scholarly journals FPN1, Targeted By Mir-17-5p, Regulates Cellular Iron Homeostasis Via activating Nrf2/Mir-17-5p/FPN1 Axis in Preclinical Model of Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5616-5616
Author(s):  
Yuanyuan Kong ◽  
Shuaikang Chang ◽  
Liangning Hu ◽  
Gege Chen ◽  
Yi Tao ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Cell Growth ◽  
Iron Homeostasis ◽  
Intracellular Iron ◽  
Myeloma Cells ◽  
Cellular Iron ◽  
Public Datasets ◽  
Cellular Iron Homeostasis

Abstract Abstract Multiple myeloma (MM) is a hematologic malignancy with lytic bone lessions, including dysregulation of iron homeostasis. Ferroportin (FPN1), the only known cellular iron exporter, has been demonstrated to have a dysregulated expression and to be associated with poor clinical outcome. However, the detailed mechanism of FPN1 in iron transportion is not clear. Our current study showed that miR-17-5p was another new FPN1 microRNA target, which had a negative correlation with FPN1 expression level in MM. We also identified that nuclear factor erythroid 2-related factor 2 (Nrf2) induced the transcription of miR-17-5p, which in turn suppressed the protein level of FPN1. Moreover, chromatin immunopreciptation (ChIP) and luciferase reporter assay revealed that Nrf2 directly inhibited the transcription of FPN1. Here, we investigated the mechanism of unbalanced Nrf2/miR-17-5p/FPN1 axis and CRISPR-mediated knockout of FPN1 function involved in iron metabolism of MM. To investigate the potential role of miR-17-5p in MM, several public datasets were analyzed, and we identified that miR-17-5p was significantly overexpressed in the progression of MM. We next examined the functional effect on cell proliferation and apoptosis, ARP1 and OCI-MY5 cells were transiently transfected with miR-17-5p mimics or inhibitors. The experiment revealed that miR-17-5p promoted proliferation, cell cycle and apoptosis resistance both in vitro and in vivo. Besides, we explored whether FPN1 could rescue the effect on cells growth by overexpressing FPN1 in MM cells co-transfecting with miR-17-5p mimics or scramble. Our results showed that FPN1 restoration partially induced an inhibitory effect on myeloma cells, confirming that miR-17-5p accumulation favored cell growth and survival by targeting FPN1. Furthermore, to investigate the regulatory mechanism of FPN1 in iron transportion, several transcription factor targeting predication algorithms were exploited, and we revealed that the FPN1 and miR-17-5p common target gene Nrf2, was significantly relative overexpressed in myeloma. We also analyzed the MM patient public datasets, including paired samples and different stages of disease, and all showed Nrf2 overexpression in relapsed MM with poor prognosis. Luciferase activity analysis verified that FPN1 was significantly inhibited when co-transfected, including truncated bodies of FPN1 promotor sequence. Meanwhile, ChIP-PCR showed that there was an interaction between Nrf2 and FPN1. Additionally, the direct interactions of Nrf2 with the miR-17-5p promoter were also confirmed, suggesting that Nrf2 bound to the miR-17-5p promoter region, thus regulating the transcription of miR-17-5p. These data indicateed that Nrf2 activation could modulate FPN1 levels directly or through miR-17-5p. To more comprehensively evaluate FPN1 in regulating iron exportation in MM, we introduced single guide (sg) RNA targeting FPN1 into MM cells stably expressing Cas9. Of note, CRISPR-mediated FPN1 knockout promoted the growth and increased intracellular iron levels. In addition, to test the requirement of iron for the growth of myeloma, two sgFPN1 depleted MM cells were treated with iron supplement Fecl3, or the iron chelator deferoxamine (DFO). Fecl3 had significantly increased intracellular iron while the effect treated with DFO was opposite. Besides, MM cell proliferation was significantly inhibited by DFO treatment, further confirming the role of intracellular iron in MM cell growth. We also analyzed the mRNA levels of signature genes involved in maintaining cellular iron homeostasis and the level of reactive oxygen species (ROS) generation during the iron-deficient or iron-abundant state, and our results showed that these genes involved in iron acquisition or iron storage were differentially expressed and influenced intracellular iron availability and ROS levels. In conclusion, our data suggested that the miR-17-5p/FPN1 axis induced by Nrf2 regulated intracellular liable iron pool, thus providing surplus iron for metabolic processes like DNA synthesis, the proliferation and growth of myeloma cells. Understanding the mechanisms on iron metabolism is important to develop new therapeutic strategies for clinical application to the treatment of MM. Funding This study was supported by grants from the National Natural Science Foundation of China (Nos. 81570190; 81670194 and 81529001). Disclosures No relevant conflicts of interest to declare.

