PU.1 Oncogene Autoregulation Loop
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Oncogene (2010) 29, 2807–2816 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00 www.nature.com/onc ORIGINAL ARTICLE Subtle distinct regulations of late erythroid molecular events by PI3K/ AKT-mediated activation of Spi-1/PU.1 oncogene autoregulation loop O Breig, O The´oleyre, A Douablin and F Baklouti mRNA Metabolism in Normal and Pathological Cells; CGMC, CNRS, Universite´ Lyon 1, Villeurbanne, France Spi-1/PU.1 oncogene is downregulated as proerythro- balance between self-renewal and differentiation in blasts undergo terminal differentiation. Insertion of the hematopoietic progenitor cells, and functions in a Friend virus upstream of the Spi-1/PU.1 locus leads to the concentration-dependent manner to promote differen- constitutive upregulation of Spi-1/PU.1, and a subsequent tiation of the lymphoid and myeloid lineages (Back block in the differentiation of the affected erythroblasts. et al., 2004; Fisher et al., 2004 and references therein). We have shown that sustained overexpression of Spi-1/ Furthermore, Spi-1/PU.1 is expressed at low levels in PU.1 also inhibits the erythroid splicing of protein 4.1R erythroid progenitor cells and subsequently downregu- exon 16, irrespective of chemical induction of differentia- lated on terminal differentiation. It acts to maintain the tion. Here, we show a positive feedback loop that couples self-renewal capacity of immature erythroid precursors constitutive phosphatidylinositol 3-kinase (PI3K)/protein and to prevent differentiation in the erythroid lineage kinase B (AKT) signaling to high expression of Spi-1/ (Back et al., 2004; Fisher et al., 2004). PU.1 in Friend erythroleukemia cells. Inhibition of PI3K/ Spi-1/PU.1 was initially discovered as a proviral AKT results in Spi-1/PU.1 downregulation in a stepwise integration site in Friend virus-induced erythroleukemia manner and induces cell differentiation. Chromatin (reviewed in Moreau-Gachelin, 2008). Friend virus immunoprecipitation assays further supported the positive complex consists of two viruses, a helper replication- autoregulatory effect of Spi-1/PU.1. Mutational analysis competent Friend murine leukemia virus (F-MuLV) indicated that Ser41, but not Ser148, is necessary for Spi- and a replication-defective spleen focus-forming virus 1/PU.1-mediated repression of hemoglobin expression, (SFFV). Friend SFFV carries a unique env gene whereas both Ser residues are required for Spi-1/PU.1 encoding a truncated form of a retroviral envelope inhibition of the erythroid splicing event. We further show glycoprotein (gp55), which is responsible for its patho- that inhibition of the erythroid transcriptional and splicing genicity (reviewed in Moreau-Gachelin, 2008). In the events are strictly dependent on distinct Spi-1/PU.1 first stage of the disease, gp55 binds and activates the phosphorylation modifications rather than Spi-1/PU.1 ex- erythropoietin receptor (Epo-R) and a short form of the pression level per se. Our data further support the fact that receptor tyrosine kinase, resulting in constitutive activa- Spi-1/PU.1 inhibits 4.1R erythroid splicing through two tion of signal transducing molecules and the develop- different pathways, and bring new insights into the extra- ment of Epo-independent erythroid hyperplasia and cellular signal impact triggered by erythropoietin on late polycythemia, due to the polyclonal expansion and erythroid regulatory program, including pre-mRNA splicing. differentiation of erythroid cells in the absence of Epo. Oncogene (2010) 29, 2807–2816; doi:10.1038/onc.2010.29; The second stage of the disease results from the published online 1 March 2010 outgrowth of Friend SFFV-infected erythroid cells that have become transformed because of integration of the Keywords: erythroleukemia; mRNA splicing; cell virus into the Spi-1 locus. This event triggers aberrant differentiation; cell signaling overexpression of the Spi-1/PU.1 protein in erythroid cells and causes a block in their differentiation and the outgrowth of transformed mouse erythroleukemia Introduction (MEL) cells (Moreau-Gachelin et al., 1989; Paul et al., 1991; Schuetze et al., 1992). Treatment of the cells with Spi-1/PU.1 is a member of the ets family of transcription polar compounds, such as dimethylsulfoxide (DMSO) factors that is expressed specifically in hematopoietic and hexamethylene bisacetamide, causes them to reenter tissues. spi-1/pu.1 gene disruption results in lethality a differentiation program best characterized by rapid (Scott et al., 1994). It is essential for controlling the downregulation of Spi-1/PU.1 and the concomitent accumulation of hemoglobin and other membrane Correspondence: Dr F Baklouti, mRNA Metabolism in Normal and erythrocyte-specific proteins (reviewed in Moreau- Pathological Cells, CGMC; CNRS UMR 5534, Universite´Lyon 1, Gachelin, 2008). Consistently, forced expression of Baˆt. Gregor Mendel, 16, rue R. Dubois, 69622 Villeurbanne Cedex, Spi-1/PU.1 blocks MEL cell differentiation (Rao et al., France. 