From the
Platelet-derived growth factor (PDGF)
It is currently not
known whether PDGF-AA, -AB, and -BB bind differently to
On-line formulae not verified for accuracy where K
On-line formulae not verified for accuracy where k
Our finding of
TGF-
In the present study,
we obtained a dissociation constant of approximately 1 µM for
The binding of
PDGF-B chain dimers to
In summary, we have shown that PDGF-AB and PDGF-BB but not PDGF-AA
bind to native and activated forms of
-Macroglobulin (
M) is a
multifunctional secreted glycoprotein that serves as a ubiquitous
proteinase inhibitor and as a binding protein for platelet-derived
growth factor (PDGF) BB and homologues of PDGF-BB secreted in culture
by macrophages. The interaction of
M with PDGF-A chain
molecules has not been addressed. This is a potentially important issue
because fibroblasts and smooth muscle cells produce PDGF-AA, whereas
macrophages produce mainly PDGF-BB. Recombinant human
I-PDGF-B chain molecules (AB and BB) bound to
plasma-derived, native human, or bovine
M and
trypsin-activated
M on Superose 6 fast protein liquid
chromatography gel filtration and on nondenaturing polyacrylamide gel
electrophoresis, whereas
I-PDGF-AA did not. Similar
results were obtained with
I-PDGF isoforms binding to
immobilized bovine
M and
M-methylamine. The same differential pattern of
unlabeled PDGF isoforms binding to
M was observed by
Western blotting of PDGF. Human lung fibroblasts secreted
M as measured by Western blotting, and
fibroblast-derived
M possessed the same differential
binding pattern for PDGF isoforms as did plasma-derived
M. The specific binding of PDGF-AB and -BB to these
fibroblasts was inhibited by native bovine
M, although
PDGF-AA binding was not affected. Native
M
preferentially blocked fibroblast chemotaxis to the PDGF-B chain
dimers. These data suggest that only PDGF-B chain dimers, such as those
produced by macrophages or released from platelets, are regulated by
M and that PDGF-AA produced by fibroblasts and smooth
muscle cells is not controlled by this cytokine-binding protein.
(
)is
a cationic polypeptide that stimulates mesenchymal cell mitogenesis and
chemotaxis(1, 2, 3, 4, 5) . PDGF
plays a role in wound healing(5) , pulmonary
fibrosis(6, 7) , acute lung injury(8) , and
atherosclerosis (9). Two chains, A and B, are the products of separate
genes (4) and dimerize to form functional PDGF-AA, -AB, and -BB
isoforms. PDGF isoforms are released by a variety of cells other than
platelets, including macrophages, which produce primarily PDGF-B chain
dimers (10-13), and mesenchymal cells (fibroblasts and smooth
muscle cells), which secrete
PDGF-AA(14, 15, 16, 17, 18) .
Two PDGF receptor subtypes, PDGF-R
and PDGF-R
, are also the
products of separate genes (19) and dimerize to form functional
,
, or
receptors(20) . The
PDGF-R
binds all three isoforms, whereas the PDGF-R
binds
only the B chain isoforms(20, 21, 22) . The
signals for mitogenesis and chemotaxis can be transduced through either
the PDGF-R
or the PDGF-R
(23, 24) , and the
PDGF-R
can be up-regulated by several cytokines
(interleukin-1
, tumor necrosis factor-
, and basic fibroblast
growth factor)(25, 26) . Thus, PDGF activity is
controlled by the relative numbers of PDGF-R
and PDGF-R
at
the cell surface.
Macroglobulin
(
M) is a 725-kDa glycoprotein that serves as a
proteinase inhibitor and as a cytokine-binding
protein(27, 28) . Proteinases are covalently entrapped
by
M, rendering the
M molecule
``receptor-recognized''(29) . Proteinase-activated
M migrates as a ``fast'' form in a
nondenaturing gel as compared with ``slow'' or native
M(27) .
M activated with
proteinases or methylamine binds to the
M receptor/low
density lipoprotein receptor-related protein (LRP) on a variety of cell
types including macrophages, fibroblasts and hepatocytes(30) .
Several cytokines bind to
M including PDGF,
transforming growth factor-
(TGF-
1 and -
2), basic
fibroblast growth factor, nerve growth factor, tumor necrosis
factor-
, and interleukin-1
(reviewed in Ref. 28). Some of
these cytokines bind apparently through different mechanisms (e.g. interleukin-1
binds covalently to the fast form of
M(31) , whereas PDGF-BB and TGF-
1 bind to
the slow and the fast form of
M
noncovalently)(32, 33) .
M
represents an extracellular level of control over PDGF activity by
regulating the amount of PDGF available to bind to cell surface PDGF
receptors(34, 35) . PDGF purified from human platelets,
which is a mixture of all three PDGF isoforms(36) , was first
reported to bind to
M covalently(37) . More
recently it was discovered that the majority of platelet-purified PDGF
and macrophage-derived PDGF binds to
M noncovalently
(32, 38). PDGF-BB binds both native slow and proteinase-activated fast
form
M, indicating that the mechanism of PDGF binding
to
M was different from that of
proteinases(32, 38, 39) . Slow form
M, which does not bind to the
M
receptor/LRP, appears to serve as an extracellular reservoir for
PDGF-BB(35) . On the other hand, proteinase-activated fast
M has been shown to mediate the clearance of PDGF-BB
through an LRP-dependent mechanism(40) .
