Yvonne W.-H. Yang

The 34 kilodalton insulin-like growth factor binding proteins in human cerebrospinal fluid and the A673 rhabdomyosarcoma cell line are human homologues of the rat BRL-3A binding protein

Three members of a family of insulin-like growth factor binding proteins have been identified by nucleotide sequencing of cDNA clones: the binding subunit of the 150 kDa IGF-binding protein complex in human serum, the 30 kDa IGF binding protein in human amniotic fluid, and a 30 kDa binding protein (BP-3A) isolated from the rat BRL-3A cell line. The present study demonstrates by molecular hybridization and immunoreactivity that the human counterpart of rat BP-3A is a 34 kDa IGF binding protein that is present in human cerebrospinal fluid and is synthesized and secreted by the A673 human rhabdomyosarcoma cell line.

Developmental regulation of insulin-like growth factor binding protein-2 in chick embryo serum and vitreous humor

The chick embryo is a useful vertebrate model for studying developmental embryogenesis. Insulin-like growth factor I (IGF-I), a potent mitogen, is thought to contribute to the general growth of the embryo as an endocrine factor, and as a paracrine factor to the development of the early embryo and of specific organs such as the eye. Recent data suggest that a family of at least six IGF binding proteins (IGFBPs) complex IGF-I and modulate its biological actions. In the present study, we examine the expression of IGFBPs in chicken serum and vitreous humor at different stages of embryonic development, and compare it with that of IGF-I. As determined by ligand blotting, the predominant IGFBP in chick serum and vitreous humor between embryonic days 4 and 22 (E4-E22) is a 30 kDa IGFBP. This IGFBP was specifically immunoprecipitated by a polyclonal antiserum raised against rat IGFBP-2, the predominant IGFBP in fetal human and rat serum. Although IGFBP-2 is present in both chick fluids at all times examined, serum IGFBP-2 increased progressively between E10-E22, whereas vitreous IGFBP-2 was highest during eye organogenesis (E4-E8). This suggests that vitreous IGFBP-2 is synthesized locally. Like serum IGFBP-2, levels of immunoreactive IGF-I in serum are higher in the second week of embryogenesis than the first. Despite this correlation, changes in IGFBP-2 do not appear to be regulated by IGF-I: (a) serum IGF-I decreases after day 15, whereas IGFBP-2 levels remain stable until hatching; (b) vitreous IGF-I, like serum IGF-I, is higher in the second week of embryogenesis, whereas vitreous IGFBP-2 is highest in the first week; (c) embryos cultured ex ovo express IGFBP-2 at E15-E19, although they lack the normal mid-embryogenesis surge in IGF-I. We conclude that vitreous IGFBP-2 is synthesized locally in the eye, and that the expression of IGFBP-2 in chick embryos is not directly regulated by IGF-I.

Regulation of Gene Expression of Rat Insulin-Like Growth Factor Binding Proteins 1 and 2

Virtually all of the insulin-like growth factors (IGFs) in extracellular fluids and cell culture medium occur complexed to specific IGF-binding proteins (IGFBPs).1,2 The IGFBPs are a family of proteins that bind IGF-I and IGF-II but are unrelated to IGF receptors. Four IGFBPs have been cloned from human and rat sources,1–8 and partial protein sequence information is available forafifthlGFBP.9–11 OthermembersoftheIGFBPfamilyundoubtedlyexist,12butspecific assignment must await amino acid or nucleotide sequencing.

Characterization and Cloning of a Rat Insulin-Like Growth Factor Binding Protein

The insulin-like growth factors, IGF-I and IGF-II, occur complexed to specific binding proteins in blood and other extracellular fluids.1 IGF-binding protein complexes of 150 kDa predominate in adult human and rat serum2,3, and also have been observed in human and porcine milk 4,5, and human fibroblast conditioned media6,7. Acid pH irreversibly dissociates the 150 kDa binding protein complex into an ~40 kDa acid-stable binding subunit.1 Recently, Baxter8 provided evidence for the existence of a second subunit of ~100 kDa that is unstable at acid pH and does not bind IGFs.

Insulin-Like Growth Factors in Fetal Growth

The insulin-like growth factors, IGF-I and IGF-II, are single-chain polypeptides chemically related to insulin that are synthesized in multiple fetal and adult tissues, and stimulate DMA synthesis and cell differentiation (1,2). IGF-I is regulated by growth hormone and nutritional factors, and promotes bone elongation in childhood (2). Based on results initially obtained in rats, IGF-II was proposed to play a role in fetal growth and development. This paper will review some of the evidence in support of this hypothesis, and discuss its applicability to human fetal development.

Biosynthesis of Rat Insulinlike Growth Factor II in Intact Cells and Cell-Free Translation

Dulak and Temin(1) first reported that the BRL-3A cell line established from normal rat liver secreted a family of polypeptides, which they termed MSA, that had multiplication-stimulating activity for chick embryo fibroblasts. MSA was purified by Moses and colleagues from conditioned media using Dowex chromatography, Sephadex G-75 gel filtration in 1 M acetic acid, and preparative gel electrophoresis, and shown to appear in multiple forms of Mr 16.3K, 8.7K, and 7.1K.(2) Marquardt et al. (3) purified MSA by a different purification scheme (i.e., Bio-Gel P-10 in 1 M acetic acid and high-performance liquid chromatography), and determined the amino acid sequence of a Mr 7484-dalton form. Mr 7484 MSA appears to correspond to our Mr 7.1K species. It is identical to human IGF-II at 62 of 67 amino acid loci, establishing that BRL-MSA represents the rat homologue of IGF-II.(3)