]i, vinculin disruption from adhesion plaques, and DNA synthesis, but had no effect on PDGF-induced increase in total inositol phosphate levels. These findings suggest that INF prevents entry of quiescent BALB/c-3T3 cells into Gl by inhibiting PDGF-induced release of Ca++ from intracellular stores. 0 1988Academic Press,Inc.
INTRODUCTION:A great deal of attention potential on their
has been generated regarding the
use of INF's in the treatment of various neoplastic ability
INF's inhibit
to inhibit
cell proliferation
cell growth are not clear,
Go/G1 phase of the cell cycle (3). been shown to inhibit the major growth factor to growth factors competence (5,6).
(1,2).
Exposure of BALB/c-3T3 cells of the cell
in serum which sensitizes
Co-addition
to INF's has
cycle (4).
BALB/c-3T3 cells
poor plasma derived serum, a condition of PDGFand INF's inhibited
synthesis of p29 and ~35, as well as inhibiting tein synthesis seen dthin
The mechanismsby which
but they seem to block growth at the
serum induced activation
in platelet
diseases based
4-6 hours after
PDGFis
to respond termed
PDGF-induced
the general increase in pro-
PDGFaddition
(4).
Since the
ABBREVIATIONS: PDGF, Platelet-Derived Growth Factor, INF, a,8-interferon, DMEM, Dulbecco's Minimum Essential Medium, Ca++, calcium, FCS, Fetal Calf Serum, TPA, 12-tetradecanoylphorbol-13-acetate //Present address: Department of Chemotherapy, Glaxo Pharmaceuticals, 5 Moore Drive, Research Triangle Park, NC 27709 ODO6-291X/88 $1.50 1207
Copyright 8 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 150, No. 3, 1988
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
expression of p29 and p35 occur within likely
that early changes in cellular
PDGFaddition
may be affected
we examine the effects
indicate
of INF pretreatment
and vinculin
that INF's inhibit
of vinculin
total
to INF's.
is due to prevention
that alteration
In this study,
phosphate production,
from adhesion plaques.
PDGF-stimulated increase in [Ca++]i,
inositol
following
on three very early PDGF-induced
inositol
disruption
it seems
which occur in cells
from adhesion plaques, and DNA synthesis,
increases in total vity
functions
by exposure of cells
changes in cytoplasmic functions: tions in [Ca++]i,
1 hour of PDGFaddition,
altera-
Cur results disappearance
but not PDGF-induced
phosphates, suggesting that INF's inhibitory of PDGF-stimulated alterations
acti-
in [Ca++]i suggesting
in [Ca++]i is necessary for PDGF-stimulated mitogenesis.
METHODS:BALB/c-3T3 fibroblasts (clone A31) were grown as described previously Cells were used on 4 to 5 days after plating and were between the 6th iZ'l2th passage. [Ca++]i was measured using multiparameter digitized video fluorophore Fura- (8,9), Cells were microscopy (MDVM) and the Caitsensitive prechilled for 10 minutes at 4°C followed by a 15 minute incubation at 37'C in DMEMcontaining 5 uM Fura-2-AM. This loading condition resulted in an intracellular concentration of Fura- of 30-40 uM based on digitonin releasable absorbance. [Ca++]f value s were determined by the ratio imaging method (8), and the excitation intensity was routinely decreased by a factor of l,OOO10,000 fold (10). Vinculin distribution was assessed by immunocytochemistry DNA synthesis was examined by [3H]-thymidine incorporation (12). Total (11). inositol phosphates were quantitated in [3H]-inositol labeled cells as described previously (13). MATERIALS: PDGF(HPLC purified) was purchased from PDGFInc. (Cambridge MA) or purified as described previously (12). Fetal calf serum was obtained from H clone Laboratories (Logan UT), Fura-2-AM from Molecular Probes (Eugene OR), from American [ 3 ]-thymidine from Amersham(Chicago IL), and [3]-lnositol Radiolabeled Chemicals, Inc, (St. Louis MO). All other chemicals were from Sigma Chemical Co. (St. Louis MO). Interferon was a gift from ScheringPlough. RESULTS:Exposure of BALB/c-3T3 cells sitol for
phosphates (Table I).
