©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inhibition of Platelet-derived Growth Factor-BB-induced Fibroblast Proliferation by Plasmin-activated -Macroglobulin Is Mediated via an -Macroglobulin Receptor/Low Density Lipoprotein Receptor-related Protein-dependent Mechanism (*)

(Received for publication, July 22, 1994; and in revised form, November 28, 1994)

James C. Bonner (§) Annette Badgett Maureane Hoffman (1) Pamela M. Lindroos

From the Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 and Laboratory Services, Durham Veterans Affairs Medical Center, Department of Pathology, Duke University Medical Center, Durham, North Carolina 27705

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

alpha(2)-Macroglobulin (alpha(2)M) is a potentially important regulator of platelet-derived growth factor-BB (PDGF-BB)-stimulated cell growth due to our previous observation that PDGF-BB binds to alpha(2)M noncovalently (Bonner, J. C., Goodell, A. L., Lasky, J. A., and Hoffman, M. R.(1992) J. Biol. Chem. 267, 12837-12844). We examined the in vitro effect of native and plasmin-activated (receptor-recognized) alpha(2)M on the PDGF-BB-induced proliferation of mouse Swiss 3T3 and rat lung fibroblasts. Nondenaturing polyacrylamide gel electrophoresis showed that plasmin converted alpha(2)M to its electrophoretically ``fast'' form at a 2:1 molar ratio and that I-PDGF-BB bound both alpha(2)M and alpha(2)M-plasmin. PDGF-BB-induced growth was not affected by native alpha(2)M (0.3 µM) or plasmin (0.6 µM). The combination of plasmin and alpha(2)M (2:1 molar ratio) inhibited PDGF-BB-induced cell proliferation 80-90%. Complexes of PDGF-BBbulletalpha(2)M purified by gel filtration chromatography retained growth promoting activity, but the PDGF-BBbulletalpha(2)M-plasmin complex did not. Preincubation of fibroblasts (37 °C for 24 h) with alpha(2)M-plasmin did not change I-PDGF-BB binding or affect gene expression of the 6.5-kilobase PDGF-alpha receptor or 5.2-kilobase PDGF-beta receptor mRNA. However, preincubation with alpha(2)M-plasmin (0-4 °C for 4 h) increased I-PDGF-BB binding 2-fold, and this increase was blocked by a receptor-associated protein antagonist of the alpha(2)M-receptor/low density lipoprotein receptor-related protein. The receptor-associated protein antagonist blocked I-alpha(2)M-methylamine binding, inhibited PDGF-BB-alpha(2)M-plasmin uptake from fibroblast-cultured supernatants, and abolished the inhibitory effect of alpha(2)M-plasmin on PDGF-stimulated growth. These data suggest that inhibition of PDGF-stimulated proliferation by alpha(2)M-plasmin is mediated in part by clearance of PDGF-BB-alpha(2)M-plasmin through the lipoprotein receptor-related protein.


INTRODUCTION

Platelet-derived growth factor (PDGF) (^1)and homologues of PDGF secreted by macrophages(1, 2) , endothelial cells(3) , smooth muscle cells(4) , and fibroblasts (5) are mesenchymal cell mitogens and chemoattractants that operate in the normal processes of development, wound healing, and tissue remodeling(6) . The aberrant expression of PDGF has been postulated as a common feature in the progression of fibroproliferative diseases such as atherosclerosis (7) and pulmonary fibrosis(8, 9) . PDGF is a disulfide-linked dimer of two polypeptide chains termed A or B that give rise to functional PDGF-AA, PDGF-AB, or PDGF-BB isoforms(10) . Two subtypes of PDGF cell-surface receptors, termed PDGF-Ralpha and PDGF-Rbeta are present on cells of mesenchymal origin(5, 11) , and two related but distinct cDNAs encoding alpha and beta PDGF receptors have been cloned(12, 13) . The PDGF-A chain binds only the PDGF-Ralpha subtype, whereas the PDGF-B chain binds both PDGF-Ralpha and PDGF-Rbeta(11, 14) . Apparently, PDGF receptor dimerization, mediated by ligand binding, is required for signal transduction(15) . The precise role for each of the different PDGF isoforms and receptor subtypes is not well understood, but it has been suggested that the different subtypes of PDGF and receptors could allow for fine tuning of cellular responses, due to observations that different cell types can vary greatly in the ratio of PDGF isoforms secreted and in the receptor subtype composition that the responding target cell possesses(11) .

The biological activity of PDGF is also regulated by alpha(2)-macroglobulin (alpha(2)M)(4, 16, 17, 18, 19) . The function of alpha(2)M as a proteinase inhibitor has been well described and the mechanism whereby native or electrophoretically ``slow'' alpha(2)M covalently entraps serine, aspartic, cysteine, and metalloproteinases has been extensively studied(20, 21, 22, 23, 24) . Proteinases ``activate'' alpha(2)M through cleavage at a specific ``bait region'' which inititates a series of conformational changes in the molecule that entraps the proteinase(20) . The conformational change reveals latent receptor recognition sites on the molecule and also makes the alpha(2)M more compact; conferring ``fast'' mobility when subjected to nondenaturing gel electrophoresis as compared to the native or slow form of alpha(2)M. The irreversible triggering of the proteinase trap is mimicked by primary amines(25) , and fast alpha(2)M-proteinase or alpha(2)M-amine complex, but not native alpha(2)M, bind high affinity receptors on fibroblasts(26, 27) , hepatocytes(28) , and macrophages(29, 30) . This receptor, termed the alpha(2)M receptor/low density lipoprotein receptor-related protein (LRP), binds beta-migrating very low density lipoproteins activated with apolipoprotein E as well as fast alpha(2)Ms(31, 32) . It is synthesized as a 600-kDa precursor protein which undergoes post-translational processing into a 515-kDa ligand-binding subunit and an 85-kDa transmembrane subunit(33) . A 39-kDa protein that has been copurified with LRP, termed receptor-associated protein (RAP), can reversibly bind to the 515-kDa subunit and inhibit binding and uptake of ligands which interact with the LRP(34) .

