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, 36 (9), 1110-20

E3 Ligase FLRF (Rnf41) Regulates Differentiation of Hematopoietic Progenitors by Governing Steady-State Levels of Cytokine and Retinoic Acid Receptors

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E3 Ligase FLRF (Rnf41) Regulates Differentiation of Hematopoietic Progenitors by Governing Steady-State Levels of Cytokine and Retinoic Acid Receptors

Xin Jing et al. Exp Hematol.

Abstract

Objective: FLRF (Rnf41) gene was identified through screening of subtracted cDNA libraries form murine hematopoietic stem cells and progenitors. Subsequent work has revealed that FLRF acts as E3 ubiquitin ligase, and that it regulates steady-state levels of neuregulin receptor ErbB3 and participates in degradation of IAP protein BRUCE and parkin. The objective of this study was to start exploring the role of FLRF during hematopoiesis.

Materials and methods: FLRF was overexpressed in a murine multipotent hematopoietic progenitor cell line EML, which can differentiate into almost all blood cell lineages, and in pro-B progenitor cell line BaF3. The impact of FLRF overexpression on EML cell differentiation into myeloerythroid lineages was studied using hematopoietic colony-forming assays. The interaction of FLRF with cytokine receptors and receptor levels in control cells and EML and BaF3 cells overexpressing FLRF were examined with Western and immunoprecipitation.

Results: Remarkably, overexpression of FLRF significantly attenuated erythroid and myeloid differentiation of EML cells in response to cytokines erythropoietin (EPO) and interleukin-3 (IL-3), and retinoic acid (RA), and resulted in significant and constitutive decrease of steady-state levels of IL-3, EPO, and RA receptor-alpha (RARalpha) in EML and BaF3 cells. Immunoprecipitation has revealed that FLRF interacts with IL-3, EPO, and RARalpha receptors in EML and BaF3 cells, and that FLRF-mediated downregulation of these receptors is ligand binding-independent.

Conclusions: The results of this study have revealed new FLRF-mediated pathway for ligand-independent receptor level regulation, and support the notion that through maintaining basal levels of cytokine receptors, FLRF is involved in the control of hematopoietic progenitor cell differentiation into myeloerythroid lineages.

