Anti-apoptotic effect of insulin-like growth factor (IGF)-I and its receptor in porcine preimplantation embryos derived from in vitro fertilization and somatic cell nuclear transfer

Insulin-like growth factor (IGF)-I is a receptor-mediated autocrine and/or paracrine growth and/or survival factor for mammalian embryo development. It is known to promote the growth and development of mouse preimplantation embryos. The present study was designed to investigate the effects of IGF-I (50 ng/ml), anti-IGF-I receptor antibody (50 ng/ml) and their combination on porcine preimplantation embryo development. Furthermore, the mechanism underlying the embryotropic effects of IGF-I was evaluated by monitoring the incidence of apoptosis and expression of apoptosis-related genes. In both in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos, culturing with IGF-I increased the rate of blastocyst formation and this embryotrophic effect was neutralized by culturing with IGF-I along with anti-IGF-I receptor (IGF-IR) antibody. Culturing IVF and SCNT embryos with IGF-I significantly increased the number of total cells in blastocysts and decreased the number of apoptotic nuclei. These effects of IGF-I were also neutralized by culturing with IGF-I along with anti-IGF-IR antibody. Expression of the anti-apoptotic Bcl-2 gene was increased, while expression of the pro-apoptotic Bax was decreased in both IVF and SCNT embryos cultured with IGF-I. In both IVF and SCNT embryos, anti-IGF-IR antibody along with IGF-I neutralized the effect of IGF-I on expression of Bcl-2 and Bax genes. In conclusion, the present study demonstrated that IGF-I through its specific receptors improved the developmental competence of IVF and SCNT embryos by decreasing the incidence of apoptosis and regulating apoptosis-related genes in porcine preimplantation embryos.     

Differential expression of the two GH genes during embryonic development of rainbow trout Oncorhynchus mykiss in relation with the IGFs system

The Growth hormone (GH)/insulin-like growth factor (IGF) system promotes embryonic growth in higher vertebrates. Such a system exists in salmonids, but exhibits an additional level of complexity resulting from a recent whole genome tetraploidisation. Thus, two nonallelic GH genes are present in the trout genome. Although the two GH genes are similar, the possibility remains that the two genes have evolved separately, acquiring a distinct expression pattern. In this study, using whole mounted in situ hybridisation, we observed a one stage delay between the appearance of GH-2 (Stage 22) and GH-1 (Stage 23) soon after pituitary formation (Stage 21). In addition, by double in situ hybridisation, we clearly evidenced two types of somatotroph, one expressing only GH-2 and the other type both GH-1 and GH-2 at Stage 24. Consequently, at this stage more cells expressed GH-2 than GH-1 as confirmed by quantitative RT-PCR. However at hatching, as in adult, the difference between the expression of the two GH genes was no longer observed. In addition, our immunohistochemical studies did not show any delay between the expression of the mRNA and its translation as a protein at Stage 24. A comparison of the expression pattern of the IGF system components (IGF-1, IGF-2, and the receptor type I) determined by real time RT-PCR, have shown an IGF-1 mRNA increase concomitantly to the appearance of GH expression. On the whole, our results demonstrate a differential regulation of GH-1 and GH-2 genes in rainbow trout embryo. The relationship observed between the expression of different component of the GH/IGF system seems to indicate that this system could be functional early on during embryonic development.     

Rainbow trout follicular maturational competence acquisition is associated with an increased expression of follicle stimulating hormone receptor and insulin-like growth factor 2 messenger RNAs

Post-vitellogenic female rainbow trout (Oncorhynchus mykiss) were assayed in vitro for follicular maturational competence (FMC). Ovarian follicles were stimulated with a range of concentrations of partially purified gonadotropin. The efficient concentration for 50% germinal vesicle breakdown (GVBD) was calculated and used as an indicator of FMC. Before in vitro assay, ovarian tissue was sampled in order to quantify mRNA abundance of specific genes in the ovarian follicle by real-time PCR. In addition, maturation-inducing steroid (MIS, 17, 20 beta-dihydroxy-4-pregnen-3-one) and estradiol (E2) plasma levels were measured by radioimmunoassay. The mRNA expression of several genes such as luteinizing hormone receptor (LH-r), follicular stimulating hormone receptor (FSH-r), insulin-like growth factor 1 (IGF1), insulin-like growth factor 2 (IGF2), insulin-like growth factor receptor 1a (IGF-r1a), and 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD) that are putatively expressed in the preovulatory ovary, was studied in females of varying FMC using real-time PCR. FMC acquisition is characterized by an increase of MIS circulating levels and a concomitant drop of E2 levels. At the ovarian level, no significant variation of LH-r, 20 beta-HSD, IGF1, and IGF-r1a mRNA abundance was observed among females of varying FMC. In contrast, FSH-r and IGF2 mRNA levels were significantly higher in females exhibiting high FMC. In addition, correlation analyses showed that IGF2 and FSH-r, mRNA levels were positively correlated with FMC. These results indicate that FMC acquisition is associated with an increased expression of these gene products that may be useful markers of FMC.     

Regulation of Ligand and Shear Stress-induced Insulin-like Growth Factor 1 (IGF1) Signaling by the Integrin Pathway

Mechanical loading of the skeleton, as achieved during daily movement and exercise, preserves bone mass and stimulates bone formation, whereas skeletal unloading from prolonged immobilization leads to bone loss. A functional interplay between the insulin-like growth factor 1 receptor (IGF1R), a major player in skeletal development, and integrins, mechanosensors, is thought to regulate the anabolic response of osteogenic cells to mechanical load. The mechanistic basis for this cross-talk is unclear. Here we report that integrin signaling regulates activation of IGF1R and downstream targets in response to both IGF1 and a mechanical stimulus. In addition, integrins potentiate responsiveness of IGF1R to IGF1 and mechanical forces. We demonstrate that integrin-associated kinases, Rous sarcoma oncogene (SRC) and focal adhesion kinase (FAK), display distinct actions on IGF1 signaling; FAK regulates IGF1R activation and its downstream effectors, AKT and ERK, whereas SRC controls signaling downstream of IGF1R. These findings linked to our observation that IGF1 assembles the formation of a heterocomplex between IGF1R and integrin β3 subunit indicate that the regulation of IGF1 signaling by integrins proceeds by direct receptor-receptor interaction as a possible means to translate biomechanical forces into osteoanabolic signals.     

Alteration of the insulin-like growth factor axis during in vitro differentiation of the human osteosarcoma cell line HOS 58

The insulin-like growth factors I and II (IGF-I, IGF-II), their receptors, and high affinity binding proteins (IGFBPs) represent a family of cellular modulators that play essential roles in the development and differentiation of cells and tissues including the skeleton. Recently, the human osteosarcoma cell line HOS 58 cells were used as an in vitro model of osteoblast differentiation characterized by (i) a rapid proliferation rate in low-density cells that decreased continuously with time of culture and (ii) an increasing secretion of matrix proteins during their in vitro differentiation. In the present paper, HOS 58 cells with low cell density at early time points of the in vitro differentiation (i) displayed a low expression of IGF-I and -II; (ii) synthesized low levels of IGFBP-2, -3, -4, and -5, but (iii) showed high expression levels of both the type I and II IGF receptors. During the in vitro differentiation of HOS 58 cells, IGF-I and -II expressions increased continuously in parallel with an upregulation of IGFBP-2, -3, -4, and -5 whereas the IGF-I receptor and IGF-II/M6P receptor mRNA were downregulated. In conclusion, the high proliferative activity in low cell density HOS 58 cells was associated with high mRNA levels of the IGF-IR, but low concentrations of IGFBP-2. The rate of proliferation of HOS 58 cells continuously decreased during cultivation in parallel with a decline in IGF-IR expression, but increase of mitoinhibitory IGFBP-2. These data are indicative for a role of the IGF axis during the in vitro differentiation of HOS 58 cells.     

