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J. Biol. Chem., Vol. 275, Issue 24, 17933-17936, June 16, 2000

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ACCELERATED PUBLICATION
Activation of Platelet-transforming Growth Factor -1 in the Absence of Thrombospondin-1^*

Mustapha Abdelouahed, Anna Ludlow^§, Georg Brunner^¶, and Jack Lawler

From the  Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, the ^¶ Department of Cancer Research, Fachklinik Hornheide, University of Münster, D-48157 Münster, Germany, and ^§ the School of Biological Sciences, Division of Cells, Immunology and Development, University of Manchester, Manchester M13 9PT, United Kingdom

Received for publication, February 28, 2000, and in revised form, April 5, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Thrombospondin-1 (TSP-1) has been shown to bind and activate transforming growth factor-1 (TGF-1). This observation raises the possibility that TSP-1 helps to sequester TGF-1 in platelet  granules and activates TGF-1 once both proteins are secreted. Herein, we evaluated the level of active and latent TGF-1 in the plasma and in the supernatant of thrombin-treated platelets from TSP-1 null and wild-type mice on two genetic backgrounds (C57BL/6 and 129Sv). The plasminogen activator inhibitor-1/luciferase bioassay and an immunological assay were used to determine active and latent TGF-1. No significant differences were observed in the levels of active and latent TGF-1 in the supernatant of thrombin-treated platelets from TSP-1 null and wild-type mice. Active and latent TGF-1 were significantly increased in the plasma and platelets of C57BL/6 mice as compared with 129Sv mice. In addition, there was an increase of plasma level of latent TGF-1 in TSP-1 null mice as compared with wild-type mice on the C57BL/6 background but not on the 129Sv background. No active TGF-1 was observed in the plasma of either TSP-1 null and wild-type mice. These data indicate that TSP-1 does not function as a chaperon for TGF-1 during platelet production and does not activate significant quantities of secreted TGF-1 despite a vast excess in the number of TSP-1 molecules as compared with TGF-1 molecules. Because platelet releasates from TSP-1 null mice contain active TGF-1, we suggest that other important mechanisms of physiological activation of TGF-1 probably exist in platelets.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Transforming growth factor- (TGF-1, -2, and -3)¹ are mammalian cytokines with a wide range of biological effects (1). They are involved in the regulation of development, proliferation, angiogenesis, inflammation, extracellular matrix production, integrin expression, protease activity, and apoptosis (2). They play a pathologic role in inflammation and fibrotic diseases such as nephrosclerosis (3). Mice null for either, TGF-1, -2, and -3 do not survive beyond a few days or weeks (4-8). Surviving pups of animals null for TGF-1 exhibit dysregulated myelopoiesis and a wasting syndrome characterized by an inflammatory response targeting the heart, lung, pancreas, stomach, liver, and striated muscle that has been attributed to an autoimmune process (9, 10). Overexpression of TGF-1 causes lethality in utero or just after birth (11).

TGF-1 is synthesized by cells in a latent form that must be activated to be recognized by cell-surface receptors and to trigger biological responses (2). Small latent TGF-1 is a dimeric complex of ~100 kDa, composed of two identical chains in which an amino-terminal 278 amino acid latency-associated peptide (LAP) is noncovalently associated with the carboxyl-terminal 112 amino acid active peptides (12). This latent complex is the product of a single gene. Prior to secretion, LAP is enzymatically cleaved from the active peptide, and the integrity and latency of the secreted complex are presumably maintained via electrostatic interactions (13). Latent TGF-1 can exist as a large complex in which it is associated with a latent TGF-1-binding protein (LTBP). LTBP has features in common with extracellular matrix proteins and targets latent TGF-1 to the matrix (2). The latency of TGF-1 is dependent on the presence of LAP; the presence of LTBP is neither necessary nor sufficient for prevention of activation (14).

Physiological mechanisms of activation of TGF-1 are not well understood (12), although proteolytic processing by plasmin, exposure to reactive oxygen species, and binding to _v6 integrin may participate in TGF-1 activation (2, 15). Interaction of latent TGF-1 with thrombospondin-1 (TSP-1) results in activation of latent TGF-1 (16-19). TSP-1 is a trimer of disulfide-linked 180-kDa subunits found at high concentrations in platelet  granules and also produced by a number of other cell types (20). It is an adhesive protein with a number of domains available for binding to cell surface or matrix proteins. TSP-1-deficient mice are viable and exhibit subtle abnormalities in development (21). The adult mice exhibit increased inflammatory cell infiltrates and epithelial cell hyperplasia in the lungs, suggesting that TSP-1 is involved in normal lung homeostasis (21). TSP-1 purified from human platelets has been shown to contain TGF-1 (22). TSP-1 also activates TGF-1 in cell culture assays when added to endothelial cells. The site in TSP-1 responsible for latent TGF-1 activation has been localized to the type 1 repeats, especially the K⁴¹²RFK⁴¹⁵ sequence located between the first and second type 1 repeats of TSP-1 (16). More recently, Ribeiro et al. (23) showed that the TSP-1 sequence KRFK binds LAP through interactions that involve a specific sequence at the amino terminus of LAP (L⁵⁴SKL⁵⁷). The binding of TSP-1 to LAP appears to induce a conformational change that renders the TGF-1 active.

Mechanisms controlling conversion of the latent complex to the active state are key regulators of TGF-1 activity. TSP-1 is the first activator of TGF-1 shown to function in natural, untreated, nondiseased tissues in vivo (24). The previous observations that purified platelet TSP-1 contains associated TGF-1 (16-19), raises the possibility that TSP-1 activates platelet TGF-1 or serves as a carrier for TGF-1 during  granules formation. Here, we evaluated and compared the level of active and latent TGF-1 in the plasma and in platelet  granules of TSP-1 null and wild-type mice using the plasminogen activator inhibitor-1/luciferase bioassay (25) and the commercial TGF-1 enzyme-linked immunosorbent assay (ELISA) kit (Genzyme, Boston, MA). We observed that (i) there is no active TGF-1 in the plasma of both TSP-1 null and wild-type mice, (ii) there is an increase in the level of plasma latent TGF-1 in TSP-1 null C57BL/6 mice compared with wild-type, and (iii) there is no significant difference in the level of active and latent TGF-1 in platelets from TSP-1 null and wild-type mice. Despite the absence of TSP-1 in TSP-1 null mice, active TGF-1 is observed in the platelet releasates. These data suggest that TSP-1 is not the only physiological activator of TGF-1 in platelet  granules and that TSP-1 does not play a role of carrier for TGF-1 during platelet biosynthesis.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Animals-- TSP-1 null animals were generated by homologous recombination in 129Sv-derived ES cells, as described previously (21).

Preparation and Stimulation of Mouse Platelets-- Mice were anesthetized with 2.5% avertin. Blood was drawn by periorbital insertion of a heparinized capillary tube (~1.5 cm in length). Blood from five to eight TSP-1 null and wild-type (C57BL/6 or 129Sv) mice was drawn into tubes containing acid-citrate dextrose and pooled separately. Platelets were isolated by differential centrifugation and washed in pH 6.5 buffer containing 0.102 M NaCl, 3.9 mM K₂HPO₄, 3.9 mM Na₂HPO₄, 22 mM NaH₂PO₄, and 5.5 mM glucose. The platelets were resuspended in 15 mM Tris-HCl (pH 7.6), 0.14 M NaCl, 5 mM glucose, and 2 mM CaCl₂. The platelet counts were determined and platelet concentration was adjusted to 1.8 × 10⁵ platelets/µl. Platelets were activated with human thrombin (0.5 unit/ml; Sigma) for 3 min under constant stirring. Platelet aggregates were centrifuged at 3,000 × g, and the releasates of thrombin-stimulated platelets were collected and stored at 20 °C for further quantification of total and active TGF-1 by both the TGF-1 ELISA kit and the plasminogen activator inhibitor-1/luciferase bioassay (25). In other experiments, thrombin-activated platelets are treated with 10 mM EDTA for 10 min before the collection of the releasate of thrombin-stimulated platelets.

Quantification of Total and Active TGF-1 by ELISA Kit-- Total and active TGF-1 concentration in the plasma and in the supernatant of thrombin-treated platelets from TSP-1 null and wild-type mice were assayed by a sandwich TGF-1 ELISA kit (Genzyme, Boston, MA) according to the manufacturer's specifications. Plasma and supernatant of thrombin-treated platelets were thawed and divided into two pools. In the first pool, acid activation was required to convert latent to active TGF-1 and to record detectable levels of total TGF-1. By contrast, no acid activation is used in the second pool; the TGF-1 levels in the samples are therefore representative of active TGF-1 in the plasma or in the supernatant of thrombin-treated platelets. Plasma (10 µl) and supernatant of thrombin-treated platelets (50 µl) were added to sample diluent with or without 1 N HCl for 60 min at 4 °C followed by neutralization with 1 N NaOH if samples were activated with HCl. Samples were plated on microtiter plates coated with anti-TGF-1 antibody and incubated at 37 °C for 60 min. After vigorous washing, wells were incubated with a second biotin-conjugated anti-TGF-1 antibody, and the peroxidase reaction was initiated. A standard curve was constructed using serial dilutions of human TGF-1 (Genzyme) as standard. TGF-1 levels in samples were compared with known standards and read as nanograms per milliliter.

