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BACKGROUND - In glioblastoma (GBM), Id1 serves as a functional marker for self-renewing cancer stem-like cells. We investigated the mechanism by which cyclooxygenase-2 (Cox-2)-derived prostaglandin E2 (PGE2) induces Id1 and increases GBM self-renewal and radiation resistance.
METHODS - Mouse and human GBM cells were stimulated with dimethyl-PGE2 (dmPGE2), a stabilized form of PGE2, to test for Id1 induction. To elucidate the signal transduction pathway governing the increase in Id1, a combination of short interfering RNA knockdown and small molecule inhibitors and activators of PGE2 signaling were used. Western blotting, quantitative real-time (qRT)-PCR, and chromatin immunoprecipitation assays were employed. Sphere formation and radiation resistance were measured in cultured primary cells. Immunohistochemical analyses were carried out to evaluate the Cox-2-Id1 axis in experimental GBM.
RESULTS - In GBM cells, dmPGE2 stimulates the EP4 receptor leading to activation of ERK1/2 MAPK. This leads, in turn, to upregulation of the early growth response1 (Egr1) transcription factor and enhanced Id1 expression. Activation of this pathway increases self-renewal capacity and resistance to radiation-induced DNA damage, which are dependent on Id1.
CONCLUSIONS - In GBM, Cox-2-derived PGE2 induces Id1 via EP4-dependent activation of MAPK signaling and the Egr1 transcription factor. PGE2-mediated induction of Id1 is required for optimal tumor cell self-renewal and radiation resistance. Collectively, these findings identify Id1 as a key mediator of PGE2-dependent modulation of radiation response and lend insight into the mechanisms underlying radiation resistance in GBM patients.
© The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: email@example.com.
Transcriptional regulators are recurrently altered through translocations, deletions, or aberrant expression in acute myeloid leukemia (AML). Although critically important in leukemogenesis, the underlying pathogenetic mechanisms they trigger remain largely unknown. Here, we identified that Id1 (inhibitor of DNA binding 1) plays a pivotal role in acute myeloid leukemogenesis. Using genetically modified mice, we found that loss of Id1 inhibited t(8;21) leukemia initiation and progression in vivo by abrogating protein kinase B (AKT)1 activation, and that Id1 interacted with AKT1 through its C terminus. An Id1 inhibitor impaired the in vitro growth of AML cells and, when combined with an AKT inhibitor, triggered even greater apoptosis and growth inhibition, whereas normal hematopoietic stem/progenitor cells were largely spared. We then performed in vivo experiments and found that the Id1 inhibitor significantly prolonged the survival of t(8;21)(+) leukemic mice, whereas overexpression of activated AKT1 promoted leukemogenesis. Thus, our results establish Id1/Akt1 signaling as a potential therapeutic target in t(8;21) leukemia.
© 2015 by The American Society of Hematology.
Valvular Interstitial Cells (VICs) are a common substrate for congenital and adult heart disease yet the signaling mechanisms governing their formation during early valvulogenesis are incompletely understood. We developed an unbiased strategy to identify genes important in endocardial epithelial-to-mesenchymal transformation (EMT) using a spatial transcriptional profile. Endocardial cells overlaying the cushions of the atrioventricular canal (AVC) and outflow tract (OFT) undergo an EMT to yield VICs. RNA sequencing (RNA-seq) analysis of gene expression between AVC, OFT, and ventricles (VEN) isolated from chick and mouse embryos at comparable stages of development (chick HH18; mouse E11.0) was performed. EMT occurs in the AVC and OFT cushions, but not VEN at this time. 198 genes in the chick (n=1) and 105 genes in the mouse (n=2) were enriched 2-fold in the cushions. Gene regulatory networks (GRN) generated from cushion-enriched gene lists confirmed TGFβ as a nodal point and identified NF-κB as a potential node. To reveal previously unrecognized regulators of EMT four candidate genes, Hapln1, Id1, Foxp2, and Meis2, and a candidate pathway, NF-κB, were selected. In vivo spatial expression of each gene was confirmed by in situ hybridization and a functional role for each in endocardial EMT was determined by siRNA knockdown in a collagen gel assay. Our spatial-transcriptional profiling strategy yielded gene lists which reflected the known biology of the system. Further analysis accurately identified and validated previously unrecognized novel candidate genes and the NF-κB pathway as regulators of endocardial cell EMT in vitro.