Cryo-EM Structure Reveals Polymorphic Ligand-bound States of IGF1R

Type 1 insulin-like growth factor receptor (IGF1R) plays an important role in regulating cellular metabolism and cell growth and has been identified as an anticancer drug target. Although previous studies have revealed some structures of IGF1R with different ligands, the continuous dynamic conformation change remains unclear. Here, we report 10 distinct structures (7.9–3.6 Å) of IGF1R bound to IGF1 or insulin to reveal the polymorphic conformations of ligand-bound IGF1R. These results showed that the α-CT², disulfide bond (C670-C670′), and FnIII-2 domains had the most flexible orientations for the conformational change that occurs when ligands bind to the receptor. In addition, we found one special conformation (tentatively named the diverter-switch state) in both complexes, which may be one of the apo-IGF1R forms under ligand-treatment conditions. Hence, these results illustrated the mechanism of how different ligands could bind to human IGF1R and provided a rational template for drug design.     

Cross‐talk between IGF‐1 and estrogen receptors attenuates intracellular changes in ventral spinal cord 4.1 motoneuron cells because of interferon‐gamma exposure

Insulin‐like growth factor‐1 (IGF‐1) is a neuroprotective growth factor that promotes neuronal survival by inhibition of apoptosis. To examine whether IGF‐1 exerts cytoprotective effects against extracellular inflammatory stimulation, ventral spinal cord 4.1 (VSC4.1) motoneuron cells were treated with interferon‐gamma (IFN‐γ). Our data demonstrated apoptotic changes, increased calpain:calpastatin and Bax:Bcl‐2 ratios, and expression of apoptosis‐related proteases (caspase‐3 and ‐12) in motoneurons rendered by IFN‐γ in a dose‐dependent manner. Post‐treatment with IGF‐1 attenuated these changes. In addition, IGF‐1 treatment of motoneurons exposed to IFN‐γ decreased expression of inflammatory markers (cyclooxygenase‐2 and nuclear factor‐kappa B:inhibitor of kappa B ratio). Furthermore, IGF‐1 attenuated the loss of expression of IGF‐1 receptors (IGF‐1Rα and IGF‐1Rβ) and estrogen receptors (ERα and ERβ) induced by IFN‐γ. To determine whether the protective effects of IGF‐1 are associated with ERs, ERs antagonist ICI and selective siRNA targeted against ERα and ERβ were used in VSC4.1 motoneurons. Distinctive morphological changes were observed following siRNA knockdown of ERα and ERβ. In particular, apoptotic cell death assessed by TUNEL assay was enhanced in both ERα and ERβ‐silenced VSC4.1 motoneurons following IFN‐γ and IGF‐1 exposure. These results suggest that IGF‐1 protects motoneurons from inflammatory insult by a mechanism involving pivotal interactions with ERα and ERβ.

     

Insulin-like Growth Factor-2 Enhances Functions of Antigen (Ag)-specific Regulatory B Cells

Regulatory B cells (Bregs) are important in immune regulation. The factors that regulate Breg functions are less clear. Insulin-like growth factor 2 (IGF2) is capable of inducing hematopoietic stem cell differentiation. This study aimed to investigate the role of IGF2 in the development of Bregs and the enhancement of their function. In this study, the expression of IGF1 receptor (IGF1R) and IGF2R in ovalbumin (OVA)-specific B cells (OVAsBCs) was assessed by real time RT-PCR and Western blotting. The release of interleukin (IL)-10 from OVAsBCs and OVAsBC proliferation were assessed by enzyme-linked immunoassay and proliferation assay. The role of IGF2 in enhancing the function of OVAsBCs was tested with an intestinal allergic inflammation mouse model. The results showed that OVAsBCs expressed high levels of IGF2R. Exposure to both IGF2 and a specific antigen (Ag), OVA, markedly enhanced the expression of IL-10 in OVAsBCs as well as enhanced the IL-10⁺ OVAsBC proliferation. The concurrent exposure to IGF2 and specific Ag markedly induced the IL-10 promoter DNA demethylation via activating the STAT5 pathway. IGF2 also enhanced both the OVAsBC proliferation in vivo and the effect of Ag-specific immunotherapy on inhibiting allergic inflammation in the intestine. We conclude that OVAsBCs express high levels of IGF2R and that IGF2 increases the expression of IL-10 in OVAsBCs and enhances OVAsBC proliferation and the inhibitory effect on allergic inflammation.     

