Alterations of the phosphoinositide 3-kinase and mitogen-activated protein kinase signaling pathways in the erythropoietin-independent Spi-1/PU.1 transgenic proerythroblasts

Abstract

During the cell transformation processes leading to erythroleukemia, erythroid progenitors often become erythropoietin (Epo)-independent for their proliferation. The biochemical events that could lead an erythroleukemic cell to growth factorindependence were investigated using spi-1 transgenic poerythroblasts. Spi-1/ PU.1 is a myeloid and B-cell transcription factor of the ETS family and is activated by insertional mutagenesis during Friend erythroleukemia. Its overexpression in proerythroblasts induces their differentiation arrest without altering their erythropoietin requirement for proliferation (HS1 cells). At a later step, genetic alterations most probably occur allowing spi-1 transgenic poerythroblasts to proliferate in the absence of erythropoietin (HS2 cells). The signaling transduction pathways in HS1 and HS2 proerythroblasts were analyzed. The authors have previously shown that the Jak/STAT pathway was not activated in Epo-independent cells, but remained sensitive to Epo stimulation. In the present study, it is shown that the Epo-independent proliferation of HS2 cells requires active phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways. In these cells, PI3K was constitutively associated with the molecular adapters Grb2 and Gab1, and with the phosphatases SHP-2 and SHIP. Moreover, PI3K activity was correlated with the constitutive phosphorylation of serine-threonine protein kinase (AKT) in HS2 cells. Lastly, a constitutive activation of the MAPKs extracellular signal-regulated kinases (ERK1/2) in HS2 cells was observed that occurs in a PI3K-independent manner, but depends strictly on the activity of the protein kinase C (PKC). These results suggest that constitutive activations of PI3K/AKT and PKC/MAPK pathways can act in synergy to lead a proerythroblast to proliferate without Epo. IntroductionErythropoietin (Epo) is involved in the survival, proliferation, and differentiation of erythroid progenitor cells. 1 The binding of Epo to its receptor (EpoR) induces the activation of a variety of downstream signaling pathways such as the Jak2/STAT5, the Shc/Ras/ MAPK, and the PI3K/AKT (serine-threonine protein kinase) signaling cascades. Upon Epo stimulation the EpoR-associated Janus kinase 2 (Jak2) 2 is activated and phosphorylates the transcriptional activator STAT5. Activation of this pathway could be involved in both proliferation 3 and differentiation of the proerythroblast. 4 The activation of the MAPKs' extracellular signal-related kinases (ERKs) 5 appears mainly involved in the proliferation of the erythroid cells, whereas activation of the stress-activated protein kinase (JNK/SAPK) 6 and the p38 MAPK 7 would be associated with Epo-induced differentiation. 8 A third signal transduction pathway activated by Epo involves the PI3K. The p85 regulatory subunit of the PI3K becomes associated through its SH2 domains to a phosphorylated tyrosine of the EpoR, 3 and the PI3K can induce activation of the cell survival protein AKT/PKB. 9 p85 also associates with several tyrosine-p...

Interleukin-1 beta Induces MUC2 Gene Expression and Mucin Secretion via Activation of PKC-MEK/ERK,and PI3K in Human Airway Epithelial Cells

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Spi-1/PU.1 participates in erythroleukemogenesis by inhibiting apoptosis in cooperation with Epo signaling and by blocking erythroid differentiation

“…In contrast, preleukemic proerythroblasts derived from spi-1 transgenic mice as used in the present study are dependent on Epo for their survival and growth, and Epo signaling is normal in that cells. 16,39 We show that reduction in Spi-1 expression was sufficient to release the arrest in differentiation, establishing that Spi-1 overexpression is the sole causal event responsible for erythroid differentiation blockage. In addition, reduction of Spi-1 levels triggered apoptotic death of a part of the preleukemic population/proerythroblasts, indicating that Spi-1 is involved in the survival of preleukemic proerythroblasts despite an active Epo signaling.…”

Subtle distinct regulations of late erythroid molecular events by PI3K/AKT-mediated activation of Spi-1/PU.1 oncogene autoregulation loop

“…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 (Nishigaki et al, 2006 and references therein). Proliferation of Epo-independent proerythroblastic cells (called HS2 cells), derived from transgenic mice overexpressing Spi-1/ PU.1, requires active PI3K/AKT and mitogen-activated protein kinase pathways (Barnache et al, 2001). Consistently, Epo initiates a transcriptional program that leads to significant change of expression levels in over 580 genes, the majority of which are apparently regulated in a PI3K-dependent manner (Sivertsen et al, 2006).…”