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Molecular Endocrinology, doi:10.1210/me.2006-0522
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Molecular Endocrinology 21 (5): 1246-1257
Copyright © 2007 by The Endocrine Society

Regulation of Insulin-Like Growth Factor (IGF) Bioactivity by Sequential Proteolytic Cleavage of IGF Binding Protein-4 and -5

Lisbeth S. Laursen, Kasper Kjaer-Sorensen, Mikkel H. Andersen and Claus Oxvig

Department of Molecular Biology, University of Aarhus, DK-8000 Århus C, Denmark

Address all correspondence and requests for reprints to: Claus Oxvig, Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark. E-mail: co{at}mb.au.dk.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The biological activity of IGF-I and -II is controlled by six binding proteins (IGFBPs), preventing the IGFs from interacting with the IGF receptor. Proteolytic cleavage of IGFBPs is one mechanism by which IGF can be released to bind the receptor. The IGFBPs are usually studied individually, although the presence of more than one of the IGFBPs in most tissues suggests a cooperative function. Thus, the IGFBPs are part of regulatory networks with proteolytic enzymes in one end and the IGF receptor in the other end. We have established a model system that allows analysis of the dynamics between IGF, IGFBP-4 and -5, the IGF receptor, and the proteolytic enzyme PAPP-A, which specifically cleaves both IGFBP-4 and -5. We demonstrate different mechanisms of IGF release from IGFBP-4 and –5: cooperative binding to IGF is observed for the proteolytic fragments of IGFBP-5, but not fragments of IGFBP-4. Furthermore, we find that PAPP-A-mediated IGF-dependent cleavage of IGFBP-4 is inhibited by IGFBP-5, which sequesters IGF from IGFBP-4, and that cleavage of both IGFBP-4 and -5 is required for the release of bioactive IGF. Finally, we show that cell surface-localized proteolysis of IGFBP-4 represents the final regulatory step of efficient IGF delivery to the receptor. Our data define a regulatory system in which molar ratios between the IGFBPs and IGF and between the different IGFBPs, sequential proteolytic cleavage of the IGFBPs, and surface association of the activating proteinase are key elements in the regulation of IGF receptor stimulation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IGF-I AND -II ARE regulators of cell proliferation, migration, and differentiation through stimulation of the IGF receptor (IGF receptor type 1) (1). The biological activities of the IGFs are modulated by a family of six IGF binding proteins (IGFBP-1 to -6), which bind the IGFs with higher affinity than the receptor (2, 3). In many systems, proteolytic cleavage of the IGFBPs in the central domain causes the release of bioactive IGF (4). However, a number of recent experiments have shown that recombinantly expressed N- and C-terminal domains of IGFBPs both bind IGF (5, 6), and studies based on mutagenesis (7, 8) in combination with recent structural data (9, 10, 11) have identified the residues of these domains involved in IGF binding. But details of how the naturally occurring fragments resulting from proteolysis interact with IGF and how the IGFs are released from the fragments are limited. Indeed, recent crystal structures have questioned whether IGF is actually released from proteolytically cleaved IGFBP-4, because a direct interaction between the N- and C-terminal fragment in a ternary complex with IGF was shown (11, 12). Furthermore, the IGF binding affinities reported for recombinantly expressed domains of variable length differ, emphasizing the importance of analyzing natural IGFBP fragments generated by proteolytic cleavage (11, 12, 13).

Pregnancy-associated plasma protein-A (PAPP-A) is the founding member of a newly recognized family of the metzincin superfamily of metalloproteinases, the pappalysins (14, 15). PAPP-A has been shown to function in the IGF system by specific cleavage of both IGFBP-4 (16) and IGFBP-5 (17) at a single site in the central domain, resulting in the generation of two fragments of similar molecular weight (18). Of particular importance, PAPP-A-mediated proteolysis of IGFBP-4 strictly depends on the presence of IGF (17).

IGFBP-5 either enhances or inhibits the biological effects of IGF, depending on the system analyzed (19). The mechanisms by which IGFBP-5 potentiates effects of IGF remain uncertain, but have been suggested to involve interactions of IGFBP-5 with extracellular matrix proteins (20, 21). Interestingly, IGFBP-4 is generally reported to inhibit IGF activity, but it was recently shown that targeted deletion of the mouse gene encoding IGFBP-4 causes an impairment of growth (22).

Most tissues express more than one of the IGFBPs at the same time and, typically, the binding proteins are present in a molar excess of IGF (23). Thus, the relative affinities and binding kinetics of the individual binding proteins toward IGF, their relative expression levels, and the presence of specific proteolytic activity generating binding protein fragments determine to which binding proteins IGF is bound. Specifically, IGFBP-4 and -5 are coexpressed in several tissues by different cell types, including osteoblasts (24), granulosa cells (25), and vascular smooth muscle cells (26, 27). In tissues of these cells, PAPP-A proteolytic activity is believed to be a regulator of IGF activity (28, 29, 30).

Here, we first establish a model allowing experimental analysis of the regulatory network that includes the IGF receptor, its ligands IGF-I and -II, IGFBP-4 and -5, and PAPP-A. Analysis of receptor stimulation, binding protein proteolysis, and properties of the proteolytic fragments allows us to suggest a mechanism of IGF release, in which IGFBP-4 and -5 are sequentially cleaved and IGFBP-4 is critical in the final release of bioactive IGF causing receptor stimulation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Proteolytic Release of Bioactive IGF Differs between IGFBP-4 and -5
To allow quantification of bioactive IGF after PAPP-A-mediated IGFBP proteolysis, we stably transfected and cloned human embryonic kidney 293T cells to express high levels of the IGF receptor. Using the clone 293-IGFR(clone H) (Fig. 1AGo), a dose-dependent increase in IGF receptor phosphorylation after stimulation for 30 min with IGF-I (1–20 nM) was obtained (Fig. 1BGo). Importantly, because no decrease in receptor phosphorylation was observed over a stimulation period of 30 min (Fig. 1CGo), the increase in IGF receptor phosphorylation can be used as a measure of the release of bioactive IGF caused by proteolysis during such period of time.