Insulin-like growth factor-1 receptor transactivation modulates the inflammatory and proliferative responses of neurotensin in human colonic epithelial cells

Neurotensin (NT) is a gastrointestinal neuropeptide that modulates intestinal inflammation and healing by binding to its high-affinity receptor NTR1. The dual role of NT in inflammation and healing is demonstrated in models of colitis induced by Clostridium difficile toxin A and dextran sulfate sodium, respectively, and involves NF-κB-dependent IL-8 expression and EGF receptor-mediated MAPK activation in human colonocytes. However, the detailed signaling pathways involved in these responses remain to be elucidated. We report here that NT/NTR1 coupling in human colonic epithelial NCM460 cells activates tyrosine phosphorylation of the insulin-like growth factor-1 receptor (IGF-1R) in a time- and dose-dependent manner. NT also rapidly induces Src tyrosine phosphorylation, whereas pretreatment of cells with the Src inhibitor PP2 before NT exposure decreases NT-induced IGF-1R phosphorylation. In addition, inhibition of IGF-1R activation by either its specific antagonist AG1024 or siRNA against IGF-1 significantly reduces NT-induced IL-8 expression and NF-κB-dependent reporter gene expression. Pretreatment with AG1024 also inhibits Akt activation and apoptosis induced by NT. Silencing of Akt expression by siRNA also substantially attenuates NT-induced IL-8 promoter activity and NF-κB-dependent reporter gene expression. This is the first report to indicate that NT transactivates IGF-1R and that this response is linked to Akt phosphorylation and NF-κB activation, contributing to both pro-inflammatory and tissue repair signaling pathways in response to NT in colonic epithelial cells. We propose that IGF-1R activation represents a previously unrecognized key pathway involved in the mechanisms by which NT and NTR1 modulate colonic inflammation and inflammatory bowel disease.     

Insulin‐like growth factor 1 receptor signaling induced by supraphysiological doses of IGF‐1 in human peripheral blood lymphocytes

Insulin‐like growth factor‐1 (IGF‐1) mediates some of growth hormone anabolic functions through its receptor, IGF‐1R. Following ligand binding, intracellular signaling pathways are activated favouring proliferation, cell survival, tissue growth, development, and differentiation. IGF‐1 is included in the World Anti‐Doping Agency Prohibited List. While the evidence for IGF‐1 as performance‐enhancing substrate in healthy humans is still weak, clinical studies demonstrated that the endogenous growth hormone/IGF‐1 excess is associated with cardiovascular implications. Previously, we demonstrated that human peripheral blood lymphocytes represent a suitable system to identify a gene signature, related to dihydrotestosterone or IGF‐1 abuse, independent from the type of sport. In addition, in a proteomic study, we demonstrated that dihydrotestosterone hyperdosage affects cell motility and apoptosis. Here, we investigate the doping action of IGF‐1 by means of a differential proteomic approach and specific protein arrays, revealing an active cytoskeletal reorganization mediated by Stat‐1; moreover, IGF‐1 stimulation produces a sustained activation of different signaling pathways as well as an overproduction of cytokines positively related to immune response and inflammation. In conclusion, these data indicate that, following IGF‐1 hyperdosage, circulating peripheral blood lymphocytes could be more prone to transendothelial migration.     