PDGFbulletalpha(2)M complexes have been isolated from plasma (17) and from macrophage supernatants(18) . PDGF binds both native and proteinase- or amine-activated forms of alpha(2)M(35) , and PDGF-stimulated fibroblast proliferation (19) and chemotaxis (36) are inhibited by native alpha(2)M at concentrations above 0.3 µM. Below this concentration, the native form has no significant effect on the biological properties of PDGF. alpha(2)M activated with methylamine synergistically enhances the growth promoting activity of PDGF purified from human platelets (19) . alpha(2)M also binds and modulates the biological activities of several other growth factors, including transforming growth factor-beta (TGF-beta)(37) , tumor necrosis factor-alpha (TNF-alpha) (38) , basic fibroblast growth factor(39) , interleukin-1beta (IL-1beta) (40) , interleukin-6 (IL-6)(41) , nerve growth factor (NGF)(42) , and vascular endothelial growth factor(43) . The action of native and activated forms of alpha(2)M in regulating this wide spectrum of growth factors has been reviewed(44) . However, the precise role of alpha(2)M in regulating growth factor activity is poorly understood.

The potential modulatory activity of proteinase-activated alpha(2)M on PDGF-stimulated mitogenesis has not yet been investigated. Because methylamine-modified and proteinase-modified fast alpha(2)Ms are both receptor recognized by fibroblasts, we hypothesized that both would possess similar biological activities with regard to modulation of PDGF-stimulated cell growth. On the contrary, we report that plasmin-activated fast alpha(2)M inhibited the proliferation of Swiss mouse 3T3 fibroblasts and rat lung fibroblasts induced by PDGF-BB, and this inhibitory effect was blocked by the RAP antagonist of LRP. We hypothesize that, in extravascular tissues, native (nonreceptor recognized) alpha(2)M serves as a latent reservoir for PDGF-BB, whereas proteinase-activated (receptor recognized) alpha(2)M serves to clear PDGF-BB through the LRP.


MATERIALS AND METHODS

Reagents and Cells

Bovine alpha(2)M (Boehringer Mannheim) was dialyzed against 100 volumes of distilled water to precipitate fast alpha(2)M as has been described previously (35) . Slow alpha(2)M was converted to fast alpha(2)M by incubation with 25 mM methylamine (Tris-HCl, 50 mM, pH 8.0) overnight at 25 °C, or by incubation with a 4:1 molar excess of plasmin overnight at 37 °C. Excess methylamine was removed from alpha(2)Mbulletmethylamine complexes by dialysis against 100 volumes of 50 mM Tris-HCl, pH 8.2, at 4 °C. Excess plasmin was removed from alpha(2)Mbulletplasmin complexes by Superose 6 FPLC gel filtration chromatography (see below). alpha(2)M preparations were tested for PDGF contamination as described previously(19) . A 39-kDa glutathione S-transferase receptor-associated protein (RAP) antagonist of the LRP was the kind gift of Dr. Dudley Strickland (American Red Cross, Rockville, MD)(33) . Swiss mouse 3T3 fibroblasts were purchased from American Type Culture Collection (Rockville, MD). Early passage rat lung fibroblasts were isolated and characterized as described elsewhere(19) .

Gel Electrophoresis

Electrophoresis of the PDGF-BB-alpha(2)M mixtures was performed by nondenaturing gel electrophoresis, due to our previous observation that PDGF-BB is dissociated from alpha(2)M under denaturing conditions(35) . Ten µg of alpha(2)M was mixed with I-PDGF-BB (2 ng) and incubated overnight at 37 °C in a final volume of 25 µl. Samples were mixed with 10 µl of 6 times nondenaturing sample buffer containing Tris-borate EDTA (TBE) glycerol, 1% xylene cyanol, and 1% bromphenol blue and electrophoresed on a native 6% gel (Novex, Encinitas, CA). 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-BB bound to alpha(2)M.

Superose 6 FPLC Gel Filtration Chromatography

PDGF-BBbulletalpha(2)M and PDGF-BBbulletalpha(2)M-plasmin complexes were routinely prepared by incubating 100 ng of human recombinant I-PDGF-BB with 1 mg of alpha(2)M or alpha(2)M-plasmin for 24 h at 37 °C. These mixtures were isolated by loading onto a gel filtration, molecular weight exclusion column (Superose 6 FPLC, Pharmacia LKB Biotechnol) equilibrated in phosphate-buffered saline, pH 7.5, operating at a flow rate of 0.5 ml/min.

Northern Analysis for PDGF-alpha and -beta Receptor mRNA

Fibroblasts were grown to confluence in 150-cm^2 flasks in 10% FBS-DMEM and then rendered quiescent for 24 h in serum-free defined medium (SFDM: Ham's F-12 with HEPES, CaCl(2), and 0.25% BSA supplemented with an insulin-transferrin-selenium mixture purchased from Boehringer Mannheim Biochemicals). Fibroblasts were then treated with alpha(2)M or alpha(2)M-plasmin for 24 h at 37 °C. Collection of total RNA and Northern analysis was performed as described previously(59) . The human cDNA probes for the PDGF-Ralpha subunit and PDGF-Rbeta subunit, kindly provided by Dr. Carl-Henrik Heldin (Ludwig Institute for Cancer Research, Uppsala, Sweden), were labeled with a random primed DNA labeling kit (Boehringer Mannheim).

