©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Native and Activated Forms of -Macroglobulin Increase Expression of Platelet-derived Growth Factor -Receptor in Vascular Smooth Muscle Cells
EVIDENCE FOR AUTOCRINE TRANSFORMING GROWTH FACTOR-beta ACTIVITY (*)

(Received for publication, August 1, 1995; and in revised form, October 24, 1995)

Alissa M. Weaver (1)(§) Gary K. Owens (2) Steven L. Gonias (1) (3)(¶)

From the  (1)Departments of Biochemistry, (2)Molecular Physiology and Cellular Biophysics, and (3)Pathology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Cellular response to platelet-derived growth factor AA (PDGF-AA) is mediated exclusively by the PDGF alpha-receptor. Vascular smooth muscle cells (VSMCs) in culture typically express very low levels of alpha-receptor. In this study, we demonstrate that the proteinase inhibitor and cytokine carrier alpha(2)-macroglobulin (alpha(2)M) increases rat VSMC PDGF alpha-receptor expression. PDGF alpha-receptor mRNA levels increased 3-fold by 6 h and were sustained at that level through 24 h in VSMCs treated with 280 nM methylamine-modified alpha(2)M (alpha(2)M-MA), a form of activated alpha(2)M. PDGF beta-receptor mRNA levels were unchanged in the same time period. In I-PDGF-AA binding experiments, treatment of VSMCs with alpha(2)M-MA increased the maximum binding capacity (B(max)) from 1.9 to 9.2 fmol/mg of cell protein without affecting binding affinity (K 80 pM). alpha(2)M-MA also increased the VSMC response to PDGF-AA as determined by tyrosine phosphorylation of a 170-kDa band, corresponding in mass to the PDGF alpha-receptor. The native form of alpha(2)M was comparable to alpha(2)M-MA in its ability to increase PDGF-AA binding to VSMCs and tyrosine phosphorylation of the 170-kDa band. Recombinant and proteolytic alpha(2)M derivatives were used to demonstrate that alpha(2)M increases PDGF alpha-receptor expression by binding VSMC-secreted cytokine(s) and interrupting an autocrine loop that ordinarily suppresses alpha-receptor expression in these cells. Transforming growth factor-beta-neutralizing antibody mimicked the activity of alpha(2)M, increasing the binding capacity of VSMCs for PDGF-AA. This study demonstrates that VSMC PDGF alpha-receptor expression and responsiveness to PDGF-AA are regulated by autocrine transforming growth factor-beta activity, potentially other autocrine growth factors, and alpha(2)M.


INTRODUCTION

Vascular smooth muscle cell (VSMC) (^1)gene expression is regulated by extracellular mediators synthesized by cells within the blood vessel wall or transferred from the plasma, especially with blood vessel injury(1, 2, 3) . Isoforms of the platelet-derived growth factor (PDGF) family originate from both sources and are important regulators of VSMC growth and phenotype(4, 5) . The three PDGF isoforms, PDGF-AA, PDGF-AB, and PDGF-BB, are formed by homodimerization or heterodimerization of the PDGF A-chain and B-chain, which are separate gene products(4, 5, 6, 7) . The activities of PDGF are mediated by two separate tyrosine kinase domain-containing receptors, the 170-kDa alpha-receptor and the 180-kDa beta-receptor(6, 7, 8) . PDGF receptor activation results from dimerization; in this process, PDGF stabilizes the receptor dimer, allowing autophosphorylation(7, 9, 10) . PDGF-BB can bind to both the alpha-receptor and beta-receptor; however, PDGF-AA binds only the alpha-receptor(5) . Thus, one mechanism underlying the differential responsiveness of cells to PDGF-BB and PDGF-AA may be the independent regulation of expression of the PDGF alpha-receptor and beta-receptor.

PDGF-BB is a strong VSMC mitogen and chemoattractant(4, 11, 12, 13) . The VSMC response to PDGF-AA is less well defined. VSMCs in culture demonstrate minimal mitogenic responses to PDGF-AA, probably reflecting low levels of alpha-receptor expression(12, 13, 14, 15) . There is also evidence that the PDGF alpha-receptor and beta-receptor activate nonidentical signal transduction pathways(10, 16) . In VSMCs from spontaneously hypertensive rats, PDGF-AA stimulates protein synthesis despite the absence of a strong mitogenic response(10, 17) . PDGF-AA inhibits VSMC migration in Boyden's chamber assays, while PDGF-BB promotes migration (18) . Thus, PDGF receptors are not entirely redundant and may differentially regulate VSMC physiology.

Numerous studies have documented the ability of VSMCs in culture to respond to exogenously added cytokines and mediators by regulating expression of PDGF chains and receptors. Exogenous TGF-beta1 down-regulates expression of the PDGF alpha-receptor (19) while increasing synthesis of the PDGF A-chain(19, 20) . Basic fibroblast growth factor (bFGF) is the only cytokine reported to up-regulate PDGF alpha-receptor expression in VSMCs(13) . Other agents reported to increase PDGF A-chain synthesis in VSMCs include angiotensin II(15, 21) , arginine vasopressin(15) , and thrombin(22) .

alpha(2)-Macroglobulin (alpha(2)M) is a large (M(r) 718,000) homotetrameric proteinase inhibitor and cytokine carrier that has been implicated in the regulation of VSMC growth(23) . alpha(2)M exists in at least two well characterized conformations(24) . The native form of alpha(2)M expresses proteinase inhibitory activity, but is not recognized by cellular receptors. When native alpha(2)M reacts with proteinases, it undergoes a major structural rearrangement to form the ``activated'' conformation. The identical conformational change is also induced by reacting native alpha(2)M with small primary amines, such as methylamine, that modify the alpha(2)M thiol ester bonds(25) . Methylamine-modified alpha(2)M (alpha(2)M-MA) and alpha(2)M-proteinase complexes are recognized by alpha(2)M receptors equivalently(24) , bind cytokines similarly(26, 27) , and lack proteinase inhibitory activity(24) , justifying the frequent use of alpha(2)M-MA as a model of the activated alpha(2)M conformation. One alpha(2)M receptor has been characterized and shown to be identical to low density lipoprotein receptor-related protein (LRP)(28, 29) .

alpha(2)M may regulate cellular growth and physiology by at least two mechanisms, the first of which involves cytokine carrier activity. By selectively binding specific cytokines, alpha(2)M may alter the cytokine milieu and thereby alter cellular phenotype(30) . Cytokine binding to alpha(2)M is conformation-specific. TGF-beta2 binds with equal affinity to native alpha(2)M and activated alpha(2)M, while TGF-beta1 and PDGF-BB bind with higher affinity to activated alpha(2)M(30) . In contrast, PDGF-AA does not bind to alpha(2)M at all (31) . In the rat, there are two homologues of human alpha(2)M, a constitutively expressed protein, alpha(1)M, and an acute-phase reactant, alpha(2)M(24) . The two rat alpha-macroglobulins and human alpha(2)M demonstrate similar growth factor-binding properties(32) .