Figure 1
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  		Fig. 1. Assessment of IGF Receptor Stimulation over Time

A, A cloned cell line with high expression of the IGF receptor, 293-IGFR(clone H), was obtained by stably transfecting 293T cells with IGF receptor cDNA. Flow cytometry of nontransfected 293T cells (left and center panels), and 293-IGFR(clone H) cells (right panel) is shown. A primary antibody (mAb 24–55) against the IGF receptor and secondary fluorescein isothiocyanate-conjugated antibodies were used. B, 293-IGFR(clone H) cells were treated (30 min) with increasing concentrations of IGF-I, as indicated, and the degree of IGF receptor phosphorylation was determined by Western blotting using a phosphotyrosine-specific antibody (mAb PY99) (upper panel). In a control experiment (lower panel), the level of IGF receptor in the same samples was monitored using an IGR receptor-specific antibody (mAb CT-1). The positions of the ß-subunit of the mature IGF receptor (IGFRß) and the unprocessed receptor (proIGFR) are indicated, according to previous experiments (47 ). The position of IRS1 is tentative. C, A similar experiment in which the concentration of IGF-I was kept constant (10 nM), and the time period of stimulation was varied, as indicated.

 
We treated 293-IGFR(clone H) cells with IGF, variable amounts of IGFBP, and catalytic amounts of PAPP-A for up to 30 min. The molar amounts of reagents were carefully determined by means of amino acid analysis of the purified proteins. In cells treated with IGFBP in excess of IGF-I, a substantially higher level of receptor phosphorylation was observed with IGFBP-4 compared with IGFBP-5 (Fig. 2AGo). The difference in receptor phosphorylation becomes even more pronounced when the level of receptor phosphorylation is correlated with the degree of binding protein proteolysis, because proteolysis of IGFBP-5 progressed much more rapidly than proteolysis of IGFBP-4 (Fig. 2AGo). The more efficient release of IGF-I from IGFBP-4 could be a result of at least two different mechanisms: 1) PAPP-A-mediated IGFBP-4 proteolysis depends strictly on the presence of IGF, whereas proteolysis of IGFBP-5 is independent of IGF-I. Thus, in contrast to IGFBP-5, cleavage of IGFBP-4 invariably causes release of IGF, or 2) proteolytic fragments of IGFBP-5 bind IGF with higher affinity than fragments of IGFBP-4, resulting in less efficient generation of bioactive IGF from IGFBP-5/IGF.


Figure 2
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  		Fig. 2. PAPP-A-Mediated Proteolytic Release of IGF from IGFBP-4 and -5

A, Cells [293-IGFR(clone H)] were stimulated with IGF (10 nM) in the presence of IGFBP-4 (40 nM) or -5 (40 nM), and PAPP-A (0.6 nM). The degree of IGF receptor phosphorylation at different time points was determined and normalized to the level of receptor phosphorylation with IGF-I (10 nM) alone (left panel). The data represent an average of three independent experiments ± 2 SDs. In parallel, the degree of binding protein cleavage was determined at different time points (right panel). Trace amounts of radiolabeled IGFBP-4 or -5 were added to allow quantification by autoradiography following separation of cleaved and intact protein by SDS-PAGE (17 ). The amount of remaining intact binding protein at each time point was plotted, as indicated. The data shown are representative of three independent experiments with similar results. B, A similar experiment, in which the concentrations of IGFBP-4 and -5 were 10 nM.

 
A similar experiment was carried out using equimolar amounts of binding proteins and IGF (Fig. 2BGo). Under these conditions, only a minor difference in receptor phosphorylation was observed for IGFBP-4 and -5. However, the difference in proteolysis was even more pronounced in this experiment, suggesting that the difference in IGF requirement for proteolysis of IGFBP-4 and -5 cannot alone be responsible for the difference in IGF release from cleaved IGFBP-4 and -5.

Analysis of PAPP-A-Generated IGFBP Fragments by Surface Plasmon Resonance
To further characterize the mechanisms involved in proteolytic release of IGF from IGFBP-4 and -5, we purified binding protein fragments generated by PAPP-A cleavage (Fig. 3AGo). To confirm cleavage at a single site in the central domain (17, 18) and to identify the purified fragments, N-terminal sequence analysis was carried out before and after the chromatographic separation (data not shown). The integrity and purity of the purified fragments was further verified by SDS-PAGE (Fig. 3BGo) and Western blotting (Fig. 3CGo).


Figure 3
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  		Fig. 3. Purification of Proteolytic Fragments of IGFBP-4 and -5

A, Separation by reversed-phase HPLC of proteolytic fragments of IGFBP-4 (left panel) and -5 (right panel) generated by cleavage with PAPP-A. Monitoring was at 280 nm. B, SDS-PAGE of intact and cleaved IGFBP-4 (lanes 1–4) and -5 (lanes 5–8), as indicated. The Coomassie-stained gel shows uncleaved proteins (lanes 1 and 5), nonpurified comigrating fragments before separation (lanes 2 and 6), and C-terminal (lanes 3 and 7) and N-terminal (lanes 4 and 8) fragments after separation by HPLC. The positions of intact (i) and proteolytic cleavage fragments (c) are indicated. C, The same samples were analyzed by Western blotting to verify the identities of the purified fragments of IGFBP-4 and -5 using a monoclonal antibody (mAb 9E10) specific for the C-terminal c-myc tag. C-term, C-terminal; N-term, N-terminal.

 
The binding kinetics for the interactions between the intact binding proteins or their isolated fragments and immobilized IGF-I and -II were analyzed by surface plasmon resonance (Fig. 4Go and Table 1Go). Dissociation constants for the interactions of intact IGFBP-5 showed high-affinity binding to IGF-I (KD = 0.3 nM), and even tighter binding to IGF-II (KD = 0.04 nM). In both cases, a very fast association rate and a slow dissociation rate suggested that highly stable complexes were formed. The dissociation constants for the interactions between both of the IGFBP-5 fragments and IGF-I and -II were reduced several fold compared with intact IGFBP-5. Interestingly, however, the C-terminal fragments of IGFBP-5 retain a relatively high affinity for IGF-I compared with IGF-II (KD = 21 nM vs. KD = 183 nM), whereas the N-terminal fragment of IGFBP-5 bound IGF-I weaker than IGF-II (KD = 49 nM vs. KD = 7 nM).


Figure 4
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  		Fig. 4. Binding of IGF-I to Intact Binding Proteins and Proteolytic Fragments

Interactions with immobilized IGF-I were analyzed by surface plasmon resonance using intact IGFBP-4 and -5 and their purified proteolytic fragments generated by PAPP-A, as indicated. Analyte concentrations were 50, 25, 12.5, 6.25, 3.13, and 1.56 nM of intact binding proteins, or 200, 100, 50, 25, 12.5, and 6.25 nM of proteolytic fragments, except for the N-terminal fragment of IGFBP-4, for which the two lowest concentrations were not used. The analytes were injected over an IGF-I surface with a flow rate of 20 µl/min for 4 min followed by a 5-min disassociation phase. The sensograms shown are representative of two to four experiments. Resulting kinetic parameters are presented in Table 1Go. C-term, C-terminal; N-term, N-terminal; s, sec.

 

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  		Table 1. Kinetic Constants of Interactions of IGFBP-4 and -5 and Their Proteolytic Fragments with Immobilized IGF-I and -II

 
Intact IGFBP-4 showed a more than 10-fold higher affinity for IGF-II (KD = 0.2 nM) than IGF-I (KD= 4.5 nM). The stronger binding to IGF-II was also reflected in the affinities measured for the N-terminal fragment of IGFBP-4. In contrast to IGFBP-5, low-affinity binding was observed for the C-terminal fragment of IGFBP-4 toward IGF-I, but the C-terminal fragments of IGFBP-4 and -5 showed similar affinities toward IGF-II (Table 1Go).

In summary, the affinity of the proteolytic fragments of IGFBP-4 and -5 generated by PAPP-A showed a 20- to 1000-fold reduction in affinity for IGF-I and -II compared with the uncleaved binding proteins. Importantly, some of the proteolytic fragments retained binding affinities comparable to those measured for the IGF receptor toward IGF-I (KD = 4.45 nM) and IGF-II (KD = 23 nM), respectively (31), suggesting that IGF binding to these fragments may influence binding of IGF to the receptor.

Inhibition of IGF Receptor Phosphorylation by Proteolytic Fragments of IGFBP-4 and -5
To analyze the effect of the proteolytic fragments on IGF receptor stimulation, cells were treated with different combinations of IGF and intact binding proteins or purified fragments of these, and the degree of receptor phosphorylation was measured (Fig. 5Go). Surprisingly, all of the four proteolytic fragments inhibited receptor stimulation, the IGFBP-5 fragments being more efficient than fragments of IGFBP-4. Of particular interest, the presence of both N- and C-terminal fragments of IGFBP-5 inhibited receptor phosphorylation by 70%. A similar cooperative effect was not observed with the fragments of IGFBP-4. This suggests that the presence of both of the IGFBP-5 fragments may stabilize the interaction with IGF, explaining the slow proteolytic release of IGF from cleaved IGFBP-5 compared with IGFBP-4 (Fig. 2Go). We therefore conclude that proteolysis results in destabilization of the IGFBP-IGF complex, but not necessarily immediate release of IGF.


Figure 5
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  		Fig. 5. Inhibition of IGF Receptor Phosphorylation by Binding Protein Fragments

Cells [293-IGFR(clone H)] were stimulated (10 min) with IGF-I (10 nM) in the absence or presence of intact binding proteins (BP), or N-terminal (N) and C-terminal (C) proteolytic fragments alone or in combination. The experiments were carried out with IGFBP-4 and -5 and fragments hereof, as indicated, and the total concentration of binding proteins and fragments was 60 nM. The degree of IGF receptor stimulation is expressed relative to the level obtained with IGF-I alone. Plotted values are averages of three independent experiments ± 2 SDs. No inhibition of IGF receptor phosphorylation was observed when the period of stimulation was increased to 30 min.