I-PDGF-AA and -BB Receptor Assay

Rat lung fibroblasts or Swiss 3T3 fibroblasts were rendered quiescent for 24 h with SFDM. Plates of cells were either exposed to alpha(2)M, alpha(2)M-plasmin, or media alone for an additional 24 h prior to assay. The plates were then cooled to 0-4 °C for 15-30 min, the media aspirated, and the cells washed twice with ice-cold binding medium (Ham's F-12 with HEPES, CaCl(2), and 0.25% BSA). I-PDGF-BB (DuPont NEN) or I-PDGF-AA (Biomedical Technologies, Stoughton, MA) was diluted to 100 ng/ml in binding buffer. Binding assays were performed by adding increasing amounts (0.5-20 ng/ml) of I-PDGF-BB or -AA to cells in duplicate wells in a final volume of 0.2 ml/well. After incubating on an oscillating platform at 0-4 °C for 3 h, the cells were washed three times with ice-cold binding buffer, the cell-bound radioactivity solubilized with 1% Triton-X containing 0.1% BSA and 0.1 N NaOH, and the radioactivity measured on a -counter.

alpha(2)-Macroglobulin Receptor Assay

Fibroblasts were plated as described above for the PDGF binding assay. Once confluent, the cells were rendered quiescent for 24 h in SFDM. Cultures were then cooled on ice for 15-30 min before aspirating the 10% FBS and washing three times with a serum-free-binding medium. Fibroblasts were incubated in binding medium (1 ml/well) at 4 °C for 3-4 h, then washed three more times before adding human I-alpha(2)M-methylamine (custom iodinated by DuPont NEN, 8 mCi/mg) at a final concentration of 0.1 nM in the absence or presence of unlabeled bovine alpha(2)M, alpha(2)M-plasmin, or the RAP antagonist of the alpha(2)M receptor. Following a 3-4-h incubation at 4 °C, the medium was aspirated and the cells were washed three times with cold binding medium. The cell-bound radioactivity was extracted with solubilization buffer (1% Triton X-100, 0.1 N NaOH) and counted on a -counter.

IbulletPDGF-BBbulletalpha(2)M-Plasmin Uptake

Native alpha(2)M or plasmin-activated alpha(2)M (20 µg) was incubated for 1 h at 37 °C with IbulletPDGF-BB (2 ng) in a final volume of 50 µl. These complexes were then purified by Superose 6 FPLC as described above and the column fractions eluting between 20-28 min were pooled and diluted in SFDM to a final concentration of 1 nM (alpha(2)M) in 20 ml. The complexes were then added to confluent lung fibroblasts (10 times 10^6/150-cm^2 flask) that had been rendered quiescent in SFDM for 24 h. Some cultures of fibroblasts were pretreated with 100 nM RAP for 1 h prior to adding IbulletPDGF-BBbulletalpha(2)M-plasmin complex to block uptake. The medium was collected at 0, 4, and 12 h post-treatment, centrifuged 1400 times g to remove nonadherent cells, and the supernatant filtered (0.2 µm acrodisc, Gelman Sciences). The filtrate was concentrated to 1 ml using 30-kDa cutoff membrane (Amicon, Beverly, MA) and dialyzed on the Amicon apparatus against 100 ml of deionized water three times. After concentrating again to 1 ml, the sample was lyophilized using a Speed Vac (Savant, Inc.) and reconstituted in 20 µl of water and 6 µl of Tris-borate EDTA native sample buffer. The samples were electrophoresed on 5% native gels, dried, and autoradiographed as described above under ``Electrophoresis.'' In another set of experiments, I-PDGF in the absence or presence of alpha(2)M slow and fast forms was bound at 0-4 °C for 3 h to confluent cultures of fibroblasts in 150-cm^2 dishes prior to washing the cultures and warming to 37 °C to measure release of radioactivity into the medium. The supernatants were then run on a 4-20% SDS-polyacrylamide gel (Novex) along with standard I-PDGF-BB, dried, and autoradiographed to assess the molecular weight of the released radioactivity. This experiment allowed us to determine whether alpha(2)M fast forms facilitated recycling of intact PDGF following uptake via the LRP.

Cell Proliferation Assay

Fibroblasts suspended in 10% FBS-DMEM were seeded 2 ml/well in 12-well tissue culture plates at 20,000/well and allowed to proliferate for 24 h in 5% CO(2) humidified air at 37 °C. These subconfluent cultures were washed two times with SFDM, and fresh SFDM alone or containing increasing concentrations of human recombinant PDGF-BB (UpState Biotechnology, Lake Placid, NY) was added back to the wells. Parallel incubations were coincubated with alpha(2)M, plasmin, or a mixture of alpha(2)M and plasmin. In another experiment, fractions from the Superose 6 FPLC column containing purified PDGF-BBbulletalpha(2)M or PDGF-BBbulletalpha(2)M-plasmin complex were added to the cultures. After 3 days of incubation, the growth medium was aspirated and the cells harvested by trypsinization and enumerated using a Coulter Counter (Coulter Electronics, Hialeah, FL).


RESULTS

Fibroblast Proliferation Induced by PDGF-BB Is Inhibited by A Mixture of alpha(2)M and Plasmin or by Purified alpha(2)M-Plasmin Complex

We first sought to determine the molar ratio of plasmin:alpha(2)M that would convert all of the slow form alpha(2)M to electrophoretically fast, receptor-recognized alpha(2)M. Increasing concentration of plasmin progressively converted alpha(2)M slow form to its electrophoretically fast conformation with complete conversion occurring at a 2:1 molar ratio of plasmin to alpha(2)M (Fig. 1A). I-PDGF-BB bound to both native slow alpha(2)M and fast plasmin-activated alpha(2)M (Fig. 1B). The PDGF-BB-stimulated proliferation of rat lung fibroblasts and Swiss 3T3 fibroblasts was inhibited in the presence of a mixture of 0.3 µM alpha(2)M and 0.6 µM plasmin (Fig. 2). alpha(2)M or plasmin alone at these same concentrations did not affect PDGF-BB-induced growth. Further experiments were performed on rat lung fibroblasts with PDGF-BBbulletalpha(2)M and PDGF-BBbulletalpha(2)M-plasmin complexes purified by Superose 6 FPLC chromatography. alpha(2)M and alpha(2)M-plasmin bound approximately equal amounts of I-PDGF-BB (Fig. 3). However, no mitogenic activity was observed at the molecular weight of the PDGFbulletalpha(2)M-plasmin complex, while the PDGF-BBbulletalpha(2)M complex stimulated the proliferation of rat lung fibroblasts by as much as 40% above the number of cells obtained from SFDM alone (Fig. 3).


