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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 5 2382-2389
Copyright © 2004 by The Endocrine Society

The Role of the Acid-Labile Subunit in Regulating Insulin-Like Growth Factor Transport across Human Umbilical Vein Endothelial Cell Monolayers

Louis D. Payet, Sue M. Firth and Robert C. Baxter

Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia

Address all correspondence and requests for reprints to: Robert C. Baxter, Ph.D., D.Sc., Kolling Institute of Medical Research, Department of Molecular Medicine, Royal North Shore Hospital, University of Sydney (E25), St. Leonards, Sydney, New South Wales 2065, Australia. E-mail: robaxter{at}med.usyd.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have established a human umbilical vein endothelial cell (HUVEC) monolayer system to study the role of complex formation with IGF binding protein (IGFBP) and acid-labile subunit (ALS) on the transendothelial transport of IGF. Incubation with recombinant human IGFBP-3 alone did not retard 125I-IGF-I or -II transport, but addition of ALS caused marked inhibition. Transport of 125I-des(1–3)IGF-I was more rapid than 125I-IGF-I, suggesting the presence of some endogenous IGFBPs, although these were undetectable by affinity labeling of cells or medium. In the presence of ALS, recombinant human IGFBP-5 also retarded IGF transport, although significantly less than IGFBP-3, despite their similar ternary complex formation. In contrast, IGFBP-3 mutated in its ALS-binding domain was not inhibitory. To study IGF transport by pregnancy-proteolyzed IGFBP-3, we prepared [Tyr31]monoiodoIGF-I, the only iodoIGF-I form that reacts normally with proteolyzed IGFBP-3. In the presence of ALS, IGFBP-3 isolated by immunoaffinity chromatography from second-trimester pregnancy serum significantly retarded IGF transport, but to a lower extent than IGFBP-3 isolated from normal serum, despite normal ALS binding. This study demonstrates the key role of ALS in regulating transendothelial IGF transport, but indicates that other factors are also involved. Our data suggest that pregnancy-proteolyzed IGFBP-3, despite forming normal ternary complexes, is less effective than intact IGFBP-3 in retarding IGF egress from the circulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE IGFs (IGF-I and IGF-II) are growth-regulatory peptides that exhibit both mitogenic and anabolic effects in many cell types. The IGFs in the serum are regulated by their binding to a group of proteins called the insulin-like growth factor binding proteins (IGFBPs), of which six have been identified (IGFBP-1 to -6). Members of the IGFBP family are characterized both by their high IGF binding affinities and also their sequence homology within both the amino-terminal and carboxyl-terminal domains (1, 2, 3). Within the circulation, the predominant IGFBP is IGFBP-3. While the IGFs bind to IGFBP-3 to form binary complexes, a majority of circulating IGFs are associated with IGFBP-3 and a third protein, the acid-labile subunit (ALS), in ternary complexes (4). More recently, it was discovered that IGFBP-5 may also interact with ALS in the presence of IGFs to form ternary complexes (5) and that these complexes exist in the human circulation (6).

Previous studies in vivo have demonstrated that IGFs bound in IGFBP-3 ternary complexes in the serum have a longer circulating half-life than free or binary-complexed IGFs (7). It is thought that IGFs within the ternary complex are prevented from leaving the circulation due to the size of the complex, reducing their bioavailability but providing the body with a potentially rapidly mobilized pool of IGFs (8). Proteolysis of IGFBP-3 in ternary complexes may be a mechanism contributing to the dissociation and release of the IGFs (9, 10, 11).

Proteolysis of IGFBP-3 has been associated with a variety of conditions in which mobilization of serum IGFs may be advantageous. For example, the increase in IGFBP-3 proteolysis after surgery has been suggested to enhance the release of IGFs from the serum, increasing their tissue access and thus modulating the catabolic state induced by surgery (12, 13, 14). The proteolysis of IGFBP-3 was first identified in pregnancy serum (ps) in which the serum IGFBP-3 is totally proteolyzed by 8 wk of pregnancy (15, 16). This proteolytic event has been speculated to increase the fetus’s access to maternal IGFs, thus promoting fetal development and growth (17). Interestingly, although the IGFBP-3 in ps is proteolyzed, most of the serum IGFs are still found within ternary complexes, with only a small increase in the amount of free IGFs in the serum (18, 19). However, there is little experimental data explicitly demonstrating what effect serum proteolysis of IGFBP-3 has on IGF bioavailability.