Pretreatment
15 minutes at 37'C did not affect
sit01 phosphates (Table I).
to PDGFresulted
No
observed when cells were treated
of cells
increase in total
cellular
activity
inositol
inositol
1208
to
phosphates (data not shown).
to INF's prior
to PDGFdoes not
receptor and subsequent alterations
necessary for production
ino-
phosphates was
with INF's alone while exposure of cells
These data suggest that exposure of cells PDGFbinding to its
with 10,000 U/ml of INF's
PDGF-stimulated increases in total
10% FCS caused a 300%increase in total
inhibit
in an increase in ino-
of inositol
in receptor
phosphates.
or
BIOCHEMICAL
Vol. 150, No. 3, 1988
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Accumulation
of
inositol
1
phosphates
in
Balb/c-3T3
cells
InsP2
InsPl
InsP3
Pretreat
Addition
cpm
C SEX
cpm?
Vehicle
Vehicle
3073
(-
199 (.
Vehicle
PDGF
19575
? 1062
2436
k
122
1573
+ 103
Interferon
PDGF
18634
t
2452
t
111
1507
f
183
1797
SEM
+ InsP4
cpm 1. SEM
20
857 ?
52
56
Confluent, quiescent Balb/c-3T3 cells were labelled overnight with 5 pCi/ml of [3B]-inositol in inositol free and serum starved media. Cells were pretreated for 15 min (37'C; Ph 7.4) with vehicle or Interferon (10,000 U/ml) in the pre37'C) with vehicle (0.1% BSA), sence of 10 r&i LiCl and then challanged (15 min, of PDGF (20 nglml). Individual inositol phosphates were separated and collected by an ion exchange chromatography as described previously (13). The data are the mean f SEM of triplicate determinations and are representitive of 5 separate experiments. Blank (t=O) levels of radioactivity were not subtracted from the values presented.
Addition increase lity
in
of PDGF to BALB/c-3T3 [Ca++]i
of PDGF to induce
of BALB/c-3T3 The
which
basal
increased
cells
level
of
within
10,000
(Figure
inhibited
U/ml
competence
followed
[Ca++]i
cells
20 seconds
1) or
1,000
increases
was found
after
of PDGF U/ml
in many cells
in a 75% increase
in these
90 seconds
by a rapid,
to be necessary
and growth
to PDGF resulted
PDGF-stimulated
is
has been hypothesized
to 350 nM within
to baseline
cells
(data
Exposure now shown)
for
the
(12,14). [Ca++]i
Exposure (Figure
INF's
[Ca++]i of cells
returned to either
substantially
[Ca++]f.
100
75
q q
PDGF aj3lNF ‘5 PDGF
50
25
0
-El
Figure 1. Effect of INF pretreatment (15 minutes, 37'C), on PDGF-induced alterations in [Ca++]. Exposure of cells to INF's inhibited by 87% PDGFinduced increases in 1' Ca'+]i. Data is average of four experiments with three cells observed/experiment (n=12).
1209
abi-
to be 175 nM and was
PDGF addition.
addition.
in
in
transient
1).
Vol. 150, No. 3, 1988
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
IFN WI
n
L
400
U/ml
IFN bc3 200
l-PA
10’
IFN WI
400
U/ml
IFN W
200
U/ml
I 0
20
15’
U/ml
15’
13
1 1 II L 15
a0
60
40
100
% Cells Without Vlnculin Adhesion Plaques Figure 2. Effect oE INF pretreatment or TPA (150 tit)-induced disruption of inhibited PDGF but not TPA disappearance Mean of four experiments with 100-150
We have previOUSly as well very
as a number
rapid,
vinculin was found
reversible, from
adhesion
to be highly
= f ii k 5 [ i
described
of other
(15 minutes, 37-C), on PDGF (20 rig/ml) INF vinculin from adhesion plaques. of vinculin from adhesion plaques. cells examined/treatment/experiment.
that
addition
PDGF responsive
time,
temperature,
plaques
(7,11,12).
correlated
with
of PDGF to BALB/c-3T3 cell
types,
is
accompanied
and dose-dependent The change PDGF-induced
by
disruption
in vinculin entry
cells,
of cells
distribution into
200 aPlNF (10,000 Ulml)‘~ PDGF
180
aPlNF (1,000 U/ml) ‘s PDGF 160 140 120
5 3 i 8
100
8
80
Figure 3. Effect of INF pretreatment stimulated DNA synthesis ([3Hl-thymidine dependent inhibition of PDGF-stimulated ments, each done in triplicate.