 
IGFBP-5 Inhibits Proteolysis of IGFBP-4 by Sequestering IGF
Binding analyses (Fig. 4Go and Table 1Go) showed tighter binding of IGF to IGFBP-5 than IGFBP-4, a difference that can be ascribed mainly to a very high association rate for the interaction between IGF and IGFBP-5. This suggests that when IGFBP-4 and -5 are present in excess of IGF, the majority of IGF will be bound to IGFBP-5. The preference of IGF for IGFBP-5 potentially influences the mechanism involved in proteolytic release of IGF, as cleavage of IGFBP-4 by PAPP-A strictly requires that it binds IGF (17). To test experimentally whether IGF in solution initially is bound to IGFBP-5, we took advantage of the IGF dependency of PAPP-A-mediated IGFBP-4 cleavage and the fact that IGFBP-5 is also cleaved by PAPP-A, but independent of IGF (17). We found that in the presence of both binding proteins in excess of IGF, PAPP-A preferably cleaves IGFBP-5 (Fig. 6Go). Almost no cleavage of IGFBP-4 was observed until more than half of the IGFBP-5 was cleaved, demonstrating that IGFBP-4 competes for IGF only with the fragments of IGFBP-5, in agreement with the binding data (Table 1Go). Accordingly, the cleavage of IGFBP-5 was not influenced by the presence of IGFBP-4 (Fig. 6Go). Furthermore, saturating the binding proteins with IGF reversed the inhibitory effect of IGFBP-5 on IGFBP-4 proteolysis (data not shown). We therefore conclude that IGFBP-5 inhibits proteolysis of IGFBP-4 by sequestering IGF from IGFBP-4, implicating that cleavage of IGFBP-4 occurs only after IGFBP-5 has been cleaved.


Figure 6
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  		Fig. 6. IGFBP-5 Inhibits Proteolysis of IGFBP-4 by Sequestering IGF

The cleavage by PAPP-A (0.6 nM) of IGFBP-4 and -5 alone or together, as indicated, was analyzed over time. IGF-I was used at a concentration of 10 nM, and the concentration of each of the binding proteins was 20 nM when used together, or 40 nM when used alone. In each experiment, proteolytic cleavage of one of the two binding proteins was monitored by the addition of trace amounts of radiolabeled IGFBP-4 or -5, indicated by an asterisk. Quantification of cleavage was carried out by autoradiography after separation of cleaved and intact protein by SDS-PAGE. The level of remaining intact binding protein at each time point was plotted. The data shown are representative of three independent experiments with similar results.

 
Sequential Cleavage of IGFBP-5 and -4 Is Required for the Proteolytic Release of IGF
If proteolysis of IGFBP-4 occurs only after cleavage of IGFBP-5, cleavage of both binding proteins is required for IGF to become available to the IGF receptor. To test this hypothesis, the release of IGF by the cleavage of wild-type IGFBP-4 or -5 and variants of IGFBP-4 or -5 mutated to resist proteolytic activity (18) was analyzed. Release of IGF was measured as the ability to cause receptor stimulation. Proteinase-resistant IGFBP-5 completely blocked the release of bioactive IGF from wild-type IGFBP-4 (Fig. 7AGo), an effect that was due to a lack of both IGFBP-5 and -4 proteolysis (Fig. 7BGo). In addition, proteinase-resistant IGFBP-4 and wild-type IGFBP-5 also almost completely blocked IGF release (Fig. 7AGo), an effect that results from the lack of IGFBP-4 proteolysis, because the IGFBP-4 mutant showed no effect on proteolysis of IGFBP-5 (Fig. 7BGo). This demonstrates that for the release of IGF, cleavage of both IGFBP-4 and -5 is required. Stimulation of the cells with IGF-I in the presence of PAPP-A and a combination of wild-type IGFBP-4 and -5 caused receptor phosphorylation that was equal to the phosphorylation after stimulation in the presence of IGFBP-4 alone, and slightly increased in comparison with stimulation in the presence of IGFBP-5 (Fig. 7AGo). In conclusion, the requirement of IGF for proteolysis of IGFBP-4 in combination with a higher affinity of IGFBP-5 for IGF makes proteolysis of IGFBP-4 the final regulatory step in the release of bioactive IGF.


Figure 7
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  		Fig. 7. Release of Bioactive IGF Requires Proteolytic Cleavage of both IGFBP-4 and -5

A, Cells were stimulated (30 min) with IGF-I (10 nM) in the absence or presence of wild-type (BP4 and BP5) or mutated (BP4m and BP5m) binding proteins alone or in combination, as indicated. The concentration of each of the binding proteins was 20 nM when used together, or 40 nM when used alone. PAPP-A (PA) was included in some experiments at a concentration of 0.6 nM. The mutated binding proteins were constructed to completely resist proteolysis by PAPP-A [BP4m, R126A/R128A/K134A/K136A and BP5m, K128A (18 )]. A control experiment without IGF is also shown (PBS). The degree of IGF receptor stimulation is expressed relative to the level with IGF-I alone. Plotted values are averages of three independent experiments ± 2 SDs. B, Mixtures of wild-type and proteinase resistant IGFBP mutants were incubated with PAPP-A (0.6 nM) in the presence of IGF-I (10 nM). The concentration of each of the binding proteins was 20 nM. Proteolytic cleavage of wild-type IGFBP-4 or -5 was monitored by the addition of trace amounts of radiolabeled protein, indicated by an asterisk (*), followed by SDS-PAGE and autoradiography, to allow plotting of the level of remaining intact binding protein at each time point. The data shown are representative of three independent experiments with similar results.

 
Cell Surface-Localized IGFBP Proteolysis Enhances Receptor Stimulation
Previous data show that PAPP-A is able to bind to the cell surface by means of an unknown heparan sulfate proteoglycan (32), and chromatographic analysis suggested that it binds with high affinity (33). In turn, proteolysis at the cell surface in close proximity to the IGF receptor has been hypothesized to be an effective mechanism by which stimulation of the IGF receptor is regulated (32). To test the effect of cell-localized proteolysis on IGF receptor activation, we compared the effect of wild-type PAPP-A with a mutated PAPP-A variant unable to bind the cell surface, PAPP-A(P2SCR3) (32). In this mutant, the heparan sulfate-binding SCR3 module in the C terminus of PAPP-A is replaced with the corresponding sequence of PAPP-A2, which is unable to bind to the cell surface. An observed difference in the ability of wild-type PAPP-A and PAPP-A(P2SCR3) to cause receptor stimulation can therefore be ascribed to the ability vs. lack of ability to bind to the cell surface.