Effects of insulin and insulin‐like growth factor 1 on osteoblast proliferation and differentiation: differential signalling via Akt and ERK

Insulin and insulin‐like growth factor 1 (IGF‐1) are evolutionarily conserved hormonal signalling molecules, which influence a wide array of physiological functions including metabolism, growth and development. Using genetic mouse studies, both insulin and IGF‐1 have been shown to be anabolic agents in osteoblasts and bone development primarily through the activation of Akt and ERK signalling pathways. In this study, we examined the temporal signalling actions of insulin and IGF‐1 on primary calvarial osteoblast growth and differentiation. First, we observed that the IGF‐1 receptor expression decreases whereas insulin receptor expression increases during osteoblast differentiation. Subsequently, we show that although both insulin and IGF‐1 promote osteoblast differentiation and mineralization in vitro, IGF‐1, but not insulin, can induce osteoblast proliferation. The IGF‐1‐induced osteoblast proliferation was mediated via both MAPK and Akt pathways because the IGF‐1‐mediated cell proliferation was blocked by U0126, an MEK/MAPK inhibitor, or LY294002, a PI3‐kinase inhibitor. Osteocalcin, an osteoblast‐specific protein whose expression corresponds with osteoblast differentiation, was increased in a dose‐ and time‐dependent manner after insulin treatment, whereas it was decreased with IGF‐1 treatment. Moreover, insulin treatment dramatically induced osteocalcin promoter activity, whereas IGF‐1 treatment significantly inhibited it, indicating direct effect of insulin on osteocalcin synthesis. Copyright © 2012 John Wiley & Sons, Ltd.     

SIRT1 is involved in adrenocortical cancer growth and motility

Adrenocortical cancer (ACC) is a rare tumour with unfavourable prognosis, lacking an effective treatment. This tumour is characterized by IGF-II (insulin-like growth factor II) overproduction, aromatase and ERα (oestrogen receptor alpha) up-regulation. Previous reports suggest that ERα expression can be regulated by sirt1 (sirtuin 1), a nicotinamide adenine dinucleotide (NAD+)-dependent class III histone deacetylases that modulates activity of several substrates involved in cellular stress, metabolism, proliferation, senescence, protein degradation and apoptosis. Nevertheless, sirt1 can act as a tumour suppressor or oncogenic protein. In this study, we found that in H295R and SW13 cell lines, sirt1 expression is inhibited by sirtinol, a potent inhibitor of sirt1 activity. In addition, sirtinol is able to decrease ACC cell proliferation, colony and spheroids formation and to activate the intrinsic apoptotic mechanism. Particularly, we observed that sirtinol interferes with E2/ERα and IGF1R (insulin growth factor 1 receptor) pathways by decreasing receptors expression. Sirt1 involvement was confirmed by using a specific sirt1 siRNA. More importantly, we observed that sirtinol can synergize with mitotane, a selective adrenolitic drug, in inhibiting adrenocortical cancer cell growth. Collectively, our data reveal an oncogenic role for sirt1 in ACC and its targeting could implement treatment options for this type of cancer.     

MicroRNA-100 regulates IGF1-receptor expression in metastatic pancreatic cancer cells

