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.     

A novel cardiomyocyte-enriched microRNA, miR-378, targets insulin-like growth factor 1 receptor: implications in postnatal cardiac remodeling and cell survival

Postnatal cardiac remodeling is characterized by a marked decrease in the insulin-like growth factor 1 (IGF1) and IGF1 receptor (IGF1R) expression. The underlying mechanism remains unexplored. This study examined the role of microRNAs in postnatal cardiac remodeling. By expression profiling, we observed a 10-fold increase in miR-378 expression in 1-week-old neonatal mouse hearts compared with 16-day-old fetal hearts. There was also a 4-6-fold induction in expression of miR-378 in older (10 months) compared with younger (1 month) hearts. Interestingly, tissue distribution analysis identified miR-378 to be highly abundant in heart and skeletal muscles. In the heart, specific expression was observed in cardiac myocytes, which was inducible by a variety of stressors. Overexpression of miR-378 enhanced apoptosis of cardiomyocytes by direct targeting of IGF1R and reduced signaling in Akt cascade. The inhibition of miR-378 by its anti-miR protected cardiomyocytes against H(2)O(2) and hypoxia reoxygenation-induced cell death by promoting IGF1R expression and downstream Akt signaling cascade. Additionally, our data show that miR-378 expression is inhibited by IGF1 in cardiomyocytes. In tissues such as fibroblasts and fetal hearts, where IGF1 levels are high, we found either absent or significantly low miR-378 levels, suggesting an inverse relationship between these two factors. Our study identifies miR-378 as a new cardioabundant microRNA that targets IGF1R. We also demonstrate the existence of a negative feedback loop between miR-378, IGF1R, and IGF1 that is associated with postnatal cardiac remodeling and with the regulation of cardiomyocyte survival during stress.     

MicroRNA-21 Promotes Glioblastoma Tumorigenesis by Down-regulating Insulin-like Growth Factor-binding Protein-3 (IGFBP3)

Despite advances in surgery, imaging, chemotherapy, and radiation, patients with glioblastoma multiforme (GBM), the most common histological subtype of glioma, have an especially dismal prognosis; >70% of GBM patients die within 2 years of diagnosis. In many human cancers, the microRNA miR-21 is overexpressed, and accumulating evidence indicates that it functions as an oncogene. Here, we report that miR-21 is overexpressed in human GBM cell lines and tumor tissue. Moreover, miR-21 expression in GBM patient samples is inversely correlated with patient survival. Knockdown of miR-21 in GBM cells inhibited cell proliferation in vitro and markedly inhibited tumor formation in vivo. A number of known miR-21 targets have been identified previously. By microarray analysis, we identified and validated insulin-like growth factor (IGF)-binding protein-3 (IGFBP3) as a novel miR-21 target gene. Overexpression of IGFBP3 in glioma cells inhibited cell proliferation in vitro and inhibited tumor formation of glioma xenografts in vivo. The critical role that IGFBP3 plays in miR-21-mediated actions was demonstrated by a rescue experiment, in which IGFBP3 knockdown in miR-21KD glioblastoma cells restored tumorigenesis. Examination of tumors from GBM patients showed that there was an inverse relationship between IGFBP3 and miR-21 expression and that increased IGFBP3 expression correlated with better patient survival. Our results identify IGFBP3 as a novel miR-21 target gene in glioblastoma and suggest that the oncogenic miRNA miR-21 down-regulates the expression of IGFBP3, which acts as a tumor suppressor in human glioblastoma.     

The Reciprocal Regulation of γ-Synuclein and IGF-I Receptor Expression Creates a Circuit That Modulates IGF-I Signaling

Insulin-like growth factor (IGF) system plays important roles in carcinogenesis and maintenance of the malignant phenotype. Signaling through the IGF-I receptor (IGF-IR) has been shown to stimulate the growth and motility of a wide range of cancer cells. γ-Synuclein (SNCG) is primarily expressed in peripheral neurons but also overexpressed in various cancer cells. Overexpression of SNCG correlates with tumor progression. In the present study we demonstrated a reciprocal regulation of IGF-I signaling and SNCG expression. IGF-I induced SNCG expression in various cancer cells. IGF-IR knockdown or IGF-IR inhibitor repressed SNCG expression. Both phosphatidylinositol 3-kinase and mitogen-activated protein kinase were involved in IGF-I induction of SNCG expression. Interestingly, SNCG knockdown led to proteasomal degradation of IGF-IR, thereby decreasing the steady-state levels of IGF-IR. Silencing of SNCG resulted in a decrease in ligand-induced phosphorylation of IGF-IR and its downstream signaling components, including insulin receptor substrate (IRS), Akt, and ERK1/2. Strikingly, SNCG physically interacted with IGF-IR and IRS-2. Silencing of IRS-2 impaired the interaction between SNCG and IGF-IR. Finally, SNCG knockdown suppressed IGF-I-induced cell proliferation and migration. These data reveal that SNCG and IGF-IR are mutually regulated by each other. SNCG blockade may suppress IGF-I-induced cell proliferation and migration. Conversely, IGF-IR inhibitors may be of utility in suppressing the aberrant expression of SNCG in cancer cells and thereby block its pro-tumor effects.     

Multiple Roles of the SO42?/Cl?/OH? Exchanger Protein Slc26a2 in Chondrocyte Functions

Mutations in the SO₄²⁻/Cl⁻/OH⁻ exchanger Slc26a2 cause the disease diastrophic dysplasia (DTD), resulting in aberrant bone development and, therefore, skeletal deformities. DTD is commonly attributed to a lack of chondrocyte SO₄²⁻ uptake and proteoglycan sulfation. However, the skeletal phenotype of patients with DTD is typified by reduction in cartilage and osteoporosis of the long bones. Chondrocytes of patients with DTD are irregular in size and have a reduced capacity for proliferation and terminal differentiation. This raises the possibility of additional roles for Slc26a2 in chondrocyte function. Here, we examined the roles of Slc26a2 in chondrocyte biology using two distinct systems: mouse progenitor mesenchymal cells differentiated to chondrocytes and freshly isolated mouse articular chondrocytes differentiated into hypertrophic chondrocytes. Slc26a2 expression was manipulated acutely by delivery of Slc26a2 or shSlc26a2 with lentiviral vectors. We demonstrate that slc26a2 is essential for chondrocyte proliferation and differentiation and for proteoglycan synthesis. Slc26a2 also regulates the terminal stage of chondrocyte cell size expansion. These findings reveal multiple roles for Slc26a2 in chondrocyte biology and emphasize the importance of Slc26a2-mediated protein sulfation in cell signaling, which may account for the complex phenotype of DTD.     

A Crayfish Insulin-like-binding Protein: ANOTHER PIECE IN THE ANDROGENIC GLAND INSULIN-LIKE HORMONE PUZZLE IS REVEALED*

Across the animal kingdom, the involvement of insulin-like peptide (ILP) signaling in sex-related differentiation processes is attracting increasing attention. Recently, a gender-specific ILP was identified as the androgenic sex hormone in Crustacea. However, moieties modulating the actions of this androgenic insulin-like growth factor were yet to be revealed. Through molecular screening of an androgenic gland (AG) cDNA library prepared from the crayfish Cherax quadricarinatus, we have identified a novel insulin-like growth factor-binding protein (IGFBP) termed Cq-IGFBP. Based on bioinformatics analyses, the deduced Cq-IGFBP was shown to share high sequence homology with IGFBP family members from both invertebrates and vertebrates. The protein also includes a sequence determinant proven crucial for ligand binding, which according to three-dimensional modeling is assigned to the exposed outer surface of the protein. Recombinant Cq-IGFBP (rCq-IGFBP) protein was produced and, using a “pulldown” methodology, was shown to specifically interact with the insulin-like AG hormone of the crayfish (Cq-IAG). Particularly, using both mass spectral analysis and an immunological tool, rCq-IGFBP was shown to bind the Cq-IAG prohormone. Furthermore, a peptide corresponding to residues 23–38 of the Cq-IAG A-chain was found sufficient for in vitro recognition by rCq-IGFBP. Cq-IGFBP is the first IGFBP family member shown to specifically interact with a gender-specific ILP. Unlike their ILP ligands, IGFBPs are highly conserved across evolution, from ancient arthropods, like crustaceans, to humans. Such conservation places ILP signaling at the center of sex-related phenomena in early animal development.     

叶酸缺乏致胎鼠宫内发育迟缓及肝脏胰岛素生长因子 系统表达变化

目的：叶酸是一种水溶性B 族维生素,在体内氨基酸与核苷酸代谢中起重要作用, 是胎儿生长发育所必须的营养素。本文通 过建立叶酸缺乏的孕鼠模型,探讨叶酸缺乏对胎鼠宫内发育的影响,并研究胎鼠肝脏组织中胰岛素生长因子（IGF）系统的表达变 化。方法：雌性C57BL/6J 小鼠叶酸缺乏组6 只、正常对照组6 只,分别饲以不含叶酸和含2 mg 叶酸/kg 的纯合饲料。四周后与雄 鼠交配,于怀孕第13.5 天（13.5 dpc）对孕鼠剖腹取胎,观察和评价胎鼠发育指标,并对宫内发育迟缓（IUGR）比率进行统计。用 Real-time PCR 法检测胎鼠肝脏组织中胰岛素生长因子Ⅰ（IGFⅠ）、胰岛素生长因子Ⅰ受体（IGFⅠ R）、胰岛素生长因子Ⅱ（IGF Ⅱ）、胰岛素生长因子Ⅱ受体（IGFⅡR）、胰岛素生长因子结合蛋白1（IGFBP-1）和胰岛素生长因子结合蛋白3（IGFBP-3）mRNA的 相对表达水平。结果：叶酸缺乏组雌鼠合笼前每日体重增长量降低,13.5 dpc胎鼠吸收胎和死胎比率升高,胎重下降,IUGR 比率显 著升高,差异有统计学意义（P<0.05）;叶酸缺乏组胎鼠肝脏组织中IGFⅡ和IGFⅡR mRNA 的相对表达水平均低于正常对照组 （P<0.05）,IGFⅠ、IGFⅠR、IGFBP-1 和IGFBP-3 mRNA的相对表达水平两组间没有差异（P>0.05）。结论：叶酸缺乏会导致小鼠孕 中期胎鼠IUGR 比率升高及胎肝IGFⅡ和IGFⅡR mRNA 的表达水平降低,提示叶酸缺乏对IGF系统基因的调控,可能与胎鼠IUGR 发生机制有关。     

Selective Disruption of Insulin-like Growth Factor-1 (IGF-1) Signaling via Phosphoinositide-dependent Kinase-1 Prevents the Protective Effect of IGF-1 on Human Cancer Cell Death

Insulin-like growth factor-1 (IGF-1) signaling system exerts a broad antiapoptotic function and plays a crucial role in resistance to anticancer therapies. Exposure of MCF-7 breast cancer cells to IGF-1 rapidly and transiently induced tyrosine phosphorylation and activation of phosphoinositide-dependent kinase-1 (PDK1). This was paralleled by Akt/protein kinase B and protein kinase C-ζ phosphorylation, at Thr³⁰⁸ and Thr⁴¹⁰, respectively. IGF-1 treatment also enhanced PDK1 interaction with IGF-1 receptor (IGF-1R) in intact MCF-7 cells. Pulldown assays revealed that PDK1 bound IGF-1R in vitro and that the region encompassing amino acids 51–359 of PDK1 was necessary for the interaction. Synthetic peptides corresponding to IGF-1R C terminus amino acids 1295–1337 (C43) and to PDK1 amino acids 114–141 reduced in vitro IGF-1R/PDK1 interaction in a concentration-dependent manner. Loading of fluoresceinated-C43 (fluorescein isothiocyanate (FITC)-C43) into MCF-7 cells significantly reduced IGF-1R/PDK1 interaction and phosphorylation of PDK1 substrates. Moreover, FITC-C43 intracellular loading reverted the protective effect of IGF-1 on growth factor deprivation-induced cell death. Finally, the inhibition of IGF-1R/PDK1 interaction and signaling by FITC-C43 was accompanied by 2-fold enhanced killing capacity of cetuximab in human GEO colon adenocarcinoma cells and was sufficient to restore cell death in cetuximab-resistant cell clones. Thus, disruption of PDK1 interaction with IGF-1R reduces IGF-1 survival effects in cancer cells and may enhance cell death by anticancer agents.     

Genetic analysis of type-1 insulin-like growth factor receptor signaling through insulin receptor substrate-1 and -2 in pancreatic beta cells

Signaling by receptor tyrosine kinases regulates pancreatic β cell function. Inactivation of insulin receptor (InsR), IGF1 receptor (Igf1r), or Irs1 in β cells impairs insulin secretion. Conversely, Irs2 ablation impairs β cell replication. In this study, we examined aspects of the Igf1r regulatory signaling cascade in β cells. To examine genetically the involvement of Irs1 and Irs2 in Igf1r signaling, we generated double mutant mice lacking Igf1r specifically in pancreatic β cells in an Irs1- or Irs2-null background. We show that Igf1r/Irs1 double mutants do not differ phenotypically from Irs1 single mutants and exhibit hyperinsulinemia, while maintaining normal β cell mass and glucose tolerance. In contrast, lack of Igf1r function in β cells aggravates the consequences of Irs2 ablation in double mutants and results in lethal diabetes by 6 weeks of age. This additivity of phenotypic manifestations indicates that Irs2 serves a pathway that is largely independent of Igf1r signaling. Consistent with the view that the latter is the InsR pathway, we show that combined β cell-specific knock-out of both Insr and Igf1r results in a phenocopy of double mutants lacking Igf1r and Irs2. We conclude that Igf1r signals primarily through Irs1 and affects insulin secretion, whereas β cell proliferation is mainly regulated by InsR using Irs2 as a downstream signaling effector. The insulin and IGF pathways appear to control β cell functions independently and selectively.     

A Novel Glycoengineered Bispecific Antibody Format for Targeted Inhibition of Epidermal Growth Factor Receptor (EGFR) and Insulin-like Growth Factor Receptor Type I (IGF-1R) Demonstrating Unique Molecular Properties

In the present study, we have developed a novel one-arm single chain Fab heterodimeric bispecific IgG (OAscFab-IgG) antibody format targeting the insulin-like growth factor receptor type I (IGF-1R) and the epidermal growth factor receptor (EGFR) with one binding site for each target antigen. The bispecific antibody XGFR is based on the “knob-into-hole” technology for heavy chain heterodimerization with one heavy chain consisting of a single chain Fab to prevent wrong pairing of light chains. XGFR was produced with high expression yields and showed simultaneous binding to IGF-1R and EGFR with high affinity. Due to monovalent binding of XGFR to IGF-1R, IGF-1R internalization was strongly reduced compared with the bivalent parental antibody, leading to enhanced Fc-mediated cellular cytotoxicity. To further increase immune effector functions triggered by XGFR, the Fc portion of the bispecific antibody was glycoengineered, which resulted in strong antibody-dependent cell-mediated cytotoxicity activity. XGFR-mediated inhibition of IGF-1R and EGFR phosphorylation as well as A549 tumor cell proliferation was highly effective and was comparable with a combined treatment with EGFR (GA201) and IGF-1R (R1507) antibodies. XGFR also demonstrated potent anti-tumor efficacy in multiple mouse xenograft tumor models with a complete growth inhibition of AsPC1 human pancreatic tumors and improved survival of SCID beige mice carrying A549 human lung tumors compared with treatment with antibodies targeting either IGF-1R or EGFR. In summary, we have applied rational antibody engineering technology to develop a heterodimeric OAscFab-IgG bispecific antibody, which combines potent signaling inhibition with antibody-dependent cell-mediated cytotoxicity induction and results in superior molecular properties over two established tetravalent bispecific formats.     

Pregnancy-associated Plasma Protein A (PAPP-A) Modulates the Early Developmental Rate in Zebrafish Independently of Its Proteolytic Activity

Pregnancy-associated plasma protein-A (PAPP-A) is a large metalloproteinase specifically cleaving insulin-like growth factor (IGF) binding proteins, causing increased IGF bioavailability and, hence, local regulation of IGF receptor activation. We have identified two highly conserved zebrafish homologs of the human PAPP-A gene. Expression of zebrafish Papp-a, one of the two paralogs, begins during gastrulation and persists throughout the first week of development, and analyses demonstrate highly conserved patterns of expression between adult zebrafish, humans, and mice. We show that the specific knockdown of zebrafish papp-a limits the developmental rate beginning during gastrulation without affecting the normal patterning of the embryo. This phenotype is different from those resulting from deficiency of Igf receptor or ligand in zebrafish, suggesting a function of Papp-a outside of the Igf system. Biochemical analysis of recombinant zebrafish Papp-a demonstrates conservation of proteolytic activity, specificity, and the intrinsic regulatory mechanism. However, in vitro transcribed mRNA, which encodes a proteolytically inactive Papp-a mutant, recues the papp-a knockdown phenotype as efficiently as wild-type Papp-a. Thus, the developmental phenotype of papp-a knockdown is not a consequence of lacking Papp-a proteolytic activity. We conclude that Papp-a possesses biological functions independent of its proteolytic activity. Our data represent the first evidence for a non-proteolytic function of PAPP-A.     

Biochemical Characterization of Individual Human Glycosylated pro-Insulin-like Growth Factor (IGF)-II and big-IGF-II Isoforms Associated with Cancer

Insulin-like growth factor II (IGF-II) is a major embryonic growth factor belonging to the insulin-like growth factor family, which includes insulin and IGF-I. Its expression in humans is tightly controlled by maternal imprinting, a genetic restraint that is lost in many cancers, resulting in up-regulation of both mature IGF-II mRNA and protein expression. Additionally, increased expression of several longer isoforms of IGF-II, termed “pro” and “big” IGF-II, has been observed. To date, it is ambiguous as to what role these IGF-II isoforms have in initiating and sustaining tumorigenesis and whether they are bioavailable. We have expressed each individual IGF-II isoform in their proper O-glycosylated format and established that all bind to the IGF-I receptor and both insulin receptors A and B, resulting in their activation and subsequent stimulation of fibroblast proliferation. We also confirmed that all isoforms are able to be sequestered into binary complexes with several IGF-binding proteins (IGFBP-2, IGFBP-3, and IGFBP-5). In contrast to this, ternary complex formation with IGFBP-3 or IGFBP-5 and the auxillary protein, acid labile subunit, was severely diminished. Furthermore, big-IGF-II isoforms bound much more weakly to purified ectodomain of the natural IGF-II scavenging receptor, IGF-IIR. IGF-II isoforms thus possess unique biological properties that may enable them to escape normal sequestration avenues and remain bioavailable in vivo to sustain oncogenic signaling.     

Androgen Receptor Splice Variant AR3 Promotes Prostate Cancer via Modulating Expression of Autocrine/Paracrine Factors

Deregulation of androgen receptor (AR) splice variants has been implicated to play a role in prostate cancer development and progression. To understand their functions in prostate, we established a transgenic mouse model (AR3Tg) with targeted expression of the constitutively active and androgen-independent AR splice variant AR3 (a.k.a. AR-V7) in prostate epithelium. We found that overexpression of AR3 modulates expression of a number of tumor-promoting autocrine/paracrine growth factors (including Tgfβ2 and Igf1) and expands prostatic progenitor cell population, leading to development of prostatic intraepithelial neoplasia. In addition, we showed that some epithelial-mesenchymal transition-associated genes are up-regulated in AR3Tg prostates, suggesting that AR3 may antagonize AR activity and halt the differentiation process driven by AR and androgen. This notion is supported by our observations that the number of Ck5⁺/Ck8⁺ intermediate cells is increased in AR3Tg prostates after castration, and expression of AR3 transgene in these intermediate cells compromises prostate epithelium regeneration upon androgen replacement. Our results demonstrate that AR3 is a driver of prostate cancer, at least in part, through modulating multiple tumor-promoting autocrine/paracrine factors.     

Insulin-like Growth Factor I (IGF-I)-induced Chronic Gliosis and Retinal Stress Lead to Neurodegeneration in a Mouse Model of Retinopathy

Insulin-like growth factor I (IGF-I) exerts multiple effects on different retinal cell types in both physiological and pathological conditions. Despite the growth factor''s extensively described neuroprotective actions, transgenic mice with increased intraocular levels of IGF-I showed progressive impairment of electroretinographic amplitudes up to complete loss of response, with loss of photoreceptors and bipolar, ganglion, and amacrine neurons. Neurodegeneration was preceded by the overexpression of genes related to retinal stress, acute-phase response, and gliosis, suggesting that IGF-I altered normal retinal homeostasis. Indeed, gliosis and microgliosis were present from an early age in transgenic mice, before other alterations occurred, and were accompanied by signs of oxidative stress and impaired glutamate recycling. Older mice also showed overproduction of pro-inflammatory cytokines. Our results suggest that, when chronically increased, intraocular IGF-I is responsible for the induction of deleterious cellular processes that can lead to neurodegeneration, and they highlight the importance that this growth factor may have in the pathogenesis of conditions such as ischemic or diabetic retinopathy.