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S Mohan and DJ Baylink

Traditionally, binding proteins are known to regulate the activity of ligands by prolonging their half-life, and insulin-like growth factor (IGF)-binding proteins (IGFBPs) are no exception to this. The IGFBP family contains six high-affinity members with variable functions and mechanisms of actions. In addition to functioning as simple carrier proteins, IGFBPs in serum function to regulate the endocrine actions of IGFs by regulating the amount of IGF available to bind to signaling IGF-I receptors, whereas locally produced IGFBPs act as autocrine/paracrine regulators of IGF action. Furthermore, recent in vitro and in vivo findings that IGFBPs function independently of the IGFs as growth modulators are particularly exciting. Regarding the role of IGFBPs as ligand-independent growth modulators, our recent data that IGFBP-5 stimulates markers of bone formation in osteoblasts lacking functional IGFs provide evidence that IGFBP-5 itself is a growth factor that can act independently of IGFs to regulate bone formation. In terms of the mechanism by which certain IGFBPs mediate their effects in a ligand-independent manner, the binding of IGFBP to its putative receptor on the cell membrane may stimulate the signaling pathway independent of an IGF receptor, to mediate the effects of IGFBPs in certain target cell types. IGFBPs may also exert IGF-independent effects by transcriptional activation of genes by IGFBPs transported into the nucleus via their nuclear localization signal. In conclusion, IGFBPs are unusually pleotrophic molecules with functions ranging from the traditional role of prolonging the half-life of the IGFs to functioning as growth factors independent of the IGFs. In this regard, it was surprising to find that the human genome contains only about 35 000 genes. One mechanism to account for such complexity with a relatively small number of genes is strikingly illustrated by the multifunctional IGFBP class of proteins.

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S Mohan and D J Baylink

Although it is well established that there is considerable inter-individual variation in the circulating levels of IGF-I in normal, healthy individuals and that a genetic component contributes substantially to this variation, the direct evidence that inter-individual variation in IGF-I contributes to differences in peak bone mineral density (BMD) is lacking. To examine if differences in IGF-I expression could contribute to peak BMD differences, we measured skeletal changes at days 23 (prepubertal), 31 (pubertal) and 56 (postpubertal) in mice with haploinsufficiency of IGF-I (+/−) and corresponding control mice (+/+). Mice (MF1/DBA) heterozygous for the IGF-I knockout allele were bred to generate +/+ and +/− mice (n=18–20 per group). Serum IGF-I was decreased by 23% (P<0.001) in mice with IGF-I haploinsufficiency (+/−) group at day 56 compared with the control (+/+) group. Femoral bone mineral content and BMD, as determined by dual energy X-ray absorptiometry, were reduced by 20% (P<0.001) and 12% respectively in the IGF-I (+/−) group at day 56 compared with the control group. The peripheral quantitative computed tomography measurements at the femoral mid-diaphysis revealed that periosteal circumference (7%, P<0.01) and total volumetric BMD (5%, P<0.05) were decreased significantly in the +/− group compared with the +/+ group. Furthermore, serum IGF-I showed significant positive correlations with both areal BMD (r=0.55) and periosteal circumference (r=0.66) in the pooled data from the +/+ and +/− groups. Our findings that haploinsufficiency of IGF-I caused significant reductions in serum IGF-I level, BMD and bone size, together with the previous findings, are consistent with the notion that genetic variations in IGF-I expression could, in part, contribute to inter-individual differences in peak BMD among a normal population.

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D Byun, S Mohan, DJ Baylink and X Qin

Our previous findings suggest that binding of IGF binding protein-4 (IGFBP-4) to IGFs is essential for the inhibitory effect of IGFBP-4 on the activity of IGFs, both in vitro and in vivo. Therefore, understanding the structural determinants of IGF binding in IGFBP-4 is important to the general understanding of the biology of the IGF system. This study sought to further localize the IGF binding domain and to evaluate the role of Cys residues in IGF binding. Our data revealed that full-length IGFBP-4 peptides lacking the residues Leu(72)-Ser(91) or Leu(72)-His(74) or Gly(75)-Ser(91) failed to bind to IGF-I or IGF-II, whereas deletion of the residue Leu(72) or residues Met(80)-Ser(91) led to a 2- to 3-fold reduction in IGF-I and IGF-II binding activity. The IGF-I and IGF-II binding activities were dramatically reduced by the single mutation, Cys9/Arg (>25-fold), and to a lesser degree, by the single mutation, Cys12/Arg (the first N-terminal Cys residue was designated Cys1). The mutation Cys17/Ser or Cys18/Tyr or Cys20/Ser each resulted in a similar but moderate ( approximately 5-fold) reduction in IGF-II binding activity. The IGF-I binding activity was also dramatically reduced by the mutation Cys18/Tyr, and to a lesser extent, by the mutation Cys17/Ser or Cys20/Ser. These data suggest: 1) the IGF-I and IGF-II binding domain in IGFBP-4 involves a hydrophobic motif (Leu(72)-Met(80)) located in the distal part of the conserved N-terminal region, and 2) the N-terminal Cys residues (Cys9 and Cys12) are more critical than the C-terminal Cys residues (Cys17 and Cys20) in affecting the IGF-I and IGF-II binding. Based on these data, we speculate that the structural determinants of IGF-I and IGF-II binding in IGFBP-4 are very similar, if not identical.

