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ABSTRACT: Global deletion of the Igfbp2 gene results in the suppression of bone turnover. To investigate the role of insulin-like growth factor-binding protein-2 (IGFBP-2) in regulating osteoclast differentiation, we cultured Igfbp2(-/-) bone marrow cells and found a reduction in the number of osteoclasts and impaired resorption. Addition of full-length IGFBP-2 restored osteoclast differentiation, fusion, and resorption. To determine the molecular domains of IGFBP-2 that were required for this effect to be manifest, Igfbp2(-/-) bone marrow cells were transfected with constructs in which the heparin-binding (HBD) or the IGF-binding domains of IGFBP-2 were mutated. We found that both domains were necessary for osteoclastogenesis because expression of the mutated forms of either domain failed to support the formation of functionally mature osteoclasts. To discern the mechanism by which IGFBP-2 regulates osteoclast formation, PTEN abundance and phosphorylation status as well as AKT responsiveness to IGF-I were analyzed. Igfbp2(-/-) cells had elevated levels of PTEN and phospho-PTEN compared with controls. Expression of wild-type IGFBP-2 reduced the level of PTEN to that of wild-type cells. Cells expressing the IGF-binding mutant showed suppression of PTEN and phospho-PTEN equivalent to the wild-type protein, whereas those expressing the IGFBP-2 HBD mutant showed no PTEN suppression. When the ability of IGF-I to stimulate AKT activation, measured by Thr³⁰⁸ and Ser⁴⁷³ phosphorylation, was analyzed, stimulation of Ser⁴⁷³ in response to IGF-I in preosteoclasts required the presence of intact IGFBP-2. This effect was duplicated by the addition of a CK2 inhibitor that prevents the phosphorylation of PTEN. In contrast, in fully differentiated osteoclasts, stimulation of Thr³⁰⁸ phosphorylation required the presence of intact IGFBP-2. We conclude that IGFBP-2 is an important regulator of osteoclastogenesis and that both the heparin- and the IGF-binding domains of IGFBP-2 are essential for the formation of fully differentiated and functional osteoclasts.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 10/2011; 27(2):390-400. · 6.04 Impact Factor
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ABSTRACT: IGF-I is structurally related to proinsulin and when administered to human subjects it enhances insulin sensitivity. However because of its growth promoting properties and its relationship to growth hormone, it has been proposed as a etiologic factor in the development of diabetic complications. This review discusses recently published data regarding the ability of hyperglycemia to sensitize cells that are capable of dedifferentiating to the growth promoting effects of IGF-I. Under normoglycemic conditions vascular smooth muscle and endothelial cells are cystostatic and stimulation of the IGF-I receptor activates the adaptor protein IRS-1 which leads to PI-3 kinase pathway activation. Following exposure to hyperglycemia these cell types undergo a signaling switch whereby an entirely different mechanism is utilized to activate both the PI-3 kinase and the MAP pathways. This leads to increased cell proliferation and migration. This molecular mechanism involves the coordinate regulation of signaling molecules and scaffolding proteins. Activation of this alternative signaling mechanism is directly linked to the stimulation of pathophysiologic processes that are involved in the pathogenesis of both diabetic retinopathy and atherosclerosis. Inhibition of activation of these intermediates has been shown to attenuate glucose induced pathophysiologic changes and results in the inhibition of both atherosclerotic lesion progression and diabetic retinopathy. In summary, hyperglycemia induces a signaling switch in vascular endothelial and smooth muscle cells that results in enhanced sensitivity to the growth promoting effects of IGF-I. This may be an important variable for determining the progression of atherosclerosis in poorly controlled diabetes and in the development of retinopathy.
Current diabetes reviews 06/2011; 7(4):235-45.