To investigate the transport of IGFs out of the circulation, we have developed and characterized a model to study the effect of the ternary complex on IGF transport across an endothelial cell monolayer and have used a monoiodinated form of IGF-I to investigate how this is affected by IGFBP-3 proteolysis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Human umbilical vein endothelial cells (HUVECs) were obtained from the American Type Culture Collection (Manassas, VA). Heparin (tissue culture grade), 3H-inulin, BSA, and endothelial cell growth factor supplement were purchased from Sigma-Aldrich (St. Louis, MO). Opaque 6.5-mm-diameter tissue culture chambers containing 0.4-µm pores were obtained from Costar (Burning, NY), and Cell-Tak cell and tissue adhesive was purchased from BD Biosciences (San Jose, CA). Recombinant human (rh)IGF-I was a generous gift from Genentech, Inc. (South San Francisco, CA); des(1–3)IGF-I and IGF-II were purchased from Gropep (Adelaide, South Australia, Australia). Ham’s F12 medium was obtained from Trace Scientific (Melbourne, Victoria, Australia). The 5-µm C18 300-Å HPLC column was purchased from Phenomenex (Torrance, CA); 30-kDa molecular mass cut-off centrifugal concentrators were obtained from Pall Filtron Corp. (Northborough, MA), and G-50 Sephadex was purchased from Amersham Biosciences (Uppsala, Sweden). Hybond C nitrocellulose membrane and antirabbit horseradish peroxidase-conjugated Fab fragments (from donkey) were purchased from Amersham Biosciences UK (Bucks, UK). AffiGel-10 was obtained from Bio-Rad (Richmond, CA). rhIGFBP-3, rhIGFBP-5, and an IGFBP-3 mutant with reduced binding to ALS (IGFBP-3mut) were produced in human 911 retinoblastoma cells by an adenoviral expression system (20, 21, 22). hALS was purified from serum as previously described (4).

Cell growth and transwell experiments

HUVECs were grown in Ham’s F12 minimal medium supplemented with 10% fetal calf serum, 100 µg/ml heparin, 30 µg/ml endothelial cell growth factor supplement, and 5.5 mM glutamine. All experiments were conducted using cells between passages 5–10. For the HUVEC monolayer transport studies, 6.5-mm transwells were precoated with Cell-Tak (20 µl per insert) and washed once with PBS. HUVECs were seeded at 5 x 104 cells per transwell and were grown for 2 wk until confluent with media changed every 3 d. The cells were then washed three times with 1 ml Ham’s F12 + 1% BSA [serum-free (SF) medium] and then used in the transport experiments.

125I-IGF transport experiments

IGF-I, des(1–3)IGF-I, and IGF-II (5 µg) were iodinated with 1 mCi Na 125I by the standard chloramine-T method, and unincorporated Na 125I was removed by gel filtration chromatography. 125I-IGF-I was coincubated with various treatments in SF media for 3 h in a final volume of 150 µl before addition to the HUVEC monolayer. Fresh SF medium (700 µl) was added to the lower chamber of the transwell, and the 125I-IGF-I-containing medium was added to the upper transwell insert. Samples of 20 µl were taken from the bottom chamber at 2 and 4 h with replenishment by fresh SF media. Samples were counted on a Wallac model 1261 {gamma}-counter (Wallac, Turku, Finland). Similar experiments were carried out using 125I-IGF-II.

Transport properties of the HUVEC monolayer

125I-BSA was prepared by iodinating 5 µg BSA with 1 mCi Na 125I using the standard chloramine-T method, and unincorporated Na 125I was removed by gel filtration chromatography. 3H-inulin (0.25 µCi) or 125I-BSA [100,000 counts per minute (cpm)] was added to the transwells with or without confluent HUVEC monolayers. The molecular masses of inulin and BSA are approximately 5,000 and 66,000, respectively. Samples (25 µl) were taken from the bottom chamber every 30 min with media replacement. Permeability coefficients were calculated by first determining the flux at each specific time point where flux = (cpm in basal chamber)/(initial cpm per milliliter in upper chamber)/(area of the filter)(area = 0.33 cm2). Permeability constants were determined by the slope of linear regression lines of the flux vs. time as previously described (23).

Integrity of 125I-IGF-I

The integrity of 125I-IGF-I after its transport across the HUVEC monolayer was assessed by trichloroacetic acid (TCA) precipitability. 125I-IGF-I alone or as part of binary or ternary IGFBP-3 complexes was added to the HUVEC monolayer as described above. After 4 h incubation, samples of each transwell and corresponding chamber were precipitated in the presence of 10% ice-cold TCA. The pellet was washed in cold 10% TCA and then counted in a Wallac model 1261 {gamma}-counter. Unprecipitated samples were also counted to determine total radioactivity present.

Purification of serum IGFBP-3

With the approval of the institutional human research ethics committee, serum samples were obtained from nonpregnant female volunteers [nonpregnancy serum (ns)] and from healthy women in the second trimester of pregnancy (ps). Serum from each group was pooled, and 5 ml of each of the pools was purified by immunoaffinity chromatography on a column containing Affigel-10 conjugated to an anti-IGFBP-3 antibody as described previously (24). Fractions containing the eluted IGFBP-3 were then depleted of IGFs by loading the eluted IGFBP-3 onto a centrifugal concentrator with a 30-kDa molecular mass cut-off and flushing the sample eight times with 5 ml of 1 M acetic acid. Samples were neutralized with three buffer exchanges of 100 mM H2NaPO4 (pH 6.8) and the concentration of IGFBP-3 was determined by human-specific IGFBP-3 RIA (25). To assess the depletion of IGFs, increasing amounts of the purified IGFBP-3 were incubated with 125I-ALS in the presence and absence of 50 ng IGF-I. Complexes were precipitated with anti-IGFBP-3 antiserum. In the absence of IGF-I, the amount of 125I-ALS precipitated was less than 4% compared with 36% in the presence of IGF-I.

Preparation of [Tyr31]monoiodo-IGF-I

Monoiodo125I-IGF-I was prepared as previously described (26) with the following modifications. IGF-I (20 µg) was iodinated using 0.25 mCi of Na 125I by the standard chloramine-T method. The 125I-IGF-I, containing a mixture of IGF-I iodoforms, was loaded onto a reverse-phase C18 HPLC column that was preequilibrated with 50 ml HPLC running buffer (0.1 M Tris, 0.05 M phosphoric acid, 1 mM EDTA, 30% ethanol, pH 7.0). Mono-iodinated forms of IGF-I were eluted using a 30–55% linear ethanol gradient over 70 min at a flow rate of 0.67 ml/min. One-minute fractions were collected and counted. As previously described (26), [Tyr31]monoiodo-IGF-I was the second major peak eluted off the column.

Ternary complex assays

Ternary complex formation assays were performed as previously described (27). Briefly, purified IGFBPs (IGFBP-3 from ns, ps, rhIGFBP-3, or rhIGFBP-5) was added to 10,000 cpm of 125I-IGF-I and 200 ng ALS. Samples were made up to 300 µl using SF media and then incubated for 3 h at 22 C. ALS binding complexes were then precipitated by incubating samples with 1 µl anti-ALS antiserum for 3 h followed by the addition of 5 µl goat antirabbit serum. One milliliter of cold polyethylene glycol, 60 g/liter in 0.15 M NaCl, was then added to all samples. Samples were incubated at 22 C for 15 min, centrifuged, and the supernatants decanted. Pellets were counted on a {gamma}-counter.

Proteolysis assays and Western immunoblots

Fifty microliters of 24-h-HUVEC-conditioned media were incubated with 100 ng IGFBP-3 for 4 and 24 h at 37 C. Laemmli buffer was added to all samples, and samples were then heated for 5 min at 95 C and then electrophoresed on a 12% sodium dodecyl sulfate polyacrylamide gel. Proteins were then transferred to a nitrocellulose membrane and blocked with Tris-buffered saline containing 1% BSA. IGFBP-3 was detected using a rabbit anti-IGFBP-3 antiserum and a donkey antirabbit horseradish peroxidase conjugated antibody using standard techniques.

Statistical analysis

Statistical analysis was carried out using Statview 5.0 PPC (Abacus Concepts Inc., Berkeley, CA). Time series were analyzed by repeated-measures ANOVA, and differences between groups were evaluated by Fisher’s protected least significant difference test with a significant difference defined as P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Before conducting transport experiments, we determined that the confluent HUVECs had formed an integral monolayer, with tight junctions between the paracellular gap junctions, by calculating the rate of transport of 3H-inulin through the transwell surface in the presence or absence of HUVEC cells (Fig. 1AGo). After 120 min, 33% of the 3H-inulin had moved through the empty transwell micropores, compared with 11% in the presence of a confluent HUVEC monolayer. 125I-BSA transport was more strongly inhibited by the HUVEC monolayer, with only 4% of the BSA being transported across the monolayer by 120 min. Permeability coefficients (PC) were calculated for the transport of molecules through the endothelial monolayer. In the absence of cells, the transwells had a PC of 1.56 ± 0.15 x 10–5 cm/sec for inulin, reduced to 4.77 ± 0.58 x 10–6 cm/sec in the presence of a confluent HUVEC monolayer. PC values for both inulin and BSA (2.19 ± 0.52 x 10–6 cm /sec) in the presence of a HUVEC monolayer were similar to previously published values (28, 29, 30), confirming the integrity of the monolayers.



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  		FIG. 1. Assessment of HUVEC monolayer integrity. A, 3H-inulin was added to transwells in the presence ({blacksquare}) or absence ({square}) of confluent HUVEC monolayers. Samples were taken from the lower transwell chamber at 30, 60, and 120 min and counted. Similar experiments were carried out using 125I-BSA in the presence of confluent HUVEC monolayers (•). B, 3H-inulin was added to transwells in the presence ({blacksquare}) or absence ({square}) of confluent HUVEC monolayers. Samples were taken from the lower transwell chamber at 30, 60, 90, 120, and 240 min and counted. Similar experiments were carried out using 125I-IGF-I in the presence of confluent HUVEC monolayers ({circ}). Data are shown as mean values ± SD for two experiments with three replicates each.