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PM Jehle, DR Jehle, S Mohan and BO Bohm

Osteopenia has been ascribed to diabetics without residual insulin secretion and high insulin requirement. However, it is not known if this is partially due to disturbances in the IGF system, which is a key regulator of bone cell function. To address this question, we performed a cross-sectional study measuring serum levels of IGF-I, IGF-binding protein-1 (IGFBP-1), IGFBP-3, IGFBP-4 and IGFBP-5 by specific immunoassays in 52 adults with Type 1 (n=27) and Type 2 (n=25) diabetes mellitus and 100 age- and sex-matched healthy blood donors. In the diabetic patients, we further determined serum levels of proinsulin, intact parathyroid hormone (PTH), 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3 and several biochemical bone markers, including osteocalcin (OSC), bone alkaline phosphatase (B-ALP), carboxy-terminal propeptide of type I procollagen (PICP), and type I collagen cross-linked carboxy-terminal telopeptide (ICTP). Urinary albumin excretion was ascertained as a marker of diabetic nephropathy. Bone mineral density (BMD) of hip and lumbar spine was determined by dual-energy X-ray absorptiometry. Data are presented as means+/-s.e.m. Differences between the experimental groups were determined by performing a one-way analysis of variance (ANOVA), followed by Newman-Keuls test. Correlations between variables were assessed using univariate linear regression analysis and partial correlation analysis. Type 1 diabetics showed significantly lower IGF-I (119+/-8 ng/ml) and IGFBP-3 (2590+/-104 ng/ml) but higher IGFBP-1 levels (38+/-10 ng/ml) compared with Type 2 patients (170+/-13, 2910+/-118, 11+/-3 respectively; P<0.05) or healthy controls (169+/-5, 4620+/-192, 3.5+/-0.4 respectively; P<0.01). IGFBP-5 levels were markedly lower in both diabetic groups (Type 1, 228+/-9; Type 2, 242+/-11 ng/ml) than in controls (460+/-7 ng/ml,P<0. 01), whereas IGFBP-4 levels were similar in diabetics and controls. IGF-I correlated positively with IGFBP-3 and IGFBP-5 and negatively with IGFBP-1 and IGFBP-4 in all subjects. Type 1 patients showed a lower BMD of hip (83+/-2 %, Z-score) and lumbar spine (93+/-2 %) than Type 2 diabetics (93+/-5 %, 101+/-5 % respectively), reaching significance in the female subgroups (P<0.05). In Type 1 patients, BMD of hip correlated negatively with IGFBP-1 (r=-0.34, P<0.05) and IGFBP-4 (r=-0.3, P<0.05) but positively with IGFBP-5 (r=0.37, P<0. 05), which was independent of age, diabetes duration, height, weight and body mass index, as assessed by partial correlation analysis. Furthermore, biochemical markers indicating bone loss (ICTP) and increased bone turnover (PTH, OSC) correlated positively with IGFBP-1 and IGFBP-4 but negatively with IGF-I, IGFBP-3 and IGFBP-5, while the opposite was observed with bone formation markers (PICP, B-ALP) and vitamin D3 metabolites. In 20 Type 2 patients in whom immunoreactive proinsulin could be detected, significant positive correlations were found between proinsulin and BMD of hip (r=0.63, P<0.005), IGF-I (r=0.59, P<0.01) as well as IGFBP-3 (r=0.49, P<0.05). Type 1 and Type 2 patients with macroalbuminuria showed a lower BMD of hip, lower IGFBP-5 but higher IGFBP-4 levels, suggesting that diabetic nephropathy may contribute to bone loss by a disturbed IGF system. In conclusion, the findings of this study support the hypothesis that the imbalance between individual IGF system components and the lack of endogenous proinsulin may contribute to the lower BMD in Type 1 diabetics.