Quantification of Total and Active TGF-1 by the Plasminogen Activator Inhibitor-1/Luciferase Bioassay-- Total and active TGF-1 concentration in the supernatant of thrombin-treated platelets from TSP-1 null and wild-type mice were assayed using the plasminogen activator inhibitor-1/luciferase (PAI/L) assay, first described by Abe et al. (25). This assay is based on the ability of TGF-1 to induce plasminogen activator inhibitor-1 (PAI-1) expression in mink lung epithelial cells (MLECs) transfected with a construct containing a truncated PAI-1 promoter fused to a firefly luciferase reporter gene. Transfected MLECs were a generous gift from Dr. D. B. Rifkin (New York University Medical Center). Cells were maintained in high glucose (4,500 mg/liter) Dulbecco's modified Eagle's medium (Life Technologies, Inc., Paisley, UK) supplemented with 5% fetal calf serum, 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, and 200 µg/ml Geneticin (G418-sulfate) (Calbiochem-Novabiochem Ltd., Nottingham, United Kingdom (UK)). Cells were cultured in a humid atmosphere at 37 °C, 5% CO₂, and passaged maximally 30 times. As described above, the supernatants of thrombin-treated platelets were divided into two pools. This time, however, the first pool was diluted in serum-free Dulbecco's modified Eagle's medium containing 0.1% pyrogen-poor bovine serum albumin (Pierce & Warriner UK Ltd., Chester, UK) and heat-activated for 10 min at 80 °C. As before, the second pool remained untreated so as to measure the amount of active TGF-1 present. MLECs were trypsinized and washed and the cell density adjusted to 1.6 × 10⁵ cells/ml before plating 100 µl/well into a 96-well tissue culture plate (Falcon, Becton Dickenson, Oxford, UK). Cells were incubated for 3-4 h to allow for optimal attachment to the plastic. Following aspiration of the growth medium from the attached cells, 100 µl of the sample was added in triplicate. Cells and samples were then incubated for 14-16 h at 37 °C, 5% CO₂. Following incubation, all wells were checked microscopically for cell viability before washing twice with 100 µl of phosphate-buffered saline. Cells were then lysed using 100 µl/well of 1 × lysis buffer (Promega, Southampton, UK) and incubated with agitation at room temperature for 20 min. Forty-five microliters of the cell lysates were transferred to an opaque Microlite^TM 1 Microtiter® read plate (Dynex Technologies Ltd., West Sussex, UK). Lysates were analyzed for luciferase activity using an MLX Luminometer (Dynex Technologies Ltd.) following the injection of 110 µl/well of substrate solution (20 mM tricine, 1.07 mM Mg(CO)₃Mg(OH)₂·5H₂O, 2.67 mM MgSO₄, 0.1 mM EDTA, 33.3 mM dithiothreitol, 750 µM ATP, and 800 µM luciferin (Promega, Southampton, UK)). The flash of light obtained upon mixing the lysates with the substrate was recorded as relative light units. The mean values of the triplicates were then converted into concentrations of TGF-1 in picograms per milliliter using a standard curve obtained with human recombinant TGF-1 (R & D Systems, Abingdon, UK).

Statistical Analysis-- Data are expressed as the mean ± S.E. Statistical evaluation of the data was performed using the unpaired t test, considering p values <0.05 as significant.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Since TGF-1 binds to TSP-1 with high affinity (16-19), we hypothesized that TSP-1 may function as a carrier protein for TGF-1. To test this hypothesis, we assayed the quantity of TGF-1 that is secreted from wild-type and TSP-1 null platelets in response to thrombin. As shown in Fig. 1, the level of total TGF-1 is equivalent in the supernatants from the wild-type and TSP-1 null platelets. We have confirmed this result by employing two different assays for TGF-1 and two different strains of TSP-1 null mouse. In our hands, the bioassay consistently gives higher values than the TGF-1 ELISA kit; however, the relative levels of active TGF-1 as compared with total TGF-1 are consistent between the two assays.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Active TGF-1 is present in the supernatant of thrombin-treated platelets from TSP-1 null mice. Mice platelets from TSP-1 null and wild-type mice were activated with 0.5 unit/ml human thrombin for 3 min, platelet suspensions were centrifuged, and platelet supernatants were removed. Total and active TGF-1 were assayed in the supernatant of thrombin-treated platelets from TSP-1 null and wild-type C57BL/6 (A) and 129Sv (B) mice by a TGF-1 ELISA kit and the plasminogen activator inhibitor-1/luciferase bioassay. Results are expressed as the means ± S.E. of duplicate determinations of four to six separate experiments (ELISA) and three to four separate experiments (Bioassay).