After preincubation of cells for 5 min with buffer containing 0.6 nM PAPP-A, no reduction in the concentration of PAPP-A in the buffer could be detected (data not shown), demonstrating that only a very small fraction of PAPP-A had become bound to the surface. Based on the accuracy of the determination of the PAPP-A concentration, we conservatively estimate this fraction to be less than 5%. Yet, wild-type PAPP-A more efficiently caused receptor stimulation than PAPP-A(P2SCR3) after the addition of IGFBP-4/IGF-I (Fig. 8AGo). This was apparent at all tested ratios of IGFBP-4 to IGF with an increasing relative effect of surface proteolysis at increasing IGFBP-4:IGF-I ratios. The observed difference in receptor stimulation was not an effect of a difference in proteolytic activity of wild-type PAPP-A and the non-cell surface-binding PAPP-A(P2SCR3), because their activity against IGFBP-4 could not be distinguished (Fig. 8BGo). The effect of surface proteolysis was highest at a molar IGFBP-4 to IGF-I ratio of 4:1 (stimulation increased by 83%), but the effect of surface proteolysis observed at equimolar amounts of IGFBP-4 and IGF-I was still apparent. The molar amount of IGF-I released is approximately the same at all three ratios of IGFBP-4 to IGF-I (Fig. 8BGo). However, at the 4:1 ratio, IGF-I released by proteolysis has a relatively high probability of binding an intact IGFBP-4 molecule rather than the IGF receptor, unless the cleavage occurs in proximity to the receptor. Hence, the effect of surface proteolysis is more pronounced at the higher IGFBP-4 to IGF-I ratio. It should be stressed that the difference in stimulation obtained with wild-type PAPP-A and PAPP-A(P2SCR3) is caused by the small fraction of the wild-type protein that is bound to the cell surface. No effect of PAPP-A surface binding on IGF receptor phosphorylation was observed in the absence of IGFBP-4 (data not shown), suggesting that in addition to increasing the probability that released IGF binds the IGF receptor rather than becomes inactivated by another binding protein, cleavage in the proximity to the receptor may involve IGF delivery to the receptor.


Figure 8
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  		Fig. 8. Surface-Localized PAPP-A Activity Enhances IGF Receptor Stimulation

A, Cells were preincubated (5 min) with 0.6 nM PAPP-A, 0.6 nM PAPP-A(P2SCR3), a variant unable to bind to the cell surface, or buffer, and then stimulated (30 min) with IGF-I (10 nM) and increasing concentrations of IGFBP-4 (10, 20, or 40 nM, as indicated by the ratios). The degree of receptor phosphorylation is expressed relative to the phosphorylation measured with 10 nM IGF-I alone. The values are averages of three independent experiments ± 2 SDs. A significant increase in the stimulation with wild-type PAPP-A over PAPP-A(P2SCR3) was observed. At the IGFBP-4 to IGF-I ratio of 1:1, the increase was 33% (P < 0.02), at the 2:1 ratio, the increase was 58% (P < 0.005), and at the 4:1 ratio, the increase was 83% (P < 0.005). B, Comparison of IGFBP-4 proteolysis mediated by PAPP-A (solid symbols) and PAPP-A(P2SCR3) (open symbols). The amounts of IGF-I and IGFBP-4 were as specified in panel A. Trace amounts of radiolabeled IGFBP-4 were added to the reactions, and samples were taken out at various time points from 0–30 min. The degree of cleavage was determined by autoradiography after separation of intact and cleaved IGFBP-4 by SDS-PAGE. The experiments shown are representative of three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The biological activity of IGF is regulated by a unique extracellular regulatory network including six binding proteins (IGFBPs) and proteolytic enzymes capable of inactivating the IGFBPs. The expression of more than one type of binding protein in most tissues suggests that individual IGFBPs have different roles in the regulation of IGF activity. Differences in IGF affinities, different kinetics of IGF release from the binding proteins after proteolytic cleavage, and different proteolytic enzymes that differ in substrate specificity all represent possible mechanisms that allow fine tuning of IGF receptor stimulation. In this study, we have begun to address how stimulation of the IGF receptor is controlled by the molecular network that includes the IGF receptor, its ligands IGF-I and -II, IGFBP-4 and -5, and PAPP-A. Of importance, IGFBP-4 and -5 are both substrates of PAPP-A. However, cleavage of IGFBP-4, but not IGFBP-5, requires bound IGF.

Initially, we observed an unexpected difference in the release kinetics of IGF after proteolysis of IGFBP-4 and -5, respectively. We found that proteolysis of IGFBP-4, but not IGFBP-5, correlated well with IGF receptor stimulation, most likely caused by the observed difference in how the N- and C-terminal fragments cooperate in the binding of IGF after proteolytic cleavage. In combination with a higher affinity of IGFBP-5 for IGF, this suggests different roles for IGFBP-4 and -5 in the regulation of IGF bioactivity. Proteolysis of IGFBP-5 is fast, does not depend on the presence of IGF, and is reported to be mediated by many different proteinases, including PAPP-A (17), PAPP-A2 (34), ADAM-9 (35), and C1s (36), whereas proteolysis of IGFBP-4 strictly depends on IGF and may be mediated only by PAPP-A in vivo (28). Additionally, efficient cell surface-mediated proteolytic release of IGF from IGFBP-4 is observed. We therefore propose a model in which IGF initially is found associated with IGFBP-5. In this model, cleavage of IGFBP-5 is the initial step in the release of IGF and allows free IGF to interact with IGFBP-4, which, in turn, is responsible for the final delivery step, involving its cleavage by PAPP-A in proximity to the cell surface.