Figure 1: [I]PDGF-BB binds to native and plasmin-activated alpha(2)M. Panel A, Native alpha(2)M (10 µg) was treated with plasmin for 24 h at 37 °C prior to loading onto a nondenaturing 6% Tris-borate-EDTA gel as described under ``Materials and Methods.'' Increasing plasmin concentrations resulted in a progressive increase in receptor-recognized, electrophoretically fast (F) alpha(2)M with complete conversion occurring at a 2:1 molar ratio of plasmin to alpha(2)M. Panel B, autoradiography of TBE gel demonstrating that alpha(2)M and alpha(2)M-plasmin bound nearly equivalent amounts of I-PDGF-BB. Native and plasmin-activated alpha(2)M (10 µg) were incubated with I-PDGF-BB (2 ng) at 37 °C for 24 h and electrophoresis was performed as described above.




Figure 2: PDGF-BB-induced fibroblast proliferation is inhibited by a combination of alpha(2)M and plasmin. The proliferation of rat lung fibroblasts (panel A) and Swiss 3T3 fibroblasts (panel B) was measured as described under ``Materials and Methods.'' Subconfluent fibroblast cultures were treated with increasing concentration of PDGF-BB in the absence or presence of alpha(2)M (0.3 µM), plasmin (0.6 µM), or a combination of alpha(2)M and plasmin (1:2 molar ratio) in serum-free defined medium. After 3 days in culture the cells were removed from the plates by trypsin treatment and enumerated with an electronic particle counter. Native alpha(2)M (closed circles) or plasmin (open triangles) had no significant affect on PDGF-stimulated cell growth as compared to PDGF-BB alone (open circles, dashed line), while the combination of alpha(2)M and plasmin (closed triangles) inhibited PDGF-BB-stimulated proliferation. Results are the mean of six separate experiments (S.E. < 5% of the mean). Each assay was performed in triplicate wells.




Figure 3: The PDGF-BBbulletalpha(2)M complex retains mitogenic activity, while the PDGF-BBbulletalpha(2)M-plasmin complex does not. One mg of alpha(2)M or alpha(2)M-plasmin was incubated with 200 ng of nonradioactive PDGF-BB for 24 h at 37 °C prior to loading onto a Superose 6 FPLC column and elution in PBS, pH 7.4. Column fractions were diluted 1:1 with serum-free defined medium and tested for mitogenic activity on lung fibroblasts in a 3-day proliferation assay as described under ``Materials and Methods.'' The equivalent binding of I-PDGF-BB (2 ng) to either alpha(2)M or alpha(2)M-plasmin (100 µg) was determined on Superose 6 FPLC in an identical manner as described above, and the column fractions were -counted. A, PDGF-BB bound alpha(2)M-plasmin (open triangles, dashed line) but the PDGF-BB/alpha(2)M-plasmin did not elicit mitogenesis of rat lung fibroblasts (solid line, closed circles). A peak of mitogenic activity was apparent at the approximate molecular mass of uncomplexed PDGF-BB. B, the PDGF-BBbulletalpha(2)M complex stimulated fibroblast proliferation by as much as 40% above control cells maintained in SFDM alone. Arrows indicate the elution of molecular weight mass markers: 1, blue dextran (V), 2, thyroglobulin (669 kDa, also marks the elution of alpha(2)M as determined by ELISA), 3, apoferritin (440 kDa), 4, beta-amylase (200 kDa), 5, BSA (66 kDa), 6, carbonic anhydrase (29 kDa), and 7, cytochrome (12.4 kDa). The data are typical of four separate experiments.



Preincubation with alpha(2)M and alpha(2)M-Plasmin at 37 °C Does Not Alter PDGF Receptor Gene Expression or Binding of I-PDGF-BB to Cell-surface PDGF Receptors

We first postulated that alpha(2)M-plasmin, which is receptor recognized (see Fig. 4), inhibited PDGF-BB-stimulated proliferation through down-regulation of either the PDGF-Ralpha or the PDGF-Rbeta on fibroblasts. Preincubation with alpha(2)M-plasmin for 24 h at 37 °C did not affect expression of the 6.5-kb PDGF-Ralpha gene on Swiss 3T3 cells, nor did it significantly change the gene expression of the 5.2-kb PDGF-Rbeta mRNA on rat lung fibroblasts (data not shown). Furthermore, preincubation of fibroblasts with alpha(2)M or alpha(2)M-plasmin for 24 h at 37 °C did not alter I-PDGF-BB or I-PDGF-AA binding on lung fibroblasts or Swiss 3T3 fibroblasts. However, preincubation of rat lung fibroblasts with methylamine-modified alpha(2)M increased I-PDGF-AA-binding sites 3-fold, indicating that this fast form of alpha(2)M up-regulated the PDGF-alpha receptor subtype (Table 1).