M. This is a potentially important issue, because the
precise biological role of each PDGF isoform remains unclear. Although
activated macrophages secrete mainly PDGF-BB, fibroblasts and smooth
muscle cells produce PDGF-AA(14, 16, 38) .
However, all of these cell types secrete
M(28, 35) . Herein, we report that
M slow and fast forms selectively bind to PDGF-B chain
isoforms and do not bind to PDGF-AA. Furthermore,
M
preferentially blocks PDGF-B chain isoform binding to cell surface
receptors on fibroblasts and inhibits the chemotactic activity of
PDGF-AB and -BB for fibroblasts. These findings suggest that PDGF-B
chain dimers secreted by macrophages or released by degranulating
platelets are regulated by
M, whereas PDGF-AA secreted
by fibroblasts and smooth muscle cells is not controlled by this
cytokine-binding protein.
Reagents and Cells
PDGF-AA, -AB, and -BB were
purchased from Upstate Biotechnology, Inc. (Lake Placid, NY).
Plasma-derived bovine M (Boehringer Mannheim,
Indianapolis, IN) was dialyzed against 100 volumes of water to
precipitate fast
M as has been described
previously(32) . Human plasma-derived
M was the
kind gift of Steven Gonias (University of Virginia Health Sciences
Center). Slow
M was converted to fast
M by incubation with 25 mM methylamine
(Tris/HCl, 50 mM, pH 8.0) overnight at 25 °C or with a 4:1
molar excess of trypsin for 10 min at room temperature followed by
treatment with soybean trypsin inhibitor (2 mol/1 mol trypsin). Excess
methylamine was removed from
M-methylamine complexes
by dialysis against 100 volumes of 50 mM Tris/HCl (pH 8.2) at
4 °C. Human lung fibroblasts (CCD 16 Lu) were purchased from
American Type Culture Collection. Human fibroblast-derived
M was isolated as described below.
Superose 6 FPLC Gel Filtration
Chromatography
I-PDGF-AA, -AB, or
-BB
M complexes were routinely prepared by
incubating 1 ng of human recombinant
I-PDGF-AA, -AB
(Biomedical Technologies, Stoughton, MA), or
I-PDGF-BB
(Amersham Corp.) with 100 µg of bovine
M or
M-trypsin in 500 µl binding buffer (Ham's
F-12 with HEPES and CaCl
containing 0.25% BSA) for 24 h at
37 °C. These mixtures were separated on a gel filtration molecular
weight exclusion column (Superose 6 FPLC, Pharmacia Biotech Inc.)
equilibrated in phosphate-buffered saline (pH 7.5) operating at a flow
rate of 0.5 ml/min, and fractions were counted on a
-counter. In
some experiments, the time course of
I-PDGF-BB and
M was studied, and rates of association and
dissociation were analyzed as described
elsewhere(41, 42) .
Gel Electrophoresis
Electrophoresis of the I-PDGF-AA, -AB, or -BB/
M mixtures was
performed by nondenaturing polyacrylamide gel electrophoresis, due to
our previous observation that PDGF-BB is dissociated from
M under denaturing conditions(32) . 20 µg
of bovine or human
M was mixed with
I-PDGF isoforms (1 ng) and incubated overnight at 37
°C in a final volume of 50 µl. Samples were mixed with 20
µl of 6
nondenaturing sample buffer containing Tris borate
EDTA, glycerol, 1% xylene cyanol, and 1% bromphenol blue and
electrophoresed on a native 6% gel (Novex, Encinitas, CA) for 4 h at
125 V/12 mA. Native gels were fixed in 40% methanol/10% acetic acid,
stained with Coomassie blue, destained with 25% methanol, and
dehydrated using a gel dryer. Dried gels were exposed to
autoradiographic film (Amersham Corp.) to visualize
I-PDGF isoforms bound to
M.
Western Blot for PDGF Isoforms Bound to
Unlabeled recombinant
human PDGF-AA, -AB, or -BB (150 ng) was mixed with native bovine
-Macroglobulin
M (50 µg) in a final volume of 30 µl of
binding buffer and incubated for 4 h at 37 °C.
PDGF
M complexes were separated by native gel
electrophoresis as described above and transferred to Immobilon-P
blotting paper (Millipore Corp., Bedford, MA) using a semi-dry
apparatus for 2 h at 352 mA. The Immobilon-P membrane was blocked with
a 5% solution of nonfat dry milk in PBS. 10 µg/ml of goat
anti-human polyclonal PDGF antibody that recognizes all three PDGF
isoforms (53) (40017, Collaborative Biomedical, Bedford, MA) was
incubated with the membrane overnight in 1% milk/PBS. After washing in
PBST, the blot was incubated with a 1:1,000 dilution of horseradish
peroxidase-conjugated rabbit anti-goat antibody (Dakopatts,
Carpinteria, CA) for 45 min at room temperature with shaking. The blot
was developed using an ECL luminol kit (Amersham Corp.), and
immunoreactive PDGF was visualized with hyperfilm (Amersham Corp.).