(15
minutes,
tncorporation). DNA synthesis.
1210
37"C),
of
on PDGF (10 ng/ml)TNF displayed a dose Mean of two experi-
S
a
BIOCHEMICAL
Vol. 150, No. 3, 1988
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
phase and occurs via Ca++-dependent proteolysis Phorbol esters (e.g.
(11).
of adhesion plaque vinculin
TPA) have also been found to cause loss of vin-
from adhesion plaques, but by a mechanismdistinct
culin
We therefore
(15).
examined the effect
on PDGFand TPA-stimulated
of pretreatment of vinculin
At very low concentrations
(Figure 2). inhibit
disruption
from that of PDGF of cells within
INF's
from adhesion plaques
(200-400 U/ml), INF's were able to
PDGF, but not TPA, induced disruption
of vinculin
from adhesion pla-
ques. INF's were also found to inhibit following
addition
dose dependent:
incorporation
of PDGF(Figure 3). 50% inhibition
of [3H]-thymidine
The inhibition
into DNA
caused by INF's was
was seen at 1,000 U/ml and total
inhibition
at
10,000 U/ml. DISCUSSION: Our findings demonstrate that INF's block very early changes in cytoplasmic production
functions of inositol
in [Ca++]i,
vinculin
PDGF-stimulated
induced by PDGFin BALBLB/c-3T3 cells. phosphates is not affected, disruption
but PDGF-induced increases
from adhesion plaques, and entry
S phase (DNA synthesis)
are all
The data are consistent
with the idea that INF-mediated inhibition
step distal
to activation
prevented by pretreatment
of Phospholipase C resulting
in [Ca++]I
in the presence of normal phosphotidyl
sis are nst clear but may involve to the Ins (1,4,5)P3 trisphosphate
receptor to its
the endoplasmic reticulum,
inhibition
(4,5) bisphosphate hydroly-
of inositol
receptor but inhibition
or rapid efflux
trisphosphate
exposure to PDGF(4),
binding
normal binding of
of release of Ca++ from
of Ca++ from the cell The findings
following
its
that INF's also Inhibit synthesis seen in
as well as DNA synthesis (4 and this
suggest that these PDGF-stimulated alterations
may depend on an increase in [Ca++li. findings
occurs at a
in phosphotidyllnositol
of p29 and ~35, the general increase in protein
cells following study),
of cells with INF's.
on the endoplasmic reticulum,
release from the endoplasmic reticulum. induction
into
The mechanismsby which INF's prevent PDGF-stimulated increases
hydrolysis.
inonitol
of cells
functions
In support of this hypothesis are the
that INF's also block PDGF-induced disruption 1211
in cellular
of vinculin
from adhe-
Vol. 150, No. 3, 1988
BIOCHEMICAL
sion plaques and disaggregatin Ca++-dependent process (ll), disruption inhibit
of vinculin
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of stress fibers while the latter
block PDGF-mediated mitogenesis by preventing
cell
of the recep-
PDGF-induced alterations
to phosphotidylinositol
properties
do not
Thus, our data suggest that INF's
and support the hypothesis that alterations
many changes in cellular
PDGF-induced
INF's apparently
nor autophosphorylation
tor (W.J. Pledger, personal communication).
sis,
The former is a
depends on prior
from adhesion plaques (7).
binding of PDGFto its receptor
[Ca++]i at a step distal
(16).
of
(4,5) bisphosphate hydrolyin [Ca++li are necessary for
induced by PDGFas well as stimulation
of
growth by PDGF.
ACKNOWLEDGEMENTS: This work was supported by grants from the American Heart Association (AHA 861299, NC-AHA86-87-02) and from NIH (AG07218). We thank Marcy Stephens for performing the [3H]-thymidine assays.
