Figure 5
Both ND EBs exposed to excess glucocorticoids (by costimulation with erythropoietin and glucocorticoids) and PV EBs expressing the dominant negative GRβ isoform exhibit impaired functional GR/STAT-5 nuclear interaction. Erythroblasts were generated at day 10 of HEMA (Prol) and analyzed either as such or after growth factor deprivation for 4 hours (GFD) followed by treatment with erythropoietin (EPO; 3 U/mL) and dexamethasone (DXM; 10−6M), alone or in combination. (A) Erythroblasts obtained from 1 ND and 1 PV patient were immunoprecipitated (IP) with either STAT-5– or GRα-specific antibodies and the immunoprecipitations were analyzed by WB with either anti–STAT-5 or anti-GRα, as loading control, or with anti–STAT-5pY and anti-GRβ antibody. Similar results were obtained in 3 additional experiments, each with a separate donor. (B) EMSA with STAT-5–specific labeled probes of nuclear extract of EBs from 1 ND and 1 PV patient with (+) and without (−) DXM for 24 hours. Lane 1 is labeled probe only; lane 2, labeled probe with ND EBs without DXM; lane 3, labeled probe with ND EBs treated with DXM; lane 4, labeled probe with PV EBs treated with DXM; and lane 5, labeled probes with PV EBs that competed with excessive unlabeled probe. The position of the STAT-5–bound and –free probe is indicated by arrows. Similar results were obtained in 3 additional experiments. (C) WB analyses for GILZ and GAPDH (as loading control) expression of EBs generated at day 10 of HEMA (Prol) from ND or PV as indicated and analyzed either as such (Prol) or after growth factor deprivation for 4 hours (GFD) followed by treatment with erythropoietin (EPO; 3 U/mL) and DXM (10−6M), alone or in combination. Similar results were obtained in 3 additional experiments, each with a separate donor. Murine 293T cells overexpressing GILZ were analyzed as positive control. (D) Proposed model for development of erythrocytosis because of inhibition of GR/STAT-5 interactions by exposure to excess glucocorticoids and expression of the dominant negative GRβ isoform. In patients chronically stimulated with glucocorticoids who express GRα only, costimulation with erythropoietin and dexamethasone impairs regulation of GR/STAT-5–responsive genes by inhibiting STAT-5 phosphorylation and formation of GR/STAT-5 tetrameric complexes (A) and reducing the DNA-binding activity of STAT-5 (B). In PV, STAT-5 is constitutively phosphorylated by JAK2V617F (A) but cannot form complexes with GRα because this protein is largely retained in the nucleus as GRβ heterodimer (B). Therefore, in PV EBs, the DNA-binding activity is reduced (B), and expression of GR/STAT-5–responsive genes, such as GILZ, is also defective (C).

Both ND EBs exposed to excess glucocorticoids (by costimulation with erythropoietin and glucocorticoids) and PV EBs expressing the dominant negative GRβ isoform exhibit impaired functional GR/STAT-5 nuclear interaction. Erythroblasts were generated at day 10 of HEMA (Prol) and analyzed either as such or after growth factor deprivation for 4 hours (GFD) followed by treatment with erythropoietin (EPO; 3 U/mL) and dexamethasone (DXM; 10−6M), alone or in combination. (A) Erythroblasts obtained from 1 ND and 1 PV patient were immunoprecipitated (IP) with either STAT-5– or GRα-specific antibodies and the immunoprecipitations were analyzed by WB with either anti–STAT-5 or anti-GRα, as loading control, or with anti–STAT-5pY and anti-GRβ antibody. Similar results were obtained in 3 additional experiments, each with a separate donor. (B) EMSA with STAT-5–specific labeled probes of nuclear extract of EBs from 1 ND and 1 PV patient with (+) and without (−) DXM for 24 hours. Lane 1 is labeled probe only; lane 2, labeled probe with ND EBs without DXM; lane 3, labeled probe with ND EBs treated with DXM; lane 4, labeled probe with PV EBs treated with DXM; and lane 5, labeled probes with PV EBs that competed with excessive unlabeled probe. The position of the STAT-5–bound and –free probe is indicated by arrows. Similar results were obtained in 3 additional experiments. (C) WB analyses for GILZ and GAPDH (as loading control) expression of EBs generated at day 10 of HEMA (Prol) from ND or PV as indicated and analyzed either as such (Prol) or after growth factor deprivation for 4 hours (GFD) followed by treatment with erythropoietin (EPO; 3 U/mL) and DXM (10−6M), alone or in combination. Similar results were obtained in 3 additional experiments, each with a separate donor. Murine 293T cells overexpressing GILZ were analyzed as positive control. (D) Proposed model for development of erythrocytosis because of inhibition of GR/STAT-5 interactions by exposure to excess glucocorticoids and expression of the dominant negative GRβ isoform. In patients chronically stimulated with glucocorticoids who express GRα only, costimulation with erythropoietin and dexamethasone impairs regulation of GR/STAT-5–responsive genes by inhibiting STAT-5 phosphorylation and formation of GR/STAT-5 tetrameric complexes (A) and reducing the DNA-binding activity of STAT-5 (B). In PV, STAT-5 is constitutively phosphorylated by JAK2V617F (A) but cannot form complexes with GRα because this protein is largely retained in the nucleus as GRβ heterodimer (B). Therefore, in PV EBs, the DNA-binding activity is reduced (B), and expression of GR/STAT-5–responsive genes, such as GILZ, is also defective (C).

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal