Main

Children with CRF usually do not attain an adult height consistent with their genetic potential(1). The GH-IGF-growth plate chondrocyte axis plays an important role in linear growth. Among other effects, GH raises serum IGF levels and stimulates growth plate chondrocyte proliferation, with subsequent linear growth; an important role for IGF in this process is suggested by the observation that exogenous IGF-I stimulates linear growth in humans, and both exogenous IGF-I and IGF-II stimulate tibial epiphyseal widening in rats(1–6). Although growth-retarded children with CRF have normal or high serum levels of GH, IGF-I, and IGF-II(1, 7–9), serum bioactivity is low(10, 11), a factor that may contribute to poor linear growth of these children. This low bioactivity may be due to an excess of high affinity IGF binding sites in CRF serum that compete with cell surface IGF receptors for IGF binding. This hypothesis is supported by the observation that passing CRF serum across an IGF-II affinity column, which should remove excess high affinity IGF binding sites, markedly increases serum bioactivity(11).

IGF-I and -II are ≈7-kD proteins found in serum and other body fluids tightly bound to a family of at least six IGFBPs(12–14). In serum from healthy adults and children, most IGFs circulate in the ≈150-kD serum fractions in a ternary complex composed of one IGF peptide, a single 41- or 38-kD form of glycosylated IGFBP-3, and an 86-kD acid-labile subunit(14–16). The remaining IGFs are found in the ≈35-kD serum fractions bound primarily to IGFBPs other than IGFBP-3. In CRF, there is an excess of IGFBPs in the ≈35-kD serum fractions, which probably accounts for the excess IGF binding sites and the low bioactivity of CRF serum(17).

IGFBP-1, IGFBP-2, and IGFBP-3 levels are high in CRF serum, with IGFBP-1 and IGFBP-2 clearly contributing to the excess of IGFBPs in the ≈35-kD serum fractions(9, 11, 17–27). In growth-retarded children with CRF, the high IGFBP-1 and IGFBP-2 levels correlate inversely with height standard deviation score(26, 28), suggesting that these IGFBPs may contribute to growth failure. Further support for this hypothesis comes from studies showing that IGFBP-1 injected daily for 2 wk significantly inhibits both GH- and IGF-I-induced weight gain and tibial epiphyseal widening in hypophysectomized rats(4).

High IGFBP-3 levels in CRF sera are due to a 29-kD IGFBP-3 form, IGFBP-329, in the ≈35-kD serum fractions(17, 27); it is not clear whether IGFBP-329 is intact nonglycosylated IGFBP-3 or a glycosylated IGFBP-3 fragment. The 41- and 38-kD forms of intact, glycosylated IGFBP-3 are not detected in the ≈35-kD serum fractions; they are detected only in the≈150-kD fractions, along with additional IGFBP-329(27). The ≈35-kD fractions of PD collected from dialyzed children also contain a 29-kD IGFBP-3 form. This form is almost certainly identical to IGFBP-329 found in the ≈35-kD serum fractions, because IGFBP-329 present in these serum fractions should easily cross the peritoneal membrane into the peritoneal space. Although final proof of identity awaits sequence analysis of the 29-kD IGFBP-3 forms in serum and PD, these two forms will be referred to as IGFBP-329 in this report. IGFBP-329 in the ≈35-kD fractions is clearly the predominant IGFBP-3 form in PD; levels of the 41- and 38-kD forms of intact IGFBP-3 are quite low in PD, because large serum proteins and protein complexes, such as those circulating at ≈150 kD in the case of intact IGFBP-3, are relatively excluded by the peritoneal membrane from entering the peritoneal space(27). Thus, IGFBP-329 is the primary IGFBP-3 form that escapes the circulation and comes into contact with target tissues. Others have also reported excess IGFBP-3 in the low molecular weight fractions of CRF serum; they hypothesized that this excess IGFBP-3 is likely the major inhibitor of IGF action in CRF, because IGFBP-3 is the most abundant serum IGFBP. However, bioactivity of this excess IGFBP-3 was never directly tested(11, 19, 29).

