Interaction of ovine somatomedin-C/IGF-I and IGF-I with specific IGF-I receptors on cultured muscle-derived fibroblasts

1987 ◽  
Vol 116 (2) ◽  
pp. 186-192 ◽  
Author(s):  
M. V. Dodson ◽  
B. A. Mathison ◽  
K. L. Hossner
Keyword(s):  
Molecular Weight ◽  
Binding Sites ◽  
Competitive Binding ◽  
Cross Linking ◽  
Type I ◽  
Somatomedin C ◽  
Receptor Complexes ◽  
Igf I ◽  
Relative Molecular Weight ◽  
Igf Receptor

Abstract. Binding of 125I-insulin-like growth factor-I and 125I-ovine somatomedin-C/IGF-I to monolayer cultures of muscle-derived ovine fibroblasts is described. Preliminary competitive binding experiments indicate that ovine fibroblasts possess independent cell surface receptors for IGF-I. Affinity of rIGF-II for IGF-I binding sites is minimal; rIGF-II binds to Type I IGF receptors at 1/1000 the strength of IGF-I. Insulin binds to the Type I IGF receptor at 1/100 the strength of IGF-I, whereas ovine somatomedin-C/IGF-I displays equivalent IGF-I binding as evidenced by overlapping competition of ovine somatomedin-C/IGF-I for 125IIGF-I binding sites. Results from disuccinimidyl suberate cross-linking of 125I-IGF-I to muscle-derived ovine fibroblasts in the presence of related polypeptides verified the competitive binding data. Under reducing conditions, 125I-IGF-I: receptor complexes migrated to a relative molecular weight of approximately 135 000 daltons. Specific 125I-IGF-I binding was completely inhibited by 10−8 mol/l IGF-I, 7.2 × 10−8 mol/l ovine somatomedin-C/IGF-I, and 10−6 mol/l insulin and partially inhibited by 7.2 × 10−9 mol/l ovine somatomedinC/IGF-I and 6.5 × 10−8 mol/l rIGF-II. 125I-ovine somatomedin-C/IGF-I: receptor complexes also migrated at a relative molecular weight of 135 000 daltons. No migratory band was observed at 250 000 to 260 000 daltons with either 125I-IGF-I or 125I-ovine somatomedin-C/IGF-I indicating that little labelled moiety bound to the Type II IGF receptor. Based on these preliminary competitive binding studies and cross-linking data, we conclude that ovine somatomedin-C/IGF-I is primarily interacting with the Type I IGF membrane receptor on ovine skeletal muscle fibroblasts.

Differential binding of 125I-IGF-I preparations to human fibroblast monolayers

1988 ◽  
Vol 118 (4) ◽  
pp. 513-520 ◽  
Author(s):  
C. A. Conover ◽  
P. Misra ◽  
R. L. Hintz ◽  
R. G. Rosenfeld
Keyword(s):  
Binding Sites ◽  
Human Fibroblast ◽  
Type I ◽  
Type Ii ◽  
Low Concentrations ◽  
Type I Igf Receptor ◽  
Igf I ◽  
Differential Binding ◽  
Igf Receptor ◽  
Binding Curves

Abstract. Specific, high affinity binding of 125I-IGF-I to the type I IGF receptor on human fibroblast monolayers was not altered by varying feeding schedules, serum lots, washing procedures, or incubation times and temperatures. However, markedly different competitive binding curves were obtained when different iodinated IGF-I preparations were used. Five of six radioligands bound preferentially to the type I IGF receptor on human fibroblast monolayers, with 50% displacement at 4–8 μg/l unlabelled IGF-I; with one radioligand a paradoxical 20–200% increase in 125I-IGF-I binding was observed at low concentrations of unlabelled IGF-I, while concentrations as high as 100 μg/l IGF-I failed to displace this radioligand. The latter binding pattern cannot be accounted for by 125I-IGF-I binding to the type II IGF receptor. These data indicate that various radioligands may have preferential affinities for different IGF-I binding sites on human fibroblast monolayers.

