The effect of insulin-like growth factor (IGF) and of human serum on steps in proteoglycan synthesis

1981 ◽  
Vol 97 (4) ◽  
pp. 503-507 ◽  
Author(s):  
Avivah Silbergeld ◽  
Rivka Mamet ◽  
Zvi Laron ◽  
Zvi Nevo
Keyword(s):  
Growth Factor ◽  
Human Serum ◽  
Chondroitin Sulphate ◽  
Proteoglycan Synthesis ◽  
Dependent Manner ◽  
Insulin Like Growth Factor ◽  
Intact Cartilage ◽  
Normal Human ◽  
Sugar Chains ◽  
Dose Dependent

Abstract. Embryonic chick pelvic cartilages were incubated in the presence of insulin like growth factor (IGF) (1–100 μU/ml), as well as normal human serum (5%), with radiolabelled precursors of proteoglycan (PG) synthesis: L-[3-3H]serine, D-[6-3H]glucosamine and [35S]Na2SO4. IGF alone (1–15 μU/ml), stimulated in a dose-dependent manner D-[6-3H]glucosamine incorporation into tissue-bound and soluble isolated glycosaminoglycan (GAG) chains. L-[3-3H]serine incorporation into PG molecules was not stimulated by IGF (1–100 μU/ml), despite the increase in the uptake of this precursor into intact cartilage. [35S]Na2SO4 incorporation was unaffected by IGF. Serum promoted the uptake of all three precursors into tissue-bound glycosaminoglycans. It was postulated that IGF could stimulate proteoglycan synthesis not only by elongating existing chondroitin sulphate chains but also by increased synthesis of other sugar chains e.g. keratan sulphate and oligosaccharides.

scholarly journals Regulation of Insulin-Like Growth Factor-Binding Protein-5 by Insulin-Like Growth Factor I and Interleukin-1α in Ovine Articular Chondrocytes*

Endocrinology ◽  
1998 ◽  
Vol 139 (5) ◽  
pp. 2356-2362 ◽  
Author(s):  
Damir Sunic ◽  
Julian D. McNeil ◽  
Timothy E. Rayner ◽  
Dennis L. Andress ◽  
David A. Belford
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Articular Chondrocytes ◽  
Critical Role ◽  
Limited Proteolysis ◽  
Cartilage Matrix ◽  
Proteoglycan Synthesis ◽  
Dependent Manner ◽  
Insulin Like Growth Factor ◽  
Igf I

Abstract Insulin-like growth factors (IGFs) contribute to the maintenance of the cartilage matrix by stimulating proteoglycan synthesis. In contrast, interleukin-1 (IL-1), an inflammatory cytokine, suppresses the synthesis of proteoglycans. In pathological conditions the chondrocytes’ responsiveness to IGF-I is decreased, and elevated levels of IGF-binding proteins (IGFBPs) have been implicated as a possible cause. The aim of this study was to investigate the effects of IGF-I and IL-1 on IGFBP production by ovine articular chondrocytes (OAC) and the roles of these IGFBPs in the regulation of proteoglycan synthesis. As revealed by Western ligand and immunoblotting, OACs secreted IGFBP-2 and a 24-kDa IGFBP in culture medium under basal conditions. Exposure of the cells to IGF-I for 48 h resulted in the appearance of IGFBP-5 in the medium. Des(1–3)IGF-I, an IGF-I analog with reduced affinity for IGFBPs, also increased the level of IGFBP-5, but to a lesser extent than IGF-I, whereas LR3IGF-I, which has virtually no affinity for IGFBPs, had no effect on IGFBP-5. Furthermore, IGFBP-5 underwent a time-dependent limited proteolysis when incubated with OAC-conditioned medium, degrading into 22- and 16-kDa fragments. The degradation of IGFBP-5 was significantly inhibited by IGF-I, but not by des(1–3)IGF-I or LR3IGF-I. Basic fibroblast growth factor, transforming growth factor-β, and platelet-derived growth factor had no effect on OAC IGFBPs. However, IL-1α increased the IGFBP-5 level in a dose-dependent manner, showing maximum activity at 200 U/ml. Furthermore, IL-1α, but not IGF-I, induced IGFBP-5 messenger RNA expression, as assessed by Northern blot analysis. Coincubation of IGF-I with IL-1α resulted in a substantially increased IGFBP-5 protein level, suggesting a synergism between the mechanisms of action of these two factors. Des(1–3)IGF-I and LR3IGF-I were 10 times more potent than IGF-I in stimulating proteoglycan synthesis, indicating inhibition of IGF-I activity by endogenous IGFBPs. IL-1α reduced the IGF-I bioactivity, but had no effect on the activities of the IGF-I analogs, thus implying that locally produced IGFBPs, particularly IGFBP-5, which was substantially increased when IGF-I and IL-1α were coincubated, mediated the reduction of the IGF-I activity. Our results demonstrate that IGF-I and IL-1α synergistically increase the level of IGFBP-5 in OAC by inhibiting the proteolysis and stimulating the expression of IGFBP-5, respectively. Furthermore, the attenuation of IGF-I-stimulated proteoglycan synthesis by IL-1α in OAC appears to be mediated by chondrocyte IGFBPs. We conclude that locally produced IGFBPs, in particular IGFBP-5, may play a critical role in the regulation of cartilage matrix degradation in inflammatory and degenerative arthritides.

