Insulin-Like Growth Factor (IGF) Binding Protein-4 Is Both a Positive and Negative Regulator of IGF Activity in Vivo

Abstract IGFs are required for normal prenatal and postnatal growth. Although actions of IGFs can be modulated by a family of IGF-binding proteins (IGFBPs) in vitro, these studies have identified a complicated pattern of stimulatory and inhibitory IGFBP effects, so that understanding relevant aspects of IGFBP action in vivo has been limited. Here we have produced a null mutation of one specific IGFBP, IGFBP-4, which is coexpressed with IGF-II early in development. Surprisingly, mutation of IGFBP-4, believed from in vitro studies to be exclusively inhibitory, leads to a prenatal growth deficit that is apparent from the time that the IGF-II growth deficit first arises, which strongly suggests that IGFBP-4 is required for optimal IGF-II-promoted growth during fetal development. Mice encoding a mutant IGFBP-4 protease (pregnancy-associated plasma protein-A), which facilitates IGF-II release from an inactive IGF-II/IGFBP-4 complex in vitro, are even smaller than IGFBP-4 mutant mice. However, the more modest IGFBP-4 growth deficit is completely restored in double IGFBP-4/pregnancy-associated plasma protein-A-deficient mice. Taken together these results indicate not only that IGFBP-4 functions as a local reservoir to optimize IGF-II actions needed for normal embryogenesis, but also establish that IGFBP-4 proteolysis is required to activate most, if not all, IGF-II mediated growth-promoting activity.

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Heparin-binding mechanism of the IGF2/IGF-binding protein 2 complex

Journal of Molecular Endocrinology ◽

10.1530/jme-13-0184 ◽

2014 ◽

Vol 52 (3) ◽

pp. 345-355 ◽

Cited By ~ 11

Author(s):

Jacob Lund ◽

Mads T Søndergaard ◽

Cheryl A Conover ◽

Michael T Overgaard

Keyword(s):

Bone Matrix ◽

Receptor Activation ◽

Heparin Binding ◽

Igf Binding Proteins ◽

Skeletal Mass ◽

Igf Binding ◽

Heparin Affinity ◽

Positively Charged

IGF1 and IGF2 are potent stimulators of diverse cellular activities such as differentiation and mitosis. Six IGF-binding proteins (IGFBP1–IGFBP6) are primary regulators of IGF half-life and receptor availability. Generally, the binding of IGFBPs inhibits IGF receptor activation. However, it has been shown that IGFBP2 in complex with IGF2 (IGF2/IGFBP2) stimulates osteoblast function in vitro and increases skeletal mass in vivo. IGF2 binding to IGFBP2 greatly increases the affinity for 2- or 3-carbon O-sulfated glycosaminoglycans (GAGs), e.g. heparin and heparan sulfate, which is hypothesized to preferentially and specifically target the IGF2/IGFBP2 complex to the bone matrix. In order to obtain a more detailed understanding of the interactions between the IGF2/IGFBP2 complex and GAGs, we investigated heparin-binding properties of IGFBP2 and the IGF2/IGFBP2 complex in a quantitative manner. For this study, we mutated key positively charged residues within the two heparin-binding domains (HBDs) in IGFBP2 and in one potential HBD in IGF2. Using heparin affinity chromatography, we demonstrate that the two IGFBP2 HBDs contribute differentially to GAG binding in free IGFBP2 and the IGF2/IGFBP2 protein complex. Moreover, we identify a significant contribution from the HBD in IGF2 to the increased IGF2/IGFBP2 heparin affinity. Using molecular modeling, we present a novel model for the IGF2/IGFBP2 interaction with heparin where all three proposed HBDs constitute a positively charged and surface-exposed area that would serve to promote the increased heparin affinity of the complex compared with free intact IGFBP2.

