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Biomedical subjects

S Mohan

Publications and source records attributed to S Mohan.

At least 145 records · Page 8Linked to original sources
PubMed1996

Intact insulin-like growth factor binding protein-5 (IGFBP-5) associates with bone matrix and the soluble fragments of IGFBP-5 accumulated in culture medium of neonatal mouse calvariae by parathyroid hormone and prostaglandin E2-treatment.

We examined the distribution of insulin-like growth factor binding proteins (IGFBPs) in cultured neonatal mouse calvariae. IGFBP-3 and -4 were predominantly found in the conditioned medium. IGFBP-2 was partitioned between conditioned medium and bone and extracellular matrix (BECM), while intact (31-kDa) IGFBP-5 was most abundant in BECM extracts. After treatment with parathyroid hormone (PTH, 10(-8) M) or prostaglandin E2 (PGE2, 10(-6) M), immunoreactive IGFBP-5 accumulated in the conditioned medium in a 21-kDa form which did not bind IGF-I on Western ligand blots. PTH and PGE2 did not alter the level of steady-state IGFBP-5 mRNA, nor markedly stimulate IGFBP-5 synthesis in the calvariae, and thus accumulation of 21-kDa IGFBP-5 was largely due to release from BECM. This accumulation of truncated IGFBP-5 in the conditioned medium was not dependent on osteoclastic bone resorption, since it was not blocked by calcitonin or a bisphosphonate which inhibited PTH- and PGE2-stimulated 45Ca-release. The conditioned medium from PTH- or PGE2-treated cultures degraded recombinant human IGFBP-5 into lower molecular weight fragments. Addition of IGF-1 at 10(-8) M into the culture resulted in accumulation of native 31-kDa IGFBP-5. However, even in the presence of IGF-1, the native IGFBP-5 was degraded and the 21-kDa product accumulated in the culture medium. These results suggested a possible proteolytic mechanism for 21-kDa IGFBP-5 accumulation, responsive to PTH and PGE2. Aprotinin, leupeptin, cystatin, and bestatin did not inhibit the effects of PTH and PGE2 in the cultures. The localization of IGFBP-5 in BECM and its release and proteolysis induced by PTH and PGE2 could play a role in the local regulation of bone metabolism.

Animals↗
PubMed1996

Evaluation of signal transduction mechanisms for the mitogenic effects of prostaglandin E2 in normal human bone cells in vitro.

Prostaglandin E2 (PGE2) is one of the most potent stimulators of bone formation in vivo. In these studies, we investigated the mechanism(s) underlying PGE2 effects on human bone formation by evaluating the effects of PGE2 on normal human bone cell (HBC) proliferation in vitro. Cell proliferation of normal HBCs was increased by PGE2 as measured by increased [3H]thymidine incorporation after 18 h and increased cell number after 48 h of treatment. The effect of PGE2 to stimulate cell proliferation was biphasic, with a maximum stimulation between 0.01 and 1.0 nM PGE2 in different experiments. At higher concentrations of PGE2 (0.1 microM), HBC proliferation was inhibited. Signal transduction for PGE2 has been reported to include both protein kinase A (PKA) and protein kinase C (PKC) pathways. In these studies, concentrations of PGE2 which stimulated cell proliferation did not increase cyclic adenosine monophosphate (cAMP) production. However, higher concentrations of PGE2 increased cAMP production (7- to 12-fold at 1-10 microM) and inhibited cell proliferation. Because stimulators of PKC, such as phorbol esters, have been reported to stimulate cell proliferation, the action of PKC inhibitors were tested. Both staurosporine and sangivamysin (PKC inhibitors) totally abrogated the effect of PGE2 to stimulate cell proliferation. Additional studies revealed that PGE2 increased 45Ca uptake in a dose-dependent manner with a peak response occurring between 1 and 10 nM PGE2 concentrations in different experiments. Furthermore, when the calcium channel blocker, verapamil, was added to HBC cultures treated with PGE2, the stimulation of 45Ca uptake and cell proliferation by PGE2 was completely blocked. These data suggest that PGE2 increases cell proliferation through activation of a verapamil-sensitive calcium channel. In conclusion, these data are consistent with a model in which stimulation of HBC proliferation by low doses of PGE2 is mediated by an enhancement of phospholipase C, which results in both an increase in PKC activity and an increase in intracellular calcium influx.