Ras Promotes Production of Reactive Oxygen Species in Normal Human CD34+ Cells: Role in Promoting Cell Survival and Protein Phosphorylation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3797-3797
Author(s):  
Paul S Hole ◽  
Lorna Pearn ◽  
Philip E James ◽  
Alan Burnett ◽  
Richard L Darley ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Growth Factor ◽  
Cell Survival ◽  
Protein Phosphorylation ◽  
Dependent Manner ◽  
Oxygen Species ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Normal Human ◽  
Dose Dependent

Abstract Activating RAS mutations are one of the most frequent molecular abnormalities associated with acute myeloid leukemia (AML) and have also been linked to induction of reactive oxygen species (ROS). ROS play an important role in immune defense but also regulate intracellular signaling and are contributing factors in several cancer models especially those associated with oncogenic Ras activity. Here we report that constitutively active Ras expression strongly promotes production of ROS in human CD34+ cells and that this is linked to growth-factor independent survival and hyper-phosphorylation of kinases in these cells. Expression of H-RasG12V in human CD34+ cells was achieved by retroviral infection using a vector co-expressing green fluorescent protein (GFP). Using luminol-based chemiluminescence, we found that mutant Ras induced constitutive production of superoxide anions (O2−) in CD34+ cells (6.3±2.1 fold greater than controls; p<0.01). Electron paramagnetic resonance spectroscopy confirmed the presence of O2−. Hydrogen peroxide (H2O2) (which forms via dismutation of O2minus;) was also elevated (2.7±0.1μM H2O2 vs <0.5μM in controls, measured using Amplex Red). Superoxide (and H2O2) can be produced via the NOX enzyme family of oxidases. NOX inhibitor treatment dramatically suppressed O2− production induced by mutant Ras (100±0.1% inhibition with diphenyleneiodonium, p<0.001) whereas Rotenone, a mitochondrial O2− inhibitor had no effect, suggesting NOX proteins are the predominant source of ROS in these cells. Consistent with this, Ras expressing cells showed greater accumulation of the NOX2 regulatory proteins Rac, p47phox and p67phox in the plasma membrane. Ras also strongly promoted the survival of human CD34+ cells following incubation for 48 hours in medium without growth factors or serum (Ras 59±3.3% viable vs control 26±3.9%; p<0.001, as determined by Annexin V and 7-AAD staining). Growth factor-independent survival of Ras-expressing (but not control) cells decreased significantly (p<0.001; ANOVA) in a cell density-dependent manner, suggesting the presence of autocrine/paracrine pro-survival factors. In order to determine whether these factors could influence the survival of control cells in a paracrine fashion, control CD34+ progenitors (expressing GFP) were co-cultured with an increasing proportion of cells expressing mutant Ras (co-expressing DsRed), enabling analysis of individual cell populations in mixed culture by flow cytometry. Ras expressing cells significantly promoted survival of co-cultured control cells in a ‘dose’-dependent manner; control cells 33±1.5% viable in 1:1 co-culture vs 21±5.1% when cultured alone (p<0.01, ANOVA). In addition, initial data shows treatment with catalase suppressed control cell survival under co-culture conditions. This suggests that the presence of H2O2 is necessary in mediating this pro-survival effect. Ras-expressing cells also demonstrated constitutive phosphorylation of a wide variety of proteins associated with growth and survival including Akt, PDK-1 and PKC which have previously shown to be a critical effectors of Ras in CD34+cells (Darley et al, Blood, 2007). H2O2 promotes protein phosphorylation via phosphatase inhibition (Rhee, Science, 2007), suggesting that phosphorylation of these molecules could be dependent on the high levels of H2O2 produced by Ras CD34+ cells. In support of this, we found that the phosphorylation of PKC in Ras (but not control) cells increased in a density-dependent manner. Treatment with catalase reduced pPKC levels in mutant Ras expressing cells in a dose-dependent manner, supporting a role for H2O2 in promoting protein phosphorylation. In summary, these data show for the first time that Ras strongly promotes ROS production via NOX family proteins in normal human progenitor cells and that ROS are likely to play a key role in promoting cell survival and phosphorylation of intracellular proteins.