Copyright © 2013 Elsevier Ltd. All rights reserved.
The Id family of genes encodes negative regulators of basic helix-loop-helix transcription factors and has been implicated in diverse cellular processes such as proliferation, apoptosis, differentiation, and migration. However, the specific role of Id1 in lung injury has not been investigated. Bleomycin has been widely used to generate animal models of acute lung injury and fibrogenesis. In this study we found that, on bleomycin challenge, Id1 expression was significantly up-regulated in the lungs, predominantly in endothelial cells, as revealed by double immunolabeling and quantitative flow cytometric analysis. Mice with Id1 loss-of-function (Id1(-/-)) displayed increased vascular permeability and endothelial apoptosis in the lungs after bleomycin-induced injury. Cultured Id1(-/-) lung microvascular endothelial cells also showed decreased survival when exposed to bleomycin. We detected a decrease in the level of Bcl-2, a primary anti-apoptotic protein, in Id1(-/-) endothelial cells, suggesting that down-regulated Bcl-2 may promote endothelial apoptosis in the lung. Therefore, we propose that Id1 plays a crucial role in promoting endothelial survival in the adult lung on injury. In addition, bleomycin-exposed Id1(-/-) mice showed increased lung collagen accumulation and fibrogenesis, suggesting that Id1 up-regulation in the lung may play a critical role in lung homeostasis.
Longstanding diabetes causes renal injury with early dropout of podocytes, albuminuria, glomerular and tubulointerstitial fibrosis, and progressive renal failure. The renal pathology seems to be driven, in part, by TGF-beta and is associated with a loss of renal bone morphogenic protein-7 (BMP-7) expression. Here, the hypothesis that maintenance of renal (especially podocyte) BMP-7 by transgenic expression reduces diabetic renal injury was tested. Diabetic mice that expressed the phosphoenolpyruvate carboxykinase promoter-driven BMP-7 transgene and nondiabetic, transgenic mice as well as diabetic and nondiabetic wild-type controls were studied for up to 1 yr. Transgenic expression of BMP-7 in glomerular podocytes and proximal tubules prevents podocyte dropout and reductions in nephrin levels in diabetic mice. Maintenance of BMP-7 also reduces glomerular fibrosis and interstitial collagen accumulation as well as collagen I and fibronectin expression. Diabetic wild-type mice develop progressive albuminuria, which is substantially reduced in transgenic mice. These effects of the BMP-7 transgene occur without changing renal TGF-beta levels. It is concluded that maintenance of renal BMP-7 during the evolution of diabetic nephropathy reduces diabetic renal injury, especially podocyte dropout. The findings also establish a role for endogenous glomerular BMP-7 as an autocrine regulator of podocyte integrity in vivo.
Pancreatic beta-cell-type-specific transcription of the insulin gene is principally regulated by a single cis-acting DNA sequence element, termed the insulin control element (ICE), which is found within the 5'-flanking region of the gene. The ICE activator is a heteromeric complex composed of an islet alpha/beta-cell-specific factor associated with the ubiquitously distributed E2A-encoded proteins (E12, E47, and E2-5). We describe the isolation and characterization of a cDNA for a protein present in alpha and beta cells, termed INSAF for insulin activator factor, which binds to and activates ICE-mediated expression. INSAF was isolated from a human insulinoma cDNA library. Transfection experiments demonstrated that INSAF activates ICE expression in insulin-expressing cells but not in non-insulin-expressing cells. Cotransfection experiments showed that activation by INSAF was inhibited by Id, a negative regulator of basic helix-loop-helix (bHLH) protein function. INSAF was also shown to associate in vitro with the bHLH protein E12. In addition, affinity-purified INSAF antiserum abolished the formation of the activator-specific ICE-binding complex. Immunohistochemical studies indicate that INSAF is restricted in terms of its expression pattern, in that INSAF appears to be detected only within the nuclei of islet pancreatic alpha and beta cells. All of these data are consistent with the proposal that INSAF is either part of the ICE activator or is antigenically related to the specific activator required for insulin gene transcription.