Glucose regulation of the IGF response system in chondrocytes: induction of an IGF-I-resistant state

1999 ◽  
Vol 276 (4) ◽  
pp. R1164-R1171 ◽  
Author(s):  
K. M. Kelley ◽  
T. R. Johnson ◽  
J. Ilan ◽  
R. W. Moskowitz
Keyword(s):  
Glucose Concentration ◽  
Northern Analysis ◽  
Type I ◽  
Mrna Levels ◽  
Protein Levels ◽  
Receptor Mrna ◽  
Response System ◽  
Type I Igf Receptor ◽  
Igf I ◽  
Igf Receptor

Nonresponsiveness to the growth-stimulatory actions of insulin-like growth factor (IGF)-I in chondrocytes has been reported in a number of disease states associated with impaired glucose metabolism. Primary rabbit chondrocytes were investigated for changes in their IGF response system [type-I IGF receptor and IGF-binding protein (IGFBP) expression] and in their ability to mount a synthetic response to IGF-I [as35S-labeled proteoglycan ([35S]PG) production] in media containing varying ambient glucose concentrations. Whereas basal [35S]PG synthetic rate was unaffected by glucose concentration, synthetic responsiveness to IGF-I was lost in media containing <5 mmol/l glucose or in media containing a “diabetic” glucose concentration (25 mmol/l). IGFBP expression, as measured by Northern analysis of mRNA levels and Western ligand blotting of secreted protein levels, was not significantly altered in the different glucose media, nor were there any differences in the cell surface localization of IGFBPs as assessed by affinity cross-linking with 125I-labeled IGF-I, suggesting that IGFBPs do not induce the IGF-I resistance. The nonresponsiveness to IGF-I in reduced glucose occurred with 25–50% reductions in steady-state levels of IGF type-I receptor mRNA and protein. A significant correlation between IGF receptor mRNA level and synthetic response to IGF-I was observed between 0 and 10 mmol/l glucose concentrations, suggesting that the loss of responsiveness in reduced glucose is manifested at the level of transcription and/or receptor mRNA stability. In contrast, nonresponsiveness to IGF-I in chondrocytes in diabetic glucose concentrations occurred without changes in receptor mRNA and protein levels, suggesting that IGF-I resistance was due to post-ligand-binding receptor defects. It is proposed that IGF-I resistance in chondrocytes subjected to inappropriate glucose levels may constitute an important pathogenic mechanism in degenerative cartilage disorders.

scholarly journals Dynamic Stretching of Fibrillar Collagen Enhances Cross-linking by Transglutaminas

2019 ◽  
Vol 4 (4) ◽  
pp. 2473011419S0038
Author(s):  
Nicolas Shealy ◽  
James Rex ◽  
Amy Bradshaw ◽  
Christopher Gross
Keyword(s):  
Molecular Weight ◽  
Type I Collagen ◽  
Cross Linking ◽  
Type I ◽  
Fibrillar Collagen ◽  
Collagen Gels ◽  
Cross Link ◽  
Dynamic Stretching ◽  
Insoluble Collagen ◽  
Static Conditions

Category: Basic Sciences/Biologics Introduction/Purpose: New approaches to improve tendon repair after injury are an active area of research. Critical properties of tendons are governed by the production and assembly of fibrillar collagens. Cross-linking of fibrillar collagen is a primary factor in determining the function and mechanical properties of the collagen fibers comprising Enzymatic cross-linking by lysyl oxidase in the telopeptide domain of collagen I and III is one determinant of collagen fibril assembly and is the best characterized biochemical cross-link. Transglutaminase catalyzes the modification of lysine residues that in turn form an n-e-glutamyl lysine bond between proteins in the extracellular space. We hypothesize that transglutaminase-dependent modification of collagen in tendons is also a principal determinant of tendon strength and function and is dependent upon tension. Methods: 3-D collagen gels were generated from acid solubilized type I collagen with telopeptides (Advanced BioMatrix). Collagen gels were plated and loaded into a MechanoCulture FX apparatus (CellScale). Gels were subjected to a 10% stretch for 24 hrs at 37°C at 2hz (dynamic) or no stretch, static controls. Gels exposed to enzymatic cross-linking were incubated with either 2.4 ng of recombinant Transglutaminase 2 (Axxora) in a 10 mM Ca2+ solution. Inhibition and labeling of transglutaminase substrates was performed by incubation of collagen gels with 0.2 mM aminopentyl biotinamide in DMSO. Soluble collagen was separated from insoluble collagen by centrifugation at 10,000G. Insoluble fractions were boiled in SDS-Laemmli buffer prior to separation by SDS-PAGE. Collagen in soluble and insoluble fractions was evaluated by Coomassie stain whereas transglutaminase modification was detected via western blot using streptavidin conjugated horse radish peroxidase to detect biotinylated proteins. Results: Evaluation of collagen gels subjected to dynamic versus static stretch revealed minor differences in insoluble collagen incorporation in the two conditions. Notably, higher molecular weight cross-linked forms of collagen appeared to be higher in dynamic versus static gels. In the presence of transglutaminase, differences in higher molecular weight cross-linked forms of collagen, beta-bands, were also detected. Finally, incorporation of biotinylated transglutaminase substrate into collagen alpha bands was enriched in dynamic versus static cultures. Hence, preliminary results support a differential role for transglutaminase modification in collagen under cyclic tension versus static conditions. Conclusion: A better understanding of the role of dynamic stretching and differential tension in the regulation of collagen cross- link formation is predicted to contribute to improved strategies to treat injured tendons.

