An In Vivo Functional Screen Uncovers miR-150-Mediated Regulation of Hematopoietic Injury Response

SummaryHematopoietic stem and progenitor cells are often undesired targets of chemotherapies, leading to hematopoietic suppression requiring careful clinical management. Whether microRNAs control hematopoietic injury response is largely unknown. We report an in vivo gain-of-function screen and the identification of miR-150 as an inhibitor of hematopoietic recovery upon 5-fluorouracil-induced injury. Utilizing a bone marrow transplant model with a barcoded microRNA library, we screened for barcode abundance in peripheral blood of recipient mice before and after 5-fluorouracil treatment. Overexpression of screen-candidate miR-150 resulted in significantly slowed recovery rates across major blood lineages, with associated impairment of bone marrow clonogenic potential. Conversely, platelets and myeloid cells from miR-150 null marrow recovered faster after 5-fluorouracil treatment. Heterozygous knockout of c-myb, a conserved target of miR-150, partially phenocopied miR-150-forced expression. Our data highlight the role of microRNAs in controlling hematopoietic injury response and demonstrate the power of in vivo functional screens for studying microRNAs in normal tissue physiology

The Erythropoietin Receptor Stimulates Rapid Cycling and Formation of Larger Red Cells During Mouse and Human Erythropoiesis [preprint]

Erythroid terminal differentiation entails cell divisions that are coupled to progressive decreases in cell size. EpoR signaling is essential for the survival of erythroid precursors, but it is unclear whether it has other functions in these cells. Here we endowed mouse precursors that lack the EpoR with survival signaling, finding that this was sufficient to support their differentiation into enucleated red cells, but that the process was abnormal. Precursors underwent fewer and slower cell cycles and yet differentiated into smaller red cells. Surprisingly, EpoR further accelerated cycling of early erythroblasts, the fastest cycling cells in the bone marrow, while simultaneously increasing their cell size. EpoR-mediated formation of larger red cells was independent of the established pathway regulating red cell size by iron through Heme-regulated eIF2α kinase (HRI). We confirmed the effect of Epo on red cell size in human volunteers, whose mean corpuscular volume (MCV) increased following Epo administration. This increase persisted after Epo declined and was not the result of increased reticulocytes. Our work reveals a unique effect of EpoR signaling on the interaction between the cell cycle and cell growth. Further, it suggests new diagnostic interpretations for increased red cell volume, as reflecting high Epo and erythropoietic stress

Forkhead Box Transcription Factor FOXO3a Regulates Estrogen Receptor Alpha Expression and Is Repressed by the Her-2/neu/Phosphatidylinositol 3-Kinase/Akt Signaling Pathway

The expression status of the estrogen receptor alpha (ERα) and that of the epidermal growth factor receptor Her-2/neu frequently correlate inversely in breast cancers. While ERα-dependent cancers respond to antiestrogen therapy, Her-2/neu-overexpressing cancers typically display resistance to antiestrogens and poor prognosis. In this report we have explored the mechanism linking the loss of expression of ERα in breast cancer cells with overexpression of Her-2/neu, which signals constitutively via a phosphatidylinositol 3-kinase (PI3K)/Akt kinase pathway. We identify for the first time the Forkhead box protein FOXO3a (formerly termed FKHRL-1), which is inactivated by Akt, as a key regulator of ERα gene transcription. In breast cancer cell lines, expression of ERα was correlated with active FOXO3a levels. Ectopic FOXO3a expression induced ERα protein levels and promoter activity, while a dominant negative FOXO3a decreased ERα levels. By using transient transfection, mobility shift assays, and site-directed mutagenesis, two major functional Forkhead binding sites were identified in the human ERα promoter B. A chromatin immunoprecipitation assay confirmed FOXO3a binding at these two sites. Ectopic FOXO3a induced estrogen response element-driven reporter activity and expression of ERα target genes. The constitutively activated myristylated Akt reduced ERα expression, whereas agents that negatively affect the PI3K/Akt pathway, i.e., wortmannin, celecoxib, and the green tea polyphenol epigallocatechin-3 gallate, induced ERα. Thus, FOXO3a represents an important intracellular mediator of ERα expression, suggesting possible therapeutic intervention strategies for Her-2/neu-overexpressing refractory breast tumors

HSC myeloablative stress response is not affected by deletion of all piwi genes.

<p>(A) White blood cells (WBC) are responsive to 5FU in both piwi triple knockout (blue circle) and control (black triangle) donor-derived cells. (B) Piwi triple knockout (blue circle) and control (black triangle) donor-derived cells (CD45.2⁺) show similar response kinetics following 5FU, measured up to 31 days after 5FU treatment. (C-E) Ratio of donor/recipient (CD45.2⁺/CD45.2⁻ ) derived cells from <i>Piwi</i> triple knockout (blue circle) and control (black triangle) donors in mature cell populations of recipient peripheral blood. (C) T-cell (CD3⁺), (D) B-cell (B220⁺), and (E) myeloid cell (CD11b⁺) lineages. 5FU injection point is indicated as a black arrow on each graph. Data is the average of 10 triple <i>Piwi</i> mutant injected recipients and 9 control injected recipients. Each data point represents the mean ±SEM of 8–10 recipient mice, per donor genotype.</p

Deletion of all three piwi genes causes increased HSC frequency, but does not affect progenitor or committed cell type numbers.

<p>Piwi-triple-knockout mature blood cell numbers are unchanged compared to control mice (WT, HET), as determined by (A) Complete Blood Cell Count (CBC) for white blood cells (WBC), neutrophils (NE), lymphocytes (LY), red blood cells (RBC) and (B) FACS analysis of committed bone marrow cell types (A: WT, n = 3; HET, n = 4; triple KO, n = 4; B: WT, n = 2; HET, n = 3, triple KO, n = 3). (C) Colony Forming Unit (CFU) assays show similar total myeloid/erythroid progenitor-derived colony numbers in control and triple <i>Piwi</i> mutant bone marrow. Colony assays used total bone marrow from 3 individual mice for each genotype. Bone marrow samples were plated in triplicate and total colonies were counted at days 7–10. Data shown as the average total colony count for each genotype. (D) In the triple <i>Piwi</i> mutant, the percentage of bone marrow progenitors (CMP, MEP and GMP) are only moderately increased compared to control mice progenitors. (E) HSC percentages are significantly increased in <i>Piwi</i> triple knockout bone marrow. (F) Total bone marrow cellularity of the triple knockout mice is almost two-fold reduced compared to control mice. (C-F: Control, n = 4; triple KO, n = 4). <i>P</i> values calculated using two-tailed, unequal variance Student's <i>t</i> test.</p