Western Blot for Human Fibroblast-derived
CCD 16 Lu fibroblasts
grown to confluence in 150-cm-Macroglobulin
flasks containing 10% fetal
bovine serum/DMEM were rendered quiescent in SFDM for 24 h prior to
adding fresh SFDM for an additional 24 h. This 24 h
``fibroblast-conditioned'' medium was concentrated to 0.5 ml
using an PM-30 43-mm membrane (Amicon, Inc., Beverly, MA) and loaded
onto a Superose 6 FPLC column operating at 0.5 ml/min in PBS. Column
fractions were screened for immunoreactive human
M by
enzyme-linked immunosorbent assay as described previously(32) .
The fractions corresponding to the elution time of standard
plasma-derived
M (20-28 min) were pooled and
concentrated to 500 µl using a 25-mm PM-30 membrane. These
fractions were vacuum-evaporated using a Speed vac (Savant) to dryness
and reconstituted in 50 µl of water. 20 µl of sample was added
to 5 µl of Tris borate EDTA sample buffer and electrophoresed as
described under ``Materials and Methods.'' After running, the
gel was equilibrated in 1
carbonate transfer buffer for 1 h
along with 4 sheets of Whatman filter paper. Immobilon-P blotting paper
(Millipore) was soaked in methanol 15 min and then equilibrated in
transfer buffer for 1 h. Fibroblast-derived proteins were transferred
using a semi-dry apparatus for 2 h at 352 mA. The Immobilon-P membrane
was blocked with a 5% solution of nonfat dry milk in PBS with shaking
at room temperature. After washing the blot three times for 15 min in
PBS/Tween, a horse radish peroxidase-conjugated anti-
M
antibody (AHP-053P, Serotec, Kidlington, UK) was incubated with the
membrane for 45 min at room temperature in 1% milk/PBS. The blot was
developed using an ECL luminol kit (Amersham Corp.), and immunoreactive
M was visualized with hyperfilm (Amersham Corp.).
The receptor assay for I-PDGF Isoforms Binding to Immobilized
M
I-PDGF isoforms binding immobilized bovine
M was a modification of the method used by Webb and
co-workers for
I-TGF-
1 binding
M
(43).
M was diluted in PBS and adsorbed to 96-well
plates (Immulon-4, Dynatech Labs, Inc., Chantilly, VA) using 1 µg
of
M/100 µl/well for 4 h at room temperature. The
wells were washed three times with PBST and blocked with 1 mg/ml BSA in
PBS for 16 h at 4 °C. After washing the plate three times in PBST,
I-PDGF-AA, -AB, or -BB (diluted to the same specific
radioactivity with the appropriate nonradioactive isoform) was added to
wells in binding buffer (Ham's F-12 with HEPES, CaCl
,
and 0.25% BSA) and incubated at room temperature for 1 h. The wells
were again washed three times with PBST, and bound
I-PDGF
was recovered with 0.1 M NaOH, 2% SDS for 5 h at room
temperature. The solubilized
I-PDGF
M complexes were transferred
to polypropylene tubes, and radioactivity was measured on a
-counter.
Human (CCD 16 Lu) fibroblasts were grown to
confluency in 10% fetal bovine serum/DMEM and rendered quiescent for 24
h with SFDM that consisted of binding buffer supplemented with an
insulin/transferrin/selenium mixture (Boehringer Mannheim). The plates
were then cooled to 0-4 °C for 15-30 min, the media
were aspirated, and the cells were washed twice with ice-cold binding
buffer (Ham's F-12 with HEPES, CaClI-PDGF-AA, -AB, and -BB Receptor
Assay
, and 0.25% BSA).
I-PDGF-BB (Amersham Corp.) was iodinated by the
Bolton-Hunter method (51, 52) and possessed an average
specific activity of 25 µCi/µg.
I-PDGF-AB or
I-PDGF-AA (Biomedical Technologies) was iodinated using
the chloramine-T method (51, 52) and possessed an
average specific activity of 80 µCi/µg or 140 µCi/µg,
respectively. All of these radiolabeled isoforms were bioactive and
bound to CCD 16 Lu human lung fibroblasts in culture with >70%
specific binding and K
values ranging
from 0.2 to 0.6 nM. Radiolabeled PDGF isoforms (0.03
nM) were incubated with increasing concentration of native
bovine
M for 1 h at 37 °C, then cooled to
0-4 °C, and added to chilled cultures of fibroblasts for 3 h
on an oscillating platform at 0-4 °C. After the cells were
washed three times with ice-cold binding buffer, the cell-bound
radioactivity solubilized with 1% Triton X-100 containing 0.1% BSA and
0.1 N NaOH, and the radioactivity was measured on a
-counter.
Chemotaxis Assay
CCD 16 Lu fibroblasts were plated
at 28 10
cells/cm
and cultured in 10%
fetal bovine serum/DMEM until confluent. Cells rendered quiescent in
SFDM for 24 h were liberated with 0.5% trypsin (Life Technologies,
Inc.) in PBS/1 mM EDTA. The trypsin was inactivated by
dilution into a 7
volume of DMEM (Life Technologies, Inc.)
containing 1% BSA (RIA Grade, Sigma). Cells were washed twice in
chemotaxis buffer (DMEM/0.5% BSA, RIA grade, pH 7.4) and resuspended at
a final concentration of 3.5
10
cells/ml.
