(Received for publication, August 1, 1995; and in revised form, October 24, 1995)
From the
Cellular response to platelet-derived growth factor AA (PDGF-AA)
is mediated exclusively by the PDGF -receptor. Vascular smooth
muscle cells (VSMCs) in culture typically express very low levels of
-receptor. In this study, we demonstrate that the proteinase
inhibitor and cytokine carrier
-macroglobulin
(
M) increases rat VSMC PDGF
-receptor expression.
PDGF
-receptor mRNA levels increased 3-fold by 6 h and were
sustained at that level through 24 h in VSMCs treated with 280 nM methylamine-modified
M (
M-MA), a
form of activated
M. PDGF
-receptor mRNA levels
were unchanged in the same time period. In
I-PDGF-AA
binding experiments, treatment of VSMCs with
M-MA
increased the maximum binding capacity (B
) from
1.9 to 9.2 fmol/mg of cell protein without affecting binding affinity (K
80 pM).
M-MA also increased the VSMC response to PDGF-AA as
determined by tyrosine phosphorylation of a 170-kDa band, corresponding
in mass to the PDGF
-receptor. The native form of
M was comparable to
M-MA in its
ability to increase PDGF-AA binding to VSMCs and tyrosine
phosphorylation of the 170-kDa band. Recombinant and proteolytic
M derivatives were used to demonstrate that
M increases PDGF
-receptor expression by binding
VSMC-secreted cytokine(s) and interrupting an autocrine loop that
ordinarily suppresses
-receptor expression in these cells.
Transforming growth factor-
-neutralizing antibody mimicked the
activity of
M, increasing the binding capacity of
VSMCs for PDGF-AA. This study demonstrates that VSMC PDGF
-receptor expression and responsiveness to PDGF-AA are regulated
by autocrine transforming growth factor-
activity, potentially
other autocrine growth factors, and
M.
Vascular smooth muscle cell (VSMC) ()gene expression
is regulated by extracellular mediators synthesized by cells within the
blood vessel wall or transferred from the plasma, especially with blood
vessel injury(1, 2, 3) . Isoforms of the
platelet-derived growth factor (PDGF) family originate from both
sources and are important regulators of VSMC growth and
phenotype(4, 5) . The three PDGF isoforms, PDGF-AA,
PDGF-AB, and PDGF-BB, are formed by homodimerization or
heterodimerization of the PDGF A-chain and B-chain, which are separate
gene products(4, 5, 6, 7) . The
activities of PDGF are mediated by two separate tyrosine kinase
domain-containing receptors, the 170-kDa
-receptor and the 180-kDa
-receptor(6, 7, 8) . PDGF receptor
activation results from dimerization; in this process, PDGF stabilizes
the receptor dimer, allowing
autophosphorylation(7, 9, 10) . PDGF-BB can
bind to both the
-receptor and
-receptor; however, PDGF-AA
binds only the
-receptor(5) . Thus, one mechanism
underlying the differential responsiveness of cells to PDGF-BB and
PDGF-AA may be the independent regulation of expression of the PDGF
-receptor and
-receptor.
PDGF-BB is a strong VSMC mitogen
and chemoattractant(4, 11, 12, 13) .
The VSMC response to PDGF-AA is less well defined. VSMCs in culture
demonstrate minimal mitogenic responses to PDGF-AA, probably reflecting
low levels of -receptor
expression(12, 13, 14, 15) . There
is also evidence that the PDGF
-receptor and
-receptor
activate nonidentical signal transduction
pathways(10, 16) . In VSMCs from spontaneously
hypertensive rats, PDGF-AA stimulates protein synthesis despite the
absence of a strong mitogenic response(10, 17) .
PDGF-AA inhibits VSMC migration in Boyden's chamber assays, while
PDGF-BB promotes migration (18) . Thus, PDGF receptors are not
entirely redundant and may differentially regulate VSMC physiology.
Numerous studies have documented the ability of VSMCs in culture to
respond to exogenously added cytokines and mediators by regulating
expression of PDGF chains and receptors. Exogenous TGF-1
down-regulates expression of the PDGF
-receptor (19) while
increasing synthesis of the PDGF A-chain(19, 20) .
Basic fibroblast growth factor (bFGF) is the only cytokine reported to
up-regulate PDGF
-receptor expression in VSMCs(13) . Other
agents reported to increase PDGF A-chain synthesis in VSMCs include
angiotensin II(15, 21) , arginine
vasopressin(15) , and thrombin(22) .
-Macroglobulin (
M) is a large (M
718,000) homotetrameric proteinase
inhibitor and cytokine carrier that has been implicated in the
regulation of VSMC growth(23) .
M exists in at
least two well characterized conformations(24) . The native
form of
M expresses proteinase inhibitory activity,
but is not recognized by cellular receptors. When native
M reacts with proteinases, it undergoes a major
structural rearrangement to form the ``activated''
conformation. The identical conformational change is also induced by
reacting native
M with small primary amines, such as
methylamine, that modify the
M thiol ester
bonds(25) . Methylamine-modified
M
(
M-MA) and
M-proteinase complexes are
recognized by
M receptors equivalently(24) ,
bind cytokines similarly(26, 27) , and lack proteinase
inhibitory activity(24) , justifying the frequent use of
M-MA as a model of the activated
M
conformation. One
M receptor has been characterized
and shown to be identical to low density lipoprotein receptor-related
protein (LRP)(28, 29) .
M may
regulate cellular growth and physiology by at least two mechanisms, the
first of which involves cytokine carrier activity. By selectively
binding specific cytokines,
M may alter the cytokine
milieu and thereby alter cellular phenotype(30) . Cytokine
binding to
M is conformation-specific. TGF-
2
binds with equal affinity to native
M and activated
M, while TGF-
1 and PDGF-BB bind with higher
affinity to activated
M(30) . In contrast,
PDGF-AA does not bind to
M at all (31) . In
the rat, there are two homologues of human
M, a
constitutively expressed protein,
M, and an
acute-phase reactant,
M(24) . The two rat
-macroglobulins and human
M demonstrate similar
growth factor-binding properties(32) .
The second mechanism
whereby M may regulate cellular growth and alter
cellular phenotype involves direct binding to cellular receptors. In
cultured mouse peritoneal macrophages, activated
M
induces rapid signal transduction responses that have been attributed
to a receptor other than LRP(33, 34) . In rat VSMCs,
activated
M induces a rapid increase in inositol
1,4,5-trisphosphate and a delayed mitogenic response; native
M does not cause either
response(23, 35) .
M derivatives that
retain receptor-binding activity but lack cytokine carrier activity
increase inositol 1,4,5-trisphosphate levels and DNA synthesis in
VSMCs, similarly to activated
M(35) ,
suggesting a mechanism that requires direct binding of
M to a VSMC receptor.
Although M
is found in the plasma at high concentration (2-5
µM), under normal conditions, transfer of
M into the blood vessel wall is probably limited due
to its size and acidic (5.4) isoelectric point(24) . In
atherosclerosis, endothelial injury allows enhanced penetration of
plasma proteins(1) . Monocytes and macrophages synthesize and
secrete
M(24, 36) , providing a
plasma-independent source of this protein in the developing atheroma.
Thus, we consider it important to determine how
M, in
each of its conformations, regulates VSMC physiology. In this
investigation, we demonstrate that both native
M and
activated
M increase PDGF
-receptor expression by
rat VSMCs. This activity is entirely due to the ability of
M to bind cytokines secreted by VSMCs.
TGF-
-neutralizing antibody mimics the activity of
M, suggesting for the first time that TGF-
functions to regulate the PDGF
-receptor within the context of an
autocrine pathway.
M regulates this VSMC autocrine
pathway and thereby determines cellular responsiveness to PDGF-AA.
A 142-amino
acid recombinant polypeptide corresponding to the C-terminal
receptor-binding domain of rat M (rRBD) was prepared
according to the method of Salvesen et al.(40) with
minor modifications. The rat
M cDNA in pcDV1 was
obtained from the American Type Culture Collection. Digestion with BamHI yielded a 1.5-kilobase fragment that was used as a
template for polymerase chain reaction. A 484-base pair sequence
encoding the C-terminal region of the rat
M subunit
was amplified as described by Salvesen et al.(40) ,
except for the use of a different 5`-primer,
5`-TTCATATGGAGGCAGAAGGAGAAGCG-3`, in order to introduce an NdeI restriction site. The polymerase chain reaction product
was cloned into pET25b(+) and transformed into Escherichia
coli BL21(DE3) for expression. rRBD was purified as described
previously(40) . rRBD and the papain-derived 18-kDa fragment
completely inhibited specific
I-
M-MA
binding to VSMCs; the IC
values were 24 and 180 nM for rRBD and the 18-kDa fragment, respectively (data not shown).
Monoclonal antibody 1D11.16, which neutralizes TGF-1,
TGF-
2, and TGF-
3, was obtained from Genzyme Corp. (Cambridge,
MA). RC20 anti-phosphotyrosine antibody was obtained from Transduction
Laboratories (Lexington, KY). The cDNA probes for the PDGF
-receptor and
-receptor were kindly provided by Dr. D.
Bowen-Pope (University of Washington, Seattle).
Figure 1:
Regulation of the PDGF -receptor
and
-receptor by
M-MA. VSMCs were incubated with
280 nM
M-MA in SFM at 37 °C for the
specified times. Total RNA was isolated and subjected to Northern blot
analysis. A shows a representative blot probed for PDGF
-receptor mRNA. B shows the same blot probed for PDGF
-receptor mRNA. C shows a composite of four separate
experiments. In each experiment, the PDGF
-receptor mRNA level was
quantitated by PhosphorImager analysis and standardized for load by
comparison to glyceraldehyde-3-phosphate dehydrogenase mRNA levels.
Results represent the means ± S.E. kb,
kilobases.
Fig. 1C shows a composite
of results from four separate experiments examining changes in PDGF
-receptor mRNA levels in response to
M-MA by
Northern blot analysis. The results are standardized for load by
comparison with glyceraldehyde-3-phosphate dehydrogenase mRNA. By 6 h,
PDGF
-receptor mRNA was increased by >3-fold. The level of
-receptor mRNA was then stable for up to 24 h. In control
experiments, PDGF
-receptor mRNA levels were unchanged when VSMCs
were maintained for up to 24 h in SFM that was not supplemented with
M-MA (data not shown).
Figure 2:
Specific binding of I-PDGF-AA to VSMCs treated with
-MA.
VSMCs were incubated with 280 nM
M-MA in SFM
at 37 °C for 10 h. The cells were then chilled to 4 °C, and
binding of
I-PDGF-AA was examined. A shows
specific PDGF-AA binding isotherms for cells treated with
M-MA (
) and untreated control cultures (
). B shows the Scatchard transformations for the same
data.
Figure 3:
Tyrosine phosphorylation of VSMC proteins
in response to PDGF-AA. VSMC cultures were treated with
M-MA for the indicated times. The control cultures
were not
M-MA-treated. The cells were then exposed to
PDGF-AA at 37 °C for 0 (no exposure), 5, or 8 min. Reactions were
terminated by the addition of ice-cold solubilization buffer. Cellular
protein concentrations were assayed, and equal amounts (100 µg)
were loaded in each lane of the gel. Phosphotyrosine-containing
proteins were detected by Western blotting. The arrow marks
the migration of the apparent PDGF
-receptor autophosphorylation
product.
VSMC cultures that were treated with M-MA, but not
stimulated with PDGF-AA, showed no consistent change in tyrosine
phosphorylation pattern (n = 3). This result indicates
that
M-MA does not have an independent sustained
effect on VSMC tyrosine phosphorylation at 6-20 h. Furthermore,
the level of PDGF-AA synthesized by VSMCs is insufficient to cause a
detectable increase in
-receptor phosphorylation in the
M-MA-treated cultures.
Upon stimulation with
exogenous PDGF-AA, M-MA-treated cells demonstrated a
pattern of tyrosine phosphorylation similar to that observed in the
control cultures; however, the magnitude of the response was
substantially increased. The 170-kDa species showed the most notable
increase in band intensity. Optimal responsiveness to PDGF-AA was
observed at 6 and 10 h following addition of
M-MA. At
20 h, the response to PDGF-AA was somewhat decreased, but still greater
than that observed in control cultures (no
M-MA
treatment).
Figure 4:
Mechanism for M-induced
up-regulation of the PDGF
-receptor. VSMCs were incubated with SFM
(control), 280 nM
M-MA, 280 nM native
M, 280 nM 600-kDa derivative, 250
nM 18-kDa fragment, or 250 nM rRBD at 37 °C for
10 h. The cells were then chilled to 4 °C, and
I-PDGF-AA (0.2 nM) was added to each well (with
or without nonradiolabeled PDGF-AA). Specific
I-PDGF
binding was determined.
Figure 5:
Effects of native M and
the 600-kDa derivative on the VSMC response to PDGF-AA. VSMCs were
incubated with vehicle (lane A), 280 nM native
M (lane B), or 280 nM 600-kDa
fragment (lane C) for 10 h at 37 °C. The cells were then
exposed to PDGF-AA at 37 °C for 5 min. Reactions were terminated by
the addition of ice-cold solubilization buffer. Cellular protein
concentrations were assayed, and equal amounts were loaded in each lane
of the gel. Phosphotyrosine-containing proteins were detected by
Western blotting. The arrow marks the migration of the
apparent PDGF
-receptor autophosphorylation
product.
In the pericellular spaces, numerous cytokines form a
signaling language that determines cellular phenotype and
behavior(45) . Because cells have the ability to integrate
numerous signals, their overall response reflects a balance between
supporting and opposing activities. Extracellular molecules that alter
the balance of cytokines in the pericellular spaces will affect
cellular phenotype. Examples include soluble receptors for nerve growth
factor-, tumor necrosis factor-
, and colony-stimulating
factor-1; cytokine-binding proteins such as insulin-like growth
factor-binding protein; components of the extracellular matrix; and
M(46) . Compared with other cytokine-binding
molecules,
M is unique in that it exists in different
conformations, each with different affinity for specific
cytokines(24, 30) . In its activated conformation,
M may also regulate cellular phenotype, independently
of cytokines, by direct interaction with cellular
receptors(33, 34, 35) . Thus, the full
spectrum of activities expressed by
M toward a
specific cell type may be complex. While our laboratory and others have
contributed numerous studies regarding the biochemistry of
M/cytokine interactions, the biological implications
of such interactions have been largely uncharacterized.
In this
investigation, we demonstrate that M regulates
expression of the PDGF
-receptor by VSMCs in culture. Our
experiments with various
M derivatives suggest that
the regulation of the PDGF
-receptor results from the ability of
M to bind cytokines. Since the experiments were
performed in serum-free medium, the critical interaction must have
involved
M and one or more cytokines secreted by the
VSMCs themselves. Thus, we propose a model in which
M
interrupts an autocrine regulatory loop whereby VSMC-secreted
cytokine(s) ordinarily suppress PDGF
-receptor expression. A
second less likely explanation for our results is that plasma growth
factors were copurified in active form with
M and able
to increase PDGF
-receptor expression despite the rigorous
M purification method. This possibility was ruled out
experimentally with bFGF, the only cytokine reported to increase PDGF
-receptor expression in VSMCs to date(13) .
Interleukin-1
increases PDGF
-receptor expression in
fibroblasts; however, interleukin-1
binds exclusively to activated
M, and the resulting complex is covalent and inactive
unless treated with thioredoxin to dissociate disulfide
linkages(47, 48, 49) .
Many biological
activities have been described for activated forms of
M, which are not duplicated by native
M. Bonner et al.(50) showed that
M-MA increases PDGF-AA binding to fibroblasts 3-fold,
while native
M has no effect. Activated
M also regulates prostaglandin E
synthesis
and phorbol 12-myristate 13-acetate-induced superoxide anion production
in mouse peritoneal macrophages(51, 52) . In each of
these studies, mechanism was not probed; however, the activity of
activated
M might be explained by cytokines that bind
with high affinity only to activated
M or by direct
binding of activated
M to cellular receptors. A unique
aspect of the present work is the demonstration of similar activity for
native
M and activated
M. In the
plasma, native
M is present at high concentration
(2-5 µM). In contrast, activated
M
is typically present at only trace levels due to the efficiency of the
LRP-dependent hepatic clearance pathway(24) . Native
M is probably the predominant form of
M found in the blood vessel wall since formation of
activated
M depends on generation of local proteinase
activity. Furthermore, activated
M is rapidly taken up
by LRP that is expressed by a number of blood vessel wall cells,
including macrophages, fibroblasts, and
VSMCs(23, 24, 29) . Thus, we propose that
native
M is the primary form of the protein to
consider as a potential regulator of PDGF
-receptor expression in vivo and in serum-supplemented cell culture medium.
Battegay et al.(19) demonstrated that exogenous
TGF- decreases expression of the PDGF
-receptor in VSMCs.
M binds TGF-
1 with moderate affinity (K
= 300 and 80 nM for native
M and
M-MA, respectively) and
TGF-
2 with high affinity (K
= 10 and
13 nM for native
M and
M-MA,
respectively)(30) . Thus, we considered TGF-
isoforms as
candidates for the VSMC autocrine loop that regulates PDGF
-receptor expression. TGF-
-neutralizing antibody increased
PDGF-AA binding to VSMCs comparably to
M. This result
extends the work of Battegay et al.(19) by
demonstrating that TGF-
regulates VSMC PDGF
-receptor
expression not only when added exogenously, but within the context of
an autocrine loop. Although the spectrum of cytokines that interact
with
M in VSMC cultures may be complex, the
TGF-
-binding activity of
M is sufficient to
account for the observed regulation of PDGF
-receptor expression.
Transfection of colon carcinoma cells with an antisense expression
construct for TGF- increases anchorage-independent growth and
reduces integrin
expression, suggesting a role for
autocrine TGF-
activity in these
processes(53, 54) . Autocrine TGF-
activity may
also be important in regulating macrophage nitric oxide
synthesis(55) . Thus, autocrine TGF-
influences cellular
functions other than PDGF
-receptor expression and cell types
other than VSMCs. We propose that
M has the potential
to regulate each of these processes through the mechanism that is
described here.
Activated forms of M are weak
mitogens that combine with TGF-
1 to induce synergistic delayed
mitogenic responses (23, 35) . The mitogenic activity
of activated
M does not appear to be due to cytokine
carrier activity, but instead to direct interaction with a VSMC
receptor(35) . Native
M is inactive as an
independent mitogen and does not affect the mitogenic activity of
TGF-
1(22, 35) . Therefore, increased expression
of the PDGF
-receptor cannot have been responsible for the
previously observed synergistic, growth-promoting activities of
activated
M and TGF-
1. In this study, we obtained
evidence for an increased mitogenic response to PDGF-AA in VSMCs
treated with native
M. However, despite a 4.8-fold
increase in PDGF-AA-binding capacity, PDGF-AA remained a weak VSMC
mitogen. Thus, the functional consequences of VSMC PDGF
-receptor
up-regulation remain to be determined. One obvious possibility is that
PDGF-AA will enhance the VSMC response to other mitogenic stimuli to a
greater degree after up-regulation of the
-receptor. PDGF-AA may
also affect other important VSMC parameters, such as protein synthesis
and cellular migration(10, 17, 18) .
PDGF
isoforms play an important role in the development of atherosclerosis;
PDGF-AA, which is synthesized by VSMCs, has been implicated in this
process(1, 2, 3, 4, 5) .
PDGF -receptor mRNA is readily detected in extracts of total RNA
from normal rat carotid arteries(56) . Thus, it is possible
that the VSMC autocrine pathway by which TGF-
regulates PDGF
-receptor expression is more effective in cell culture than in
vivo. If this is correct, then the response of VSMCs in culture to
PDGF-AA may be artificially reduced. In rat carotid arteries injured by
balloon angioplasty, TGF-
1 levels are increased within 24
h(57) ; however, PDGF
-receptor expression decreases after
2 weeks(56) . TGF-
in the neointima is produced primarily
by VSMCs (57) and may be at least partially responsible for the
loss of the PDGF
-receptor. Whether the lag phase between
up-regulation of TGF-
1 expression and loss of the PDGF
-receptor reflects the TGF-
-neutralizing activity of
M is an interesting point for future investigation.
In summary, this study has demonstrated an autocrine pathway by
which PDGF -receptor expression is regulated in cultured VSMCs.
While more than one cytokine may be involved, isoforms of the TGF-
family are strongly implicated by neutralizing antibody experiments.
M functions as a regulator of the pathway by limiting
delivery of VSMC cytokines to cellular receptors, thereby controlling
the level of PDGF
-receptor expression without directly binding to
VSMC receptors. Together, the components of this pathway may be
important in modulating VSMC phenotype and cellular response to
PDGF-AA.