Analysis of Variance↗
PubMed1996

Growth factors for bone growth and repair: IGF, TGF beta and BMP.

Current research is reviewed regarding the actions of three growth factor systems on bone formation: insulin-like growth factors (IGFs), transforming growth factor-betas (TGF betas), and bone morphogenetic proteins (BMPs). Each growth factor family consists of multiple related growth factor genes. TGF betas and BMPs 2-7 are subfamilies of a larger TGF beta superfamily. IGFs, TGF betas and BMPs are produced by osteoblasts and other bone cells and affect osteoblast proliferation and differentiation. They are also incorporated into mineralized bone matrix and retain activity when extracted from bone. Various hormones, growth factors, and mechanical stress influence bone cell production of IGFs, TGF betas, and BMPs. Thus these growth factors may function in local regulation of bone formation. Currently there is much interest in the function of IGF binding proteins, which are also produced by bone cells, in regulating IGF activities in bone. Recently, mechanisms for activation of the TGF beta serine/threonine kinase receptors have been investigated, and receptors for BMPs have been identified which are structurally related to TGF beta receptors. In vivo studies are discussed which demonstrate the applicability of IGFs, TGF betas and BMPs to increasing bone formation systemically, promoting fracture healing and inducing bone growth around implants.

Animals↗
PubMed1996

Insulin-like growth factor (IGF) system components in human prostatic cancer cell-lines: LNCaP, DU145, and PC-3 cells.

BACKGROUND: Evidence has been accumulating that in many tumors, insulin-like growth factors (IGFs) promote cancer cell growth in an autocrine/paracrine manner via the IGF-I receptor. In an effort to understand the role of IGFs in prostate cancer cell growth, we characterized the IGF system components produced by human prostatic cancer cell-lines, LNCaP, DU145, and PC-3, grown in serum-free medium. METHODS: IGFs, their receptors, and IGF binding proteins (IGFBPs) produced by the three human prostate cell lines were characterized by reverse transcriptase-polymerase chain reaction (RT-PCR), radioimmunoassay (RIA), Western ligand blot, Western immunoblot, and Northern blot analyses. RESULTS: mRNA for IGF-II and receptors for IGF-I and IGF-II were detected in all three cell-lines by RT-PCR. In contrast to the published study, only LNCaP cells expressed a trace amount of IGF-I mRNA. RIA on conditioned media collected from these cells revealed that all three cell-lines produced measurable IGF-II but not IGF-I. Western Ligand blot, Western immunoblot, and Northern blot analyses revealed that LNCaP, DU145, and PC-3 cells expressed IGFBP-2, IGFBP-2/-3/-4/-6, and IGFBP-2/-3/-4/-5/-6, respectively. IGF-II stimulated [3H]thymidine incorporation into DNA in DU145 and PC-3 cells significantly although the effect was small. DNA synthesis in PC-3 cells but not in LNCaP and DU145 cells was significantly inhibited by the IGF-I receptor-specific monoclonal antibody. CONCLUSION: Theses results suggest potentially important roles of IGFs and IGFBPs in prostate cancer cell growth, and that in particular, IGF-II may play a critical role in prostate cancer cell growth.

Antibodies, Monoclonal↗
PubMed1996

Insulin-like growth factor system components and the coupling of bone formation to resorption.

In the adult skeleton, bone formation is regulated by an event referred to as the coupling of bone formation to resorption (i.e. formation is quantitatively linked to resorption), which is thought to be mediated in part by osteogenic growth factor molecules produced by bone cells. Of the various osteogenic growth factors identified in bone, there is sufficient evidence to document an important role for the insulin-like growth factor (IGF) system in mediating this coupling process. Studies on the basic aspects of the IGF system reveal that it is complex and involves a number of components which include the inhibitory IGF binding protein (IGFBP)-4 and stimulatory IGFBP-5 and their corresponding proteases. This complex regulatory scheme of the IGF component system, together with the finding that all of the IGF system components are regulated in bone cells by both local and systemic effectors of bone metabolism, underscores the importance of the IGFs in mediating coupling of bone formation to resorption. In addition, a number of in vitro and in vivo findings also suggest that the IGF system could play a role in the positive (net gain of bone) or negative (net loss of bone) uncoupling of bone formation to resorption.

