JBC Transcription and Nuclear Factor Monoclonals

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Originally published In Press as doi:10.1074/jbc.M102793200 on August 23, 2001

J. Biol. Chem., Vol. 276, Issue 45, 41668-41674, November 9, 2001
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alpha 2-Macroglobulin: a New Component in the Insulin-like Growth Factor/Insulin-like Growth Factor Binding Protein-1 Axis*

Melissa WestwoodDagger §, John D. Aplin||, Ilse A. Collinge**, Andrew Gill**, Anne WhiteDagger §, and J. Martin GibsonDagger **

From the Dagger  Endocrine Sciences, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, United Kingdom, the § School of Biological Sciences, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, United Kingdom, the || Academic Unit of Obstetrics & Gynaecology, University of Manchester, Research Floor, St. Mary's Hospital, Whitworth Park, Manchester M13 0JH, United Kingdom, and the ** Department of Diabetes & Endocrinology, Hope Hospital, Salford M6 8HD, United Kingdom

Received for publication, March 29, 2001, and in revised form, August 21, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Insulin-like growth factors (IGFs) are crucial for many aspects of development, growth, and metabolism yet control of their activity by IGF-binding proteins (IGFBPs) remains controversial. The effect of IGFBP-1 depends on its phosphorylation status; phosphorylated IGFBP-1 inhibits IGF actions whereas the nonphosphorylated isoform is stimulatory. In order to understand this phenomenon, we purified phosphorylated IGFBP-1 from normal human plasma by immunoaffinity chromatography. Unexpectedly, the resulting preparation enhanced IGF-stimulated 3T3-L1 fibroblast proliferation, due to the presence of a co-purified protein of approx 700 kDa. Matrix-assisted laser desorption ionization-mass spectrometry and Western immunoblotting analysis identified this co-purified protein as alpha 2-macroglobulin (alpha 2M). Anti-alpha 2M antibodies co-immunoprecipitated IGFBP-1 from human plasma and from 125I-IGFBP-1·alpha 2M complexes formed in vitro. The 125I-IGFBP-1/alpha 2M association could be inhibited with excess unlabeled IGFBP-1. Surface plasmon resonance analysis indicated that alpha 2M preferentially associates with the phosphorylated isoform of IGFBP-1 and that when complexed to alpha 2M, IGFBP-1 can still bind IGF-I. These findings have functional significance since alpha 2M protects IGFBP-1 from proteolysis and abrogates the inhibitory effect of phosphorylated IGFBP-1 on IGF-I stimulated 3T3-L1 cell proliferation. We conclude that alpha 2M is a binding protein of IGFBP-1 which modifies IGF-I/IGFBP-1 actions resulting in enhanced IGF effects. In line with its role in regulating the clearance and activity of other growth factors, we predict that alpha 2M has a novel and important role in controlling the transport and biological activity of IGFs.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The insulin-like growth factors (IGFs)1 have a key role in the metabolism, development, growth, and maintenance of many tissues and organs (1). IGF bioavailability is controlled by a number of binding proteins (IGFBPs) and one of these, IGFBP-1, is capable of either inhibiting (2-4) or potentiating (5-9) IGF activity at the cellular level.

IGFBP-1 inhibits IGF actions by competing with the type 1 IGF receptor for IGF binding, however, the mechanism by which IGFBP-1 enhances IGF activity is less certain. Early work to address this phenomenon resulted in the isolation of two IGFBP-1 isoforms from amniotic fluid, which had similar physicochemical properties but markedly different effects on IGF activity (5). IGFBP-1 association with the cell surface was suggested as an explanation of these findings since only the stimulatory isoform was found to bind to cell membranes. It is now known that IGFBP-1 binds to alpha 5beta 1 integrin via its RGD site and disruption of this interaction leads to inhibition of the subsequent cellular response (6). Polymerization of IGFBP-1 was also postulated as a mechanism for enhancing IGF action (7) and this has been confirmed recently (8).

Many studies have also focused on the influence of phosphorylation in relation to IGFBP-1 effects on IGF activity. The inhibitory isoform purified by Busby et al. (5) was subsequently shown to be phosphorylated (9) whereas the stimulatory preparation contained nonphosphorylated IGFBP-1. Highly phosphorylated IGFBP-1, which is the only form found in plasma (10) has a high affinity for IGF-I (9, 11) and can therefore inhibit IGF-I actions by sequestering it from cell surface receptors. Nonphosphorylated IGFBP-1, which has a relatively low affinity for IGF (9, 11) is thought to allow more IGF/IGF receptor interactions and this hypothesis has been supported by numerous in vitro studies (9, 12-14). However, it is unclear whether in vivo alteration of IGFBP-1 phosphorylation status represents an important mechanism for regulating IGF bioavailability in the non-pregnant adult, since non- and lesser phosphorylated isoforms of IGFBP-1 are only present at high concentrations during pregnancy (10, 15).

In the light of the above findings, we were surprised to observe that phosphorylated IGFBP-1 purified from plasma could enhance IGF-I stimulated cell proliferation. Further biochemical analysis led to the discovery that IGFBP-1 in plasma is associated with the homotetrameric glycoprotein alpha 2-macroglobulin (alpha 2M). This paper describes our characterization of the alpha 2M/IGFBP-1 association and its functional impact on the IGF axis.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Purification of IGFBP-1 from Plasma-- Immunoaffinity chromatography was used to isolate IGFBP-1 from normal human plasma. Monoclonal antibody 6303 (a kind gift of Medix Biochemica, Kauniainen, Finland) was coupled to Sephacryl S-300 (16) at 1 mg/ml to form the immunoaffinity matrix. A 10-ml column was equilibrated for 24 h at 4 °C by the application of PBS, 0.25% bovine serum albumin, 0.1% Tween 20 at a flow rate of 5 ml/h.

250 ml of plasma was recirculated through the column for 72 h at a flow rate of 3.75 ml/h, the column was washed with 100 ml of Tris buffer, pH 8.0 (50 mM Tris, 0.5 M NaCl, 0.1% Tween 20), and then the bound peptide was eluted by application of 0.1 M hydrochloric acid. 10 × 1-ml fractions were collected into tubes containing 200 µl of 1 M Tris, pH 9.0, and analyzed for IGFBP-1 by radioimmunoassay (10). Fractions containing >100 µg/liter IGFBP-1 were pooled and concentrated by centrifugation through Centricon 10 filters (Amicon, Stonehouse, Gloucestershire, UK).

Biochemical Characterization of IGFBP-1 Phosphorylation Status-- IGFBP-1 phosphorylation status was determined using our previously described method of immunoprecipitation followed by n-octylglucoside electrophoresis and Western ligand blotting (10). Samples were incubated with anti-IGFBP-1 (6303) antibody at 4 °C overnight and then anti-mouse IgG antibody (Sac Cel; IDS, Tyne & Wear, United Kingdom) was added for 1 h at room temperature. Bound antibody was separated by centrifugation for 10 min and the precipitated proteins were washed in PBS, 0.25% bovine serum albumin, 0.1% Tween 20 prior to resuspension in gel loading buffer. All samples were then boiled for 5 min. Electrophoresis was performed using stacking (4%) and resolving (12%) gels containing 20 mM non-ionic detergent n-octylglucoside. Following overnight transfer onto nitrocellulose membranes, proteins were revealed by incubation with 150,000 cpm/ml 125I IGF-I (4 h at 25 °C) and autoradiography.

Peptides-- alpha 2-Macroglobulin was obtained as a gift from Dr. Claus Oxvig, University of Aarhus, Denmark (17), and also purchased from Sigma. Recombinant human IGFBP-1 and recombinant IGF-I were the kind gift of Dr. V. Quarmby (Genentech Inc, San Francisco, CA). Phosphorylated and nonphosphorylated IGFBP-1 preparations were also purchased from Sigma (Dorset, UK). IGFBP-1 and IGF-I were iodinated to a specific activity of 70 and 100 µCi/µg, respectively.

Protein Digestion and Analysis by MALDI-MS-- The IGFBP-1 preparation purified by immunoaffinity chromatography (10 µl) was analyzed by SDS-PAGE and visualized with a silver stain. Bands of interest were excised from the gel and subjected to digestion with trypsin in accordance with the method of Shevchenko et al. (18). Digests were analyzed by matrix-assisted laser desorption ionization (MALDI)-MS with a VG Tofspec E mass spectrometer. The resulting peptide masses were mapped with the ProFound Internet peptide data base search site.

Immunoprecipitation and Western Immunoblotting-- Samples were incubated with polyclonal anti-alpha 2M (Sigma) or monoclonal anti-IGFBP-1 (6303) antibodies overnight at 4 °C and then 25 µl of protein-A-Sepharose CL-4B (Zymed Laboratories Inc.) for 2 h at room temperature. The immune complexes were pelleted by centrifugation, washed (4 times) with PBS, 1% Triton X-100, 0.1% SDS, 0.25% bovine serum albumin and added to 35 µl of nonreducing SDS loading buffer (0.1 M Tris-Cl, pH 6.8, 2% SDS, 10% glycerol, 0.02% bromphenol blue). Immunoprecipitated proteins were electrophoresed on 4-12% gradient SDS gels, blotted onto nitrocellulose, and probed with a polyclonal anti-human alpha 2M antibody. Bound antibody was detected by an anti-rabbit IgG antibody linked to horseradish peroxidase followed by ECL.