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Selection of the Dominant Follicle and Insulin-Like Growth Factor (IGF)-Binding Proteins: Evidence that Pregnancy-Associated Plasma Protein A Contributes to Proteolysis of IGF-Binding Protein 5 in Bovine Follicular Fluid

Endocrinology ◽

10.1210/en.2002-220657 ◽

2003 ◽

Vol 144 (2) ◽

pp. 437-446 ◽

Cited By ~ 49

Author(s):

G. M. Rivera ◽

J. E. Fortune

Keyword(s):

Proteolytic Activity ◽

Plasma Protein ◽

Follicular Fluid ◽

Binding Proteins ◽

Protein A ◽

Positive Control ◽

Dominant Follicle ◽

Igf Binding Proteins ◽

Igf Binding ◽

Igf Binding Protein

Development of a dominant follicle is associated with decreased intrafollicular low molecular weight IGF-binding proteins (namely IGFBP-2, -4, and -5) and increased proteolysis of IGFBP-4 by pregnancy-associated plasma protein A (PAPP-A). In addition to IGFBP-4 proteolytic activity, bovine follicular fluid contains strong proteolytic activity for IGFBP-5, but not for IGFBP-2. Here we show that the IGFBP-5 protease present in bovine follicular fluid is a neutral/basic pH-favoring, Zn2+ metalloprotease very similar to the previously described IGFBP-4 protease. We hypothesized that immunoneutralization and immunoprecipitation with anti-PAPP-A antibodies would result in abrogation of the IGFBP-4, but not the IGFBP-5, proteolytic activity in follicular fluid. As expected, anti-PAPP-A antibodies were able to neutralize and precipitate the IGFBP-4, but not the IGFBP-5, proteolytic activity of human pregnancy serum, which was used as a positive control for PAPP-A. Surprisingly, immunoneutralization and immunoprecipitation of follicular fluid from bovine preovulatory follicles with anti-PAPP-A antibodies abrogated both IGFBP-4 and IGFBP-5 proteolysis. Quantitative results derived from phosphorimaging revealed a complete inhibition of both IGFBP-4 and -5 proteolysis by follicular fluid incubated for 2 or 5 h in the presence of anti-PAPP-A antibodies. After 18 h of incubation, anti-PAPP-A antibodies still inhibited IGFBP-5 degradation, although with an efficiency lower than that for IGFBP-4 degradation. Both proteolytic activities have identical electrophoretic mobility, and a single band (∼400 kDa) was detected by Western immunoblotting of bovine follicular fluid with anti-PAPP-A antibodies. Proteolysis of IGFBP-5 was readily detectable in follicular fluid from dominant follicles and was negligible in subordinate follicles from the same cohort. These results suggest that an active intrafollicular IGFBP-4/-5 proteolytic system, in which PAPP-A is the major protease involved, is an important determinant of follicular fate.

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Analysis of Pregnancy-Associated Plasma Protein A Production in Human Adult Cardiac Progenitor Cells

BioMed Research International ◽

10.1155/2013/190178 ◽

2013 ◽

Vol 2013 ◽

pp. 1-8 ◽

Cited By ~ 7

Author(s):

Piera D’Elia ◽

Vittoria Ionta ◽

Isotta Chimenti ◽

Francesco Angelini ◽

Fabio Miraldi ◽

...

Keyword(s):

Progenitor Cells ◽

Plasma Protein ◽

Protein A ◽

Therapeutic Effects ◽

Cardiac Progenitor Cells ◽

Cardiac Cell ◽

Igf Binding Proteins ◽

Human Adult ◽

Igf Binding ◽

Cardiac Cell Therapy

IGF-binding proteins (IGFBPs) and their proteases regulate IGFs bioavailability in multiple tissues. Pregnancy-associated plasma protein A (PAPP-A) is a protease acting by cleaving IGFBP2, 4, and 5, regulating local bioavailability of IGFs. We have previously shown that IGFs and IGFBPs are produced by human adult cardiac progenitor cells (haCPCs) and that IGF-1 exerts paracrine therapeutic effects in cardiac cell therapy with CPCs. Using immunofluorescence and enzyme immunoassays, we firstly report that PAPP-A is produced and secreted in surprisingly high amounts by haCPCs. In particular, the homodimeric, enzymatically active, PAPP-A is secreted in relevant concentrations in haCPC-conditioned media, while the enzymatically inactive PAPPA/proMBP complex is not detectable in the same media. Furthermore, we show that both homodimeric PAPP-A and proMBP can be detected as cell associated, suggesting that the previously described complex formation at the cell surface does not occur easily, thus positively affecting IGF signalling. Therefore, our results strongly support the importance of PAPP-A for the IGFs/IGFBPs/PAPP-A axis in CPCs biology.