scholarly journals Zinc partitions IGFs from soluble IGF binding proteins (IGFBP)-5, but not soluble IGFBP-4, to myoblast IGF type 1 receptors

2004 ◽  
Vol 180 (2) ◽  
pp. 227-246 ◽  
Author(s):  
RH McCusker ◽  
J Novakofski
Keyword(s):  
Cell Surface ◽  
Binding Sites ◽  
Affinity Binding ◽  
Igf Binding Proteins ◽  
Igf I ◽  
Igf Binding ◽  
Igf Receptor ◽  
Igf Binding Protein

Zinc (Zn(2+)), a multifunctional micronutrient, was recently shown to lower the affinity of cell-associated insulin-like growth factor (IGF) binding protein (IGFBP)-3 and IGFBP-5 for both IGF-I and IGF-II, but to increase the affinity of the cell surface type 1 IGF receptor (IGF-1R) for the same two ligands. However, there is a need for data concerning the effects of Zn(2+) on soluble IGFBPs and the type 2 IGF receptor (IGF-2R). In the current work, we demonstrate that Zn(2+) affects the affinity of IGFBP-5 secreted by myoblasts but not IGFBP-4. Zn(2+), at physiological levels, depressed binding of both IGF-I and IGF-II to IGFBP-5, affecting (125)I-IGF-I more than (125)I-IGF-II. Both (125)I-IGF-I and (125)I-IGF-II bound to high and low affinity sites on IGFBP-5. Zn(2+) converted the high affinity binding sites of IGFBP-5 into low affinity binding sites. An IGF-I analog, (125)I-R(3)-IGF-I, did not bind to the soluble murine IGFBP-5. Zn(2+) also decreased the affinity of the IGF-2R on L6 myoblasts. In contrast, Zn(2+) increased IGF-I, IGF-II and R(3)-IGF-I binding to the IGF-1R by increasing ligand binding affinity on both P(2)A(2a)-LISN and L6 myoblasts. Soluble IGFBP-5 and IGFBP-4 depressed the binding of (125)I-IGF-I and (125)I-IGF-II to the IGF-1R, but did not affect binding of (125)I-R(3)-IGF-I. By depressing the association of the IGFs with soluble IGFBP-5, Zn(2+) partitioned (125)I-IGF-I and (125)I-IGF-II from soluble IGFBP-5 onto cell surface IGF-1Rs. This effect is not seen when soluble L6-derived IGFBP-4 is present in extracellular fluids. We introduce a novel mechanism by which the trace micronutrient Zn(2+) may alter IGF distribution, i.e. Zn(2+) acts to increase IGF-1R binding at the expense of IGF binding to soluble IGFBP-5 and the IGF-2R.

Immunoreactive and receptor-active insulin-like growth factor-I (IGF-I) and IGF-binding protein in blood plasma from the freshwater fish Macquaria ambigua (golden perch)

1993 ◽  
Vol 136 (2) ◽  
pp. 191-198 ◽  
Author(s):  
T. A. Anderson ◽  
L. R. Bennett ◽  
M. A. Conlon ◽  
P. C. Owens
Keyword(s):  
Binding Protein ◽  
Size Exclusion ◽  
Type I ◽  
Factor I ◽  
Type I Igf Receptor ◽  
Receptor Assay ◽  
Igf I ◽  
Igf Binding ◽  
Igf Receptor ◽  
Igf Binding Protein