Abstract

Patients with pancreatic adenocarcinoma have the lowest 5 year survival rate and yearly rates of incidence are nearly equal to the mortality rates. Long term cure rates by standard therapies are disappointing owing to disseminated disease at diagnosis and chemotherapeutic resistance. New therapeutic targets are necessary to decrease the progression of pancreatic cancer and the ability to identify targets specific to metastasis would improve patient care. We evaluated the levels of microRNA of metastatic and non-metastatic cell lines. The expression levels of microRNAs and mRNAs were determined using microarray analysis to examine and compare five pancreatic cancer cell lines, two that can metastasize in vivo (S2VP10 and S2CP9) and three that do not metastasize (MiaPaCa2, Panc-1 and ASPC-1). MicroRNA analysis indicated an increase in miR-100 and a decrease in miR-138 expression in metastatic cancer cells. Microarray analysis of different expressions of mRNAs in metastatic and non-metastatic pancreatic cell lines also indicated significantly increased insulin growth factor-1 receptor (IGF1-R) expression in metastatic pancreatic cancer cell lines compared to non-metastatic pancreatic cancer cell lines. To confirm microarray analysis results, western blot and immunocytochemistry were performed. Western blot revealed that IGF1-R expression exhibited in metastatic cancer cell lines a seven-fold increase compared to non-metastatic cell lines. In addition, downstream expressions of the proteins, GRB2 and phosphorylated PI3K, also were increased in aggressive cancer cell lines. Immunocytochemistry confirmed the linkage of IGF1-R to miR-100, because cells transfected with miR-100 inhibitor showed a decrease in IGF1-R. Cells transfected with a miR-138 mimic, however, did not affect IGF1-R expression.     

Insulin-like Growth Factor (IGF) I Down-Regulates Type 1 IGF Receptor (IGF 1R) and Reduces the IGF I Response in A549 Non-Small-Cell Lung Cancer and Saos-2/B-10 Osteoblastic Osteosarcoma Cells

The insulin-like growth factor type 1 receptor (IGF 1R) mediates the acute metabolic effects of IGF I as well as IGF I-stimulated cell proliferation and protection from apoptosis. IGF binding proteins (IGFBPs) can modulate these responses. We, therefore, investigated whether intrinsic IGFBPs interfere with IGF I-induced regulation of IGF 1R expression and with the biological response to IGF I in two human tumor cell lines, the non-small-cell lung cancer cell line A549 and the osteoblastic osteosarcoma cell line Saos-2/B-10. We compared the growth rates, IGFBP production, IGF I binding characteristics, IGF 1R protein and mRNA levels, and the acute IGF I response (stimulation of glycogen synthesis) after pretreatment of the cells in serum-free medium with or without added IGF I or medium supplemented with 5% fetal calf serum (FCS). In contrast to A549 cells, which produce IGF I and significant amounts of IGFBPs, survival and proliferation of Saos-2/B-10 cells, which do not produce IGF I or significant amounts of IGFBPs, depended on the addition of exogenous IGF I. IGF I increased the concentration of IGFBP-2 and -3 and decreased the concentration of IGFBP-4 in the medium of A549 cells. As compared to FCS, IGF I pretreatment in both cell lines decreased the number of specific IGF I binding sites, down-regulated total and membrane IGF 1R protein, and largely reduced or abolished the acute IGF I response without affecting IGF 1R mRNA levels. The data suggest that the IGF 1R protein of the two cell lines is translationally and/or posttranslationally down-regulated by its ligand in the presence and in the absence of locally produced IGFBPs and that the cell lines have retained this negative feedback to counteract IGF I stimulation.     

Rosuvastatin reduces microglia in the brain of wild type and ApoE knockout mice on a high cholesterol diet; implications for prevention of stroke and AD

IGFBP2 is overexpressed in the most common brain tumor, glioblastoma (GBM), and its expression is inversely correlated to GBM patient survival. Previous reports have demonstrated a role for IGFBP2 in glioma cell invasion and astrocytoma development. However, the function of IGFBP2 in the restricted, self-renewing, and tumorigenic GBM cell population comprised of tumor-initiating stem cells has yet to be determined. Herein we demonstrate that IGFBP2 is overexpressed within the stem cell compartment of GBMs and is integral for the clonal expansion and proliferative properties of glioma stem cells (GSCs). In addition, IGFBP2 inhibition reduced Akt-dependent GSC genotoxic and drug resistance. These results suggest that IGFBP2 is a selective malignant factor that may contribute significantly to GBM pathogenesis by enriching for GSCs and mediating their survival. Given the current dearth of selective molecular targets against GSCs, we anticipate our results to be of high therapeutic relevance in combating the rapid and lethal course of GBM.