Adult↗
PubMed1996

Retinoic acid regulates insulin-like growth factor-binding protein expression in human osteoblast cells.

Retinoic acid (RA) regulates the growth and differentiation of numerous cells types and plays a key role in skeletal development. Previous studies have demonstrated that insulin-like growth factors (IGFs) are important local regulators of bone cell proliferation and differentiation and that IGF-binding proteins (IGFBPs) modulate their activities. In an attempt to test the hypothesis that RA mediates its effects on bone cells in part by regulating IGFBP expression, we first examined the effect of RA on IGFBP expression in human osteoblast model systems and then compared these responses to the effects of RA on IGFBP expression in human skin fibroblasts. The most dramatic effect of RA on IGFBPs++ in all cell types tested was to increase IGFBP-6 messenger RNA (mRNA) abundance more than 1000% of the control value. Significant effects on IGFBP-5 mRNA abundance were also found, with maximal reductions to 35% of control within 24 h of treatment. In addition, RNA maximally increased IGFBP-3 and -4 mRNA to 580% and 390% of the control value, respectively, in SaOS-2 cells, but had variable effects on IGFBP-3 and -4 mRNA levels in human bone cells, U2-OS, and human skin fibroblasts. The levels of the 24-, 29- to 32-, and 38- to 42 kDa IGFBPs in the conditioned medium of RA-treated cultures increased, as determined by ligand blot analysis, whereas the amount of IGFBP-5 was reduced, as determined by RIA. Cycloheximide abolished the RA-stimulated increase in IGFBP-6 mRNA and reduced baseline IGFBP-5 mRNA levels, but did not affect RA-modulated mRNA levels of the other IGFBPs. RA modestly increased the stabilities of all four IGFBP mRNAs, which could contribute to the observed increases in IGFBP-3 and IGFBP-4 mRNA levels; however, the 217% increase in the IGFBP-5 mRNA half- life in the presence of RA could not contribute to the reduction in mRNA levels. In addition, the small increase in the IGFBP-6 mRNA half-life could not account for the 1900% increase in the mRNA level. These data suggest that RA stimulated changes in IGFBP-5 and -6 mRNA levels may in part be mediated by alterations in transcription or other early posttranscription regulatory mechanisms. In conclusion, RA significantly regulates IGFBP expression in human osteoblast cells.

Cells, Cultured↗
PubMed1996

Acute effect of brief low- and high-intensity exercise on circulating insulin-like growth factor (IGF) I, II, and IGF-binding protein-3 and its proteolysis in young healthy men.

We measured circulating levels of the GH insulin-like growth factor (IGF) system in response to brief exercise of different intensities. Ten males (mean age 28 +/- 5 yr) were studied on three separate occasions: once under resting conditions (control) and once each performing 10 min of low- or high-intensity exercise. Blood samples were assayed by RIA for GH, IGF-I and -II, IGF-binding protein-3 (IGFBP-3), and IGFBP-3 proteolytic activity. After 10 min of low-intensity exercise, IGF-I and IGFBP-3 had increased over preexercise baseline by 7.7 +/- 2.7% (P < 0.05) and 12.5 +/- 3.3% (P < 0.004), respectively. After 10 min of high-intensity exercise, all measured components of the IGF system were increased: IGF-I by 13.3 +/- 3.2% (P < 0.002), IGF-II by 15.7 +/- 3.1 (P < 0.01), and IGFBP-3 by 23 +/- 6% (P < 0.001). IGFBP-3 proteolytic activity also was increased (44 +/- 14% above baseline, P < 0.05). GH reached its peak 10 min after the cessation of high-intensity exercise, unlike the earlier peaks of IGF-I and II. In summary: 1) brief exercise leads to small but significant increases in circulating IGF-I, IGF-II, IGFBP-3, and IGFBP-3 proteolysis; and 2) these responses may be influenced by exercise intensity. The IGF responses seem to be unrelated to GH. Acute exercise-induced proteolysis of IGFBP-3 may contribute to anabolic effects of physical activity by increasing the bioavailability of IGF-I.

Adult↗
PubMed1996

Physical fitness, endurance training, and the growth hormone-insulin-like growth factor I system in adolescent females.

