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Thrombopoietin regulates c-Myb expression by modulating micro RNA 150 expression

  • Charlene F. Barroga
    Affiliations
    Department of Medicine and Division of Hematology/Oncology, University of California, San Diego School of Medicine, San Diego, Calif., USA
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  • Hang Pham
    Affiliations
    Department of Medicine and Division of Hematology/Oncology, University of California, San Diego School of Medicine, San Diego, Calif., USA
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  • Kenneth Kaushansky
    Correspondence
    Offprint requests to: Kenneth Kaushansky, M.D., University of California, San Diego, Hematology Division, Department of Pediatrics and Medicine, 9500 Gilman Drive, San Diego, CA 92103-8811
    Affiliations
    Department of Medicine and Division of Hematology/Oncology, University of California, San Diego School of Medicine, San Diego, Calif., USA
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Open ArchivePublished:September 24, 2008DOI:https://doi.org/10.1016/j.exphem.2008.07.001

      Objective

      Mice harboring c-Myb hypomorphic mutations display enhanced thrombopoiesis because of increased numbers of megakaryocytes and their progenitors. Thrombopoietin induces these same effects, which lead us to hypothesize that the hormone acts through modulation of c-Myb expression, as c-Myb levels falls during thrombopoietin-induced megakaryocyte (MK) maturation. Micro RNAs (miRs) downregulate gene expression by binding to the 3′ untranslated region (UTR) of specific messenger RNAs (mRNAs); we noted that the 3′UTR of c-Myb contains four miR-150 binding sites.

      Materials and Methods

      We used quantitative reverse transcriptase polymerase chain reaction, Western blotting, and reporter gene analyses to assess the response of c-Myb to thrombopoietin stimulation and to gain of and loss of miR-150 expression.

      Results

      We found that thrombopoietin reduced c-Myb mRNA and protein levels within 7 hours in megakaryocytes and UT7/thrombopoietin (TPO) cells. Using a reporter gene containing the c-Myb 3′UTR region, including its four miR150 binding sites, we found that expression of miR150 reduced luciferase expression to 50% of baseline at 24 hours and to 25% at 48 hours in UT7/TPO cells. Quantitative polymerase chain reaction and Western blotting also revealed that miR-150 reduced endogenous c-Myb mRNA and protein to 50% in UT7/TPO cells, and to 65% in mature megakaryocytes. Converse experiments utilizing anti-miR150 increased luciferase activity twofold over control anti-miR. Finally, TPO increased miR150 expression 1.8-fold within 24 hours and 3.4-fold within 48 hours.

      Conclusions

      These findings establish that miR150 downmodulates c-Myb levels, and because TPO affects miR150 expression, our results indicate that, in addition to affecting MK progenitor cell growth, TPO downmodulates c-Myb expression through induction of miR-150.
      Thrombopoiesis, the process by which hematopoietic stem cells ultimately differentiate into platelet-producing megakaryocytes, is characterized by a series of developmental decisions dependent on expression of several lineage-specific transcription factors, hematopoietic growth factors, and supportive marrow stromal and endothelial cells [
      • Kaushansky K.
      The molecular mechanisms that control thrombopoiesis.
      ]. One of the transcription factors that influences megakaryocyte development is c-Myb, a gene which was discovered as the cellular homologue of the transforming gene of two avian retroviruses [
      • Ramsey R.G.
      c-Myb a stem-progenitor cell regulator in multiple tissue compartments.
      ]. c-Myb recognizes the core sequence PyAACT/GG [
      • Ganter B.
      • Chao S.T.
      • Lipsick J.S.
      Transcriptional activation by the Myb proteins requires a specific local promoter structure.
      ] and is expressed in immature hematopoietic cells, where it plays an important role; fetal c-myb null mice die at E15 because of anemia [
      • Emambokus N.
      • Vegiopoulos A.
      • Harman B.
      • Jenkinson E.
      • Anderson G.
      Frampton. Progression through key stages of haemopoiesis is dependent on distinct threshold levels of c-Myb.
      ]. Several targets of c-Myb have been identified, including those in G1 and G2 phases of the cell cycle [
      • Friedman A.D.
      Runx1, c-Myb, and C/EBPalpha couple differentiation to proliferation or growth arrest during hematopoiesis.
      ,
      • Nakata Y.
      • Shetzline S.
      • Sakashita C.
      • et al.
      c-Myb contributes to G2/M cell cycle transition in human hematopoietic cells by direct regulation of cyclin B1 expression.
      ]. Recently, three different mutations of c-Myb (MybD152V, MybD384V, MybM303V) that reduce its function to 20% to 60% of normal were identified and shown to cause anemia and thrombocytosis [
      • Carpinelli M.R.
      • Hilton D.J.
      • Metcalf D.
      • et al.
      Suppressor screen in Mpl-/- mice: c-Myb mutation causes supraphysiological production of platelets in the absence of thrombopoietin signaling.
      ,
      • Sandberg M.L.
      • Sutton S.E.
      • Pletcher M.T.
      • et al.
      c-Myb and p300 regulate hematopoietic stem cell proliferation and differentiation.
      ]. The mice bearing such mutations display greatly reduced numbers of erythroid progenitors and 5- to 10-fold expanded numbers of megakaryocytic progenitors [
      • Nakata Y.
      • Shetzline S.
      • Sakashita C.
      • et al.
      c-Myb contributes to G2/M cell cycle transition in human hematopoietic cells by direct regulation of cyclin B1 expression.
      ,
      • Metcalf D.
      • Carpinelli M.R.
      • Hyland C.
      • et al.
      Anomalous megakaryocytopoiesis in mice with mutations in the c-Myb gene.
      ]. Based on these studies and others, a model has developed that places c-Myb as a switch that influences the lineage fate decision of bipotent megakaryocyte-erythroid progenitors, with high c-Myb levels favoring erythroid development, and low c-Myb levels resulting in a megakaryocytic fate [
      • Mukai H.Y.
      • Motohashi H.
      • Ohneda O.
      • Suzuki N.
      • Nagano M.
      • Yamamoto M.
      Transgene insertion in proximity to the c-myb gene disrupts erythroid-megakaryocytic lineage bifurcation.
      ]. However, while several intracellular processes have been found to influence the transcriptional activity of c-Myb, regulation of c-Myb expression has not yet been fully explored.
      Thrombopoietin (TPO), the primary humoral regulator of platelet production acts by binding to the cellular proto-oncogene c-Mpl, a homodimeric member of the type I hematopoietic cytokine receptor family [
      • Kaushansky K.
      Thrombopoietin: the primary regulator of platelet production.
      ]. Upon TPO binding, c-Mpl undergoes a conformational change allowing cross-phosphorylation and activation of JAK2, a kinase constitutively tethered to the box1 region of the two receptor cytoplasmic domains. The phosphorylation of JAK2 activates its kinase, which in turn phosphorylates tyrosine residues within the cytoplasmic domain of the receptor, and other inducibly tethered secondary signaling mediators. Many TPO-dependent signaling pathways influence cellular events, including signal transducers and activators of transcription (STAT) 3, STAT5, phosphoinositol-3-kinase (PI3K) and mitogen-activated protein kinase, which contribute to hematopoietic cell survival and proliferation (reviewed in [
      • Kaushansky K.
      • Drachman J.G.
      The molecular and cellular biology of thrombopoietin: the primary regulator of platelet production.
      ]). For example, STAT5 enhances expression of the antiapoptotic molecule BclXL [
      • Socolovsky M.
      • Fallon A.E.
      • Wang S.
      • Brugnara C.
      • Lodish H.F.
      Fetal anemia and apoptosis of red cell progenitors in Stat5a-/-5b-/- mice: a direct role for Stat5 in Bcl-X(L) induction.
      ], and PI3K reduces expression of the cell-cycle inhibitor p27 [
      • Collado M.
      • Medema R.H.
      • Garcia-Cao I.
      • et al.
      Inhibition of the phosphoinositide 3-kinase pathway induces a senescence-like arrest mediated by p27Kip1.
      ], and enhances expression of the G1 cell-cycle regulator cyclin D1 [
      • Schmidt M.
      • Fernandez de Mattos S.
      • van der Horst A.
      • et al.
      Cell cycle inhibition by FoxO forkhead transcription factors involves downregulation of cyclin D.
      ]. Because both TPO and reduced levels of c-Myb enhance thrombopoiesis, we determined whether the hormone reduces expression of the transcription factor, thereby testing the more general hypothesis that hematopoietic growth factors influence expression of lineage-specific transcription factors.
      Recently, small noncoding micro RNAs (miRs) have been described that regulate gene expression by targeting messenger RNA (mRNA) for degradation, if matched perfectly, or that cause translational repression, if slightly mismatched. miRs are transcribed as part of long precursor RNAs that are processed further [
      • Sevignani C.
      • Calin G.A.
      • Siracusa L.D.
      • Croce C.M.
      Mammalian microRNAs: a small world for fine-tuning gene expression.
      ]. Literally hundreds of miRs have been identified in the mammalian genome and there is now ample evidence that they play an important role in human gene expression, including in normal and neoplastic hematopoiesis [
      • Chen C.Z.
      • Li L.
      • Lodish H.F.
      • Bartel D.P.
      MicroRNAs modulate hematopoietic lineage differentiation.
      ,
      • Lu J.
      • Getz G.
      • Miska E.A.
      • et al.
      MicroRNA expression profiles classify human cancers.
      ,
      • Lawrie C.H.
      MicroRNAs and haematology: small molecules, big function.
      ]. More specifically, several miRs have been found to affect hematopoietic differentiation, including miR150 on B- and T-lymphocyte development [
      • Zhou B.
      • Wang S.
      • Mayr C.
      • Bartel D.P.
      • Lodish H.F.
      miR-150 a microRNA expressed in mature B and T cells, blocks early B cell development when expressed prematurely.
      ], miR221 and 222 on erythroid development [
      • Felli N.
      • Fontana L.
      • Pelosi E.
      • et al.
      MicroRNAs 221 and 222 inhibit normal erythropoiesis and erythroleukemic cell growth via kit receptor down-modulation.
      ], and miR223 and miR142 on T-cell production [
      • Chen C.Z.
      • Li L.
      • Lodish H.F.
      • Bartel D.P.
      MicroRNAs modulate hematopoietic lineage differentiation.
      ]. Using a variety of predictive algorithms, we found several potential miR target sequences in the 3′ untranslated region of the c-Myb gene. As miRs bind cooperatively to targeted 3′ untranslated regions (UTR) of the genes they regulate [
      • Lewis B.P.
      • Shih I.H.
      • Jones-Rhoades M.W.
      • Bartel D.P.
      • Burge C.B.
      Prediction of mammalian microRNA targets.
      ], the possibility arose that c-Myb expression was regulated by miR. Based upon results of our current study, we have established that TPO-induced megakaryopoiesis is characterized by downregulation of c-Myb expression, mediated at least in part by enhanced expression of miR150.

      Materials and methods

      Culture of UT7/TPO cells

      UT7/TPO cells, a primitive hematopoietic cell line, were obtained from Dr. Norio Komatsu and cultured in 10% fetal bovine serum (FBS) in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10 ng/mL recombinant human TPO. For stimulation, the TPO concentration was adjusted to 100 ng/mL, and for starvation, cells were washed three times with phosphate-buffered saline (PBS) and grown in 0.1% FBS/IMDM for 24 hours.

      Isolation of murine megakaryocytes

      Primary murine bone marrow cells were isolated (∼2–3 × 109 cells) and treated with ammonium chloride to lyse red cells and grown for 1 to 3 days in serum-free Optimem medium (Stem Cell Technologies, Vancouver, Canada) supplemented with 5% conditioned medium from TPO-producing BHK cells; immature megakaryocytes were isolated using CD41-phycoerythrin-labeled antibodies and magnetic cell sorting with phycoerythrin beads (Miltenyi Biotech, Bergisch Gladbach, Germany) while mature megakaryocytes were purified by bovine serum albumin gradient.

      Quantitative polymerase chain reaction of c-Myb and other gene transcript levels

      Cells were starved of TPO for 24 hours and then stimulated with the hormone for 1 to 48 hours; total RNA was extracted from cell lines and megakaryocytes using the RNEasy Kit (Qiagen, Chatsworth, CA, USA) and reversed transcribed using Superscript III supermix (Invitrogen, Carlsbad, CA, USA) for the SYBR Greener two-step quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR) kit for the iCycler (BioRad, Hercules, CA, USA). Q-PCR cycling conditions were 50°C for 2 minutes, 95°C for 8 minutes 30 seconds, and 45 cycles of 95°C for 15 seconds followed by 60°C for 1 minute. Melting curve analysis immediately followed Q-PCR with 95°C for 1 minute, 55°C for 1 minute, and 80 cycles of 55°C + 0.5°C/cycle for 10 seconds. c-Myb and RUNX1 values were then normalized against human β-actin or murine glyceraldehyde dehydrogenase (GAPDH). Primers for Q-PCR were: human c-Myb (forward) 5′-gtcacaaattgactgttacaacaccat-3′, (reverse) 5′-ttctactagatgagagggtgtctgagg-3′; murine c-Myb (forward) 5′-ccaagtgacgctttccagatt-3′, (reverse) 5′-ccccgacacagcatctacg-3′; murine RUNX1 (forward) 5′-tacctgggatccatcacctc-3′, (reverse) 5′-gagatggacggcagagtagg-3′; murine C/EBPβ (forward) 5′- caagctgagcgacgagtaca-3′, (reverse) 5′- agctgctccaccttcttctg-3′; murine GATA1 (forward) 5′-ttgtgaggccagagagtgtg-3′, (reverse) 5′-tccgccagagtgttgtagtg-3′; murine FOG-1 (forward) 5′-agcggtgtctgtcacaactg-3′, (reverse) 5′-ggctgtctggaggaagtttg-3′; murine Fli-1 (forward) 5′-caaccagccagtgagagtca-3′, (reverse) 5′-gccgttcttctcatccatgt-3′; human β-actin (forward) 5′-ccaaccgcgagaagatga-3′, (reverse) 5′-ccagaggcgtacagggatag-3′; murine GAPDH (forward) 5′-ggtgaaggtcggtgtgaacggat-3′, (reverse) 5′-cctggaagatggtgatgggcttc-3′. Each Q-PCR was performed in triplicate. The reported data represent the mean results from at least three independent experiments.

      Western blot analyses

      UT7/TPO cell cultures or purified megakaryocytes were lysed in 1% NP40/0.1% NaDodSO4 buffer containing complete protease inhibitor and PhosStop (Roche Applied Science, Indianapolis, IN, USA). Twenty micrograms of these cell lysates were loaded onto 4% to 15% NaDodSO4 polyacrylamide gels (BioRad), size fractionated by electrophoresis, Western-blotted onto polyvinylidene fluoride membranes (BioRad), probed with anti-c-Myb (05-175; Upstate, Millipore, Billerica, MA, USA), and stripped and reprobed with anti–β-actin (A2228, Sigma-Aldrich, St Louis, MO, USA). The relative intensity of c-Myb protein signals was determined by HP Scanjet and ImageJ software; the signal intensity was calculated by comparing each c-Myb signal to the corresponding β-actin signal and then comparing to the ratios for unstimulated cells (set at 100%). Densitometric results were then averaged and p values calculated, and a representative Western blot displayed in the corresponding figure.

      Q-miR PCR using Taqman miR assays

      Specific miR-specific RT primer and PCR primers were obtained for miRs that were modulated by TPO in UT7/TPO cells and had target sites in the c-Myb 3′UTR, including miR-150 and miR-195 (ABI, Foster City, CA, USA). RNA for miR quantitation was obtained using the miRvana Isolation Kit (Ambion/ABI) for small RNA species. Additional purification and enrichment of small RNA from total RNA was achieved by differential ethanol concentrations and sequential binding onto the glass fiber filter according to manufacturer's recommendations. The QmiR assays consist of a two-step RT-PCR for quantification of small amounts of miR (from 1–10 ng total RNA); the first step is RT of miR using miR-specific looped RT primers to generate cDNA, and the second step is PCR using miR-specific forward and reverse primers and a miR-specific Taqman MGB probe. The assay detects only mature, active miR and not the inactive precursor miR, with single-base discrimination. Q-RT-PCR for RNU-6B served as a control for miR quantitation.

      Construction of the c-Myb 3′UTR luciferase reporter vectors

      miR binding sites in the 3′UTR of c-Myb were determined using the TargetScan 4.0 program (Whitehead Bioinformatics and Research Computing; http://www.targetscan.org/). The 3′UTR of human c-Myb (NM_005375.2 nt 2151-3285) was cloned by PCR amplification from UT7 cDNA using a Mlu I-tailed forward primer (5′-ccgacgcgtcagaacacttcaagttgacttgg-3′) and a Hind III–tailed reverse primer (5′-cccaagcttgctacaaggcagtaagtacaccgtc-3′). The 1.2-kb PCR product was digested with Hind III and MluI and cloned into Hind III/Mlu I digested pMIR-Report-luc vector (Ambion/ABI), which drives luciferase expression from the CMV promoter. The fidelity of the resulting construct, pCMV-luc-3′UTRcMyb, was verified by restriction analysis and DNA sequencing with luc forward primer (5′-gaggtagatgagatgtga-3′) and a polyA reverse primer (5′-aggcgattaagttgggta-3′). pMIR-Report-luc (pCMV-luc) served as a control plasmid.

      Transfection of UT7 cells

      UT7/TPO cells (4 × 106) were transduced by nucleofection with 1 μg luciferase reporter plasmid together with 2 μg miR-150, miR-195, or miR Negative Control #1 (Ambion), and anti–miR-150, anti–miR-195 or anti-miR Negative Control #1 (Ambion), and 250 ng pMXGFP using Nucleofector Reagent R and program T-024 (Amaxa, Gaithersburg, MD, USA). Cells were cultured in 10% FBS in IMDM for 24 hours. Transfection was monitored by immunofluorescence microscopy and fluorescein-activated cell sorting for green fluorescein protein. After 24 hours, cells were washed with 10% FBS in IMDM and split into subcultures for RNA extraction or cultured further for up to 48 hours in 0 to 100 ng/mL TPO.

      Reporter gene expression assays

      UT7/TPO cell cultures or purified megakaryocytes were lysed with 1× Lysis Buffer (Roche) and assayed for luciferase activity using Steady-Glo (Promega, Madison, WI, USA) reagents and read in a luminometer (Wallac 1420 Victor plate reader, Perkin Elmer, Waltham, MA, USA).

      Results

      TPO regulates c-Myb mRNA expression and protein function in UT7/TPO cells and primary murine megakaryocytes

      Based on the inverse relationship between the level of c-Myb expression and that of megakaryopoiesis [
      • Carpinelli M.R.
      • Hilton D.J.
      • Metcalf D.
      • et al.
      Suppressor screen in Mpl-/- mice: c-Myb mutation causes supraphysiological production of platelets in the absence of thrombopoietin signaling.
      ,
      • Sandberg M.L.
      • Sutton S.E.
      • Pletcher M.T.
      • et al.
      c-Myb and p300 regulate hematopoietic stem cell proliferation and differentiation.
      ], and the profound effects of TPO on megakaryocyte and platelet development [
      • Kaushansky K.
      Thrombopoietin: the primary regulator of platelet production.
      ], we hypothesized that the hormone regulates expression of c-Myb. Using Q-RT-PCR we found that TPO reduces c-Myb expression in UT7/TPO cells, which closely represent the megakaryocytic lineage. A detailed study of c-Myb expression in these cells revealed that TPO treatment reduced c-Myb mRNA expression twofold within 3 hours (p ≤ 0.03) (Fig. 1A ). To verify these changes occur in primary cells, we treated freshly isolated murine megakaryocytes with TPO and found similar results; following 2 days of treatment with TPO, megakaryocyte c-Myb levels fell by ∼15-fold, compared to a twofold increase in RUNX1, no change in GATA1, and a fourfold increases in FOG1 and Fli1 (Fig. 1B and C). To determine if these changes are reflective of direct effects of TPO, we performed a shorter time course analysis, and used Western blotting to confirm the changes also occur at the level of protein expression. We found that TPO treatment reduced c-Myb protein expression to 51% of baseline within 7 hours in UT7/TPO and to 65% of baseline in murine megakaryocytes (Fig. 2A and B). Densitometric analysis of the c-Myb to β-actin ratio revealed that reduction of c-Myb protein levels were significant from 5 hours onward in both UT7/TPO cells (p ≤ 0.01) (n = 3) and murine megakaryocytes (p ≤ 0.01). To test if these changes in c-Myb mRNA and protein levels resulted in a corresponding reduction in c-Myb function we introduced a c-Myb reporter gene into UT7/TPO cells, in which the three c-Myb binding sites in the c-mim1 promoter drives luciferase activity (cMim-luc; kindly provided by Ness [
      • Ness S.A.
      • Kowenz-Leutz E.
      • Casini T.
      • Graf T.
      • Leutz A.
      Myb and NF-M: combinatorial activators of myeloid genes in heterologous cell types.
      ]). We found that TPO reduced reporter gene activity to 62% of baseline at 5 hours (p = 0.038) (Fig. 2C). In contrast, when the mim1 promoter orientation was reversed, TPO did not change luciferase expression (data not shown). While these changes in c-Myb protein expression and function seem modest, it did not escape our attention that a ∼50% reduction in c-Myb expression in vivo results in an ∼10-fold increase in the number of mature progenitors committed to the megakaryocyte lineage [
      • Carpinelli M.R.
      • Hilton D.J.
      • Metcalf D.
      • et al.
      Suppressor screen in Mpl-/- mice: c-Myb mutation causes supraphysiological production of platelets in the absence of thrombopoietin signaling.
      ,
      • Sandberg M.L.
      • Sutton S.E.
      • Pletcher M.T.
      • et al.
      c-Myb and p300 regulate hematopoietic stem cell proliferation and differentiation.
      ].
      Figure thumbnail gr1
      Figure 1Expression of c-Myb and other megakaryocytic transcription factors in megakaryocytic cell lines and mature megakaryocytes. UT7/thrombopoietin (TPO) cells (A) were starved for 24 hours and then stimulated with 100 ng/mL TPO for up to 48 hours. RNA was extracted and c-Myb levels were assessed by quantitative reverse transcriptase polymerase chain reaction, with β-actin as internal control. Each assay was performed in triplicate. c-Myb levels significantly declined (p ≤ 0.03) for all time points with TPO stimulation (n = 3). Immature murine megakaryocytes (B, C) were induced to differentiate over 2 days with TPO. RNA was harvested from immature and mature megakaryocytes, and the levels of RUNX1, GATA1, FOG1, Fli1, C/EBPβ, and c-Myb were assessed by quantitative real-time polymerase chain reaction with glyceraldehyde dehydrogenase (GAPDH) as an internal control. Results shown are the relative values of signal intensity for the various transcription factors compared to internal controls. Each experiment was performed in triplicate and results compared using the t-test for paired values. Results represented in B and C were performed together, but are shown on separate figures due to the range of scales.
      Figure thumbnail gr2
      Figure 2Thrombopoietin (TPO) reduces c-Myb protein and function in UT7/TPO cells and in murine megakaryocytes. (A) UT7/TPO cells were starved overnight and then stimulated with 100 ng/mL TPO for 0 to 48 hours, cell lysates were prepared, size fractionated, transferred to polyvinylidene fluoride membranes and probed for c-Myb. Densitometric analysis revealed a significant decrease in c-Myb protein levels at 5 hours to 48 hours (p < 0.01 for all time points). The fraction of c-Myb remaining is shown, along with a representative Western blot. (B) Mature murine megakaryocytes were produced by marrow cell culture in TPO and the cells purified by bovine serum albumin gradient and starved for 20 hours in StemPro serum-free medium. Cells were then cultured in 100 ng/mL TPO for up to 7 hours, cell lysates prepared and subjected to Western blotting for c-Myb and β-actin,. Densitometric analyses (n = 3) showed c-Myb protein levels were significantly decreased at 5 to 7 hours (p ≤ 0.01). The fraction of c-Myb remaining is shown, along with a representative Western blot. (C) The c-Myb responsive reporter construct c-mim-luc (mim1Luc), which contains three c-Myb response elements was introduced into UT7/TPO cells and following overnight starvation was cultured an additional 5 hours with TPO or control culture medium. Sham transfected cells served as a control (C). Data represent the mean of three experiments. The difference between TPO and no TPO in c-mim1-luc transduced cultures achieved statistical significance (p = 0.038).

      c-Myb RNA and protein levels are regulated by miR species

      To begin to explore the molecular mechanism(s) by which TPO affects c-Myb mRNA expression, we hypothesized that the hormone might act to alter the levels of miRs that target the 3′UTR of c-Myb. Previous work by others using microarrays had demonstrated several miR changes in TPO-stimulated hematopoietic cells [
      • Garzon R.
      • Pichiorri F.
      • Palumbo T.
      • et al.
      MicroRNA fingerprints during human megakaryocytopoiesis.
      ], including several for which both high and modest affinity miR binding sites in the murine and human c-Myb genes were found. Findings in our miR microarray comparing TPO-stimulated megakaryocytes vs TPO-starved megakaryocytes revealed several miRs against c-Myb were upregulated, including miR-150 and miR-195 (data not shown). In this study we focused on miR-150 and miR-195, as our in silico analysis revealed that the c-Myb 3′UTR displays two high- and two low-affinity binding sites for miR-150 and one high-affinity binding site for miR-195 (Fig. 3A ). To directly test if these miR species regulate c-Myb expression, we created a reporter gene, linking the CMV promoter–driven luciferase gene and the 1.2-kb 3′UTR of c-Myb (pCMV-luc-3′UTRcMyb; Fig. 3A) and introduced the construct into UT7/TPO cells together with either miR-150, miR-195, anti–miR-150, anti–miR-195 or the corresponding controls. We found that miR-150 significantly downregulated luciferase activity to 40% of baseline 24 hours after cotransfection with pCMV-luc-3′UTRcMyb (Fig. 3B) compared to a miR-negative control (p ≤ 0.002). miR195 exerted a significant, but quantitatively lesser effect. Luciferase activity in cells transfected with a control luc plasmid lacking the 3′UTR of c-Myb was not modulated by introduction of miR-150 (Fig. 3B, column 1 vs column 4). Because UT7/TPO cells express miR-150 at baseline, we performed converse experiments utilizing anti-miRs, which inhibit expression of endogenous miRs. We found that when cotransfected with pCMV-luc-3′UTRcMyb, anti–miR-150 significantly upmodulated luciferase activity to 180% of baseline compared to an anti–miR-negative control (p = 0.0001) (Fig. 3C, column 1 vs column 3), or to a significantly lesser extent in an otherwise identical reporter construct that lacks the 3′UTR of c-Myb (p = 0.016) (Fig. 3C, column 1 vs column 4). However, in contrast to our results with miR195, anti–miR-195 had little effect on luciferase activity (Fig. 3C, column 2 vs column 3). Both miR-negative control and anti–miR negative control did not display nonspecific effects, as transfection of pCMV-luc-3′UTRcMyb alone or in combination with either miR-negative control or anti–miR-negative control resulted in similar luciferase activities (data not shown).
      Figure thumbnail gr3
      Figure 3miR150 affects a c-Myb 3′ untranslated region (UTR) reporter gene. (A) The pCMV-luc-3′UTRc-Myb reporter gene is illustrated [with the list of miR binding sites]. The construct was generated by introducing the 1.2-kb 3′UTR of c-Myb into pMIR-Report-luc. (B) pCMV-luc-3′UTRc-Myb was transfected into UT7/thrombopoietin (TPO) cells with miR150, miR195, or a negative miR control. After 24 hours, luciferase activity was assessed in duplicate cultures. Values reported are the mean ± standard error of mean of three separate experiments and reported as relative luciferase activity between the various conditions. The same vector, but without the 3′UTR of c-Myb (pCMV-luc), were also utilized as controls, and results are shown. (C) pCMV-luc-3′UTRc-Myb was transfected into UT7/TPO cells with anti-miR150, anti-miR195 or a negative anti-miR control. After 24 hours, luciferase activity was assessed in duplicate cultures and is reported as the mean ± standard error of mean of three separate experiments. The same controls as in (B) were performed. pCMV-luc-3′UTRc-Myb was also transfected into the cells with miR-negative control and anti–miR-negative control, and these gave the same values as the vector without the 3′UTRcMyb (data not shown).
      Introduction of miRs and anti-miRs into UT7/TPO cells also provided us the opportunity to monitor their effects on endogenous c-Myb mRNA and protein levels. Using Q-PCR, we found that endogenous c-Myb mRNA was significantly downregulated to 55% of baseline upon transfection of miR-150 compared to the miR negative control (p = 0.008) (Fig. 4A ), with a lesser effect of miR195, while the essential megakaryocytic transcription factor, RUNX1, remained unaltered (Fig. 4B). Western blotting of these cell lysates revealed that c-Myb protein expression was downregulated to 30% of baseline following transduction with miR-150, but not with the miR-negative control or miR195 (Fig. 4C).
      Figure thumbnail gr4
      Figure 4miR150 affects c-Myb expression. The same cultures assessed for reporter gene activity in B were also assayed for expression of the endogenous c-Myb (A, C) and RUNX-1 (B) genes using quantitative reverse transcriptase polymerase chain reaction (A, B) and quantitative Western blotting (C).