The second mechanism whereby alpha(2)M may regulate cellular growth and alter cellular phenotype involves direct binding to cellular receptors. In cultured mouse peritoneal macrophages, activated alpha(2)M induces rapid signal transduction responses that have been attributed to a receptor other than LRP(33, 34) . In rat VSMCs, activated alpha(2)M induces a rapid increase in inositol 1,4,5-trisphosphate and a delayed mitogenic response; native alpha(2)M does not cause either response(23, 35) . alpha(2)M derivatives that retain receptor-binding activity but lack cytokine carrier activity increase inositol 1,4,5-trisphosphate levels and DNA synthesis in VSMCs, similarly to activated alpha(2)M(35) , suggesting a mechanism that requires direct binding of alpha(2)M to a VSMC receptor.

Although alpha(2)M is found in the plasma at high concentration (2-5 µM), under normal conditions, transfer of alpha(2)M into the blood vessel wall is probably limited due to its size and acidic (5.4) isoelectric point(24) . In atherosclerosis, endothelial injury allows enhanced penetration of plasma proteins(1) . Monocytes and macrophages synthesize and secrete alpha(2)M(24, 36) , providing a plasma-independent source of this protein in the developing atheroma. Thus, we consider it important to determine how alpha(2)M, in each of its conformations, regulates VSMC physiology. In this investigation, we demonstrate that both native alpha(2)M and activated alpha(2)M increase PDGF alpha-receptor expression by rat VSMCs. This activity is entirely due to the ability of alpha(2)M to bind cytokines secreted by VSMCs. TGF-beta-neutralizing antibody mimics the activity of alpha(2)M, suggesting for the first time that TGF-beta functions to regulate the PDGF alpha-receptor within the context of an autocrine pathway. alpha(2)M regulates this VSMC autocrine pathway and thereby determines cellular responsiveness to PDGF-AA.


MATERIALS AND METHODS

Reagents

Human alpha(2)M was purified from plasma according to the method of Imber and Pizzo(37) . alpha(2)M-MA was prepared by reacting native alpha(2)M with 200 mM methylamine HCl at pH 8.2 for 10 h. alpha(2)M-MA demonstrated increased mobility by nondenaturing PAGE, as expected for activated forms of alpha(2)M(24) . Proteolytic derivatives of alpha(2)M-MA were prepared by digesting alpha(2)M-MA with papain according to the method of Sottrup-Jensen et al.(38) . The products, including an 18-kDa peptide from the C terminus of each alpha(2)M subunit and the residual 600-kDa derivative, were purified by chromatography on Ultrogel AcA-22. The 18-kDa peptide includes the domain that binds LRP (38) and activates the putative alpha(2)M signaling receptor(34) . The 600-kDa derivative binds growth factors, but is not receptor-recognized (38, 39) .

A 142-amino acid recombinant polypeptide corresponding to the C-terminal receptor-binding domain of rat alpha(1)M (rRBD) was prepared according to the method of Salvesen et al.(40) with minor modifications. The rat alpha(1)M cDNA in pcDV1 was obtained from the American Type Culture Collection. Digestion with BamHI yielded a 1.5-kilobase fragment that was used as a template for polymerase chain reaction. A 484-base pair sequence encoding the C-terminal region of the rat alpha(1)M subunit was amplified as described by Salvesen et al.(40) , except for the use of a different 5`-primer, 5`-TTCATATGGAGGCAGAAGGAGAAGCG-3`, in order to introduce an NdeI restriction site. The polymerase chain reaction product was cloned into pET25b(+) and transformed into Escherichia coli BL21(DE3) for expression. rRBD was purified as described previously(40) . rRBD and the papain-derived 18-kDa fragment completely inhibited specific I-alpha(2)M-MA binding to VSMCs; the IC values were 24 and 180 nM for rRBD and the 18-kDa fragment, respectively (data not shown).

Monoclonal antibody 1D11.16, which neutralizes TGF-beta1, TGF-beta2, and TGF-beta3, was obtained from Genzyme Corp. (Cambridge, MA). RC20 anti-phosphotyrosine antibody was obtained from Transduction Laboratories (Lexington, KY). The cDNA probes for the PDGF alpha-receptor and beta-receptor were kindly provided by Dr. D. Bowen-Pope (University of Washington, Seattle).

Radioiodinated Proteins

alpha(2)M was radioiodinated by the IODO-BEAD method as described by the manufacturer (Pierce). Specific activities ranged from 0.1 to 0.4 µCi/µg. I-PDGF-AA (150-170 µCi/µg) was obtained from Biomedical Technologies (Stoughton, MA).

Vascular Smooth Muscle Cell Culture

VSMCs were isolated from Sprague-Dawley rat aortas by enzymatic digestion as described previously(23) . Cells were cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium (Life Technologies, Inc.) and Ham's F-12 medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT) and L-glutamine (0.68 mM). At subconfluence, cells were passaged with 0.05% trypsin, 0.02% EDTA (Life Technologies, Inc.). Cultures were maintained humidified at 37 °C in 5% CO(2), 95% air. For experiments, VSMCs were plated at 3 times 10^3 cells/cm^2 and grown to confluence. The cells were then growth-arrested by culturing for 4 days in defined serum-free medium (SFM) containing a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with glutamine (0.68 mM), insulin (500 nM), transferrin (5 µg/ml), ascorbic acid (0.2 mM), and selenium (38 nM). This medium has been shown to maintain VSMCs in a quiescent noncatabolic state and to promote expression of VSMC-specific contractile proteins(41) .

RNA Isolation and Northern Blot Analyses

Total RNA was isolated from VSMC cultures using Trizol reagent (Life Technologies, Inc.) according to the manufacturer's instructions. Equal amounts of RNA (20 µg) were denatured with glyoxal, subjected to 1% agarose gel electrophoresis, and electrotransferred to Zeta-Probe membranes (Bio-Rad). cDNA probes for the PDGF alpha-receptor, the PDGF beta-receptor, and glyceraldehyde-3-phosphate dehydrogenase were labeled with [alpha-P]dCTP (DuPont NEN) using the random primers DNA labeling system (Life Technologies, Inc.) and hybridized with the membranes at 42 °C. The membranes were washed twice with 5 times SSPE, 1% SDS at 22 °C and twice with 0.1 times SSPE, 1% SDS at 65 °C. Specific hybridization was quantitated by PhosphorImager analysis or by exposure to x-ray film (X-Omat AR, Eastman Kodak Co.).