Overexpression of mitochondrial ferritin causes cytosolic iron depletion and changes cellular iron homeostasis

Blood ◽  
2005 ◽  
Vol 105 (5) ◽  
pp. 2161-2167 ◽  
Author(s):  
Guangjun Nie ◽  
Alex D. Sheftel ◽  
Sangwon F. Kim ◽  
Prem Ponka
Keyword(s):  
Iron Homeostasis ◽  
Rna Binding ◽  
Binding Activity ◽  
Intracellular Iron ◽  
Free Radical Damage ◽  
Iron Regulatory Proteins ◽  
Cellular Iron ◽  
Cellular Iron Uptake ◽  
A Cell ◽  
Cellular Iron Homeostasis

AbstractCytosolic ferritin sequesters and stores iron and, consequently, protects cells against iron-mediated free radical damage. However, the function of the newly discovered mitochondrial ferritin (MtFt) is unknown. To examine the role of MtFt in cellular iron metabolism, we established a cell line that stably overexpresses mouse MtFt under the control of a tetracycline-responsive promoter. The overexpression of MtFt caused a dose-dependent iron deficiency in the cytosol that was revealed by increased RNA-binding activity of iron regulatory proteins (IRPs) along with an increase in transferrin receptor levels and decrease in cytosolic ferritin. Consequently, the induction of MtFt resulted in a dramatic increase in cellular iron uptake from transferrin, most of which was incorporated into MtFt. The induction of MtFt caused a shift of iron from cytosolic ferritin to MtFt. In addition, iron inserted into MtFt was less available for chelation than that in cytosolic ferritin and the expression of MtFt was associated with decreased mitochondrial and cytosolic aconitase activities, the latter being consistent with the increase in IRP-binding activity. In conclusion, our results indicate that overexpression of MtFt causes a dramatic change in intracellular iron homeostasis and that shunting iron to MtFt likely limits its availability for active iron proteins.

The Relationship of Myeloma Cells, Stromal Cells and Monocytes Under Bone Marrow Microenvironment

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4987-4987
Author(s):  
Hiroshi Ikeda ◽  
Yuka Aoki ◽  
Nasanori Nojima ◽  
Hiroshi Yasui ◽  
Toshiaki Hayashi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Myeloma Cells ◽  
Growth And Survival

Abstract Abstract 4987 The Bone marrow (BM) microenvironment plays crucial role in pathogenesis of Multiple myeloma(MM). Myeloma cells contacts with bone marrow stromal cells (BMSCs), which secrete factors/cytokines, promoting tumor cell growth and survival. Paracrine secretion of cytokines(i. e., interleukin-6 (IL-6) insulin-like growth factor-1, inflammatory protein-1a) in BM stromal cells promotes multiple myeloma cell proliferation and protects against drug-induced cytotoxicity. These cytokines provide stimulatory signals for multiple myeloma growth and survival. Bone involvement is a common feature in MM patient, solid and hematologic cancers. MM localizes to the bone in nearly all patients ranges between 40% and 75%. Disease-related skeletal complications result in significant morbidity due to pain, pathologic fractures and spinal cord compression. The bone microenvironment creates a supportive niche for tumor growth. Osteoclasts and bone marrow stromal cells, along with extracellular matrix and cytokines stimulate tumor cell proliferation and confer chemoresistance. Therefore, the reciprocal interactions between tumor cells, osteoclasts, osteoblasts, and bone marrow stromal cells present an important. In current study, monocyte can directly promote mesenchymal stem cells osteogenic differentiation through cell contact interactions, thus resulting in the production of osteogenic factors by the monocytes. This mechanism is mediated by the activation of STAT3 signaling pathway in the mesechymal stem cells that leads to the upregulation of Osteoblasts-associated genes such as Runx2 and alkaline phosphatase (ALP), and the down-regulation of inhibitors such as DKK1 to drive the differentiation of mesechymal stem cells into osteoblasts. In this study, we examined the role of monocyte, component of BM cells, as a potential niche component that supports myeloma cells. We investigated the proliferation of MM cell lines cultured alone or co-cultured with BM stromal cells, monocytes, or a combination of BM stromal cells and monocytes. Consistently, we observed increased proliferation of MM cell lines in the presence of either BM stromal cells or monocytes compared to cell line-only control. Furthermore, the co-culture of BM stromal cells plus monocytes induced the greatest degree of proliferation of myeloma cells. In addition to increased proliferation, BMSCs and monocytes decreased the rate of apoptosis of myeloma cells. Our results therefore suggest that highlights the role of monocyte as an important component of the BM microenvironment. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Direct Activation of Genes Involved in Intracellular Iron Use by the Yeast Iron-Responsive Transcription Factor Aft2 without Its Paralog Aft1

2005 ◽  
Vol 25 (15) ◽  
pp. 6760-6771 ◽  
Author(s):  
Maïté Courel ◽  
Sylvie Lallet ◽  
Jean-Michel Camadro ◽  
Pierre-Louis Blaiseau
Keyword(s):  
Iron Homeostasis ◽  
Regulatory Elements ◽  
Intracellular Iron ◽  
Transcription Analysis ◽  
Dna Microarray Data ◽  
Cellular Iron ◽  
Surface Iron ◽  
Transcription Activators ◽  
Responsive Element ◽  
Cellular Iron Homeostasis

ABSTRACT The yeast Saccharomyces cerevisiae contains a pair of paralogous iron-responsive transcription activators, Aft1 and Aft2. Aft1 activates the cell surface iron uptake systems in iron depletion, while the role of Aft2 remains poorly understood. This study compares the functions of Aft1 and Aft2 in regulating the transcription of genes involved in iron homeostasis, with reference to the presence/absence of the paralog. Cluster analysis of DNA microarray data identified the classes of genes regulated by Aft1 or Aft2, or both. Aft2 activates the transcription of genes involved in intracellular iron use in the absence of Aft1. Northern blot analyses, combined with chromatin immunoprecipitation experiments on selected genes from each class, demonstrated that Aft2 directly activates the genes SMF3 and MRS4 involved in mitochondrial and vacuolar iron homeostasis, while Aft1 does not. Computer analysis found different cis-regulatory elements for Aft1 and Aft2, and transcription analysis using variants of the FET3 promoter indicated that Aft1 is more specific for the canonical iron-responsive element TGCACCC than is Aft2. Finally, the absence of either Aft1 or Aft2 showed an iron-dependent increase in the amount of the remaining paralog. This may provide additional control of cellular iron homeostasis.

HFE Downregulates Iron Uptake From Transferrin and Induces Iron-Regulatory Protein Activity in Stably Transfected Cells

Blood ◽  
1999 ◽  
Vol 94 (11) ◽  
pp. 3915-3921 ◽  
Author(s):  
H.D. Riedel ◽  
M.U. Muckenthaler ◽  
S.G. Gehrke ◽  
I. Mohr ◽  
K. Brennan ◽  
...  
Keyword(s):  
Transferrin Receptor ◽  
Iron Uptake ◽  
Iron Homeostasis ◽  
Hereditary Hemochromatosis ◽  
Regulatory Protein ◽  
Intracellular Iron ◽  
Iron Storage ◽  
Iron Regulatory Proteins ◽  
Cellular Iron

Hereditary hemochromatosis (HH) is a common autosomal-recessive disorder of iron metabolism. More than 80% of HH patients are homozygous for a point mutation in a major histocompatibility complex (MHC) class I type protein (HFE), which results in a lack of HFE expression on the cell surface. A previously identified interaction of HFE and the transferrin receptor suggests a possible regulatory role of HFE in cellular iron absorption. Using an HeLa cell line stably transfected with HFE under the control of a tetracycline-sensitive promoter, we investigated the effect of HFE expression on cellular iron uptake. We demonstrate that the overproduction of HFE results in decreased iron uptake from diferric transferrin. Moreover, HFE expression activates the key regulators of intracellular iron homeostasis, the iron-regulatory proteins (IRPs), implying that HFE can affect the intracellular “labile iron pool.” The increase in IRP activity is accompanied by the downregulation of the iron-storage protein, ferritin, and an upregulation of transferrin receptor levels. These findings are discussed in the context of the pathophysiology of HH and a possible role of iron-responsive element (IRE)-containing mRNAs.

scholarly journals Crucial function of vertebrate glutaredoxin 3 (PICOT) in iron homeostasis and hemoglobin maturation

2013 ◽  
Vol 24 (12) ◽  
pp. 1895-1903 ◽  
Author(s):  
Petra Haunhorst ◽  
Eva-Maria Hanschmann ◽  
Lars Bräutigam ◽  
Oliver Stehling ◽  
Bastian Hoffmann ◽  
...  
Keyword(s):  
Iron Metabolism ◽  
Iron Homeostasis ◽  
Regulatory Protein ◽  
Iron Regulation ◽  
Iron Starvation ◽  
Intracellular Iron ◽  
Cellular Iron ◽  
Cellular Iron Uptake ◽  
S Proteins