We examined the relationship between physical fitness and circulating components of the GH-insulin-like growth factor I (IGF-I) system [i.e. GH, GH-binding protein (GHBP), IGF-I, and IGF-binding proteins 1-5 (IGFBP-1 through-5)] in adolescent females (age range, 15-17 yr). The study consisted of 1) a cross-sectional protocol (n = 23) in which GH-IGF-I components were correlated with fitness, as estimated by thigh muscle volume and maximal O2 uptake; and 2) a prospective study in which fitness, GH-IGF-I system components, and osteocalcin were examined before and after a 5-week period of endurance-type training (control, n = 6; trained, n = 10). The cross-sectional analysis revealed significant (P < 0.05) positive correlations between fitness and 1) mean 12-h overnight GH levels, 2) GHBP, and 3) IGF-I. Muscle volume was negatively correlated with both IGFBP-2 and -4. The prospective training study was associated with 1) increases in circulating osteocalcin (39 +/- 14%; P < 0.007), and 2) decreases in IGF-I (-14 +/- 5%; P < 0.05) and IGFBP-5 (-10 +/- 4%; P < 0.04). Unexpectedly, IGFBP-3 fell in both control (-8 +/- 2%; P < 0.01) and trained subjects (-5 +/- 3%; P < 0.05), and GHBP was reduced only among control subjects (-10 +/- 7%; P < 0.04). In summary, fitter adolescent girls tended to have increased mean serum GH, GHBP, and IGF-I. In contrast, brief endurance training led to increases in muscle mass and serum osteocalcin that were not accompanied by increases in GH or IGF-I. In fact, training may, in the short term, have led to a catabolic state hormonally expressed by reductions in IGF-I and IGFBP-5.

Adolescent↗
PubMed1996

Recombinant synthesis of insulin-like growth factor-binding protein-4 (IGFBP-4): Development, validation, and application of a radioimmunoassay for IGFBP-4 in human serum and other biological fluids.

Insulin-like growth factor-binding protein-4 (IGFBP-4), like the five other IGFBPs present in human serum, acts as a transport protein for insulin-like growth factor I (IGF-I) and IGF-II and modulates their biological effects. To investigate the role of IGFBP-4 in the physiology of the IGF system, we developed a sensitive RIA for IGFBP-4 employing, as antigen, tracer, and standard, recombinant human IGFBP-4 (rhIGFBP-4) expressed in Escherichia coli as a fusion protein with glutathione S-transferase and affinity purified with glutathione-derivatized resin. Antibody against the rhIGFBP-4 fusion protein was raised in guinea pigs; tracer and standard were provided by the rhIGFBP-4 moiety that had been cleaved from the rhIGFBP-4 fusion protein and repurified by reverse phase high pressure liquid chromatography. We report that both IGFBP-4 purified from PC3 human prostate cell-conditioned medium and rhIGFBP-4 bound IGF and migrated in electrophoresis gels in an identical manner; that in gel permeation chromatography, rhIGFBP-4 coeluted with the IGFBP-4 present in human serum; and that both are equally immunoreactive with the IGFBP-4 antiserum. Employing this IGFBP-4 RIA, we determined that no IGFBP other than IGFBP-4 reacted with the IGFBP-4 antiserum, and that recovery of IGFBP-4 from serum samples exceeded 90% when exogenous IGFBP-4 was added and was unaffected by the addition of IGFs or by repeated freezing and thawing of the sample. We employed this IGFBP-4 RIA to demonstrate an increase in IGFBP-4 in TE85 human osteosarcoma cell-conditioned medium after treatment with dibutyryl cAMP, PTH, and 1,25-dihydroxyvitamin D3, agents known to increase the IGFBP-4 messenger ribonucleic acid level. Application of this RIA to the measurement of IGFBP-4 in human serum revealed that the circulating level of IGFBP-4 in 41 individuals in the 61-87 yr age group (546 +/- 135 microgram/L) was 35% higher than that in 24 individuals in the 23-40 yr age group (404 +/- 156 microgram/L). The mean circulating level of PTH was also 20% higher in the 61-87 yr group compared to that in the 23-40 yr group (P < 0.01). In addition, serum IGFBP-4 amounts showed a significant positive correlation with age (r = 0.54; P < 0.001) and serum PTH (r = 0.26; P < 0.01). These data validate this IGFBP-4 RIA and illustrate its utility in illuminating the physiological mechanisms that regulate IGFBP-4 in vivo and influence its effects on the IGFs in both normal and abnormal pathology and in aging.