It should be emphasized that PAPP-A is capable of cleaving both IGFBP-4 and -5, but that other proteinases may contribute to the cleavage of IGFBP-5 within tissues. Furthermore, is has been found that IGFBP-5 can be associated with extracellular matrix, and that this reduces its affinity toward IGF (37). Even though this reduction is less dramatic than the reduction caused by proteolysis, such mechanism may function alone in the absence of IGFBP-4. In the presence of IGFBP-4, extracellular matrix-mediated release of IGF may function together with IGFBP-4 and PAPP-A. Protection of IGFBP-5 proteolysis by extracellular matrix components with the ability to bind IGFBP-5 has also been proposed. In our hands, however, neither osteopontin (20) nor vitronection (21) in molar excess of IGFBP-5 showed any effect on IGFBP-5 cleavage mediated by PAPP-A (Kjaer-Sorensen, K., L. S. Laursen, and C. Oxvig, unpublished observations).

A critical function of IGFBP-4 in the final delivery of IGF is further supported by the phenotype of the IGFBP-4 knockout mouse, which, in contrast to the IGFBP-3 and -5 knockout mice, shows an impairment of growth (22). In addition, systemic administration of IGFBP-4 has been shown to increase the local bioavailability of IGF in bone (38).

X-ray crystallographic analyses of the interactions between separately expressed N-terminal domains of IGFBP-4 and -5 have suggested that even though such domains may provide most of the binding energy of the IGFBP-IGF complexes, residues of IGF known to be important for receptor binding are not shielded in such artificial complexes (9, 11). In contrast, the nuclear magnetic resonance data of the C-terminal of IGFBP-6 (39) and full-length IGFBP-2 (40) in complex with IGF show that these residues are partially covered by the C-terminal domain. Based on these observations, IGF in complex with the N-terminal domain might still be able to bind to the IGF receptor, whereas IGF in complex with the C-terminal domain is not. Analyses by surface plasmon resonance showed that both N- and C-terminal proteolytic fragments generated by PAPP-A are able to bind IGF (Fig. 4Go and Table 1Go), and even though the measured binding affinities vary, both fragments are able to prevent IGF receptor stimulation. This points at a potentially important role for the part of the central domains, present in natural PAPP-A-generated fragments, but not in the artificial domains, in masking the IGF receptor binding sites. The inhibition of fragments on IGF receptor stimulation was transient, most likely due to a much lower stability of the IGF-IGFBP fragment complex compared with IGF in complex with the intact binding proteins, in accordance with the binding affinities (Table 1Go).

An interesting difference between the proteolytic fragments of IGFBP-4 and -5 was the finding that a combination of the N- and C-terminal fragments of IGFBP-5 was more efficient in antagonizing IGF receptor stimulation than the individual fragments alone (Fig. 5Go). Thus, the proteolytic IGFBP-5 fragments complement each other in the binding of IGF. This property is not shared by the fragments of IGFBP-4, explaining the slower release of IGF from IGFBP-5 after proteolytic cleavage (Fig. 2Go). A similar effect of separate N- and C-terminal IGFBP fragments binding to IGF in a cooperative manner has been reported for IGFBP-3 (5). However, no direct interaction was found between the N- and C-termini of IGFBP-3 in the absence of IGF. The lack of increased IGF inhibition in the presence of both the N- and C-terminal fragments of IGFBP-4 observed here was rather surprising in the light of the recent crystal structure (11, 12), which demonstrates a direct interaction between the truncated N- and C-terminal domains of IGFBP-4. Such interaction may therefore not stabilize a ternary complex between IGF and natural fragments generated by proteolysis, or a similar interaction does not occur with these fragments. Our analyses of receptor stimulation were carried out using IGF-I. Based on the affinity measurements of Table 1Go, a similar sequence of cleavage would be expected with IGF-II. It is interesting that KD for the interaction between IGF-II of the C-terminal fragment of IGFBP-5 is relatively high, but how this will affect the abilities of the N- and C-terminal IGFBP-5 fragments to complement each other in IGF binding cannot be predicted.

We further showed that the rate of proteolytic release of IGF is efficiently controlled by the concentration of IGFBP (Fig. 8Go). An interesting parallel can be drawn to earlier studies on other growth factors, in which the cellular outcome of growth factor signaling was shown to be regulated by the duration of the signaling stimulus. For instance, transient Erk phosphorylation leads to cell proliferation, whereas sustained Erk phosphorylation induces neuronal differentiation of PC12 cells (41). Regulation of IGF release by the concentration of IGFBP may represent a similar mechanism. Additionally, the relative concentrations of the individual IGFBPs may also potentially be involved in such regulation. A dominating concentration of IGFBP-5 would lead to a slow release of IGF from IGFBP-5 as a result of constitutive cleavage of this binding protein. This is reflected by the common observation that IGFBP-5 is often found to be present as proteolytic fragments in conditioned media. In contrast, a dominating concentration of IGFBP-4 has the potential to cause a highly regulated and locally concentrated short boost of IGF.

An interesting model system for further substantiating the hypothesis of different IGF release kinetics is the proliferation vs. differentiation of myoblasts, a system in which high levels of IGFBP-4 are observed during the proliferative phase, and an increase in the IGFBP-5 concentration has been reported to correlate with differentiation (42). In this system, the addition of exogenous IGFBP-5 inhibits the IGF effect on cell proliferation but increases the differentiation in an IGF-dependent manner (43). Recently, PAPP-A has also been shown to induce both proliferation and differentiation in myoblasts in an IGF-dependent manner (44).