Experiments were performed using 48-microwell chemotaxis chambers
(NeuroProbe, Cabin John, MD). PDGF isoforms were incubated in the
absence or presence of native bovine
M for 1 h at 37
°C and were then added to the bottom wells. Polycarbonate
polyvinylpropylene-free membranes with a mean pore diameter of 8 mm
(Poretics Corp., Livermore, CA) were coated with 0.07% gelatin (Type A,
275 Bloom; Fisher). Cells were allowed to migrate for 4 h at 37 °C.
At the end of the incubation period, the cells on the membranes were
fixed in methanol, stained with Diff-Quick
(American
Scientific Products, MacGraw Park, IL), and mounted on glass slides,
and the cells on top of the membranes were removed with a wet swab. The
number of migrating cells was determined by counting the cells that
migrated to the bottom of the filter in 10 predetermined high power
fields (HPF) (100
magnification) and expressed as the mean
± S.E. cells/HPF.
Selective Binding of B Chain PDGF Isoforms to
Plasma-derived Bovine
The binding of M
I-PDGF isoforms to slow and fast conformations of
M was studied using several different methods. As
shown in Fig. 1, >90% of
I-PDGF-BB (1 ng) bound
to 100 µg of slow bovine
M on a gel filtration
(Superose 6 FPLC) column. Two peaks of immunoreactive
M were determined by enzyme-linked immunosorbent assay
(data not shown) and corresponded to the molecular mass of
M (
720 kDa, peak b in Fig. 1) or
aggregates of
M (peak a in Fig. 1) that
eluted with the void volume of the column. Under identical conditions,
50% of
I-PDGF-AB bound to plasma-derived
M, whereas <10% of
I-PDGF-AA eluted
at the molecular mass of
M (Fig. 1). Because
PDGF is a basic protein, we investigated whether or not another basic
protein, lysozyme, might interfere with the PDGF-BB-
M
interaction. A 100-fold molar excess of lysozyme/
M did
not inhibit
I-PDGF-BB binding to
M as
determined on Superose 6 FPLC (data not shown). Mixtures of
I-PDGF isoforms and
M were also
separated by nondenaturing gel electrophoresis (Fig. 2). In this
experiment, native slow
M was compared with
trypsin-activated fast
M.
I-PDGF-BB and
-AB bound both slow and fast forms, whereas
I-PDGF-AA did
not bind either conformation (Fig. 2). Is is important to note
that all of these iodinated PDGF isoforms bound to CCD 16 Lu
fibroblasts with high affinity (
I-PDGF-AA K
= 0.3-0.6 nM,
I-PDGF-AB K
=
0.2-0.5 nM, and
I-PDGF-BB K
= 0.1-0.5 nM)
(data not shown). However, to further investigate the possibility that
iodination of PDGF isoforms might alter the relative binding properties
of PDGF to
M and thereby account for the differential
binding of PDGF isoforms to
M, nonradioactive PDGF
isoforms were incubated with
M, and the
PDGF
M complexes were separated by nondenaturing
electrophoresis prior to performing a Western blot for PDGF using a
polyclonal antibody that recognized all PDGF isoforms(53) . The
PDGF-B chain dimers preferentially bound to
M as
determined by Western blotting for PDGF (Fig. 3) and thus
confirmed the results obtained with the radiolabeled PDGF isoforms ( Fig. 1and Fig. 2).
Figure 1:
Binding of plasma-derived native slow
form of bovine M to
I-PDGF-AB and -BB
but not
I-PDGF-AA. 100 µg of bovine
M was mixed with 1 ng of recombinant
I-PDGF-AA, -AB, or -BB in 500 µl of binding buffer
and incubated for 1 h at 37 °C prior to loading on a Superose 6
FPLC column.
I-PDGF-BB (
) and
I-PDGF-AB (dashed line) coeluted with two
immunoreactive peaks of human
M (not shown) that are
represented by arrows at peaks a and b. Peak b represents 725-kDa uncomplexed
M,
whereas peak a represents aggregates of
M at
the void volume of the column (>1000 kDa). Less than 10% of
I-PDGF-AA (solid line) bound to
M, and >90% eluted at the molecular mass of free
PDGF (
30 kDa), designated peak c. In contrast, >90% of
I-PDGF-BB bound to
M. The column was
calibrated with standard molecular mass markers: blue dextran
(V
), thyroglobulin (669 kDa), apoferritin (440 kDa),
-amylase (200 kDa), BSA (66 kDa), carbonic anydrase (29 kDa), and
cytochrome c (12.4 kDa).
Figure 2:
Nondenaturing gel electrophoresis of
plasma-derived native slow and trypsin-activated fast bovine
M incubated with
I-PDGF-AA, -AB, and
-BB. 20 µg of bovine
M or
M-trypsin was incubated with 1 ng
I-PDGF-AA, -AB, or -BB in 50 µl of binding buffer for
1 h at 37 °C prior to separation on a 6% Tris borate gel as
described under ``Materials and Methods.'' Each isoform (AA, AB, and BB) was incubated in the
absence of
M as a control (lanes 1) or the
presence of native
M (lanes 2) or
M-trypsin (lanes 3). Panel A,
Coomassie blue-stained protein demonstrated the separation of slow (S) and fast (F) forms of
M. Panel B, autoradiography of the dried gel demonstrated binding
of
I-PDGF-AB and -BB to slow and fast forms of
M but no detectable binding of
I-PDGF-AA. High specific binding of all three
radiolabeled isoforms was observed in radioligand binding assays with
human lung fibroblasts (see Fig. 5).
