Role of erythropoietin receptor signaling in Friend virus-induced erythroblastosis and polycythemia

Abstract

Friend virus is an acutely oncogenic retrovirus that causes erythroblastosis and polycythemia in mice. Previous studies suggested that the Friend virus oncoprotein, gp55, constitutively activates the erythropoietin receptor (EPOR), causing uncontrolled erythroid proliferation. Those studies showed that gp55 confers growth factor independence on an interleukin-3 (IL-3)-dependent cell line (Ba/F3) when the EPOR is coexpressed. Subsequently, we showed that a truncated form of the stem-cell kinase receptor (sf-STK) is required for susceptibility to Friend disease. Given the requirement for sf-STK, we sought to establish the in vivo significance of gp55-mediated activation of the EPOR. We found that the cytoplasmic tyrosine residues of the EPOR, and signal transducer and activator of transcription-5 (STAT5), which acts through these sites, are not required for Friend virus-induced erythroblastosis. The EPOR itself was required for the development of erythroblastosis but not for gp55-mediated erythroid proliferation. Interestingly, the murine EPOR, which is required for gp55-mediated Ba/F3-cell proliferation, was dispensable for erythroblastosis in vivo. Finally, gp55-mediated activation of the EPOR and STAT5 are required for Friend virus-induced polycythemia. These results suggest that Friend virus activates both sf-STK and the EPOR to cause deregulated erythroid proliferation and differentiation.

Figure 1.

The distal EPOR and STAT5 are not required for Friend virus-induced erythroblastosis. (A) Photograph of spleens from FVA-infected mice. Mouse strains are indicated at the top. Infection with Friend virus is indicated at the bottom (FVA). (B) Photomicrograph of spleen from Friend virus-infected EporHM mouse, stained with hematoxylin and eosin. Original magnification × 400. (C) Spleen weights of FVA-infected mice in grams. The mouse strains are indicated at the bottom. Stat5 (-/-) denotes the Stat5a^-/-;Stat5b^-/- strain. The spleen weights of uninfected mice are provided as a control (no FV). Error bars represent standard deviation (Stat5 (-/-), EporH, EporHM, P < .001). (D) Percent transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive cells in the spleens of FVA-infected mice (Stat5 (-/-), P = .07; EporH, P > .10; EporHM, P = .10).

Figure 2.

The EPOR is not required for gp55-mediated erythroid proliferation. GFP-positive Fv2^s/s;Epor^-/- fetal liver cells were transplanted into Fv2^r/r recipients. (A) Flow cytometry was performed 4 weeks after transplantation to assess bone marrow reconstitution. Three subpanels correspond to granulocytes (i), platelets (ii), and erythrocytes (iii). The percentage of GFP-positive cells is indicated by the number above the line. (B-G) Photomicrographs of FVA-infected spleens. The sections in panels B, D, and F were stained with hematoxylin and eosin. The sections in panels C and E were stained with anti-GFP antibody and counterstained with hematoxylin. The section in panel G was stained with Rauscher anti-gp69/71 antibody. Original magnifications × 20 (B-C), × 400 (D-E), and × 100 (F-G).

Figure 3.

The murine EPOR is not required for Friend virus-induced erythroblastosis. (A) Southern blot of genomic DNA from the progeny of Epor^-/-;Tg(EPOR) × Balb/cByJ Epor^+/- mice. The parents contained different targeted mutations of the Epor,, which are indicated by the arrowheads (Epor KO-1 and Epor KO-2). Bands corresponding to the germ-line band and the human EPOR transgene (Tg(EPOR)), are indicated. Genotypes: lane 1, Epor^+/-;Tg(EPOR); lane 2, wild type; lane 3, Epor^-/-;Tg(EPOR). (B) Spleen weights of FVA-infected mice in grams. Genotypes are indicated at the bottom. Epor^+/+ and Epor^+/- genotypes are grouped together as (+/^). Error bars represent standard deviations.

Figure 4.

STAT5 activation is required for Friend virus-induced polycythemia. Hematocrits of different strains of mice are shown at baseline (pre-FVP) and 3 to 4 weeks after infection with FVP (post-FVP). The mouse strains and genotypes are indicated at the top. In each panel, the pre-FVP hematocrits are shown on the left and the post-FVP hematocrits on the right with a line connecting the values. Low hematocrit values were occasionally caused by internal bleeding.

Figure 5.

FVP constitutively activates JAK2 and STAT5. Whole-cell extracts of Friend virus-infected erythroblasts were immunoprecipitated with anti-JAK2 (top group) or anti-STAT5A (bottom group) antibody and Western blotted. The antibody used for the Western blot is indicated to the left of the panels. The strain of mice, strain of Friend virus, and stimulation with erythropoietin are indicated at the top.

Figure 6.

Mechanism of Friend virus-induced erythroblastosis and polycythemia. gp55 of the anemia-(gp55_A) or polycythemia-inducing (gp55_P) strains of Friend virus activates sf-STK, causing uncontrolled erythroid proliferation and erythroblastosis. gp55_P activates the EPOR, JAK2, and STAT5, causing deregulated erythroid differentiation and polycythemia.