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

1996 ◽  
Vol 134 (5) ◽  
pp. 591-601 ◽  
Author(s):  
Thierry Chevalley ◽  
Donna D Strong ◽  
Subburaman Mohan ◽  
David J Baylink ◽  
Thomas A Linkhart
Keyword(s):  
Cell Proliferation ◽  
Growth Factor ◽  
Binding Proteins ◽  
Mrna Levels ◽  
Insulin Like Growth Factor ◽  
Human Osteoblast ◽  
Normal Human ◽  
Normal Human Osteoblast ◽  
Dose Dependent ◽  
Igfbp 3

Chevalley T, Strong DD, Mohan S, Baylink DJ, Linkhart TA. Evidence for a role for insulin-like growth factor binding proteins in glucocorticoid inhibition of normal human osteoblast-like cell proliferation. Eur J Endocrinol 1996;134:591–601. ISSN 0804–4643 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. Thomas A. Linkhart, Mineral Metabolism (151), Pettis VA Hospital, 11201 Benton Street, Loma Linda, CA 92357, USA

Selective insulin-like growth factor-I antagonist inhibits mouse embryo development in a dose-dependent manner

2010 ◽  
Vol 93 (8) ◽  
pp. 2621-2626 ◽  
Author(s):  
Jose Inzunza ◽  
Olle Danielsson ◽  
Parameswaran Grace Lalitkumar ◽  
Olle Larsson ◽  
Magnus Axelson ◽  
...  
Keyword(s):  
Growth Factor ◽  
Embryo Development ◽  
Mouse Embryo ◽  
Dependent Manner ◽  
Insulin Like Growth Factor ◽  
Factor I ◽  
Mouse Embryo Development ◽  
Growth Factor I ◽  
Dose Dependent

Effects of recombinant bovine somatotrophin, insulin-like growth factor-I and insulin on the proliferation of bovine granulosa cells in vitro

1993 ◽  
Vol 139 (1) ◽  
pp. 67-75 ◽  
Author(s):  
J. G. Gong ◽  
D. McBride ◽  
T. A. Bramley ◽  
R. Webb
Keyword(s):  
Growth Factor ◽  
Granulosa Cells ◽  
Ovarian Follicles ◽  
Dependent Manner ◽  
Insulin Like Growth Factor ◽  
Factor I ◽  
Igf I ◽  
Dose Dependent ◽  
Bovine Somatotrophin