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Differences in the association of insulin-like growth factor-I (IGF-I) and IGF-I variants with rat, sheep, pig, human and chicken plasma-binding proteins

Journal of Endocrinology ◽

10.1677/joe.0.1400475 ◽

1994 ◽

Vol 140 (3) ◽

pp. 475-482 ◽

Cited By ~ 14

Author(s):

A P D Lord ◽

S E P Bastian ◽

L C Read ◽

P E Walton ◽

F J Ballard

Keyword(s):

Binding Proteins ◽

Rat Plasma ◽

Size Exclusion ◽

Free Form ◽

Igf Binding Proteins ◽

Igf I ◽

Exclusion Chromatography ◽

Igf Binding

Abstract Associations between labelled insulin-like growth factors (IGFs) and IGF-binding proteins in plasma have been compared in the rat, sheep, human, pig and chicken. The IGFs tested were recombinant human IGF-I, the truncated variant, des(1–3)IGF-I, and LR3IGF-I, an extended form that had been engineered so as to minimize interactions with IGF-binding proteins. Marked species differences were demonstrated, notably that the IGF-I variants which exhibited extremely weak binding in rat plasma bound significantly in plasma from the other species. This result was shown both by size-exclusion chromatography of labelled IGFs added to plasma, in which the extent of variant IGF-I binding decreased in the order sheep>human>pig=chicken>rat, and by competition for labelled IGF-I binding in vitro, in which the order was pig=chicken>sheep>human>rat. Notwithstanding these differences, the two IGF-I variants showed only slight between-species binding differences when tested with purified rat, sheep and human IGF-binding protein-3. Ligand blotting experiments with plasma from the five species similarly showed a consistent pattern in that IGF-I binding was much greater than des(1–3)IGF-I binding, which in turn was greater than LR3 IGF-I binding. These experiments suggest first that IGF-binding properties measured after the removal of endogenous IGFs do not always reflect the situation with untreated plasma or in vivo, and secondly, the increased potencies of des(1–3)IGF-I and LR3 IGF-I in rat growth studies that have been ascribed to higher concentrations of these peptides in the free form cannot necessarily be extended to other species. Journal of Endocrinology (1994) 140, 475–482

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Author Correction: A common variant of the pregnancy-associated plasma protein-A (PAPPA) gene encodes a protein with reduced proteolytic activity towards IGF-binding proteins

Scientific Reports ◽

10.1038/s41598-019-53957-x ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

J. A. Bøtkjær ◽

P. R. Noer ◽

Claus Oxvig ◽

Claus Yding Andersen

Keyword(s):

Proteolytic Activity ◽

Plasma Protein ◽

Binding Proteins ◽

Protein A ◽

Common Variant ◽

Igf Binding Proteins ◽

Igf Binding

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Activity of IGF-binding proteins in seminal plasma of bulls: Effects on fertilization capacity in vivo and in vitro

Theriogenology ◽

10.1016/s0093-691x(98)90726-9 ◽

1998 ◽

Vol 49 (1) ◽

pp. 373

Author(s):

G. Schmelzinger ◽

J. Schwartz ◽

T. Grupp ◽

H.-D. Reichenbach ◽

E. Wolf

Keyword(s):

Seminal Plasma ◽

Binding Proteins ◽

Igf Binding Proteins ◽

Igf Binding ◽

Fertilization Capacity

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Prednisolone reduces the ability of serum to activate the IGF1 receptor in vitro without affecting circulating total or free IGF1

Acta Endocrinologica ◽

10.1530/eje-12-0518 ◽

2013 ◽

Vol 168 (1) ◽

pp. 1-8 ◽

Cited By ~ 10

Author(s):

Jan Frystyk ◽

Anders J Schou ◽

Carsten Heuck ◽

Henrik Vorum ◽

Mikkel Lyngholm ◽

...

Keyword(s):

Receptor Activation ◽

Specific Cell ◽

Serum Samples ◽

Double Blind ◽

Igf Binding Proteins ◽

Activation Assay ◽

Igf Binding ◽

Igf1 Receptor

ObjectiveEnd-point bioassays based on thymidine or sulfate incorporation have demonstrated that glucocorticoid (GC) treatment inhibits serum IGF1 action, but the mechanism is unknown as serum IGF1 concentrations have been reported to either increase or remain unchanged.AimTo investigate whether GC treatment affects the ability of serum to activate the IGF1 receptor (IGF1R) in vitro (i.e. bioactive IGF1), using a specific cell-based IGF1 kinase receptor activation assay.Subjects and methodsTwenty children with stable asthma (age 7.7–13.8 years) treated for 1 week with 5 mg prednisolone in a randomized, double-blind, placebo-controlled crossover study. Non-fasting serum samples were collected in the afternoon after each 7-day period and assayed for bioactive IGF1, free IGF1, total IGFs, IGF-binding proteins (IGFBPs), and insulin.ResultsPrednisolone treatment reduced IGF1 bioactivity by 12.6% from 2.22±0.18 to 1.94±0.15 μg/l (P=0.01) compared with placebo. In contrast, no changes were observed for (μg/l; placebo vs prednisolone) total IGF1 (215±27 vs 212±24), free IGF1 (1.50±0.16 vs 1.43±0.17), total IGF2 (815±26 vs 800±31), IGFBP3 (3140±101 vs 3107±95), IGFBP2 (238±21 vs 220±19), IGFBP1 (32±6 vs 42±10), or IGFBP1-bound IGF1 (24±5 vs 26±7). Insulin remained unchanged as did IGFBP levels as estimated by western ligand blotting. Prednisolone had no direct effects on IGF1R phosphorylation.ConclusionsOur study gives evidence that GC treatment induces a circulating substance that is able to inhibit IGF1R activation in vitro without affecting circulating free or total IGF1. This may be one of the mechanisms by which GC inhibits IGF1 action in vivo. However, the nature of this circulating substance remains to be identified.

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IGF-binding protein-4: biochemical characteristics and functional consequences

Journal of Endocrinology ◽

10.1677/joe.0.1780177 ◽

2003 ◽

Vol 178 (2) ◽

pp. 177-193 ◽

Cited By ~ 68

Author(s):

R Zhou ◽

D Diehl ◽

A Hoeflich ◽

H Lahm ◽

E Wolf

Keyword(s):

Biological Fluids ◽

Biological Significance ◽

Structural Similarity ◽

Cell Types ◽

Cellular Growth ◽

Igf Binding Proteins ◽

Wide Range ◽

Igf Binding

IGFs have multiple functions regarding cellular growth, survival and differentiation under different physiological and pathological conditions. IGF effects are modulated systemically and locally by six high-affinity IGF-binding proteins (IGFBP-1 to -6). Despite their structural similarity, each IGFBP has unique properties and exhibits specific functions. IGFBP-4, the smallest IGFBP, exists in both non-glycosylated and N-glycosylated forms in all biological fluids. It is expressed by a wide range of cell types and tIssues, and its expression is regulated by different mechanisms in a cell type-specific manner. IGFBP-4 binds IGF-I and IGF-II with similar affinities and inhibits their actions under almost all in vitro and in vivo conditions. In this review, we summarize the available data regarding the following aspects of IGFBP-4: genomic organization, protein structure-function relationship, expression and its regulation, as well as IGF-dependent and -independent actions. The biological significance of IGFBP-4 for reproductive physiology, bone formation, renal pathophysiology and cancer is discussed.

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Expression of the IGFBP-2 gene in post-implantation rat embryos

Development ◽

10.1242/dev.114.1.59 ◽

1992 ◽

Vol 114 (1) ◽

pp. 59-66 ◽

Cited By ~ 2

Author(s):

T.L. Wood ◽

R.D. Streck ◽

J.E. Pintar

Keyword(s):

Cell Types ◽

Tail Bud ◽

Igf Binding Proteins ◽

Surface Ectoderm ◽

Genes Encoding ◽

Mantle Layer ◽

Igf Binding ◽

Post Implantation

The insulin-like growth factors (IGFs) stimulate mitogenesis in a variety of cell types both in vitro and in vivo. These effects are mediated by both IGF receptors and a family of IGF binding proteins (IGFBPs), which are found complexed with the IGFs in serum and tissue fluids. Here we compare the sites of expression during early rat embryogenesis of the genes encoding the RGD-containing IGF binding protein IGFBP-2 and IGF-II. At all ages from early post-implantation through mid-gestation, the expression of IGFBP-2 was highly complementary to IGF-II. IGFBP-2 mRNA was detected throughout the epiblast of the egg cylinder as early as e7, when IGF-II expression was restricted to trophectoderm and other extraembryonic cells. As gastrulation proceeded, IGFBP-2 expression ceased as IGF-II expression began in the newly formed embryonic and extra-embryonic mesoderm, but was retained in other epiblast derivatives including the surface ectoderm and neuroectoderm, throughout its rostral-caudal extent. By e10-e11, IGFBP-2 expression in neuroectoderm was restricted to the rostral brain of the primary neural tube and was found in the new population of neuroepithelium formed in the tail bud during secondary neurulation. IGFBP-2 expression remained high in the ventricular layer of the rostral brain into mid-gestation ages but decreased or disappeared as cells entered the mantle layer and began to express the neurofilament-related gene alpha-internexin. IGFBP-2 mRNA was abundant in surface ectoderm, particularly that of the branchial arches, and all ectodermal placodes.(ABSTRACT TRUNCATED AT 250 WORDS)

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A common variant of the pregnancy-associated plasma protein-A (PAPPA) gene encodes a protein with reduced proteolytic activity towards IGF-binding proteins

Scientific Reports ◽

10.1038/s41598-019-49626-8 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Jane Alrø Bøtkjær ◽

Pernille Rimmer Noer ◽

Claus Oxvig ◽

Claus Yding Andersen

Keyword(s):

Plasma Protein ◽

Binding Proteins ◽

Specific Activity ◽

Protein A ◽

P Value ◽

Surface Binding ◽

Cleavage Rate ◽

Igf Binding Proteins ◽

Single Nucleotide ◽

Igf Binding

Abstract Pregnancy-associated plasma protein-A (PAPP-A) is a key regulator of insulin-like growth factor (IGF) bioactivity, by releasing the IGFs from their corresponding IGF-binding proteins (IGFBPs). The minor allele of the single nucleotide polymorphism (SNP), rs7020782 (serine < tyrosine), in PAPPA has previously been associated with recurrent pregnancy loss as well as with significant reduced levels of PAPP-A protein in human ovarian follicles. The aim of the present study was to reveal a possible functional effect of the rs7020782 SNP in PAPPA by comparing recombinant PAPP-A proteins from transfected human embryonic kidney 293 T cells. The proteolytic cleavage of IGFBP-4 was shown to be affected by the rs7020782 SNP in PAPPA, showing a significantly reduced cleavage rate for the serine variant compared to the tyrosine variant (p-value < 0.001). The serine variant also showed a trend towards reduced cleavage rates, that was not significant, towards IGFBP-2 and IGFBP-5 compared to the tyrosine variant. No differences were found when analysing cell surface binding, complex formation between PAPP-A and STC2 or proMBP, nor when analysing STC1 inhibition of PAPP-A-mediated IGFBP-4 cleavage. Regulation of IGF bioactivity in reproductive tissues is important and the rs7020782 SNP in PAPPA may disturb this regulation by altering the specific activity of PAPP-A.

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