Binding protein proteolysis at a distance from the cell surface may not result in stimulation of the IGF receptor, because released IGF may become inactivated by uncleaved binding protein before it is brought close enough to the cell surface by diffusion. However, we have previously shown that PAPP-A is capable of binding to an unknown cell surface heparan sulfate proteoglycan by means of the SCR3 module located in the C-terminal end of the protein (32). We therefore hypothesized that proteolysis of IGFBP-4 by surface-bound PAPP-A is more likely to cause release of IGF in proximity to the IGF receptor and consequently receptor stimulation. To test this hypothesis, we here compared the efficiency of wild-type PAPP-A and a PAPP-A variant that lacks the ability of surface binding (Fig. 8Go). This experiment demonstrated that PAPP-A surface binding significantly increased receptor stimulation, in particular when IGFBP-4 is present in molar excess of IGF. At a IGFBP-4 to IGF-I ratio of 4:1, surface association causes an almost 2-fold increase in receptor stimulation compared with the mutant, which lacks the ability to bind the cell surface. Importantly, this additional stimulatory potential of wild-type PAPP-A was caused by the small fraction of PAPP-A that had become associated with the cell surface, estimated to be less than 5%. PAPP-A cell surface binding therefore represents an additional mechanism of regulation, by which different cells in the microenvironment may regulate stimulation of the IGF receptor dependent on their ability to bind PAPP-A. Potentially, the effect in vivo of surface association may be more pronounced than in our model system, because tissues most likely contain much less PAPP-A that is not surface bound.

Different roles of individual binding proteins have implications for the regulation of IGF bioactivity in many tissues, because most tissues are reported to express more than one of the binding proteins. Our experiments show that in a system comprised of both IGFBP-4 and -5, relative affinities of the intact IGFBPs and their proteolytic fragments toward IGF, the relative concentrations of the different IGFBPs, and the ratio between IGFBP and IGF play important roles in the regulation of IGF activity. In particular, we find that IGFBP-4 and -5 have different roles in coordinating the release of IGF, and that IGFBP-4 is responsible for the final delivery of IGF to the IGF receptor.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Expression Constructs, Cell Culture, and Transfection
Human embryonic kidney 293T cells (293tsA1609neo) were maintained in high-glucose DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, nonessential amino acids, and gentamicin (Invitrogen, Carlsbad, CA). For transient transfection, 2.5 x 106 cells were plated onto 6-cm dishes and were transfected 18 h later by calcium phosphate coprecipitation using 5–10 µg plasmid DNA prepared by QIAprep Spin Kit (QIAGEN, Chatsworth, CA). Expression constructs for transient transfection were pcDNA3.1-PAPP-A, encoding mature PAPP-A (45), PAPP-A/PAPP-A2(chim6), encoding PAPP-A(P2SCR3), in which the SCR3 module of PAPP-A was replaced with the corresponding sequence of PAPP-A2 (32), and a variant of each of IGFBP-4 (R126A/R128A/K134A/K136A) and IGFBP-5 (K128A), both mutated to resist PAPP-A proteolytic activity (18). Culture supernatants were harvested 48 h post transfection and cleared by centrifugation. Concentrations of PAPP-A and mutated PAPP-A were determined by ELISA (46). For expression of wild-type binding proteins, cDNAs encoding c-myc-tagged IGFBP-4 (14) and -5 (17) were cloned into the pcDNA3.1/Hygro(+) expression vector (Invitrogen), 293T cells were transfected with FspI-linearized (IGFBP-4) or SspI-linearized (IGFBP-5) plasmids, and populations of cells were cultured in medium containing 0.19 mg/ml hygromycin B (Invitrogen). Cells were maintained in serum-free medium (CD 293, Invitrogen) before harvesting to facilitate purification. A cell line stably expressing the IGF receptor, 293-IGFR(clone H), was generated by transfecting 293T cells with FspI-linearized vector containing the IGF receptor cDNA (47) cloned into the BamHI site of pcDNA 3.1 Hygro(+) (Invitrogen). Several clones were selected, cultured, and analyzed by flow cytometry on a Beckman-Coulter Cytomics FC 500 MPL instrument using mAb 24–55 (GroPep, Adelaide, Australia) as primary antibody and fluorescein isothiocyanate-conjugated goat antimouse IgG (Zymed Laboratories, South San Francisco, CA) as the secondary antibody.

Generation and Purification of Fragments of IGFBP-4 and IGFBP-5
To generate microgram quantities of proteolytic fragments of IGFBP-4 and -5, 50 µg purified (17) IGFBP-4 or -5 was digested (37 C for 6 h) with purified rPAPP-A (1 µg) immobilized to recombinant protein G-agarose (Invitrogen) by means of a monoclonal antibody (mAb 234–5) (48). Cleavage of IGFBP-4 was performed in the presence of IGF-I (1 µg) (Diagnostic Systems Laboratories, Webster, TX), and IGFBP-5 cleavage was performed in the absence of IGF. Complete cleavage of the binding proteins was verified by SDS-PAGE, and the PAPP-A-generated fragments were separated by reversed-phase HPLC on a 4 x 250 mm column packed with Nucleosil C4 500-7 (Macherey-Nagel, Düren, Germany). The column was eluted at 0.5 ml/min using a linear gradient formed from 0.1% (vol/vol) trifluoroacetic acid (solvent A) and 0.075% (vol/vol) trifluoroacetic acid in 90% acetonitrile (solvent B), increasing the amount of solvent B with 1.75%/min. The column was equilibrated with 10% solvent B, operated at 50 C, and the separation was monitored at 280 nm. The N- and C-terminal fragments were identified by Edman degradation after separation by SDS-PAGE (10–20%), blotting onto a polyvinylidene fluoride membrane, staining by Coomassie Brilliant Blue, and excision of the relevant bands. The identity of the fragments was further verified by Western blotting using monoclonal anti-c-myc (9E10) against the C-terminal c-myc tag. The amounts of purified fragments were quantified by quantitative amino acid analysis.

