Academic literature on the topic 'Igf1r knockout'

Abstract Although TSH stimulates all aspects of thyroid physiology IGF-I signaling through a tyrosine kinase-containing transmembrane receptor exhibits a permissive impact on TSH action. To better understand the importance of the IGF-I receptor in the thyroid in vivo, we inactivated the Igf1r with a Tg promoter-driven Cre-lox system in mice. We studied male and female mice with thyroidal wild-type, Igf1r+/−, and Igf1r−/− genotypes. Targeted Igf1r inactivation did transiently reduce thyroid hormone levels and significantly increased TSH levels in both heterozygous and homozygous mice without affecting thyroid weight. Histological analysis of thyroid tissue with Igf1r inactivation revealed hyperplasia and heterogeneous follicle structure. From 4 months of age, we detected papillary thyroid architecture in heterozygous and homozygous mice. We also noted increased body weight of male mice with a homozygous thyroidal null mutation in the Igf1r locus, compared with wild-type mice, respectively. A decrease of mRNA and protein for thyroid peroxidase and increased mRNA and protein for IGF-II receptor but no significant mRNA changes for the insulin receptor, the TSH receptor, and the sodium-iodide-symporter in both Igf1r+/− and Igf1r−/− mice were detected. Our results suggest that the strong increase of TSH benefits papillary thyroid hyperplasia and completely compensates the loss of IGF-I receptor signaling at the level of thyroid hormones without significant increase in thyroid weight. This could indicate that the IGF-I receptor signaling is less essential for thyroid hormone synthesis but maintains homeostasis and normal thyroid morphogenesis.

Medulloblastoma (Med) is the most common malignant brain tumor in children. The role of ESR2 [estrogen receptor (ER)-β] in promoting Med growth was comprehensively examined in three in vivo models and human cell lines. In a novel Med ERβ-null knockout model developed by crossing Esr2−/− mice with cerebellar granule cell precursor specific Ptch1 conditional knockout mice, the tumor growth rate was significantly decreased in males and females. The absence of Esr2 resulted in increased apoptosis, decreased B-cell lymphoma 2 (BCL2), and IGF-1 receptor (IGF1R) expression, and decreased levels of active MAPKs (ERK1/2) and protein kinase B (AKT). Treatment of Med in Ptch1+/−Trp53−/− mice with the antiestrogen chemotherapeutic drug Faslodex significantly increased symptom-free survival, which was associated with increased apoptosis and decreased BCL2 and IGF1R expression and signaling. Similar effects were also observed in nude mice bearing D283Med xenografts. In vitro studies in human D283Med cells metabolically stressed by glutamine withdrawal found that 17β-estradiol and the ERβ selective agonist 2,3-bis(4-hydroxyphenyl)-propionitrile dose dependently protected Med cells from caspase-3-dependent cell death. Those effects were associated with increased phosphorylation of IGF1R, long-term increases in ERK1/2 and AKT signaling, and increased expression of IGF-1, IGF1R, and BCL2. Results of pharmacological experiments revealed that the cytoprotective actions of estradiol were dependent on ERβ and IGF1R receptor tyrosine kinase activity and independent of ERα and G protein-coupled estrogen receptor 1 (G protein coupled receptor 30). The presented results demonstrate that estrogen promotes Med growth through ERβ-mediated increases in IGF1R expression and activity, which induce cytoprotective mechanisms that decrease apoptosis.