REFERENCES 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11)
12) 13) 14) 15) 16)
Sokawa, Y., Watanabe, Y., Watanabe, Y., and Kawade, Y. (1977) Nature (Land) 268, 236-238. Lundgren, E., Larsson, I., Miorner, II., and Strannengard, 0. (1979) J. Gem Virol. 42, 589-595. Creasey, A.A., Bartholomew, J.C., and Merigan, T.C. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 1741-1745. Lin, s.L., Kikuchi, T., Pledger, W.J., and Tamm,I. (1986) Science 233, 356-358. Pledger, W.J., Stiles, C.D., Antoniades, H.N., and Scher, C.D. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 4481-4485. Leof, E.B., Wharton, W., Van Wyk, J.J., and Pledger, W.J. (1982) Exp. Cell Res. 141, 107-115. Herman, B. and Pledger, W.J. (1985) J. Cell Biol. 101, 1031-1040. DiGuiseppi, J., Inman, R., Ishihara, A., Jacobson,K;, and Herman, B. (1985) Biotech. 2, 394-403. Gynkiewicz, G., Poenie, M., and Tsien, R. (1985) J. Biol. Chem.260, 3440-3450. Becker, P.L., and Fay, F.S. (1987) Amer. J. Physiol. 253, C613-C618. Herman, B., Harrington, M.A., Olashaw, N.E., and Pledger, W.J. (1986) J. Cell. Physiol. 126, 115-125. Herman, B., Roe, M.W., Ha,rris, C., Wray, B. and Clemmons, D. (1987) Cell Motil. Cytoskel. s, 91-105. Hepler, J.R., Nakahata, N., Lovenberg, T.W., DiGuiseppi, J., Herman, B., Earp, H.S., and Harden, T.K. (1987) J. Biol. Chem.262, 2951-2956. Zagari, M., Stephens, M., Earp, H.S., and Herman, B. (1988) J. Biol. Chem. submitted. DiGuiseppi, J., Zagari, M., and Herman, E. (1986) J. Cell Btol. 103, 268a. Lin, S.L., Pledger, W.J., and Tamm,I. in The Biology of the Interferon System, W.E. Stewart II and H. Schellekensxds., Elsevier, Amsterdam, e press.
1212
Vol. 150, No. 3, 1988 February 15; 1988
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1207-1212
INHIBITION OF EARLYPLATELET-DERIVEDGROWTH FACTORRESPONSES IN BALB/C-3T3 CELLSBY INTERFERON M. Zagari",
J.R. Hepler@, C. Harris/l,
and B. Herman*+
*Laboratories for Cell Biology, Department of Cell Biology and Anatomy, @Programin Neurobiology and @Pharmacology, +Lineberger Cancer Research Center, University of North Carolina at Chapel Hill, CB d 7090, Chapel Hill, NC 27599 Received January 6, 1988 SUMMARY:Stimulation of total inositol phosphate production, alteration of cytosolic free calcium ([Ca++]f), vinculin disruption from adhesion plaques, and DNA synthesis caused by PDGFwere examined in normal and INF pretreated density arrested BALB/c-3T3 fibroblasts. In normal cells, PDGFcaused an increase in total inositol phosphates, a rapid, transient increase in [Ca++]i, disappearance of vinculin from adhesion plaques, and stimulation of DNA synthesis. Pretreatment of cells with INF inhibited PDGF-stimulated increases in [Ca++]i, vinculin disruption from adhesion plaques, and DNA synthesis, but had no effect on PDGF-induced increase in total inositol phosphate levels. These findings suggest that INF prevents entry of quiescent BALB/c-3T3 cells into Gl by inhibiting PDGF-induced release of Ca++ from intracellular stores. 0 1988Academic Press,Inc.
INTRODUCTION:A great deal of attention potential on their
has been generated regarding the
use of INF's in the treatment of various neoplastic ability
INF's inhibit
to inhibit
cell proliferation
cell growth are not clear,
Go/G1 phase of the cell cycle (3). been shown to inhibit the major growth factor to growth factors competence (5,6).
(1,2).
Exposure of BALB/c-3T3 cells of the cell
in serum which sensitizes
Co-addition
to INF's has
cycle (4).
BALB/c-3T3 cells
poor plasma derived serum, a condition of PDGFand INF's inhibited
synthesis of p29 and ~35, as well as inhibiting tein synthesis seen dthin
The mechanismsby which
but they seem to block growth at the
serum induced activation
in platelet
diseases based
4-6 hours after
PDGFis
to respond termed
PDGF-induced
the general increase in pro-
PDGFaddition
(4).