Western Blotting
Proteins separated by SDS-PAGE were blotted onto a PVDF membrane, blocked with 2% Tween 20, and equilibrated in 50 mM Tris, 500 mM NaCl, 0.1% Tween 20, pH 9.0 (TST). Primary antibodies [anti-c-myc, mAb 9E10; antiphosphotyrosine, mAb PY99 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); anti-IGF receptor, mAb CT-1 (GroPep)] were diluted in TST containing 0.5% fetal bovine serum, and blots were incubated overnight at room temperature. Incubation with peroxidase-conjugated secondary antibodies (P260; DAKO, Carpinteria, CA) diluted in TST was done for 1 h at room temperature. The blots were developed using enhanced chemiluminescence (ECL Plus; Amersham Biosciences,), and images were captured and analyzed using a Kodak Image Station 1000 and the Kodak 1D software (version 3.6; Eastman Kodak, Rochester, NY).

Binding Analyses
Surface plasmon resonance analyses were carried out on a BIAcore 3000 system (BIAcore). IGF-I and -II (Diagnostic Systems Laboratories) were immobilized on a CM5 biosensor chip (BIAcore). A reference surface, to which no ligand was bound, was included on the chip. Ligands (12.5 µg/ml) contained in 50 mM sodium acetate, pH 4.75, were injected onto the activated CM5 surface at 5 µl/min. The running buffer was 10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.005% surfactant P20, pH 7.4 [HEPES-buffered saline (HBS)]. The surfaces were coupled to final resonance values of approximately 200 (IGF-I) or 300 (IGF-II) response units and were deactivated using ethanolamine (1 M). Various concentrations of analyte (50, 25, 12.5 6.25, or 3.125 nM of intact binding proteins or 200, 100, 50, 25, or 12.5 nM of binding protein fragments) were injected during the association phase for 4 min (20 µl/min), using HBS as the running buffer. The dissociation phase, initiated by passage of HBS alone, was carried out over a period of 5 min. The biosensor surfaces were regenerated by two injections (60 sec) of 0.1 M HCl. Samples were injected in duplicate, and the experiments were performed using two separately prepared chips and protein of at least two independent rounds of preparation. The kinetic data were analyzed using the BIAevaluation software version 3.0 (BIAcore). The 1:1 Langmuir binding model describing a 1:1 binding between analyte and ligand was locally fitted to the data.

Measurement of IGF Bioactivity
IGF receptor stimulation was quantitated by measuring the degree of phosphorylation of the ß-subunit of the receptor. 293-IGFR(clone H) cells (300,000 per well) were transferred 48 h before stimulation to 24-well tissue culture plates. The medium was changed to DMEM with 1% fetal bovine serum 24 h after seeding. Immediately before stimulation, the cells were gently rinsed in PBS containing CaCl2 (0.1 mg/liter) and MgCl2 (0.1 mg/liter), pH 7.4. The cells were stimulated with IGF-I and combinations of IGFBP-4, IGFBP-5, and PAPP-A for 3–30 min. The stimulated cells were lysed on ice with 50 mM HEPES (pH 7.9), 150 mM NaCl, 10 mM EDTA, 1% Triton-X-100, 4 mM sodium orthovanadate, 10 mM sodium fluoride, supplemented with phenylmethylsulfonylfluoride (1 mM), leupeptin (2 µg/ml), aprotinin (2 µg/ml), and iodoacetamide (100 µM) for 10 min. The lysates were cleared by centrifugation for 20 min at 4 C. Western blotting was used to quantitate ß-subunit phosphorylation. Quantification of band intensities was carried out using the Kodak Image Station 1000 and the Kodak 1D software (version 3.6).

Measurement of PAPP-A Proteolytic Activity
The proteolytic activity of PAPP-A while bound to the surface of cells was analyzed. Cells [293-IGFR(clone H), 300.000 per well] were transferred to 24-well plates 48 h before analysis. The medium was changed 24 h after seeding to DMEM with 1% fetal bovine serum. Just before the cleavage analysis, the serum medium was removed, and the cells were rinsed gently in PBS containing CaCl2 (0.1 mg/liter) and MgCl2 (0.1 mg/liter), pH 7.4 (buffer A). PAPP-A (0.6 nM) or PAPP-A(P2SCR3) (0.6 nM) contained in 100 µl buffer A was added to the cells allowing cell surface binding of PAPP-A for 5 min before the addition of IGFBP-4 or -5 (10–40 nM) in complex with IGF-I (10 nM). To estimate the amount of PAPP-A attached to the cell surface, a parallel experiment was carried out in which samples were taken out for the determination of PAPP-A concentration by ELISA (46). To allow quantification of cleavage, trace amounts (~0.6 nM) of radiolabeled IGFBP-4 or -5 was added to the reaction mixture (17). Samples were taken out at different time points (0–30 min), and the reaction mixtures were analyzed by nonreducing SDS-PAGE (10–20%) followed by autoradiography. The degree of cleavage was determined by measuring band intensities with a Typhoon TRIO imaging system (Amersham Biosciences).


   FOOTNOTES
 
This work was supported by the Novo Nordisk Foundation and the Danish Medical Research Council.

Disclosure Statement: The authors have nothing to disclose.

First Published Online February 20, 2007

Abbreviations: HBS, HEPES-buffered saline; IGFBP, IGF binding protein; mAb, monoclonal antibody; PAPP-A, pregnancy- associated plasma protein-A; TST, Tris-NaCl-Tween 20.

Received for publication December 5, 2006. Accepted for publication February 15, 2007.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals