Insulin-like growth factor binding proteins-3 and -5 form sodium dodecyl sulfate-stable multimers

Insulin-like growth factor binding proteins (IGFBPs) are important modulators of IGF actions. IGFBP-3 and IGFBP-5 can bind to the extracellular matrix of a number of cell types. We now describe a new posttranslational structural modification of IGFBP-3 and IGFBP-5, which could play a role in determining their localization. We incubated radioiodinated forms of all six IGFBPs in the presence of a redox buffer consisting of 10 mM reduced glutathione and 0.2 mM oxidized glutathione. Under these conditions IGFBP-3 and IGFBP-5, but not the other IGFBPs, formed high molecular weight disulfide-linked multimers. Heparin and a peptide encompassing the high-affinity heparin-binding site in the C-terminal portion of IGFBP-3 were capable of blocking the multimerization of IGFBP-3. IGFBP-3, but not IGFBP-1, was shown to be able to self-associate non-covalently, which could be a requisite first step in the formation of covalent multimers. The self-association of IGFBP-3 required the high-affinity heparin-binding site in the C-terminal portion of the molecule.     

Growth hormone receptor activity is stimulated by insulin-like growth factor binding protein 5 in rat osteosarcoma cells

Osteoblast-like UMR-106.01 rat osteosarcoma cells express high affinity growth hormone (GH) receptors (GHRs). Because osteoblasts secrete insulin-like growth factor binding protein-5 (IGFBP-5), we evaluated whether it also modulates GH binding and GHR expression in UMR cells. Human recombinant intact IGFBP-5 stimulated 125I-hGH binding in a dose-dependent manner (dose range 300-3000 ng/ml), inducing an increase to 193.6 +/- 2.1% of control binding at 3000 ng/ml (P < 0.001). Carboxy-truncated IGFBP-5 also stimulated GH binding but with less potency (125 +/- 2.7% of control at 3000 ng/ml, P < 0.01). GHRs identified by chemical crosslinking of 125I-hGH to cell monolayers increased after treatment with IGFBP-5 and decreased in response to insulin-like growth factor-I (IGF-I). GHR mRNA levels, as quantitated by a solution hybridization RNAse protection assay, increased up to 3 to 7-fold in a time-dependent manner by intact IGFBP-5 but not by carboxy-truncated IGFBP-5. An antiserum to IGFBP-5 reduced basal GH binding to 56.7 +/- 4.3% of control value at a concentration of 0.5% (P < 0.001), showing that IGFBP-5 produced by the cells is a strong regulator of GH binding. IGFBP-5 antiserum also decreased GH binding to 85.9 +/- 0.9% of IGFBP-5 stimulated value (P < 0.001), showing the specificity of IGFBP-5 stimulation. To determine whether the GHR upregulation was physiologically significant, cell proliferation was evaluated after coincubation of IGFBP-5 with low, non-stimulatory concentrations of GH. IGFBP-5 (1000 ng/ml) induced cell proliferation to 116.2 +/- 3.2% of control levels, and coincubation with hGH at 10 ng/ml induced an increase to 133.3 +/- 0.1% of control levels. We conclude that exogenous and endogenous IGFBP-5 upregulate GHR mRNA levels and GH binding and this interaction potentiates GH-stimulated mitogenesis in osteoblastic cells.     

Insulin-like growth factor receptors and binding proteins

The insulin-like growth factor receptors are integral membrane proteins and demonstrate separate, but important effects on the regulation of cellular processes. The IGF-I receptor signals multiple cascades via its inherent tyrosine kinase activity. The IGF-II/M-6-P receptor on the other hand is primarily involved in targeting of enzymes to various subcellular compartments. In contrast, the insulin-like binding proteins are secreted by the cells and accumulate in the extracellular matrix or on the external surface of the cell. They are also involved in regulating cellular processes more indirectly. They modulate the interactions of the IGFs with their receptors, and in addition, may have some IGF-independent effects probably by direct interaction with integrin and other cell membrane receptor proteins. The recent studies, as outlined in this review, strongly suggest an important, if not essential role for the IGF system in normal physiology and disease states. The challenge now is to define the mechanisms involved in these effects. More studies are required to fully understand the post-receptor mechanism involved in IGF-I receptor signal transduction and the mechanisms whereby the IGFBPs exert their interesting effects. Understanding these mechanisms will enable investigators to create new therapeutic modalities for diseases that are affected by the IGF system.     