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ABSTRACT: Insulin-like growth factor-1 (IGF-1) plays a central role in cellular growth, differentiation, survival, and cell cycle progression. It is expressed early during development and its effects are mediated through binding to a tyrosine kinase receptor, the insulin-like growth factor-1 receptor (IGF-1R). In the circulation, the IGFs bind to IGF-binding proteins (IGFBPs), which determine their bioavailability and regulate the interaction between the IGFs and IGF-1R. Studies in animal models and in humans have established critical roles for IGFs in skeletal growth and development. In this review we present new and old findings from mouse models of the IGF system and discuss their clinical relevance to normal and pathological skeletal physiology.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 12/2010; 25(12):2543-52. · 6.04 Impact Factor
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ABSTRACT: Hyperglycemia has been shown to induce the p66shc expression leading to increased reactive oxygen species (ROS) generation and apoptosis. In the present study, we demonstrated that hyperglycemia induced p66shc expression in vascular smooth muscle cells. This induction was associated with an increase in apoptosis as assessed by the increase of capspase-3 enzymatic activity, cleaved caspase-3 protein, and the number of dead cells. The ability of IGF-I to inhibit apoptosis was also attenuated. Further studies showed that hyperglycemia-induced p66shc inhibited IGF-I-stimulated phosphoinositide (PI)-3 kinase and AKT activation. Mechanistic studies showed that knockdown of p66shc enhanced IGF-I-stimulated SHPS-1/p85, p85/SHP-2, and p85/Grb2 association, all of which are required for PI-3 kinase/AKT activation. These responses were attenuated by overexpression of p66shc. IGF-I-stimulated p85 and AKT recruitment to the cell membrane fraction was altered in the same manner. Disruption of p66shc-Src interaction using either a blocking peptide or by expressing a p66shc mutant that did not bind to Src rescued IGF-I-stimulated PI-3 kinase/AKT activation as well as IGF-I-dependent cell survival. Although the highest absolute level of ROS was detected in p66shc-overexpressing cells, the relative increase in ROS induced by hyperglycemia was independent of p66shc expression. Taken together, our data suggest that the increase in p66shc that occurs in response to hyperglycemia is functioning to inhibit IGF-I-stimulated signaling and that the incremental increase in SMC sensitivity to IGF-I stimulation that occurs in response to p66shc induction of ROS is not sufficient to overcome the inhibitory effect of p66shc on Src kinase activation.
Endocrinology 08/2010; 151(8):3611-23. · 4.46 Impact Factor
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ABSTRACT: Age-related osteoporosis is accompanied by an increase in marrow adiposity and a reduction in serum insulin-like growth factor-1 (IGF-1) and the binding proteins that stabilize IGF-1. To determine the relationship between these proteins and bone marrow adiposity, we evaluated the adipogenic potential of marrow-derived mesenchymal stromal cells (MSCs) from mice with decreased serum IGF-1 due to knockdown of IGF-1 production by the liver or knock-out of the binding proteins. We employed 10-16-week-old, liver-specific IGF-1-deficient, IGFBP-3 knock-out (BP3KO) and acid-labile subunit knock-out (ALSKO) mice. We found that expression of the late adipocyte differentiation marker peroxisome proliferator-activated receptor gamma was increased in marrow isolated from ALSKO mice. When induced with adipogenic media, MSC cultures from ALSKO mice revealed a significantly greater number of differentiated adipocytes compared with controls. MSCs from ALSKO mice also exhibited decreased alkaline-phosphatase positive colony size in cultures that were stimulated with osteoblast differentiation media. These osteoblast-like cells from ALSKO mice failed to induce osteoclastogenesis of control cells in co-culture assays, indicating that impairment of IGF-1 complex formation with ALS in bone marrow alters cell fate, leading to increased adipogenesis.
Journal of Biological Chemistry 12/2009; 285(7):4709-14. · 4.77 Impact Factor
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Shoshana Yakar,
Clifford J Rosen,
Mary L Bouxsein,
Hui Sun,
Wilson Mejia,
Yuki Kawashima,
Yingjie Wu,
Kelly Emerton,
Valerie Williams,
Karl Jepsen, [......],
David Hwang,
Patricia Pennisi,
Jan Frystyk,
Yves Boisclair,
John Pintar,
Héctor Jasper,
Horacio Domene,
Pinchas Cohen, David Clemmons,
Derek LeRoith
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ABSTRACT: Serum insulin-like growth factor (IGF) -1 is secreted mainly by the liver and circulates bound to IGF-binding proteins (IGFBPs), either as binary complexes or ternary complexes with IGFBP-3 or IGFBP-5 and an acid-labile subunit (ALS). The purpose of this study was to genetically dissect the role of IGF-1 circulatory complexes in somatic growth, skeletal integrity, and metabolism. Phenotypic comparisons of controls and four mouse lines with genetic IGF-1 deficits-liver-specific IGF-1 deficiency (LID), ALS knockout (ALSKO), IGFBP-3 (BP3) knockout, and a triply deficient LID/ALSKO/BP3 line-produced several novel findings. 1) All deficient strains had decreased serum IGF-1 levels, but this neither predicted growth potential or skeletal integrity nor defined growth hormone secretion or metabolic abnormalities. 2) IGF-1 deficiency affected development of both cortical and trabecular bone differently, effects apparently dependent on the presence of different circulating IGF-1 complexes. 3) IGFBP-3 deficiency resulted in increased linear growth. In summary, each IGF-1 complex constituent appears to play a distinct role in determining skeletal phenotype, with different effects on cortical and trabecular bone compartments.