ABSTRACT The presence of insulin-like growth factor-I (IGF-I)-related molecules and IGF-binding factors in blood from golden perch, Macquaria ambigua, an Australian native freshwater fish, was investigated. Serum was acidified to dissociate IGF and IGF-binding protein complexes that might be present, and fractionated by size-exclusion high-performance liquid chromatography at pH 2·8. Fractions were neutralized and their activities assessed by (i) an immunoassay for mammalian IGF-I which also detects chicken IGF-I but in which all known forms of IGF-II react very poorly, (ii) a receptor assay for IGF-II in which all known forms of IGF-I react poorly, and (iii) a type-I IGF receptor assay in which mammalian IGF-I and IGF-II polypeptides are almost equivalent. No IGF-II-like activity was detected. Three peaks of IGF-I-like activity were detected by IGF-I immunoassay and type-I IGF receptor assay. The major peak of activity was similar in molecular size to human IGF-binding protein-3, 45–55 kDa ('large IGF'), and a minor peak of activity which was similar in size to mammalian IGFs, 7·5 kDa. A third peak of activity was observed eluting at a time which indicates that it is a smaller molecule than any previously described IGF. The large IGF was temperature-sensitive, but was not a binding protein for 125I-labelled human IGF-I (hIGF-I). This material therefore was able to bind to anti-hIGF-I antibodies and to human type-I IGF receptors, and may represent the fish equivalent of mammalian prepro-IGFs. The two smallest forms of IGF activity identified by IGF-I radioimmunoassay and type-I radioreceptor assay following acidic size-exclusion chromatography were able to stimulate protein synthesis by L-6 myoblasts in culture, although large IGF did not. When fresh (but not frozen and thawed) golden perch serum was incubated with 125I-labelled hIGF-I and then fractionated by size-exclusion liquid chromatography at pH 7·4 through Sephadex G-100, the radioactivity became associated with a complex, intermediate in size between free IGF-I and the major IGF-binding protein in human serum. The association of 125I-labelled hIGF-I with the complex was inhibited by the presence of unlabelled hIGF-I in the incubation. These studies show that receptor-active, immunoreactive and bioactive IGF-I-like activity is present in golden perch serum, and demonstrate the presence of an IGF-I-binding factor in this species. Journal of Endocrinology (1993) 136, 191–198

scholarly journals Activation of Peroxisome Proliferator-Activated Receptor γ (PPARγ) by Rosiglitazone Suppresses Components of the Insulin-Like Growth Factor Regulatory System in Vitro and in Vivo

Endocrinology ◽  
2007 ◽  
Vol 148 (2) ◽  
pp. 903-911 ◽  
Author(s):  
B. Lecka-Czernik ◽  
C. Ackert-Bicknell ◽  
M. L. Adamo ◽  
V. Marmolejos ◽  
G. A. Churchill ◽  
...  
Keyword(s):  
Regulatory System ◽  
Peroxisome Proliferator ◽  
Type I ◽  
Peroxisome Proliferator Activated Receptor ◽  
Igf I ◽  
Stromal Cell Line ◽  
Igf Receptor

Rosiglitazone (Rosi) belongs to the class of thiazolidinediones (TZDs) that are ligands for peroxisome proliferator-activated receptor γ (PPARγ). Stimulation of PPARγ suppresses bone formation and enhances marrow adipogenesis. We hypothesized that activation of PPARγ down-regulates components of the IGF regulatory system, leading to impaired osteoblast function. Rosi treatment (1 μm) of a marrow stromal cell line (UAMS-33) transfected with empty vector (U-33/c) or with PPARγ2 (U-33/γ2) were analyzed by microarray. Rosi reduced IGF-I, IGF-II, IGFBP-4, and the type I and II IGF receptor (IGF1R and IGF2R) expression at 72 h in U-33/γ2 compared with U-33/c cells (P &lt; 0.01); these findings were confirmed by RT-PCR. Rosi reduced secreted IGF-I from U-33/γ2 cells by 75% (P &lt; 0.05). Primary marrow stromal cells (MSCs) extracted from adult (8 months) and old (24 months) C57BL/6J (B6) mice were treated with Rosi (1 μm) for 48 h. IGF-I, IGFBP-4, and IGF1R transcripts were reduced in Rosi-treated MSCs compared with vehicle (P &lt; 0.01) and secreted IGF-I was also suppressed (P &lt; 0.05). B6 mice treated with Rosi (20 mg/kg·d) for short duration (i.e. 4 d), and long term (i.e. 7 wk) had reduced serum IGF-I; this was accompanied by markedly suppressed IGF-I transcripts in the liver and peripheral fat of treated animals. To determine whether Rosi affected circulating IGF-I in humans, we measured serum IGF-I, IGFBP-2, and IGFBP-3 at four time points in 50 postmenopausal women randomized to either Rosi (8 mg/d) or placebo. Rosi-treated subjects had significantly lower IGF-I at 8 wk than baseline (−25%, P &lt; 0.05), and at 16 wk their levels were reduced 14% vs. placebo (P = 0.15). We conclude that Rosi suppresses IGF-I expression in bone and liver; these changes could affect skeletal acquisition through endocrine and paracrine pathways.

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