Insulin-Like Growth Factor Binding Proteins--an Update.

The insulin-like growth factor (IGF) system is essential for normal growth and development, and its perturbation is implicated in a number of diseases. IGF activity is finely regulated by a family of six high-affinity IGF binding proteins (IGFBPs). 1GFBPs usually inhibit IGF actions but may enhance them under certain conditions. Additionally, IGFBPs bind non-IGF ligands in the extracellular space, cell membrane, cytoplasm and nucleus, thereby modulating cell proliferation, survival and migration in an IGF-independent manner. IGFBP activity is regulated by transcriptional mechanisms as well as by post-translational modifications and proteolysis. Understanding the balance between the various actions of IGFBPs in vivo may lead to novel insights into disease processes and possible IGFBP-based therapeutics.

Insulin-like growth factor binding protein-6 interacts with the thyroid hormone receptor α1 and modulates the thyroid hormone-response in osteoblastic differentiation.

Insulin-like growth factor binding protein-6 (IGFBP-6) is a member of the insulin-like growth factor binding protein family, which has both Insulin-like growth factor-dependent and independent effects on cell growth. In previous studies, we have shown that recombinant IGFBP-6 could be translocated into the cell nucleus. But the effect in the nucleus of IGFBP-6 is not clear. In the present study, we use multiple methodologies including Glutathione S-transferase pull-down assay, co-immunoprecipitation, fluorescence resonance energy transfer to demonstrate that IGFBP-6 can directly interact with thyroid hormone receptor alpha 1 (TRα1) in vitro and in vivo. We also demonstrate that the DNA-binding domains and Ligand-binding domains of TRα1 and N-terminal domains and C-terminal domains of IGFBP-6 are involved in the interaction. This interaction also can block the formation of TR: retinoid X receptor heterodimers. Furthermore, immunofluorescence co-localization studies show IGFBP-6 and TRα1 could co-localize in the nucleus of the cells. Reporter gene experiment shows that IGFBP-6 negatively regulates the growth hormone promoter activity induced by ligand activated TRα1. Moreover, real-time RT-PCR demonstrates that IGFBP-6 could inhibit the osteocalcin mRNA transcription induced by Triiodothyronine (3,3',5-Triiodo-L-thyronine, T3) in osteoblastic cells. Finally, alkaline phosphatase activity was significantly decreased in osteoblastic cells when the cells were transfected with IGFBP-6 in the presence of T3. In conclusion, these studies provide evidence that overexpression of IGFBP-6 suppresses osteoblastic differentiation regulated by TR in the present of T3.

A novel interaction between insulin-like growth factor binding protein-6 and the vitamin D receptor inhibits the role of vitamin D3 in osteoblast differentiation.

Insulin-like growth factor binding protein-6 (IGFBP-6) is a secreted glycoprotein that reduces the bioavailability of IGFs. It has both IGF-dependent and -independent effects on cell growth, however the mechanisms responsible for its IGF-independent actions of IGFBP-6 are not fully understood. In previous studies, we have shown that recombinant IGFBP-6 can be internalized and translocated to the nucleus. The present study shows that IGFBP-6 interacts with the vitamin D receptor (VDR). Physical interactions between IGFBP-6 and the VDR were confirmed by GST pulldown and co-immunoprecipitation assays. We also determined that the interaction binding sites were on the C-terminal region of the VDR. This interaction can influence retinoid X receptor (RXR):VDR heterodimerization. Furthermore, immunofluorescence colocalization studies showed that IGFBP-6 colocalized with the VDR predominantly in the cell's nucleus. Inductions of osteocalcin and growth hormone promoter activities by 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) were significantly decreased when cells were co-transfected with IGFBP-6 and the VDR compared with cells transfected with the VDR only. Moreover, we found that alkaline phosphatase activity (ALP, a general marker of osteoblast differentiation) was significantly decreased in osteoblast-like cells when they were transfected with IGFBP-6 in the presence of 1,25(OH)(2)D(3). No obvious difference in ALP activity was observed when cells were transfected with IGFBP-6 and endogenous VDR was knocked down by siRNA. These results demonstrate that IGFBP-6 inhibits osteoblastic differentiation mediated by 1,25(OH)(2)D(3) and the VDR through interacting with the VDR and inhibiting its function. This is a novel mechanism for IGFBP-6.