Figure 3:
Western blotting of PDGF isoforms bound to
native bovine M.
M (50 µg) was
incubated in the absence or presence of nonradioactive recombinant
human PDGF-AA, -AB, or -BB (150 ng) in 30 µl of binding buffer for
4 h prior to nondenaturing gel electrophoresis and Western blotting for
PDGF as described under ``Materials and Methods.'' A
polyclonal anti-human PDGF antibody that recognized both A and B chain
PDGF molecules was used in the Western blot. These data support the
observations made with
I-PDGF isoforms binding
M in Figs. 1 and 2, showing the differential binding
of PDGF isoforms (BB > AB > AA) to
M. No immunoreactivty was demonstrated for
M in the absence of PDGF (control).
Binding of
We had previously reported that rat alveolar
macrophages secrete a homologue of human plasma-derived
I-PDGF Isoforms to
M Secreted by Human Fibroblasts in
Culture
M that serves as a binding protein for PDGF-BB, the
primary isoform produced by these phagocytes(38) . However, the
isoform produced by fibroblasts is PDGF-AA(16) , and, due to our
observations in Fig. 1, 2, and 3, it seemed likely that PDGF-AA
would not bind to
M produced by fibroblasts in
culture. Under serum-free conditions, human lung fibroblasts (CCD 16
Lu) secreted
500 ng of
M/10
cells/24
h in culture as determined by an enzyme-linked immunosorbent assay that
employed an anti-
M antibody specific for human
M (data not shown). The human anti-
M
antibody did not cross react with bovine
M, and thus
we did not detect residual
M from fetal bovine serum
that was used in the intial culturing of these fibroblasts. This
fibroblast
M eluted at the molecular weight of
plasma-derived
M on a Superose 6 FPLC column (data not
shown). The human fibroblast-derived
M was detected by
Western blotting and nearly all of the protein migrated as the native
slow (non-receptor-recognized) form on a nondenaturing gel (Fig. 4A).
I-PDGF-BB and -AB but not
I-PDGF-AA bound to native and trypsin-activated forms of
human fibroblast-derived
M (Fig. 4B).
Figure 4:
Selective binding of I-PDGF-B chain isoforms to human fibroblast-derived
M. Confluent cultures of CCD 16 Lu fibroblasts grown
in 10% fetal bovine serum/DMEM were rendered quiescent in SFDM for 24
h, washed twice with fresh SFDM, and incubated for another 24 h prior
to collecting the ``conditioned'' medium. The medium was
concentrated, and
M was isolated by Superose 6 FPLC
gel filtration prior to separation on a 6% Tris borate EDTA gel and
Western blotting as described under ``Materials and
Methods.'' The Western blot employed a primary anti-human
M antibody that did not cross-react with bovine
M and thus any residual
M from the
fetal bovine serum upon culturing was not detected. The
fibroblast-derived
M was converted to its fast form
with a 4:1 molar ratio of trypsin:
M. Panel A,
in Western blots, human plasma-derived
M (lane
1) and plasma-derived
M-trypsin (lane 2)
were run as standards for the identification of slow (S) and
fast (F) forms of human fibroblast-derived
M
in triplicate samples that were untreated (lanes 3) or
trypsin-treated (lanes 4). Panel B, 10 µg of
native, slow (S) fibroblast-derived
M or
trypsin-activated fast (F) fibroblast-derived
M was incubated with 0.1 ng of
I-PDGF-AA, -AB, or -BB for 1 h at 37 °C prior to
separation on a 6% Tris borate EDTA gel. The autoradiographic signal
shows each isoform (AA, AB, and BB) in the
absence of
M as a control (lanes 1) or in the
presence of native fibroblast-derived
M (lanes
2) or trypsin-activated fibroblast-derived
M (lanes 3). Barely detectable binding was observed for
I-PDGF-AA to slow
M, whereas binding
I-PDGF-AB and -BB to both slow and fast
fibroblast-derived
Ms was
evident.
Specific Binding of PDGF-B Chain Molecules to
Immobilized Bovine
An immobilized
M
M system was utilized to investigate the specific
binding of PDGF isoforms to native and methylamine-modified bovine
M. Nonspecific binding was determined in the presence
of 2000 ng/ml of the appropriate nonradioactive PDGF isoform.
I-PDGF-BB specifically bound immobilized
M and
M-methylamine in a
concentration-dependent fashion, whereas
I-PDGF-AB
specific binding was 50-70% less than PDGF-BB binding, and
I-PDGF-AA binding was barely detectable (i.e. specific binding of this isoform was less than 10% of the total
bound
I-PDGF-AA) (Fig. 5). In comparison, only
I-PDGF-AB and -BB gave reliable specific binding results
(
40% specific binding) to immobilized
M. To
ensure that the radiolabeled PDGF-AA was biologically active, we
routinely performed binding assays on confluent cultures of CCD 16 Lu
fibroblasts in parallel with the immobilized
M assay
with the same batch of
I-PDGF-AA and observed high
specific binding (>70%) for all radiolabeled isoforms to fibroblast
cultures (Fig. 6). Increasing concentrations of nonradioactive
PDGF isoforms were coincubated with
I-PDGF-BB in the
immobilized
M assay (Fig. 7). Nonradioactive
PDGF-BB was the most potent competitor of its radiolabeled counterpart
for binding to immobilized
M, followed by PDGF-AB and
PDGF-AA in decreasing order of potency. As an negative control,
I-PDGF-BB did not bind specifically to plates that were
coated with BSA alone (i.e. binding was identical in the
absence or presence of excess nonradioactive PDGF-BB) (data not shown).
