CD71 up-regulation is an EpoR-activated stress response that requires the high-intensity, graded p-Stat5 signal.

<p>(A) Erythropoietic stress causes up-regulation of cell-surface CD71 on spleen EryA erythroblasts (CD71^highTer119^highFSC^high). At t = 0, Balb/C mice were injected with Epo subcutaneously (300 U/ml, left panel) or placed in an hypoxia chamber (11% oxygen, right panel). Spleen was harvested at the indicated time points. Cell-surface CD71 was measured using flow-cytometry in EryA. Data points are MFI ± sem for three to six mice per time point, expressed as relative fluorescence units. (B) A dose/response curve in vivo of CD71 up-regulation in response to stress levels of Epo. Balb/C mice were injected with a single subcutaneous Epo dose as indicated. Spleen was harvested at t = 24 h post-injection, and EryA CD71 was measured by flow cytometry. Data points are MFI ± sem of three to eight mice per Epo dose, pooled from two independent experiments, and expressed relative to CD71 levels at t = 0. Bone-marrow EryA CD71 increased similarly. (C–E) EpoR-HM mice fail to up-regulate CD71 and to increase erythropoietic rate in response to stress levels of Epo. Adult EpoR-HM mice or strain-matched wild-type mice were injected with a single Epo dose (100 U/25 g mouse) or with saline control. Cell surface CD71 (C, D) and hematocrit (E) were measured at the indicated time points. (C) Representative CD71 flow-cytometry histograms of spleen EryA, in either wild-type (WT) or EpoR-HM (HM) mice, injected with either saline or Epo, at 24 h post-injection. Two mice are shown for each condition. (D) Summary of data measured as in (C), for HM and WT mice injected with either Epo or saline, at 18 and 24 h post-injection. Each datapoint represents CD71 levels (MFI) in spleen EryA from a single mouse. Two to five mice are shown per time point/genotype combination. (E) Hematocrit response to Epo injection. The hematocrit increases in the wild-type, but not in the EpoR-HM mouse. (F) Rescue of stress-induced CD71 up-regulation in EpoR-HM fetal liver cells after rescue of high-intensity graded p-Stat5 signaling by transduction with high levels of FLAG-Stat5. EpoR-HM or wild-type fetal liver erythroblasts were transduced with either FLAG-Stat5 or FLAG-Stat5Y694, as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001383#pbio-1001383-g005" target="_blank">Figure 5A</a>. Cells were incubated overnight in the presence of stress-levels of Epo (0.2 U/ml; this is 6- to 10-fold the Epo basal levels), and were then labeled and analyzed for CD71, Ter119, and FLAG expression by flow-cytometry. Datapoints are CD71 (MFI ± sem) for each FLAG vertical gate, as illustrated in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001383#pbio-1001383-g005" target="_blank">Figure 5B</a>, following correction for background fluorescence by subtracting the corresponding fluorescence for each FLAG gate of samples transduced with FLAG-Stat5Y694F.