©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Differential Binding and Regulation of Platelet-derived Growth Factor A and B Chain Isoforms by -Macroglobulin (*)

James C. Bonner (1)(§), Alvaro R. Osornio-Vargas (2)

From the (1)Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 and the (2)Division of Basic Investigation, National Cancer Institute, Mexico City, Mexico District Federal 14000

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

-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.


INTRODUCTION

Platelet-derived growth factor (PDGF)()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) .

It is currently not known whether PDGF-AA, -AB, and -BB bind differently to 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.


MATERIALS AND METHODS

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 -BBM 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 -Macroglobulin

Unlabeled recombinant human PDGF-AA, -AB, or -BB (150 ng) was mixed with native bovine M (50 µg) in a final volume of 30 µl of binding buffer and incubated for 4 h at 37 °C. PDGFM 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 -Macroglobulin

CCD 16 Lu fibroblasts grown to confluence in 150-cm 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.).

I-PDGF Isoforms Binding to Immobilized M

The receptor assay for 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-PDGFM complexes were transferred to polypropylene tubes, and radioactivity was measured on a -counter.

I-PDGF-AA, -AB, and -BB Receptor Assay

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, CaCl, 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.


RESULTS

Selective Binding of B Chain PDGF Isoforms to Plasma-derived Bovine M

The binding of 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 PDGFM 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 ofI-PDGF Isoforms to M Secreted by Human Fibroblasts in Culture

We had previously reported that rat alveolar macrophages secrete a homologue of human plasma-derived 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 M

An immobilized 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 forI-PDGF-BB Binding Bovine M

We attempted to assess the relative binding affinities of PDGF isoforms for 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:

On-line formulae not verified for accuracy

where K is the dissociation constant, k is the on constant, and k is the off constant, and

On-line formulae not verified for accuracy

where k 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-BBM 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-BBM 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 M Inhibits the Binding of PDGF-AB and -BB but Not PDGF-AA to Fibroblasts

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 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 M

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 [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).




DISCUSSION

Our finding of 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.

TGF- 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).

In the present study, we obtained a dissociation constant of approximately 1 µM for I-PDGF-BB binding to native M through analysis of time course association and dissociation data (Fig. 8). The Kthat 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.

The binding of PDGF-B chain dimers to 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.

In summary, we have shown that PDGF-AB and PDGF-BB but not PDGF-AA bind to native and activated forms of 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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: NIEHS, P. O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-541-0766; Fax: 919-541-4133.

The abbreviations used are: PDGF, platelet-derived growth factor; PDGF-R, platelet-derived growth factor receptor; 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.