Adult↗
PubMed1996

Growth hormone (GH) treatment of GH-deficient children increases serum levels of insulin-like growth factors (IGFs), IGF-binding protein-3 and -5, and bone alkaline phosphatase isoenzyme.

To investigate the contribution of the insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) to the regulation of bone growth in 10 GH-deficient Japanese children receiving recombinant GH therapy, we determined the percent increase from pretreatment levels of serum IGF-I, IGF-II, IGFBP-3, IGFBP-5, and bone-specific alkaline phosphatase isoenzyme (B-ALP). For 10 children between 6-13 yr of age, serum IGF-I and IGF-II were increased after 1 month of treatment by 53% and 7%, respectively; after 12 months of therapy, IGF levels remained elevated at 51% and 17%, respectively. Serum IGFBP-3 and IGFBP-5 were also increased after 1 month of GH therapy by 17% and 13% respectively; after 12 months of therapy, they remained elevated at 22% and 15%, respectively. After 12 months of treatment, the bone formation marker B-ALP was also elevated to 23% greater than pretreatment levels. The elevation of IGF-I induced by GH was significantly correlated with the increases in IGFBP-3 (r = 0.735; P < 0.0001) and IGFBP-5 (r = 0.795; P < 0.0001), and the elevation of B-ALP was also significantly positively correlated with the increases in IGF-I, IGF-II, IGFBP-3, and IGFBP-5 (r = 0.544, P < 0.0001; r = 0.268, P = 0.0399; r = 0.414, P = 0.0010; and r = 0.500, P < 0.0001, respectively). Our data are consistent with the anabolic effect on bone growth of GH treatment being mediated by IGF-II, IGFBP-3, and IGFBP-5 as well as by IGF-I. This is the first evidence that GH treatment increases IGF-II in GH-deficient children. This finding was probably the result of application of a valid assay that measures IGF-II without interference of IGFBPs.

Adolescent↗
PubMed1996

Evidence for a role for insulin-like growth factor binding proteins in glucocorticoid inhibition of normal human osteoblast-like cell proliferation.

Glucocorticoids (GCs) inhibit bone formation in vivo and inhibit osteoblast proliferation and collagen synthesis in vitro. These effects may be mediated by alterations in the insulin-like growth factor (IGF) system. In the present study of normal human osteoblast-like (HOB) cells, we tested the hypothesis that dexamethasone (Dex) inhibits IGF anabolic activity in bone by altering expression of IGF binding proteins (IGFBPs), particularly by decreasing expression of IGFBP-5 and IGFBP-3 (which enhance IGF activity) and increasing expression of IGFBP-4 (which inhibits IGF actions). Dexamethasone treatment caused a dose-dependent inhibition of HOB cell proliferation (69 +/- 4% of control at 10(-8) mol/l Dex) in seven separate experiments. Dexamethasone decreased IGFBP-5 mRNA levels to 20-30% of control (10(-8) and 10(-7) mol/l for 24 h). In six of six HOB preparations, 10(-8) mol/l Dex decreased IGFBP-5 mRNA levels (35 +/- 7% of control) and this effect was time dependent. Dexamethasone also decreased IGFBP-3 mRNA levels (74 +/- 9% of control in three HOB preparations). Dexamethasone decreased secretion of 29-31-kD IGFBP-5 and 38-42-kD IGFBP-3 proteins, determined by Western ligand blot and IGFBP-5 immunoblot, and induced a dose-dependent decrease in IGFBP-3 and IGFBP-5 secretion determined by specific radioimmunoassays. The effects of Dex on IGFBP-4 mRNA and on secretion of 25-kD IGFBP-4 levels were inconsistent between different cell preparations. Results suggest that GC inhibition of IGFBP-5 and IGFBP-3 production could decrease IGF activities and contribute to GC inhibition of bone formation.

Base Sequence↗
PubMed1996

Insulin-like growth factor-binding protein-6 produced by human PC-3 prostate cancer cells: isolation, characterization and its biological action.

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.

Blotting, Western↗
PubMed1995

Studies on the mechanisms by which insulin-like growth factor (IGF) binding protein-4 (IGFBP-4) and IGFBP-5 modulate IGF actions in bone cells.