Since the
ABBREVIATIONS: PDGF, Platelet-Derived Growth Factor, INF, a,8-interferon, DMEM, Dulbecco's Minimum Essential Medium, Ca++, calcium, FCS, Fetal Calf Serum, TPA, 12-tetradecanoylphorbol-13-acetate //Present address: Department of Chemotherapy, Glaxo Pharmaceuticals, 5 Moore Drive, Research Triangle Park, NC 27709 ODO6-291X/88 $1.50 1207
Copyright 8 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 150, No. 3, 1988
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
expression of p29 and p35 occur within likely
that early changes in cellular
PDGFaddition
may be affected
we examine the effects
indicate
of INF pretreatment
and vinculin
that INF's inhibit
of vinculin
total
to INF's.
is due to prevention
that alteration
In this study,
phosphate production,
from adhesion plaques.
PDGF-stimulated increase in [Ca++]i,
inositol
following
on three very early PDGF-induced
inositol
disruption
it seems
which occur in cells
from adhesion plaques, and DNA synthesis,
increases in total vity
functions
by exposure of cells
changes in cytoplasmic functions: tions in [Ca++]i,
1 hour of PDGFaddition,
altera-
Cur results disappearance
but not PDGF-induced
phosphates, suggesting that INF's inhibitory of PDGF-stimulated alterations
acti-
in [Ca++]i suggesting
in [Ca++]i is necessary for PDGF-stimulated mitogenesis.
METHODS:BALB/c-3T3 fibroblasts (clone A31) were grown as described previously Cells were used on 4 to 5 days after plating and were between the 6th iZ'l2th passage. [Ca++]i was measured using multiparameter digitized video fluorophore Fura- (8,9), Cells were microscopy (MDVM) and the Caitsensitive prechilled for 10 minutes at 4°C followed by a 15 minute incubation at 37'C in DMEMcontaining 5 uM Fura-2-AM. This loading condition resulted in an intracellular concentration of Fura- of 30-40 uM based on digitonin releasable absorbance. [Ca++]f value s were determined by the ratio imaging method (8), and the excitation intensity was routinely decreased by a factor of l,OOO10,000 fold (10). Vinculin distribution was assessed by immunocytochemistry DNA synthesis was examined by [3H]-thymidine incorporation (12). Total (11). inositol phosphates were quantitated in [3H]-inositol labeled cells as described previously (13). MATERIALS: PDGF(HPLC purified) was purchased from PDGFInc. (Cambridge MA) or purified as described previously (12). Fetal calf serum was obtained from H clone Laboratories (Logan UT), Fura-2-AM from Molecular Probes (Eugene OR), from American [ 3 ]-thymidine from Amersham(Chicago IL), and [3]-lnositol Radiolabeled Chemicals, Inc, (St. Louis MO). All other chemicals were from Sigma Chemical Co. (St. Louis MO). Interferon was a gift from ScheringPlough. RESULTS:Exposure of BALB/c-3T3 cells sitol for
phosphates (Table I).
Pretreatment
15 minutes at 37'C did not affect
sit01 phosphates (Table I).
to PDGFresulted
No
observed when cells were treated
of cells
increase in total
cellular
activity
inositol
inositol
1208
to
phosphates (data not shown).
to INF's prior
to PDGFdoes not
receptor and subsequent alterations
necessary for production
ino-
phosphates was
with INF's alone while exposure of cells
These data suggest that exposure of cells PDGFbinding to its
with 10,000 U/ml of INF's
PDGF-stimulated increases in total
10% FCS caused a 300%increase in total
inhibit
in an increase in ino-
of inositol
in receptor
phosphates.
or
BIOCHEMICAL
Vol. 150, No. 3, 1988
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Accumulation
of
inositol
1
phosphates
in
Balb/c-3T3
cells
InsP2
InsPl
InsP3
Pretreat
Addition
cpm
C SEX
cpm?
Vehicle
Vehicle
3073
(-
199 (.
Vehicle
PDGF
19575
? 1062
2436
k
122
1573
+ 103
Interferon
PDGF
18634
t
2452
t
111
1507
f
183
1797
SEM
+ InsP4
cpm 1. SEM
20
857 ?
52
56
Confluent, quiescent Balb/c-3T3 cells were labelled overnight with 5 pCi/ml of [3B]-inositol in inositol free and serum starved media. Cells were pretreated for 15 min (37'C; Ph 7.4) with vehicle or Interferon (10,000 U/ml) in the pre37'C) with vehicle (0.1% BSA), sence of 10 r&i LiCl and then challanged (15 min, of PDGF (20 nglml). Individual inositol phosphates were separated and collected by an ion exchange chromatography as described previously (13). The data are the mean f SEM of triplicate determinations and are representitive of 5 separate experiments. Blank (t=O) levels of radioactivity were not subtracted from the values presented.