 
To demonstrate the integrity of the HUVEC monolayer for longer periods under experimental conditions, a similar experiment was performed over 4 h (Fig. 1BGo). In the absence of cells, the rate of 3H-inulin moving through the transwell micropores became nonlinear as the percentage transported approached 40–50% (between 90 and 240 min). This is probably due to an equilibrium being approached between the transwell and the lower chamber that is reflected by the fact that almost half of 3H-inulin was in the bottom chamber by 4 h. However, in the presence of an intact HUVEC monolayer, the rate of 3H-inulin transport remained linear throughout the 4-h sampling period. The transport of 125I-IGF-I alone was also linear and significantly slower than 3H-inulin, indicating the extended integrity of the monolayer under experimental conditions. The PC values for the transport of inulin and IGF-I through the monolayer were 4.75 ± 0.61 x 10–6 cm/sec (similar to that calculated in Fig. 1AGo) and 2.55 ± 0.94 x 10–6 cm/sec, respectively. Interestingly, the PC values for IGF-I (molecular weight, 7650) and BSA (molecular weight, ~66,000) are not significantly different, suggesting that the IGF-I may in part form binary complexes with endogenous IGFBPs.

The integrity of 125I-IGF-I (added alone or as preformed IGFBP-3 binary and ternary complexes) after its transport across the HUVEC monolayer was assessed by TCA precipitability. After 4-h incubation, 85–89% of 125I-IGF-I in the transwell was TCA precipitable compared with 82–86% of 125I-IGF-I in the bottom chamber. This indicates that there was minimal degradation of 125I-IGF-I over 4 h and after transport across the cell monolayer.

The transport of IGF-I and IGF-II across the HUVEC monolayer alone or when associated with IGFBP-3 and ALS was assessed (Fig. 2Go). In the absence of added IGFBPs, 24% of the 125I-IGF-I or -II had been transported across the HUVEC monolayer after 4 h. Preincubating 125I-IGF-I or 125I-IGF-II with IGFBP-3 before addition to the HUVEC monolayer had no significant effect on the rate of IGF transport (P > 0.05). Similarly, preincubating either IGF with ALS did not affect the rate of transport (data not shown). However, when 125I-IGF-I or 125I-IGF-II was preincubated with both ALS and IGFBP-3 and then added to the HUVEC monolayer, the rate of IGF transport was significantly reduced to 13 and 14%, respectively, over 4 h (both P < 0.0001). Thus, the IGF ternary complexes formed by IGF-I or -II, IGFBP-3, and ALS impede the passage of the IGFs across the monolayer. 125I-IGF-II appeared to be transported slightly faster than 125I-IGF-I when both were in ternary complexes, but this difference was not significant.



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  		FIG. 2. The effect of IGFBP-3 and ALS on IGF-I (A) or IGF-II (B) transport. 125I-IGF alone (•), or after preincubation for 3 h with 10 ng IGFBP-3 ({blacksquare}) or 10 ng IGFBP-3 plus 100 ng ALS ({square}), was added to 0.4-µm transwell containing confluent HUVEC monolayers. Samples were taken for counting from the bottom chamber at 2 and 4 h with replenishment of medium. Data are shown as mean values ± SD for three replicates in a single experiment, which was performed three times with similar results.

 
The importance of ternary complex formation was further investigated by using a variant of IGFBP-3, mutated in the IGFBP-3 carboxyl-terminal basic domain at residues 228–232. This mutant (IGFBP-3mut) still retains normal IGF-I affinity but has a 90% reduced affinity for ALS in the presence of IGF-I (21). 125I-IGF-I was incubated with IGFBP-3mut in the presence or absence of ALS and the effect on IGF-I transport examined (Fig. 3AGo). At 4 h, 28.0 ± 1.6% of the 125I-IGF-I had been transported under control conditions, and incubating the tracer with both ALS and IGFBP-3(mut) had no effect on 125I-IGF-I transport (28.0 ± 2.7%). By comparison, 125I-IGF-I in ternary complexes formed by wild-type IGFBP-3 significantly reduced the rate of 125I-IGF-I transport (15.3 ± 3.4%, P < 0.0001). Thus, the formation of the ternary complex is an essential step in inhibiting IGF-I transport across the monolayer.