Affinity Labeling and Cross-linking-- Complex formation was carried out in a reaction mixture containing 10 µg of alpha 2M or 10 µl of plasma and 1 nM 125I-IGFBP-1 in 200 µl of PBS, 0.2% Triton X-100 for 4 h at 37 °C. The associated proteins were cross-linked according to the protocol of Vaughan and Vale (19); BS3 (Pierce; final concentration 0.5 mM) was added for 30 min at room temperature followed by 10 µl of 2.5 M glycine to stop the reaction. In some experiments, these reactions were performed in the presence of excess unlabeled IGFBP-1. A 20-µl aliquot of each sample was added to 2 × nonreducing SDS loading buffer and the remainder was immunoprecipitated with an antibody to halpha 2M or hIGFBP-1.

Immunoprecipitation and Analysis of Affinity Labeled Complexes-- Cross-linked affinity labeled complexes were incubated overnight at 4 °C with either 10 µl of rabbit anti-halpha 2M or 10 µl of mouse anti-IGFBP-1. Antibody-bound complexes were precipitated by the addition of 25 µl of protein-A-Sepharose CL-4B and analyzed on 4-12% SDS gradient gels followed by autoradiography.

Surface Plasmon Resonance Analysis-- IGFBP-1 (20 µg/ml of phosphorylated or non-phosphorylated isoform diluted in HBS buffer (10 mM Hepes, 150 mM NaCl, 3.4 mM EDTA, 0.05% P20, pH 7.4) was immobilized to BIACore CM5 sensor chips using standard amine coupling procedures (20).

All experiments were performed at 25 °C and a constant flow rate of 15 µl/min. Immediately before injection of ligand, the surface of the IGFBP-1 sensor chip was preconditioned using 100 mM HCl, ensuring equal conditions for analysis of all samples. A 10-µl injection of ligate (200 ng/ml IGF-I or 200 µg/ml alpha 2M diluted in HBS buffer) was passed across the immobilized IGFBP-1 and the binding profile recorded. Dissociation of bound ligate from immobilized ligand, initiated by flowing buffer across the sensor surface, was monitored for 500 s. This was followed by a regeneration phase (35 µl of 100 mM HCl) to dissociate the remaining ligand from the binding protein and provide a ligate free surface for subsequent interaction analyses.

Proteolysis of IGFBP-1-- 125I-IGFBP-1 was incubated with 1 µg of chymotrypsin in the absence or presence of 1-10 µg of alpha 2M in a final volume of 20 µl of PBS, 0.5 mM CaCl2 for 16 h at 37 °C. Following addition of 2 × SDS loading buffer, samples were subjected to 10% SDS-PAGE followed by autoradiography.

Cell Culture-- 3T3-L1 fibroblasts (American Type Culture Collection (Manassas, VA)) and mouse embryo fibroblasts (generously provided by Professor Willnow, Berlin, Germany) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine, 5 µg/ml gentamicin, and 100 µg/ml streptomycin at 37 °C in 5% CO2.

Activation of alpha 2M-- alpha 2M was activated by reaction with 300 mM methylamine as previously reported (21). Tris borate native electrophoresis (22) demonstrated differing mobility between the activated and native isoforms indicating that conformational change and thus activation had occurred. This was confirmed by comparing uptake of 125I-alpha 2M and 125I-activated alpha 2M (iodinated by chloramine T to a specific activity of 0.4 µCi/µg) in mouse embryo fibroblast cells expressing the LRP receptor which only recognizes alpha 2M in the activated form. Briefly, cells plated at 2 × 105 were serum starved for 3 h before the addition of 3 nM 125I-alpha 2M or 125I-activated alpha 2M. After 10-360 min incubation at 37 °C, cells were washed 2 times with PBS and then incubated for 4 min at 25 °C with EDTA, trypsin, 0.2 mg/ml proteinase K. The resulting cell suspension was centrifuged at 13,000 rpm; membrane bound versus incorporated 125I-alpha 2M was determined by counting the supernatant and solubilized cell pellet, respectively.