The FASEB Journal 11/2008; 23(3):709-19. · 5.71 Impact Factor
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ABSTRACT: IGF-I and insulin are structurally related polypeptides that mediate a similar pattern of biological effects via receptors that display considerably homology. Administration of recombinant human IGF-I (rhIGF-I) has been proven to improve glucose control and liver and muscle insulin sensitivity in patients with type 2 diabetes mellitus (DM). The effect of rhIGF-I treatment was evaluated in a mouse model of type 2 DM (MKR mouse), which expresses a dominant-negative form of the human IGF-I receptor under the control of the muscle creatine kinase promoter specifically in skeletal muscle. MKR mice have impaired IGF-I and insulin signaling in skeletal muscle, leading to severe insulin resistance in muscle, liver, and fat, developing type 2 DM at 5 wk of age. Six-week-old MKR mice were treated with either saline or rhIGF-I for 3 wk. Blood glucose levels were decreased in response to rhIGF-I treatment in MKR mice. rhIGF-I treatment also increased body weight in MKR with concomitant changes in body composition such as a decrease in fat mass and an increase in lean body mass. Insulin, fatty acid, and triglyceride levels were not affected by rhIGF-I, nor were insulin or glucose tolerance in MKR mice. Hyperinsulinemic-euglycemic clamp analysis demonstrated no improvement in overall insulin sensitivity. Pyruvate and glutamine tolerance tests proved that there was a decrease in the rate of glucose appearance in MKR mice treated with rhIGF-I, suggesting a reduction in the gluconeogenic capacity of liver, kidney, and small intestine. Taken together these results demonstrate that the improvement of the hyperglycemia was achieved by inhibition of gluconeogenesis rather than an improvement in insulin sensitivity. Also, these results suggest that a functional IGF-I receptor in skeletal muscle is required for IGF-I to improve insulin sensitivity in this mouse model of type 2 DM.
Endocrinology 07/2006; 147(6):2619-30. · 4.46 Impact Factor
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Clifford J Rosen,
Cheryl L Ackert-Bicknell,
Martin L Adamo,
Kathy L Shultz,
Janet Rubin,
Leah Rae Donahue,
Lindsay G Horton,
Krista M Delahunty,
Wesley G Beamer,
Jennifer Sipos, David Clemmons,
Tracy Nelson,
Mary L Bouxsein,
Mark Horowitz
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ABSTRACT: Targeted gene studies have demonstrated the importance of insulin-like growth factor-I (IGF-I) for osteoblast (OB) differentiation and the acquisition of peak bone mineral density (BMD). The skeletal response to allelic differences in IGF-I expression can also be measured in vivo, using congenic mice. We created a congenic strain with reduced (approximately 20%) circulating IGF-I (C3H.B6-6T [6T]) by backcrossing a small genomic region (30 cM) of Chromosome 6 (Chr6) from C3H/HeJ (C3H) onto a C57Bl/6J (B6) background. 6T female mice have lower serum IGF-I (P<0.001 vs. B6) but similar growth hormone (GH) and serum IGF binding protein (IGFBP) concentrations as B6. At 16 weeks of age, congenics have greater body fat (P<0.02 vs. B6) despite less total body weight, and exhibit smaller femoral cross-sectional size (P=0.001), reduced cortical thickness (P<0.001) and lower trabecular BV/TV (P<0.05) than B6. 6T mice also have suppressed serum leptin (P<0.01), but compared to B6 have similar markers of bone resorption (i.e., urine CTx and serum TRAP 5B). At 8 weeks of age, skeletal IGF-I mRNA from long bones was reduced by 40% (P<0.05) as were liver mRNA transcripts (i.e., 50%, P<0.01). Osteoblast progenitors from the bone marrow of 6T mice formed less colony forming unit fibroblasts by crystal violet staining than B6 (P<0.007) and had significantly reduced alkaline phosphatase-positive colonies than B6(P<0.0001). In addition, staining of bone marrow with oil red O revealed greater numbers of adipocytes in 6T than B6. Several candidate genes in the Chr6 QTL were excluded by lack of strain-related expression differences in bone, but genes positively regulating adipocyte differentiation including Alox 5 and PPAR-gamma require further study as either "pathway" or candidate genes. In summary, allelic differences in a QTL on Chr6 result in altered IGF-I gene expression, changes in OB lineage allocation, and reduced peak bone mass. Congenic mice are useful models not only for mapping genes related to bone mass but also for elucidating the biology underlying various skeletal phenotypes associated with more subtle manipulation of the mouse genome.