Figure 5:
Immobilized plasma-derived bovine
M preferentially binds
I-PDGF-BB and
I-PDGF-AB. A
I-PDGF radioligand binding
assay was performed with bovine
M (A) or
M-methylamine (B) adhered to 96-well plastic
dishes as described under ``Materials and Methods.''
Nonspecific binding was determined in the presence of 2000 ng/ml
nonradioactive PDGF isoform. Immobilized
M or
M-methylamine bound 3-4 times more
I-PDGF-BB (
) as compared with
I-PDGF-AB (
).
I-PDGF-AA specific
binding was not significant (
). The data are representative of
a typical experiment performed in quadruplicate (S.E. < 10% of the
mean) and repeated four separate times for
M and
M-methylamine.
Figure 6:
Comparison of specific I-PDGF isoform binding to immobilized bovine
M and 16 Lu human lung fibroblasts in culture. To
verify the biological activity of radiolabeled PDGF isoforms, parallel
2-h incubations were conducted with quiescent, confluent human
fibroblasts (open bars) or immobilized
M (hatched bars) as described under ``Materials and
Methods.'' Total binding was determined in the presence of 0.03
nM radiolabeled PDGF and nonspecific binding was determined in
the presence of 500 ng/ml PDGF isoform at 0-4 °C for binding
to fibroblasts or 2000 ng/ml PDGF isoform for immobilized
M binding at 25 °C. All three radiolabeled PDGF
isoforms possessed high specific binding for fibroblast cultures
(>70%), but only radiolabeled PDGF-B chain dimers specifically bound
immobilized
M (
40% specific binding). The
specific binding data shown are the means ± S.E. of four
separate experiments, each performed in
quadruplicate.
Figure 7:
Competition of nonradioactive PDGF
isoforms for the binding of I-PDGF-BB to immobilized
M. Binding of radiolabeled PDGF isoforms to
immobilized
M was performed as described under
``Materials and Methods.''
I-PDGF-BB (0.25
nM) was incubated with increasing concentrations of
nonradioactive PDGF-AA (
), PDGF-AB (
), or PDGF-BB (
)
for 3 h at 25 °C. PDGF-BB and -AB inhibited
I-PDGF-BB
binding in a concentration-dependent manner that was maximal (
40%
inhibition of total binding) at
250 nM PDGF. In contrast,
PDGF-AA inhibited
I-PDGF-BB binding by <10% at 250
nM and concentrations as high as 3 µM PDGF-AA
were required to inhibit
I-PDGF-BB binding by 40%. The
data are representative of a typical experiment performed in
quadruplicate (S.E. <10% of the mean) and repeated four separate
times for
M.
Dissociation Constant for
We attempted to assess the relative
binding affinities of PDGF isoforms for I-PDGF-BB Binding Bovine
M
M by analyzing
time course association and dissociation binding data from a Superose 6
FPLC column. Association and dissociation experiments were performed as
described previously (32). Using the method described by
Limbird(42) , we defined:
is the dissociation constant, k
is the on constant, and k
is the off constant, and
is the slope of the association
regression plot, k
is the on constant, and k
is the slope of dissociation regression plot
divided by -2.303. The concentration of PDGF radioligand is
represented by [D], and that of
M
is represented by [R] in the first equation. In the
second equation, [M] represents the molar
concentration of PDGF. Representative plots of association and
dissociation for
I-PDGF-BB binding native bovine
M are shown in Fig. 8. A dissociation constant
of 1.1 µM was calculated based on these data.
Figure 8:
Rate of association and dissociation of I-PDGF-BB binding to bovine
M.
Association was measured by incubating bovine
M (100
µg) with
I-PDGF-BB at increasing time points at 37
°C prior to isolation of the
I-PDGF-BB
M complex by FPLC gel
filtration chromatography. Dissociation was measured by incubating
I-PDGF-BB with
M for 24 h at 37 °C
and then incubating aliqouts of the FPLC-isolated
I-PDGF-BB
M complex for increasing
times prior to a second FPLC separation and isolation of the
M peak (20-28 min zone, see Fig. 1). Association
data (A) and dissociation data (B) were fitted to a
first order model for estimation of association constant and the
dissociation rate, respectively (see ``Results'' for details
of calculated binding constants). A dissociation constant (K)
of 1.1 µM was estimated for
I-PDGF-BB
binding to
M.