Studies presented here describe the purification of IGFBP-329 from the ≈35-kD fractions of PD and demonstrate that IGFBP-329 is a glycosylated IGFBP-3 fragment. Additional studies examine the ability of this fragment to bind, and to interfere with the biologic effects of, IGF-I and IGF-II.

METHODS

Patient samples. Sera were obtained from children with CRF who had the following characteristics: 1) irreversible renal insufficiency (GFR < 40 mL/min/1.73 m2); 2) height <5 percentile for chronologic age; 3) >2.5 y of age; 4) Tanner stage 1; 5) normal serum albumin; and 6) no evidence for other causes of growth failure. Some of these children also provided PD samples collected as described previously(27). All samples were obtained using a protocol approved by the Baylor College of Medicine Institutional Review Board for Research Involving Human Subjects. Blood was obtained by peripheral venipuncture and allowed to clot, after which the serum was separated and stored along with the PD at -80 °C until assay.

Endo F. One-microliter aliquots of sera pooled from five children with CRF were adjusted to pH 5 and preincubated with either 25 μL of water (control) or 25 μL (24 mU) of Endo-F (Calbiochem, San Diego, CA) at 37 °C for 3 h. Reactions were terminated by adding 25 μL of a 4-fold concentrated solution of Laemmli sample buffer without reducing agent. Each sample was heated to 95 °C for 5 min and then analyzed by IGFBP-3 immunoblot and [125I]IGF ligand blot as described below.

IGFBP-3 immunoblot and [125I]IGF ligand blot. The equivalent of 1 μL of untreated or Endo-F-treated CRF sera, or aliquots from individual steps in the purification of IGFBP-329, were separated by 12% SDS-PAGE under nonreducing conditions and then transferred to a nitrocellulose membrane as described previously(30). IGFBP-3 immunoblot analysis was performed on membranes as described previously(27, 31);αIGFBP-3g1(27, 32), a kind gift of Dr. Ron Rosenfeld (Oregon Health Science Center, Portland, OR), was diluted 1:1000 and served as IGFBP-3 antibody. For [125I]IGF ligand blot analysis, IGF-I and IGF-II were iodinated by a modified chloramine-T method(33); nitrocellulose membranes were then probed with 2× 106 cpm each of [125I]IGF-I and [125I]IGF-II as described previously(22, 27).

Purification of IGFBP-329. In all purification steps, an immunoradiometric assay detected the presence of IGFBP-3 forms in individual samples (Diagnostic Systems Laboratories, Inc., Webster, TX), and the size of the IGFBP-3 forms was determined by immunoblot. PD (≈150 mL) was concentrated ≈5-fold using a Centriprep 10 filter(Amicon, Beverly, MA) and then separated on a 2.5 × 110-cm column of Sephacryl S-300 at pH 7.5. Fractions containing proteins of ≈35 kD, which included IGFBP-329 but no 41- or 38-kD forms of intact IGFBP-3, were pooled. These fractions were recycled overnight at 4 °C across an affinity column consisting of 1 mg of human IGFBP-3 antibody (Diagnostic Systems) bound to 2 mL of Affi-Gel 10 beads (Bio-Rad). After washing with 50 mL of PBS, the column was eluted in 1% acetic acid. The eluant was concentrated to 2 mL using a Centriprep 10 filter and then gel filtered through a 0.9 × 110-cm column of Sephadex G-75, using 1% acetic acid as the mobile phase. Fractions containing IGFBP-329 were pooled and lyophilized; purity of IGFBP-329 was confirmed by India ink staining as described previously(34).

