Disrupted Signaling in a Mutant J2E Cell Line That Shows Enhanced Viability, but Does Not Proliferate or Differentiate, with Erythropoietin

Abstract

The immature erythroid J2E cell line proliferates and terminally differentiates following erythropoietin stimulation. In contrast, the mutant J2E-NR clone does not respond to erythropoietin by either proliferating or differentiating. Here we show that erythropoietin can act as a viability factor for both the J2E and J2E-NR lines, indicating that erythropoietin-initiated maturation is separable from the prevention of cell death. The inability of J2E-NR cells to mature in response to erythropoietin was not due to a defect in the erythropoietin receptor sequence, although surface receptor numbers were reduced. Both the receptor and Janus kinase 2 were phosphorylated after erythropoietin stimulation of J2E-NR cells. However, protein interactions with the erythropoietin receptor and Grb2 were restricted in the mutant cells. Subsequent investigation of several other signaling molecules exposed numerous alterations in J2E-NR cells; phosphorylation changes to phosphatidylinositol 3-kinase, phospholipase C␥, p120 GAP, and mitogen-activated protein kinases (p42 and p44) observed in erythropoietin-stimulated J2E cells were not seen in the J2E-NR line. These data indicate that some pathways activated during erythropoietin-induced differentiation may not be essential for the prevention of apoptosis.

Dominant action of mutated erythropoietin receptors on differentiation in vitro and erythroleukemia development in vivo

“…Moreover, epo-initiated viability and proliferation were inhibited in these cells (Figure 5b,c). Since J2E-NR cells do not proliferate or di erentiate, but remain alive, in response to epo (Klinken and Nicola, 1990;Tilbrook et al, 1996a), a dose response curve was generated to test the e ects of the D321 receptor on the viability of J2E-NR cells. Figure 5d shows that epo-induced viability of cells with D321 receptor was enhanced compared with J2E-NR cells.…”

“…These phosphorylated tyrosines act as docking sites for signaling proteins including Signal Transducer and Activator of Transcription 5 (STAT 5), the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) and phosphatases SHP-1 and SHP-2 (Damen et al, 1995a,b;Gobert et al, 1996;Klingmuller et al, 1995Klingmuller et al, , 1996Klingmuller et al, , 1997Penta and Sawyer, 1995;Quelle et al, 1996;Sharlow et al, 1997;Tauchi et al, 1995Tauchi et al, , 1996Yi et al, 1995). Although Shc and Grb2 bind to the epo receptor and stimulate the Ras/Raf/MAP-kinase pathway (Barber et al, 1997;Bittorf et al, 1994;Carroll et al, 1991;Cutler et al, 1993;Gobert et al, 1995;He et al, 1995;Miura et al, 1994b;Tilbrook et al, 1996a;Torti et al, 1992), their precise binding locations on the receptor have not been identi®ed.…”

“…Despite being immortalized at the proerythroblast/basophilic erythroblast stage of erythroid development, J2E cells still respond to epo in vitro by di erentiating biochemically with hemoglobin production, and morphologically by undergoing cellular alterations which result in a proportion of cells enucleating to form reticulocytes (Bus®eld et al, 1993a,b;Bus®eld and Klinken, 1992;Klinken et al, 1988). In addition, exposure to epo promotes cell division and maintains viability in the absence of serum (Chappell et al, 1997;Tilbrook et al, 1996a). Despite being transformed, epo signaling in J2E cells appears perfectly normal ± these cells express the characteristic 1000 epo receptors on the surface of erythroid cells (Klinken et al, 1988) and the receptor, JAK2, STAT5, ras-GAP, PI-3 kinase and MAP-kinase all show typical phosphorylation kinetics after hormonal stimulation (Tilbrook et al, 1996a,b).…”

“…These cells produce a rapid, fatal erythroleukemia when introduced into mice, but the pathogenesis can be ameliorated by ex vivo epo treatment (Farr et al, 1995). In contrast with J2E cells, the mutant J2E-NR subclone remains alive, does not mature or divide in response to epo (Klinken and Nicola, 1990;Tilbrook et al, 1996a), due to a lack of the tyrosine kinase lyn . Additionally, the J2E-NR cells contain 300 ± 500 surface epo receptors (Klinken and Nicola, 1990).…”

Prevention of apoptosis in J2E erythroid cells by erythropoietin: involvement of JAK2 but not MAP kinases

“…Studies on Friend virus-infected erythroid cells and the epo-dependent HCD-57 line have shown that epo retards DNA breakdown and maintains cell viability Bondurant, 1988, 1990;Spivak et al, 1991). Our recent studies have indicated that distinct epo-induced signalling pathways may exist in cells which lead to cell division, maturation and maintenance of viability (Tilbrook et al, 1996a). In addition, IL-2, IL-3 and GM ± CSF transmit signals that promote cell survival, independent of initiating cell division (Okuda et al, 1994;Inhorn et al, 1995;Kinoshita et al, 1995;Boise et al, 1995).…”