[3H]Thymidine Uptake Assay-- Cells were serum starved for 24 h before the addition of IGF-I (10 ng/ml) ± IGFBP-1 (40 ng/ml) and/or 100 µg/ml activated alpha 2M. 20 h later, [methyl-3H]thymidine was added to a final concentration of 0.25 µCi/ml and after a further 4 h, cells were washed twice with PBS and once with 10% trichloroacetic acid. The cells were incubated with 10% trichloroacetic acid for 2 h at 4 °C and solubilized with 0.1 M NaOH and counted on a beta -counter using Optiphase HiSafe liquid scintillant.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of IGFBP-1 Purified from Plasma on IGF-I Stimulated [3H]Thymidine Uptake by 3T3-L1 Fibroblasts-- IGF-I (10 ng/ml) caused a 1.5-fold increase (p < 0.005) in [3H]thymidine uptake by 3T3-L1 fibroblasts in mid-log growth (Fig. 1). This was unaffected by nonphosphorylated IGFBP-1 added at a 1:1 molar ratio (40 ng/ml; Fig. 1). Contrary to our expectations, however, 40 ng/ml of a preparation of phosphorylated IGFBP-1 isolated from normal human plasma, enhanced the effect of IGF-I by 5-fold (p < 0.0005; Fig. 1). Furthermore, the IGFBP-1 preparation also increased [3H]thymidine uptake independently of IGF-I (130% increase over control; p < 0.001) whereas npIGFBP-1 alone had no effect on cell proliferation.


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  		Fig. 1.   A, the effect of IGF-I ± nonphosphorylated (np) IGFBP-1 or phosphorylated (p) IGFBP-1 on [3H]thymidine uptake by 3T3-L1 fibroblasts. IGF-I (10 ng/ml) ± 40 ng/ml np- or pIGFBP-1 was incubated with serum-starved cells for 20 h before the addition of 0.25 µCi/ml [3H]thymidine for a further 4 h. Uptake is expressed as percentage increase over control and is shown as the mean (± S..D) of three experiments performed in triplicate. B, the effect of 3T3-L1 cells on the phosphorylation status of IGFBP-1. 40 ng/ml of a preparation of phosphorylated IGFBP-1 purified from normal human plasma was incubated with 3T3-L1 fibroblasts or BeWo choriocarcinoma cells. After 24 h medium was harvested, immunoprecipitated with anti-IGFBP-1 monoclonal antibody 6303, and subjected to n-octylglucoside electrophoresis and Western ligand blotting with 125I-IGF-I. The phosphorylation pattern of recombinant IGFBP-1 and IGFBP-1 from normal plasma, amniotic fluid (AF) and decidualized endometrial cells (decidua CM) is shown for comparison

Enhancement of IGF action by the presence of phosphorylated IGFBP-1 was unexpected since this high affinity isoform was previously shown to be inhibitory (9) (11). This was not due to the presence of co-purified IGF-I since measurement of IGF-I in the purified IGFBP-1 preparation indicated that levels were below the detection limit of our radioimmunoassay (0.8 ng/ml;data not shown). We therefore questioned if the 3T3-L1 cells had dephosphorylated the IGFBP-1 to produce the nonphosphorylated isoform that is thought to enhance IGF activity. The phosphorylation status of IGFBP-1 was monitored by n-octylglucoside electrophoresis/Western ligand blotting before and after exposure to 3T3-L1 cells. Fig. 1B shows that 3T3-L1 cells do not dephosphorylate IGFBP-1 because the nonphosphorylated isoform could not be detected in the medium harvested from these cells after 24 h. The control in this experiment was the BeWo choriocarcinoma cell line, which has placental alkaline phosphatase and converts phosphorylated IGFBP-1 to the fully dephosphorylated form.

Protein Co-purified with IGFBP-1 Is Identified as alpha 2-Macroglobulin-- These results suggested that a protein which had co-purified with IGFBP-1 might be enhancing IGF-I action on target cells. Indeed, increased IGF-I stimulated [3H]thymidine uptake was observed in response to a plasma-derived preparation that had been depleted of IGFBP-1 by immunoprecipitation (data not shown). Proteins in the IGFBP-1 preparation were therefore isolated from silver-stained SDS-polyacrylamide gels, treated with trypsin, and subjected to MALDI-MS analysis. The predominant contaminating component was identified using the Pro-Found data base as alpha 2-macroglobulin.

IGFBP-1/alpha 2M Are Associated in Plasma-- Western immunoblotting with an anti-human alpha 2M antibody confirmed the presence of alpha 2M in the IGFBP-1 preparation purified from plasma (Fig. 2A). Immunoprecipitates of human plasma with an anti-human IGFBP-1 antibody, contained a high molecular weight protein that co-migrated with human alpha 2M, providing further proof of the association between IGFBP-1 and alpha 2M (Fig. 2B).