Native
A
radioligand binding assay was performed on CCD 16 Lu human lung
fibroblasts using each of the three radiolabeled PDGF isoforms at a
concentration of 0.03 nM (Fig. 9). All three PDGF
isoforms bound to these cells with high specific binding (see Fig. 6). In this experiment, each radiolabeled PDGF isoform was
incubated with increasing concentrations of M Inhibits the
Binding of PDGF-AB and -BB but Not PDGF-AA to Fibroblasts
M for 1 h
at 37 °C prior to chilling on ice for 30 min and then addition to
cultures of fibroblasts at 0-4 °C. As shown in Fig. 9,
native bovine
M blocked the binding of
I-PDGF-BB (IC
=
0.3
µM
M) and
I-PDGF-AB binding
(IC
=
0.8 µM
M).
I-PDGF-AA binding was not inhibited by
M.
Figure 9:
Native plasma-derived bovine
M inhibits the binding of
I-PDGF-AB and
-BB but not
I-PDGF-AA to cell surface receptors on 16 Lu
fibroblasts. Radiolabeled PDGF isoforms (0.03 nM) were
incubated with increasing concentrations of native bovine
M for 1 h at 37 °C in the absence of fibroblasts,
then cooled to 0-4 °C, and added to chilled cultures of
confluent, quiescent fibroblasts for 3 h prior to solubilization as
described under ``Materials and Methods.'' All radiolabeled
PDGF isoforms bound to fibroblast cultures with high specific binding
(see Fig. 6). Native
M inhibited the binding of
I-PDGF-AB (
) and -BB (
) in a
concentration-dependent manner but had no discernible effect on the
binding of
I-PDGF-AA (
). The data are the means
± S.E. of an experiment performed in triplicate that was typical
of four separate experiments.
Inhibition of PDGF-stimulated Chemotaxis by
CCD 16 Lu fibroblasts were allowed
to migrate to a concentration of PDGF that induced a maximal
chemotactic response (PDGF-AA, 0.06 nM; PDGF-AB and -BB, 0.13
nM). All three isoforms were also mitogenic and induced a
2-3-fold increase in [M
H]thymidine
incorporation as compared to SFDM alone (data not shown). Prelimimary
chemotaxis experiments defined a typical bell-shaped, dose-dependent
chemotaxis curve for each of the PDGF isoforms(3) . The mean
(±S.E.) number of fibroblasts migrating to PDGF-AA, -AB, and -BB
was 6.1 ± 0.1, 11.9 ± 2.3, and 21.2 ± 6.7
cells/HPF, respectively. In the presence of 1.4 µM native
bovine
M, PDGF-AA, -AB, and -BB stimulated the
chemotaxis of 4.3 ± 0.9, 5.3 ± 0.5, and 8.6 ± 3.4
cells/HPF. The number of cells that migrated in chemotaxis buffer alone
was 0.7 ± 0.2 cells/HPF. Thus, PDGF-AB- and PDGF-BB-stimulated
chemotaxis was inhibited by 50% at 0.3 µM
M, whereas PDGF-AA-induced chemotaxis was
inhibited
25% only at the highest concentration of
M tested (Fig. 10). Checkerboard analysis
demonstrated that the responses were chemotactic rather than
chemokinetic, because equal concentrations of a particular PDGF isoform
in the upper and lower wells of the chemotaxis chamber did not induce
any significant migration (data not shown). Native
M,
which does not bind the
M receptor/LRP, was not
chemotactic or chemokinetic over the concentration range used (i.e. mean ± S.E. number of fibroblasts in triplicate experiments
that migrated in the presence of 1.4 µM
M
was 0.2 ± 0.2 cells/HPF). However, methylamine- and
trypsin-activated
Ms, which are receptor-recognized,
possessed chemotactic activity and thus were not used in further
studies for modulation of PDGF-stimulated chemotaxis (data not shown).
Figure 10:
Native plasma-derived bovine
M preferentially inhibits the chemotaxis of 16 Lu
fibroblasts to PDGF-B chain isoforms. Chemotaxis of fibroblasts was
performed as described under ``Materials and Methods''.
PDGF-AA, -AB, or -BB was incubated with increasing concentrations of
bovine
M for 1 h at 37 °C prior to adding to
chemotaxis chambers. Native
M alone was not
chemotactic for fibroblasts. PDGF-AB (
) and -BB (
) induced
chemotaxis maximally at 0.13 nM, and
M
inhibited migration by 50% at 0.3 µM
M.
PDGF-AA (
) induced chemotaxis maximally at 0.06 nM,
and
M suppressed chemotaxis by
25% at 1.4
µM
M. The data shown are the means
± S.E. from a representative experiment assayed in triplicate
and expressed as the percentages of maximal chemotaxis of each PDGF
isoform (in the absence of
M).
M interacting only with
PDGF-B chain-containing dimers and not PDGF-AA could be significant in
the selective control of the three PDGF isoforms secreted during normal
tissue repair processes or during the pathogenesis of
fibroproliferative diseases. The evolutionary significance of two
distinct PDGF chains (A and B) and three different PDGF isoforms
remains unclear, because all PDGF dimers are potent mitogens and
chemoattractants(21, 22, 24) . One critical
level of control over PDGF signaling that has been studied in depth is
the relative abundance of PDGF-R
and PDGF-R
at the cell
surface(21, 22, 44, 45, 46) .
Another aspect relating to the existence of multiple PDGF isoforms is
the fact that certain cell types express both PDGF-A and PDGF-B chain
genes but preferentially produce one isoform over another. For example,
macrophages produce mainly PDGF-BB(10, 11, 12) ,
although smooth muscle cells and fibroblasts secrete
PDGF-AA(14, 15, 16, 17, 18) .
Macrophages, fibroblasts, and smooth muscle cells all produce
M (35). In light of our observations in the present
study, taken together with our previous observation that the majority
of macrophage-derived PDGF is complexed with
M(38) , we propose that macrophage-derived
PDGF-BB is tightly regulated by
M, whereas
fibroblast-derived PDGF-AA is not regulated by this binding protein.
isoforms have also been reported to bind differentially to
M. Danielpour and Sporn observed that
M inhibited the ability of TGF-
2 to suppress the
growth of a lung epithelial cell line but not that of
TGF-
1(47) . LaMarre and co-workers found that, although
M binds both TGF-
1 and TGF-
2, it
preferentially counteracted the mitoinhibitory effect of TGF-
2 on
hepatocytes(48) . A later study by Crookston and colleagues
determined that TGF-
2 bound to
M with a higher
affinity than TGF-
1(33) . Our findings clearly demonstrate
that
M binds PDGF isoforms with differential
selectivity; PDGF-BB is bound more than PDGF-AB, which is bound more
than PDGF-AA. Therefore, it appears that the regulation of the PDGF
isoform system by
M is similar to regulation of the
TGF-
isoform system by
M (i.e. both
native and activated forms of the binding protein preferentially bind
to one growth factor isoform in each system).
I-PDGF-BB binding to native
M
through analysis of time course association and dissociation data (Fig. 8). The K
that we obtained
for PDGF-BB binding
M was similar to measurements by
Crookston and co-workers (i.e. in the micromolar
range)(33) . We were not able to obtain a reliable K
for PDGF-AB binding to
M, although binding was evident. Through extrapolation
of the data in Fig. 7we estimated a K
between 2 and 6 µM for PDGF-AB binding
M. PDGF-AA binding
M was not evident
using the gel filtration and nondenaturing gel systems employed in this
study (Figs. 1-4). Some specific binding of
I-PDGF-AA to immobilized
M-methylamine
was detected (Fig. 5), but this was a low percentage of the total
binding (<10%). Likewise, PDGF-AA competed somewhat for the binding
of
I-PDGF-BB to immobilized
M (Fig. 7), but 30 nM of PDGF-AA reduced bound
I-PDGF-BB by <10%. In an earlier study, we reported
that PDGF-AA and -AB competed for PDGF-BB binding to
M(32) , but this inhibition required as much as
3 µM of PDGF-AA to reduce
I-PDGF-BB binding
as determined by gel filtration chromatography. This amount of PDGF-AA
is far in excess of the concentrations of PDGF isoforms that were
employed in the competition assay in Fig. 7. Thus, it is possible
that PDGF-AA could bind to
M with an extremely weak
affinity and we could not detect binding by the conventional methods
used in this study. However, the interaction between PDGF-AA and
M is probably negligible when compared with the
binding of PDGF-AB and -BB to
M.
M is reminiscent of another
PDGF-binding protein, termed SPARC (secreted protein, rich in
cysteine)(49) . SPARC, like
M, inhibits the
binding of radiolabeled PDGF-AB and PDGF-BB but not PDGF-AA to human
dermal fibroblasts in a dose-dependent manner. Furthermore, PDGF-AB and
-BB bind to SPARC in a reversible, noncovalent manner(49) , and
this has also been reported for PDGF-AB and -BB binding to
M(32) . Unlike
M, SPARC is an
extracellular matrix protein that is up-regulated following vascular
injury along with PDGF-B chain expresssion, and thus SPARC has been
proposed to regulate PDGF-B chain activity in
atherosclerosis(49) . Unlike SPARC,
M is
activated to a receptor-recognized fast form by proteinases and serves
as a clearance mechanism for PDGF-BB via the liver
M
receptor/LRP system (50) or via the
M
receptor/LRP system on fibroblasts and macrophages in extravascular
spaces(35, 40) . Thus, it appears that at least two
secreted glycoproteins are important in the regulation of PDGF-B chain
dimers that are released from platelets or secreted by activated tissue
macrophages. The isoform produced by fibroblasts and smooth muscle
cells (PDGF-AA) is not regulated by either of these binding proteins.
M purified from
plasma or isolated from cultured lung fibroblast supernatants. PDGF-AB
and -BB binding to cell surface receptors on fibroblasts and the
chemotaxis of these cells is inhibited selectively by
M, whereas PDGF-AA binding and PDGF-AA-induced
chemotaxis are not affected by
M. Because macrophages
secrete mainly PDGF-B chain dimers and mesenchymal cells (fibroblasts
and smooth muscle cells) produce PDGF-AA, it appears that the system
involving
M and its receptor (LRP) allows for
discrimination of macrophage and mesenchymal cell PDGF signals during
tissue repair processes or during the pathogenesis of
fibroproliferative diseases.
M,
-macroglobulin; LRP, low density lipoprotein
receptor-related protein; SFDM, serum-free defined medium; FPLC, fast
protein liquid chromatography; DMEM, Dulbecco's modified
Eagle's medium; TGF-
, transforming growth factor
; BSA,
bovine serum albumin; PBS, phosphate-buffered saline; HPF, high power
field; SPARC, secreted protein, rich in cysteine; PBST,
phosphate-buffered saline with Tween 20.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.