Insulin-like growth factor binding protein production in bovine retinal endothelial cells

Retinopathy is the most frequent microangiopathic complication in diabetes. Many circulating hormones and locally produced mitogenic factors have been involved. Bovine retinal endothelial cells (BRECs) were cultured to investigate if insulin, insulin-like growth factors (IGFs), IGF binding proteins (IGFBPs), and a chronic high-glucose condition could control endothelial cell growth. Specific IGF-I receptors with two binding sites with high (Kd 0.03 nmol/L) and low (Kd 1.3 nmol/L) affinity were found when analyzing families of displacement curves between IGF-I versus IGF-I and IGF-I versus insulin. However, IGFs failed to be mitogenic factors in these cells. This could be explained by an inhibitory effect due to the presence of specific IGFBPs with a molecular weight between 24 and 43 kd. Using Western blot and immunoblot analysis, Northern blot study, and specific radioimmunoassay (RIA), these IGFBPs have been identified as IGFBP-3, -2, -5, and -4. Insulin, which does not bind to IGFBPs, was a potent mitogenic factor in these cells at a high concentration (10 nmol/L), suggesting a cross-reaction to IGF-I receptor. These IGFBPs, except the 24-kd form (IGFBP-4), were modulated by both IGF-I and IGF-II, with a maximum effect at 100 and 10 nmol/L, respectively. This regulation on IGFBPs was IGF-I receptor-independent. In fact, (1) IGFBP mRNA levels were not modified after stimulation with 100 nmol/L IGF-I, (2) 100 nmol/L IGF plus an equimolar concentration of alpha IR3 did not affect IGFBP production, (3) Des(1-3)IGF-I had no effect on IGFBP modulation, whereas at 10 nmol/L it enhanced BREC thymidine cell incorporation, and (4) 100 nmol/L insulin, which at this concentration can cross-react with the IGF-I receptor, did not modify the IGFBP pattern. Chronic exposure (4 weeks) of BRECs to 25 mmol/L glucose had no effect on cell growth. However, after 3 weeks, we observed a decreased IGFBP detection, and addition of 100 nmol/L IGF-I did not change IGFBP levels and did not modify cell growth. Conversely, BRECs grown in regular medium for 4 weeks showed increased IGFBP production. In conclusion, we showed that conditions mimicking hyperinsulinemia, rather than high levels of IGFs, could regulate BREC growth and that the IGF-I analog, Des(1-3), even with reduced affinity for IGFBPs but in part capable of binding to IGFBP-3, significantly stimulated BRECs growth only at 10 nmol/L. IGF actions are modulated by locally produced endothelial IGFBPs, and in turn, these endothelial IGFBPs are regulated, via in IGF-I receptor-independent mechanism, by the presence of IGFs. The autoregulatory IGF system together with the direct glucose modulation of IGFBPs could contribute in diabetic subjects to the retinal endothelial cell growth and metabolism through local changes in IGF bioavailability.     

Insulin-like growth factor binding protein-1 induces insulin release in the rat

Injections of human insulin-like growth factor binding protein (hIGFBP-1) are reported to induce hyperglycemia in the rat, suggesting that IGFBP-1 acutely regulates glucose homeostasis. We now report the effects on glucose and insulin levels of administering recombinant (r) hIGFBP-1. In a series of studies, normal and streptozotocin (STZ) diabetic male Wistar rats (180-210 g), fasted for 6 or 16 h, were injected with rhIGFBP-1 (i.v., 80-500 microg/rat). rhIGFBP-1 did not affect blood glucose acutely but did stimulate insulin release in normal rats (5 min post injection; PBS, 103.5 +/- 8.5; rhIGFBP-1 (500 microg), 166.8 +/- 15.7; rhIGFBP-1 (100 microg); 151.4 +/- 14.1% initial). rhIGFBP-1 pretreatment, in normal and diabetic rats, reduced the hypoglycemic response to rhIGF-I (diabetic rats after 20 min: PBS, 103.4 +/- 11.4; BP-1 (500 microg) +/- rhIGF-I (50 microg), 97.6 +/- 3.6; rhIGF-I, 48.2 +/- 4.3% initial) but did not affect the hypoglycemic response to des(1-3)IGF-I or insulin (0.5 U/kg). These studies show that rhIGFBP-1 causes insulin release, has a minimal effect on blood glucose, and inhibits the hypoglycemic effect of rhIGF-I. These data suggest that endogenous IGF-I tonically suppresses insulin secretion and imply that aberrant IGFBP levels or reduced IGF-I bioactivity may lead to chronic hyperinsulinemia.     