      TPO regulates miR150 expression

      Previous results establish that miR-150 can significantly alter c-Myb expression. To test whether miR-150 responds to TPO stimulation, we used a miR-specific Q-PCR assay and tested the effects of the hormone on its expression. We found that TPO increased miR-150 expression to 144% of baseline at 3 hours, 183% at 24 hours, and 340% at 48 hours in UT7/TPO cells (Fig. 5). In contrast, erythropoietin did not affect miR-150 levels in UT7/EPO cells (data not shown).
      Figure thumbnail gr5
      Figure 5Thrombopoietin (TPO) affects miR-150 expression. A quantitative reverse transcriptase polymerase chain reaction assay for miR-150 using the RNU6B control microRNA as an internal control was developed and used to assess the miR-150 cellular response to stimulation with thrombopoietin. We found that UT-7/TPO cells stimulated for 3 to 48 hours with the hormone upregulated miR-150 expression substantially and significantly. Results represent the mean ± standard error of mean of three independent experiments with assays performed in triplicate.

      Discussion

      Several features of the biology of Tpo and c-Myb are consistent with the hypothesis that the two genes lie on the same genetic pathway and that TPO reduces c-Myb expression. For example, the 10-fold increase in hematopoietic stem cells found in the hypomorphic c-mybM303V animals matches closely the ∼8-fold reduction in these cells seen in c-mpl–/– mice [
      • Solar G.P.
      • Kerr W.G.
      • Zeigler F.C.
      • et al.
      Role of c-mpl in early hematopoiesis.
      ] or the ∼15-fold decrease in stem cell expansion seen when normal marrow cells are transplanted into tpo–/– mice [
      • Fox N.E.
      • Priestley G.V.
      • Papayannopoulou Th
      • Kaushansky K.
      Thrombopoietin (TPO) expands hematopoietic stem cells (HSCs) in vivo.
      ]. Moreover, the level of c-Myb mRNA in TPO-stimulated CD41+ cord blood–derived cells is 7.4-fold lower than seen in burst-forming unit erythroid or CD34+ cells [
      • Balduini A.
      • D'Apolito M.
      • Arcelli D.
      • et al.
      Cord blood expanded CD41+cells: identification of novel components of megakaryocytopoiesis.
      ]. In the present study, we sought to mechanistically link TPO and c-Myb expression. We found TPO suppresses c-Myb transcription and protein levels to 40% to 65% of resting levels in UT7/TPO cells and mature megakaryocytes within a few hours of its addition to TPO-depleted cells; while the direct effect of TPO on c-Myb may seem modest, this level is physiologically relevant because the c-MybM303V mice, which display anemia and thrombocytosis, are characterized by a ∼50% reduction in c-Myb function.
      Several lines of evidence suggest that c-Myb enhances erythropoiesis and is a negative regulator of megakaryopoiesis [
      • Vegiopoulos A.
      • Garcia P.
      • Emambokus N.
      • Frampton J.
      Coordination of erythropoiesis by the transcription factor c-Myb.
      ]. Based on our finding that TPO affects c-myb mRNA levels, we next determined if this is due to miR-induced modulation of c-myb mRNA stability and/or translation efficiency. To test the effect of various miR species, we generated and introduced into UT7/TPO cells a reporter construct that reflects posttranscriptional modulation of c-Myb; we found that luciferase activity was significantly reduced by miR-150, and that endogenous miR-150 regulates c-Myb as an anti-miR150 increased reporter gene activity. Next, we assessed the effects of miR-150 on c-Myb mRNA and protein levels in UT7/TPO cells; we found that miR-150 reduced c-Myb mRNA levels to 55% of control levels, and c-Myb protein to 30% of control levels. These results were found in multiple experiments and strongly suggest that miR-150 affects both c-Myb mRNA stability and translation efficiency.
      Regulation of c-Myb transcription is complex, dependent on stage of cell differentiation and cell cycle, and levels of [Ca++]i, c-Myb, Ets1, GATA1, nuclear factor-κB and E2F [
      • Schaefer A.
      • Magócsi M.
      • Stöcker U.
      • Fandrich A.
      • Marquardt H.
      Ca2+/calmodulin-dependent and -independent down-regulation of c-myb mRNA levels in erythropoietin-responsive murine erythroleukemia cells: the role of calcineurin.
      ,
      • Guerra J.
      • Withers D.A.
      • Boxer L.M.
      Myb binding sites mediate negative regulation of c-myb expression in T- cell lines.
      ,
      • Bloch A.
      • Liu X.M.
      • Wang L.G.
      Regulation of c-myb expression in ML-1 human myeloblastic leukemia cells by c-ets-1 protein.
      ,
      • Bartunek P.
      • Kralova J.
      • Blendinger G.
      • Dvorak M.
      • Zenke M.
      GATA-1 and c-myb crosstalk during red blood cell differentiation through GATA-1 binding sites in the c-myb promoter.
      ,
      • Lauder A.
      • Castellanos A.
      • Weston K.
      c-Myb transcription is activated by protein kinase B (PKB) following interleukin 2 stimulation of T cells and is required for PKB-mediated protection from apoptosis.
      ]. Cell context is critical; c-Myb itself upregulates c-Myb expression in fibroblasts, but downmodulates itself in T cells [
      • Guerra J.
      • Withers D.A.
      • Boxer L.M.
      Myb binding sites mediate negative regulation of c-myb expression in T- cell lines.
      ]. In addition, once transcription initiates, it can stall within intron 1, a step that is controlled during B-cell development [
      • Bender T.P.
      • Thompson C.B.
      • Kuehl W.M.
      Differential expression of c-myb mRNA in murine B lymphomas by a block to transcription elongation.
      ]. Once transcribed and translated into protein, c-Myb is subject to further regulation, including lysine acetylation [
      • Sano Y.
      • Ishii S.
      Increased affinity of c-Myb for CREB-binding protein (CBP) after CBP-induced acetylation.
      ] and SUMOylation [
      • Verger A.
      • Perdomo J.
      • Crossley M.
      Modification with SUMO. A role in transcriptional regulation.
      ], which increase and decrease its transcriptional function, respectively. Thus, our results add another dimension to the regulation of c-Myb expression, modulation by a miR, miR-150.
      Our results with miR-150 during megakaryopoiesis differ from that of Garzon and colleagues [
      • Garzon R.
      • Pichiorri F.
      • Palumbo T.
      • et al.
      MicroRNA fingerprints during human megakaryocytopoiesis.
      ], who found using a miR gene array that miR-150 was downmodulated 5.3-fold during TPO-supported development of human megakaryocytes from primitive CD34+ bone marrow hematopoietic progenitors. While these results appear opposite in direction to our own, levels of the miR were not monitored throughout the developmental period. Thus it is possible that expression of miR-150 is biphasic, high in primitive hematopoietic cells, dropping down as cells begin to differentiate, and then rising again in response to lineage-specific signals. Alternately, the differences in the two studies might relate to use of a gene array in one, and specific Q-RT-PCR analyses in our current work.
      Taken together, our results, based on both gain-of-function and loss-of-function strategies, have identified a direct mechanistic link between TPO-induced miR-150 expression and c-Myb downmodulation during megakaryopoiesis. This novel molecular pathway could thus account for some of the favorable effects of TPO on platelet production. These studies also establish that hematopoietic growth factors can influence transcription factor expression through modulation of miR species.