Bone 12/2004; 35(5):1046-58. · 4.02 Impact Factor
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ABSTRACT: IGF binding protein-5 (IGFBP-5) is an important trophic factor for controlling the actions of IGF-I in human dermal fibroblasts and porcine aortic smooth muscle cells. When IGFBP-5 is associated with extracellular matrix, it acts to enhance the cell growth response to IGF-I. The amount of IGFBP-5 within the extracellular matrix is related in part to the amount that is present in conditioned medium, which is related to its rate of synthesis and degradation. A serine protease that degrades IGFBP-5 is present in the conditioned medium of both of these cell types. Because the IGFBP-5 protease activity that is secreted by fibroblasts has been shown to be due to the complement components C1r and C1s, these studies were undertaken to determine whether smooth muscle cells also secreted these proteases and to identify some of the factors that regulate their secretion by both cell types. Both smooth muscle cells and human fibroblasts were shown to release C1r and C1s into conditioned medium. Both C1r and C1s were detected as activated forms, as determined by SDS-PAGE using reducing conditions. The addition of increasing concentrations of either IL-1beta or TNFalpha resulted in increased synthesis of C1s by fibroblasts and smooth muscle cells, and they each increased C1r release. TNFalpha (50 ng/ml) and IL-1beta (20 ng/ml) resulted in maximum stimulation of release of both proteases. In contrast dexamethasone (10(-7) M) had no effect on C1s release and stimulated C1r release only by smooth muscle cells. To determine the physiological significance of this increase in C1r and C1s, the amount of IGFBP-5 protease activity that was present in conditioned medium was determined before and after exposure to TNFalpha, IL-beta, and dexamethasone. All three compounds resulted in an increase in the amount of IGFBP-5 proteolytic activity. Dexamethasone inhibited the release of C(1) inhibitor from fibroblasts, and this contributed to the net increase in proteolytic activity. TNFalpha inhibited the smooth muscle cell DNA synthesis response to IGF-I, but the effect of IGF-I was partially restored by the addition of C1 inhibitor. In conclusion, both C1r and C1s are released by cultured fibroblasts, and the release of each into fibroblast or porcine smooth muscle cells medium is stimulated by TNFalpha and IL-1beta. This increase results in a net increase in IGFBP-5 proteolysis, which has the potential to modify IGF-I and IGFBP-5 actions.
Endocrinology 07/2003; 144(6):2489-95. · 4.46 Impact Factor
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ABSTRACT: IGF binding protein-5 (IGFBP-5) and vitronectin are matricellular proteins that are produced by smooth muscle cells and modulate their responsiveness to IGF-I. These studies were conducted to determine if vitronectin bound IGFBP-5 with high affinity and if this altered the ability of either protein to modify cellular responsiveness to IGF-I. Solution binding assays were used to determine that vitronectin bound to IGFBP-5 with high affinity. This binding was inhibitable with glycosaminoglycans. Synthetic peptides that contained four distinct regions of the IGFBP-5 sequence were used in competitive binding assays to determine the regions of IGFBP-5 that were necessary for vitronectin binding. The regions that mediated the interaction were determined to be between amino acids 201 and 218 and between amino acids 131 and 141. Mutation of specific basic residues within these regions resulted in significant reduction in vitronectin binding and residues R134, R136, K138, K139, R201, and K202 were shown to be the most important. When the combination of IGFBP-5 and IGF-I was added to smooth muscle cells that had been plated on a vitronectin-enriched matrix, the smooth muscle cell DNA synthesis and migration responses to IGF-I plus vitronectin were enhanced. In contrast, if mutant forms of IGFBP-5 that did not bind to vitronectin were used, the responses were decreased. These effects appeared to be due to modulation of IGF-I action because the addition of a mutant form of IGFBP-5 that did not bind to IGF-I had no additional effect over and above that noted with vitronectin alone. These findings suggest that localization of IGFBP-5 within the extracellular matrix by vitronectin results in modification of cellular responsiveness to IGF-I and that vitronectin may be an important component of the extracellular matrix that modulates IGFBP-5 actions.
Endocrinology 02/2002; 143(1):30-6. · 4.46 Impact Factor
