Intact IGFBP-3 Research Articles

Tissue IGFBP-3 proteolysis: Contrasting pathophysiology to that in the circulation

Endogenous IGFBP-3 has been examined in the circulation and in four different extravascular fluids in normal healthy adults and in patients with psoriasis or arthritis. In all of these cases there was no apparent increase of IGFBP-3 protease activity in the circulation. In contrast, endogenous IGFBP-3 from normal skin interstititial fluid and synovial fluid from healthy adults was found to be predominantly in the 29 kDa proteolytically modified form. This indicated that in these extravascular fluids in normal healthy adults a protease was active which was similar, if not identical, to that found in the circulation in pregnancy and other conditions. This was confirmed by the fragmentation of recombinant IGFBP-3 when incubated with these fluids. When the skin interstitial fluid or synovial fluid were taken from abnormal tissues (psoriasis in the former and osteoarthritis or rheumatoid arthritis in the latter) there was a considerable reduction in the amount of endogenous IGFBP-3 in the ‘clipped’ form and a reduction in the protease activity. In psoriatic lesions, this reduction in IGFBP-3 protease activity was shown to be due to the presence of an inhibitor in the interstitial fluid but not in the circulation. In both peritoneal and follicular fluid, the ratio of intact to fragmented IGFBP-3 appeared to relate to the oestrogen status. In peritoneal fluid there was a decrease in intact IGFBP-3 during the late proliferative/early secretory phase of the endometrial cycle. In the ovary there was an increase in the amount of fragmented IGFBP-3 in the follicular fluid from the dominant follicle in comparison with atretic follicles from the same ovary. There is normally little proteo-lysis of IGFBP-3 in the circulation but this increases in many conditions where there is increased metabolic activity. The same enzyme(s) appear to be active in many extravascular fluids but under very different regulation. The activity in these extravascular fluids is normally high but can be decreased with local tissue inflammation; this decrease appears to be mediated by the induction of a local inhibitor.

Growth hormone and parathyroid hormone stimulate IGFBP-3 in rat osteoblasts.

Osteoblast-like cells prepared from calvaria of newborn rats produce insulin-like growth factor (IGF) I and several insulin-like growth factor binding proteins (IGFBPs) in vitro. Among the IGFBPs found in conditioned cell culture medium, IGFBP-3 is the most abundant. Intact IGFBP-3, as assessed by 125I-labeled IGF-II ligand blot analysis, is more abundant in culture media of cells exposed to growth hormone (GH) or to parathyroid hormone (PTH), both at 5 x 10(-9) mol/l, for 24 h. At the same time, concentrations of IGF-I are increased in media of cells exposed to PTH but not to GH, compared with hormone-free control cultures. IGFBP-3 mRNA is increased in osteoblasts exposed to PTH or to GH but not in response to 5 x 10(-9) mol/l IGF-I. PTH exerts a rapid (within 2 h) stimulatory effect on IGF-I and IGFBP-3 production, both at the message and peptide levels, whereas GH increases only IGFBP-3, both at the message and peptide levels (after 24 h). We conclude that IGF-I does not mediate increased IGFBP-3 production by rat osteoblasts in response to GH and PTH.

Vascular smooth muscle cells synthesize two forms of insulin-like growth factor binding proteins which are regulated differently by the insulin-like growth factors.

Vascular smooth muscle cells (SMC) synthesize insulin-like growth factor-I (IGF-I), which is a mitogen for this cell type in vitro. Since IGF binding proteins (IGFBP) modulate IGF bioactivity, we determined which IGFBPs were secreted by porcine SMC. Porcine SMC secreted 34,000 and 24,000 M(r) forms of IGFBPs which were identified as IGFBP-2 and IGFBP-4, respectively, by immunoblotting. Northern blot analysis showed single transcripts of 1.6 kb and 2.4 kb for IGFBP-2 and IGFBP-4, respectively. Secretion of IGFBP-2 was not regulated to a significant degree, with insulin, IGF-II, IGF-I, forskolin, and dibutyryl cyclic adenosine monophosphate (cAMP) inducing minimal changes in IGFBP-2 secretion of less than 30% by radioimmunoassay (RIA). Insulin increased (2.8 +/- 0.1-fold) the abundance of IGFBP-4 protein in conditioned media (CM) and increased IGFBP-4 mRNA levels. Growth factors for SMC such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), and transforming growth factor beta-1 (TGF beta-1) were without effect on either IGFBP-2 or -4. IGF-I treatment decreased the amount of IGFBP-4 present in CM, but a corresponding decrease in IGFBP-4 mRNA levels was not observed. In order to determine if IGFBP-4 could modulate IGF-I bioactivity, IGFBP-4 was added to pSMCs with and without IGF-I. IGF-I alone (20 ng/ml) induced a 1.6 to threefold increase in 3H-thymidine incorporation. Addition of IGFBP-4 (between 50 and 250 ng/ml) to cultures containing IGF-I (20 ng/ml) had no effect on DNA synthesis compared to that observed with IGF-I alone, while 500 ng/ml consistently caused a small decrease (15 +/- 5%; mean +/- SE). Immunoblotting of the CM obtained at the end of the 3H-thymidine assay showed a loss of intact IGFBP-4 in the cultures containing IGF-I. This corresponded with an increase in the abundance of a 16,000 M(r) immunoreactive fragment that did not bind IGF-I. Coincubation with insulin had no effect on the amount of IGFBP-4 that was converted to fragment, suggesting that the reaction was dependent upon IGF-I binding to IGFBP-4. In contrast, addition of IGFBP-4 (500 ng/ml) to human fibroblast cultures with IGF-I (20 ng/ml) almost completely inhibited the stimulatory effect of IGF-I on DNA synthesis and no increase in fragment was detected in the CM. In summary, SMC secrete IGFBP-2 and IGFBP-4, both of which have been shown to regulate IGF-mediated DNA synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Insulin-like growth factor binding protein-3 protease activity in the urine of children with chronic renal failure

The insulin-like growth factors, IGF-1 and IGF-II, are polypeptides that potentiate cellular growth. In addition to binding to specific cell surface receptors, the IGFs bind with high affinity to a family of proteins, the insulin-like growth factor binding proteins (IGFBPs). Serum and urine IGFBP patterns are altered in individuals with chronic renal failure (CRF). We recently reported that the urinary IGFBP pattern of CRF patients is unique for increased insulin-like growth factor binding protein-1 (U-IGFBP-1) levels. In this study, we used western ligand blotting (WLB), western immunoblotting (WIB), and radioimmunoassay (RIA) to further evaluate serum and urine IGFBP profiles of children with CRF (n = 14). Five patients with CRF displayed decreased serum IGFBP-3 profiles by WLB. Serum IGFBP-3 WIB profiles were remarkable for 30- and 20-kDa fragments of IGFBP-3 not seen in control serum. Serum IGFBP-3 levels, as determined by RIA, were slightly elevated. Serum levels of IGFBP-2 also were increased, although not at a level reaching statistical significance. WLB of CRF urine revealed a large increase in U-IGFBP-1 and a complete absence of urinary IGFBP-3. Recent studies of serum from pregnant women and seminal plasma have demonstrated a similar absence of intact IGFBP-3, due to the presence of a specific IGFBP-3 protease. To evaluate whether an IGFBP-3 protease accounts for the absence of intact U-IGFBP-3 in children with CRF, urine and serum samples from individuals with CRF and controls were tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative Use of Immunological Methods & Ligand Blotting of Insulin-like Growth Factor Binding Proteins in Serum and Other Biological Fluids

There is controversy as to which methodology most accurately detects the Insulin-like growth factor binding proteins (IGFBPs). We have compared immunological and ligand binding techniques to assess the IGFBPs in serum from non-pregnant women and women in late pregnancy, fetal cord serum and amniotic fluid. A specific radioimmunoassay for IGFBP-3 measured mean levels of 3.5 and 3.7μg/ml for non-pregnancy and pregnancy serum, respectively, 0.7μg/ml for fetal cord serum and 5.1μg/ml for amniotic fluid. In contrast, Western-ligand blot of fluid showed IGFBP-3 as a 40 kDa doublet in non-pregnancy serum and fetal cord serum, unlike pregnancy serum or amniotic fluid, where IGFBP-3 was undetectable by standard blot analysis. However, analysis of fluids at 25-50 times more volume showed that IGFBP-3 was detectable as a 40 kDa doublet in pregnancy serum and amniotic fluid. The molecular mass of immunoreactive IGFBP-3 was examined by Western-immunoblot, probed with antibody α IGFBP-31l. As in Westem-ligand blot, intact IGFBP-3 (40 kDa) was easily detected in non-pregnancy serum and fetal cord serum, but required greater volumes of sample for detection in pregnancy serum and amniotic fluid. In addition, smaller immunoreactive fragments were found in all fluids: a faint 27 and 22 kDa band was found in non-pregnancy serum and fetal cord serum, with more dominant bands of 31-27, 26, 25, 24 and 22 kDa in pregnancy serum and 31-26, 25, 22, 19-17 kDa in amniotic fluid. To address the obvious discrepancies between ligand and immunological analysis, samples were assessed for IGFBP-3 protease activity. Non-pregnancy serum proteolyzed 14% of the substrate, pregnancy serum 61%, fetal cord serum 33%, and amniotic fluid 50-61%. Thus, fluids with apparently low IGFBP-3 by Western-ligand blot, but high IGFBP-3 by RIA, showed high levels of protease activity. Previously, IGFBP-proteases have been shown to degrade IGFBP to smaller fragments with a reduced affinity for IGF. We conclude that the discrepancy between ligand binding techniques and immunological techniques can be explained, at least in part, by the presence of IGFBP-protease activity.

Insulin-like growth factor-binding protein-3 proteolysis is induced after elective surgery.

The insulin-like growth factors (IGFs) are bound to several binding proteins (IGFBPs) that appear to regulate IGF transport, receptor binding, and action. The concentrations of these peptides are altered by catabolic conditions. To determine if IGF-I and IGFBP levels change after surgery, sera were obtained from 16 patients before and after cholecystectomy. Immunoreactive IGF-I measured in plasma samples from which IGFBPs had been extracted did not change postoperatively. In contrast, IGF-I determined in unextracted samples increased roughly 3-fold postoperatively, presumably due to changes in IGFBPs. Two days postoperatively, IGFBP-3 levels, determined by ligand blot, averaged 36% of preoperative values, whereas levels of IGFBP-2 and a 24,000 mol wt IGFBP did not change significantly. Similarly, by immunoblot, intact IGFBP-3 was decreased 84.2 +/- 20.2%, and a 31,000 mol wt IGFBP-3 fragment increased 57.5 +/- 47.4% postoperatively. Coincubation of postoperative, but not preoperative, sera with control sera resulted in a significant decrease in IGFBP-3 and production of proteolytic fragments. IGFBP-3 proteolytic activity in postoperative sera was markedly inhibited by antipain, Na-p-tosyl-L-lysine chloromethyl ketone, phenylmethylsulfonylfluoride, aprotinin, o-phenanthroline, and EDTA, but not by leupeptin or N-tosyl-L-phenylalanine chloromethyl ketone. This pattern of inhibition is consistent with a metal-dependent trypsin-like serine protease. We speculate that proteolysis of IGFBP-3 may alter tissue uptake of IGF-I and thereby help to counteract the catabolic state caused by surgery.

Evaluation of the Developmental and Nutritional Changes in Porcine Insulin-Like Growth Factor-Binding Protein-1 and -2 Serum Levels by Immunoassay*

Porcine serum contains five insulin-like growth factor-binding proteins (IGFBPs), whose regulation has been studied by ligand blotting. To more accurately quantify changes in two specific forms of IGFBP a heterologous RIA for porcine (p) IGFBP-2 was developed, and IGFBP-1 levels were analyzed by immunoblotting. By RIA, postnatal hypophysectomy caused a 7-fold increase in serum pIGFBP-2 levels compared to controls (2,622 +/- 378 vs. 382 +/- 10 ng/ml, respectively). Fetal pIGFBP-2 levels were higher at 110 vs. 45 days gestation (1,074 +/- 214 vs. 418 +/- 30 ng/ml, respectively), rose to 1,905 +/- 167 ng/ml within 12 h after birth, then decreased to 1,010 +/- 10 ng/ml at 48 h. By immunoblot analysis, bovine IGFBP-2 antiserum reacted with a 34,000 mol wt (Mr) IGFBP and did not react with other forms of IGFBP detected by ligand blotting. Serum levels of the 34,000 Mr IGFBP, as detected by ligand blot analysis, are decreased when neonatal pigs are fasted for 48 h. In contrast, by RIA, pIGFBP-2 concentrations increased 4-fold. Immunoblots of these sera showed two lower Mr (22,000 and 14,000 Mr) bands that did not bind either [125I]IGF-I or [125I]IGF-II and were distinct from a smaller (20,000 Mr) IGFBP which bound only [125I]IGF-II. These two bands were increased in serum of 48-h fasted compared to fed piglets, suggesting that they are proteolytic fragments of pIGFBP-2. In vitro incubation of 48-h fasted pig serum with intact IGFBP-2 failed to reveal proteolytic fragments, indicating that the IGFBP-2 fragments were not generated by a protease that was released into the serum. Analysis performed with human IGFBP-1 antiserum revealed a 29,000 Mr immunoreactive band whose abundance was increased by either postnatal hypophysectomy or fasting. No fragments of IGFBP-1 were found in any serum tested. We conclude that heterologous antibodies can be used to identify and quantify IGFBP-1 and IGFBP-2 in porcine serum. Changes in pIGFBP-2 levels measured during fasting are due to the combination of changes in intact 34,000 Mr IGFBP-2 and smaller non-IGF-binding fragments. Changes in levels of specific forms of IGFBP as well as the presence of fragments have the potential to modulate the transport of IGF-I and -II out of the vasculature.