Figure 4: alpha(2)M-plasmin and a 39-kDa RAP antagonist compete for the specific binding of I-alpha(2)M-methylamine to fibroblasts. Confluent quiescent cultures of rat lung fibroblasts (panel A) or Swiss 3T3 fibroblasts (panel B) were chilled to 0-4 °C for 30 min and assayed for specific binding of I-alpha(2)M-methylamine as described under ``Materials and Methods.'' Increasing concentration of alpha(2)M-plasmin (open circles) and the 39-kDa RAP antagonist (closed triangles) were added immediately prior to the addition of 0.1 nMI-alpha(2)M-methylamine. A 200-fold molar excess of native alpha(2)M did not inhibit the specific binding of I-alpha(2)M-methylamine (closed squares). The RAP antagonist inhibited specific binding (IC = 0.1-0.2 nM) with about a 5-fold lesser potency than that of alpha(2)M-plasmin (IC = 0.5-1.0 nM). The data are representative of four experiments each performed in triplicate.





Preincubation with alpha(2)M-Plasmin, But Not alpha(2)M, at 0-4 °C Increases Cell-surface Binding of I-PDGF-BB That Is Blocked by the RAP Antagonist of LRP

As an alternate hypothesis, we postulated that alpha(2)M-plasmin inhibited PDGF-BB-stimulated growth via the LRP on fibroblasts. We first demonstrated that both fibroblast types possessed LRP and that RAP antagonized alpha(2)M-plasmin binding (Fig. 4). The specific binding of I-alpha(2)M-methylamine to rat lung fibroblasts and Swiss 3T3 cells was inhibited in a concentration-dependent manner by alpha(2)M-plasmin (IC between 0.2-1 nM). Similarly, the 39-kDa RAP antagonist inhibited I-alpha(2)M-methylamine binding (IC between 0.5-1 nM) for both types of fibroblasts. Maximal inhibition of binding by the antagonist was reached at approximately 10 nM RAP, a 100-fold molar excess over the concentration of I-alpha(2)M-methylamine used in the assay (Fig. 4). The RAP antagonist did not inhibit the specific binding of I-PDGF-BB to rat lung fibroblasts (Fig. 5). We then preincubated fibroblasts with alpha(2)M-plasmin at 0-4 °C for 4 h, which prevents internalization of LRP-bound complex(34) , prior to performing the PDGF receptor assay in order to determine if this receptor-recognized form of alpha(2)M would increase the binding of I-PDGF-BB at the cell surface. alpha(2)M-plasmin, but not native alpha(2)M, increased specific I-PDGF-BB binding 2-fold (Fig. 6). Importantly, this increased binding was observed only at concentrations of I-PDGF-BB above 0.3 nM (>10 ng/ml), i.e. at concentrations of radioligand above saturation of PDGF cell-surface receptors. These data indicated that I-PDGF-BB bound to LRP-bound alpha(2)M-plasmin, but only when PDGF cell-surface receptors (predominantly PDGF-Rbeta) were saturated.


Figure 5: The 39- kDa alpha(2)M RAP antagonist does not compete for I-PDGF-BB binding to fibroblasts. Increasing concentrations of PDGF-BB or the RAP alpha(2)M-receptor antagonist were added with 1 ng/ml I-PDGF-BB to confluent rat lung fibroblasts rendered quiescent in SFDM for 24 h. Receptor binding was assayed as described under ``Materials and Methods.'' These data show that RAP is a specific antagonist for the LRP and does not directly interfere with PDGF-BB binding to its cell-surface receptor. The data are representative of four experiments each performed in triplicate.




Figure 6: Preincubation of fibroblasts with alpha(2)M-plasmin at 0-4 °C increases the specific binding of I-PDGF-BB. Confluent, quiescent rat lung fibroblasts were incubated with alpha(2)M or alpha(2)M-plasmin, with or without the RAP antagonist, for 4 h on ice prior to washing the cells three times with ice-cold binding buffer and performing the radioligand binding assay for I-PDGF-BB (20 ng/ml) as described under ``Materials and Methods.'' alpha(2)M-plasmin, but not native alpha(2)M, caused a 2-fold increase in the specific binding of I-PDGF-BB that was significant (p < 0.01 paired Student's t test), but only at radioligand concentrations >10 ng/ml. The increase in I-PDGF-BB binding was blocked by co-incubation with a 100-fold excess of the RAP antagonist. These data indicate that I-PDGF-BB binds to surface-bound alpha(2)M-plasmin, but only when the cell-surface PDGF receptors are saturated. Data are the mean ± S.E. of four separate experiments each performed in triplicate.



Uptake of the IbulletPDGF-BBbulletalpha(2)M-Plasmin Complex and Inhibition of PDGF-BB-stimulated Growth by alpha(2)M-Plasmin Is Blocked by the RAP Antagonist of the LRP