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  		FIG. 3. Ternary complexes containing mutant IGFBP-3 or IGFBP-5. A, 125I-IGF-I alone ({diamondsuit}) or preincubated for 3 h with 100 ng ALS ({blacksquare}), 10 ng IGFBP-3 ({circ}), 10 ng IGFBP-3mut ({triangleup}), 10 ng IGFBP-3 plus 100 ng ALS (•), or 10 ng IGFBP-3mut plus 100 ng ALS ({blacktriangleup}) was added to transwells containing confluent HUVEC monolayers. B, 125I-IGF-I alone ({diamondsuit}) or preincubated for 3 h with 10 ng IGFBP-3 ({square}), 10 ng IGFBP-5 ({circ}), IGFBP-3 plus ALS ({blacktriangleup}), or IGFBP-5 plus ALS ({triangleup}) was added to transwells containing confluent HUVEC monolayers. Samples were taken for counting from the bottom chamber at 2 and 4 h with media replenishment. Data are shown as mean values ± SD for three replicates in a single experiment, which was performed three times with similar results. C, Increasing amounts of either IGFBP-3 ({blacksquare}) or IGFBP-5 (•) was incubated with 125I-IGF-I in the presence of 100 ng ALS for 3 h at 22 C. Ternary complexes were then precipitated using anti-ALS antiserum.

 
It has been demonstrated in our laboratory that IGFBP-5, like IGFBP-3, is able to bind to ALS in the presence of IGF-I or IGF-II and form into a ternary complex (5). To compare the effect of the IGFBP-5 ternary complex on IGF-I transport, 125I-IGF-I was preincubated with either IGFBP-3 or IGFBP-5 in the presence or absence of ALS (Fig. 3BGo). As with IGFBP-3 (27.4 ± 2.7% transport at 4 h), the IGFBP-5 binary complex (26.3 ± 1.7%) had no effect on 125I-IGF-I transport (28.2 ± 2.1%). However, the addition of ALS to either of the IGFBP-3 or IGFBP-5 binary complexes significantly inhibited the transport of the 125I-IGF-I across the HUVEC monolayer (P < 0.0001). Interestingly, the inhibition of IGF-I transport by the IGFBP-5 ternary complex (21.6 ± 0.9% transport at 4 h) was significantly less than that caused by the IGFBP-3 ternary complex (18.6 ± 0.7%; P = 0.005). This difference was not due to decreased ternary complex formation by IGFBP-5 compared with IGFBP-3 because their ability to bind ALS in the presence of IGF-I was identical (Fig. 3CGo).

Endothelial cells express IGFBPs, and these may influence the transport of 125I-IGF-I across cell monolayers (31, 32). To determine whether any secreted HUVEC IGFBPs were inhibiting the transport of IGFs across the endothelial monolayer, the transport of des(1–3)IGF-I, an IGF-I analog with reduced IGFBP binding affinity, was investigated (Fig. 4AGo). After 4 h, 28.2 ± 1.9% of the 125I-des(1, 2, 3)IGF-I had been transported across the HUVEC monolayer, and coincubation of the tracer with IGFBP-3 (27.1 ± 1.2%) had no effect. However, in comparison with 125I-IGF-I (23.3 ± 1.7%), 125I-des(1–3)IGF-I was transported significantly faster (P = 0.0025). This suggests that the HUVECs express some IGFBPs that may influence the transport of the IGFs in this system. Because IGFBP-3 alone did not affect 125I-IGF-I transport in this model, it is possible that the slight decrease in IGF-I transport rate relative to des(1–3)IGF-I may be due to IGF-I binding to endogenous cell surface or medium IGFBPs. However, 125I-IGF-I affinity-labeling of cells or culture medium under the conditions of the transport experiments, and analysis by SDS-PAGE and autoradiography, did not reveal any endogenous IGFBPs (data not shown), suggesting that, if present, their concentration must be low. Alternatively, the increase in des(1–3)IGF-I transport may be related to its smaller molecular size or decreased negative charge compared with IGF-I. Bastian et al. (33) showed that the transport of LR3IGF-I, an IGF-I analog that is larger than IGF-I, is significantly reduced compared with IGF-I. These authors also suggested that this may be due to charge differences between the two molecules.



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  		FIG. 4. Possible influence of endogenous IGFBPs or proteases. A, 125I-IGF-I ({diamondsuit}), 125I-des(1–3)IGF-I ({circ}), or 125I-des(1–3)IGF-I preincubated with 10 ng IGFBP-3 (•) was added to transwells containing confluent HUVEC monolayers. Samples were taken for counting from the bottom chamber at 2 and 4 h. Data are shown as mean values ± SD for three replicates in a single experiment, which was performed three times with similar results. B, HUVEC culture medium conditioned for 24 h (50 µl) was incubated at 37 C with 50 ng IGFBP-3 for 4 h (lane D) or 24 h (lane F). Similar treatments were prepared using IGFBP-3 incubated with unconditioned media for 4 h (lane C) and 24 h (lane E). Conditioned medium alone (50 µl, lane A) and 50 ng of untreated IGFBP-3 (lane B) are shown as controls. All samples were separated by SDS-PAGE and immunoblotted with an anti-IGFBP-3 antibody. Relative migration distances of molecular mass standards are indicated in kilodaltons on the left.

