Insulin-like growth factor (IGF) signalling is required for early dorso-anterior development of the zebrafish embryo

The insulin-like growth factor (IGF) signalling pathway has been highly conserved in animal evolution and, in mammals and Xenopus, plays a key role in embryonic growth and development, with the IGF-1 receptor (IGF-1R) being a crucial regulator of the signalling cascade. Here we report the first functional role for the IGF pathway in zebrafish. Expression of mRNA coding for a dominant negative IGF-1R resulted in embryos that were small in size compared to controls and had disrupted head and CNS development. At its most extreme, this phenotype was characterized by a complete loss of head and eye structures, an absence of notochord and the presence of abnormal somites. In contrast, up-regulation of IGF signalling following injection of IGF-1 mRNA, resulted in a greatly expanded development of anterior structures at the expense of trunk and tail. IGF-1R knockdown caused a significant decrease in the expression of Otx2, Rx3, FGF8, Pax6.2 and Ntl, while excess IGF signalling expanded Otx2 expression in presumptive forebrain tissue and widened the Ntl expression domain in the developing notochord. The observation that IGF-1R knockdown reduced expression of two key organizer genes (chordin and goosecoid) suggests that IGF signalling plays a role in regulating zebrafish organizer activity. This is supported by the expression of IGF-1, IGF-2 and IGF-1R in shield-stage zebrafish embryos and the demonstration that IGF signalling influences expression of BMP2b, a gene that plays an important role in zebrafish pattern formation. Our data is consistent with a common pathway for integration of IGF, FGF8 and anti-BMPs in early vertebrate development.     

Neural and head induction by insulin-like growth factor signals.

Evidence is presented for a new pathway participating in anterior neural development. It was found that IGF binding protein 5 (IGFBP-5), as well as three IGFs expressed in early embryos, promoted anterior development by increasing the head region at the expense of the trunk in mRNA-injected Xenopus embryos. A secreted dominant-negative type I IGF receptor (DN-IGFR) had the opposite effect. IGF mRNAs led to the induction of ectopic eyes and ectopic head-like structures containing brain tissue. In ectodermal explants, IGF signals induced anterior neural markers in the absence of mesoderm formation and DN-IGFR inhibited neural induction by the BMP antagonist Chordin. Thus, active IGF signals appear to be both required and sufficient for anterior neural induction in Xenopus.     

Insulin-like growth factor signaling regulates zebrafish embryonic growth and development by promoting cell survival and cell cycle progression

Although much is known about the global effects of insulin-like growth factor 1 receptor (IGF1R)-mediated signaling on fetal growth and the clinical manifestations resulting from IGF/IGF1R deficiencies, we have an incomplete understanding of the cellular actions of this essential pathway during vertebrate embryogenesis. In this study, we inhibited IGF1R signaling during zebrafish embryogenesis using antisense morpholino oligonucleotides or a dominant-negative IGF1R fusion protein. IGF1R inhibition resulted in reduced embryonic growth, arrested development and increased lethality. IGF1R-deficient embryos had significant defects in the retina, inner ear, motoneurons and heart. No patterning abnormalities, however, were found in the brain or other embryonic tissues. At the cellular level, IGF1R inhibition increased caspase 3 activity and induced neuronal apoptosis. Coinjection of antiapoptotic bcl2-like mRNA attenuated the elevated apoptosis and rescued the retinal and motoneuron defects, but not the developmental arrest. Subsequent cell cycle analysis indicated an increased percentage of cells in G1 and a decreased percentage in S phase in IGF1R-deficient embryos independent of apoptosis. These results provide novel insight into the cellular basis of IGF1R function and show that IGF1R signaling does not function as an anteriorizing signal but regulates embryonic growth and development by promoting cell survival and cell cycle progression.     

Longevity effect of IGF‐1R+/− mutation depends on genetic background‐specific receptor activation

Growth hormone (GH) and insulin-like growth factor (IGF) signaling regulates lifespan in mice. The modulating effects of genetic background gained much attention because it was shown that life-prolonging effects in Snell dwarf and GH receptor knockout vary between mouse strains. We previously reported that heterozygous IGF-1R inactivation (IGF-1R^+/−) extends lifespan in female mice on 129/SvPas background, but it remained unclear whether this mutation produces a similar effect in other genetic backgrounds and which molecules possibly modify this effect. Here, we measured the life-prolonging effect of IGF-1R^+/− mutation in C57BL/6J background and investigated the role of insulin/IGF signaling molecules in strain-dependent differences. We found significant lifespan extension in female IGF-1R^+/− mutants on C57BL/6J background, but the effect was smaller than in 129/SvPas, suggesting strain-specific penetrance of longevity phenotypes. Comparing GH/IGF pathways between wild-type 129/SvPas and C57BL/6J mice, we found that circulating IGF-I and activation of IGF-1R, IRS-1, and IRS-2 were markedly elevated in 129/SvPas, while activation of IGF pathways was constitutively low in spontaneously long-lived C57BL/6J mice. Importantly, we demonstrated that loss of one IGF-1R allele diminished the level of activated IGF-1R and IRS more profoundly and triggered stronger endocrine feedback in 129/SvPas background than in C57BL/6J. We also revealed that acute oxidative stress entails robust IGF-1R pathway activation, which could account for the fact that IGF-1R^+/− stress resistance phenotypes are fully penetrant in both backgrounds. Together, these results provide a possible explanation why IGF-1R^+/− was less efficient in extending lifespan in C57BL/6J compared with 129/SvPas.     

Expression profile of IGF system during lung injury and recovery in rats exposed to hyperoxia: a possible role of IGF-1 in alveolar epithelial cell proliferation and differentiation

Although several studies have shown that an induction of insulin-like growth factor (IGF) components occurs during hyperoxia-mediated lung injury, the role of these components in tissue repair is not well known. The present study aimed to elucidate the role of IGF system components in normal tissue remodeling. We used a rat model of lung injury and remodeling by exposing rats to > 95% oxygen for 48 h and allowing them to recover in room air for up to 7 days. The mRNA expression of IGF-I, IGF-II, and IGF-1 receptor (IGF-1R) increased during injury. However, the protein levels of these components remained elevated until day 3 of the recovery and were highly abundant in alveolar type II cells. Among IGF binding proteins (IGFBPs), IGFBP-5 mRNA expression increased during injury and at all the recovery time points. IGFBP-2 and -3 mRNA were also elevated during injury phase. In an in vitro model of cell differentiation, the expression of IGF-I and IGF-II increased during trans-differentiation of alveolar epithelial type II cells into type-I like cells. The addition of anti-IGF-1R and anti-IGF-I antibodies inhibited the cell proliferation and trans-differentiation to some extent, as evident by cell morphology and the expression of type I and type II cell markers. These findings demonstrate that the IGF signaling pathway plays a critical role in proliferation and differentiation of alveolar epithelium during tissue remodeling.     

R1507, an anti-insulin-like growth factor-1 receptor (IGF-1R) antibody, and EWS/FLI-1 siRNA in Ewing's sarcoma: convergence at the IGF/IGFR/Akt axis

A subset of patients with Ewing's sarcoma responds to anti-insulin-like growth factor-1 receptor (IGF-1R) antibodies. Mechanisms of sensitivity and resistance are unknown. We investigated whether an anti-IGF-1R antibody acts via a pathway that could also be suppressed by small interfering (si) RNA against the EWS/FLI-1 fusion protein, the hallmark of Ewing's sarcoma. The growth of two Ewing's sarcoma cell lines (TC-32 and TC-71) was inhibited by the fully human anti-IGF-1R antibody, R1507 (clonogenic and MTT assays). TC-32 and TC-71 cells express high levels of IGF-2, while RD-ES and A4573 Ewing's cell lines, which were less responsive to R1507 in our assays, express low or undetectable IGF-2, respectively. TC-71 cells also expressed high levels of IGF-1R, and R1507 decreased steady-state levels of this receptor by internalization/degradation, an effect which was associated with a decrease in p-IGF-1R, p-IRS-1, and p-Akt. EWS/FLI-1 siRNA also decreased p-Akt, due to its ability to increase IGF-BP3 levels and subsequently decrease IGF-1 and IGF-2 levels, thus inhibiting signaling through p-IGF-1R. This inhibition correlated with growth suppression and apoptosis. The attenuation of Akt activation was confirmed in TC-71 and HEK-293 (human embryonic kidney) cells by transfecting them with IGF-1R siRNA. We conclude that antibodies and siRNA to IGF-1R, as well as siRNA to EWS/FLI-1, act via intersecting IGF/IGF-1R signals that suppress a common point in this pathway, namely the phosphorylation of Akt.     

Suppression of IGF‐I signals in neural stem cells enhances neurogenesis and olfactory function during aging

Downregulation of insulin-like growth factor (IGF) pathways prolongs lifespan in various species, including mammals. Still, the cellular mechanisms by which IGF signaling controls the aging trajectory of individual organs are largely unknown. Here, we asked whether suppression of IGF-I receptor (IGF-1R) in adult stem cells preserves long-term cell replacement, and whether this may prevent age-related functional decline in a regenerating tissue. Using neurogenesis as a paradigm, we showed that conditional knockout of IGF-1R specifically in adult neural stem cells (NSC) maintained youthful characteristics of olfactory bulb neurogenesis within an aging brain. We found that blocking IGF-I signaling in neural precursors increased cumulative neuroblast production and enhanced neuronal integration into the olfactory bulb. This in turn resulted in neuro-anatomical changes that improved olfactory function. Interestingly, mutants also displayed long-term alterations in energy metabolism, possibly related to IGF-1R deletion in NSCs throughout lifespan. We explored Akt and ERK signaling cascades and revealed differential regulation downstream of IGF-1R, with Akt phosphorylation preferentially decreased in IGF-1R^−/− NSCs within the niche, and ERK pathway downregulated in differentiated neurons of the OB. These challenging experimental results were sustained by data from mathematical modeling, predicting that diminished stimulation of growth is indeed optimal for tissue aging. Thus, inhibiting growth and longevity gene IGF-1R in adult NSCs induced a gain-of-function phenotype during aging, marked by optimized management of cell renewal, and enhanced olfactory sensory function.     

Repression of malignant tumor progression upon pharmacologic IGF1R blockade in a mouse model of insulinoma

NVP-AEW541, a specific ATP-competitive inhibitor of the insulin-like growth factor-1 receptor (IGF1R) tyrosine kinase, has been reported to interfere with tumor growth in various tumor transplantation models. We have assessed the efficacy of NVP-AEW541 in repressing tumor growth and tumor progression in the Rip1Tag2 transgenic mouse model of pancreatic β-cell carcinogenesis. In addition, we have tested NVP-AEW541 in Rip1Tag2;RipIGF1R double-transgenic mice which show accelerated tumor growth and increased tumor malignancy compared with Rip1Tag2 single-transgenic mice. Previously, we have shown that high levels of IGF-2, a high-affinity ligand for IGF1R, are required for Rip1Tag2 tumor cell survival and tumor growth. Unexpectedly, treatment of Rip1Tag2 mice with NVP-AEW541 in prevention and intervention trials neither did affect tumor growth nor tumor cell proliferation and apoptosis. Yet, it significantly repressed progression to tumor malignancy, that is, the rate of the transition from differentiated adenoma to invasive carcinoma. Treatment of Rip1Tag2;RipIGF1R double-transgenic mice resulted in moderately reduced tumor volumes and increased rates of tumor cell apoptosis. Sustained expression of IGF-2 and of the IGF-2-binding form of insulin receptor (IR-A) in tumor cells suggests a compensatory role of IR-A upon IGF1R blockade. The results indicate that inhibition of IGF1R alone is not sufficient to efficiently block insulinoma growth and imply an overlapping role of IGF1R and insulin receptor in executing mitogenic and survival stimuli elicited by IGF-2. The reduction of tumor invasion upon IGF1R blockade on the other hand indicates a critical function of IGF1R signaling for the acquisition of a malignant phenotype.     

Transactivation of CXCR4 by the insulin-like growth factor-1 receptor (IGF-1R) in human MDA-MB-231 breast cancer epithelial cells

In the multimolecular environment in tissues and organs, cross-talk between growth factor and G protein-coupled receptors is likely to play an important role in both normal and pathological responses. In this report, we demonstrate transactivation of the chemokine receptor CXCR4 by the growth factor insulin-like growth factor (IGF)-1 is required for IGF-1-induced cell migration in metastatic MDA-MB-231 cells. The induction of chemotaxis in MDA-MB-231 cells by IGF-1 was inhibited by pretreatment of the cells with pertussis toxin (PTX) and by RNAi-mediated knockdown of CXCR4. Transactivation of the CXCR4 pathway by IGF-1 occurred independently of CXCL12, the chemokine ligand of CXCR4. Neither CXCR4 knockdown nor PTX had any effect on the ability of IGF-1 to activate IGF-1R, suggesting that CXCR4 and G proteins are activated subsequent to, or independently of, phosphorylation of IGF-1R by IGF-1. Coprecipitation studies revealed the presence of a constitutive complex containing IGF-1R, CXCR4, and the G protein subunits, G(i)alpha2 and Gbeta, and stimulation of MDA-MB-231 cells with IGF-1 led to the release of G(i)alpha2 and Gbeta from CXCR4. Based on our findings, we propose that CXCR4 constitutively forms a complex with IGF-1R in MDA-MB-231 cells, and that this interaction allows IGF-1 to activate migrational signaling pathways through CXCR4, G(i)alpha2 and Gbeta.     

胰岛素样生长因子IGF系统与鱼类性腺的研究进展

IGFs系统包含3个配体(IGF-1、IGF-2、IGF-3)、2个受体(IGF-1R、IGF-2R)和6个IGF结合蛋白(IGFBP).生殖和生长是生物体最基本的特征,两者既密切相关又相互区别,胰岛素样生长因子(IGFs)是生长轴和生殖轴相交联的关键因子.最近研究表明:鱼类性腺的发育及成熟伴随着细胞分化和组织生长,传统的生长因子IGF-1、IGF-2和最近发现的IGF-3,对鱼类性腺发挥着重要作用.本文重点介绍鱼类特有的配体IGF-3的结构,鱼类IGFs系统的信号通路及其与鱼类性腺的相关性研究进展.     

Insulin and Insulin-like Growth Factor-1 Receptors Act as Ligand-specific Amplitude Modulators of a Common Pathway Regulating Gene Transcription

Insulin and insulin-like growth factor-1 (IGF-1) act on highly homologous receptors, yet in vivo elicit distinct effects on metabolism and growth. To investigate how the insulin and IGF-1 receptors exert specificity in their biological responses, we assessed their role in the regulation of gene expression using three experimental paradigms: 1) preadipocytes before and after differentiation into adipocytes that express both receptors, but at different ratios; 2) insulin receptor (IR) or IGF1R knock-out preadipocytes that only express the complimentary receptor; and 3) IR/IGF1R double knock-out (DKO) cells reconstituted with the IR, IGF1R, or both. In wild-type preadipocytes, which express predominantly IGF1R, microarray analysis revealed ∼500 IGF-1 regulated genes (p < 0.05). The largest of these were confirmed by quantitative PCR, which also revealed that insulin produced a similar effect, but with a smaller magnitude of response. After differentiation, when IR levels increase and IGF1R decrease, insulin became the dominant regulator of each of these genes. Measurement of the 50 most highly regulated genes by quantitative PCR did not reveal a single gene regulated uniquely via the IR or IGF1R using cells expressing exclusively IGF-1 or insulin receptors. Insulin and IGF-1 dose responses from 1 to 100 nm in WT, IRKO, IGFRKO, and DKO cells re-expressing IR, IGF1R, or both showed that insulin and IGF-1 produced effects in proportion to the concentration of ligand and the specific receptor on which they act. Thus, IR and IGF1R act as identical portals to the regulation of gene expression, with differences between insulin and IGF-1 effects due to a modulation of the amplitude of the signal created by the specific ligand-receptor interaction.     

Targeting Insulin-Like Growth Factor 1 Receptor Inhibits Pancreatic Cancer Growth and Metastasis

Pancreatic cancer is one of the most lethal cancers. Increasing incidence and mortality indicates that there is still much lacking in detection and management of the disease. This is partly due to a lack of specific symptoms during early stages of the disease. Several growth factor receptors have been associated with pancreatic cancer. Here, we have investigated if an RNA interference approach targeted to IGF-IR could be effective and efficient against pancreatic cancer growth and metastasis. For that, we evaluated the effects of IGF-1R inhibition using small interfering RNA (siRNAs) on tumor growth and metastasis in HPAC and PANC-1 pancreatic cancer cell lines. We found that silencing IGF-1R inhibits pancreatic cancer growth and metastasis by blocking key signaling pathways such AKT/PI3K, MAPK, JAK/STAT and EMT. Silencing IGF-1R resulted in an anti-proliferative effect in PANC-1 and HPAC pancreatic cancer cell lines. Matrigel invasion, transwell migration and wound healing assays also revealed a role for IGF-1R in metastatic properties of pancreatic cancer. These results were further confirmed using Western blotting analysis of key intermediates involved in proliferation, epithelial mesenchymal transition, migration, and invasion. In addition, soft agar assays showed that silencing IGF-1R also blocks the colony forming capabilities of pancreatic cancer cells in vitro. Western blots, as well as, flow cytometric analysis revealed the induction of apoptosis in IGF-1R silenced cells. Interestingly, silencing IGF-1R also suppressed the expression of insulin receptor β. All these effects together significantly control pancreatic cancer cell growth and metastasis. To conclude, our results demonstrate the significance of IGF-1R in pancreatic cancer.     

Hydrogen peroxide inhibits activity of the IGF-1 receptor kinase

IGF-1 receptor (IGF1R) is a transmembrane tyrosine kinase, which is indispensable for cellular growth and differentiation. Using a recombinant GST-tagged cytosolic fragment of IGF1R (GST-IGFK), we now show that oxidation by low doses (50 muM) of hydrogen peroxide markedly inhibits maximum phosphate incorporation in autophosphorylation and substrate phosphorylation assays. A similar inhibition was observed on the activity of intact IGF1R after treatment of T-47D cells. These results are in sharp contrast to the positive influence of hydrogen peroxide on the highly homologous insulin receptor kinase, which was assayed for comparison. This reciprocal influence of physiologically relevant doses of hydrogen peroxide may have important implications on signal transduction of the closely related receptors for insulin and IGF-1.     

Insulin-like growth factor 1 receptor signaling regulates skin development and inhibits skin keratinocyte differentiation

The insulin-like growth factor 1 receptor (IGF-1R) is a multifunctional receptor that mediates signals for cell proliferation, differentiation, and survival. Genetic experiments showed that IGF-1R inactivation in skin results in a disrupted epidermis. However, because IGF-1R-null mice die at birth, it is difficult to study the effects of IGF-1R on skin. By using a combined approach of conditional gene ablation and a three-dimensional organotypic model, we demonstrate that IGF-1R-deficient skin cocultures show abnormal maturation and differentiation patterns. Furthermore, IGF-1R-null keratinocytes exhibit accelerated differentiation and decreased proliferation. Investigating the signaling pathway downstream of IGF-1R reveals that insulin receptor substrate 2 (IRS-2) overexpression compensates for the lack of IGF-1R, whereas IRS-1 overexpression does not. We also demonstrate that phosphatidylinositol 3-kinase and extracellular signal-regulated kinase 1 and 2 are involved in the regulation of skin keratinocyte differentiation and take some part in mediating the inhibitory signal of IGF-1R on differentiation. In addition, we show that mammalian target of rapamycin plays a specific role in mediating IGF-1R impedance of action on keratinocyte differentiation. In conclusion, these results reveal that IGF-1R plays an inhibitory role in the regulation of skin development and differentiation.     

Gefitinib resistance in HCC mahlavu cells: upregulation of CD133 expression, activation of IGF-1R signaling pathway, and enhancement of IGF-1R nuclear translocation

Hepatocellular carcinoma (HCC) is the major form of primary liver cancer which accounts for more than half million deaths annually worldwide. While the incidence of HCC is still on the rise, options of treatment are limited and the overall survival rate is poor. The acquisition of cancer drug resistance remains one of the key hurdles to successful treatment. Clearly, a thorough understanding of the underlying mechanisms is needed for new strategies to design novel treatments and/or to improve the current therapies. In the present study, we examined the expression of cancer stem cell (CSC) marker CD133, the activation of insulin-like growth factor 1 receptor (IGF-1R) signaling, and the nuclear translocation of IGF-1R in HCC Mahlavu cells under the treatment of gefitinib, a cancer drug that inhibits epidermal growth factor receptor (EGFR) pathway. Our results demonstrated that Mahlavu cells exhibited strong gefitinib resistance and the CD133 expression level was dramatically increased (from 3.88% to 32%) after drug treatment. In addition, the gefitinib treated cells displayed increased levels of phosphorylation in IGF-1R and Akt, indicating the intensified activation of this cancer-associated signaling pathway. Moreover, we revealed that IGF-1R underwent nuclear translocation in gefitinib treated cells using confocal microscopy. The IGF-1R nuclear translocation was enhanced under gefitinib treatment and appeared in a dose-dependent manner. Our findings suggest that increased IGF-1R nuclear translocation after gefitinib treatment may contribute to the drug resistance and IGF1-R activation, which might also associate with the upregulation of CD133 expression.