Because LRP-bound alpha(2)M-plasmin increased the specific binding of I-PDGF-BB to fibroblasts, we proposed that PDGF-BBbulletalpha(2)M-plasmin complex could be taken up through the LRP and that the mitoinhibitory effect of alpha(2)M-plasmin on PDGF-BB-stimulated fibroblast growth could be blocked by the RAP antagonist. We were unable to show uptake of the IbulletPDGF-BBbulletalpha(2)M-plasmin complex in the 3-day cell proliferation assay due to the low number of cells in the assay (20,000/well at time 0) and the relatively low specific activity of I-PDGF-BB bound to alpha(2)M. While uptake of I-alpha(2)M-MA could have been assessed in the cell proliferation assay, we sought to copurify I-PDGF-BB bound to alpha(2)M by autoradiography of a nondenaturing gel and visualize alpha(2)M by protein staining the same gel. Therefore, the IbulletPDGF-BBbulletalpha(2)M or the IbulletPDGF-BBbulletalpha(2)M-plasmin complex was diluted to 1 nM in 20 ml of SFDM, and these complexes were added to confluent lung fibroblasts (10 times 10^6/150-cm^2 flask) in the absence or presence of 100 nM RAP. The IbulletPDGF-BBbulletalpha(2)M-plasmin complex was taken up by rat lung fibroblasts in culture after 12 h, and this uptake was blocked by RAP (Fig. 7). Furthermore, in the 3-day cell proliferation assay, the RAP antagonist blocked alpha(2)M-plasmin-induced inhibition of PDGF-BB-stimulated rat lung fibroblast proliferation, while RAP alone did not affect PDGF-BB-induced proliferation (Fig. 8). In another experiment, we investigated the fate of I-PDGF-BB bound to alpha(2)M-plasmin or alpha(2)M-methylamine following uptake, since we previously reported that alpha(2)M-methylamine synergistically enhanced PDGF-stimulated growth of fibroblasts and this contrasted with our present observation of alpha(2)M-plasmin inhibition of PDGF-stimulated growth(19) . I-PDGF-BB was incubated with rat lung fibroblasts at 0-4 °C for 3 h in the absence or presence of 0.3 µM alpha(2)M-plasmin or alpha(2)M-methylamine, then the cells were rinsed with binding buffer and allowed to warm to 37 °C to allow uptake of growth factor. At various time points (0.5, 1, 2, and 3 h) the supernatants were removed and run on 4-20% SDS-polyacrylamide gel electrophoresis to determine the amount of radioactivity released by the cells and to determine if either fast form of alpha(2)M was mediating recycling of intact 30-kDa PDGF-BB. Radioactivity possessing a molecular mass below that of PDGF-BB (30 kDa) was released in a time-dependent manner from fibroblasts that had internalized surface-bound I-PDGF-BB (30 min, 2,333 ± 168 cpm; 1 h, 6,641 ± 170 cpm; 2 h, 20,916 ± 1,293 cpm; and 3 h, 22, 725 ± 781 cpm). The presence of alpha(2)M-plasmin or alpha(2)M-methylamine did not significantly alter the amount of radioactivity released into the medium nor was any intact I-PDGF-BB released (i.e. all released radioactivity possessed a molecular mass below 30 kDa). These data indicated that neither alpha(2)M-plasmin nor alpha(2)M-methylamine mediate recycling of biologically active PDGF.


Figure 7: The uptake of IbulletPDGF-BBbulletalpha(2)M-plasmin complex by fibroblasts in culture is inhibited by RAP. Confluent cultures of rat lung fibroblasts grown in 10% FBS-DMEM in 150-cm^2 flasks were rendered quiescent with SFDM for 4-6 h at 37 °C and then treated with BSA-free SFDM alone or supplemented with 100 nM RAP for an additional 1 h prior to adding 1 nMIbulletPDGF-BBbulletalpha(2)M or IbulletPDGF-BBbulletalpha(2)M-plasmin complex. These complexes were prepared by incubating 20 µg of alpha(2)M or alpha(2)M-plasmin with 0.3 ng of I-PDGF-BB in a final volume of 100 µl for 4 h at 37 °C prior to diluting in 10 ml BSA-free SFDM and adding to 10 ml of pre-existing medium (with or without RAP) on the cultures. The final concentration of alpha(2)M was 1 nM. At various time points the media was removed, concentrated with a 100-kDa cutoff filter, lyophilized, reconstituted to 50 µl, and electrophoresed on a nondenaturing gel as described under ``Materials and Methods.'' Following electrophoresis, the gels were dried and stained with Coomassie Blue to visualize alpha(2)M slow (S) and fast (F) forms (panel A). The same gel was exposed to autoradiographic film to visualize I-PDGF-BB bound to S and F forms of alpha(2)M (panel B). The IbulletPDGF-BBbulletalpha(2)M-plasmin complex, but not the native complex, was taken up by the fibroblasts, and this uptake was blocked the RAP antagonist.




Figure 8: The RAP antagonist blocks inhibition of PDGF-BB-induced growth by alpha(2)M-plasmin. Subconfluent cultures of rat lung fibroblasts in 10% FBS-DMEM were washed three times with SFDM and treated with PDGF-BB (10 ng/ml) in the absence or presence of alpha(2)M (0.3 µM), plasmin (0.6 µM), or a combination of both (2:1 molar ratio of plasmin to alpha(2)M) as described in Fig. 2. A 100-fold molar excess of RAP antagonist significantly blocked the growth inhibitory effect of the alpha(2)M-plasmin mixture on PDGF-BB-stimulated growth (**p < 0.01 paired Student's t test). Data are expressed as the percent increase in cell number above that obtained in SFDM without PDGF-BB and are the mean ± S.E. from six separate experiments each performed in triplicate. All treatments shown contain 10 ng/ml PDGF-BB.




DISCUSSION

alpha(2)M has been described as a potential modulator of several growth factors and cytokines. Its precise function in regulating growth factor activity appears to vary greatly depending on the specific growth factor, the conformation of alpha(2)M to which the growth factor is bound, and the biological function studied. In general, alpha(2)M could serve several regulatory roles, including 1), inactivation and clearance of growth factors, 2) potentiation of growth factor activity, 3) protection of growth factors against proteolytic inactivation, and 4) as a latent extracellular reservoir of growth factors. In this study we report that alpha(2)M activated by plasmin is a negative growth regulator of PDGF-stimulated fibroblast proliferation in vitro, and this appears to be related to the clearance of PDGF-BBbulletalpha(2)M-plasmin complex through an LRP-dependent mechanism. In contrast, the PDGF-BBbulletalpha(2)M complex, which does not bind LRP or PDGF receptors(18, 19) , retained growth-promoting activity. This is likely explained by our previous observation that PDGF is bound to alpha(2)M in a reversible, noncovalent manner, and PDGF-BB dissociates from alpha(2)M with a half-time of about 2 h(35) . PDGF-BB bound to alpha(2)M does not bind PDGF-Ralpha or PDGF-Rbeta, apparently because the receptor-recognized sites on the PDGF molecule are masked by the alpha(2)M molecule (18) . Thus, PDGF-BB was probably released from the PDGF-BBbulletalpha(2)M complex during the course of the 3-day growth assay to bind cell-surface PDGF receptors (Fig. 3). The relative low affinity binding of PDGF-BB to alpha(2)M in the micromolar range (47) as compared to PDGF-BB binding either the PDGF-Ralpha or PDGF-Rbeta in the nanomolar range (Fig. 5) supports our hypothesis that native alpha(2)M serves as a reservoir of latent PDGF-BB that can be released to bind high affinity PDGF cell-surface receptors on mesenchymal cells.

