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A three-step pathway comprising PLZF/miR-146a/CXCR4 controls megakaryopoiesis

Nature Cell Biology volume 10, pages 788–801 (2008)Cite this article

Abstract

MicroRNAs (miRNAs or miRs) regulate diverse normal and abnormal cell functions. We have identified a regulatory pathway in normal megakaryopoiesis, involving the PLZF transcription factor, miR-146a and the SDF-1 receptor CXCR4. In leukaemic cell lines PLZF overexpression downmodulated miR-146a and upregulated CXCR4 protein, whereas PLZF knockdown induced the opposite effects. In vitro assays showed that PLZF interacts with and inhibits the miR-146a promoter, and that miR-146a targets CXCR4 mRNA, impeding its translation. In megakaryopoietic cultures of CD34+ progenitors, PLZF was upregulated, whereas miR-146a expression decreased and CXCR4 protein increased. MiR-146a overexpression and PLZF or CXCR4 silencing impaired megakaryocytic (Mk) proliferation, differentiation and maturation, as well as Mk colony formation. Mir-146a knockdown induced the opposite effects. Rescue experiments indicated that the effects of PLZF and miR-146a are mediated by miR-146a and CXCR4, respectively. Our data indicate that megakaryopoiesis is controlled by a cascade pathway, in which PLZF suppresses miR-146a transcription and thereby activates CXCR4 translation.

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Figure 1: Enhanced PLZF expression in K562 cells is associated with miR-146a downmodulation and CXCR4 protein upregulation.
Figure 2: PLZF, miR-146a and CXCR4 expression in purified HPCs induced to Mk differentiation.
Figure 3: PLZF binds and represses miR-146a promoter, whereas miR-146a interacts with CXCR4 mRNA-3´UTR and inhibits mRNA translation.
Figure 4: Ectopic miR-146a expression in Mk cells downregulates CXCR4 protein expression and impairs Mk cell growth, differentiation and maturation and Mk progenitor clonogenesis.
Figure 5: AntagomiR-146a transfection in Mk cells upregulates CXCR4 protein expression and stimulates Mk cell growth, differentiation and maturation.
Figure 6: Inhibition of PLZF expression by siRNA treatment impairs Mk cell growth, differentiation-maturation and Mk progenitor clonogenesis.
Figure 7: Inhibition of CXCR4 expression by siRNA treatment impairs Mk cell growth, differentiation-maturation and Mk progenitor clonogenesis.
Figure 8: Restored miR-146a expression in K562-PLZF cells blocks CXCR4 translation and impairs Mk differentiation induced by PDB.

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Acknowledgements

We are grateful to M. Fontana, V. Michetti and M. Blasi for editorial assistance. We thank G. Loreto for graphics and A. M. Cerio for technical support. This work was supported by Italy National Grants (Progetto Oncotecnologico, Programma Nazionale Italia-USA in Oncologia) to C.P., institutional grants from the Italian Ministry of Health (Alleanza Contro il Cancro, Programma Straordinario di Ricerca Oncologica 2006) to U.T. and C.L. and Associazione Italiana per la Ricerca sul Cancro (AIRC) to R.F. and F.G.

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Authors and Affiliations

  1. Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, 00161, Italy

    Catherine Labbaye, Isabella Spinello, Maria Teresa Quaranta, Elvira Pelosi, Luca Pasquini, Eleonora Petrucci, Mauro Biffoni, Eugenia Rosa Nuzzolo, Ugo Testa & Cesare Peschle

  2. Department of Cellular Biotechnologies and Hematology, Division of Hematology, 'La Sapienza' University, Rome, 00161, Italy

    Robin Foà

  3. San Pietro FBF Centro Ricerche AFaR, Rome, 00100, Italy

    Ercole Brunetti

  4. Department of General Pathology, University of Perugia, Perugia, 06122, Italy

    Monia Billi & Francesco Grignani

  5. IRCCS MultiMedica, Milan, 20138, Italy

    Cesare Peschle

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  1. Catherine Labbaye
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Contributions

C.L., I.S. M.T.Q., E.P., L.P., E.P., M.B., E.R.N., R.F. and U.T. performed experiments; E.B. provided cord-blood samples; F.G. revised the paper. C.L. and C.P. planned and designed the experiments and wrote the paper.

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Correspondence to Catherine Labbaye or Cesare Peschle.

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Labbaye, C., Spinello, I., Quaranta, M. et al. A three-step pathway comprising PLZF/miR-146a/CXCR4 controls megakaryopoiesis. Nat Cell Biol 10, 788–801 (2008). https://doi.org/10.1038/ncb1741

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