 
Various factors may influence IGF transport across the HUVEC monolayer, including endogenous proteases that might act on added or endogenous IGFBPs. Proteolysis of IGFBP-3 is a possible mechanism by which the IGFs may be released from the binary and ternary complexes that they form with IGFBP-3 (10, 34, 35). To determine whether recombinant IGFBP-3 was being proteolyzed by secreted proteases, IGFBP-3 was incubated in HUVEC-conditioned medium and then analyzed by Western immunoblotting (Fig. 4BGo). No HUVEC-secreted IGFBP-3 was detectable in the 24-h conditioned medium (lane A). Incubating IGFBP-3 for 4 h with either HUVEC-conditioned media or unconditioned media did not result in any degradation of the IGFBP-3 (lanes C and D), although some proteolytic degradation of the IGFBP-3 was seen by 24 h. Because the maximum time that the transport studies were carried out was 4 h, proteolysis of the added recombinant IGFBP-3 during these experiments was not likely to influence IGF transport.

Even if IGFBP-3 is not proteolyzed by endothelial cell proteases, circulating IGFBP-3 complexes in which the IGFBP-3 is proteolyzed (for example, in pregnancy) might differ in their delivery of IGFs to the tissues. To examine this in the HUVEC model, we purified IGFBP-3 from both ns and ps by immunoaffinity chromatography (Fig. 5AGo). ps IGFBP-3 forms into ternary complexes, normally with only one specific iodoform of 125I-IGF-I, [Tyr31]-125I-monoiodoIGF-I (24, 36), because iodination on the other tyrosine residues (Tyr24, Tyr60) changes IGF-I reactivity toward proteolyzed IGFBP-3. [Tyr31]-125I-monoiodoIGF-I was purified from an iodinated IGF-I mixture by reverse-phase HPLC using an ethanol gradient (Fig. 5BGo). This yielded three major radioactive peaks (peaks I, II, and III) that correspond to the three different monoiodo-forms of IGF-I, iodo-[Tyr24], [Tyr31], and [Tyr60], as previously reported (26). The different monoiodinated IGF-I forms were tested for their ability to form into ternary complexes with either ns or ps IGFBP-3 (Fig. 5CGo). All three monoiodo-forms of IGF-I formed ternary complexes with equal efficiency to ns IGFBP-3; however, only one of these monoiodo-forms of IGF-I, peak II, formed ternary complexes equally with both ns and ps IGFBP-3, confirming that this peak contains the desired [Tyr31]-125I-monoiodoIGF-I.



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  		FIG. 5. Comparison of complexes containing IGFBP-3 from ps or ns. A, Western blot analysis of IGFBP-3 purified from ns (lane N) and ps (lane P). After immunoaffinity chromatography, samples were separated by SDS-PAGE and immunoblotted with an anti-IGFBP-3 antibody. Relative migration distances of molecular mass standards are indicated in kilodaltons on the left. B, Purification and characterization of monoiodinated IGF-I forms. An iodinated IGF-I mixture was fractionated by reverse-phase HPLC using a 30–55% ethanol gradient. The three main peaks are designated fraction I, II, and III. C, Ternary complex assays were performed using the three peak fractions separated by HPLC. Increasing concentrations of IGFBP-3 purified from ps (open symbols) or ns (closed symbols) was incubated with 10,000 cpm of fraction I ({square}, {blacksquare}), II ({circ}, •), or III ({triangleup}, {blacktriangleup}) and 100 ng ALS. Ternary complexes were immunoprecipitated using ALS antisera. Binding curves shown are representatives of three repeat experiments. (D) [Tyr31]-125I-monoiodoIGF-I alone ({triangleup}) or preincubated for 3 h with 10 ng ns IGFBP-3 ({circ}), 10 ng ps IGFBP-3 ({square}), 10 ng ns IGFBP-3 plus 100 ng ALS (•), or 10 ng ps IGFBP-3 plus 100 ng ALS ({blacksquare}) was added to transwells containing confluent HUVEC monolayers. Samples were taken for counting from the bottom chamber at 2 and 4 h with media replacement. Data are shown as mean values ± SD for three replicates in a single experiment, which was performed four times with similar results.