Soluble IGF binding assay. IGFBP-3 forms used include nonglycosylated human IGFBP-3 expressed in Escherichia coli(IGFBP-3E.coli) which was generously provided by Dr. C. Maack(Celtrix, Richmond, CA), intact recombinant human IGFBP-3 expressed in Chinese hamster ovary cells (IGFBP-3CHO; Genentech, South San Francisco, CA) and IGFBP-329. These three IGFBP-3 forms were evaluated for their ability to bind IGFs in a soluble IGF binding assay as described previously(35). Briefly, triplicate aliquots of either IGFBP-3E.coli, IGFBP-3CHO, or IGFBP-329 in 50 mM Tris-HCl, 0.5% BSA, were incubated overnight at 4 °C with[125I]IGF and various concentrations of unlabeled IGF. The dose of IGFBP-3 was based on a preliminary titration experiment to determine a concentration near but not at the plateau of maximal [125I]IGF binding. One percent activated charcoal containing 0.2 mg/mL protamine sulfate was added, and the samples then centrifuged at 4 °C to separate bound from free IGF(36, 37). Binding in buffer alone was subtracted from the total bound radioactivity to determine specific IGF binding activity.

Cell culture. Human chondrosarcoma cells (SW1353) were obtained from the American Type Culture Collection (Rockville, MD). These cells were maintained in DMEM supplemented with 10% iron-supplemented calf serum(Hyclone, Logan, UT).

Bovine dermal fibroblasts (GM06034) purchased from the Human Genetic Mutant Cell Repository (Camden, NJ) were cultured in DMEM supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 4 mM glutamine, and containing 10% fetal bovine serum. Cultures were used between passages 9 and 15. Cell number was determined in triplicate wells with an automatic cell counter (Coulter Electronics, Hialeah, FL).

IGF binding to fibroblast monolayers. Binding assays were performed directly on confluent cell monolayers (24-multiwell plates) as described previously(34, 35, 38). Fibroblast cultures were washed three times with cold HEPES binding buffer, pH 7.4, 0.5% BSA, and then incubated with [125I]IGF-I (25 000 cpm) along with either IGFBP-3E.coli, IGFBP-3CHO, IGFBP-329, or no additives at 15 °C for 2.5 h. Nonspecific binding, defined as the amount of [125I]IGF-I bound in the presence of excess IGF-I (250 ng/mL), was less than 1% of total counts added and was subtracted from total binding to determine specific binding.

AIB uptake. α-[methyl-3H]AIB was purchased from DuPont NEN (Boston, MA). [3H]AIB uptake was determined in bovine fibroblasts as described previously(34, 35, 38). Fibroblasts were plated at≈30 000 cells/cm2 and grown to confluency (4-5 d) in 24-multiwell plates (Costar, Cambridge, MA). Monolayers were preincubated with either IGFBP-3CHO, IGFBP-E.coli, IGFBP-329 or no additives for 72 h at 37 °C and then washed three times with Hanks' balanced salt solution containing 1.75 g/liter NaHCO3, 20 mM HEPES, pH 7.4, and 1% BSA (Hanks' balanced salt solution buffer). The medium was changed to Hanks' balanced salt solution buffer with or without experimental additives, and the monolayers were incubated at 37 °C for 6 h.[3H]AIB was added, and the incubation was continued for 12 min. Cultures were placed on ice and quickly washed four times with cold PBS + 0.5% BSA. Monolayers were solubilized in 0.25 N NaOH, and aliquots were taken for liquid scintillation counting. Results are expressed as percentage of total counts in the incubation medium that were taken up by the cells.

Thymidine incorporation into chondrosarcoma cells. Cell proliferation was determined by measuring the incorporation of[3H]thymidine into DNA. Chondrosarcoma cells (2000 cells/well) were plated in 50 μL of DMEM containing 0.1% BSA (DMEM/BSA) in 96-well culture plates and incubated for 24 h, after which 50 μL of DMEM/BSA with or without protein additives were added, and the cells were incubated for an additional 18 h. The cells were then pulse-labeled for 4 h with 0.25 μCi of[3H]methylthymidine (DuPont NEN). Radioactive medium was removed, and the cell layer was rinsed three times with PBS; parallel studies with trichloroacetic precipitation showed that the pool of trichloroacetic acid-soluble radioactivity in the rinsed cells was insignificant. After the cell layer was extracted with 0.2 M NaOH, the extract was transferred to scintillation vials and mixed with 2 mL of scintillation fluid, and the amount of [3H]thymidine was determined by liquid scintillation counting.