Binding of I-PDGF-AA to VSMCs

Quiescent VSMC cultures were incubated with native alpha(2)M, alpha(2)M-MA, other alpha(2)M derivatives, or antibody 1D11.16 for 10 h at 37 °C. The cells were then chilled to 4 °C and washed with ice-cold binding medium (Ham's F-12 medium supplemented with 25 mM HEPES, 0.25% (w/v) bovine serum albumin, 1 mM CaCl(2), pH 7.4). Different concentrations of I-PDGF-AA, with or without a 50-fold molar excess of nonradiolabeled PDGF-AA (Promega, Madison, WI), were added to the cultures. Incubations were conducted on an oscillating platform for 4 h, a time predetermined to establish apparent equilibrium. The cells were then washed twice with ice-cold binding medium and twice with ice-cold 20 mM sodium phosphate, 150 mM NaCl, 1 mM CaCl(2), and 0.25% (w/v) bovine serum albumin, pH 7.4. Cell-associated radioactivity was recovered by solubilizing the cells for 5 min in 1% Triton X-100, 0.25% (w/v) bovine serum albumin and measured in a -counter. Cellular protein was determined in parallel cultures for each experiment using the bicinchoninic acid assay (Sigma). The protein detected in each well deviated from the mean by an average of 4-9% in reported experiments. Specific binding was determined by subtracting the radioactivity bound in the presence of nonradiolabeled PDGF-AA from that measured in the absence of nonradiolabeled PDGF-AA. Binding isotherms were fit to the equation for a rectangular hyperbola by nonlinear regression. The same data were analyzed by Scatchard transformation. The reported K(D) and B(max) values represent the means ± S.E.

Phosphotyrosine Western Blots

Quiescent VSMCs were incubated with alpha(2)M-MA for 6, 10, or 20 h; with native alpha(2)M for 10 h; or with the 600-kDa derivative for 10 h. The cultures were then equilibrated in 1 mM sodium orthovanadate in 1:1 Dulbecco's modified Eagle's medium/Ham's F-12 medium at 37 °C for 15 min. Cells were stimulated with PDGF-AA for 0, 5, or 8 min and then solubilized in 1% Nonidet P-40, 0.25% (w/v) sodium deoxycholate, 50 mM Tris-HCl, 150 mM NaCl, 1 mM EGTA, 1 mM sodium orthovanadate, 200 nM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml E-64, 70 µg/ml aprotinin, 0.7 mM benzamidine, 10 µg/ml soybean trypsin inhibitor, 60 µg/ml leupeptin, pH 7.4. Equal amounts of solubilized cellular protein were subjected to SDS-PAGE under reducing conditions on 4-12% gradient gels and electrotransferred to nitrocellulose (Immobilon-NC, Millipore Corp., Bedford, MA). Membranes were probed with a 1:10,000 dilution of RC20 anti-phosphotyrosine antibody. Antibody binding to immobilized proteins was detected by enhanced chemiluminescence (Amersham Corp.).

alpha(2)M Activation in VSMC Cultures

Native I-alpha(2)M (280 nM) was incubated for 10 h at 37 °C with quiescent VSMCs or in wells containing no cells. The culture conditions were identical to those used in the PDGF-AA binding experiments. At the end of the incubation, medium was gently separated from cells and from the wells without cells. Radioactivity recovery in the medium was determined. The percentage of the recovered radioactivity that was precipitable in ice-cold 10% trichloroacetic acid was measured. Samples of medium from the VSMC cultures and from wells without cells were analyzed by nondenaturing PAGE according to the method of Van Leuven et al.(42) . In this PAGE system, alpha(2)M that is activated by proteinase or primary amines demonstrates increased mobility.

bFGF Copurification with alpha(2)M

I-bFGF (15 µCi) was incubated with 100 ml of fresh human plasma. The concentration of bFGF was 100 pM. After 30 min, the plasma was subjected to our standard alpha(2)M purification method, the steps of which are as follows: (i) dialysis against 20 volumes of deionized H(2)O for 4 h with three changes, (ii) chromatography on zinc-Sepharose, and (iii) chromatography on Ultrogel AcA-22. After water dialysis, the plasma was subjected to centrifugation at 600 times g for 5 min to remove fibrinogen and other precipitated proteins. The soluble plasma components were then dialyzed against 0.1 M sodium phosphate, 0.8 M NaCl, pH 6.5, for chromatography. The plasma was applied to the zinc-Sepharose column, and the resin was washed with 4 column volumes of 0.1 M sodium phosphate, 0.8 M NaCl, pH 6.5, followed by 4 column volumes of 20 mM sodium phosphate, 150 mM NaCl, pH 6.0. alpha(2)M was eluted with a continuous gradient formed by 20 mM sodium phosphate, 150 mM NaCl, pH 6.0 (lead buffer), and 10 mM sodium acetate, 150 mM NaCl, pH 5.0. alpha(2)M elutes at pH 5.35. At each step of the procedure, recovery of I-bFGF was determined by measuring radioactivity.

[^3H]Thymidine Incorporation

Relative rates of DNA synthesis were compared by measuring [^3H]thymidine incorporation into trichloroacetic acid-precipitable material. VSMC cultures were incubated with native alpha(2)M for 10 h or maintained in SFM without alpha(2)M for the same time. The cultures were then washed and treated with 0.6 nM PDGF-AA for 24 h. [^3H]Thymidine (2 µCi/ml) was added for an additional 24 h. The cultures were washed with phosphate-buffered salt solutions and with trichloroacetic acid as described(23) . Radioactivity was then recovered in NaOH and quantitated in a scintillation counter.


RESULTS

Activated alpha(2)M Up-regulates PDGF alpha-Receptor mRNA

alpha(2)M may modulate cellular activities by binding cytokines (30) or by direct interaction with cellular receptors(33) . Since only activated alpha(2)M binds cellular receptors and many cytokines bind with higher affinity to activated alpha(2)M, initial experiments were conducted with alpha(2)M-MA. Quiescent VSMCs were incubated with alpha(2)M-MA (280 nM) in SFM. RNA was harvested at different times, ranging from 0.5 to 24 h. As shown in the representative experiment (Fig. 1A), a low level of PDGF alpha-receptor mRNA was detected in the untreated cells. In response to alpha(2)M-MA, PDGF alpha-receptor mRNA levels increased with time. PDGF beta-receptor mRNA levels were not significantly altered by alpha(2)M-MA in the same time period (n = 3; representative experiment shown in Fig. 1B).


Figure 1: Regulation of the PDGF alpha-receptor and beta-receptor by alpha(2)M-MA. VSMCs were incubated with 280 nM alpha(2)M-MA in SFM at 37 °C for the specified times. Total RNA was isolated and subjected to Northern blot analysis. A shows a representative blot probed for PDGF alpha-receptor mRNA. B shows the same blot probed for PDGF beta-receptor mRNA. C shows a composite of four separate experiments. In each experiment, the PDGF alpha-receptor mRNA level was quantitated by PhosphorImager analysis and standardized for load by comparison to glyceraldehyde-3-phosphate dehydrogenase mRNA levels. Results represent the means ± S.E. kb, kilobases.



Fig. 1C shows a composite of results from four separate experiments examining changes in PDGF alpha-receptor mRNA levels in response to alpha(2)M-MA by Northern blot analysis. The results are standardized for load by comparison with glyceraldehyde-3-phosphate dehydrogenase mRNA. By 6 h, PDGF alpha-receptor mRNA was increased by >3-fold. The level of alpha-receptor mRNA was then stable for up to 24 h. In control experiments, PDGF alpha-receptor mRNA levels were unchanged when VSMCs were maintained for up to 24 h in SFM that was not supplemented with alpha(2)M-MA (data not shown).

alpha(2)M Increases PDGF-AA Binding to VSMC

To determine whether alpha(2)M-MA increases expression of the functional cell-surface PDGF alpha-receptor, binding of I-PDGF-AA to VSMCs was examined. Quiescent VSMCs were incubated with 280 nM alpha(2)M-MA for 10 h or in SFM without alpha(2)M. Radioligand binding was studied at 4 °C. Fig. 2shows that specific and saturable binding of I-PDGF-AA was demonstrated in both the alpha(2)M-MA-treated and untreated cultures (representative experiment). Table 1summarizes the binding parameters derived from four separate experiments. The K(D) was unchanged by treating the cells with alpha(2)M-MA. In contrast, alpha(2)M-MA increased the maximum PDGF-AA-binding capacity (B(max)) by 4.8-fold. These results indicate that alpha(2)M-MA increases expression of the functional PDGF alpha-receptor within the plasma membrane without changing the affinity of the receptor for PDGF-AA.


Figure 2: Specific binding of I-PDGF-AA to VSMCs treated with alpha(2)-MA. VSMCs were incubated with 280 nM alpha(2)M-MA in SFM at 37 °C for 10 h. The cells were then chilled to 4 °C, and binding of I-PDGF-AA was examined. A shows specific PDGF-AA binding isotherms for cells treated with alpha(2)M-MA (circle) and untreated control cultures (bullet). B shows the Scatchard transformations for the same data.





Activated alpha(2)M Increases the VSMC Response to PDGF-AA

The PDGF alpha-receptor is a tyrosine kinase; receptor ligation results in receptor autophosphorylation and phosphorylation of other cellular proteins(5) . Tyrosine phosphorylation is therefore a sensitive index of cellular response to PDGF-AA. Quiescent VSMCs were incubated with alpha(2)M-MA for 6, 10, or 20 h and then stimulated with 3 nM PDGF-AA for 0, 5, or 8 min. Equal amounts of solubilized cellular protein were analyzed by phosphotyrosine Western blotting. As shown in Fig. 3, VSMCs that were not treated with alpha(2)M-MA responded to PDGF-AA. Within 5 min, a number of bands showed increased tyrosine phosphorylation compared with the base-line state (time 0). The 170-kDa band (marked with an arrow) was apparent only after PDGF-AA exposure; this band corresponds to the mass of the PDGF alpha-receptor.


Figure 3: Tyrosine phosphorylation of VSMC proteins in response to PDGF-AA. VSMC cultures were treated with alpha(2)M-MA for the indicated times. The control cultures were not alpha(2)M-MA-treated. The cells were then exposed to PDGF-AA at 37 °C for 0 (no exposure), 5, or 8 min. Reactions were terminated by the addition of ice-cold solubilization buffer. Cellular protein concentrations were assayed, and equal amounts (100 µg) were loaded in each lane of the gel. Phosphotyrosine-containing proteins were detected by Western blotting. The arrow marks the migration of the apparent PDGF alpha-receptor autophosphorylation product.



VSMC cultures that were treated with alpha(2)M-MA, but not stimulated with PDGF-AA, showed no consistent change in tyrosine phosphorylation pattern (n = 3). This result indicates that alpha(2)M-MA does not have an independent sustained effect on VSMC tyrosine phosphorylation at 6-20 h. Furthermore, the level of PDGF-AA synthesized by VSMCs is insufficient to cause a detectable increase in alpha-receptor phosphorylation in the alpha(2)M-MA-treated cultures.

Upon stimulation with exogenous PDGF-AA, alpha(2)M-MA-treated cells demonstrated a pattern of tyrosine phosphorylation similar to that observed in the control cultures; however, the magnitude of the response was substantially increased. The 170-kDa species showed the most notable increase in band intensity. Optimal responsiveness to PDGF-AA was observed at 6 and 10 h following addition of alpha(2)M-MA. At 20 h, the response to PDGF-AA was somewhat decreased, but still greater than that observed in control cultures (no alpha(2)M-MA treatment).

Mechanism for alpha(2)M-induced Up-regulation of the PDGF alpha-Receptor

I-PDGF-AA binding provided a sensitive and quantitative index of alpha-receptor expression by VSMCs treated with alpha(2)M-MA. Therefore, this technique was used to probe the mechanism responsible for the activity of alpha(2)M-MA. Since alpha(2)M-MA increased the B(max) for PDGF-AA without affecting the K(D), changes in PDGF alpha-receptor expression were detected by examining binding of a single I-PDGF-AA concentration (0.2 nM). As shown in Fig. 4, I-PDGF-AA binding was increased by native alpha(2)M and by the 600-kDa derivative. The concentration of native alpha(2)M chosen for this experiment (280 nM) was <10% of the plasma alpha(2)M concentration(24) . Since native alpha(2)M and the 600-kDa derivative do not bind to alpha(2)M receptors, the increase in PDGF alpha-receptor expression must have resulted from the cytokine-binding activity of alpha(2)M. To confirm this, VSMCs were treated with the proteolytically derived 18-kDa fragment or with the corresponding recombinant preparation, rRBD. These derivatives bind to alpha(2)M receptors and mimic the receptor-binding activities of activated alpha(2)M, without binding cytokines(34, 35, 38, 40) . Neither preparation increased I-PDGF-AA binding to VSMCs. Table 2summarizes the properties of the various alpha(2)M derivatives and their effects on PDGF-AA binding. We hypothesized that alpha(2)M affects PDGF-AA binding to VSMCs by inhibiting VSMC-secreted cytokine(s) that normally down-regulate PDGF alpha-receptor expression and/or by potentiating the activity of VSMC-secreted cytokines that up-regulate PDGF alpha-receptor expression.


Figure 4: Mechanism for alpha(2)M-induced up-regulation of the PDGF alpha-receptor. VSMCs were incubated with SFM (control), 280 nM alpha(2)M-MA, 280 nM native alpha(2)M, 280 nM 600-kDa derivative, 250 nM 18-kDa fragment, or 250 nM rRBD at 37 °C for 10 h. The cells were then chilled to 4 °C, and I-PDGF-AA (0.2 nM) was added to each well (with or without nonradiolabeled PDGF-AA). Specific I-PDGF binding was determined.





alpha(2)M Activation in VSMC Cultures

Formation of activated alpha(2)M in vivo depends on local production of extracellular proteinases(24) . Furthermore, accumulation of activated alpha(2)M may be limited by cells that express LRP(24, 29) . Thus, although activated alpha(2)M binds many cytokines with higher affinity than native alpha(2)M(30) , the likelihood that alpha(2)M will accumulate at high concentration in the VSMC microenvironment in vivo is much greater for the native form. We therefore undertook experiments to determine whether native alpha(2)M requires activation by VSMC-secreted proteinases in order to regulate PDGF alpha-receptor expression in vitro. I-Labeled native alpha(2)M (280 nM) was incubated with VSMCs or in wells without cells for 10 h at 37 °C. Total radioactivity recovery in the medium was equivalent in the VSMC cultures and in the wells without cells (n = 3; data not shown). To determine whether a fraction of the alpha(2)M was internalized and degraded by the cells, as might be expected if the alpha(2)M was activated by cellular proteinases, radioactivity in the medium was subjected to trichloroacetic acid precipitation; 4.6 ± 0.7 and 3.7 ± 0.2% of the radioactivity was trichloroacetic acid-soluble in the VSMC culture medium and in the wells without cells, respectively. The difference was not statistically significant at the p < 0.10 level. Nondenaturing PAGE analysis, with Coomassie staining, of the VSMC culture medium showed that all of the alpha(2)M remained in the native conformation. These results indicate that minimal activation of native alpha(2)M occurs in the VSMC cultures and that the regulation of the PDGF alpha-receptor by native alpha(2)M occurs without prior alpha(2)M activation.

I-bFGF Does Not Copurify with alpha(2)M from Human Plasma

bFGF has been reported to up-regulate PDGF alpha-receptor levels in VSMCs(13) . Dennis et al.(43) showed that bFGF binds to alpha(2)M, but only the activated form. These investigators also demonstrated that bFGF, which is bound to activated alpha(2)M, is inactive. Our laboratory confirmed the work of Dennis et al.(43) , showing that bFGF does not bind to native alpha(2)M with significant affinity(30) ; however, to completely exclude the possibility that bFGF copurifies with human alpha(2)M (a possible explanation for the PDGF alpha-receptor regulation results), I-bFGF was added to fresh human plasma. Recovery of I-bFGF at each step of the purification was determined (Table 3). A substantial fraction of the I-bFGF precipitated during dialysis against deionized H(2)O, a step typically used to decrease the concentration of plasma fibrinogen. Essentially all of the remaining I-bFGF coeluted with the flow-through fraction of the plasma during zinc affinity chromatography. The amount of radioactivity coeluting with alpha(2)M from the zinc-Sepharose column was barely above background and represented at most 0.04% of the initial I-bFGF. Although 2 times 10^7 cpm were present in the initial plasma preparation, insufficient radioactivity was present for detection in elution fractions from the Ultrogel AcA-22 column. These studies confirm that plasma bFGF is not copurified with alpha(2)M by our methods and thus is not responsible for the regulation of PDGF alpha-receptor expression.



PDGF-AA Response in Native alpha(2)M-treated Cultures

Having demonstrated that native alpha(2)M increases PDGF-AA binding to VSMCs without prior activation by cellular proteinases, experiments were conducted to determine whether native alpha(2)M increases VSMC protein phosphorylation in response to PDGF-AA. VSMCs were treated with 280 nM native alpha(2)M for 10 h at 37 °C. Similar incubations were also conducted with the 600-kDa derivative. The VSMC cultures were then stimulated with 3 nM PDGF-AA, and tyrosine phosphorylation of cellular proteins was detected by Western blotting. Native alpha(2)M and the 600-kDa derivative increased the VSMC response to PDGF-AA as measured by tyrosine phosphorylation of a number of bands and primarily the 170-kDa band, which is the putative PDGF alpha-receptor autophosphorylation product (Fig. 5). These studies demonstrate that cytokine binding by native alpha(2)M and the 600-kDa derivative increases VSMC responsiveness to PDGF-AA.


Figure 5: Effects of native alpha(2)M and the 600-kDa derivative on the VSMC response to PDGF-AA. VSMCs were incubated with vehicle (lane A), 280 nM native alpha(2)M (lane B), or 280 nM 600-kDa fragment (lane C) for 10 h at 37 °C. The cells were then exposed to PDGF-AA at 37 °C for 5 min. Reactions were terminated by the addition of ice-cold solubilization buffer. Cellular protein concentrations were assayed, and equal amounts were loaded in each lane of the gel. Phosphotyrosine-containing proteins were detected by Western blotting. The arrow marks the migration of the apparent PDGF alpha-receptor autophosphorylation product.



TGF-beta-neutralizing Antibody Mimics the Activity of alpha(2)M

TGF-beta1 is known to down-regulate PDGF alpha-receptor expression in cultured VSMCs (19) without affecting PDGF beta-receptor expression(44) . Human and rat VSMCs express TGF-beta(21) . Therefore, TGF-beta was considered a likely candidate to be involved in the alpha(2)M-regulated VSMC autocrine loop that controls PDGF alpha-receptor expression. Studies from our laboratory have shown that TGF-beta1 and TGF-beta2 bind to alpha(2)M with moderate and high affinity, respectively(30) . To test the hypothesis that binding of VSMC-secreted TGF-beta could be responsible for the observed increase in PDGF alpha-receptor expression, experiments were performed with TGF-beta-neutralizing antibody. Antibody 1D11.16 (50 µg/ml), which recognizes TGF-beta isoforms 1-3, was incubated with the VSMC cultures for 10 h. I-PDGF-AA binding was then measured. The antibody increased binding of I-PDGF-AA by 6-fold, a response that was equivalent to that measured with alpha(2)M-MA in side-by-side cultures (Table 4). Although these studies do not prove that TGF-beta binding is responsible for the activity of alpha(2)M, the results strongly suggest the involvement of TGF-beta in an autocrine pathway that limits PDGF alpha-receptor expression by VSMCs in culture. Furthermore, these studies demonstrate that extracellular proteins, such as specific antibodies and alpha(2)M, can interfere with this autocrine pathway and significantly alter VSMC phenotype without directly binding to VSMC receptors.



PDGF-AA as a Mitogen in alpha(2)M-treated Cultures

We previously demonstrated that native alpha(2)M does not independently promote [^3H]thymidine incorporation in VSMCs(23, 35) , a result that was confirmed in the present study. Thus, we were able to test whether native alpha(2)M enhances the VSMC mitogenic response to PDGF-AA. In quiescent VSMC cultures that were not pretreated with native alpha(2)M, 0.6 nM PDGF-AA increased [^3H]thymidine incorporation by 107 ± 16% (n = 4) compared with controls (cultures that did not receive alpha(2)M or PDGF-AA). In VSMC cultures that were pretreated with 280 nM native alpha(2)M for 10 h, PDGF-AA increased [^3H]thymidine incorporation by 263 ± 38% (n = 4) compared with controls. The response of native alpha(2)M-pretreated VSMCs to PDGF-AA was significantly greater than that observed with VSMCs that were not alpha(2)M-pretreated (p < 0.05); however, the extent of [^3H]thymidine incorporation was still minimal. Thus, although native alpha(2)M substantially increases the VSMC response to PDGF-AA, as determined by tyrosine phosphorylation, PDGF-AA remains a weak mitogen in the absence of other VSMC growth promoters.


DISCUSSION

In the pericellular spaces, numerous cytokines form a signaling language that determines cellular phenotype and behavior(45) . Because cells have the ability to integrate numerous signals, their overall response reflects a balance between supporting and opposing activities. Extracellular molecules that alter the balance of cytokines in the pericellular spaces will affect cellular phenotype. Examples include soluble receptors for nerve growth factor-beta, tumor necrosis factor-alpha, and colony-stimulating factor-1; cytokine-binding proteins such as insulin-like growth factor-binding protein; components of the extracellular matrix; and alpha(2)M(46) . Compared with other cytokine-binding molecules, alpha(2)M is unique in that it exists in different conformations, each with different affinity for specific cytokines(24, 30) . In its activated conformation, alpha(2)M may also regulate cellular phenotype, independently of cytokines, by direct interaction with cellular receptors(33, 34, 35) . Thus, the full spectrum of activities expressed by alpha(2)M toward a specific cell type may be complex. While our laboratory and others have contributed numerous studies regarding the biochemistry of alpha(2)M/cytokine interactions, the biological implications of such interactions have been largely uncharacterized.

In this investigation, we demonstrate that alpha(2)M regulates expression of the PDGF alpha-receptor by VSMCs in culture. Our experiments with various alpha(2)M derivatives suggest that the regulation of the PDGF alpha-receptor results from the ability of alpha(2)M to bind cytokines. Since the experiments were performed in serum-free medium, the critical interaction must have involved alpha(2)M and one or more cytokines secreted by the VSMCs themselves. Thus, we propose a model in which alpha(2)M interrupts an autocrine regulatory loop whereby VSMC-secreted cytokine(s) ordinarily suppress PDGF alpha-receptor expression. A second less likely explanation for our results is that plasma growth factors were copurified in active form with alpha(2)M and able to increase PDGF alpha-receptor expression despite the rigorous alpha(2)M purification method. This possibility was ruled out experimentally with bFGF, the only cytokine reported to increase PDGF alpha-receptor expression in VSMCs to date(13) . Interleukin-1beta increases PDGF alpha-receptor expression in fibroblasts; however, interleukin-1beta binds exclusively to activated alpha(2)M, and the resulting complex is covalent and inactive unless treated with thioredoxin to dissociate disulfide linkages(47, 48, 49) .

Many biological activities have been described for activated forms of alpha(2)M, which are not duplicated by native alpha(2)M. Bonner et al.(50) showed that alpha(2)M-MA increases PDGF-AA binding to fibroblasts 3-fold, while native alpha(2)M has no effect. Activated alpha(2)M also regulates prostaglandin E(2) synthesis and phorbol 12-myristate 13-acetate-induced superoxide anion production in mouse peritoneal macrophages(51, 52) . In each of these studies, mechanism was not probed; however, the activity of activated alpha(2)M might be explained by cytokines that bind with high affinity only to activated alpha(2)M or by direct binding of activated alpha(2)M to cellular receptors. A unique aspect of the present work is the demonstration of similar activity for native alpha(2)M and activated alpha(2)M. In the plasma, native alpha(2)M is present at high concentration (2-5 µM). In contrast, activated alpha(2)M is typically present at only trace levels due to the efficiency of the LRP-dependent hepatic clearance pathway(24) . Native alpha(2)M is probably the predominant form of alpha(2)M found in the blood vessel wall since formation of activated alpha(2)M depends on generation of local proteinase activity. Furthermore, activated alpha(2)M is rapidly taken up by LRP that is expressed by a number of blood vessel wall cells, including macrophages, fibroblasts, and VSMCs(23, 24, 29) . Thus, we propose that native alpha(2)M is the primary form of the protein to consider as a potential regulator of PDGF alpha-receptor expression in vivo and in serum-supplemented cell culture medium.

Battegay et al.(19) demonstrated that exogenous TGF-beta decreases expression of the PDGF alpha-receptor in VSMCs. alpha(2)M binds TGF-beta1 with moderate affinity (K(D) = 300 and 80 nM for native alpha(2)M and alpha(2)M-MA, respectively) and TGF-beta2 with high affinity (K(D) = 10 and 13 nM for native alpha(2)M and alpha(2)M-MA, respectively)(30) . Thus, we considered TGF-beta isoforms as candidates for the VSMC autocrine loop that regulates PDGF alpha-receptor expression. TGF-beta-neutralizing antibody increased PDGF-AA binding to VSMCs comparably to alpha(2)M. This result extends the work of Battegay et al.(19) by demonstrating that TGF-beta regulates VSMC PDGF alpha-receptor expression not only when added exogenously, but within the context of an autocrine loop. Although the spectrum of cytokines that interact with alpha(2)M in VSMC cultures may be complex, the TGF-beta-binding activity of alpha(2)M is sufficient to account for the observed regulation of PDGF alpha-receptor expression.

Transfection of colon carcinoma cells with an antisense expression construct for TGF-beta increases anchorage-independent growth and reduces integrin alpha(5) expression, suggesting a role for autocrine TGF-beta activity in these processes(53, 54) . Autocrine TGF-beta activity may also be important in regulating macrophage nitric oxide synthesis(55) . Thus, autocrine TGF-beta influences cellular functions other than PDGF alpha-receptor expression and cell types other than VSMCs. We propose that alpha(2)M has the potential to regulate each of these processes through the mechanism that is described here.

Activated forms of alpha(2)M are weak mitogens that combine with TGF-beta1 to induce synergistic delayed mitogenic responses (23, 35) . The mitogenic activity of activated alpha(2)M does not appear to be due to cytokine carrier activity, but instead to direct interaction with a VSMC receptor(35) . Native alpha(2)M is inactive as an independent mitogen and does not affect the mitogenic activity of TGF-beta1(22, 35) . Therefore, increased expression of the PDGF alpha-receptor cannot have been responsible for the previously observed synergistic, growth-promoting activities of activated alpha(2)M and TGF-beta1. In this study, we obtained evidence for an increased mitogenic response to PDGF-AA in VSMCs treated with native alpha(2)M. However, despite a 4.8-fold increase in PDGF-AA-binding capacity, PDGF-AA remained a weak VSMC mitogen. Thus, the functional consequences of VSMC PDGF alpha-receptor up-regulation remain to be determined. One obvious possibility is that PDGF-AA will enhance the VSMC response to other mitogenic stimuli to a greater degree after up-regulation of the alpha-receptor. PDGF-AA may also affect other important VSMC parameters, such as protein synthesis and cellular migration(10, 17, 18) .

PDGF isoforms play an important role in the development of atherosclerosis; PDGF-AA, which is synthesized by VSMCs, has been implicated in this process(1, 2, 3, 4, 5) . PDGF alpha-receptor mRNA is readily detected in extracts of total RNA from normal rat carotid arteries(56) . Thus, it is possible that the VSMC autocrine pathway by which TGF-beta regulates PDGF alpha-receptor expression is more effective in cell culture than in vivo. If this is correct, then the response of VSMCs in culture to PDGF-AA may be artificially reduced. In rat carotid arteries injured by balloon angioplasty, TGF-beta1 levels are increased within 24 h(57) ; however, PDGF alpha-receptor expression decreases after 2 weeks(56) . TGF-beta in the neointima is produced primarily by VSMCs (57) and may be at least partially responsible for the loss of the PDGF alpha-receptor. Whether the lag phase between up-regulation of TGF-beta1 expression and loss of the PDGF alpha-receptor reflects the TGF-beta-neutralizing activity of alpha(2)M is an interesting point for future investigation.

In summary, this study has demonstrated an autocrine pathway by which PDGF alpha-receptor expression is regulated in cultured VSMCs. While more than one cytokine may be involved, isoforms of the TGF-beta family are strongly implicated by neutralizing antibody experiments. alpha(2)M functions as a regulator of the pathway by limiting delivery of VSMC cytokines to cellular receptors, thereby controlling the level of PDGF alpha-receptor expression without directly binding to VSMC receptors. Together, the components of this pathway may be important in modulating VSMC phenotype and cellular response to PDGF-AA.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants CA-53462 and HL-19242. 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.

§
Supported in part by Medical Scientist Training Program Grant GM 07267.

To whom correspondence should be addressed: Depts. of Pathology and Biochemistry, University of Virginia Health Sciences Center, P. O. Box 214, Charlottesville, VA 22908. Tel.: 804-924-9192; Fax: 804-924-8060; slg2t@virginia.edu.

(^1)
The abbreviations used are: VSMC, vascular smooth muscle cell; PDGF, platelet-derived growth factor; TGF-beta, transforming growth factor-beta; bFGF, basic fibroblast growth factor; alpha(2)M, alpha(2)-macroglobulin; alpha(1)M, alpha(1)-macroglobulin; alpha(2)M-MA, methylamine-modified alpha(2)M; LRP, low density lipoprotein receptor-related protein; PAGE, polyacrylamide gel electrophoresis; rRBD, recombinant receptor-binding domain of alpha(2)M; SFM, defined serum-free medium.


REFERENCES

  1. Ross, R. (1993) Nature 362, 801-809 [Medline]
  2. Jackson, C. L., and Schwartz, S. M. (1992) Hypertension (Dallas) 20, 713-736
  3. Raines, E. W., and Ross, R. (1993) Br. Heart J. 69, S30-S37 [Medline]
  4. Ross, R., Raines, E. W., and Bowen-Pope, D. F. (1986) Cell 46, 155-169 [Medline]
  5. Heldin, C.-H. (1992) EMBO J. 11, 4251-4259 [Medline]
  6. Westermark, B., and Heldin, C.-H. (1993) Acta Oncol. (Stockh.) 32, 101-105
  7. Claesson-Welsh, L. (1994) J. Biol. Chem. 269, 32023-32026 [Medline]
  8. Seifert, R. A., Hart, C. E., Philips, P. E., Forstrom, J. W., Ross, R., Murray, M. J., and Bowen-Pope, D. F. (1989) J. Biol. Chem. 264, 8771-8778 [Medline]
  9. Heldin, C.-H., Ernlund, A., Rorsman, C., and Ronnestrand, L. (1989) J. Biol. Chem. 264, 8905-8912 [Medline]
  10. Jensen, R. A., Beeler, J. F., Heidaran, M. A., and LaRochelle, W. J. (1992) Biochemistry 31, 10887-10892 [Medline]
  11. Inui, H., Kitami, Y., Tani, M., Kondo, T., and Inagami, T. (1994) J. Biol. Chem. 269, 30546-30552 [Medline]
  12. Grainger, D. J., Witchell, C. M., Weissberg, P. L., and Metcalfe, J. C. (1994) Cardiovasc. Res. 28, 1238-1242 [Medline]
  13. Schollmann, C., Grugel, R., Tatje, D., Hoppe, J., Folkman, J., Marme, D., and Weich, H. A. (1992) J. Biol. Chem. 267, 18032-18039 [Medline]
  14. Inui, H., Kitami, Y., Kondo, T., and Inagami, T. (1993) J. Biol. Chem. 268, 17045-17050 [Medline]
  15. Stouffer, G. A., Shimizu, R. T., Turla, M. B., and Owens, G. K. (1993) Am. J. Physiol. 264, C390-C395
  16. Matsui, T. (1991) Jpn. Circ. J. 55, 1027-1035 [Medline]
  17. Inui, H., Kondo, T., and Inagami, T. (1992) Biochem. Biophys. Res. Commun. 188, 524-530 [Medline]
  18. Koyama, N., Morisaki, N., Saito, Y., and Yoshida, S. (1992) J. Biol. Chem. 267, 22806-22812 [Medline]
  19. Battegay, E. J., Raines, E. W., Seifert, R. A., Bowen-Pope, D. F., and Ross, R. (1990) Cell 63, 515-524 [Medline]
  20. Majack, R. A., Majesky, M. W., and Goodman, L. V. (1990) J. Cell Biol. 111, 239-247 [Medline]
  21. Weber, H., Taylor, D. S., and Molloy, S. J. (1994) J. Clin. Invest. 93, 788-798 [Medline]
  22. Kanthou, C., Parry, G., Wijelath, E., Kakkar, V. V., and Demoliou-Mason, C. (1992) FEBS Lett. 314, 143-148 [Medline]
  23. Stouffer, G. A., LaMarre, J., Gonias, S. L., and Owens, G. K. (1993) J. Biol. Chem. 268, 18340-18344 [Medline]
  24. Sottrup-Jensen, L. (1987) in The Plasma Proteins (Putnam, F. W., ed) Vol. 5, pp. 192-291, Academic Press, Inc., Orlando, FL
  25. Gonias, S. L., Reynolds, J. A., and Pizzo, S. V. (1982) Biochim. Biophys. Acta 705, 306-314
  26. Hall, S. W., LaMarre, J., Marshall, L. B., Hayes, M. A., and Gonias, S. L. (1992) Biochem. J. 281, 569-575 [Medline]
  27. Crookston, K. P., Webb, D. J., LaMarre, J., and Gonias, S. L. (1993) Biochem. J. 293, 443-450 [Medline]
  28. Moestrup, S. K., and Gliemann, J. (1989) J. Biol. Chem. 264, 15574-15577 [Medline]
  29. Strickland, D. K., Ashcom, J. D., Williams, S., Burgess, W. H., Migliorini, M., and Argraves, W. S. (1990) J. Biol. Chem. 265, 17401-17404 [Medline]
  30. Crookston, K. P., Webb, D. J., Wolf, B. B., and Gonias, S. L. (1994) J. Biol. Chem. 269, 1533-1540 [Medline]
  31. Bonner, J. C., and Osornio-Vargas, A. R. (1995) J. Biol. Chem. 270, 16236-16242 [JBC][Medline]
  32. Webb, D. J., Crookston, K. P., Figler, N. L., LaMarre, J., and Gonias, S. L. (1995) Biochem. J. 312, 579-586
  33. Misra, U. K., Chu, C. T., Rubenstein, D. S., Gawdi, G., and Pizzo, S. V. (1993) Biochem. J. 290, 885-891 [Medline]
  34. Misra, U. K., Chu, C. T., Gawdi, G., and Pizzo, S. V. (1994) J. Biol. Chem. 269, 12541-12547 [Medline]
  35. Webb, D. J., Hussaini, I. M., Weaver, A. M., Atkins, T. L., Chu, C. T., Pizzo, S. V., Owens, G. K., and Gonias, S. L. (1996) Eur. J. Biochem. , in press
  36. Hovi, T., Mosher, D. F., and Vaheri, A. (1977) J. Exp. Med. 145, 1580-1589 [Medline]
  37. Imber, M. J., and Pizzo, S. V. (1981) J. Biol. Chem. 256, 8134-8139 [Medline]
  38. Sottrup-Jensen, L., Gliemann, J., and Van Leuven, F. (1986) FEBS Lett. 205, 20-24 [Medline]
  39. LaMarre J., Hayes, M. A., Wollenberg, G. K., Hussaini, I., Hall, S. W., and Gonias, S. L. (1991) J. Clin. Invest. 87, 39-44 [Medline]
  40. Salvesen, G., Quan, L. T., Enghild, J. J., Snipas, S., Fey, G. H., and Pizzo, S. V. (1992) FEBS Lett. 313, 198-202 [Medline]
  41. Libby, P., and O'Brien, K. V. (1983) J. Cell. Physiol. 115, 217-223 [Medline]
  42. Van Leuven, F., Cassiman, J.-J., and Van den Berghe, H. (1981) J. Biol. Chem. 256, 9016-9022 [Medline]
  43. Dennis, P. A., Saksela, O., Harpel, P., and Rifkin, D. B. (1989) J. Biol. Chem. 264, 7210-7216 [Medline]
  44. Agrotis, A., Saltis, J., and Bobik, A. (1994) Clin. Exp. Pharmacol. Physiol. 21, 145-148 [Medline]
  45. Sporn, M. B., and Roberts, A. B. (1988) Nature 332, 217-218 [Medline]
  46. Flaumenhaft, R., and Rifkin, D. B. (1992) Mol. Biol. Cell 3, 1057-1065 [Medline]
  47. Lindroos, P. M., Coin, P. G., Osornio-Vargos, A. R., and Bonner, J. C. (1996) Am. J. Respir. Cell Mol. Biol. , in press
  48. Borth, W., and Luger, T. A. (1989) J. Biol. Chem. 264, 5818-5825 [Medline]
  49. Borth, W., Scheer, B., Urbansky, A., Luger, T. A., and Sottrup-Jensen, L. (1990) J. Immunol. 145, 3747-3754 [Medline]
  50. Bonner, J. C., Badgett, A., Hoffman, M., and Lindroos, P. M. (1995) J. Biol. Chem. 270, 6389-6395 [Medline]
  51. Hoffman, M., Pizzo, S. V., and Weinberg, J. B. (1988) Agents Actions 25, 360-367
  52. Hoffman, M., Feldman, S. R., and Pizzo, S. V. (1983) Biochim. Biophys. Acta 760, 421-423
  53. Wu, S., Theodorescu, D., Kerbel, R. S., Wilson, J. K. V., Mulder, K. M., Humphrey, L. E., and Brattain, M. G. (1995) J. Cell Biol. 116, 187-196 [Medline]
  54. Wang, D., Zhou, G, Birkenmeier, T. M., Gong, J., Sun, L., and Brattain, M. G. (1995) J. Biol. Chem. 270, 14154-14159 [JBC][Medline]
  55. Lysiak, J. J., Hussaini, I. M., Webb, D. J., Glass, W. F., Allietta, M. A., and Gonias, S. L. (1995) J. Biol. Chem. 270, 21919-21927 [JBC][Medline]
  56. Majesky, M. W., Reidy, M. A., Bowen-Pope, D. F., Hart, C. E., Wilcox, J. N., and Schwartz, S. M. (1990) J. Cell Biol. 111, 2149-2158 [Medline]
  57. Majesky, M. W., Lindner, V., Twardzik, D. R., Schwartz, S. M., and Reidy, M. A. (1991) J. Clin. Invest. 88, 904-910 [Medline]

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