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B. S. SETTY, MAN MOHAN SINGH, S. R. CHOWDHURY and AMIYA B. KAR

SUMMARY

Sodium and potassium levels were determined in the rat endometrium and uterine washings during normal and 'delayed' implantation. Both endometrium and uterine washings of normal rats differed from those of 'delayed' animals in their electrolyte concentrations. A dose of oestradiol dipropionate (1 μg/rat) capable of inducing implantation in 'delayed' rats (ovariectomized and maintained on progesterone) did not evoke any significant changes in electrolyte concentration of either the endometrium or uterine washings. These findings are discussed in the light of a hypothesis regarding delayed implantation in rats.

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N Srinivasan, D Edwall, T A Linkhart, D J Baylink and S Mohan

Abstract

The PC-3 human prostatic carcinoma cell line has been extensively used as a model for studies on the regulation of prostate tumor cell proliferation. Because of the importance of IGF-binding proteins (IGFBPs) in the control of IGF activities that regulate cell proliferation in normal and malignant cell types, we undertook studies to characterize the IGFBPs produced by PC-3 prostate tumor cells in culture. We previously found, using an IGF-I affinity column for purification and a polyethylene glycol (PEG) precipitation assay for IGFBP detection, that PC-3 cells in culture produced a single predominant IGFBP, IGFBP-4, which inhibits IGF activities. We now present evidence that PC-3 cells also produce IGFBP-6 in abundant quantity; in the previous study this was apparently not detected in the IGF-I-bound fraction with the PEG precipitation and Western ligand blot assays. In the current study, IGF-II affinity purification of IGFBPs produced by PC-3 cells, followed by C8 HPLC reverse-phase chromatography using a shallow acetonitrile gradient, produced two major protein peaks. N-terminal amino acid sequence of peak 1 was identical to that of IGFBP-6 while that of peak 2 was identical to that of IGFBP-4. Characterization of purified IGFBP-6 from PC-3 cells revealed properties which are distinct from other IGFBPs. PEG did not precipitate the complex of 125I-IGF-II/IGFBP-6 while it precipitated the complexes between 125I-IGF-II and other IGFBPs. Indeed, IGFBP-6 decreased the amount of 125I-IGF-II tracer in the PEG precipitate in a dose-dependent manner. Also, the binding of IGFBP-6 with 125I-IGF-II was poor in Western ligand blots compared with other IGFBPs. In studies on IGFBP-6 actions, IGFBP-6 completely inhibited IGF-II-induced [3H]thymidine incorporation in MC3T3-E1 mouse osteoblast cells while it had only minimal inhibitory effects on IGF-I-induced [3H]thymidine incorporation. This differential effect is associated with the fact that IGFBP-6 has greater affinity for IGF-II than IGF-I. The results of this study indicated that (1) Western ligand blotting is not sensitive for identification of IGFBP-6, (2) the unique behavior of IGFBP-6 in the PEG assay system necessitates the use of charcoal adsorption procedure for IGFBP-6 activity detection and (3) PC-3 cells should provide a useful model system for studying regulation of IGFBP-6 expression and the role of IGFBP-6 in modulating IGF actions.

Journal of Endocrinology (1996) 149, 297–303

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MK Lindberg, SL Alatalo, JM Halleen, S Mohan, JA Gustafsson and C Ohlsson

There are two known estrogen receptors, estrogen receptor-alpha (ER alpha) and estrogen receptor-beta (ER beta), which may mediate the actions of estrogen. The aim of the present study was to compare fat content, skeletal growth and adult bone metabolism in female mice lacking ER alpha (ERKO), ER beta (BERKO) or both ERs (DERKO). We demonstrate that endogenous estrogens decrease the fat content in female mice via ER alpha and not ER beta. Interestingly, the longitudinal bone growth was decreased in ERKO, increased in BERKO, but was intermediate in DERKO females, demonstrating that ER alpha and ER beta exert opposing effects in the regulation of longitudinal bone growth. The effects on longitudinal bone growth were correlated with similar effects on serum levels of IGF-I. A complex regulation of the trabecular bone mineral density (BMD), probably caused by a disturbed feedback regulation of estrogen and testosterone, was observed in female ER-inactivated mice. Nevertheless, a partial functional redundancy for ER alpha and ER beta in the maintenance of the trabecular BMD was observed in the female mice at 60 days of age. Thus, ER alpha and ER beta may have separate effects (regulation of fat), opposing effects (longitudinal bone growth) or partial redundant effects (trabecular BMD at 60 days of age), depending on which parameter is studied.