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  		Fig. 2.   A, Western immunoblot demonstrating presence of alpha 2M in the IGFBP-1 preparation purified from human plasma. 10 µl of IGFBP-1 preparation (lane 1) and 10 µg of activated alpha 2M (lane 2) were electrophoresed on SDS 4-12% polyacrylamide gradient gels, blotted onto nitrocellulose, and probed with a polyclonal antibody to alpha 2M. An anti-rabbit IgG-horseradish peroxidase antibody and ECL were used for visualization. B, immunoprecipitation of alpha 2M from human plasma using an antibody to IGFBP-1. Plasma (10 µl) was incubated with an anti-IGFBP-1 antibody (6303) overnight and immune complexes were precipitated with protein A-Sepharose CL-4B. The resulting peptides were electrophoresed on SDS 4-12% polyacrylamide gradient gels, blotted to nitrocellulose, and probed with an antibody to human alpha 2M. Lane 1 shows a high molecular weight species which co-migrates with activated alpha 2M (10 µg; lane 2).

Characterization of IGFBP-1/alpha 2M Complexes Formed in Vitro-- IGFBP-1/alpha 2M binding in solution was assessed by incubating 125I-IGFBP-1 with human plasma (10 µl) for 4 h, fixing the resulting complexes with the cross-linking agent BS3, and SDS-PAGE analysis both before and after immunoprecipitation with an anti-alpha 2M antibody. Fig. 3A demonstrates that 125I-IGFBP-1 can associate with a high molecular weight species in plasma which can be immunoprecipitated with an antibody to alpha 2M. This complex co-migrated with the labeled species seen as the result of 125I-IGFBP-1 incubation with alpha 2M (10 µg). The high molecular weight 125I-IGFBP-1/human plasma-binding protein and the 125I-IGFBP-1·alpha 2M complexes could also be precipitated by an antibody to IGFBP-1 (Fig. 3B) although not by protein A-Sepharose CL-4B alone. The radioactive species migrating to ~30 kDa represents uncomplexed 125I-IGFBP-1. 125I-IGFBP-1 also formed high molecular weight complexes with activated alpha 2M (data not shown).


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  		Fig. 3.   Affinity labeling of human plasma-binding proteins and alpha 2M with 125I-IGFBP-1. A, human plasma (10 µl; lanes 1 and 3, or alpha 2M (10 µg; lanes 2 and 4) was incubated with 125I-IGFBP-1 for 4 h and then for 30 min with the cross-linking agent BS3. An aliquot of each sample was removed for SDS-PAGE analysis (lanes 1 and 2) and the remainder was immunoprecipitated with anti-alpha 2M and protein A-Sepharose CL-4B (lanes 3 and 4). Samples were electrophoresed on SDS 4-12% polyacrylamide gradient gels and labeled proteins were visualized by autoradiography. B, 125I-IGFBP-1/human plasma-binding protein (lane 2) and the 125I-IGFBP-1·alpha 2M complexes (lane 3) were immunoprecipitated by an anti-IGFBP-1 antibody and visualized as described above. 125I-IGFBP-1 alone (lane 1) and 125I-IGFBP-1 incubated with protein A only (lane 4) were included as control samples. C, 125I-IGFBP-1 was incubated with 10 µl of alpha 2M in the absence (lane 1) or presence of increasing concentrations (lane 2, 10 times; lane 3, 100 times; lane 4, 1000 times) of unlabeled IGFBP-1. Samples were treated with anti-alpha 2M antibody followed by protein A-Sepharose CL-4B and precipitated proteins were analyzed by 4-12% gradient SDS-PAGE and autoradiography.

Specificity of IGFBP-1/alpha 2M association was confirmed by competition studies in which 125I-IGFBP-1 was cross-linked to alpha 2M in the presence of increasing concentrations of unlabeled IGFBP-1. Fig. 3C shows that excess unlabeled IGFBP-1 (10-1000-fold) decreased binding of 125I-IGFBP-1 to alpha 2M.

Surface Plasmon Resonance Analysis of IGFBP-1/alpha 2M Association-- Surface plasmon resonance was used to investigate further the association of IGFBP-1 and alpha 2M; using standard amine coupling procedures, phosphorylated or nonphosphorylated IGFBP-1 was immobilized on a BIAcore sensor chip CM5 and then exposed to alpha 2M. The sensorgram depicted in Fig. 4A shows binding of alpha 2M to phosphorylated IGFBP-1. alpha 2M was applied at a concentration sufficient to saturate the immobilized IGFBP-1, based on a 1:1 binding ratio. After binding alpha 2M, the chip was further exposed to IGF-I and Fig. 4A shows that IGFBP-1 can still bind IGF-I in the presence of alpha 2M. Nonphosphorylated IGFBP-1, however, does not bind to alpha 2M (Fig. 4B) although IGF-I binding of this isoform is evident.