ABSTRACT Ribosomal S6 kinases (S6Ks) have been depicted as critical effectors downstream of growth factor pathways, which play an important role in the regulation of protein synthesis by phosphorylating the ribosomal protein, S6. The goal of this study was to determine whether S6Ks regulate heart size, are critical for the induction of cardiac hypertrophy in response to a pathological or physiological stimulus, and whether S6Ks are critical downstream effectors of the insulin-like growth factor 1 (IGF1)-phosphoinositide 3-kinase (PI3K) pathway. For this purpose, we generated and characterized cardiac-specific S6K1 and S6K2 transgenic mice and subjected S6K1−/−, S6K2−/−, and S6K1−/− S6K2−/− mice to a pathological stress (aortic banding) or a physiological stress (exercise training). To determine the genetic relationship between S6Ks and the IGF1-PI3K pathway, S6K transgenic and knockout mice were crossed with cardiac-specific transgenic mice overexpressing the IGF1 receptor (IGF1R) or PI3K mutants. Here we show that overexpression of S6K1 induced a modest degree of hypertrophy, whereas overexpression of S6K2 resulted in no obvious cardiac phenotype. Unexpectedly, deletion of S6K1 and S6K2 had no impact on the development of pathological, physiological, or IGF1R-PI3K-induced cardiac hypertrophy. These studies suggest that S6Ks alone are not essential for the development of cardiac hypertrophy.

Abstract Despite progress in developing defined conditions for human embryonic stem cell (hESC) cultures, little is known about the cell-surface receptors that are activated under conditions supportive of hESC self-renewal. A simultaneous interrogation of 42 receptor tyrosine kinases (RTKs) in hESCs following stimulation with mouse embryonic fibroblast (MEF) conditioned medium (CM) revealed rapid and prominent tyrosine phosphorylation of insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R); less prominent tyrosine phosphorylation of epidermal growth factor receptor (EGFR) family members, including ERBB2 and ERBB3; and trace phosphorylation of fibroblast growth factor receptors. Intense IGF1R and IR phosphorylation occurred in the absence of MEF conditioning (NCM) and was attributable to high concentrations of insulin in the proprietary KnockOut Serum Replacer (KSR). Inhibition of IGF1R using a blocking antibody or lentivirus-delivered shRNA reduced hESC self-renewal and promoted differentiation, while disruption of ERBB2 signaling with the selective inhibitor AG825 severely inhibited hESC proliferation and promoted apoptosis. A simple defined medium containing an IGF1 analog, heregulin-1β (a ligand for ERBB2/ERBB3), fibroblast growth factor-2 (FGF2), and activin A supported long-term growth of multiple hESC lines. These studies identify previously unappreciated RTKs that support hESC proliferation and self-renewal, and provide a rationally designed medium for the growth and maintenance of pluripotent hESCs.

There is interest in targeting protein quality control pathways that ensure refolding or elimination of misfolded proteins to kill cancer cells. Osorio et al. identified a role for AIRAPL (an ortholog of AIP-1, which is involved in protein quality control pathways in Caenorhabditis elegans) in myeloproliferative neoplasms, a group of diseases in which the production of blood cells in the bone marrow is increased. Mice lacking Zfand2b (the gene encoding AIRAPL) developed hematological symptoms characteristic of myeloproliferative neoplasms, such as splenomegaly and expansion of various myeloid lineages. Western blot analysis of bone marrow from Zfand2b–/– mice revealed an increase in the total abundance and phosphorylation of insulin/insulin-like growth factor 1 receptor (IGF1R), a kinase that activates cell growth and proliferation. In HEK-293T cells, overexpression of AIRAPL reduced IGF1R abundance in a proteasome-dependent manner. Furthermore, exogenous AIRAPL interacted with pro-IGF1R rather than mature IGF1R, and knockout of endogenous AIRAPL resulted in an increase in the abundance of mature IGF1R, suggesting that AIRAPL triggered the proteasomal degradation of pro-IGF1R before it could undergo processing to the mature form. Igf1r haploinsufficiency in Zfand2b–/– mice or treatment of Zfand2b–/– mice with a kinase inhibitor of IGF1R prevented or attenuated the development of hematological symptoms seen in Zfand2b–/– mice. In addition, hematological symptoms were improved in mice that are a model of a specific type of myeloproliferative neoplasm upon treatment with an IGF1R kinase inhibitor or transduction with bone marrow cells from Igf1r+/+ mice (but not with that from Igf1r+/– mice). In samples from individuals with various types of myeloproliferative neoplasms, AIRAPL was not detected and both pro- and mature IGF1R were increased in abundance. Thus, AIRAPL limits the processing of IGF1R into its mature form in hematopoietic stem cells and thus the production of myeloid cells. These results also suggest that IGF1R inhibitors could be used in combination with existing therapies for myeloproliferative neoplasms (see LaFave and Levine). F. G. Osorio, C. Soria-Valles, O. Santiago-Fernández, T. Bernal, M. Mittelbrunn, E. Colado, F. Rodríguez, E. Bonzon-Kulichenko, J. Vázquez, M. Porta-de-la-Riva, J. Cerón, A. Fueyo, J. Li, A. R. Green, J. M. P. Freije, C. López-Otín, Loss of the proteostasis factor AIRAPL causes myeloid transformation by deregulating IGF-1 signaling. Nat. Med. 22, 91–96 (2016). [PubMed]L. M. LaFave, R. L. Levine, Targeting a regulator of protein homeostasis in myeloproliferative neoplasms. Nat. Med. 22, 20–21 (2016). [PubMed]

Hematopoietic stem cells (HSCs) are responsible for long-term maintenance and regeneration of the hematopoietic system. Loss of long-term (LT)-HSC function is a major contributor to decline in hematopoietic function with aging, leading to increased risk of infection, poor vaccination response, and increased susceptibility to hematologic malignancies. A number of LT-HSC-intrinsic alterations and LT-HSC-extrinsic changes in the bone marrow (BM) microenvironment have been associated with functional decline in aged LT-HSCs. Outstanding questions in the field are which of these mechanisms cause, rather than are a consequence of, physiological aging and what mechanism(s) represent key therapeutic targets to extend healthy LT-HSC function in older age? Previously, we took the novel approach of examining LT-HSC frequency and function in mice at a wide array of ages with the rationale that interventions to extend LT-HSC function will be most effective starting at or before the age of onset of functional hematopoietic decline. We found that canonical markers of LT-HSC aging significantly accumulate by middle age (9-12mo) in C57BL/6 mice, including increased phenotypic LT-HSC frequency, reduced regenerative capacity, myeloid lineage bias at both transcriptional and functional levels, increased gH2.AX staining, and loss of polarity of CDC42 and tubulin. Furthermore, we found by reciprocal transplantation studies of young LT-HSCs into middle-aged recipient mice and middle-aged LT-HSCs into young recipient mice that LT-HSC-extrinsic changes in the middle-aged BM microenvironment were necessary and sufficient to cause LT-HSC aging. By transcriptome analysis, we identified decreased IGF1 signaling in LT-HSCs as a candidate mechanism causing LT-HSC aging. Here, we systematically identify mesenchymal stromal cells (MSCs) as the major local producer of IGF1 in the BM of young mice and determine that this production is diminished by middle age. To evaluate the specific effect of MSC-produced IGF1 on LT-HSCs, we co-cultured LT-HSCs with Igf1conditional knockout MSCs, which was found to cause increased differentiation of LT-HSCs toward myeloid progenitor cells. In vivo, reduced IGF signaling in the BM microenvironment was modeled by transplantation of wild-type BM cells into Igf1 conditional knockout recipients, which phenocopied myeloid-biased hematopoiesis as observed in middle-aged mice. A similar myeloid-biased hematopoiesis phenotype was observed upon transplant of Igf1r conditional knockout LT-HSCs into wild-type recipients, supporting a model of direct communication between MSCs and LT-HSCs via IGF1 signaling. To determine whether restoration of IGF1 signaling had the capacity to rejuvenate middle-aged LT-HSCs, we applied short-term (24hr) in vitro treatment of recombinant IGF1. IGF1 treatment restored polarity of CDC42 and tubulin, decreased gH2.AX staining, increased in vivo regenerative capacity, and increased lymphoid differentiation potential of middle-aged LT-HSCs at the transcriptional and functional levels. Furthermore, increasing local IGF1 levels in the middle-aged BM microenvironment by transient intrafemoral injection (once per week for 4 wks) restored mature B cell frequency and decreased mature myeloid cell frequency in the BM and peripheral blood, rebalancing the composition of hematopoietic cells to that observed in young mice. At a mechanistic level, short-term IGF1 stimulation of middle-aged LT-HSCs was found to induce phosphorylation of IGF1R and the downstream signaling mediator AKT, and transcriptionally activate PI3K/AKT target genes. Open chromatin profiling by ATAC-seq revealed altered chromatin organization in middle-aged LT-HSCs in response to IGF1 stimulation, altering chromatin accessibility at promoters of genes enriched in intracellular and protein localization, regulation of transport and sequence-specific DNA binding, and enrichment of regulatory regions containing E2F2 binding sites. In summary, our data strongly support that decreased local production of IGF1 in the BM microenvironment causes many hallmarks of LT-HSC aging. We propose that restoration of local IGF1 signaling, or its downstream target pathways, represents an attractive prophylactic strategy for extending hematopoietic healthspan into older age. Disclosures Trowbridge: Fate Therapeutics: Patents & Royalties: patent license.