Insulin-like growth factor binding protein production in human follicular thyroid carcinoma cells

OBJECTIVE: The study investigated the value of using national or regional data bases to examine care in a specific hospital. DATA SOURCES: The following data sources were included: (1) the results of the 1992 HCFA analysis of the index hospital for patients hospitalized in fiscal year 1990; (2) the 1989 Medicare Provider Analysis and Review (MEDPAR) file; and (3) clinical information from bypass surgery patients in Wisconsin and from the index hospital. PRINCIPAL FINDINGS: The assessment of the mortality rates in the index hospital for all conditions combined and for CABG patients differed depending on what data base was used and how the data were analysed. The national data were most useful in establishing that the coding practices for all patients and the mortality rate for intra-aortic balloon patients differed between the index hospital and other hospitals. The regional clinical data base for bypass surgery patients was used to establish that the high mortality rates for intra-aortic balloon patients were due to patient selection. CONCLUSIONS: National claims data must be analysed carefully before applying results to an individual hospital. Even a careful analysis is more for raising questions about care at a specific hospital rather than for reaching definitive conclusions.     

Developmental pattern of fetal growth hormone, insulin-like growth factor I, growth hormone binding protein and insulin-like growth factor binding protein-3

AIMS: To evaluate the developmental pattern of fetal growth hormone (GH), insulin-like growth factor I (IGF-I), GH binding protein (GHBP) and IGF binding protein-3 (IGF-3); to determine the implications for fetal growth. METHODS: Serum GH, IGF-I, GHBP and IGFBP-3 were measured in 53 fetuses, 41 aged 20-26 weeks (group A) and 12 aged 31-38 weeks (group B). Fetal blood samples were obtained by direct puncture of the umbilical vein in utero. Fetal blood samples were taken to rule out beta thalassaemia, chromosome alterations, mother to fetus transmissible infections, and for maternal rhesus factor. GHBP was determined by gel filtration chromatography of serum incubated overnight with 125I-GH. GH, IGF-I and IGFBP-3 were determined by radioimmunoassay. RESULTS: Fetal serum GH concentrations in group A (median 29 micrograms/l, range 11-92) were significantly higher (P < 0.01) than those of group B (median 16.7 micrograms/l, range 4.5-29). IGF-I in group A (median 20 micrograms/l, range 4.1-53.3) was significantly lower (P < 0.01) than in group B (median 75.2 micrograms/l, range 27.8-122.3). Similarly, IGFBP-3 concentrations in group A (median 950 micrograms/l, range 580-1260) were significantly lower than those of group B (median 1920 micrograms/l, range 1070-1770). There was no significant difference between GHBP values in group A (median 8.6%, range 6.6-12.6) and group B (median 8.3%, range 6-14.3). Gestational age correlated positively with IGF-I concentrations (P < 0.0001) and IGFBP-3 (P < 0.0001) and negatively with GH (P < 0.0001). GHBP values did not correlate with gestational age. Multiple regression analysis showed a negative correlation between GH:IGF-I ratio and fetal growth indices CONCLUSIONS: The simultaneous evaluation of fetal GH, IGF-I, IGFBP-3 and GHBP suggests that the GH-IGF-I axis might already be functional in utero. The progressive improvement in the efficiency of this axis in the last part of gestation does not seem to be due to an increase in GH receptors.     