Insulin-like growth factor binding protein-6 (IGFBP-6) interacts with DNA-end binding protein Ku80 to regulate cell fate.

Insulin-like growth factor binding protein-6 (IGFBP-6) is a growth inhibitory protein that regulates the availability of insulin-like growth factors (IGFs). We recently reported that IGFBP-6 exerts intracellular actions via its translocation to the nucleus. We now show that IGFBP-6 co-purifies by tandem-affinity with nuclear proteins involved in DNA stability and repair such as Ku80, Ku70, histone H2B and importin-alpha. Furthermore, this report shows that IGFBP-6 and Ku80 interact specifically using two active binding sites for Ku80 in IGFBP-6. One of the binding sites [196RKR199], as part of the NLS-sequence in IGFBP-6 also binds importin-alpha which may selectively compete with Ku80 regulating its trafficking to the nucleus. Moreover, IGFBP-6 co-localized with Ku80 based on a cell cycle pattern. Overexpression of IGFBP-6 increased the nuclear Ku80 in mitotic cells and reduced it post-mitosis. It is known that if highly expressed IGFBP-6 induces apoptosis and in our model, the down-regulation of Ku80 by specific siRNAs enhanced the apoptotic effect caused by the IGFBP-6 overexpression. This study demonstrates that IGFBP-6 alters cell survival by potentially regulating the availability of Ku80 for the DNA-repair process. This action represents a novel mechanism by which growth inhibitory proteins such as IGFBP-6 regulate cell fate.

Co-expression and purification of recombinant human insulin-like growth factor II and insulin-like growth factor binding protein-6 in Pichia pastoris yeast.

For the preparation of the complex of IGF-II and IGFBP-6, a co-expression vector containing two copies of human IGF-II and IGFBP-6 expression cassette was constructed with alcohol oxidase (AOX1) promoter and secretion signal sequence of alpha-factor, and transformed to Pichia pastoris yeast. Through a purification procedure involving anion-exchange chromatography and gel filtration, a complex of IGF-II with IGFBP-6 was obtained. An additional C-terminal sequence of IGFBP-6 (CS-BP6) was found to be bound to this complex. Dynamic light scattering showed that this complex was very stable and homogenous in solution. Western blotting based on non-reducing Tricine-SDS-PAGE indicated that IGF-II expression coupled with IGFBP-6 might significantly avoid the mispairing of disulfide bonds compared with the IGF-II expressed alone.

The N-terminal subdomain of insulin-like growth factor (IGF) binding protein 6. Structure and interaction with IGFs.

Insulin-like growth factor binding proteins (IGFBPs) modulate the activity and distribution of insulin-like growth factors (IGFs). IGFBP-6 differs from other IGFBPs in being a relatively specific inhibitor of IGF-II actions. Another distinctive feature of IGFBP-6 is its unique N-terminal disulfide linkages; the N-domains of IGFBPs 1-5 contain six disulfides and share a conserved GCGCC motif, but IGFBP-6 lacks the two adjacent cysteines in this motif, so its first three N-terminal disulfide linkages differ from those of the other IGFBPs. The contributions of the N- and C-domains of IGFBP-6 to its IGF binding properties and their structure-function relationships have been characterized in part, but the structure and function of the distinctive N-terminal subdomain of IGFBP-6 are unknown. Here we report the solution structure of a polypeptide corresponding to residues 1-45 of the N-terminal subdomain of IGFBP-6 (NN-BP-6). The extended structure of the N-terminal subdomain of IGFBP-6 is very different from that of the short two-stranded beta-sheet of the N-terminal subdomain of IGFBP-4 and, by implication, the other IGFBPs. NN-BP-6 contains a potential cation-binding motif; lanthanide ion binding was observed, but no significant interaction was found with physiologically relevant metal ions like calcium or magnesium. However, this subdomain of IGFBP-6 has a higher affinity for IGF-II than IGF-I, suggesting that it may contribute to the marked IGF-II binding preference of IGFBP-6. The extended structure and flexibility of this subdomain of IGFBP-6 could play a role in enhancing the rate of ligand association and thereby be significant in IGF recognition.

IGFBP-6 five years on; not so 'forgotten'?

Insulin-like growth factor binding protein (IGFBP)-6 is unique among IGFBPs for its IGF-II binding specificity. IGFBP-6 inhibits growth of a number of IGF-II-dependent cancers, including rhabdomyosarcoma, neuroblastoma and colon cancer. Although the major action of IGFBP-6 appears to be inhibition of IGF-II actions, a number of studies suggest that it may also have IGF-independent actions. Gene array studies show regulation of IGFBP-6 in many circumstances that are consistent with antiproliferative actions. However, other studies show the opposite, so that IGFBP-6 may be acting as a counter-regulator in these situations or it may have other as yet undetermined actions. Both the N-terminal and C-terminal domains of IGFBP-6 contribute to high affinity IGF binding, and the C-terminal domain appears to confer its IGF-II specificity. The three-dimensional structure of the C-domain of IGFBP-6 contains a thyroglobulin type 1 fold, and the IGF-II binding site is located in the proximal half of this domain adjacent to the glycosaminoglycan binding site. Future studies are needed to further delineate the putative IGF-independent actions of IGFBP-6 and to build on the structural information to enhance our understanding of this IGFBP. This is particularly significant since IGFBP-6 provides an attractive basis for therapy of IGF-II-dependent tumors.

C-terminal domain of insulin-like growth factor (IGF) binding protein 6: conformational exchange and its correlation with IGF-II binding.

Insulin-like growth factor binding proteins (IGFBPs) function as carriers and regulators of the insulin-like growth factors (IGF-I and -II). Within the family of six binding proteins, IGFBP-6 is unique in having a 20-100-fold higher affinity for IGF-II over IGF-I and appears to act primarily as an inhibitor of IGF-II actions. We have recently determined the solution structure of the C-terminal domain of IGFBP-6 (C-BP-6), which shows the presence of substantial flexible regions, including three loop regions. In this paper, we report results from (15)N relaxation measurements carried out in both the laboratory and rotating frames. Analysis of conventional (15)N relaxation data (R(1), R(2), and steady-state (15)N-[(1)H] nuclear Overhauser effect) indicated that there was a considerable number of residues involved in conformational/chemical exchange. Measurements of off-resonance (15)N R(1)(rho) in the rotating frame and (15)N relaxation dispersion using an in- and antiphase coherence-averaged Carr-Purcell-Meiboom-Gill sequence were thus carried out to gain further insight into the solution dynamics of C-BP-6. Although the off-resonance (15)N relaxation data showed no clear evidence for residues undergoing microsecond motion, the (15)N relaxation dispersion data allowed us to identify 15 residues that clearly exhibit submilli- to millisecond motion. A good correlation was observed between residues exhibiting motion at submilli- to millisecond time scales and those affected by IGF-II binding, as identified through the perturbation of nuclear magnetic resonance (NMR) spectra of C-BP-6 following IGF-II addition. A complete NMR relaxation study of C-BP-6 dynamics in complex with IGF-II was hampered by peak broadening and disappearance of C-BP-6 in the presence of IGF-II. Nonetheless, current results strongly suggest possible conformation switching or population shifting between pre-existing conformations in C-BP-6 upon binding to IGF-II.

C-terminal domain of insulin-like growth factor (IGF) binding protein-6: structure and interaction with IGF-II.

IGFs are important mediators of growth. IGF binding proteins (IGFBPs) 1-6 regulate IGF actions and have IGF-independent actions. The C-terminal domains of IGFBPs contribute to high-affinity IGF binding and modulation of IGF actions and confer some IGF-independent properties, but understanding how they achieve this has been constrained by the lack of a three-dimensional structure. We therefore determined the solution structure of the C-domain of IGFBP-6 using nuclear magnetic resonance (NMR). The domain consists of a thyroglobulin type 1 fold comprising an alpha-helix followed by a loop, a three-stranded antiparallel beta-sheet incorporating a second loop, and finally a disulfide-bonded flexible third loop. The IGF-II binding site on the C-domain was identified by examining NMR spectral changes upon complex formation. It consists of a largely hydrophobic surface patch involving the alpha-helix, the first beta-strand, and the first and second loops. The site was confirmed by mutagenesis of several residues, which resulted in decreased IGF binding affinity. The IGF-II binding site lies adjacent to surfaces likely to be involved in glycosaminoglycan binding of IGFBPs, which might explain their decreased IGF affinity when bound to glycosaminoglycans, and nuclear localization. Our structure provides a framework for understanding the roles of IGFBP C-domains in modulating IGF actions and conferring IGF-independent actions, as well as ultimately for the development of therapeutic IGF inhibitors for diseases including cancer.

Structure-function relationships of insulin-like growth factor binding protein 6 (IGFBP-6) and its chimeras.

Insulin-like growth factor binding protein 6 (IGFBP-6) is a high-affinity IGFBP with substantially greater affinity for insulin-like growth factor-II (IGF-II) than IGF-I. IGFBP-6(3) is a chimera which has a 20 amino acidC -terminal portion of IGFBP-6 switched with the homologous area of IGFBP-3, P3. Unlike IGFBP-4(3), in which the P3 region was exchanged for the homologous region of IGFBP-4 (P4), IGFBP-6(3) does not bind to endothelial cells. Double mutations were made with the P3 region exchanged as well as a second area differing from IGFBP-3 to form IGFBP-6(3)A and IGFBP-6(3)B, by replacing this area with the homologous region of IGFBP-3. Neither [(125)I]IGFBP-6(3)A nor IGFBP-6(3)B specifically bound to endothelial cells. However, each double mutant competed for [(125)I]IGFBP-3 binding to cultured cells. In the perfused heart, transendothelial transport of IGFBP-6 and IGFBP-6(3) was only 25% of similar transendothelial transport of perfused IGFBP-3. We conclude that chimeras of IGFBP-6 and IGFBP-3(6) clearly differ from IGFBP-4(3) in their ability to bind specifically to endothelial cells and in their capacity to undergo transendothelial transportation in the perfused heart.

Expression of growth factors in Dictyostelium discoideum.

Growth factors and their binding proteins are important proteins regulating mammalian cell proliferation and differentiation so there is considerable interest in producing them as recombinant proteins, especially in hosts that do not already produce a complex mixture of growth factors. Many growth factors require post-translational modifications making them unsuitable for production in Escherichia coli or other prokaryotes. Since several expression vector systems have been recently developed for foreign protein production in the cellular slime mould, Dictyostelium discoideum, we attempted to use two of these systems to express human insulin-like growth factor binding protein 6 (hIGFBP6) and bovine beta-cellulin (bBTC) as secreted proteins. Although both proteins were successfully produced in stably transformed amoebae, no secretion was detected in spite of several attempts to facilitate this occurring.

Effect of IGFBP-derived peptides on incorporation of(35)SO(4)into proteoglycans.

18 amino acid peptides from the C-terminal region of IGFBP-3, -5 (P3, P5), increased the incorporation of(35)SO(4)into proteoglycans in endothelial cells with greater stimulation in large vessel than microvessel cells. The homologous region of IGFBP-6 (P6) also stimulated sulfate uptake, but less potently than P3 and P5. P6 variants were synthesized with one or two amino acids changed to the basic amino acid in the equivalent position of P3. The P6 variants with one additional basic amino acid behaved similarly to P6. The P6 mutant with two altered amino acids was equipotent to P3. P3F, a scrambled version of P3 was less effective than P3. P3, P5, P6, P3F and all P6 variants all stimulated glucose uptake, which occurred only in microvessel cells. P1, P2, P4, and equimolar intact IGFBP-3 stimulated neither glucose uptake nor sulfate incorporation. Thus, C-terminal basic portions of IGFBP-3, -5 and -6 alter two specific functions of endothelial cells with sufficient differences to suggest mediation by distinct mechanisms.

O-glycosylation of insulin-like growth factor (IGF) binding protein-6 maintains high IGF-II binding affinity by decreasing binding to glycosaminoglycans and susceptibility to proteolysis.

Insulin-like growth factor binding protein-6 (IGFBP-6) is an O-linked glycoprotein which specifically inhibits insulin-like growth factor (IGF)-II actions. The effects of O-glycosylation of IGFBP-6 on binding to glycosaminoglycans and proteolysis, both of which reduce the IGF binding affinity of other IGFBPs were studied. Binding of recombinant human nonglycosylated (n-g) IGFBP-6 to a range of glycosaminoglycans in vitro was approximately threefold greater than that of glycosylated (g) IGFBP-6. When bound to glycosaminoglycans, IGFBP-6 had approximately 10-fold reduced binding affinity for IGF-II. Exogenously added n-gIGFBP-6 but not gIGFBP-6 also bound to partially purified rat PC12 phaeochromocytoma membranes. Binding of n-gIGFBP-6 was inhibited by increasing salt concentrations, which is typical of glycosaminoglycan interactions. O-glycosylation also protected human IGFBP-6 from proteolysis by chymotrypsin and trypsin. Proteolysis decreased the binding affinity of IGFBP-6 for IGF-II, even with a relatively small reduction in apparent molecular mass as observed with chymotrypsin. Analysis by ESI-MS of IGFBP-6 following limited chymotryptic digestion showed that a 4.5-kDa C-terminal peptide was removed and peptide bonds involved in the putative high affinity IGF binding site were cleaved. The truncated, multiply cleaved IGFBP-6 remained held together by disulphide bonds. In contrast, trypsin cleaved IGFBP-6 in the mid-region of the molecule, resulting in a 16-kDa C-terminal peptide which did not bind IGF-II. These results indicate that O-glycosylation inhibits binding of IGFBP-6 to glycosaminoglycans and cell membranes and inhibits its proteolysis, thereby maintaining IGFBP-6 in a high-affinity, soluble form and so contributing to its inhibition of IGF-II actions.

A central domain binding site in insulin-like growth factor binding protein-5 for the acid-labile subunit.

Like insulin-like growth factor binding protein-3 (IGFBP-3), IGFBP-5 forms a ternary complex with insulin-like growth factor (IGF)-I or IGF-II, and the acid-labile subunit (ALS). The study of IGFBP-5/IGFBP-6 chimeric proteins with amino-terminal and middle domain swaps, has revealed the existence of a site in the middle domain of IGFBP-5, that binds to ALS in the absence of the IGFBP-5 carboxy-terminal domain. An IGFBP-6 chimeric protein containing the central domain of IGFBP-5 complexed efficiently with ALS, and a carboxy-terminally truncated IGFBP-5 mutant, IGFBP-5'(1-169), also bound to ALS in the presence of IGFs, although with much less potency than full length rhIGFBP-5. In contrast to the latter, IGFBP-5(1-169) preferentially formed ternary complexes with IGF-II rather than IGF-I. These results indicate that a site which binds ALS exists in IGFBP-5 mutants which lack the IGFBP-5 carboxy-terminal domain.

Generation of antisera to mouse insulin-like growth factor binding proteins (IGFBP)-1 to -6: comparison of IGFBP protein and messenger ribonucleic acid localization in the mouse embryo.

The insulin-like growth factor (IGF) system is an important regulator of fetal growth and differentiation. IGF bioavailability is modulated by IGF binding proteins (IGFBPs). We have generated six different antisera, directed to synthetic peptide fragments of mouse IGFBP-1 through -6. The specificity of the produced antisera was demonstrated by enzyme-linked immunosorbent assay, Western blotting, and by immunohistochemistry on sections of mouse embryos of 13.5 days post coitum. Specificity for the IGFBP-2 through -6 antisera also was confirmed immunohistochemically in liver and lung of corresponding gene deletion (knock-out) mutant mice and wild-type litter mates. Immunohistochemistry and messenger RNA (mRNA) in situ hybridization on sections of mouse embryos of 13.5 days post coitum revealed tissue-specific expression patterns for the six IGFBPs. The only site of IGFBP-1 protein and mRNA production was the liver. IGFBP-2, -4, and -5 protein and mRNA were detected in various organs and tissues. IGFBP-3 and -6 protein and mRNA levels were low. In several tissues, such as lung, liver, kidney, and tongue, more than one IGFBP (protein and mRNA) could be detected. Differences between mRNA and protein localization were extensive for IGFBP-3, -5, and -6, suggesting that these IGFBPs are secreted and transported. These results confirm the different spatial localization of the IGFBPs, on the mRNA and protein level. The overlapping mRNA and protein localization for IGFBP-2 and -4, on the other hand, may indicate that these IGFBPs also function in an auto- or paracrine manner.

The N-terminal disulfide linkages of human insulin-like growth factor-binding protein-6 (hIGFBP-6) and hIGFBP-1 are different as determined by mass spectrometry.

The actions of insulin-like growth factors (IGFs) are modulated by a family of six high affinity binding proteins (IGFBPs 1-6). IGFBP-6 differs from other IGFBPs in having the highest affinity for IGF-II and in binding IGF-I with 20-100-fold lower affinity. IGFBPs 1-5 contain 18 conserved cysteines, but human IGFBP-6 lacks 2 of the 12 N-terminal cysteines. The complete disulfide linkages of IGFBP-6 were determined using electrospray ionization mass spectrometry of purified tryptic peptide complexes digested with combinations of chymotrypsin, thermolysin, and endoproteinase Glu-C. Numbering IGFBP-6 cysteines sequentially from the N terminus, the first three disulfide linkages are Cys1-Cys2, Cys3-Cys4, and Cys5-Cys6. The next two linkages are Cys7-Cys9 and Cys8-Cys10, which are analogous to those previously determined for IGFBP-3 and IGFBP-5. The C-terminal linkages are Cys11-Cys12, Cys13-Cys14, and Cys15-Cys16, analogous to those previously determined for IGFBP-2. Disulfide linkages of IGFBP-1 were partially determined and show that Cys1 is not linked to Cys2 and Cys3 is not linked to Cys4. Analogous with IGFBP-3, IGFBP-5, and IGFBP-6, Cys9-Cys11 and Cys10-Cys12 of IGFBP-1 are also disulfide-linked. The N-terminal linkages of IGFBP-6 differ significantly from those of IGFBP-1 (and, by implication, the other IGFBPs), which could contribute to the distinctive IGF binding properties of IGFBP-6.

Identification of O-glycosylation sites and partial characterization of carbohydrate structure and disulfide linkages of human insulin-like growth factor binding protein 6.

The actions of insulin-like growth factors (IGFs) are modulated by a family of high-affinity binding proteins (IGFBPs), including IGFBP-6, which preferentially binds IGF-II and is O-glycosylated. Glycosylated and nonglycosylated recombinant human IGFBP-6, expressed in Chinese hamster ovary cells and Escherichia coli, respectively, were purified using IGF-II affinity chromatography and reverse-phase medium-pressure chromatography. Electrospray ionization mass spectrometry (ESMS) of glycosylated IGFBP-6 revealed considerable heterogeneity of carbohydrate composition. Major glycoforms contained 8-16 monosaccharides, including N-acetylhexosamine, hexose, and N-acetylneuraminic acid. Glycosylation sites of IGFBP-6 were identified as Thr126, Ser144, Thr145, Thr146, and Ser152 by using a combination of ESMS and Edman sequencing of tryptic fragments separated by reverse-phase high-pressure liquid chromatography. One oligosaccharide chain contained 5-6 monosaccharides, whereas the others contained 2-4 monosaccharides. Glycosylated IGFBP-6 exhibited greater resistance to proteolysis by chymotrypsin and trypsin than nonglycosylated IGFBP-6. Native disulfide bond positions in IGFBP-6 were localized by means of observed disulfide-linked tryptic fragments, revealing that there are two disulfide-linked subdomains within each of the N- and C-terminal regions and confirming a previous suggestion that the latter regions are not interconnected. A model of IGFBP-6 is developed in which these distinct domains are separated by a central region which is O-glycosylated.

Insulin-like growth factor-binding protein-6 levels are elevated in serum of children with chronic renal failure: a report of the Southwest Pediatric Nephrology Study Group.

Previous studies suggest that growth retardation in children with chronic renal failure (CRF) results in part from inhibition of insulin-like growth factor (IGF) action by excess serum IGF-binding proteins (IGFBPs). Excess IGFBPs in CRF serum include IGFBP-1, -2, and -3 and a diffuse approximately 24- to 28-kDa IGFBP band identified by [125I]IGF ligand blot. The present studies characterized this diffuse approximately 24- to 28-kDa band. Initial studies identified this band as IGFBP-6, because it was immunoprecipitated by antiserum raised against a synthetic peptide of human IGFBP-6 (hIGFBP-6). Additional [125I]IGF ligand blots found that the immunoprecipitated band was 1) recognized by [125I]IGF-II but not [125I]IGF-1, 2) more abundant in CRF than in normal serum, and 3) more abundant in serum from dialyzed than nondialyzed prepubertal CRF children. Using the hIGFBP-6 antiserum in a specific and sensitive RIA, we found that serum IGFBP-6 levels were 4.7 +/- 1.7 nmol/L in 10 normal prepubertal children, 21.4 +/- 6.1 nmol/L in 44 nondialyzed prepubertal CRF children, 73.5 +/- 14.4 nmol/L in 7 dialyzed prepubertal CRF children, and 94.6 +/- 26.2 nmol/L in 14 dialyzed pubertal CRF children. IGFBP-6 levels were also elevated in 71 nondialyzed European children with CRF. In nondialyzed CRF children, serum IGFBP-6 levels 1) correlated inversely with the glomerular filtration rate, 2) did not correlate with height SD score, and 3) were not altered by 12 months of daily recombinant hGH treatment. In summary, a specific antiserum and RIA were used to demonstrate elevated levels of intact IGF-II-binding IGFBP-6 in serum of CRF children. We postulate that the excess IGFBP-6 may modulate the action of IGF-II on target tissues.

Heparin-binding, highly basic regions within the thyroglobulin type-1 repeat of insulin-like growth factor (IGF)-binding proteins (IGFBPs) -3, -5, and -6 inhibit IGFBP-4 degradation.

MC3T3-E1 murine osteoblasts produce insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4)-degrading proteinase activity, which is inhibited by IGFBP-3 and a highly basic, C-terminal domain of IGFBP-3. Of all the other five IGFBPs, IGFBP-5 and -6 share the highest degree of homology with this domain of IGFBP-3; therefore, we investigated whether these two IGFBPs inhibit IGFBP-4 degradation. Both IGFBP-5 and IGFBP-6 inhibit the degradation of 125I-IGFBP-4 by MC3T3-E1-conditioned media, and their inhibitory effects are variably reversed by IGFs. Synthetic peptides containing highly basic, C-terminal regions of IGFBP-5 and IGFBP-6 inhibit 125I-IGFBP-4 degradation, as does an homologous IGFBP-3 peptide, yet each peptide displays a different IC50, with the IGFBP-5 peptide being the most potent and the IGFBP-6 peptide being the least potent. In contrast, a homologous, yet neutral, IGFBP-4 peptide does not inhibit 125I-IGFBP-4 proteolysis, confirming the role of basic residues in the inhibitory process. The IGFBP-3, -5, and -6 peptides, each of which contains the heparin-binding consensus sequence XBBBXXBX, bind heparin, yet the IGFBP-3 and -5 peptides bind heparin with the highest affinities, whereas the IGFBP-6 peptide binds heparin with approximately 10-fold less affinity. Consistent with these regions being involved in proteinase inhibition, heparin completely reverses their inhibitory effects on 125I-IGFBP-4 proteolysis. Together, these data demonstrate that IGFBP-3, -5, and -6 can function as IGF-reversible inhibitors of IGFBP-4 proteolysis, likely through homologous, highly basic, heparin-binding domains contained within the conserved thyroglobulin type-1 motif present in the C-termini of these IGFBPs.