1997), and results in inhibition of growth and differ- E-mail: [email protected] Received 28 July 2009; revised 27 December 2009; accepted 15 January entiation and apoptotic death of other erythroleukemia 2010; published online 1 March 2010 cell lines (Yamada et al., 1997). PI3K/AKT induces PU.1 autoregulation loop O Breig et al 2808 Binding of Epo to its receptor triggers the phosphor- short hairpin RNA-mediated downregulation of Spi-1/ ylation and activation of Epo-R-bound Janus kinase 2 PU.1 release the inhibition and lead to exon 16 inclusion (JAK2) tyrosine kinase, resulting in activation of several (Blaybel et al., 2008). downstream signaling pathways that include the phos- For many years, great effort has been made to phatidylinositol 3-kinase (PI3K)/protein kinase B understand how signals received by cells impact (PKB or AKT), the signal transducer and activator of transcription factor activity. In an attempt to connect transcription 5 (STAT5)-Bcl-XL and the extracellular the splicing event and its blocking by upregulated Spi-1/ signal-regulated kinase/mitogen-activated protein ki- PU.1 with the signaling pathways activated in MEL nase (reviewed in Ghaffari et al., 2003). In human cells, we here provide the first direct evidence for an erythroid progenitors, PI3K activation protects the cells Epo-generated signal that modulates an erythroid- from apoptosis and mediates Epo-induced proliferation specific splicing. Our results show that constitutive through downregulation of p25Kip1 expression (Bouscary PI3K/AKT sustains Spi-1/PU.1 autoregulated expres- et al., 2003). Activation of AKT serine threonine kinase of sion that inhibits protein 4.1R exon 16 splicing and PKB family is crucial for Epo-induced erythropoiesis erythroid differentiation, as evidenced by impaired (Myklebust et al., 2002; Bouscary et al., 2003 and globin gene expression. Inhibition of PI3K/AKT signal- references therein). This activation is mediated by PI3K ing blocks Spi-1/PU.1 autoregulation loop, and induces through phosphorylation of Ser473 on AKT. In this cell differentiation and 4.1R erythroid splicing activa- context of Epo signaling, AKT is a major effector tion. However, mutational targeting of specific serine downstream of PI3K, having fundamental roles in the residues of Spi-1/PU.1 led to uncouple the two Spi-1/ regulation of cell cycle, survival and differentiation PU.1 inhibitory effects on 4.1R splicing and globin gene (Bao et al., 1999; Bouscary et al., 2003). Moreover, AKT expression. Taking together, these data show subtle activation has been reported in SFFV-infected erythroleu- differential effects of Spi-1/PU.1 expression on late kemic cells, in which Epo-R is constitutively activated by erythroid-regulated events. the viral gp55 (Nishigaki et al., 2000). In fact, most of the signal transduction pathways induced in erythroid cells by Epo, including the Jak-STAT pathway, are constitutively activated in SFFV-transformed cells in the absence of Epo Results (Nishigaki et al., 2006 and references therein). Proliferation of Epo-independent proerythroblastic cells (called HS2 PI3K/AKT inhibition triggers erythroleukemia cells), derived from transgenic mice overexpressing Spi-1/ cell differentiation in the absence of DMSO PU.1, requires active PI3K/AKT and mitogen-activated Phosphatidylinositol 3-kinase complex is a protein protein kinase pathways (Barnache et al., 2001). Consis- dimer that consists of a p85 regulatory subunit and a tently, Epo initiates a transcriptional program that leads to p110 catalytic subunit. Earlier works have shown that significant change of expression levels in over 580 genes, p85 expression, phosphorylation and activity decline in the majority of which are apparently regulated in a PI3K- Friend cells after DMSO exposure for 96 h (Bavelloni dependent manner (Sivertsen et al., 2006). et al., 2000; Cataldi et al., 2000). Immunoblot analysis of The inclusion of exon 16 in mature protein 4.1R p85 subunit shows a reduction of the phosphorylated mRNA is probably the best-studied example of stage- form of p85 (P-p85) when the cells are treated with specific pre-mRNA splicing that characterizes the late 10–20 mM LY294002 or DMSO (Figure 1a). Activation erythroid development. It has been well documented of PI3K triggers the phosphorylation of its downstream that exon 16 is excluded in early erythroid progenitors target AKT. As shown in Figure 1b, the phosphorylated and included in late erythroid differentiated cells (Chasis AKT (Ser473) decreases on LY294002 treatment in a et al., 1993; Baklouti et al., 1996). This regulated splicing dose-dependent manner, suggesting that LY294002 is event underlies the production of a functional 4.1R; effective on both PI3K autophosphorylation and down- inclusion of the 21 amino acid peptide encoded by exon stream AKT phosphorylation. 16, at the N-terminus of the 10 kDa spectrin/actin- We then tested the effect of PI3K/AKT inhibition on binding domain of 4.1R is indeed essential to promote MEL SFFV cells. Cells were seeded at stationary phase in high mechanical stability and high deformability of the the presence of increasing concentrations of LY294002, mature red cell membrane.