ABSTRACT Treatment of heifers with recombinant bovine somatotrophin (BST) significantly increases the population of small ovarian follicles and peripheral concentrations of somatotrophin, insulin-like growth factor-I (IGF-I) and insulin. To investigate the possible mechanism(s) involved in the action of BST on ovarian follicles, the effects of BST, IGF-I and insulin, given alone or in combination with either FSH or LH, on the proliferation of bovine granulosa cells in vitro were examined using a serum-free culture system. Bovine granulosa cells were obtained from antral follicles classified into three size categories according to diameter: small <5 mm; medium-sized 5–10 mm and large >10 mm. The proliferation of granulosa cells was assessed by the incorporation of [3H]thymidine into the cultured cells. Both FSH and LH (1–1000 ng/ml) inhibited the proliferation of bovine granulosa cells obtained from all three size classes of follicles in a dose-dependent manner. BST, at doses ranging from 1 to 1000 ng/ml, had no effect on the proliferation of granulosa cells from small and medium-sized follicles, but inhibited the division of granulosa cells from large follicles in a dose-dependent manner. Treatment with either IGF-I (10–3000 ng/ml) or insulin (0·5–1000 ng/ml) stimulated, in a dose-dependent manner, the proliferation of granulosa cells obtained from all three size categories of follicles. No synergistic interaction between BST (30 ng/ml) and either FSH (50 ng/ml) or LH (5 ng/ml) was observed in granulosa cells from all three size classes of follicles. In contrast, physiological concentrations of both IGF-I (100 ng/ml) and insulin (1 ng/ml) acted in synergy with both FSH (50 ng/ml) and LH (5 ng/ml) to stimulate the proliferation of granulosa cells from small follicles, whilst no such synergistic interactions were observed in granulosa cells from medium-sized and large follicles. It was concluded that the increase in the number of small ovarian follicles induced by BST treatment in heifers may be mediated by increased peripheral concentrations of IGF-I and/or insulin, possibly acting in synergy with gonadotrophins. Furthermore, insulin probably acts through its own receptor rather than acting via the type-I IGF receptor, as it can stimulate the proliferation of bovine granulosa cells at physiological concentrations. Journal of Endocrinology (1993) 139, 67–75

scholarly journals Insulin-like growth factor 1 can decrease degradation and promote synthesis of proteoglycan in cartilage exposed to cytokines

1989 ◽  
Vol 260 (2) ◽  
pp. 543-548 ◽  
Author(s):  
J A Tyler
Keyword(s):  
Growth Factor ◽  
Interleukin 1 ◽  
Molecular Size ◽  
Qualitative Change ◽  
Relative Increase ◽  
Proteoglycan Synthesis ◽  
Insulin Like Growth Factor ◽  
Dose Dependent ◽  
Fold Stimulation

A model system of explanted cartilage has been used in vitro to determine whether insulin-like growth factor 1 (IGF 1), which promotes matrix formation is effective in the presence of cytokines such as interleukin 1 (IL1) and tumour necrosis factor (TNF), which induce net matrix depletion. IGF 1 induced a dose-dependent 2.5-fold stimulation of proteoglycan synthesis, with a half-maximal dose of 25 ng/ml. A similar relative increase occurred in response to IGF 1 (10-100 ng/ml) in cartilage cultured also with IL1 or TNF (5-500 pM). There was no detectable qualitative change in the average molecular size or charge of the aggregating proteoglycan synthesized by explants exposed to IGF 1 alone or with IL1 or TNF. The increased production of prostaglandin E2, which is initiated when IL1 or TNF bind to the chondrocytes, was the same in the presence or absence of IGF 1. The time taken for 50% of pre-labelled proteoglycan to be released from the explants (t1/2) increased in the presence of IGF 1 (100 ng/ml) from 21 to 32 days in control cultures and from 8 to 26 days in cartilage cultured with IL1 (50 pM). It is concluded that IGF 1 enhances the synthesis of aggregating proteoglycan in cartilage exposed to cytokines and can directly decrease both the basal and the cytokine-stimulated degradation of proteoglycan in cartilage.

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