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  		Fig. 4.   Surface plasmon resonance analysis of IGFBP-1/alpha 2M association. Association and dissociation curves for alpha 2M binding to phosphorylated (A) and nonphosphorylated (B) IGFBP-1 in the absence and presence of IGF-I. alpha 2M (200 µg/ml) was passed over the immobilized IGFBP-1 (association phase) before switching to buffer alone (dissociation phase). IGF-I (200 ng/ml) was then passed over the chip to assess IGFBP-1/IGF-I association in the presence of alpha 2M. The ordinate gives the measured signal representing the mass of protein bound (response units (RU)).

alpha 2M Protects IGFBP-1 from Proteolysis-- Since alpha 2M is a recognized protease inhibitor, one physiological consequence of the IGFBP-1/alpha 2M association could be protection of IGFBP-1 from proteolysis. 125I-IGFBP-1 was incubated with 1 µg of chymotrypsin (23) in the presence of alpha 2M (0-10 µg). Fig. 5 demonstrates that 1 µg of chymotrypsin is sufficient to proteolyse 125I-IGFBP-1 completely since there is no evidence of intact or partially fragmented IGFBP-1 following incubation for 16 h. However, in the presence of 5 µg of alpha 2M, radiolabeled proteins of molecular weights corresponding to IGFBP-1 fragments are apparent, suggesting that chymotrypsin activity is reduced by a low concentration of alpha 2M. IGFBP-1 proteolysis was completely abolished by the presence of 10 µg of alpha 2M; here the majority of 125I-IGFBP-1 was detected in high molecular weight complexes which co-migrate with the radiolabeled species observed when IGFBP-1 and alpha 2M are incubated in the absence of chymotrypsin. This suggests that in this instance, alpha 2M protects against proteolysis by associating with the substrate (IGFBP-1) rather than the protease.


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  		Fig. 5.   alpha 2M protects IGFBP-1 from proteolysis. 125I-IGFBP-1 alone is shown in lane 1. 125I-IGFBP-1 was incubated with chymotrypsin (1 µg) in the absence (lane 2) or presence of alpha 2M (5 µg, lane 3, or 10 µg, lanes 4 and 5) for 16 h at 37 °C. Samples were subjected to 10% SDS-PAGE followed by auotoradiography.

Effect of alpha 2M on IGF-I-stimulated [3H]Thymidine Uptake-- alpha 2M influences the action of other growth factors and therefore the effect of alpha 2M on IGF-I stimulated [3H]thymidine uptake by 3T3-L1 fibroblasts was investigated to determine whether alpha 2M could be responsible for the enhanced stimulation observed in the presence of plasma-derived IGFBP-1. Conversion of alpha 2M into the activated isoform, which is recognized by the alpha 2M receptor LRP (Fig. 6A (i)), was achieved by reaction with methylamine. Activated alpha 2M (100 µg/ml) but not the native isoform (100 µg/ml), was able to enhance IGF-I-stimulated [3H]thymidine uptake 2-fold (p < 0.001; Fig. 6A (ii)). Importantly, alpha 2M could also abrogate the inhibitory effect of IGFBP-1; an HPLC-purified preparation of phosphorylated IGFBP-1 could reduce IGF action (Fig. 6B, p < 0.01), however, when 100 µg/ml alpha 2M was also included in the incubation, IGF-I-stimulated [3H]thymidine uptake (p < 0.01) was enhanced. Activated alpha 2M (200 µg/ml) also had an independent effect on mitogenesis (p < 0.05), whereas phosphorylated IGFBP-1 (40 ng/ml) alone had no effect (Fig. 6B).


