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Activation of protein kinase C down-regulates IFN-gamma receptors
Vol. 144, No. 1, 1987
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 337-344
April 14, 1987
ACTIVATION
OF PROTEIN KINASE C D O W N - R E G U L A T E S
Antonella Fassio, Franca Cofano, Giorgio Cavallo,
IFN-GAMMA RECEPTORS
and Santo Landolfo
Department of Microbiology, Medical School, University of Torino, 10126 - Torino, Italy Received January 19, 1987
SUMMARY: Treatment of mouse EL-4 cells with intracellular activators of protein kinase C, namely 4 -phorbol 12-myristate 13-acetate (PMA) and diacylglycerol, resulted in 90% reduction in cell surface interferon-gamma (IFN-gamma) receptors as judged by iodinated-IFN-gamma binding. This did not seem to be due to a decreased in the receptor affinity, since that of the remaining surface receptors appeared to be singnificantly increased as shown in Scatchard plot analysis. Kinetics experiments revealed that a PMA treatment as short as 15 min was sufficient to induce a decrease of 30% of IFN-gamma receptors, whereas the highest levels of down-regulation were observed after 60-90 min. Treatment of EL-4 cells with calcium ionophore, A23187, although ineffective by itself, dramatically increased the ability of suboptimal P M A concentrations to mediate IFN-gamma receptor down-regulation. Finally, specificity studies revealed that P M A is particularly effective in decreasing the binding of IFN-gamma to T-iymphocytes. Altogether these results suggest a possible involvement of protein kinase C in the regulation of IFN-gamma receptor expression. © 1987AcaaemicPress, lnc.
Phorbol esters, tetracyclic diterpenes, are potent tumor promoting agents
that
appear
to
specific
exert pleiotropic effects on cells (1,2). Their be
mediated
intracellular
receptor,
diacylglycerol-activated, been
shown
that
(5,6). Directly
finding
that
protein
activation
of
the
Ca2+/phospholipid-dependent kinase C (3,4). Recently,
1,2-diacylglycerol,
phosphatidylinositol C
through the binding and
effects
a
product
of
it
has
the
metabolism, can also activate the protein kinase
related to the activation of this e n z y m e
is
the
PMA and 1,2-diacylglycerol can affect the expression of
Address correspondence to: Dr. Santo Landolfo, Istituto di Microbiologia, Via Santena 9, lO126-Torino, Italy. Abbreviations used in this paper: IFN-gammma, interferon-gamma; PMA, 4 ~-phorbol 12-myristate 13-acetate- diC 8. 1,2-Dioctanoyl-glycerol; BSA: bovine serum aibumin." 0006-291X/87 $1.50 337
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 144, No. 1,1987
a
variety
of
membrane
down-regulation
receptors by either up-regulation
(7)
or
(8-10).
IFN-gamma, produced by T-lymphocytes
on stimulation with antigens
(II), modulates cell activity by activating genes coding for a number of
enzymes,
as yet poorly characterized , and also by enhancing
synthesis
of cell surface components
IFN-gamma
must
cells
(ii). To achieve these
interact with specific membrane
(12,13). Although
the
initial
effects,
receptors
physicochemical
the
on
target
interaction
between IFN-gamma and its receptor has now been characterized, nothing is
known
concerning the pathway of signalling transmission from
the
receptor to the intracellular environment. In this study, we present evidence that activation kinase
C
by
ionophore,
A23187,
receptors.
These
itself
1,2-Dioctanoyl-glycerol
PMA,
down-regulates results,
down-regulates
transmembrane
its
signalling
phospho-transferase
(diC8)
the expression
of
coupled with the finding own
receptors,
apparatus
suggest
involving
a
of and
calcium
the
IFN-gamma
that a
protein
IFN-gamma
common
unique
system. MATERIALS
AND METHODS
Cells. The murine cell lines used, EL-4 and L1210, were maintained in RPMI-1640 medium supplemented with 10% FCS. To analyze the effects of protein kinase C activators, cells were resuspended at 5 x 106/ml and incubated with increasing concentrations for different times. Chemicals. PMA, 1,2-Dioctanoyl-glycerol (diC8) and calcium ion0phore A23187 were purchased from Sigma Chemical C o m p a n y (St. Louis, Mo). Iodination of IFN-qamma. E. co/i-derived recombinant MuIFN-gamma with a specific activity of 1.5 x 107U/rag of protein was kindly provided by Dr. E. Adolf, Boehringer, Ingelheim (Vienna, Austria). Thirty micrograms of MuIFN-gamma were reacted for 2 hours at 4°C with 1 mCi of l~25I-Bolton-Hunter reagent ( < 2000 Ci/mmol)(Amersham, England) in 0.25 ml of sodium borate buffer, pH 8.0, as previously described (12). The reaction was stopped by addition of glycine at a final concentration of 0.2M and the mixture applied to a 20 x 0.6 cm column of Sephadex G-lO equilibrated with phosphate-buffered saline, 0.5M NaCl, pH 7.4, containing 0.2% bovine serum albumin (BSA). The fractions containing 125I-IFN-gamma were pooled, tested for antiviral activity and stored at 4° C. The specific radioactivity of the labeled IFN ranged from 30 to 40 uCi/ug. The overall recovery of antiviral activity was greater than 80%. Competition bindinq assay.s. Cells from exponetially growing cultures, resuspended at 2 x I0 ~ cells in 0.5 ml of RPMI-1640 medium
338
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol, 144, No. 1 , 1 9 8 7
supplemented with 0.2% BSA, w e r e incubated for 2 h o u r s at 23°C with constant amounts of 1 2 5 I - I F N - g a m m a a n d increasing concentrations of unlabeled IFN-gamma. Nonspecific linear binding was determined by simultaneous addition of a 500-fold e x c e s s of unlabeled IFN-gamma. After 120 min incubation at 23°C the cells were w a s h e d four times a n d the pellets c o u n t e d for cell-bound radioactivity. Nonspecific binding, n e v e r e x c e e d i n g 15% of total counts at saturation, was subtracted from total counts for determining specific binding.
RESULTS AND DISCUSSION The initial event during I F N - g a m m a represented Although
by
the
determined
and target cell interaction is
its binding to a specific m e m b r a n e
primary
structure
receptor
of m u r i n e I F N - g a m m a
from nucleotide s e q u e n c e s
has
known
about
the
now
obtained from c D N A clones
a n d that of its receptor has n o w partially characterized is
(12,13).
biochemical
signal
been (14),
(13), nothing
transduction
in
the
intracellular compartment. In IFN-gamma
order
to a n a l y z e the effect of P M A
the
expression
doses
of P M A for 2 h at 37°C. As s h o w n in Fig. I,
as
low
as
IO-9M resulted in
25-30%
loss
treatment of
surface
IFN-gamma
receptor, as a s s e s s e d by c o m p a r i s o n with untreated
cells.
higher
At
of
receptors, EL-4 cells w e r e initially treated with increasing
concentrations with
on
P M A d o s e s (10 -8 , Io-TM) the
decrease
in
control
surface
lO 40_
o ~
.~
~
°
I.
"~..
o
°
.o,_
u
.~.
5_
:----_.o\\. ~/
,
0.15
-
\
----..-: ,
,
0.76
3.84
:-------~---~---.~-
19.2
96
~
480
IFN-7 (ng)
Fig. 1. Binding competition curves of 1251_iFN_gamma to EL-4 cel~s, untreated (O-~--O) or treated with increasing concentrations of PMA ( • .... • :10- M; z~.... Z~ :10-8M; • .... • :I0-7M). Each experiment has been repeated at least three times and one representative is reported in the figure. 339
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 144, No. 1, 1987
o-o Untreated ,-, 10 min. ~ - ~ 3 hours
40-
o
o,2, "o ._= u
•
\
p,
7'/
,
,
~3
,
064
,
52
16 -
t
,
80
400
IFN- ? (.g)
Fig. 2. Binding competition curves of 125I-IFN-gamma to EL-4 cells, untreated or treated with PMA (10-TM) for increasing times ( • .... • :I0 min; A .... ~ :3 hours; • .... • :overnight). Each experiments has been repeated at least three times and one representative is reported in the figure.
receptors a
r e a c h e d 90%, indicating that loss of I F N - g a m m a receptors is
function
that due
the to
of P M A concentration. T h e s e results,
therefore,
suggest
d e c r e a s e of I F N - g a m m a binding to the EL-4 cells was the receptor down-regulation
by PMA. This h y p o t h e s i s
mainly is
also
s u p p o r t e d by the finding that a m a r k e d increase in the affinity of the remaining receptors (KD 2.8 x 10-10M of P M A - t r e a t e d vs. 1.5 x 10-9M of untreated
cells),
Scatchard
analysis,
inhibition affinity. cells
as
indicated was
by the slope of the
observed, ruling out the
curve
At
Trypan-bleu
all doses tested, moreover, the viability of PMA
treatment
always
exceeded
95%,
as
evaluated at various times
suggesting
short
the
EL-4
judged
by
by
PMA
2). It should be
after 60added
P M A resulted in r e c o v e r y of the I F N - g a m m a binding the
complete
were
after P M A treatment. A 20-30% reduction in
90 min and persisted for several hours(Fig.
shown).
receptor
sites was already detectable at 15 min, it was maximal
of
that
dye exclusion (data not shown).
The kinetics of I F N - g a m m a receptor down-regulation
removal
the
possibility
of IFN binding could be due to a d e c r e a s e in the
after
binding
in
reversibility of the P M A effect
The time c o u r s e of the o b s e r v e d down-regulation
(data
that
sites, not
is relatively
a n d closely follows the activation of proein kinase C (Fassio & 340
Vol. 144, No. 1 , 1 9 8 7
Landolfo, has
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
unpublished
been
identified
observations). Since the major receptor for P M A as the
serine/threonine-specific
Ca2+/phospholipid-dependent
diacylglycerol-activated
protein kinase C
(6), o n e can a r g u e that its activation induces p h o s p h o r y l a t i o n I F N - g a m m a receptor a n d c o n s e q u e n t l y
its down-regulation.
As already mentioned, diacylglycerols activate
protein
replace were
treated
competition occurred diC 8
have been
kinase C (5). To see w h e t h e r
P M A in the d o w n - r e g u l a t i o n
of the
demonstrated
diacylglycerol
of I F N - g a m m a
to
could
receptors, EL-4 cells
with different doses of the synthetic diC 8 a n d u s e d binding experiments. As s h o w n in Fig. 3,
in a d o s e - d e p e n d e n t
in
down-regulation
m a n n e r w h e n increasing concentrations
w e r e added, with the maximal effect occurring at 12.5 ug/ml.
As
s h o w n by Scatchard plot analysis, increase of affinity (KD 8.5 x IO-gM of
diC8-treated
vs. 2 x IO-gM of untreated cells) in
receptors was o b s e r v e d
with
diacylglycer01
to
induce
10
2500
remaining
in accord with the results obtained with PMA.
It has b e e n recently s h o w n that intracellular synergizes
the
~
o~
0.1
~
~2ooo
~
=m "u
mobilization
transferrin
receptor
o
° ~ o
~.
~ o
Ca 2+
"~
..,
" 1500 -~
0.001
°
e~ 1000_ O. (n
I::ls 1
500
/
50
" ~
0.13
0.64
3.2
'16
80
400
IFN-'y (ng)
Fig. 3. Binding competition curves of 1251-1FN-gamma to EL-4 cells, untreated ( O .... O ) or treated with increasing concentrations of dic 8
has
( • ....
been
reported
ZX :6,25 u g / m l ;
repeated in the
• ....
•
:12,50 u g / m l ) . E a c h e x p e r J . m e n t s
at least three times and
figure. 341
one
representative
is
of
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 144, No. 1,1987
40_
o--o
o ~
+A23187 (100pM) .... PMA ( ' 0 " 9 M )
o~ 20-
0
untreated
~--~
,~
5-
m.
u)
-7// 0,15
0.76
3.84
19.2
96
480
IFN-'Y (ng) Fig. 4. Binding competition curves of 1251-IFN-gamma to EL-4 cells, untreated ( O .... O ) or treated for three hour~ with A23187 2+
.
Ca -lonophore ( zl.... Z~ : 100pM), P M A ( • .... • :i0- M) or A 2 3 1 8 7 and PMA together. E a c h e x p e r i m e n t h a s b e e n r e p e a t e d at least three t~mes a n d o n e r e p r e s e n t a t i v e is r e p o r t e d in the figure.
down-regulation modulate cells
(15). To
expression
were
treated
ionophore
themselves
as
(about
of
IO-gM
down-regulation. suggest
of
the IFN-gamma
with
increasing
could
receptor in our
concentrations
also
system, of
EL-4
Ca 2+
the
A23187 and used in binding experiments. As shown in Eig. 4,
concentrations
potency
see whether Ca 2+ mobilization
low
as
i00 pM, although not
23% down-regulation), PMA
The
as
a
very
increased
mediator
of
effective
dramatically
IFN-gamma
results obtained with diC 8 and
Ca 2+
the
receptor ionophore
that proein kinase C is involved in this down-regulation
decrease
of
affinity.
phosphatidylinositol
Diacylglycerol,
a
primary
product
in
and
of
metabolism, has, in fact, been shown to bind and
activate protein kinase C (5). Yet the use of Ca2+ionophore A23187 has revealed kinase
an C
Raising increases
apparent synergism between Ca2+mobilization and
activation of
in the down-regulation
intracellular the
Ca 2+,
although
of
membrane
ineffective
by
protein
receptors. itself,
potency of P M A in the activation of protein kinase
phosphorylation
of
down-regulation.
We
transferrin
receptor
and
its subsequent
would therefore suggest that IFN-gamma
342
C,
receptor
Vol. 144, No. 1, 1987
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
EL-4
40
R" O v,-
o-o Untreated * - * PMA (10-7M)
L 1210 ~-~ Untreated , - , PMA (10-7M)
°~o
x
E
20
O. u rO~ "O c
10
\
.D
.u_ U)
i
i
i
0.15
0.76
384
i ""-'-
19.2
ei ' - ' ' -
96
480
IFN-7
0.15
0.76
~84
19.2
96
480
(ng)
Fig. 5. Binding competition curves of 125I-IFN-gamma to EL-~ and LI210 cells untreated or treated for 3 hours with PMA (IO-~M). Each experiment has been repeated at least three times and one representative is reported in the figure.
down-regulation
also o c c u r r e d via protein kinase C activation in
our
system. Since the effect on surface I F N - g a m m a receptor evaluated cells
of
were
as
expression
was
on EL-4 cells, a T i y m p h o m a line, we decided to see w h e t h e r B-lymphocyte-, mcrophage-, fibroblast-, sensitive
to d o w n - r e g u l a t i o n
mastocytoma-origin
by PMA. Our
results
indicate
that these cell lines are m u c h less sensitive. In Fig. 5, a c o m p a r i s o n between indicate protein
EL-4 T-cells a n d LI210 B-cells is shown. therefore kinase
observation although
can
widely
particularly
that
Specificity studies
d o w n regulation of I F N - g a m m a
receptors
C activators is particularly evident on T-cells. be e x p l a i n e d by the finding that protein distributed
in
the tissues
a b u n d a n t in l y m p h o c y t e s
of
many
by
This
kinase
species,
(6). In this connection, it
C, is
may
be m e n t i o n e d that s o m e time ago Kraft et al. (16) f o u n d high levels of protein
kinase C in
EL-4 cells, a n d d e m o n s t r a t e d that P M A induces
a
translocation of protein kinase C from the cytosol to the membrane. Activation of protein kinase C by P M A causes
both
a
or
sn-l,2-diacylglycerols
d e c r e a s e in the affinity of s o m e receptors,
343
such
as
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 144, No. 1, 1987
insulin
and
such as transferrin (8). Moreover,
receptors be
epidermal growth factor (9,10), and
mediated by h y p e r p h o s p h o r y l a t i o n
protein
kinase
C
(8). Our
1,2-Dioctanoyl-glycerol receptor the
both p h e n o m e n a
of the receptors
demonstration
induce
down-regulation
extend those findings to the I F N - g a m m a
first
time
a
possible
regulation of the I F N - g a m m a
down-regulation
involvement
a p p e a r to
by the activated that
of
of
the
PMA
and
IFN-gamma
system and suggest for
of
protein
by grants from the
Italian
kinase
C
receptor.
ACKNOWLEDGMENTS This w o r k was s u p p o r t e d National
Research
Council (PF'Oncologia' No 85.02161.44, a n d
No 85.00860.52), and from the Ministry of Public Education
PF'CMI'
(40%).
REFERENCES l. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Diamond, L., O'Brien, T., and Rovera, G. (]978) Life Sci. 23, 1979-1988. Rovera, G., Santo/i, D., and Samsky, C. (1979) Proc. Natl. Acad. Sci. USA 76, 2779-2783. Castagna, M., Takui, Y., Kaibuchi, K., Sano, R., Kikkawa, U., and Nishizuka, Y. (1982) J. biol. Chem. 257, 7847-7851. Leach, K.L., James, M.L., and Blumberg, P.M. (1983) Proc. Natl. Acad. Sci. USA 80, 4208-4212. Ganong, B.R., Loomis, C.R., Hannun, Y.A., and Bell, R.M. (1986) Proc. Natl. Acad. Sci. USA 83, 1184-1188. Nishizuka, Y. (]984) Nature (London) 308, 693-697. Farrar, W.L., a n d Ruscetti, F.W. (1986) J. Immunol. 136, 1266-1273. May, W.S., Jacobs, S., and Cuatrecasas, P. (1984) Proc. Natl. Acad. Sci. USA 81, 2016-2020. Thomopoulos, P., Testa, Y., Gourdin, M., Hervy, C., Titeux, M., and Vainchenker, W. (1982) Eur. J. Biochem. 129, 389-393. Shoyab, M., DeLarco, J.E., and Todaro, G.J. (1979) Nature (London) 279, 387-391. Vilcek, J., Gray, P.W., Rinderknecht, E., and Sevastopoulos, C.G. (1985) L y m p h o k i n e s ii, 1-33. Cofano, F., Fassio, A., Cavallo, G., and Landolfo, S. (1986) J. Gen. Virol. 67, 1205-1209. Zoon, K., and Arnheiter, H. (1984) P h a r m a c o l o g y and Therapy, pp. 259-278, P e r g a m o n Press, London. Gray, P.W., and Goeddel, D.V. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 5842-5846. May, W.S., Sahyoun, N., Wolf, M., and Cuatrecasas, P. (1985) Nature 317, 549-551. Kraft, A.S., Anderson, W.B., Cooper, H.I., and Sando, J.J. (1982) J. biol. Chem. 257, 13193-13196.