The growth potentiating effects of the insulin-like growth factor (IGF)-I and IGF-II are modulated by a family of six insulin-like growth factor binding proteins (IGFBPs). Despite the similarity in amino acid sequences of the IGFBPs, their effects on the growth of bone cells differ. Studies on the molecular mechanisms for IGFBP-4 actions revealed that coincubation of bone cells with IGFBP-4 and 125I-IGF-I or 125I-IGF-II decreased the binding of both of these ligands in a dose-dependent manner. In addition, IGFBP-4 decreased the binding of IGF-I tracer to purified type I IGF receptor. These data in conjunction with data showing that IGFBP-4 had no effect on cell proliferation induced by analogs of IGF-I or IGF-II, which exhibited > 100-fold reduced affinity for binding to IGFBP-4 suggest that IGFBP-4 may inhibit IGF action by preventing the binding of ligand to its membrane receptor. In contrast to IGFBP-4, IGFBP-5 treatment increased the binding of IGF tracer to bone cells but did not increase the binding of 125I-IGF-I to type I IGF receptor. Studies on the mechanism by which IGFBP-5 increased the binding of 125I-IGF tracer to bone cells suggest that IGFBP-5 could facilitate IGF binding by a mechanism in which IGFBP-5 has cell surface binding sites independent of IGF receptors. These data in conjunction with the findings that IGFBP-5 potentiated cell proliferation even in the presence of those same IGF analogs that exhibited > 200-fold reduced affinity for binding to IGFBP-5, suggest that IGFBP-5 may in part stimulate bone cell proliferation by an IGF-independent mechanism involving IGFBP-5-specific cell surface binding sites.

Animals↗
PubMed1995

Evidence that human bone cells in culture secrete insulin-like growth factor (IGF)-II and IGF binding protein-3 but not acid-labile subunit both under basal and regulated conditions.

Insulin-like growth factors (IGFs) are found in human circulation predominantly as part of a growth hormone (GH)-dependent complex of 125-150 kD, which is composed of three subunits: IGF-I or IGF-II, an acid stable IGF binding protein (IGFBP)-3, and an acid labile subunit (ALS). Although recent studies demonstrate that a number of cell types in culture secrete IGFs and IGFBP-3, very little is known with regard to the origin of circulating ALS. To test the hypothesis that human bone cells (HBCs), which produce abundant amounts of IGF-II and IGFBP-3, also produce ALS, we measured the IGF-I, IGF-II, IGFBP-3, and ALS levels using specific radioimmunoassays (RIAs) in the conditioned medium (CM) of untransformed normal HBCs and SaOS-2 osteosarcoma cells treated with various effectors (IGF-II, osteogenic protein-1 [OP-1, bone morphogenetic protein-7] and human GH) for 48 h. No detectable levels (< 3 ng/ml) of ALS were found in the CM of various HBC types under basal conditions. In contrast, CM collected from liver explants in culture contained significant amount of ALS prepared and assayed under identical conditions. The IGF-I level was also undetectable in the CM of various HBC types. In the IGF-II (3, 30 ng/ml)-treated HBC CM, the IGFBP-3 level was increased in a dose-dependent manner but neither IGF-I nor ALS could be detected. In the SaOS-2 cell culture, OP-1 (1, 100 ng/ml) increased both IGF-II and IGFBP-3 secretion but neither ALS nor IGF-I secretion. Treatment of HBCs with GH (1, 10, 100 ng/ml) had no significant effect on the secretion of either IGF-I, IGF-II, IGFBP-3, or ALS.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

Effects of extracellular calcium on insulin-like growth factor II in human bone cells.