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  		Fig. 6.   A, confirmation of alpha 2M activation by methylamine. alpha 2M was activated by incubation with 200 mM methylamine and then analyzed by native Tris borate electrophoresis and silver staining. Activation was confirmed by comparing uptake of activated (lambda ) and native (nu ) 125I-alpha 2M by mouse embryo fibroblast cells expressing the LRP receptor, which only recognizes alpha 2M in the activated form (i). 3T3-L1 cells were then used to compare the effect of active and native alpha 2M on IGF-I activity (ii). Serum-starved cells were incubated with 10 ng/ml IGF-I ± 100 µg/ml alpha 2M for 20 h before the addition of 0.25 µCi/ml [3H]thymidine for a further 4 h. Uptake is expressed as percentage increase over control and is shown as the mean (± S.D.) of three experiments performed in triplicate. B, effect of alpha 2M and pIGFBP-1 on IGF-I stimulated [3H]thymidine uptake by 3T3-L1 fibroblasts. IGF-I (10 ng/ml) and IGFBP-1 (40 ng/ml) were incubated in the presence or absence of activated alpha 2M (100 µg/ml) and effects on [3H]thymidine uptake were determined as described above. The effect of alpha 2M (200 µg/ml) and IGFBP-1 (40 ng/ml) alone or in combination was also determined. Uptake is expressed as percentage increase over control and is shown as the mean (± S.D.) of three experiments performed in triplicate.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Analysis of the highly phosphorylated IGFBP-1 found in the circulation of non-pregnant adults has demonstrated association with alpha 2-macroglobulin, another plasma protein. alpha 2M can enhance IGF-I-stimulated proliferation of fibroblasts, even in the presence of phosphorylated IGFBP-1, however, when alpha 2M is absent, phosphorylated IGFBP-1 is inhibitory.

Highly phosphorylated IGFBP-1 has a high affinity for IGF-I (9, 11) and can therefore sequester IGF-I from its cell surface receptors. In order for IGFBP-1 enhancement of IGF action to occur, it is generally thought that IGFBP-1 must be either in the nonphosphorylated isoform, which has a lower affinity for IGF-I (5), or associated with cell membranes (6, 24). We found that increased IGF action in the presence of a preparation of IGFBP-1 purified from plasma was not the result of dephosphorylation by 3T3-L1 cells. BeWo choriocarcinoma cells, however, are able to dephosphorylate IGFBP-1 (23) and this isoform of IGFBP-1 is normally only detected in the human during pregnancy (10), raising the possibility that altering IGFBP-1 phosphorylation status may predominantly be a mechanism for regulating IGF bioavailability in pregnancy.

An early report from Clemmons and Gardner (25) suggested that a factor present in plasma was necessary for IGFBP-1 to potentiate IGF stimulated smooth muscle cell DNA synthesis. This group discerned the factor to be a macromolecule and excluded mitogens such as platelet-derived growth factor, epidermal growth factor, and fibroblast growth factor and the carrier proteins transferrin, albumin, and fibronectin, although its identity remained elusive. These studies led us to suspect that our purified preparation of plasma IGFBP-1 contained another plasma component that had co-purified on the anti-IGFBP-1 monoclonal antibody immunoaffinity column by virtue of its association with IGFBP-1 in plasma.

Radioimmunoassay of the IGFBP-1 preparation refuted our initial assumption that IGF-I was the contaminating protein and so we then used a combination of MALDI-TOF mass spectrometry and co-imunoprecipitation/Western blot studies, to identify the co-purified factor as alpha 2-macroglobulin.

Early size exclusion chromatographic studies of plasma did not detect the association between IGFBP-1 and alpha 2M. Several explanations for this are tenable. First alpha 2M is a large protein (approx 700,000) which would have been outside the molecular weight range of most chromatographic studies. Appearance of material within the void volume could have been discounted as being due to protein solubility/aggregation. Second, the association of IGFBP-1 with alpha 2M may be low affinity resulting in dissociation of the complex on size exclusion chromatography; these studies indicate a kd of 2.75 × 10-3 s-1, which supports this hypothesis. Third it is possible that some IGFBP-1 antibodies cannot recognize the peptide when bound to alpha 2M. Furthermore, initial chromatographic studies of other IGFBPs did not demonstrate associations with plasma proteins and yet it has recently been recognized that this is the case. For example, IGFBP-3 specifically binds to lactoferrin (26), transferrin (27), type I collagen (28), and fibronectin (29) although the physiological significance of these associations is as yet unclear.

alpha 2M is a homotetrameric glycoprotein that circulates at concentrations of 2-4 mg/ml (30). Each subunit contains multiple reactive sites suggesting that alpha 2M has diversified functions as a binding, carrier, and targeting protein and it may therefore be important for several aspects of IGFBP-1 function.

alpha 2M is well known as a protease inhibitor and has the unique ability of being able to inhibit proteinases from all four mechanistic classes (30). Protease cleavage of native alpha 2M results in a conformational change to form activated-alpha 2M which traps the protease so that it is sterically hindered from access to substrate. Nonproteolytic peptides trapped by alpha 2M become largely protected from exogenous proteases and our own results show that when associated with alpha 2M, IGFBP-1 is protected from proteolysis by chymotrypsin. Thus in plasma, alpha 2M may be acting as a chaperone to IGFBP-1 and this may explain why there are no reports of IGFBP-1 fragments in the circulation despite the fact that IGFBP-1 has many of the cleavage motifs displayed by other circulating IGFBPs which are proteolysed.