Statistics. Differences among groups were determined by ANOVA followed by Newman-Keuls multiple range testing. Results were considered statistically significant when p < 0.05.

RESULTS

IGFBP-329 is a glycosylated IGFBP-3 fragment. To determine whether the 29-kD form was an intact nonglycosylated IGFBP-3 or a glycosylated IGFBP-3 fragment, deglycosylation studies were performed. By immunoblot, major IGFBP-3 forms of 41, 38, 29, 24, and 18 kD were seen in pooled CRF serum (Fig. 1). Endo-F lowered the size of IGFBP-329, which was the predominant form in CRF serum, from 29 kD to ≈22 kD, indicating that IGFBP-329 is a glycosylated IGFBP-3 fragment. Endo-F also lowered the size of intact differentially glycosylated IGFBP-3 from 41 and 38 kD to 35 and 32 kD, closer to the 29-kD size of intact nonglycosylated IGFBP-3. In addition, Endo-F lowered the abundance of the 18-kD IGFBP-3 form, suggesting that it, too, is an IGFBP-3 fragment.

Figure 1
figure 1

Glycosylation state of IGFBP-3 forms in CRF serum.(Left) Sera from five children with CRF were pooled and incubated either with (+) or without (-) Endo F, separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted (IB) using the IGFBP-3 antibodyαIGFBP-3g1. Molecular mass (kD) of various glycosylated and deglycosylated IGFBP-3 forms are on the left. (Right) The same nitrocellulose filter was Western ligand blotted (WLB) with[125I]IGF-I and [125I]IGF-II. Molecular mass (kD) of various IGFBPs with high affinity for IGFs are on the right.

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IGFBP-329 has low affinity for IGF-I and IGF-II. The affinity of IGFBP-329 for IGFs was studied initially by using [125I]IGF-I and [125I]IGF-II to probe the nitrocellulose membrane used for immunoblotting. This Western ligand blot (Fig. 1) revealed a number of IGFBPs. The 41- and 38-kD IGFBP-3 forms were seen, as were their 35- and 32-kD deglycosylated counterparts. However, IGFBP-329 and its ≈22-kD deglycosylated counterpart were not seen by this method. This suggests that IGFBP-329 has a low affinity for [125I]IGFs, at least under these experimental conditions.

For further studies of IGF binding by this IGFBP-3 fragment, IGFBP-329 was purified from the ≈35-kD fractions of PD as outlined in Table 1. Purification was achieved primarily through the specific step of binding IGFBP-329 to an IGFBP-3 antibody affinity column and eluting it with 1% acetic acid. The final preparation of IGFBP-329 was pure as judged by India ink staining (Fig. 2A); additional studies found that the IGFBP-3 immunoradiometric assay estimates the amount of purified IGFBP-329 to within ≈15% of the weight of IGFBP-329 measured by microbalance. Treatment with Endo-F decreased the molecular weight of this IGFBP-329 preparation just as it did with unpurified serum IGFBP-329 (data not shown).

Table 1 Purification of IGFBP-329from PD
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Figure 2
figure 2

IGF binding characteristics of IGFBP-329.(A) Purified IGFBP-329. Three micrograms of IGFBP-329, purified to homogeneity from the 35-kD fractions of peritoneal dialysate, were separated by SDS-PAGE, transferred to nitrocellulose, and stained with India ink. Molecular mass markers, in kD, are on the right. (B) Competition for binding of [125I]IGF-I to bovine fibroblasts by IGFBP-3 forms. Bovine fibroblasts were incubated with [125I]IGF-I and the indicated concentrations of IGFBP-329, IGFBP-3CHO or IGFBP-3E.coli for 2.5 h at 15 °C. Results are mean± SD of triplicate determinations expressed as percent of maximum[125I]IGF-I binding.