344
in
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 337-344
April 14, 1987
ACTIVATION
OF PROTEIN KINASE C D O W N - R E G U L A T E S
Antonella Fassio, Franca Cofano, Giorgio Cavallo,
IFN-GAMMA RECEPTORS
and Santo Landolfo
Department of Microbiology, Medical School, University of Torino, 10126 - Torino, Italy Received January 19, 1987
SUMMARY: Treatment of mouse EL-4 cells with intracellular activators of protein kinase C, namely 4 -phorbol 12-myristate 13-acetate (PMA) and diacylglycerol, resulted in 90% reduction in cell surface interferon-gamma (IFN-gamma) receptors as judged by iodinated-IFN-gamma binding. This did not seem to be due to a decreased in the receptor affinity, since that of the remaining surface receptors appeared to be singnificantly increased as shown in Scatchard plot analysis. Kinetics experiments revealed that a PMA treatment as short as 15 min was sufficient to induce a decrease of 30% of IFN-gamma receptors, whereas the highest levels of down-regulation were observed after 60-90 min. Treatment of EL-4 cells with calcium ionophore, A23187, although ineffective by itself, dramatically increased the ability of suboptimal P M A concentrations to mediate IFN-gamma receptor down-regulation. Finally, specificity studies revealed that P M A is particularly effective in decreasing the binding of IFN-gamma to T-iymphocytes. Altogether these results suggest a possible involvement of protein kinase C in the regulation of IFN-gamma receptor expression. © 1987AcaaemicPress, lnc.
Phorbol esters, tetracyclic diterpenes, are potent tumor promoting agents
that
appear
to
specific
exert pleiotropic effects on cells (1,2). Their be
mediated
intracellular
receptor,
diacylglycerol-activated, been
shown
that
(5,6). Directly
finding
that
protein
activation
of
the
Ca2+/phospholipid-dependent kinase C (3,4). Recently,
1,2-diacylglycerol,
phosphatidylinositol C
through the binding and
effects
a
product
of
it
has
the
metabolism, can also activate the protein kinase
related to the activation of this e n z y m e
is
the
PMA and 1,2-diacylglycerol can affect the expression of
Address correspondence to: Dr. Santo Landolfo, Istituto di Microbiologia, Via Santena 9, lO126-Torino, Italy. Abbreviations used in this paper: IFN-gammma, interferon-gamma; PMA, 4 ~-phorbol 12-myristate 13-acetate- diC 8. 1,2-Dioctanoyl-glycerol; BSA: bovine serum aibumin." 0006-291X/87 $1.50 337
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 144, No. 1,1987
a
variety
of
membrane
down-regulation
receptors by either up-regulation
(7)
or
(8-10).
IFN-gamma, produced by T-lymphocytes
on stimulation with antigens
(II), modulates cell activity by activating genes coding for a number of
enzymes,
as yet poorly characterized , and also by enhancing
synthesis
of cell surface components
IFN-gamma
must
cells
(ii). To achieve these
interact with specific membrane
(12,13). Although
the
initial
effects,
receptors
physicochemical
the
on
target
interaction
between IFN-gamma and its receptor has now been characterized, nothing is
known
concerning the pathway of signalling transmission from
the
receptor to the intracellular environment. In this study, we present evidence that activation kinase
C
by
ionophore,
A23187,
receptors.
These
itself
1,2-Dioctanoyl-glycerol
PMA,
down-regulates results,
down-regulates
transmembrane
its
signalling
phospho-transferase
(diC8)
the expression
of
coupled with the finding own
receptors,
apparatus
suggest
involving
a
of and
calcium
the
IFN-gamma
that a
protein
IFN-gamma
common
unique
system. MATERIALS
AND METHODS
Cells. The murine cell lines used, EL-4 and L1210, were maintained in RPMI-1640 medium supplemented with 10% FCS. To analyze the effects of protein kinase C activators, cells were resuspended at 5 x 106/ml and incubated with increasing concentrations for different times. Chemicals. PMA, 1,2-Dioctanoyl-glycerol (diC8) and calcium ion0phore A23187 were purchased from Sigma Chemical C o m p a n y (St. Louis, Mo). Iodination of IFN-qamma. E. co/i-derived recombinant MuIFN-gamma with a specific activity of 1.5 x 107U/rag of protein was kindly provided by Dr. E. Adolf, Boehringer, Ingelheim (Vienna, Austria). Thirty micrograms of MuIFN-gamma were reacted for 2 hours at 4°C with 1 mCi of l~25I-Bolton-Hunter reagent ( < 2000 Ci/mmol)(Amersham, England) in 0.25 ml of sodium borate buffer, pH 8.0, as previously described (12). The reaction was stopped by addition of glycine at a final concentration of 0.2M and the mixture applied to a 20 x 0.6 cm column of Sephadex G-lO equilibrated with phosphate-buffered saline, 0.5M NaCl, pH 7.4, containing 0.2% bovine serum albumin (BSA). The fractions containing 125I-IFN-gamma were pooled, tested for antiviral activity and stored at 4° C. The specific radioactivity of the labeled IFN ranged from 30 to 40 uCi/ug. The overall recovery of antiviral activity was greater than 80%. Competition bindinq assay.s. Cells from exponetially growing cultures, resuspended at 2 x I0 ~ cells in 0.5 ml of RPMI-1640 medium
338
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol, 144, No. 1 , 1 9 8 7
supplemented with 0.2% BSA, w e r e incubated for 2 h o u r s at 23°C with constant amounts of 1 2 5 I - I F N - g a m m a a n d increasing concentrations of unlabeled IFN-gamma. Nonspecific linear binding was determined by simultaneous addition of a 500-fold e x c e s s of unlabeled IFN-gamma. After 120 min incubation at 23°C the cells were w a s h e d four times a n d the pellets c o u n t e d for cell-bound radioactivity. Nonspecific binding, n e v e r e x c e e d i n g 15% of total counts at saturation, was subtracted from total counts for determining specific binding.
RESULTS AND DISCUSSION The initial event during I F N - g a m m a represented Although
by
the
determined
and target cell interaction is
its binding to a specific m e m b r a n e
primary
structure
receptor
of m u r i n e I F N - g a m m a
from nucleotide s e q u e n c e s
has
known
about
the
now
obtained from c D N A clones
a n d that of its receptor has n o w partially characterized is
(12,13).
biochemical
signal
been (14),
(13), nothing
transduction
in
the
intracellular compartment. In IFN-gamma
order
to a n a l y z e the effect of P M A
the
expression
doses
of P M A for 2 h at 37°C. As s h o w n in Fig. I,
as
low
as
IO-9M resulted in
25-30%
loss
treatment of
surface
IFN-gamma
receptor, as a s s e s s e d by c o m p a r i s o n with untreated
cells.
higher
At
of
receptors, EL-4 cells w e r e initially treated with increasing
concentrations with
on
P M A d o s e s (10 -8 , Io-TM) the
decrease
in
control
surface
lO 40_
o ~
.~
~
°
I.
"~..
o
°
.o,_
u
.~.
5_
:----_.o\\. ~/
,
0.15
-
\
----..-: ,
,
0.76
3.84
:-------~---~---.~-
19.2
96
~
480
IFN-7 (ng)
Fig. 1. Binding competition curves of 1251_iFN_gamma to EL-4 cel~s, untreated (O-~--O) or treated with increasing concentrations of PMA ( • .... • :10- M; z~.... Z~ :10-8M; • .... • :I0-7M). Each experiment has been repeated at least three times and one representative is reported in the figure. 339
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 144, No. 1, 1987
o-o Untreated ,-, 10 min. ~ - ~ 3 hours
40-
o
o,2, "o ._= u
•
\
p,
7'/
,
,
~3
,
064
,
52
16 -
t
,
80
400
IFN- ? (.g)
Fig. 2. Binding competition curves of 125I-IFN-gamma to EL-4 cells, untreated or treated with PMA (10-TM) for increasing times ( • .... • :I0 min; A .... ~ :3 hours; • .... • :overnight). Each experiments has been repeated at least three times and one representative is reported in the figure.
receptors a
r e a c h e d 90%, indicating that loss of I F N - g a m m a receptors is
function
that due
the to
of P M A concentration. T h e s e results,
therefore,
suggest
d e c r e a s e of I F N - g a m m a binding to the EL-4 cells was the receptor down-regulation
by PMA. This h y p o t h e s i s
mainly is
also
s u p p o r t e d by the finding that a m a r k e d increase in the affinity of the remaining receptors (KD 2.8 x 10-10M of P M A - t r e a t e d vs. 1.5 x 10-9M of untreated
cells),
Scatchard
analysis,
inhibition affinity. cells
as
indicated was
by the slope of the
observed, ruling out the
curve
At
Trypan-bleu
all doses tested, moreover, the viability of PMA
treatment
always
exceeded
95%,
as
evaluated at various times
suggesting
short
the
EL-4
judged
by
by
PMA
2). It should be
after 60added
P M A resulted in r e c o v e r y of the I F N - g a m m a binding the
complete
were
after P M A treatment. A 20-30% reduction in
90 min and persisted for several hours(Fig.
shown).
receptor
sites was already detectable at 15 min, it was maximal
of
that
dye exclusion (data not shown).
The kinetics of I F N - g a m m a receptor down-regulation
removal
the
possibility
of IFN binding could be due to a d e c r e a s e in the
after
binding
in
reversibility of the P M A effect
The time c o u r s e of the o b s e r v e d down-regulation
(data
that
sites, not
is relatively
a n d closely follows the activation of proein kinase C (Fassio & 340
Vol. 144, No. 1 , 1 9 8 7
Landolfo, has
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
unpublished
been
identified
observations). Since the major receptor for P M A as the
serine/threonine-specific
Ca2+/phospholipid-dependent
diacylglycerol-activated
protein kinase C
(6), o n e can a r g u e that its activation induces p h o s p h o r y l a t i o n I F N - g a m m a receptor a n d c o n s e q u e n t l y
its down-regulation.
As already mentioned, diacylglycerols activate
protein
replace were
treated
competition occurred diC 8
have been
kinase C (5). To see w h e t h e r
P M A in the d o w n - r e g u l a t i o n
of the
demonstrated
diacylglycerol
of I F N - g a m m a
to
could
receptors, EL-4 cells
with different doses of the synthetic diC 8 a n d u s e d binding experiments. As s h o w n in Fig. 3,
in a d o s e - d e p e n d e n t
in
down-regulation
m a n n e r w h e n increasing concentrations
w e r e added, with the maximal effect occurring at 12.5 ug/ml.
As
s h o w n by Scatchard plot analysis, increase of affinity (KD 8.5 x IO-gM of
diC8-treated
vs. 2 x IO-gM of untreated cells) in
receptors was o b s e r v e d
with
diacylglycer01
to
induce
10
2500
remaining
in accord with the results obtained with PMA.
It has b e e n recently s h o w n that intracellular synergizes
the
~
o~
0.1
~
~2ooo
~
=m "u
mobilization
transferrin
receptor
o
° ~ o
~.
~ o
Ca 2+
"~
..,
" 1500 -~
0.001
°
e~ 1000_ O. (n
I::ls 1
500
/
50
" ~
0.13
0.64
3.2
'16
80
400
IFN-'y (ng)
Fig. 3. Binding competition curves of 1251-1FN-gamma to EL-4 cells, untreated ( O .... O ) or treated with increasing concentrations of dic 8
has
( • ....
been
reported
ZX :6,25 u g / m l ;
repeated in the
• ....
•
:12,50 u g / m l ) . E a c h e x p e r J . m e n t s
at least three times and
figure. 341
one
representative
is
of
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 144, No. 1,1987
40_
o--o
o ~
+A23187 (100pM) .... PMA ( ' 0 " 9 M )
o~ 20-
0
untreated
~--~
,~
5-
m.
u)
-7// 0,15
0.76
3.84
19.2
96
480
IFN-'Y (ng) Fig. 4. Binding competition curves of 1251-IFN-gamma to EL-4 cells, untreated ( O .... O ) or treated for three hour~ with A23187 2+
.
Ca -lonophore ( zl.... Z~ : 100pM), P M A ( • .... • :i0- M) or A 2 3 1 8 7 and PMA together. E a c h e x p e r i m e n t h a s b e e n r e p e a t e d at least three t~mes a n d o n e r e p r e s e n t a t i v e is r e p o r t e d in the figure.
down-regulation modulate cells
(15). To
expression
were
treated
ionophore
themselves
as
(about
of
IO-gM
down-regulation. suggest
of
the IFN-gamma
with
increasing
could
receptor in our
concentrations
also
system, of
EL-4
Ca 2+
the
A23187 and used in binding experiments. As shown in Eig. 4,
concentrations
potency
see whether Ca 2+ mobilization
low
as
i00 pM, although not
23% down-regulation), PMA
The
as
a
very
increased
mediator
of
effective
dramatically
IFN-gamma
results obtained with diC 8 and
Ca 2+
the
receptor ionophore
that proein kinase C is involved in this down-regulation
decrease
of
affinity.
phosphatidylinositol
Diacylglycerol,
a
primary
product
in
and
of
metabolism, has, in fact, been shown to bind and
activate protein kinase C (5). Yet the use of Ca2+ionophore A23187 has revealed kinase
an C
Raising increases
apparent synergism between Ca2+mobilization and
activation of
in the down-regulation
intracellular the
Ca 2+,
although
of
membrane
ineffective
by
protein
receptors. itself,
potency of P M A in the activation of protein kinase
phosphorylation
of
down-regulation.