Insulin-like growth factor binding protein-3 is overexpressed in senescent and quiescent human fibroblasts

Cellular insulin-like growth factor binding protein-3 (IGFBP-3) mRNA and IGFBP-3 levels in conditioned medium were consistently higher in cultures of late passage normal (old) fibroblasts and prematurely senescent fibroblasts derived from Werner syndrome (WS) during quiescence induced by serum depletion and during the renewed growth ensuing after serum repletion, compared to cultures of early passage normal (young) fibroblasts. Molar ratios of IGFBP-3/IGF-II were always higher in senescent cultures and maintained a hierarchy of old > WS > young human diploid fibroblasts. Transfection into fibroblasts of the normal full-length IGFBP-3 cDNA in an expression vector resulted in a significant reduction in colony formation compared to cells transfected with an empty expression vector (no cDNA) or with IGFBP-3 cDNA altered by a 273 base pair (bp) deletion. Addition to old and young cultures of recombinant human IGFBP-3 and IGF-I at 1:1 or 5:1 molar ratios inhibited IGF-I-mediated DNA synthesis by approximately 70-80%. These data indicate that IGFBP-3 may play an important role in the quiescent and senescent growth arrest of HDF.     

Effects of growth hormone and pregnancy on expression of growth hormone receptor, insulin-like growth factor-I, and insulin-like growth factor binding protein-2 and -3 genes in bovine uterus, ovary, and oviduct

The effects of growth hormone (GH) and pregnancy on insulin-like growth factor (IGF)-I, IGF binding protein (IGFBP)-2, and IGFBP-3 mRNA in reproductive tissues were studied in cattle. Lactating dairy cows were inseminated at estrus and treated with 25 mg/day GH (n = 8) or saline (n = 8) for 16 days. Corpus luteum (CL), ovary (CL removed), oviduct, endometrium, and myometrium were collected at the end of treatment. Messenger RNA for GH receptor, IGF-I, IGFBP-2, IGFBP-3, and actin were measured by nuclease protection assays. The CL contained more GH receptor mRNA than the other reproductive tissues examined. Expression of IGF-I mRNA was highest in myometrium, with lower amounts found in endometrium; the CL expressed the least amount of IGF-I mRNA. The IGFBP-2 mRNA was most abundant in endometrium and least abundant in CL. Expression of IGFBP-3 mRNA was detected in all reproductive tissues examined. However, endometrium, a tissue that expressed the most IGFBP-2 mRNA, had the lowest amount of IGFBP-3 mRNA. The GH receptor mRNA was decreased in cows treated with GH whereas the mRNA for IGF-I, IGFBP-2, or IGFBP-3 was not changed. In the reproductive tissues evaluated, cows that contained a conceptus at tissue collection (pregnant) had higher amounts of IGF-I mRNA than did nonpregnant cows. In summary, the level of mRNA encoding GH receptor, IGF-I, IGFBP-2, and IGFBP-3 varied within the tissues examined, suggesting that these genes may play a variety of roles in the bovine female reproductive tract. Supplemental GH failed to change the expression of IGF-I, IGFBP-2, and IGFBP-3 mRNA, possibly because of low GH receptor mRNA levels in tissues other than CL. A direct action of GH on IGF-I, IGFBP-2, or IGFBP-3 gene expression within cow reproductive tissues was not supported because the amount of IGF-I, IGFBP-2, or IGFBP-3 mRNA was not altered by GH.     

Insulin-like growth factor I and insulin-like growth factor binding protein 3 as determinants of blood hemoglobin concentration in healthy subjects

We studied the serum concentrations of IGF-I, IGF-binding protein 3 (IGFBP-3), and testosterone in relation to blood Hb in 60 healthy prepubertal or early pubertal boys twice, with a 9-mo interval. Serum IGF-I and testosterone levels were measured by RIA, and serum IGFBP-3 was measured by monoclonal immunofluorometric assay. Positive correlations were observed between the concentrations of blood Hb and serum IGF-I at the first examination (r = 0.36, p = 0.008) and Hb and IGFBP-3 at both examinations (r = 0.53, p < 0.001, and r = 0.39, p = 0.003). No association between Hb and testosterone concentrations was found. Our results show that blood Hb is positively correlated to serum IGF-I and IGFBP-3 levels, indicating indirectly the involvement of growth hormone in the regulation of physiologic Hb concentration. Because no association was found between Hb and testosterone concentrations, this may indicate that the role of androgens in erythropoiesis may be different at different stages of puberty. It is concluded that the IGF system may be involved in the rise of Hb level during early puberty.     

Insulin-like growth factors-I and -II stimulate chemotaxis of osteoblasts isolated from fetal rat calvaria

Repair and regeneration of damaged bone is believed to be regulated in part by growth factors stored in the bone matrix. These growth factors are synthesized and secreted by osteoblasts and are incorporated into the developing bone. This pool of stored growth factors is then released into the immediate area following resorption of the matrix. One of the initial steps in bone repair is the recruitment of osteoblasts to the repair site. Growth factors, such as TGF-beta and PDGF, which are present in bone matrix, have been shown to be chemotactic for osteoblasts. In this study, primary cultures of osteoblasts isolated from fetal rat calvaria were examined for chemotaxis in response to IGF-I and IGF-II. IGF-I stimulated a dose-dependent increase in osteoblast chemotaxis, while IGF-II stimulated chemotaxis maximally at the lowest concentration studied (0.1 ng/ml), and had no effect at the highest concentration studied (100 ng/ml). IGF-I and -II had no effect on osteoblast proliferation at any of the concentrations examined. These results indicate that IGFs may be playing an important role in the early stages of bone repair by stimulating osteoblast chemotaxis to the repair site.     

Insulin-like growth factor II signaling through the insulin-like growth factor II/mannose-6-phosphate receptor promotes exocytosis in insulin-secreting cells

The insulin-like growth factor II (IGF-II)/mannose-6-phosphate (M-6-P) receptor is known to participate in endocytosis as well as sorting of lysosomal enzymes and is involved in membrane trafficking through rapid cycling between cytosolic membrane compartments and the plasma membrane. Here we demonstrate that IGF-II, acting through the IGF-II/M-6-P receptor, promotes exocytosis of insulin in the pancreatic beta cell. The effect of IGF-II was evoked at nonstimulatory concentrations of glucose, was mediated by a pertussis toxin sensitive GTP-binding protein, was dependent on protein kinase C-induced phosphorylation, and was independent of changes in cytoplasmic free Ca2+ concentration. Since the applied concentration of IGF-II is within the range normally found free in circulation in humans, this novel signaling pathway for the IGF-II/M-6-P receptor is likely to be involved in modulation of insulin exocytosis under physiological conditions.     

Insulin-like growth factor I receptor prevents apoptosis and enhances neuroblastoma tumorigenesis

Autocrine stimulation of the type I insulin-like growth factor receptor (IGF-IR) by IGF-II is one mechanism that allows cancer cells to maintain unregulated growth and to resist programmed cell death (PCD). SH-SY5Y and SHEP cells are cloned human neuroblastoma (NBL) lines originating from a single primary tumor. SH-SY5Y cells, which express abundant cell surface IGF-IR and produce IGF-II, exhibit serum independent growth and resist PCD due to hypoxia and hyperosmolar conditions. In contrast, SHEP cells, which produce no IGF-II and express five-fold fewer IGF-IRs, die in serum-free media or following exposure to metabolic stressors. To better understand the roles of IGF-IR and its ligand, IGF-II, in NBL carcinogenesis, we stably transfected SHEP cells with either IGF-II or IGF-IR. Unregulated expression of IGF-II did not alter the growth characteristics of SHEP/human IGF-II transfectants. In contrast, overexpression of IGF-IR allowed SHEP/IGF-IR transfectants to survive in media supplemented only by IGF-II. IGF-IR abundance correlated in a graded fashion with resistance to PCD in response to three different death-inducing paradigms: mitogen withdrawal, hyperosmolar metabolic stress, and treatment with etoposide. Our results suggest that adjuvant therapy aimed at reducing IGF-IR abundance may enhance chemotherapy-coupled apoptosis in the treatment of NBL.     

Insulin-like growth factor binding protein -2 and -4 messenger ribonucleic acid expression in bovine ovarian follicles: effect of gonadotropins and developmental status

This work is concerned with the role of insulin-like growth factor binding protein (IGFBP)-2 and -4 in the regulation of IGF bioactivity in bovine follicles during the development of dominance. We measured the expression of IGFBP-2 and -4 messenger RNA (mRNA) in small (1-4 mm) gonadotropin-sensitive follicles and medium (4-8 mm) and large (>8 mm) gonadotropin-dependent follicles using in situ hybridization. In healthy nonatretic bovine follicles, IGFBP-2 and -4 mRNA expression was confined to granulosa and theca tissue, respectively. Moreover, during the development of follicular atresia, there were distinct changes in the temporal and spatial expression of these genes. IGFBP-2 immunoactivity was localized in granulosa tissue and the basement membrane of healthy preantral follicles, whereas IGFBP-4 immunoactivity was localized in both theca and granulosa tissue. Of particular interest was the lack of IGFBP-2 mRNA expression in large (>8 mm) gonadotropin-dependent follicles, an observation that was confirmed by the lack of immunoreactive IGFBP-2 in these follicles. The regulation of IGFBP-2 and -4 mRNA expression in granulosa and theca cells was analyzed using a serum-free cell culture system. FSH inhibited the expression of IGFBP-2 mRNA in granulosa cells, whereas LH stimulated IGFBP-4 mRNA expression in theca cells. Our results provide evidence for the existence of different roles for IGFBP-2 and -4 in the developing follicle.     

Insulin-like growth factor binding protein production by bovine and human vascular smooth muscle cells: production of insulin-like growth factor binding protein-6 by human smooth muscle

Insulin-like growth factor binding protein (IGFBP) secretory profiles were determined for vascular smooth muscle cells (VSMC) derived from bovine aorta and human aorta, pulmonary artery, and coronary artery. The bovine cells produced IGFBP-4, IGFBP-3, and an IGFBP-3 protease. IGF-I stimulated messenger RNA (mRNA) and media levels of IGFBP-3. The human cells produced IGFBP-3, IGFBP-4, and IGFBP-3 and IGFBP-4 proteases. The three human cells also produced a 30K IGFBP, shown to be IGFBP-6, based on increased affinity for IGF-II vs. IGF-I, size decrease when treated with O-glycanase, but not N-glycanase, reactivity with IGFBP-6 antiserum, presence of a 1.3-kilobase pair mRNA that hybridized to IGFBP-6 specific complementary DNA, and N-terminal amino acid sequence corresponding to IGFBP-6. In the human cells, IGF-I increased media levels of IGFBP-3 through stimulation of IGFBP-3 mRNA and dissociation of cell bound IGFBP-3, and decreased IGFBP-4 via potentiation of IGFBP-4 proteolysis. Neither the bovine nor the human aorta VSMC produced sufficient IGFBP-2 or IGFBP-2 mRNA to be detected by ligand blot and Northern analysis, as previously reported for porcine and rat aorta smooth muscle cells. The variable expression of IGFBPs and IGFBP proteases by VSMC are likely to contribute to differential vascular reactivity to the IGFs in larger arterial blood vessels.     

Competitive binding assay for determination of rat insulin-like growth factor binding protein-3

We describe a novel competitive assay for rat insulin-like growth factor (IGF)-binding protein-3 (rIGFBP-3) based on the ability of IGFBP-3 to form a ternary complex with the acid labile subunit (ALS) in the presence of IGF. Human (h)ALS was bound to test tubes pre-coated with anti-human ALS antibody. The assay depends on competition between a covalent complex of 125I-hIGF-I and hIGFBP-3, added as tracer, and hIGFBP-3 or rIGFBP-3 in standards and test samples, for binding to the immobilized hALS. Purified natural hIGFBP-3 served as standard. Human IGFBP-3 and rIGFBP-3 were able to compete for tracer binding in the presence, but not in the absence, of IGF-I. Before assay, rat serum samples were acidified to denature endogenous ALS. Standards ranged from 0.10 (lower detection limit) to 20 ng/tube. Rat serum, semipurified rIGFBP-3, human serum and purified hIGFBP-3 diluted in parallel. The level of rIGFBP-3 was 1.63+/-0.28 mg/l (mean+/-SEM) in young rats and increased to 3.41+/-0.26 mg/l (p < 0.05) in old rats (n = 5-6). Fasting for 3 days reduced rIGFBP-3 from 2.41+/-0.27 to 1.33+/-0.14 mg/l (p < 0.05). Levels of rIGFBP-3 were reduced in hypophysectomized (0.16+/-0.04 mg/l; p < 0.05) and diabetic rats (1.04+/-0.30 mg/l; p < 0.05), and normal in insulin-treated diabetic rats (2.49+/-0.04 mg/l; ns), when compared to controls (2.79+/-0.22 mg/l). Changes in levels of IGFBP-3 parallelled those of immunoreactive rALS. We conclude that this assay provides a novel method of quantitating functional IGFBP-3 in rat serum.     

Insulin-like growth factor-I and binding protein-3 in relation to childhood leukaemia

The aetiology of most cases of childhood leukaemia remains unknown, but several studies have indicated that increased birthweight and height are risk factors for the disease. Since insulin-like growth factor-I (IGF-I) mediates the effect of growth hormone and has been positively associated with prostate cancer, we have evaluated the role of this hormone and its principal binding protein, IGFBP-3, in the aetiology of childhood leukaemia. Incident cases of childhood leukaemia from those recorded by a national network of childhood oncologists were enrolled in our study. Controls were children hospitalised for acute conditions of no more than moderate severity with matching for gender, age and maternal place of residence. Blood measurements of IGF-I and IGFBP-3 were undertaken using commercially available radioimmunoassays. Serum IGF-I values decreased by about 1.7% per month, and the rate of decline was higher, though not significantly so, among cases (2.1% per month) than among controls (1.4%). There was no significant association between IGF-I and the likelihood of childhood leukaemia, but an increment of 1 microg/ml of IGFBP-3 was associated with a substantial and statistically significant reduction of childhood leukaemia by 28% (95% confidence interval 7% to 45%). Because IGFBP-3 is essentially a binding protein, we interpret our findings as indicating that bioavailable IGF-I may play an important role in the aetiology of childhood leukaemia. The much smaller quantities and the inherent instability of IGF-I in the blood in comparison to those of IGFBP-3 are likely to hinder documentation of an underlying positive association of IGF-I with the disease.     

Insulin-like growth factor binding proteins localize to discrete cell culture compartments in periosteal and osteoblast cultures from fetal rat bone

Insulin-like growth factor (IGF)-I and IGF-II are expressed at biologically effective levels by bone cells. Their stability and activity are modulated by coexpression of IGF binding proteins (IGFBPs). Secreted IGFBPs may partition to soluble, cell-associated, and matrix-bound compartments. Extracellular localization may sequester, store, or present IGFs to appropriate receptors. Of the six IGFBPs known, rat osteoblasts synthesize all but IGFBP-1. Of these, IGFBP-3, -4, and -5 mRNAs are induced by an increase in cAMP. Little is known about extracellular IGFBP localization in bone and nothing about IGFBP expression by nonosteoblastic periosteal bone cells. We compared basal IGFBP expression in periosteal and osteoblast bone cell cultures and assessed the effects of changes in cAMP-dependent protein kinase A or protein kinase C. Basal IGFBP gene expression differed principally in that more IGFBP-2 and -5 occurred in osteoblast cultures, and more IGFBP-3 and -6 occurred in periosteal cultures. An increase in cAMP enhanced IGFBP-3, -4, and -5 mRNAand accordingly increased soluble IGFBP-3, -4, and -5 and matrix-bound IGFBP-3 and -5 in both bone cell populations. In contrast, protein kinase C activators suppressed IGFBP-5 mRNA, and its basal protein levels remained very low. We also detected low Mr bands reactive with antisera to IGFBP-2, -3, and -5, suggesting proteolytic processing or degradation. Our studies reveal that various bone cell populations secrete and bind IGFBPs in selective ways. Importantly, inhibitory IGFBP-4 does not significantly accumulate in cell-associated compartments, even though its secretion is enhanced by cAMP. Because IGFBPs bind IGFs less tightly in cell-bound compartments, they may prolong anabolic effects by agents that increase bone cell cAMP.