      Acknowledgments

      This work was supported by National Institutes of Health grant R01 DK49855 and R01 CA 31615 to K.K., and 2006 and 2007 Training Grants from the American Society of Hematology to H.P.

      References

        • Kaushansky K.
        The molecular mechanisms that control thrombopoiesis.
        J Clin Invest. 2005; 115: 3339-3347
        • Ramsey R.G.
        c-Myb a stem-progenitor cell regulator in multiple tissue compartments.
        Growth Factors. 2005; 23: 253-261
        • Ganter B.
        • Chao S.T.
        • Lipsick J.S.
        Transcriptional activation by the Myb proteins requires a specific local promoter structure.
        FEBS Lett. 1999; 460: 401-410
        • Emambokus N.
        • Vegiopoulos A.
        • Harman B.
        • Jenkinson E.
        • Anderson G.
        Frampton. Progression through key stages of haemopoiesis is dependent on distinct threshold levels of c-Myb.
        EMBO J. 2003; 22: 4478-4488
        • Friedman A.D.
        Runx1, c-Myb, and C/EBPalpha couple differentiation to proliferation or growth arrest during hematopoiesis.
        J Cell Biochem. 2002; 86: 624-629
        • Nakata Y.
        • Shetzline S.
        • Sakashita C.
        • et al.
        c-Myb contributes to G2/M cell cycle transition in human hematopoietic cells by direct regulation of cyclin B1 expression.
        Mol Cell Biol. 2007; 27: 2048-2058
        • Carpinelli M.R.
        • Hilton D.J.
        • Metcalf D.
        • et al.
        Suppressor screen in Mpl-/- mice: c-Myb mutation causes supraphysiological production of platelets in the absence of thrombopoietin signaling.
        Proc Natl Acad Sci U S A. 2004; 101: 6553-6558
        • Sandberg M.L.
        • Sutton S.E.
        • Pletcher M.T.
        • et al.
        c-Myb and p300 regulate hematopoietic stem cell proliferation and differentiation.
        Dev Cell. 2005; 8: 153-166
        • Metcalf D.
        • Carpinelli M.R.
        • Hyland C.
        • et al.
        Anomalous megakaryocytopoiesis in mice with mutations in the c-Myb gene.
        Blood. 2005; 105: 3480-3487
        • Mukai H.Y.
        • Motohashi H.
        • Ohneda O.
        • Suzuki N.
        • Nagano M.
        • Yamamoto M.
        Transgene insertion in proximity to the c-myb gene disrupts erythroid-megakaryocytic lineage bifurcation.
        Mol Cell Biol. 2006; 26: 7953-7965
        • Kaushansky K.
        Thrombopoietin: the primary regulator of platelet production.
        Blood. 1995; 86: 419-431
        • Kaushansky K.
        • Drachman J.G.
        The molecular and cellular biology of thrombopoietin: the primary regulator of platelet production.
        Oncogene. 2002; 21: 3359-3367
        • Socolovsky M.
        • Fallon A.E.
        • Wang S.
        • Brugnara C.
        • Lodish H.F.
        Fetal anemia and apoptosis of red cell progenitors in Stat5a-/-5b-/- mice: a direct role for Stat5 in Bcl-X(L) induction.
        Cell. 1999; 98: 181-191
        • Collado M.
        • Medema R.H.
        • Garcia-Cao I.
        • et al.
        Inhibition of the phosphoinositide 3-kinase pathway induces a senescence-like arrest mediated by p27Kip1.
        J Biol Chem. 2000; 275: 21960-21968
        • Schmidt M.
        • Fernandez de Mattos S.
        • van der Horst A.
        • et al.
        Cell cycle inhibition by FoxO forkhead transcription factors involves downregulation of cyclin D.
        Mol Cell Biol. 2002; 22: 7842-7852
        • Sevignani C.
        • Calin G.A.
        • Siracusa L.D.
        • Croce C.M.
        Mammalian microRNAs: a small world for fine-tuning gene expression.
        Mamm Genome. 2006; 17: 189-202
        • Chen C.Z.
        • Li L.
        • Lodish H.F.
        • Bartel D.P.
        MicroRNAs modulate hematopoietic lineage differentiation.
        Science. 2004; 303: 83-86
        • Lu J.
        • Getz G.
        • Miska E.A.
        • et al.
        MicroRNA expression profiles classify human cancers.
        Nature. 2005; 435: 834-838
        • Lawrie C.H.
        MicroRNAs and haematology: small molecules, big function.
        Br J Haematol. 2007; 137: 503-512
        • Zhou B.
        • Wang S.
        • Mayr C.
        • Bartel D.P.
        • Lodish H.F.
        miR-150 a microRNA expressed in mature B and T cells, blocks early B cell development when expressed prematurely.
        Proc Natl Acad Sci U S A. 2007; 104: 7080-7085
        • Felli N.
        • Fontana L.
        • Pelosi E.
        • et al.
        MicroRNAs 221 and 222 inhibit normal erythropoiesis and erythroleukemic cell growth via kit receptor down-modulation.
        Proc Natl Acad Sci U S A. 2005; 102: 18081-18086
        • Lewis B.P.
        • Shih I.H.
        • Jones-Rhoades M.W.
        • Bartel D.P.
        • Burge C.B.
        Prediction of mammalian microRNA targets.
        Cell. 2003; 115: 787-798
        • Ness S.A.
        • Kowenz-Leutz E.
        • Casini T.
        • Graf T.
        • Leutz A.
        Myb and NF-M: combinatorial activators of myeloid genes in heterologous cell types.
        Genes Dev. 1993; 7: 749-759
        • Garzon R.
        • Pichiorri F.
        • Palumbo T.
        • et al.
        MicroRNA fingerprints during human megakaryocytopoiesis.
        Proc Natl Acad Sci U S A. 2006; 103: 5078-5083
        • Solar G.P.
        • Kerr W.G.
        • Zeigler F.C.
        • et al.
        Role of c-mpl in early hematopoiesis.
        Blood. 1998; 92: 4-10
        • Fox N.E.
        • Priestley G.V.
        • Papayannopoulou Th
        • Kaushansky K.
        Thrombopoietin (TPO) expands hematopoietic stem cells (HSCs) in vivo.
        J Clin Invest. 2002; 110: 389-394
        • Balduini A.
        • D'Apolito M.
        • Arcelli D.
        • et al.
        Cord blood expanded CD41+cells: identification of novel components of megakaryocytopoiesis.
        J Thromb Hemostat. 2006; 4: 848-860
        • Vegiopoulos A.
        • Garcia P.
        • Emambokus N.
        • Frampton J.
        Coordination of erythropoiesis by the transcription factor c-Myb.
        Blood. 2006; 107: 4703-4710
        • Schaefer A.
        • Magócsi M.
        • Stöcker U.
        • Fandrich A.
        • Marquardt H.
        Ca2+/calmodulin-dependent and -independent down-regulation of c-myb mRNA levels in erythropoietin-responsive murine erythroleukemia cells: the role of calcineurin.
        J Biol Chem. 1996; 271: 13484-13490
        • Guerra J.
        • Withers D.A.
        • Boxer L.M.
        Myb binding sites mediate negative regulation of c-myb expression in T- cell lines.
        Blood. 1995; 86: 1873-1880
        • Bloch A.
        • Liu X.M.
        • Wang L.G.
        Regulation of c-myb expression in ML-1 human myeloblastic leukemia cells by c-ets-1 protein.
        Adv Enzyme Regul. 1995; 35: 35-41
        • Bartunek P.
        • Kralova J.
        • Blendinger G.
        • Dvorak M.
        • Zenke M.
        GATA-1 and c-myb crosstalk during red blood cell differentiation through GATA-1 binding sites in the c-myb promoter.
        Oncogene. 2003; 22: 1927-1935
        • Lauder A.
        • Castellanos A.
        • Weston K.
        c-Myb transcription is activated by protein kinase B (PKB) following interleukin 2 stimulation of T cells and is required for PKB-mediated protection from apoptosis.
        Mol Cell Biol. 2001; 21: 5797-5805
        • Bender T.P.
        • Thompson C.B.
        • Kuehl W.M.
        Differential expression of c-myb mRNA in murine B lymphomas by a block to transcription elongation.
        Science. 1987; 237: 1473-1476
        • Sano Y.
        • Ishii S.
        Increased affinity of c-Myb for CREB-binding protein (CBP) after CBP-induced acetylation.
        J Biol Chem. 2001; 276: 3674-3682
        • Verger A.
        • Perdomo J.
        • Crossley M.
        Modification with SUMO. A role in transcriptional regulation.
        EMBO Rep. 2003; 4: 137-142