REFERENCES
  1. Grotendorst, G. R., Chang, T., Seppa, H. E. J., Kleinman, K. H., and Martin, G. R. (1982) J. Cell. Physiol.113, 261-266 [Medline] [Order article via Infotrieve]
  2. Seppa, H., Grotendorst, G., Seppa, S., Schiffman, E., and Martin, G. R. (1982) J. Cell Biol.92, 584-588 [Abstract]
  3. Osornio-Vargas, A. R., Bonner, J. C., Badgett, A., and Brody, A. R. (1990) Am. J. Respir. Cell Mol. Biol.3, 595-602 [Medline] [Order article via Infotrieve]
  4. Heldin, C.-H., and Westermark, B. (1990) J. Cell Sci.96, 193-196 [Medline] [Order article via Infotrieve]
  5. Raines, E. W., Bowen-Pope, D. F., and Ross, R. (1990) in Peptide Growth Factors and Their Receptors (Sporn, M. B., and Roberts, A. B., eds) Vol. I, pp. 173-262, Springer-Verlag, New York Inc., New York
  6. Martinet, Y., Rom, W. N., Grotendorst, G. R., Martin, G. R., and Crystal, R. G. (1987) N. Engl. J. Med.317, 302-309
  7. Antoniades, H. N., Bravo, M. A., Avila, R. E., Galanopoulos, T., Neville-Golden, J., Maxwell, M., and Selman, M. (1990) J. Clin. Invest.86, 1055-1064 [Medline] [Order article via Infotrieve]
  8. Marinaelli, W. A., Craig, M. D., Hemke, A., Henke, K. R., Harmon, K. R., Jessurun, J., Vaillant, P., Fox, J., Peterson, M., Chiang, L., Conklin, K., and Bitterman, P. B. (1993) Am. Rev. Respir. Dis.147, 133-147
  9. Ross, R. (1993) Nature362, 801-809 [CrossRef][Medline] [Order article via Infotrieve]
  10. Shimokado, K., Raines, E. W., Madtes, D. K., Barrett, T. B., Benditt, E. P., and Ross, R. (1985) Cell43, 277-286 [Medline] [Order article via Infotrieve]
  11. Bonner, J. C., Osornio-Vargas, A. R., Badgett, A., and Brody, A. R. (1991) Am. J. Respir. Cell Mol. Biol.5, 539-547 [Medline] [Order article via Infotrieve]
  12. Nagaoka, I., Trapnell, B. C., and Crystal, R. G. (1990) J. Clin. Invest.85, 2023-2027 [Medline] [Order article via Infotrieve]
  13. Vignaud, J.-M., Allam, M., Martinet, N., Pech, M., Plenat, F., and Martinet, Y. (1991) Am. J. Respir. Cell Mol. Biol.5, 531-538 [Medline] [Order article via Infotrieve]
  14. Sjolund, M., Hedin, U., Sejersen, T., Heldin, C., and Thyberg, J. (1988) J. Cell Biol.106, 403-413 [Abstract]
  15. Raines, E. W., Dower, S. K., and Ross, R. (1989) Science243, 393-395 [Medline] [Order article via Infotrieve]
  16. Paulsson, Y., Hammacher, A., Heldin, C.-H., and Westermark, B. (1987) Nature328, 715-717 [CrossRef][Medline] [Order article via Infotrieve]
  17. Fabisiak, J. P., Absher, M., Evans, J. N., and Kelley, J. (1992) Am. J. Physiol.263, L185-L193
  18. Lasky, J. A., Coin, P. G., Lindroos, P. M., Ostrowski, L. E., Brody, A. R., and Bonner, J. C. (1995) Am. J. Respir. Cell Mol. Biol.12, 162-170 [Abstract]
  19. Claesson-Welsh, L., Ericksson, A., Westermark, B., and Heldin, C.-H. (1989) Proc. Natl. Acad. Sci. U. S. A.86, 4917-4921 [Abstract]
  20. Heldin, C.-H., Backstrom, G., Ostman, G., Hammacher, A., Ronnstrand, L., Rubin, K., Nister, M., and Westermark, B. (1988) EMBO J.7, 1387-1393 [Abstract]
  21. Seifert, R. A., Hart, C. E., Phillips, P. E., Forstrom, J. W., Ross, R., Murray, M. J., and Bowen-Pope, D. F. (1989) J. Biol. Chem.264, 8771-8778 [Abstract/Free Full Text]
  22. Hart, C. E., Forstrom, J. W., Kelly, J. D., Seifert, R. A., Smith, R. A., Ross, R., Murray, M. J., and Bowen-Pope, D. F. (1988) Science240, 1529-1531 [Medline] [Order article via Infotrieve]
  23. Hosang, M., Rouge, M., Wipf, B., Eggimann, B., Kaufmann, F., and Hunziker, W. (1989) J. Cell. Physiol.140, 295-304 [Medline] [Order article via Infotrieve]
  24. Matsui, T., Pierce, P. H., Fleming, T. P., Greenberger, J. S., LaRochelle, W. J., Ruggiero, M., and Aaronson, S. A. (1989) Proc. Natl. Acad. Sci. U. S. A.86, 8314-8318 [Abstract]
  25. Schollmann, C., Grugel, R., Tatje, D., Hoppe, J., Folkman, J., Marme, D., and Weich, H. A. (1992) J. Biol. Chem.267, 18032-18039 [Abstract/Free Full Text]