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Purified IGFBP-329 was first tested for the ability to compete with type I IGF receptors for IGF-I binding. Bovine fibroblasts in monolayer culture were used because these cells have abundant type I IGF receptors(34, 35, 38). As shown in Figure 2B, 70% of [125I]IGF-I binding to fibroblast monolayers was inhibited by 100 nM IGFBP-329 and by only 0.5 nM IGFBP-3CHO; thus, intact IGFBP-3CHO has a ≈200-fold greater ability than IGFBP-329 to compete for IGF-I binding. In addition, IGFBP-3E.coli was almost as potent as IGFBP-3CHO and much more potent than IGFBP-329 at inhibiting[125I]IGF-I binding to fibroblast monolayers.

The ability of IGFBP-329 to bind IGFs in solution was also studied using a soluble binding assay. Preliminary studies found that[125I]IGF-I was not appreciably bound by up to 25 nM IGFBP-329, whereas ≈50% was bound by 0.2 nM IGFBP-3CHO, consistent with the ability of these two IGFBP-3 forms to compete with type I IGF receptors for IGF-I binding. Although IGF-II was bound by all IGFBP-3 forms, IGFBP-329 also has the lowest affinity for this IGF; as shown in Figure 3, IGFBP-329 binds IGF-II with a ≈4-fold lower affinity than IGFBP-3CHO and a ≈10-fold lower affinity than IGFBP-3E.coli

Figure 3
figure 3

IGF-II binding to soluble IGFBP-3. Aliquots of either IGFBP-3E.coli, IGFBP-3CHO, or IGFBP-329 in 50 mM Tris-HCl, 0.5% BSA were incubated overnight at 4 °C with[125I]IGF-II and various concentrations of unlabeled IGF-II as described previously(35). One percent activated charcoal containing 0.2 mg/mL protamine sulfate was added, and the samples were centrifuged at 4 °C to separate bound from free IGF. Binding in buffer alone was subtracted from the total bound radioactivity to determine specific IGF binding activity; data are presented as percent of maximum specific binding.

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IGFBP-329 modulates IGF-II but not IGF-I action. As shown in Figure 4, IGFBP-3E.coli and IGFBP-329 had no significant effect on the rate of thymidine incorporation into chondrosarcoma cell DNA in the absence of IGF peptides. The rate of thymidine incorporation was increased≈2-fold by 1.4 nM IGF-I; 35 nM IGFBP-3E.coli inhibited the IGF-I-mediated increase by ≈47%, but 35 nM IGFBP-329 had no effect. The rate of thymidine incorporation into chondrosarcoma cell DNA was also increased significantly by 1.4 nM IGF-II; this increase was inhibited completely by 35 nM IGFBP-329 or 35 nM IGFBP-3E.coli, was inhibited 48% by 3.5 nM IGFBP-3E.coli, and was not inhibited at all by 3.5 nM IGFBP-329. Raising IGF-II levels to 4.2 nM overcame the inhibition caused by 35 nM IGFBP-329 or 3.5 nM IGFBP-3E.coli, but 35 nM IGFBP-3E.coli still inhibited the effect of 4.2 nM IGF-II by 81% (data not shown).

Figure 4
figure 4

Effect of IGFBP-3 on thymidine incorporation into DNA of human chondrosarcoma cells. Human chondrosarcoma cells (SW1353) in 96-well culture plates were incubated for 18 h with or without IGFBP-3E.coli (A) or IGFBP-329 (B) and with or without 1.4 nM IGF-I or 1.4 nM IGF-II. Cells were then pulse-labeled for 4 h with [3H]methylthymidine, after which thymidine incorporation into DNA was measured as described in “Methods.” Thymidine incorporation into DNA is presented as mean ± SD of percent of control (no IGFBP-3 or IGF added) value; the control value is arbitrarily set at 100%. 1, Different from control, p < 0.005;2, different from control, p < 0.01; 3, different from control, p < 0.05; 4, different from 1.4 nM IGF-I, p < 0.005; 5, different from 1.4 nM IGF-II,p < 0.025; and 6, different from 1.4 nM IGF-II,p < 0.05.