TSP-1 reportedly binds to the platelet membrane after secretion when calcium is present (26). Thus, TGF-1 that is complexed with TSP-1 may also become associated with the platelet membrane. If this is the case, then the TGF-1 levels that we measured in the supernatant of the thrombin-treated wild-type platelets may be anomalously low. To determine whether this is the case, we treated the platelets with 10 mM EDTA after thrombin treatment to remove the TSP-1 from the platelet membrane (Fig. 2). Treatment of the platelets with EDTA resulted in an increase in the level of TGF-1 in the supernatants of both the wild-type and the TSP-1 null platelets (Fig. 2). Thus, while there appears to be a calcium-dependent mechanism for the association of TGF-1 to the platelet membrane, it does not require TSP-1. It is known that LAP contains an arginine-glycine-aspartate (RGD) sequence that might function to localize latent cytokine to the cell surface by binding integrins (2). Grainger et al. (27) reported indirect evidence that the RGD sequence in platelet-derived latent TGF-1 may be recognized by platelet integrins. Like several other matrix proteins, human LTBP also contains an RGD sequence; however, there are no reports that this sequence serves as an integrin ligand (2). In wild-type mice, LAP can remain associated with the TSP-1/TGF-1 complex without inhibiting the activity of TSP-1-associated TGF-1 (23).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Treatment of platelets with EDTA increases the level of TGF-1 in the supernatants of both the wild-type and the TSP-1 null platelets. Mice platelets from TSP-1 null and wild-type were activated with 0.5 unit/ml human thrombin for 3 min and treated with 10 mM EDTA for 10 min. Platelet suspensions were centrifuged and platelet supernatants were removed. Total and active TGF-1 were assayed in platelet releasates from TSP-1 null and wild-type C57BL/6 (A) and 129Sv (B) mice by a TGF-1 ELISA kit and the plasminogen activator inhibitor-1/luciferase bioassay. Results are expressed as the means ± S.E. of duplicate determinations of three to five separate experiments (ELISA) and three to four separate experiments (Bioassay).

Surprisingly, we have found that the absence of TSP-1 has no effect on the level of active TGF-1 in the supernatant of thrombin-treated platelets (Figs. 1 and 2). In the lung, pancreas, and liver, TSP-1 null mice exhibit pathologies that are similar to, although generally not as severe as, TGF-1 null mice (21, 24). The abnormalities include epithelial cell hyperplasia and focal inflammation. Treatment of the TSP-1 null mice with the TGF-1-activating peptide KRFK normalizes these abnormalities in the lung and pancreas (24). Furthermore, treatment of wild-type mice with a peptide (LSKL) that inhibits the ability of TSP-1 to activate TGF-1 induces lung and pancreas pathologies similar to those seen in the TSP-1 null mice (24). These data indicate that constitutive activation of TGF-1 by TSP-1 contributes to epithelial homeostasis in some organs. It remains possible that TSP-1 is an important activator of TGF-1 in platelets from wild-type mice and that an alternative pathway is up-regulated in the absence of TSP-1. Since active TGF-1 is present in the supernatant of thrombin-treated TSP-1 null platelets, alternative mechanisms for TGF-1 activation must be present in platelets. These mechanisms may involve the association of TGF-1 with another protein that is present in the -granules or shed from the platelet membrane after activation. Since the platelets are removed shortly after thrombin-treatment, it is unlikely that the activation of TGF-1 is due to the release of a constituent of the lysosomal compartment. It is also unlikely that the amount of thrombin used or the duration of exposure to thrombin would be sufficient to activate significant levels of TGF-1. In fact, human thrombin (0.1, 0.5 and 1 unit/ml) added to both serum and platelet releasate samples for 30 min did not increase the amount of active TGF-1, as compared with non-thrombin-treated samples, demonstrating that thrombin was not responsible for the active TGF-1 detected in our assays (data not shown). An alternative interpretation of the data is that TSP-1 is not a significant activator of TGF-1 in platelets. In wild-type platelets, the majority of TGF-1 is inactive despite a vast excess in the number of TSP-1 molecules as compared with TGF-1 molecules (12, 28). Since the type 1 repeats appear to be important for protein-protein interactions in general, it is possible that TGF-1 binding to TSP-1 is inhibited by the presence of another protein. It is also possible that post-translational modifications of TSP-1 that occur in megakaryocytes inhibit TGF-1 binding. The data clearly show that coexpression of TSP-1 and TGF-1 does not necessarily mean that TGF-1 will be activated via a TSP-1-dependent mechanism. Because TSP-1 and TGF-1 are stored and secreted together from platelet  granules, and because active TGF-1 has a short half-life, an independent spatial and temporal mechanism for regulating TGF-1 activation may be necessary. This mechanism may not be active in epithelial cells because TGF-1 and TSP-1 are constitutively secreted and a basal level of activated TGF-1 is maintained.