  26. Centrella, M., McCarthy, T. L., Kusmik, W. F., and Canalis, E. (1992) J. Clin. Invest.89, 1076-1084 [Medline] [Order article via Infotrieve]
  27. Sottrup-Jensen, L. (1987) in The Plasma Proteins (Putnam, F. W., ed) pp. 192-291, Academic Press, Orlando, FL
  28. LaMarre, J., Wollenberg, G. K., Gonias, S. L., and Hayes, M. A. (1991) Lab. Invest.65, 3-14 [Medline] [Order article via Infotrieve]
  29. Imber, M. J., and Pizzo, S. V. (1981) J. Biol. Chem.256, 8134-8139 [Abstract/Free Full Text]
  30. Strickland, D. K., Ashcom, J. D., Williams, S., Burgess, W. H., Migliorini, M., and Argraves, W. S. (1990) J. Biol. Chem.265, 17401-17404 [Abstract/Free Full Text]
  31. Borth, W., and Luger, T. A. (1989) J. Biol. Chem.264, 5818-5825 [Abstract/Free Full Text]
  32. Bonner J. C., Goodell, A. L., Lasky, J. A., and Hoffman, M. R. (1992) J. Biol Chem.267, 12837-12844 [Abstract/Free Full Text]
  33. Crookston, K. P., Webb, D. J., Wolf, B. B., and Gonias, S. L. (1994) J. Biol. Chem.269, 1533-1540 [Abstract/Free Full Text]
  34. Raines, E. W., Bowen-Pope, D. F., and Ross, R. (1984) Proc. Natl. Acad. Sci. U. S. A.81, 3424-3428 [Abstract]
  35. Bonner, J. C. (1994) Ann. N. Y. Acad. Sci.737, 324-338 [Abstract]
  36. Hart, C. E., Bailey, M., Curtis, D. A., Osborn, S., Raines, E., Ross, R., and Forstrom, J. W. (1990) Biochemistry.29, 166-172 [Medline] [Order article via Infotrieve]
  37. Huang, J. S., Huang, S. S., and Deuel, T. S. (1984) Proc. Natl. Acad. Sci. U. S. A.81, 342-346 [Abstract]
  38. Bonner, J. C., Hoffman, M., and Brody, A. R. (1989) Am. J. Respir. Cell Mol. Biol.1, 171-179 [Medline] [Order article via Infotrieve]
  39. Bonner, J. C., Badgett, A., Osornio-Vargas, A. R., Hoffman, M., and Brody, A. R. (1990) J. Cell. Physiol.145, 1-8 [Medline] [Order article via Infotrieve]
  40. Bonner, J. C., Badgett, A., Hoffman, M., and Lindroos, P. M. (1995) J. Biol. Chem.270, 6389-6395 [Abstract/Free Full Text]
  41. Gilbert, G. E., Sims, P. J., Wiedmer, T., Furie, B., Furie, B. C., and Shattil, S. J. (1991) J. Biol. Chem.266, 17261-17268 [Abstract/Free Full Text]
  42. Limbird, L. E. (1985) Cell Surface Receptors: A Short Course on Theory and Methods, pp. 51-96, Martinus Nijhoff Publishing, Boston
  43. Webb, D. J., Crookston, K. P., Hall, S. W., and Gonias, S. L. (1992) Arch. Biochem. Biophys.292, 487-492 [Medline] [Order article via Infotrieve]
  44. Grotendorst, G. R., Igarashi, A., Larson, R., Soma, Y., and Charette, M. (1991) J. Cell. Physiol.149, 235-243 [Medline] [Order article via Infotrieve]
  45. Sachinidis, A., Locher, R., Vetter, W., Tatje, D., and Hoppe, J. (1990) J. Biol. Chem.265, 10238-10243 [Abstract/Free Full Text]
  46. Siegbahn, A., Hammacher, A., Westermark, B., and Heldin, C.-H. (1990) J. Clin. Invest.85, 916-920 [Medline] [Order article via Infotrieve]
  47. Danielpour, D., and Sporn, M. B. (1990) J. Biol. Chem.265, 6973-6977 [Abstract/Free Full Text]
  48. LaMarre, J., Wollenberg, G. K., Gauldie, J., and Hayes, M. A. (1990) Lab. Invest.62, 545-551 [Medline] [Order article via Infotrieve]
  49. Raines, E. W., Lane, T. F., Iruela-Arispe, M. L., Ross, R., and Sage, E. H. (1992) Proc. Natl. Acad. Sci. U. S. A.89, 1281-1285 [Abstract]
  50. Crookston, K. P., Webb, D. J., LaMarre, J., and Gonias, S. L. (1993) Biochem. J.293, 443-450 [Medline] [Order article via Infotrieve]
  51. Fretto, L. J., Snape, A. J., Tomlinson, J. E., Seroogy, J. J., Wolf, D. L., La Rochelle, W. J., and Giese, N. A. (1993) J. Biol. Chem.268, 3625-3631 [Abstract/Free Full Text]
  52. Heidaran, M. A., Yu, J.-C., Jensen, R. A., Pierce, J. H., and Aaronson, S. A. (1992) J. Biol. Chem.267, 2884-2887 [Abstract/Free Full Text]
  53. Lasky, J. A., Coin, P. G., Lindroos, P. M., Ostrowski, L. E., Brody, A. R., and Bonner, J. C. (1995) Am. J. Respir. Cell Mol. Biol.12, 162-170 [Abstract]

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