Vol. 188, No. 2, 1992 October 30, 1992
BIOCHEMICAL
AND BlOPHYSlCAL
RESEARCH COMMUNICATIONS Pages 524-530
PL4TELlCDJCRIVEDGROWTHFACl'ORAAHOMOD~ !xIMULATRSPRoTEINSYNTEESIS RATHERTEANDNASYNTIESISINVA!XULAR sMooTBMuscLEcELlsFRoM spoNTANE(xIsLY EYPlWXNSIVERATSBUTNOTFROMNORMYlXNSIVERATS Hiroshi Department
Inui,
of Biochemistry,
Takao Kondo and Tadashi
Inagami
Vanderbilt University Nashville, TN 37232
School of Medicine,
Received September 12, 1992
Sumary: Platelet-derived growth factor (PDGF) AB and BB isoforms were potent mitogens for cultured vascular smooth muscle cells from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). PDGF-AA promotes protein synthesis in a dose-dependent manner in SHR cells, whereas DNA synthesis was stimulated only slightly. However, this isoform did not activate either DNA or protein synthesis in WKY cells. PDGF-AA stimulated tyrosine phosphorylation of its receptor protein and phospholipase C-y, in SHR,cell but not in WKY cells. These results indicate that vascular smooth muscle cell of SHR is uniquely responsive to PDGF-AA, presumably due to abnormality in receptor expression, in its hypertrophic response. 0 1992 Academic mess,
Inc.
Platelet-derived disulfide-linked
growth polypeptides
can form homodimers receptor subunits, shows high affinity preferentially
as well termed for
binds
factor
(PDGF) is
composed of
denoted
A and B chains,
as the AB heterodimer
and the A and B chains (1).
Two types
c1 and S, have been demonstrated. both A and B chains whereas
B chain
two homologous, of PDGF
The a subunit the S subunit
(2-4).
The receptor subunits appear to exist independently on cell membranes, and may dimerize in response to ligand binding. A tyrosine kinase domain exists in both types of receptor subunits, and is activated
upon the binding
of PDGF.
We have reported that PDGF-BB is a potent mitogen and stimulates tyrosine phosphorylation of various proteins in vascular smooth muscle cells (VSMC) of normotensive
Wistar-Kyoto
rats
(WKY) (5).
phosphorylation is less extensive with this isoform stimulates DNA synthesis isoform.
In contrast,
Increase hypertension characteristic (8.9).
Resink
Activation of protein tyrosine PDGF-AB than the BB isoform, although almost to the same extent as the BB
PDGF-AAdoes not act as a mitogen
in the cells.
of vascular wall thickening is a common feature of chronic (6,7). Unusually rapid growth is also recognized as a of cultured VSMC from spontaneously hypertensive rats (SHR) et
al.
(10)
0006-293X/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
reported
that
524
long
term
exposure
to
PDGF-AA
Vol.
188, No. 2, 1992
resulted
in
BIOCHEMICAL
gradual
and Owens (11)
growth
rats
of PDGF in
vascular
by
cultured
WKY in short
inactive
VSMC from SHR.
In addition, Blank cultured VSMC from
treatment with PDGF-BB. hypertrophy in cells with different
However, mechanism genetic background
that
hypertrophy
for
occurred
chronic
is not clear. We here report that than DNA synthesis in cultured completely
RESEARCH COMMUNICATIONS
in
observed
normotensive
in
AND BIOPHYSICAL
both
PDGF-AA stimulates protein VSMC from SHR whereas
DNA and protein
synthesis
synthesis rather this isoform is in
the cells
from
term experiments. MATWIALS
AND tf.EmoDs
Materials: Recombinant human PDGF-AA, -AB and -BB, and antiphosphotyrosine and anti-bovine phospholipase C-y, (PLC-yl) antibodies were products Biotechnology, Placid, Upstate Inc. (Lake NY). [Methyl-3H]ThO:midine Ci/mmol), [5-'Hluridine Ci/mmol) and (86 (26 [5,6-3H]leucine (163 Ci/mmol) were purchased from Amersham (Arlington Heights, IL), and [ "'I]-goat anti-mouse IgG antibody from ICN (Costa Mesa, CA). Cell culture: VSMC were isolated from thoracic aorta of lo-week old female SHR (n=5, systolic blood pressure 165-190) and WKY (n=5, systolic blood pressure 115-125), purchased from Taconic Farm (Germem Town, PA), according to Jones et al. (12). Cells were grown in Dulbeco's modified Eagle's medium (DMEM) with 10% fetal serum, and were fed every other days. Cells between passage 7-12 were seeded in a dish (10% cells/cm2), and used at confluence. Measurement of DNA, RNA and protein synthesis: Confluent VSMCin a 13-mm dish were cultured in a mixture (1:l) of DMEM and Ham's F-12 medium, supplemented with insulin (5 ug/ml), transferrin (5 ug/ml) and sodium serenite (5 rig/ml), for 48 h, and then exposed to PDGF in 1 ml of the medium containing 1 mg/ml bovine serum albumin (BSA). After 16 h of the exposure, [methyl-3H]thymidine or [5-3H]uridine (2 uCi/dish) was added into the medium and incubated for additional 4 h. Cells were washed with cold 20 mM HEPES-NaOHbuffer, pH 7.4, containing 130 mM NaCl, 5 mM KCl, 1 mM MgC12 and 1.5 mM CaC12 (Buffer A), and treated with 10% (w/w) trichloroacetic acid (TCA). After washing with a mixture of ethanol and ethyl ether (2:1, v/v), the TCA-insoluble fraction was solubilized with 0.5 N NaOH, and radioactivity in newly synthesized DNA or RNA in this fraction was counted. VSMC in a 13-mm dish, precultured For measurement of protein synthesis, in the mixture of DMEM and Ham's F-12 medium for 48 h, were incubated with PDGF in 1 ml of the same medium containing [5,6-'H]leucine (3 uCi/dish) and BSA (1 me/ml) for 20 h and radioactivity in the TCA-insoluble fraction was Protein content was measured as described (13) with BSA as a counted. standard. Immunoblotting: VSMC in a loo-mm dish, precultured in DMEMsupplemented with insulin (5 ug/ml), transferrin (5 ug/ml) and sodium serenite (5 rig/ml) for 48 h, were stimulated with PDGF (100 rig/ml) in Buffer A containing 1 mg/ml BSA and 10 mM glucose for 5 min. After washing with cold Buffer A containing 2 mM ethyleneglycol-bis(B-aminoethyl ether) N,N,N',N'-tetraacetate (EGTA) in place of CaC12, the cells were disrupted with 20 mM HEPES-NaOHbuffer, pH 7.4, containing 0.5% Nonidet P-40, 50 mM B-glycerophosphate, 0.1 mM orthovanadate, 10 uM molybdate, 1 mM phenylmethylsulfonyl fluoride, 10 ug/ml After centrifugation (14,000 aprotinin and 0.1 mg/ml leupeptin at 4 "C. The lysate was x g, 5 min), the supernatant was used as cell lysate. subjected to sodium dodecyl sulfate (SDS)-acrylamide gel (7.5%) in the were electroblotted to a electrophoresis, and proteins gel The membrane was treated with anti-phosphotyrosine nitrocellulose membrane. antibody in 10 mM Tris-HCl buffer, pH 7.4, containing 3% BSA, 0.9% NaCl and 0.1% Triton X-100, and tyrosine-phosphorylated proteins were detected by autoradiography with [1251]-goat anti-mouse IgG antibody. For detection
525
Vol.
188,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
of tyrosine-phosphorylated PJX-yr, the cell lysate was incubated with antibody and Protein A-Sepharose at 4 'C for 24 h. anti-phosphotyrosine Proteins immunoprecipitated were redisolved with 50 mM Tris-HCl buffer, pH 7.4, containing 2% SDS and 5% 2-mercaptoethanol, at 100 "C, subjected to electrophoresis and immunoblotted with anti-PLC-Yr antibody.
RESIJLTSAND DISCDSSION Effect of PDGF-AA, -AB and -BB on DNA synthesis was determined in cultured VSMC from SHR and WKY by pulse labeling experiments with labeled thymidine.
As shown in
Fig.
1, PDGF-BB acted
as a potent
mitogen
for
both
SHR and WKY cells, and DNA synthesis was activated in concentration-dependent manners about 16-fold in SHR cells and about 12-fold in WKY cells by 25 rig/ml PDGF-BB. PDGF-AB also stimulated DNA synthesis showing a similar profile as that observed with the BB isoform in both SHR and WKY cells. PDGF-AA was a very weak mitogen in SHR cells, and enhanced
DNA synthesis
only about 2-fold even at a concentration as high as 100 rig/ml. PDGF-AA was completely inactive in stimulating DNA synthesis.
In WKY cells, The results
obtained (5),
in WKYcells
are consistent
and are also similar
with those reported
In contrast,
in our previous
to those observed in cultured
paper
bovine VSMC (14).
m
0
20 0 PDGF
Fig.
1,
02
(rig/ml)
Stimulation
of
Stimulation and -BB (
q
25
DNA synthesis
by
PDGF
50
75
PDGF
isoforms
in
@g/ml)
SHR and
of DNA synthesis by PDGF-AA (O,@), ,m ) in SHR ( 0. A, 0 1 and WKY( 0.
100
-AB
UKY cells.
(n,A
A, NJ
)
cells
was determined with [methyl- Hlthymidine
as described in MATERIALS
Increase
PDGF
AND METHODS. Each experimental point (mean of 4 determinations, SEM
of
isoforms.
cells were incubated with PDGF-AA (0, .I, -AB (A,A) or -BB (0.m) for 20 h, and protein content per dish was determined. Each experimental point is presented as mean of 4 determinations (SEM
BIOCHEMICAL
AND BlOPHYSlCAL
RESEARCH COMMUNICATIONS Pages 524-530
PL4TELlCDJCRIVEDGROWTHFACl'ORAAHOMOD~ !xIMULATRSPRoTEINSYNTEESIS RATHERTEANDNASYNTIESISINVA!XULAR sMooTBMuscLEcELlsFRoM spoNTANE(xIsLY EYPlWXNSIVERATSBUTNOTFROMNORMYlXNSIVERATS Hiroshi Department
Inui,
of Biochemistry,
Takao Kondo and Tadashi
Inagami
Vanderbilt University Nashville, TN 37232
School of Medicine,
Received September 12, 1992
Sumary: Platelet-derived growth factor (PDGF) AB and BB isoforms were potent mitogens for cultured vascular smooth muscle cells from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). PDGF-AA promotes protein synthesis in a dose-dependent manner in SHR cells, whereas DNA synthesis was stimulated only slightly. However, this isoform did not activate either DNA or protein synthesis in WKY cells. PDGF-AA stimulated tyrosine phosphorylation of its receptor protein and phospholipase C-y, in SHR,cell but not in WKY cells. These results indicate that vascular smooth muscle cell of SHR is uniquely responsive to PDGF-AA, presumably due to abnormality in receptor expression, in its hypertrophic response. 0 1992 Academic mess,
Inc.
Platelet-derived disulfide-linked
growth polypeptides
can form homodimers receptor subunits, shows high affinity preferentially
as well termed for
binds
factor
(PDGF) is
composed of
denoted
A and B chains,
as the AB heterodimer
and the A and B chains (1).
Two types
c1 and S, have been demonstrated. both A and B chains whereas
B chain
two homologous, of PDGF
The a subunit the S subunit
(2-4).
The receptor subunits appear to exist independently on cell membranes, and may dimerize in response to ligand binding. A tyrosine kinase domain exists in both types of receptor subunits, and is activated
upon the binding
of PDGF.
We have reported that PDGF-BB is a potent mitogen and stimulates tyrosine phosphorylation of various proteins in vascular smooth muscle cells (VSMC) of normotensive
Wistar-Kyoto
rats
(WKY) (5).
phosphorylation is less extensive with this isoform stimulates DNA synthesis isoform.
In contrast,
Increase hypertension characteristic (8.9).
Resink
Activation of protein tyrosine PDGF-AB than the BB isoform, although almost to the same extent as the BB
PDGF-AAdoes not act as a mitogen
in the cells.
of vascular wall thickening is a common feature of chronic (6,7). Unusually rapid growth is also recognized as a of cultured VSMC from spontaneously hypertensive rats (SHR) et
al.
(10)
0006-293X/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
reported
that
524
long
term
exposure
to
PDGF-AA
Vol.
188, No. 2, 1992
resulted
in
BIOCHEMICAL
gradual
and Owens (11)
growth
rats
of PDGF in
vascular
by
cultured
WKY in short
inactive
VSMC from SHR.
In addition, Blank cultured VSMC from
treatment with PDGF-BB. hypertrophy in cells with different
However, mechanism genetic background
that
hypertrophy
for
occurred
chronic
is not clear. We here report that than DNA synthesis in cultured completely
RESEARCH COMMUNICATIONS
in
observed
normotensive
in
AND BIOPHYSICAL
both
PDGF-AA stimulates protein VSMC from SHR whereas
DNA and protein
synthesis
synthesis rather this isoform is in
the cells
from
term experiments. MATWIALS
AND tf.EmoDs
Materials: Recombinant human PDGF-AA, -AB and -BB, and antiphosphotyrosine and anti-bovine phospholipase C-y, (PLC-yl) antibodies were products Biotechnology, Placid, Upstate Inc. (Lake NY). [Methyl-3H]ThO:midine Ci/mmol), [5-'Hluridine Ci/mmol) and (86 (26 [5,6-3H]leucine (163 Ci/mmol) were purchased from Amersham (Arlington Heights, IL), and [ "'I]-goat anti-mouse IgG antibody from ICN (Costa Mesa, CA). Cell culture: VSMC were isolated from thoracic aorta of lo-week old female SHR (n=5, systolic blood pressure 165-190) and WKY (n=5, systolic blood pressure 115-125), purchased from Taconic Farm (Germem Town, PA), according to Jones et al. (12). Cells were grown in Dulbeco's modified Eagle's medium (DMEM) with 10% fetal serum, and were fed every other days. Cells between passage 7-12 were seeded in a dish (10% cells/cm2), and used at confluence. Measurement of DNA, RNA and protein synthesis: Confluent VSMCin a 13-mm dish were cultured in a mixture (1:l) of DMEM and Ham's F-12 medium, supplemented with insulin (5 ug/ml), transferrin (5 ug/ml) and sodium serenite (5 rig/ml), for 48 h, and then exposed to PDGF in 1 ml of the medium containing 1 mg/ml bovine serum albumin (BSA). After 16 h of the exposure, [methyl-3H]thymidine or [5-3H]uridine (2 uCi/dish) was added into the medium and incubated for additional 4 h. Cells were washed with cold 20 mM HEPES-NaOHbuffer, pH 7.4, containing 130 mM NaCl, 5 mM KCl, 1 mM MgC12 and 1.5 mM CaC12 (Buffer A), and treated with 10% (w/w) trichloroacetic acid (TCA). After washing with a mixture of ethanol and ethyl ether (2:1, v/v), the TCA-insoluble fraction was solubilized with 0.5 N NaOH, and radioactivity in newly synthesized DNA or RNA in this fraction was counted. VSMC in a 13-mm dish, precultured For measurement of protein synthesis, in the mixture of DMEM and Ham's F-12 medium for 48 h, were incubated with PDGF in 1 ml of the same medium containing [5,6-'H]leucine (3 uCi/dish) and BSA (1 me/ml) for 20 h and radioactivity in the TCA-insoluble fraction was Protein content was measured as described (13) with BSA as a counted. standard. Immunoblotting: VSMC in a loo-mm dish, precultured in DMEMsupplemented with insulin (5 ug/ml), transferrin (5 ug/ml) and sodium serenite (5 rig/ml) for 48 h, were stimulated with PDGF (100 rig/ml) in Buffer A containing 1 mg/ml BSA and 10 mM glucose for 5 min. After washing with cold Buffer A containing 2 mM ethyleneglycol-bis(B-aminoethyl ether) N,N,N',N'-tetraacetate (EGTA) in place of CaC12, the cells were disrupted with 20 mM HEPES-NaOHbuffer, pH 7.4, containing 0.5% Nonidet P-40, 50 mM B-glycerophosphate, 0.1 mM orthovanadate, 10 uM molybdate, 1 mM phenylmethylsulfonyl fluoride, 10 ug/ml After centrifugation (14,000 aprotinin and 0.1 mg/ml leupeptin at 4 "C. The lysate was x g, 5 min), the supernatant was used as cell lysate. subjected to sodium dodecyl sulfate (SDS)-acrylamide gel (7.5%) in the were electroblotted to a electrophoresis, and proteins gel The membrane was treated with anti-phosphotyrosine nitrocellulose membrane. antibody in 10 mM Tris-HCl buffer, pH 7.4, containing 3% BSA, 0.9% NaCl and 0.1% Triton X-100, and tyrosine-phosphorylated proteins were detected by autoradiography with [1251]-goat anti-mouse IgG antibody. For detection
525
Vol.
188,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
of tyrosine-phosphorylated PJX-yr, the cell lysate was incubated with antibody and Protein A-Sepharose at 4 'C for 24 h. anti-phosphotyrosine Proteins immunoprecipitated were redisolved with 50 mM Tris-HCl buffer, pH 7.4, containing 2% SDS and 5% 2-mercaptoethanol, at 100 "C, subjected to electrophoresis and immunoblotted with anti-PLC-Yr antibody.
RESIJLTSAND DISCDSSION Effect of PDGF-AA, -AB and -BB on DNA synthesis was determined in cultured VSMC from SHR and WKY by pulse labeling experiments with labeled thymidine.
As shown in
Fig.
1, PDGF-BB acted
as a potent
mitogen
for
both
SHR and WKY cells, and DNA synthesis was activated in concentration-dependent manners about 16-fold in SHR cells and about 12-fold in WKY cells by 25 rig/ml PDGF-BB. PDGF-AB also stimulated DNA synthesis showing a similar profile as that observed with the BB isoform in both SHR and WKY cells. PDGF-AA was a very weak mitogen in SHR cells, and enhanced
DNA synthesis
only about 2-fold even at a concentration as high as 100 rig/ml. PDGF-AA was completely inactive in stimulating DNA synthesis.
In WKY cells, The results
obtained (5),
in WKYcells
are consistent
and are also similar
with those reported
In contrast,
in our previous
to those observed in cultured
paper
bovine VSMC (14).
m
0
20 0 PDGF
Fig.
1,
02
(rig/ml)
Stimulation
of
Stimulation and -BB (
q
25
DNA synthesis
by
PDGF
50
75
PDGF
isoforms
in
@g/ml)
SHR and
of DNA synthesis by PDGF-AA (O,@), ,m ) in SHR ( 0. A, 0 1 and WKY( 0.
100
-AB
UKY cells.
(n,A
A, NJ
)
cells
was determined with [methyl- Hlthymidine
as described in MATERIALS
Increase
PDGF
AND METHODS. Each experimental point (mean of 4 determinations, SEM
of
isoforms.
cells were incubated with PDGF-AA (0, .I, -AB (A,A) or -BB (0.m) for 20 h, and protein content per dish was determined. Each experimental point is presented as mean of 4 determinations (SEM