We also assayed the levels of TGF-1 in the plasma of wild-type and TSP-1 null mice to determine whether there is a systemic increase in TGF-1 levels that might compensate for the lack of a TSP-1-dependent activation mechanisms that exist in epithelial tissues (21, 24). A statistically significant (p < 0.01) increase in the total TGF-1 was observed in the plasma of the TSP-1 null mice as compared with wild-type mice on the C57BL/6 background (Table I). By contrast, no difference was observed in the plasma levels of TGF-1 in the TSP-1 null mice and the wild-type mice on the 129Sv background. No active TGF-1 was found in the plasma of either TSP-1 null and wild-type mice. Free TGF-1 can interact with and be inactivated by a number of soluble or matrix molecules, including ₂-macroglobulin, decorin, betaglycan, and fucoidan (29, 30). Furthermore, TGF-1 in plasma is found almost exclusively bound to 2-macroglobulin and presumably represents activated TGF-1 that will be cleared by the liver (31). The physiological mechanisms for the increase in the level of latent TGF-1 in TSP-1 null C57BL/6 mice is currently unknown.

View this table: [in this window] [in a new window]   Table I Plasma TGF- 1 in C57BL/6 and 129Sv mice TGF-1 levels in the plasma of C57BL/6 and 129Sv mice were determined by a TGF-1 ELISA kit. Results are expressed as the means ± S.E. of two duplicate determinations of 22-23 separate experiments for C57BL/6 mice and 22-26 separate experiments for 129Sv mice.

In this study, we have shown that the levels of latent and active TGF-1 in the supernatant of thrombin-treated platelets are equivalent in the presence or absence of TSP-1. Thus, TSP-1 does not appear to function as a chaperon for TGF-1 during platelet production. The lack of correlation between TSP-1 expression and the level of active TGF-1 indicates that (i) the ability of TSP-1 to activate TGF-1 is inhibited in wild-type mice and (ii) alternative mechanisms for activation of TGF-1 are present in platelets. Elucidation of the mechanisms underlying each of the observations will have important implications for the regulation of TGF-1 activation in vivo.

	ACKNOWLEDGEMENTS

The technical assistance of Mark Duquette is greatly appreciated. We thank Dr. Daniel Rifkin for the transfected MLECs and Professor Mark W. J. Ferguson for his helpful comments on this manuscript.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant HL28749 from the NHLBI (to J. L.) and by the Biotechnology and Biological Sciences Research Council (BBSRC)-CASE studentship supported by Johnson & Johnson Medical Ltd. (to A. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pathology, Beth Israel Deaconess Medical Center, Research North, Rm. 270C, 99 Brookline Ave., Boston, MA 02215. Tel.: 617-667-1694; Fax: 617-667-3591; E-mail: lawler@mbcrr.harvard.edu.

Published, JBC Papers in Press, April 14, 2000, DOI 10.1074/jbc.C000132200

	ABBREVIATIONS

The abbreviations used are: TGF-1, transforming growth factor-; LAP, latency-associated peptide; LTBP, latent TGF-1-binding protein; TSP-1, thrombospondin-1; ELISA, enzyme-linked immunosorbent assay; MLEC, mink lung epithelial cell.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 275/24/17933    most recent C000132200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Abdelouahed, M. Articles by Lawler, J. Search for Related Content PubMed PubMed Citation Articles by Abdelouahed, M. Articles by Lawler, J. Social Bookmarking                      What's this?