Addition increase lity
in
of PDGF to BALB/c-3T3 [Ca++]i
of PDGF to induce
of BALB/c-3T3 The
which
basal
increased
cells
level
of
within
10,000
(Figure
inhibited
U/ml
competence
followed
[Ca++]i
cells
20 seconds
1) or
1,000
increases
was found
after
of PDGF U/ml
in many cells
in a 75% increase
in these
90 seconds
by a rapid,
to be necessary
and growth
to PDGF resulted
PDGF-stimulated
is
has been hypothesized
to 350 nM within
to baseline
cells
(data
Exposure now shown)
for
the
(12,14). [Ca++]i
Exposure (Figure
INF's
[Ca++]i of cells
returned to either
substantially
[Ca++]f.
100
75
q q
PDGF aj3lNF ‘5 PDGF
50
25
0
-El
Figure 1. Effect of INF pretreatment (15 minutes, 37'C), on PDGF-induced alterations in [Ca++]. Exposure of cells to INF's inhibited by 87% PDGFinduced increases in 1' Ca'+]i. Data is average of four experiments with three cells observed/experiment (n=12).
1209
abi-
to be 175 nM and was
PDGF addition.
addition.
in
in
transient
1).
Vol. 150, No. 3, 1988
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
IFN WI
n
L
400
U/ml
IFN bc3 200
l-PA
10’
IFN WI
400
U/ml
IFN W
200
U/ml
I 0
20
15’
U/ml
15’
13
1 1 II L 15
a0
60
40
100
% Cells Without Vlnculin Adhesion Plaques Figure 2. Effect oE INF pretreatment or TPA (150 tit)-induced disruption of inhibited PDGF but not TPA disappearance Mean of four experiments with 100-150
We have previOUSly as well very
as a number
rapid,
vinculin was found
reversible, from
adhesion
to be highly
= f ii k 5 [ i
described
of other
(15 minutes, 37-C), on PDGF (20 rig/ml) INF vinculin from adhesion plaques. of vinculin from adhesion plaques. cells examined/treatment/experiment.
that
addition
PDGF responsive
time,
temperature,
plaques
(7,11,12).
correlated
with
of PDGF to BALB/c-3T3 cell
types,
is
accompanied
and dose-dependent The change PDGF-induced
by
disruption
in vinculin entry
cells,
of cells
distribution into
200 aPlNF (10,000 Ulml)‘~ PDGF
180
aPlNF (1,000 U/ml) ‘s PDGF 160 140 120
5 3 i 8
100
8
80
Figure 3. Effect of INF pretreatment stimulated DNA synthesis ([3Hl-thymidine dependent inhibition of PDGF-stimulated ments, each done in triplicate.
(15
minutes,
tncorporation). DNA synthesis.
1210
37"C),
of
on PDGF (10 ng/ml)TNF displayed a dose Mean of two experi-
S
a
BIOCHEMICAL
Vol. 150, No. 3, 1988
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
phase and occurs via Ca++-dependent proteolysis Phorbol esters (e.g.
(11).
of adhesion plaque vinculin
TPA) have also been found to cause loss of vin-
from adhesion plaques, but by a mechanismdistinct
culin
We therefore
(15).
examined the effect
on PDGFand TPA-stimulated
of pretreatment of vinculin
At very low concentrations
(Figure 2). inhibit
disruption
from that of PDGF of cells within
INF's
from adhesion plaques
(200-400 U/ml), INF's were able to
PDGF, but not TPA, induced disruption
of vinculin
from adhesion pla-
ques. INF's were also found to inhibit following
addition
dose dependent:
incorporation
of PDGF(Figure 3). 50% inhibition
of [3H]-thymidine
The inhibition
into DNA
caused by INF's was
was seen at 1,000 U/ml and total
inhibition
at
10,000 U/ml. DISCUSSION: Our findings demonstrate that INF's block very early changes in cytoplasmic production
functions of inositol
in [Ca++]i,
vinculin
PDGF-stimulated
induced by PDGFin BALBLB/c-3T3 cells. phosphates is not affected, disruption
but PDGF-induced increases
from adhesion plaques, and entry
S phase (DNA synthesis)
are all
The data are consistent
with the idea that INF-mediated inhibition
step distal
to activation
prevented by pretreatment
of Phospholipase C resulting
in [Ca++]I
in the presence of normal phosphotidyl
sis are nst clear but may involve to the Ins (1,4,5)P3 trisphosphate
receptor to its
the endoplasmic reticulum,
inhibition
(4,5) bisphosphate hydroly-
of inositol
receptor but inhibition
or rapid efflux
trisphosphate
exposure to PDGF(4),
binding
normal binding of
of release of Ca++ from
of Ca++ from the cell The findings
following
its
that INF's also Inhibit synthesis seen in
as well as DNA synthesis (4 and this
suggest that these PDGF-stimulated alterations
may depend on an increase in [Ca++li. findings
occurs at a
in phosphotidyllnositol
of p29 and ~35, the general increase in protein
cells following study),
of cells with INF's.