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The ability of IGFBP-329 to potentiate IGF-I action was tested using bovine fibroblast monolayers inasmuch as previous studies found that preincubation of IGFBP-3 with these fibroblasts led to fragmentation of IGFBP-3 on the cell surface that coincided temporally with potentiation of IGF-I-stimulated [3H]AIB uptake(34, 35, 38). As shown in Figure 5, 1 nM IGF-I alone increased [3H]AIB uptake ≈3-fold; preincubation with 20 nM IGFBP-3CHO or IGFBP-3E.coli potentiated the effect of 1 nM IGF-I by 2.3- or 1.6-fold, respectively, whereas preincubation with 20 or 100 nM IGFBP-329 failed to potentiate IGF-I-stimulated [3H]AIB uptake.

Figure 5
figure 5

Effect of IGFBP-3 preincubation on IGF-I-stimulated[3H]AIB uptake. Bovine fibroblasts were preincubated without(0) or with the indicated concentrations of IGFBP-329, IGFBP-3CHO, or IGFBP-3E.coli for 72 h. Stimulation of[3H]AIB uptake by 1 nM IGF-I (hatched bars) or serum-free medium alone (open bars) was measured as described in“Methods.” Results are presented as mean ± SD of percent[3H]AIB uptake. The asterisk (*) indicates a significant effect of IGFBP-3 compared with no added IGFBP-3, p < 0.001.

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DISCUSSION

A striking excess of IGFBPs is found in the ≈35-kD fractions of serum from growth-retarded children with CRF(17, 27); because of their size, these IGFBPs may escape the circulation to modulate IGF actions at the tissue level. Among the IGFBPs in excess in the ≈35-kD CRF serum fractions is IGFBP-3. However, intact IGFBP-3 is not detected in the≈35-kD serum fractions from CRF or normal individuals, but instead is detected only in the ≈150-kD serum fractions where it has been shown to bind one IGF peptide and one acid-labile subunit protein in a ternary complex; the size of the ≈150-kD complex precludes most of the IGFBP-3 in these serum fractions from leaving the vascular space(14–17, 27). The excess IGFBP-3 present in CRF serum is primarily a 29-kD IGFBP-3 form, IGFBP-329, detected in the ≈35-kD serum fractions; this form is not detected in comparable fractions of normal sera(17, 27). In CRF sera, IGFBP-3 in the ≈150-kD fractions is more abundant than IGFBP-329 in the ≈35-kD fractions, whereas in PD, IGFBP-329 in the ≈35-kD fractions is much more abundant than IGFBP-3 in the≈150-kD fractions(27); this is consistent with the tendency of peritoneal capillary membranes to exclude larger proteins and protein complexes from the peritoneal space. Thus, IGFBP-329 is the most abundant IGFBP-3 form in contact with CRF target tissues; however, the ability of IGFBP-329 to influence basal and IGF-modulated cellular growth and metabolism has not been investigated.

Studies presented here show that IGFBP-329 is a glycosylated IGFBP-3 fragment and that, relative to intact native IGFBP-3, IGFBP-329 purified from the ≈35-kD fractions of PD has a 200-fold lower affinity for IGF-I and a 4-10-fold lower affinity for IGF-II. These binding affinities are consistent with the biologic effects of IGFBP-329, because IGFBP-329 was unable to inhibit or potentiate IGF-I effects, but could inhibit IGF-II effects at a slightly lower potency than native IGFBP-3. Levels of IGFBP-329 in the serum of adolescents with CRF are ≈150 nM(17, 27), and levels in PD, which likely did not have time to equilibrate with serum, are at least ≈15 nM(27). These levels suggest that enough IGFBP-329 is present in interstitial fluids of some individuals with CRF to influence IGF-II action on target tissues.

A role for IGF-II in cartilage growth and metabolism is suggested by reports that: 1) exogenous IGF-II stimulates widening of the tibial epiphyseal growth plate(6), 2) IGF-II is more highly expressed than IGF-I in chondrocytes of growing rodents(39, 40), and 3) IGF-II protein levels are higher than those of IGF-I in lamb and rabbit cartilage(41, 42). The ability of IGFBP-329 to inhibit IGF-II-stimulated DNA synthesis in chondrosarcoma cells suggests that IGFBP-329 may inhibit the growth of chondrocytes in the growth plates of long bones. However, IGFBP-3 levels do not correlate strongly with the degree of growth failure in CRF children(26, 28), suggesting that IGFBP-329 plays at best a minor role as an inhibitor of linear growth in CRF children. Nevertheless, IGF-II plays a role in the physiology of other cell types, such as osteoblasts and myoblasts(43, 44); it remains to be seen if IGFBP-329 can influence IGF-II effects on these and other cell types in vitro or in vivo.