Extracellular calcium concentration is critically important for normal function of the body. Recently, reports have shown that cells derived from parathyroid glands contain an extracellular calcium receptor that is responsive to changes in extracellular calcium. Bone is intimately involved in calcium homeostasis; therefore, we sought to test the hypothesis that extracellular calcium has direct effects on bone cells. Extracellular calcium was increased by the addition of varying concentrations of CaCl2 (0.4-2.0 mM) to the control medium. An increase in extracellular calcium increased cell proliferation, as assessed by 3H-thymidine incorporation, in a number of cell types including normal human bone cells derived from vertebrae (HBV155) and a number of human osteosarcoma cell lines. The increase in cell proliferation by elevated CaCl2 was dose dependent, whereas MgCl2 was not effective at the doses tested (up to 2 mM added MgCl2). To test the hypothesis that the mitogenic activity of elevated extracellular calcium involved a growth factor, levels of insulin-like growth factor II (IGF-II) were measured in the conditioned medium of HBV155 cells by radioimmunoassay after removal of binding proteins by size exclusion chromatography. The effects of an increase in extracellular calcium by 1 mM were: 1) increased culture media levels of IGF-II within 1 h of treatment, 2) the increase in IGF-II levels reached a maximum after 8 h of treatment, and 3) IGF-II levels were still elevated after 24 h of treatment. Furthermore, a blocking monoclonal antibody against IGF-II abolished the increased cell proliferation in HBV155 cells following elevation of extracellular calcium. Taken together, these findings suggest that an increase in extracellular calcium results in an increase in IGF-II which is required for the subsequent increase in cell proliferation.

Analysis of Variance↗
PubMed1995

An age-related decrease in the concentration of insulin-like growth factor binding protein-5 in human cortical bone.

The skeletal contents of insulin-like growth factor-2 (IGF-II), insulin-like growth factor binding protein-5 (IGFBP-5), and insulin-like growth factor binding protein-3 (IGFBP-3) were determined in duplicate samples of human femoral cortical bone obtained from 64 subjects (44 males and 20 females) between the ages of 20 and 64 years. The results of these quantitative measurements revealed an age-related decrease in the femoral cortical content of IGFBP-5 (r = -0.272, P = 0.031) in the total population. Although the femoral cortical content of IGF-II did not show a similar decrease with age, it could be correlated to the femoral cortical content of IGFBP-5 (r = 0.442, P < 0.001). In contrast, the femoral cortical content of IGFBP-3 did not decrease with age and could not be correlated to the femoral cortical contents of either IGFBP-5 or IGF-II. Comparisons of these results with previous measurements of insulin-like growth factor-1 (IGF-I) and transforming growth factor-beta (TGF-beta), in extracts of the same bones, showed significant cross-correlations between the femoral cortical contents of each of these growth factors and the femoral cortical contents of IGFBP-5 (r = 0.625 for IGF-I versus IGFBP-5, r = 0.554 for TGF-beta versus IGFBP-5, P < 0.001 for each) but not IGFBP-3.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

Age-related changes in IGFBP-4 and IGFBP-5 levels in human serum and bone: implications for bone loss with aging.

Osteoporosis develops because of an age-dependent imbalance between the rates of bone formation and bone resorption (i.e. bone formation rate is inadequate compared with bone resorption rate to maintain bone volume). With regard to the mechanism for the deficiency in bone formation, we propose that age-associated changes in the IGF system components contribute to an age-related decrease in the skeletal capacity for osteoblast cell proliferation. As a means of testing this hypothesis, we have measured serum levels of IGFBP-4 and IGFBP-5 since our studies have shown that the mitogenic actions of IGFs in bone cells are modulated by inhibitory IGFBP-4 and stimulatory IGFBP-5. By using newly developed and validated radioimmunoassays for measurement of IGFBP-4 and IGFBP-5, we found that the circulating level of IGFBP-4 increases with age while that of IGFBP-5 declines with age. In subjects from 23-87 years, serum IGFBP-4 concentrations showed a significant positive correlation with serum PTH while serum IGFBP-5 concentrations showed a significant positive correlation with IGF-I. These age-related changes in the serum levels of IGF system components are consistent with our previous findings of age-related decreases in the femoral cortical contents of IGF-I, IGF-II and IGFBP-5. Although the biological implications of the sequestration of IGFs in bone are unknown, we have hypothesized that the level of the IGFs in bone is a reflection of their integrated local secretion by osteoblasts. Based on our data, we now propose a model in which (a) underproduction of the stimulatory components and overproduction of an inhibitory component of the IGF system occur as a consequence of aging, and (b) these changes lead to an age-related decrease in the local (autocrine/paracrine) as well as the hormonal (endocrine) actions of the IGFs, which in aggregate could contribute to the decrease in osteoblast proliferation and the deficiency in bone formation. In conclusion, although our findings provide indirect evidence that age associated changes in IGF system components could lead to a deficit in bone formation, further studies are needed to demonstrate a cause and effect relationship between changes in bone cell production of IGF system components and the age-related uncoupling of bone formation from resorption.

Aging↗