 
The abilities of ternary complexes containing either ns IGFBP-3 or ps IGFBP-3 to inhibit IGF-I transport across the HUVEC monolayer were compared (Fig. 5DGo). [Tyr31]-125I-monoiodoIGF-I was preincubated with either ns or ps IGFBP-3 in the presence and absence of ALS before use in the transport experiments. After 4 h, 22.7 ± 1.1% of the untreated monoiodoIGF-I was transported through the HUVEC monolayer, and preincubation of the tracer with either ns (22.5 ± 1.0%) or ps (20.2 ± 0.7%) IGFBP-3 had no effect on its rate of transport. MonoiodoIGF-I, when preincubated with ns IGFBP-3 and ALS (10.7 ± 0.9%), was transported across the HUVEC monolayer at a significantly reduced rate compared with the monoiodoIGF-I alone (P < 0.0001). The ps IGFBP-3 ternary complex also inhibited the transport of monoiodoIGF-I across the monolayer compared with the tracer alone, reducing the amount transported to 16.3 ± 1.2% (P = 0.0002). However, the ps IGFBP-3 ternary complex was not as effective as the ns IGFBP-3 ternary complex in limiting IGF-I transport (P = 0.0001). Thus, whereas proteolyzed IGFBP-3 still forms a ternary complex apparently normally, this complex is less inhibitory to IGF transport than the complex containing intact IGFBP-3.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IGFBPs are believed to play an important role in directing IGFs from the circulation to their target tissues (37, 38). This extravascular IGF transport is believed to be limited by the capillary endothelial barrier, which is thus central in regulating the bioavailability of circulating IGFs (39). HUVEC monolayers have been used extensively to study transendothelial transport of various serum proteins due to the ready availability of cord tissue and their ease of isolation (29, 32). Other studies have previously used this model to investigate the transport of IGFs across an endothelial cell monolayer. Bastian et al. (40) showed that IGF transport across a HUVEC monolayer was not mediated by IGF cell surface receptors, indicating that the IGFs were transported via a paracellular route. Other studies have also indicated that, whereas the IGFs may bind to the subendothelial matrix, they are still transported across the endothelial cell monolayer through paracellular gaps (32). However, it has been reported that IGF-I bound to IGFBP-3 alone is inhibited in its rate of transport across a HUVEC monolayer (33), whereas we found no difference in the rate of IGF transport for IGF-I or IGF-II associated with IGFBP-3 or IGFBP-5 alone. It is possible that this difference is due to variable production of endogenous IGFBPs by HUVECs from different sources because our data showed no significant difference between the permeability coefficients of IGF-I and BSA, suggesting that the IGF-I may partly be in a complexed form with endogenous IGFBPs. Also, different adhesion compounds were used to attach the cells to the transwells, possibly influencing the size of the paracellular gap junctions in the confluent cell monolayer. However, the integrity of the monolayers used in these experiments was confirmed by calculating the permeability coefficient of inulin, with the values obtained being similar to previously reported values for confluent HUVEC monolayers (30, 41).

We have demonstrated in this study that the transport of IGFs across an endothelial cell monolayer is inhibited by their formation into ternary complexes with either IGFBP-3 or IGFBP-5 and ALS. This inhibition was observed after 4 h but was not obvious after 2 h, suggesting that some initial dissociation of ternary complexes may have occurred upon their dilution into the transwells. This dissociation may reflect the fact that, for technical reasons, these experiments are performed at much lower binding protein concentrations than the high ternary complex concentration (100 nM) found in the circulation. It is also possible that initial association of IGF-I with cells, Cell-Tak, or plastic may in part affect the linearity of transport rates. Whereas no other studies have used in vitro models specifically to study the transport of IGFs in the ternary complex, several in vivo studies have focused on the role of the ternary complex in reducing the rate of IGF clearance from the circulation. Studies in humans have demonstrated that free IGF-I (that is, not associated with any IGFBPs) is rapidly cleared from the circulation with a half-life of approximately 14 min (42). Earlier studies in sheep indicated that, whereas the rate of clearance of binary-complexed IGFs could be measured in minutes, the rate of clearance of ternary-complexed IGFs was measurable in hours (7). Similarly, in rats, the formation of IGF-I into the ternary complex strongly promotes the retention of IGF-I in the serum, whereas IGFs associated with IGFBPs alone are more rapidly cleared from the circulation (43). In this study, we have demonstrated that IGFs incubated with both IGFBP-3 and ALS are transported across an endothelial cell monolayer significantly more slowly than uncomplexed IGFs, supporting the idea that ternary complex formation restricts IGF movement across the capillary endothelium.

The importance of the ALS interaction with IGFBP-3 binary complex was further shown by using an IGFBP-3 mutant with low ALS affinity, which was unable to retard IGF transport in the presence of ALS, whereas the wild-type protein was able to do so. Several studies in vivo have examined the significance of ALS in the IGFBP-3/IGF serum dynamics. Mice with an ALS gene deletion had a 62% reduction in their serum IGF levels, suggesting the importance of the ternary complex in maintaining a pool of serum IGFs (44). Interestingly, the mutant mice showed only a slight reduction in growth rate and no differences in their glucose and insulin homeostasis compared with wild-type mice. Similarly, we recently showed that, whereas equimolar IGFBP-3 blocks the hypoglycemic action of injected IGF-I in rats, an equal concentration of IGFBP-3mut was considerably less effective (45), confirming that inhibition of IGF-induced hypoglycemia by IGFBP-3 depends explicitly on ternary complex formation with ALS. Thus, the bioavailability of circulating IGFs is regulated by forming complexes with IGFBP-3 and ALS.