The mechanisms by which eukaryotic cells handle and distribute the essential micronutrient iron within the cytosol and other cellular compartments are only beginning to emerge. The yeast monothiol multidomain glutaredoxins (Grx) 3 and 4 are essential for both transcriptional iron regulation and intracellular iron distribution. Despite the fact that the mechanisms of iron metabolism differ drastically in fungi and higher eukaryotes, the glutaredoxins are conserved, yet their precise function in vertebrates has remained elusive. Here we demonstrate a crucial role of the vertebrate-specific monothiol multidomain Grx3 (PICOT) in cellular iron homeostasis. During zebrafish embryonic development, depletion of Grx3 severely impairs the maturation of hemoglobin, the major iron-consuming process. Silencing of human Grx3 expression in HeLa cells decreases the activities of several cytosolic Fe/S proteins, for example, iron-regulatory protein 1, a major component of posttranscriptional iron regulation. As a consequence, Grx3-depleted cells show decreased levels of ferritin and increased levels of transferrin receptor, features characteristic of cellular iron starvation. Apparently, Grx3-deficient cells are unable to efficiently use iron, despite unimpaired cellular iron uptake. These data suggest an evolutionarily conserved role of cytosolic monothiol multidomain glutaredoxins in cellular iron metabolism pathways, including the biogenesis of Fe/S proteins and hemoglobin maturation.

scholarly journals Essential role of FBXL5-mediated cellular iron homeostasis in maintenance of hematopoietic stem cells

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Yoshiharu Muto ◽  
Masaaki Nishiyama ◽  
Akihiro Nita ◽  
Toshiro Moroishi ◽  
Keiichi I. Nakayama
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Essential Role ◽  
Iron Homeostasis ◽  
Hematopoietic Stem ◽  
Cellular Iron ◽  
Cellular Iron Homeostasis

Role of GSK3 in Multiple Myeloma Cell Growth and Survival.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2509-2509
Author(s):  
Francesco A. Piazza ◽  
Carmela Gurrieri ◽  
Gino Chioetto ◽  
Anna Colpo ◽  
Luca Bonanni ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Cell Death ◽  
Growth Factors ◽  
Cell Growth ◽  
Cell Membrane ◽  
Critical Role ◽  
Igf I

Abstract The serine-threonine GSK3 displays a crucial role in different cancer-pathogenetic pathways, including the PI3K/AKT, Wnt β-catenin and NF-κB signaling cascades, either promoting or counteracting cell survival. The aim of this study was to investigate the role of GSK3 in multiple myeloma (MM) cell growth. GSK3α and β total and phosphorylated protein levels were found differentially expressed in malignant plasma cells as compared to normal resting B-lymphocytes and to normal in vitro generated plasmablasts. Intriguingly, in freshly isolated malignant plasmacells from patients, most of GSK3 was found colocalized with Wnt receptor LRP6 and casein kinase I next to the cell membrane as compared to normal plasmacells or B-cells from other malignancies, wher it was distributed in the cytosol and in the nucleus, thus suggesting a peculiar role of this kinase in these cells. Upon stimulation of MM cells with IL-6 and IGF-I, GSK3 enzymatic activity was hampered, while stimulation with TNFα did not affect GSK3 function nor the early events in NF-κB activation. Basal and IL-6 and IGF-I driven proliferation of MM cells was slightly impaired by GSK3 blockade. Interestingly, GSK3β−/− mouse embryo fibroblasts (MEFs) proliferated slightly slower as compared to GSK3β+/+ cells; however, GSK3α inhibition and IL-6 and IGF-I stimulation, resulted in much higher proliferation of GSK3β −/− cells. Intriguingly, GSK3 inhibition with specific compounds (SB216763 and SB415286) caused a significant rescue from cell death of growth factor-deprived MM cells while resulted in reduced cell proliferation and apoptosis of MM cells grown with serum or growth factors. When GSK3 inhibitors were added to MM cell cultured with bone marrow stromal cells (BMSC), MM cells survival increased and NF-κB and β-catenin-mediated gene transcription (of IAPs and cyclinD1, respectively) was deregulated. GSK3 activity inhibition did not modify the rate of proteasome inhibitor-induced cell death in co-colture experiments with BMSC, suggesting that the sensitivity to bortezomib-induced MM cell apoptosis is independent on GSK3. Altogether, our data indicate thatGSK3 localizes on the cell membrane in primary MM cells;GSK3 is differently regulated downstream from growth factors or TNFα-induced signaling pathways in MM cells;a peculiar role of GSK3 in malignant MM cells as compared to normal MEFs with regard to cell proliferation; anda critical role of this kinase in regulating the MM microenvironment.

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