Erythropoietin Down-Regulates Stem Cell Factor Receptor (Kit) Expression in the Leukemic Proerythroblast: Role of Lyn Kinase Olivier Kosmider 1,2 , Dorothe ´ e Buet 1,2 , Isabelle Gallais 1,2 , Nicole Denis 1,2 , Franc ¸oise Moreau-Gachelin 1,2 * 1 Inserm U830, Paris, France, 2 Institut Curie, Paris, France Abstract Overexpression of the transcription factor Spi-1/PU.1 by transgenesis in mice induces a maturation arrest at the proerythroblastic stage of differentiation. We have previously isolated a panel of spi-1 transgenic erythroleukemic cell lines that proliferated in the presence of either erythropoietin (Epo) or stem cell factor (SCF). Using these cell lines, we observed that EpoR stimulation by Epo down-regulated expression of the SCF receptor Kit and induced expression of the Src kinase Lyn. Furthermore, enforced expression of Lyn in the cell lines increased cell proliferation in response to Epo, but reduced cell growth in response to SCF in accordance with Lyn ability to down-regulate Kit expression. Together, the data suggest that Epo-R/Lyn signaling pathway is essential for extinction of SCF signaling leading the proerythroblast to strict Epo dependency. These results highlight a new role for Lyn as an effector of EpoR in controlling Kit expression. They suggest that Lyn may play a central role in during erythroid differentiation at the switch between proliferation and maturation. Citation: Kosmider O, Buet D, Gallais I, Denis N, Moreau-Gachelin F (2009) Erythropoietin Down-Regulates Stem Cell Factor Receptor (Kit) Expression in the Leukemic Proerythroblast: Role of Lyn Kinase. PLoS ONE 4(5): e5721. doi:10.1371/journal.pone.0005721 Editor: Mikhail V. Blagosklonny, Roswell Park Cancer Institute, United States of America Received February 27, 2009; Accepted April 28, 2009; Published May 28, 2009 Copyright: ß 2009 Kosmider et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work was supported by the Institut National de la Sante et de la Recherche Medicale (Inserm) and Institut Curie, and by grants from Association pour la Recherche sur le Cancer, Ligue contre le Cancer (Equipe labellisee 2006 to F.M.G.), Institut National du Cancer (INCa) and Association Christelle Bouillot. OK was supported by fellowships from Inserm (poste d’accueil), Association pour la Recherche sur le Cancer and Socie ´te ´ Franc ¸aise d’He ´ matologie. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: framoreau@curie.fr Introduction Erythropoiesis is critically regulated by a number of growth factors acting through specific receptors, among which erythro- poietin (Epo) and stem cell factor (SCF) are essential factors [1]. SCF, the ligand for the Kit receptor, is mainly involved in the survival and proliferation of immature erythroid progenitors, whereas Epo is the predominant regulator preventing apoptosis at the CFU-E/proerythroblast stage of differentiation. The impor- tance of the SCF/Kit pathway during erythropoiesis was highlighted in mice with inactivating mutation in the SCF (Sl/Sl mice) or Kit gene (W/W mice) [2,3]. Mutant mice die in utero between day 14–16 of gestation with anemia and a profoundly reduced number of erythroid progenitors in fetal liver demon- strating the proliferative function mediated by Kit during early stages of erythropoiesis. Likewise, mice with null mutations in the genes encoding either Epo or EpoR die at midgestation with a severe anemia. Fetal livers from these mice contain BFU-E and CFU-E progenitors, although in reduced number, indicating that the Epo/EpoR pathway is crucial in regulating survival, proliferation and terminal differentiation of CFU-E [4]. Thus, Epo and SCF are growth factors working synergistically to support erythropoiesis, with SCF exerting a predominant role to expand early progenitors, while Epo is acting later on to sustain maturation. Signaling induced by Epo/EpoR and SCF/Kit is determined by the temporal and spatial expression of their cognate receptors at the surface of responsive cells. Kit is expressed from the earliest committed erythroid progenitor up to the basophilic erythroblastic stage of differentiation [5,6]. EpoR expression arises at the BFU-E stage, reaches a maximum at the CFU-E and proerythroblast stages and declines thereafter [7,8]. In an attempt to dissect the signaling determinants controlling the expression of EpoR and Kit, we used proerythroblastic cell lines isolated during the preleukemic step of erythroleukemia developing in spi-1 transgenic mice [9]. The spi-1 gene encodes the ETS transcription factor Spi-1/PU.1, a main player regulating the commitment of multipotent hematopoietic progenitors and the development of the B lymphoid and monocytic lineages [10–13]. Germline overexpression of the spi-1 transgene induces a differentiation arrest in the erythroid lineage at the CFU-E/ proerythroblast transition leading to severe anemia [9,14]. In response to anemia, Epo production is up-regulated [15] causing a massive expansion of proerythroblasts in the hematopoietic tissues of diseased mice. It is likely that SCF expressed by stromal cells in spleen and marrow microenvironments also contributes to the expansion of these proerythroblasts. Indeed, spi-1 transgenic proerythroblasts express both Epo and SCF receptors and can be expanded in vitro in the presence of Epo or SCF. Using cell lines established from the spleen of various diseased mice, we observed that each of these cell lines exhibited a particular growth rate in response to either Epo alone or SCF alone, and expressed EpoR and Kit in a ratio modulated by the cytokine used to sustain their proliferation. Starting from this observation, we investigated the molecular mechanisms control- ling the expression of Kit and EpoR. We show that Epo down- PLoS ONE | www.plosone.org 1 May 2009 | Volume 4 | Issue 5 | e5721