The role of IGFBP-3 proteolysis in regulating serum IGF bioavailability during pregnancy has remained contentious. Despite apparently complete proteolysis of IGFBP-3 (15, 16), IGFs and IGFBP-3 itself are transported normally in high molecular weight serum complexes (18, 46), and there is only a small increase in the proportion of SF IGFs, from 1–2% (19). Other studies, using 125I-IGF-I to examine serum IGF distribution, have found that the amount of free IGFs in ps is increased relative to ns (47, 48). However, the interpretation of these results is difficult given that different iodoforms of IGF-I bind to proteolyzed IGFBP-3 and ALS with different affinities, whereas native, noniodoinated IGF-I shows no difference in the formation of ternary complexes with either ps or ns IGFBP-3 (46). IGF-I has three tyrosine residues that can be iodinated, resulting in a heterogenous population of mono-, di-, and tri-iodinated forms of IGF-I (26). As shown here, only one of these forms, [Tyr31]monoiodo-IGF-I, forms into ternary complexes with equal efficiency with both ns- and ps-derived IGFBP-3 and thus was used in the transport experiments (24). IGFs in the ps IGFBP-3 ternary complex were transported across the cell monolayer significantly faster than IGFs in the ns IGFBP-3 ternary complex. This effect is unlikely to be an artifact resulting from errors in quantitating the purified ns and ps IGFBP-3 forms because we have previously shown that our IGFBP-3 RIA detects IGFBP-3 similarly before and after proteolysis by the ps protease (46). Although the rate of ternary complex formation is equal for both ns and ps IGFBP-3, the rate of IGF dissociation may not be equal, resulting in a larger transient pool of free IGFs that may be transported across the monolayer. Alternatively, the proteolyzed IGFBP-3 may more readily bind to other factors, such as a cell surface molecule, which may lead to the dissociation of the ternary complex and the subsequent release of the trapped IGFs. This idea is supported by the IGFBP-3 mutant, altered in residues 228–232, which, in addition to having decreased ALS affinity, also has reduced binding to cell surfaces (21). This indicates that the ALS-binding determinants and cell-binding determinants of IGFBP-3 may be located within the same region of the protein, leading to competition for binding to IGFBP-3. The proteolysis of IGFBP-3 by the serum protease in ps could increase the ability of the surface binding component to bind to IGFBP-3 and thus be a factor promoting the release of IGFs at the cell surface.

We have also shown for the first time that ternary complexes containing IGFBP-5, like those with IGFBP-3, retard IGF transport across endothelial cells. Interestingly, IGFBP-5 ternary complexes were not as efficient as IGFBP-3 complexes in restricting IGF-I transport. This is consistent with our observation that a smaller proportion of IGFBP-5 in the circulation is found in ternary complexes, compared with IGFBP-3 (6). Again, it is possible that the factor contributing to this difference may be the relative dissociation rate of IGF-I from IGFBP-3 and IGFBP-5. BIAcore analysis of IGF-I binding to IGFBP-5 and IGFBP-3 suggested that IGF-I dissociated from IGFBP-5 more rapidly than IGFBP-3, although this result was not statistically significant (49). Alternatively, IGFBP-5 may interact with the endothelial cell surface, thus contributing to the release of the IGFs bound within the ternary complex. IGFBP-5/IGF binary complexes have been reported to bind to cell surfaces, presumably via a specific cell surface receptor (50).

In summary, this study confirms in vitro the importance of the formation of the ternary complex with ALS and either IGFBP-3 or -5 to retard transendothelial transport of IGFs, with IGFBP-5 being less effective than IGFBP-3, despite their similar ternary complex formation. Whereas cell-derived proteases did not appear to affect transport in this model, IGFBP-3 already affected by protease action in ps was less effective than intact IGFBP-3 in retarding IGF transport, despite similar ternary complex formation. These observations indicate that the formation of IGFs, IGFBPs, and ALS into ternary complexes, although an important factor in the regulation of IGF transport, is not the only factor that regulates this process. Finally, this cell model may be useful to study the effect of other proteases on the stability of the ternary complex and to determine whether they also promote IGF transport.


   Acknowledgments
 
The collection of the ps samples by Mrs. Rosemary Hitchman is gratefully acknowledged.


   Footnotes
 
This work was supported by the National Health and Medical Research Council, Australia (Grant 990005 to R.C.B. and S.M.F.).

Abbreviations: ALS, Acid-labile subunit; HUVEC, human umbilical vein endothelial cell; IGFBP, IGF binding protein; IGFBP-3mut, IGFBP-3 mutant; ns, nonpregnancy serum; PC, permeability coefficients; ps, pregnancy serum; rh, recombinant human; SF, serum-free; TCA, trichloroacetic acid.

Received October 28, 2003.

Accepted February 1, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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