Our data suggest that the alpha(2)Mbulletplasmin complex does not suppress PDGF action by down-regulation of the PDGF receptor, since preincubation of fibroblasts with alpha(2)M-plasmin did not alter PDGF-Ralpha or PDGF-Rbeta gene expression. Furthermore, I-PDGF-BB binding to fibroblasts was not affected by alpha(2)M-plasmin preincubation for 24 h at 37 °C. However, preincubation of fibroblasts with alpha(2)M-plasmin at 0-4 °C for 4 h, which prevents internalization of alpha(2)M-plasminbulletLRP complex, increased the specific binding of I-PDGF-BB 2-fold. This increase in specific binding was observed only at concentrations of I-PDGF-BB above receptor saturation (0.3 nMI-PDGF-BB) (Fig. 6). Recently, Crookston and co-workers (47) reported a K(d) of 0.3 µM for PDGF-BB binding to alpha(2)M , indicating that PDGF-BB binds to alpha(2)M with an affinity 1000-fold less than PDGF-BB binding to its own receptor. In general, this is in agreement with our observation that I-PDGF-BB will bind to LRP-bound alpha(2)M-plasmin only when cell-surface PDGF receptors are occupied (i.e. at concentrations of I-PDGF-BB above 10 ng/ml). We observed that the increase in I-PDGF-BB binding following alpha(2)M-plasmin preincubation on ice was blocked by the RAP antagonist of the LRP (Fig. 6). This proved that PDGF-BB bound to LRP-bound alpha(2)M-plasmin at the cell surface, since excess concentrations of RAP did not inhibit the binding of I-PDGF-BB binding to its own receptor on fibroblasts (Fig. 5). The RAP antagonist inhibited I-alpha(2)M-methylamine binding to fibroblasts (Fig. 4), inhibited uptake of IbulletPDGF-BBbulletalpha(2)M complex by fibroblasts (Fig. 7), and abolished the inhibitory effect of alpha(2)M-plasmin on PDGF-induced proliferation (Fig. 8). Thus, we concluded that the inhibitory effect of alpha(2)M-plasmin on PDGF-BB-stimulated fibroblast growth is mediated at least in part by uptake and degradation of PDGF-BBbulletalpha(2)M-plasmin complex through the LRP.

Our findings are novel in that we have demonstrated that fibroblasts can mediate growth factor clearance via their cell-surface LRP, and this results in a mitoinhibitory effect. The concentration of alpha(2)M used in this study (300 nM) is much lower than that found in plasma (2-4 µM) (23, 24) and could be similar to concentrations found in extravascular spaces. Activated macrophages produce alpha(2)M(18) , and we have observed that human lung fibroblasts in culture spontaneously secrete 0.5 ng alpha(2)M/million cells. (^2)Thus, in vivo fibroblasts could have nanomolar levels of alpha(2)M in their immediate environment. Several other investigators have reported that activated alpha(2)Ms mediate the plasma clearance and inactivation of growth factors and cytokines, including PDGF-BB(48) , TGF-beta1(48, 49) , and TNF-alpha(38) . These studies are particularly relevant to circumstances of vascular injury, where platelets degranulate and release growth factors such as PDGF and TGF-beta. Crookston and co-workers showed that, while PDGF-BB and TGF-beta1 bound both native and methylamine-activated alpha(2)M, only the IbulletPDGF-BBbulletalpha(2)M-methylamine and IbulletTGF-beta1bulletalpha(2)M-methylamine complexes were cleared from the circulation(48) . These investigators suggested that native, non-receptor-recognized alpha(2)M is the major carrier of TGF-beta1 and PDGF-BB in the blood, whereas activated alpha(2)Ms mediate the rapid clearance of these growth factors through the liver. This is a reasonable assessment, since it has been established that TGF-beta1 and PDGF-BB bind to slow and fast alpha(2)Ms and because alpha(2)M-proteinase complexes are cleared through the liver with a half-time of minutes(28) . Our findings herein suggest that alpha(2)M and alpha(2)M-proteinase complexes could be critical mediators of growth factor clearance in extravascular compartments, particularly during inflammation and tissue repair where activated macrophages have accumulated and release numerous growth factors (e.g. PDGF (1) , TGF-beta(50) , IL-1beta(51) ), alpha(2)M(18) ,and a wide spectrum of proteolytic enzymes(52) . We reported earlier that the majority of macrophage-derived PDGF in culture supernatants is complexed to macrophage-derived homologues of plasma alpha(2)M and that the macrophage-derived PDGFbulletalpha(2)M complex does not bind to PDGF receptors, nor is it recognized by antibodies raised against PDGF(18) . However, these complexes retain PDGF-like activity for fibroblasts. Like fibroblasts, macrophages also possess LRP (29) and potentially could reclaim secreted PDGFbulletalpha(2)M complex in the presence of proteinases through an LRP autocrine loop. This hypothetical situation would be further complicated by inflammatory mediators (e.g. colony stimulating factor, interferon-, and lipopolysaccharide) which have been shown to either up-regulate or down-regulate the macrophage LRP(53, 54) .

We previously reported that alpha(2)M-methylamine enhanced the growth promoting activity of PDGF for fibroblasts(19) . A similar effect of alpha(2)M-methylamine was reported for TGF-beta1-induced mitogenesis of smooth muscle cells(55) . TGF-beta1 induces a growth response in smooth muscle cells via a PDGF-AA autocrine loop (56) and alpha(2)M-MA was recently shown to synergistically enhance the TGF-beta1-stimulated proliferation of these cells(55) . On the other hand, alpha(2)M blocks the mitoinhibitory effect of TGF-beta1 for mink lung epithelial cells(57) . We postulated that the opposing effects of alpha(2)M-plasmin and alpha(2)M-methylamine on PDGF-stimulated growth could be due to a differences in the fate of the complexes once endocytosed, i.e. whether the PDGFbulletalpha(2)M-methylamine or PDGFbulletalpha(2)M-plasmin complexes follow the same pathways of degradation or recycling to the cell surface. However, no differences were observed in the fate of I-PDGF-BB that was endocytosed by fibroblasts in the absence or presence of alpha(2)M-plasmin or alpha(2)M-methylamine. In all instances, the endocytosed radioactivity was released from fibroblasts in a time-dependent manner that was maximal at 2 h and all of the radioactivity possessed a molecular mass less than 30 kDa (i.e. degraded PDGF). Thus, the enhancement of PDGF-stimulated growth by alpha(2)M-methylamine was not due to recycling of bioactive PDGF through the LRP. Stouffer and co-workers reported that alpha(2)M-methylamine, but not native alpha(2)M, stimulated mitogenesis of smooth muscle cells. Thus, it appears that alpha(2)M-methylamine can directly induce mitogenesis, and this finding is consistent with the discovery of a second G-protein-coupled alpha(2)M-receptor by Misra and co-workers (58) that possesses signaling properties. While it is clear that alpha(2)M-methylamine and alpha(2)M-proteinase complex bind and are cleared through the LRP, possible differences in the interaction of alpha(2)M-methylamine and alpha(2)M-proteinase complex have not been investigated with regard to the G-protein-coupled alpha(2)M receptor. In the present study, we observed that alpha(2)M-methylamine, but not alpha(2)M-plasmin, up-regulated the PDGF-alpha receptor subtype (Table 1). We previously reported that up-regulation of this receptor increases the mitogenic response of fibroblasts to PDGF (59) in a manner reminiscent of that observed with the combination of alpha(2)M-methylamine and PDGF(19) ; i.e. in both cases the growth response is increased 2-3-fold above the maximal response obtained with PDGF alone. Thus, these data taken together suggest that alpha(2)M-methylamine, but not alpha(2)M-plasmin, enhance PDGF-stimulated growth through up-regulation of the PDGF-alpha receptor subtype which then increases the mitogenic activity of PDGF isoforms. It is important to note that proteinase-activated forms of alpha(2)M, but not alpha(2)M-methylamine, occur in vivo. Thus, an understanding of how alpha(2)M-proteinase complexes modulate growth factor activity should be more relevant to tissue repair and disease.

In light of our findings, it seems reasonable to speculate that native and proteinase-activated alpha(2)M differentially regulate PDGF-BB activity in extravascular tissues, i.e. native PDGFbulletalpha(2)M complexes purfied by Superose 6 FPLC chromatography retained growth-promoting activity, while PDGFbulletalpha(2)M-plasmin complexes did not (Fig. 3). Other studies of growth factor/alpha(2)M binding and clearance have been related to the plasma(38, 48, 49) . However, it is probable that alpha(2)M regulates growth factor activity in the extravascular tissues as well. Macrophages and fibroblasts both produce alpha(2)M and possess LRP(60) . Thus, we postulate that native alpha(2)M serves as a reservoir of secreted PDGF in tissues, where it can be released under conditions such as decreased pH(19) . Conversely, proteinases secreted by inflammatory cells such as macrophages and neutrophils mediate the slow to fast conformational change in alpha(2)M and could trigger the clearance of growth factors via the LRP on macrophages and fibroblasts.

In summary, we have shown that alpha(2)M-plasmin, but not alpha(2)M or plasmin alone, inhibits PDGF-BB-stimulated proliferation of rat lung fibroblasts and Swiss mouse 3T3 fibroblasts. Preincubation of alpha(2)M-plasmin complexes with these fibroblasts does not alter PDGF-Rbeta or PDGF-Ralpha gene expression. The inhibitory effect of alpha(2)M-plasmin on PDGF-induced growth is blocked by the RAP antagonist of the LRP (i.e. the alpha(2)M receptor). We hypothesize that alpha(2)M fast forms activated by proteinases function in vivo as a vehicle for PDGF clearance and inactivation via the LRP, while native, slow alpha(2)M serves as an extracellular reservoir for PDGF where it could be released in the vicinity of responding target cells.


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: National Institute of Environmental Health Sciences, P. O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-541-0766; Fax: 919-541-4133.

(^1)
The abbreviations used are: PDGF, platelet-derived growth factor; PDGF-Ralpha, platelet-derived growth factor alpha-receptor; PDGF-Rbeta, platelet-derived growth factor beta-receptor; alpha(2)M, alpha(2)-macroglobulin; alpha(2)M-MA, alpha(2)-macroglobulin-methylamine; LRP, alpha(2)-macroglobulin receptor/low density lipoprotein receptor-related protein; RAP, receptor-associated protein antagonist; TGF-beta, transforming growth factor-beta; IL-1beta, interleukin-1beta; TNF-alpha, tumor necrosis factor-alpha; FGF, fibroblast growth factor; IL-6, interleukin-6; NGF, nerve growth factor; BSA, bovine serum albumin; FPLC, fast protein liquid chromatography; SFDM, serum-free defined medium; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; cpm, counts/min..

(^2)
J. C. Bonner and P. M. Lindroos, unpublished observation.


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