Another physiological consequence of IGFBP-1/alpha 2M association may be in regulating IGF activity. alpha 2M is known to associate with several other growth factors including fibroblast growth factor (31), vascular endothelial growth factor (32), epidermal growth factor (33), transforming growth factor-beta 1 (34), and platelet-derived growth factor (35) to synergistically enhance their action on cell proliferation (36). We have found that alpha 2M also enhances IGF-I-stimulated mitogenesis and importantly, that alpha 2M can also influence how IGFBP-1 modulates this effect. In our initial studies using the IGFBP-1 preparation purified from plasma, IGF-I stimulated [3H]thymidine uptake by 3T3-L1 fibroblasts was seemingly enhanced by phosphorylated IGFBP-1 and the preparation also appeared to have an independent mitogenic effect. However, we have now demonstrated that this effect was more likely due to the presence of alpha 2M.

The mechanism behind alpha 2M enhancement of IGF-I activity is unclear, although it probably involves one of the two alpha 2M receptors, LRP/alpha 2MR, or the recently described alpha 2M signaling receptor (alpha 2MSR). The LRP/alpha 2MR is a member of the low density lipoprotein receptor superfamily (37-39) which recognizes both free and growth factor-associated alpha 2M once it has been activated by proteases or amines such as methylamine (40-42). Thus alpha 2M, through IGFBP-1, could serve to increase the concentration of IGF-I in the local environment of the cell; dissociation at or near the cell surface could release IGF-I to interact with its cell surface receptor. Our preparation of native alpha 2M was unable to enhance IGF-I activity, whereas activated alpha 2M did enhance IGF actions. These data strongly suggest that such synergy was due to alpha 2M/receptor interactions and not the presence of an additional alpha 2M-associated mitogen such as platelet-derived growth factor. Alternatively, the LRP/alpha 2MR scavenges alpha 2M complexes resulting in their rapid clearance from the circulation and thus internalization of IGFBP-1 along with alpha 2M could alleviate the inhibitory effect on paracrine or autocrine IGF-I, or, if both IGF-I and IGFBP-1 were internalized, their dissociation within an endocytic compartment could influence signal transduction in a manner analogous to that of epidermal growth factor (43-45).

A further mechanism by which alpha 2M may regulate cellular growth involves a second alpha 2M receptor (46), which also recognizes only activated alpha 2M. alpha 2M interaction with this receptor increases phosphatidylinositol 3-kinase activity (47) and elevates p21Ras-GTP (48), which may explain our finding that alpha 2M acts as an independent mitogen. However, signaling through the type 1 IGF receptor also involves activation of the phosphatidylinositol 3-kinase and Ras/Raf pathways (1) and so it is possible that alpha 2M could enhance endogenous and exogenous IGF-I actions as a result of synergy between their intracellular signaling components.

In summary, we have demonstrated that alpha 2M is a binding protein of IGFBP-1 which modifies IGFBP-1/IGF interaction. This represents a novel and potentially important mechanism for controlling the transport and biological activity of IGFs since the effect of IGFBP-1 on IGF activity depend not only on IGFBP-1 phosphorylation status, but also on whether IGFBP-1 is bound to alpha 2M and the complement of alpha 2M receptors at the cell surface.

    ACKNOWLEDGEMENTS

We gratefully acknowledge the The Royal Society, Salford Royal Hospitals NHS Trust and Wellbeing and thank all collaborators for the generous donation of reagents and cells.

    FOOTNOTES

* This work was supported by The Royal Society and the Salford Royal Hospitals NHS Trust and Wellbeing.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. Tel.: 44-161-275-5174; Fax: 44-161-275-5958; E-mail: melissa.westwood@man.ac.uk.

Published, JBC Papers in Press, August 23, 2001, DOI 1.1074/jbc.M102793200

    ABBREVIATIONS

The abbreviations used are: IGF, insulin-like growth factor; IGFBP, insulin-like growth factor-binding protein; alpha 2M, alpha 2-macroglobulin; PBS, phosphate-buffered saline; MALDI, matrix-assisted laser desorption ionization; PAGE, polyacrylamide gel electrophoresis; LRP, low-density lipoprotein receptor related protein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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