We
transferrin
receptor
and
its subsequent
would therefore suggest that IFN-gamma
342
C,
receptor
Vol. 144, No. 1, 1987
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
EL-4
40
R" O v,-
o-o Untreated * - * PMA (10-7M)
L 1210 ~-~ Untreated , - , PMA (10-7M)
°~o
x
E
20
O. u rO~ "O c
10
\
.D
.u_ U)
i
i
i
0.15
0.76
384
i ""-'-
19.2
ei ' - ' ' -
96
480
IFN-7
0.15
0.76
~84
19.2
96
480
(ng)
Fig. 5. Binding competition curves of 125I-IFN-gamma to EL-~ and LI210 cells untreated or treated for 3 hours with PMA (IO-~M). Each experiment has been repeated at least three times and one representative is reported in the figure.
down-regulation
also o c c u r r e d via protein kinase C activation in
our
system. Since the effect on surface I F N - g a m m a receptor evaluated cells
of
were
as
expression
was
on EL-4 cells, a T i y m p h o m a line, we decided to see w h e t h e r B-lymphocyte-, mcrophage-, fibroblast-, sensitive
to d o w n - r e g u l a t i o n
mastocytoma-origin
by PMA. Our
results
indicate
that these cell lines are m u c h less sensitive. In Fig. 5, a c o m p a r i s o n between indicate protein
EL-4 T-cells a n d LI210 B-cells is shown. therefore kinase
observation although
can
widely
particularly
that
Specificity studies
d o w n regulation of I F N - g a m m a
receptors
C activators is particularly evident on T-cells. be e x p l a i n e d by the finding that protein distributed
in
the tissues
a b u n d a n t in l y m p h o c y t e s
of
many
by
This
kinase
species,
(6). In this connection, it
C, is
may
be m e n t i o n e d that s o m e time ago Kraft et al. (16) f o u n d high levels of protein
kinase C in
EL-4 cells, a n d d e m o n s t r a t e d that P M A induces
a
translocation of protein kinase C from the cytosol to the membrane. Activation of protein kinase C by P M A causes
both
a
or
sn-l,2-diacylglycerols
d e c r e a s e in the affinity of s o m e receptors,
343
such
as
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 144, No. 1, 1987
insulin
and
such as transferrin (8). Moreover,
receptors be
epidermal growth factor (9,10), and
mediated by h y p e r p h o s p h o r y l a t i o n
protein
kinase
C
(8). Our
1,2-Dioctanoyl-glycerol receptor the
both p h e n o m e n a
of the receptors
demonstration
induce
down-regulation
extend those findings to the I F N - g a m m a
first
time
a
possible
regulation of the I F N - g a m m a
down-regulation
involvement
a p p e a r to
by the activated that
of
of
the
PMA
and
IFN-gamma
system and suggest for
of
protein
by grants from the
Italian
kinase
C
receptor.
ACKNOWLEDGMENTS This w o r k was s u p p o r t e d National
Research
Council (PF'Oncologia' No 85.02161.44, a n d
No 85.00860.52), and from the Ministry of Public Education
PF'CMI'
(40%).
REFERENCES l. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Diamond, L., O'Brien, T., and Rovera, G. (]978) Life Sci. 23, 1979-1988. Rovera, G., Santo/i, D., and Samsky, C. (1979) Proc. Natl. Acad. Sci. USA 76, 2779-2783. Castagna, M., Takui, Y., Kaibuchi, K., Sano, R., Kikkawa, U., and Nishizuka, Y. (1982) J. biol. Chem. 257, 7847-7851. Leach, K.L., James, M.L., and Blumberg, P.M. (1983) Proc. Natl. Acad. Sci. USA 80, 4208-4212. Ganong, B.R., Loomis, C.R., Hannun, Y.A., and Bell, R.M. (1986) Proc. Natl. Acad. Sci. USA 83, 1184-1188. Nishizuka, Y. (]984) Nature (London) 308, 693-697. Farrar, W.L., a n d Ruscetti, F.W. (1986) J. Immunol. 136, 1266-1273. May, W.S., Jacobs, S., and Cuatrecasas, P. (1984) Proc. Natl. Acad. Sci. USA 81, 2016-2020. Thomopoulos, P., Testa, Y., Gourdin, M., Hervy, C., Titeux, M., and Vainchenker, W. (1982) Eur. J. Biochem. 129, 389-393. Shoyab, M., DeLarco, J.E., and Todaro, G.J. (1979) Nature (London) 279, 387-391. Vilcek, J., Gray, P.W., Rinderknecht, E., and Sevastopoulos, C.G. (1985) L y m p h o k i n e s ii, 1-33. Cofano, F., Fassio, A., Cavallo, G., and Landolfo, S. (1986) J. Gen. Virol. 67, 1205-1209. Zoon, K., and Arnheiter, H. (1984) P h a r m a c o l o g y and Therapy, pp. 259-278, P e r g a m o n Press, London. Gray, P.W., and Goeddel, D.V. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 5842-5846. May, W.S., Sahyoun, N., Wolf, M., and Cuatrecasas, P. (1985) Nature 317, 549-551. Kraft, A.S., Anderson, W.B., Cooper, H.I., and Sando, J.J. (1982) J. biol. Chem. 257, 13193-13196.
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in