The ability of IGFBP-3 to potentiate IGF-I-stimulated [3H]AIB uptake in bovine fibroblasts is associated with processing of IGFBP-3 to lower molecular weight forms on the cell surface(34, 35, 38), suggesting that smaller molecular weight forms such as IGFBP-329 might be able to directly potentiate IGF-I action. The inability of IGFBP-329 to potentiate IGF-I action suggests that this particular IGFBP-3 fragment plays no role in the potentiation process, but it is also possible that intact IGFBP-3 must first bind to the cell surface before correctly processed IGFBP-3 forms, which may include IGFBP-329, can potentiate IGF-I action.

The origin of IGFBP-329 in CRF serum is not yet established. In normal rat serum, proteolysis of IGFBP-3 in the 150-kD complex by a cation-dependent serine protease produces a 30-kD IGFBP-3 fragment that circulates at ≈150 kD. Because of a ≈500-fold decreased affinity for IGF-I and a ≈13-fold decreased affinity for IGF-II, this 30-kD fragment is unbound to IGF peptides while it is in the serum ≈150-kD complex; presumably, IGFBP-3 proteolysis is a mechanism by which IGFs can be released from the ≈150-kD complex and be made available to target tissues(45–47). This mechanism is probably also responsible for the increased abundance of 30-kD IGFBP-3 fragments in the≈150-kD fractions of pregnancy serum and also in sera of patients with a variety of illnesses(48–50). IGFBP-329 and this 30-kD fragment are similar in size and in affinity for IGF-I and -II, suggesting that they may be identical IGFBP-3 fragments. In addition, a 29-kD IGFBP-3 form is abundant in the ≈150-kD fractions of CRF serum and is likely to be IGFBP-329(27). This suggests that IGFBP-329 arises in CRF serum due to proteolysis of IGFBP-3 in the ≈150-kD complex; indeed, IGFBP-3 proteolysis occurs in CRF serum at a rate similar to that in normal serum(51)(our unpublished observations). The accumulation of IGFBP-329 in the≈35-kD fractions of CRF serum is probably due to decreased renal clearance, similar to the mechanism behind the accumulation of parathyroid hormone fragments in CRF serum. The identity of the protease(s) which generates IGFBP-329 is currently unknown.

A number of functions have been attributed to IGFBP-3 fragments. As noted above, cell surface-associated IGFBP-3 fragments appear to potentiate IGF action(34, 35, 38), whereas in another study an IGFBP-3 fragment inhibited DNA synthesis in the absence of IGFs(52). In contrast, a 30-kD IGFBP-3 fragment purified from normal human serum had no effect on IGF-I action in PyMS rat bone cells(53). IGFBP-329 may differ from this 30-kD fragment inasmuch as they were purified from different molecular weight fractions, and because different proteases cleave IGFBP-3 at multiple sites(54, 55), suggesting that many different 30-kD IGFBP-3 fragments may exist. However, similarities in size and inability to inhibit IGF-I action suggest that these two forms are the same IGFBP-3 fragment.

In summary, IGFBP-329 appears to be a proteolytic fragment of IGFBP-3 that accumulates in CRF serum. Similar to parathyroid hormone fragments, which accumulate in CRF serum, IGFBP-329 has decreased biologic activity relative to native IGFBP-3. Although other IGFBPs present in excess in CRF serum are likely to play a more important role as inhibitors of IGF-mediated activities, such as linear growth, IGFBP-329 is present in sufficient concentration in some children with CRF to inhibit the biologic effects of IGF-II. Whether IGFBP-329 plays any role in the growth failure of children with CRF, or in the pathogenesis of other CRF processes such as renal osteodystrophy, remains to be determined.