Growth Factor-Independent Proliferation of Erythroid Cells Infected with Friend Spleen Focus-Forming Virus Is Protein Kinase C Dependent but Does Not Require Ras-GTP

Abstract

Interaction of erythropoietin (Epo) with its cell surface receptor activates signal transduction pathways which result in the proliferation and differentiation of erythroid cells. Infection of erythroid cells with the Friend spleen focus-forming virus (SFFV) leads to the interaction of the viral envelope glycoprotein with the Epo receptor and renders these cells Epo independent. We previously reported that SFFV induces Epo independence by constitutively activating components of several Epo signal transduction pathways, including the Jak-Stat and the Raf-1/mitogen-activated protein kinase (MAPK) pathways. To further evaluate the mechanism by which SFFV activates the Raf-1/MAPK pathway, we investigated the effects of SFFV on upstream components of this pathway, and our results indicate that SFFV activates Shc and Grb2 and that this leads to Ras activation. While studies with a dominant-negative Ras indicated that Ras was required for Epo-induced proliferation of normal erythroid cells, the Epo-independent growth of SFFV-infected cells can still occur in the absence of Ras, although at reduced levels. In contrast, protein kinase C (PKC) was shown to be required for the Epo-independent proliferation of SFFV-infected cells. Further studies indicated that PKC, which is thought to be involved in the activation of both Raf-1 and MAPK, was required only for the activation of MAPK, not Raf-1, in SFFV-infected cells. Our results indicate that Ras and PKC define two distinct signals converging on MAPK in both Epo-stimulated and SFFV-infected erythroid cells and that activation of only PKC is sufficient for the Epo-independent proliferation of SFFV-infected cells.Infection of erythroid cells with the Friend spleen focusforming virus (SFFV) induces a rapidly occurring erythroleukemia in susceptible adult mice due to expression of its unique envelope gene (for a review, see reference 56). The SFFV envelope glycoprotein associates with the Epo receptor at the cell surface (8, 23) and is thought to be responsible for the proliferation of SFFV-infected erythroid cells in the absence of its normal regulator, erythropoietin (Epo). Interactions between the SFFV envelope glycoprotein and the Epo receptor may alter cell growth by activating signal transduction pathways that are normally regulated by Epo.Previous studies have shown that growth factor stimulation of the Epo receptor activates several signal transduction pathways including the Jak-Stat pathway (24,44,51,53,69), the Raf-1/mitogen-activated protein kinase (MAPK) pathway (6,15,45,65,66), and components of the phosphatidylinositol 3-kinase (PI 3-kinase) pathway (14,27,28,31,32,41,43). Using the Epo-dependent HCD-57 erythroleukemia cell line, we have previously demonstrated that infection with SFFV, which abrogates the Epo dependence of HCD-57 cells (55), activates Stat DNA-binding activity in the absence of Epo (51) and constitutively activates components of the serine/threonine kinase cascade in the Raf-1/MAPK pathway, specifically, Raf-1, MAPK kinase (MEK), and MAPK (48).A...

Induction of erythroid differentiation by inhibition of Ras/ERK pathway in a Friend murine leukemia cell line

“…These ®ndings imply that basal activity of Ras/ERK signaling pathway plays a major role in preventing di erentiation and sustaining proliferative potential of these cells. Muszynski et al (1998) recently reported that Friend murine leukemia virus (FV) may elevate the basal activity of Raf-1/MAPK pathway in infected cells, which o ers an interesting hypothesis on the molecular mechanism of leukemogenesis by this virus. Consistent with this hypothesis, we observed that super-activation of Ras/ERK signaling pathway, through expression of Ras12V or MAPKKm, prevented the di erentiation induced by EPO, DMSO, or n-butyrate.…”

“…Transient activation of Jak/ STAT pathway as well as Ras/MAP kinase (MAPK) pathways after EPO-stimulation has been documented (Miura et al, 1994;Sawyer and Penta, 1996;Quelle et al, 1996;Torti et al, 1992;Todokoro et al, 1994;Tilbrook et al, 1996;Gobert et al, 1995;Nagata et al, 1997). Among various EPO-dependent cell lines, good correlation has been observed between the basal activity of these signaling pathways and the proliferative potential of the cells (Gobert et al, 1995;Tilbrook et al, 1996;Miura et al, 1994;Carroll and May, 1994;Muszynski et al, 1998).…”

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

“…35,36 However, experiments described in these reports used Spi-1 overexpression in erythroleukemic cells harboring a deregulation in Epo/EpoR signaling, 34,37,38 thus impeding a clear definition of the contribution of Spi-1 in the differentiation blockage. 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.…”

Oncogene cooperativity in Friend erythroleukemia: erythropoietin receptor activation by the env gene of SFFV leads to transcriptional upregulation of PU.1, independent of SFFV proviral insertion

“…A functional F-gp55 activated EpoR signal was required for PU.1 transcriptional upregulation in Friend erythroleukemia cells Accumulating evidence indicates that F-gp55 activated EpoR signals di er from Epo activated EpoR signals (Tarr et al, 1997;Muszynski et al, 2000;Nishigaki et al, 2000). Speci®cally F-gp55/EpoR activated PKC activity is required for proliferation of Friend erythroleukemia cells .…”

“…Panel on the right is an immunoblot analysis for PU.1 protein levels in cells following Zn treatment (+) or without Zn treatment (7). Equal amounts of total protein were loaded in each lane way, the lipid phosphatase SHIP, and PKC occur in SFFV infected erythroid cells, tyrosine phosphorylation of the EpoR and activation of JAK2 appear to not be required for proliferation of SFFV infected cells (Muszynski et al, 1998;Muszynski et al, 2000;Nishigaki et al, 2000). Activation of PI3K signaling pathways in SFFV infected cells occurs through tyrosine phosphorylated Insulin Receptor SubstrateRelated (IRS) adapter proteins (Nishigaki et al, 2000).…”