Abstract Insulin-like growth factors (IGFs) are critical regulators of cell growth, proliferation and survival. Their activities are mainly mediated through insulin-like growth factor 1 receptor (IGF1R). Both IGFs and IGF1R are commonly involved in human cancers, including leukemia. Thus, a better understanding of the role of IGF/IGF1R signaling in normal hematopoiesis will enhance our understanding of leukemogenesis. We showed previously a developmental stage-specific role of this pathway in regulating fetal (but not adult) megakaryocytic progenitors via interplay with GATA1 (Genes Dev. 24:1659-72). To determine whether IGF/IGF1R signaling also plays differential roles in fetal and adult hematopoiesis in general, we analysed Igf1r conditional knockout mice using Mx1-Cre and Vav-Cre for the adult stage, and Tie2-Cre for the fetal stage. Although both fetal and adult stages of hematopoiesis were not significantly perturbed, loss of Igf1r in fetal (but not in adult) hematopoietic cells led to a significant reduction in the number of CFUs in clonogenic assays. In adult Igf1r -null Lin-Sca-1+c-Kit+ (LSK) hematopoietic stem and progenitor cells (HSPCs), analysis of the long-term hematopoietic stem cell (LT-HSC), short-term HSC (ST-HSC) and multipotent progenitor (MPP) subsets of LSK cells revealed an increase in the percentage of LT-HSCs when compared to that of wild-type (WT) mice, consistent with a recent study (Nature. 500:345-9). These data suggest that IGF/IGF1R signaling may have a developmental stage-restricted role in fetal progenitors; in adults, this pathway may play a role in the transition from LT-HSCs to MPPs. Recently it was shown that estrogen signaling plays an important role in regulating proliferation of HSCs (Nature. 505:555-8). Since during development, both male and female fetuses are exposed to the same high level of maternal estrogen, whereas in adults, male and female hematopoietic cells are exposed to different levels of circulating estrogen, we hypothesized that adult (but not fetal) HSPCs from male and female mice might exhibit different phenotypes in response to Igf1r -loss. To test this, we analysed the adult LSK population by separating males and females. Interestingly, we found that in females, but not in males, the LSK population is significantly reduced upon Igf1r -loss. Conversely, a preliminary study in fetal liver CFU assay revealed that both Igf1r -null female and male fetuses exhibited a similar reduction in their CFUs compared to matched WT controls. To understand the female-specific role of IGF/IGF1R signaling in adult HSPCs, we analysed the cell cycle status of HSCs. Similar to the reported observation (Nature. 505:555-8), we found that in WT female adults, there were significantly more Ki67+ cycling LT-HSCs than those in males. Intriguingly, Igf1r -loss significantly reduced the percentage of cycling LT-HSCs in females to a level comparable to that of WT males, but had a neglectable effect on cycling LT-HSCs in males. Administration of estradiol (E2) revealed that in females, E2 injection led to an increase in the percentage of Ki67+ LT-HSCs and this increase was partially abolished when under the Igf1r- null background; in males, E2 injection also increased the percentage of Ki67+ LT-HSCs, although we did not observe a notable reduction in this population when under the Igf1r -null background. Overall, our data suggest that although IGF/IGF1R signaling is not essential for normal hematopoiesis, it may play a more important role under conditions (e.g., fetal development, pregnancy) when there is a higher demand for the output from the hematopoietic system; in particular, this pathway may play an important role in mediating the effect of estrogen on self-renewal and proliferation of HSPCs. Disclosures No relevant conflicts of interest to declare.

Abstract Glioblastoma (GBM) is composed of heterogeneous tumor cell populations including those with stem cell properties, termed glioma stem cells (GSCs). GSCs are innately less radiation sensitive than the tumor bulk and are believed to drive GBM formation and recurrence after repeated irradiation. However, it is unclear how GSCs adapt to escape the toxicity of repeated irradiation used in clinical practice. To identify important mediators of adaptive radioresistance, we generated radioresistant human and mouse GSCs by exposing them to repeat cycles of irradiation. Surviving subpopulations acquired strong radioresistance in vivo, which was accompanied by increased cell-cell adhesion, slower proliferation, an elevation of stemness properties and N-cadherin expression. Increasing N-cadherin expression rendered parental GSCs radioresistant, reduced their proliferation, and increased their stemness and intercellular adhesive properties. Conversely, radioresistant GSCs lost their acquired phenotypes upon CRISPR/Cas9-mediated knockout of N-cadherin. Mechanistically, elevated N-cadherin expression resulted in the accumulation of b-catenin at the cell surface, which suppressed Wnt/ b-catenin proliferative signaling, reduced neural differentiation, and protected against apoptosis through Clusterin secretion. Restoration of wild type N-cadherin, but not mutant N-cad lacking b-catenin binding region, led to induce radioresistance in N-cadherin knockout GSCs, indicating the importance of the binding between N-cadherin and b-catenin. We also demonstrated that N-cadherin upregulation was induced by radiation-induced IGF1 secretion, and the radiation resistance phenotype could be reverted with picropodophyllin (PPP), a clinically applicable blood-brain-barrier permeable IGF1 receptor inhibitor, supporting clinical translation. Moreover, the elevation of N-cad and Clusterin are related to prognosis of GBM in the TCGA dataset. In conclusion, our data indicate that IGF1R inhibitor can block the N-cadherin-mediated resistance pathway. Our study deepens our understanding of adaptive radioresistance during repeated irradiation in GBM, and validates the IGF1/N-cadherin/b-catenin/Clusterin signaling axis as a novel target for radio-sensitization, which has direct therapeutic applicability.

GH stimulates growth plate chondrogenesis and longitudinal bone growth directly at the growth plate. However, it is not clear yet whether these effects are entirely mediated by the local expression and action of IGF-1 and IGF-2. To determine whether GH has any IGF-independent growth-promoting effects, we generated TamCartIgf1rflox/flox mice. The systemic injection of tamoxifen in these mice postnatally resulted in the excision of the IGF-1 receptor (Igf1r) gene exclusively in the growth plate. TamCartIgf1rflox/flox tamoxifen-treated mice [knockout (KO) mice] and their Igf1rflox/flox control littermates (C mice) were injected for 4 weeks with GH. At the end of the 4-week period, the tibial growth and growth plate height of GH-treated KO mice were greater than those of untreated C or untreated KO mice. The systemic injection of GH increased the phosphorylation of Janus kinase 2 and signal transducer and activator of transcription 5B in the tibial growth plate of the C and KO mice. In addition, GH increased the mRNA expression of bone morphogenetic protein-2 and the mRNA expression and protein phosphorylation of nuclear factor-κB p65 in both C and KO mice. In cultured chondrocytes transfected with Igf1r small interfering RNA, the addition of GH in the culture medium significantly induced thymidine incorporation and collagen X mRNA expression. In conclusion, our findings demonstrate that GH can promote growth plate chondrogenesis and longitudinal bone growth directly at the growth plate, even when the local effects of IGF-1 and IGF-2 are prevented. Further studies are warranted to elucidate the intracellular molecular mechanisms mediating the IGF-independent, growth-promoting GH effects.