on the endoplasmic reticulum,
release from the endoplasmic reticulum. induction
into
The mechanismsby which INF's prevent PDGF-stimulated increases
hydrolysis.
inonitol
of cells
functions
In support of this hypothesis are the
that INF's also block PDGF-induced disruption 1211
in cellular
of vinculin
from adhe-
Vol. 150, No. 3, 1988
BIOCHEMICAL
sion plaques and disaggregatin Ca++-dependent process (ll), disruption inhibit
of vinculin
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of stress fibers while the latter
block PDGF-mediated mitogenesis by preventing
cell
of the recep-
PDGF-induced alterations
to phosphotidylinositol
properties
do not
Thus, our data suggest that INF's
and support the hypothesis that alterations
many changes in cellular
PDGF-induced
INF's apparently
nor autophosphorylation
tor (W.J. Pledger, personal communication).
sis,
The former is a
depends on prior
from adhesion plaques (7).
binding of PDGFto its receptor
[Ca++]i at a step distal
(16).
of
(4,5) bisphosphate hydrolyin [Ca++li are necessary for
induced by PDGFas well as stimulation
of
growth by PDGF.
ACKNOWLEDGEMENTS: This work was supported by grants from the American Heart Association (AHA 861299, NC-AHA86-87-02) and from NIH (AG07218). We thank Marcy Stephens for performing the [3H]-thymidine assays.
REFERENCES 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11)
12) 13) 14) 15) 16)
Sokawa, Y., Watanabe, Y., Watanabe, Y., and Kawade, Y. (1977) Nature (Land) 268, 236-238. Lundgren, E., Larsson, I., Miorner, II., and Strannengard, 0. (1979) J. Gem Virol. 42, 589-595. Creasey, A.A., Bartholomew, J.C., and Merigan, T.C. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 1741-1745. Lin, s.L., Kikuchi, T., Pledger, W.J., and Tamm,I. (1986) Science 233, 356-358. Pledger, W.J., Stiles, C.D., Antoniades, H.N., and Scher, C.D. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 4481-4485. Leof, E.B., Wharton, W., Van Wyk, J.J., and Pledger, W.J. (1982) Exp. Cell Res. 141, 107-115. Herman, B. and Pledger, W.J. (1985) J. Cell Biol. 101, 1031-1040. DiGuiseppi, J., Inman, R., Ishihara, A., Jacobson,K;, and Herman, B. (1985) Biotech. 2, 394-403. Gynkiewicz, G., Poenie, M., and Tsien, R. (1985) J. Biol. Chem.260, 3440-3450. Becker, P.L., and Fay, F.S. (1987) Amer. J. Physiol. 253, C613-C618. Herman, B., Harrington, M.A., Olashaw, N.E., and Pledger, W.J. (1986) J. Cell. Physiol. 126, 115-125. Herman, B., Roe, M.W., Ha,rris, C., Wray, B. and Clemmons, D. (1987) Cell Motil. Cytoskel. s, 91-105. Hepler, J.R., Nakahata, N., Lovenberg, T.W., DiGuiseppi, J., Herman, B., Earp, H.S., and Harden, T.K. (1987) J. Biol. Chem.262, 2951-2956. Zagari, M., Stephens, M., Earp, H.S., and Herman, B. (1988) J. Biol. Chem. submitted. DiGuiseppi, J., Zagari, M., and Herman, E. (1986) J. Cell Btol. 103, 268a. Lin, S.L., Pledger, W.J., and Tamm,I. in The Biology of the Interferon System, W.E. Stewart II and H. Schellekensxds., Elsevier, Amsterdam, e press.
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