Figures

Figure 1
Figure 1
The model of the maintenance and multilineage differentiation of a multipotent hematopoietic cell line EML. In the presence of SCF EML cells undergo proliferative self-renewal and remain undifferentiated, whereas in the presence of cytokines and/or stroma EML cells differentiate into erythroid, myeloid and lymphoid lineages.
Figure 2
Figure 2
The expression of FLRF protein in murine and human hematopoietic cell lines. A. Detection of mouse and human FLRF protein in EML cells, human myeloid cell line KG-1, and mouse pre-B cell line 70Z3 with rabbit α-FLRF Abs 1123 and 1124. B. Protein immunoprecipitation (IP) with α-FLRF Abs 1123 and 1124 and subsequent reciprocal Western analysis have shown that both α-FLRF Abs recognize the 37 kDa protein in EML cells and mouse pro-B cell line BaF3.
Figure 3
Figure 3
Over-expression of FLRF attenuates myelo-erythroid differenmtiation of EMl cells. A. Western analysis of FLRF protein in wt EML cells, vector control EML/pcDNA cells, and two EML cell clones over-expressing FLRF (EML/FLRF). Fifty μg of protein from each cell sample were loaded per lane. The blots were stripped and re-probed with α-actin Ab. B. Comparison of the differentiation capacity and generation of granulocyte/macrophage (CFU-GM), erythroid (BFU-E) and megakaryocytic (CFU-Meg) colonies by wild type EML, vector control EML/pcDNA cells and EML/FLRF cells. C. Comparison of the CFU-GM, BFU-E and CFU-Meg colony size generated by wild type EML, EML/pcDNA and EML/FLRF cells.
Figure 4
Figure 4
FLRF associates with and regulates basal levels of cytokine receptors in EML cells. A. Western analysis of Epo and IL-3 receptor levels in wt EML, EML/pcDNA and EML/FLRF cells. Over-expression of FLRF leads to constitutively reduced steady-state levels of Epo, IL-3Rα and IL-3β receptors in EML/FLRF cells, independent of the treatment with Epo or IL-3. Equal amounts (50 μg) of protein from each cell sample were loaded per lane. The blots were stripped and re-probed with α-actin Ab. B. Western analysis of basal levels of c-kit receptor protein in undifferentiated wt EML, EML/pcDNA and EML/FLRF cells has revealed that over-expression of FLRF does not have any effect on the levels of c-kit protein in EML cells. Protein extracts from triplicate cultures of wt EML, EML/pcDNA and EML/FLRF cells were analyzed with goat α-c-kit M-14 antibody. The blots were stripped and re-probed with α-actin Ab. C. Protein IP with IL-3R and EpoR Abs has shown that FLRF associates with EpoR and both IL-3Rα and IL-3Rβ receptor subunits in wt EML, EML/pcDNA and EML/FLRF cells, both prior to and after exposure to cytokines. D. Epo and IL-3 receptors in EML/FLRF cells are associated with increased levels of poly-ubiquitin chains prior to and after the treatment with Epo or IL-3.
Figure 4
Figure 4
FLRF associates with and regulates basal levels of cytokine receptors in EML cells. A. Western analysis of Epo and IL-3 receptor levels in wt EML, EML/pcDNA and EML/FLRF cells. Over-expression of FLRF leads to constitutively reduced steady-state levels of Epo, IL-3Rα and IL-3β receptors in EML/FLRF cells, independent of the treatment with Epo or IL-3. Equal amounts (50 μg) of protein from each cell sample were loaded per lane. The blots were stripped and re-probed with α-actin Ab. B. Western analysis of basal levels of c-kit receptor protein in undifferentiated wt EML, EML/pcDNA and EML/FLRF cells has revealed that over-expression of FLRF does not have any effect on the levels of c-kit protein in EML cells. Protein extracts from triplicate cultures of wt EML, EML/pcDNA and EML/FLRF cells were analyzed with goat α-c-kit M-14 antibody. The blots were stripped and re-probed with α-actin Ab. C. Protein IP with IL-3R and EpoR Abs has shown that FLRF associates with EpoR and both IL-3Rα and IL-3Rβ receptor subunits in wt EML, EML/pcDNA and EML/FLRF cells, both prior to and after exposure to cytokines. D. Epo and IL-3 receptors in EML/FLRF cells are associated with increased levels of poly-ubiquitin chains prior to and after the treatment with Epo or IL-3.
Figure 5
Figure 5
FLRF associates with and regulates basal levels of cytokine receptors in a panel of BaF3 pro-B progenitor cell lines. Protein IP with IL-3R, EpoR and FLRF Abs has shown that FLRF associates with IL-3 receptor in BaF3, BaF3/EpoR and BaF3/Mpl cells (A and B), and with Epo receptor in BaF3/EpoR cells (B). C. Transient over-expression of FLRF in BaF3/EpoR/FLRF and BaF3/Mpl/FLRF cells leads to reduced levels of Epo and IL-3 receptor proteins. BaF3/EpoR and BaF3/Mpl cells are mock controls. BaF3/EpoR/pcDNA and BaF3/Mpl/pcDNA are cells transfected with control vector. The blots were stripped and re-probed with α-actin Ab.
Figure 6
Figure 6. Retinoic acid further attenuates myeloid differentiation of EML cells over-expressing FLRF
Wild type EML, EML/pcDNA and EML/FLRF cells were cultured for 20 hours with SCF alone (control), or in the presence of SCF and RA (10-5 M), or SCF, RA and IL-3. The cells were than plated into agarose cultures supplemented with IL-3 to examine their differentiation into granulocyte/macrophage (CFU-GM) progenitors. A. Generation of CFU-GM colonies by control wt EML, EML/pcDNA and EML/FLRF cells (SCF), and cells cultured for 20 hours with SCF and RA, or with SCF, RA and IL-3. Treatment with RA increases the number of CFU-GM colonies produced by wt EML and EML/pcDNA cells. In contrast, RA treatment further decreased the number of CFU-GM colonies produced by EML/FLRF cells. B. Western analysis of RARα and RXR receptor proteins in untreated control wt EML, EML/pcDNA and EML/FLRF cells (SCF), and cells cultured for 20 hours with SCF and RA, or with SCF, RA and IL-3. C. Western analysis of RARα and RXR receptor proteins in control wt EML, EML/pcDNA and EML/FLRF cells, and cells cultured for 20 hours with IL-3. D. Transient over-expression of FLRF in four BaF3 cell clones (BaF3/FLRF 1-4) decreased the amount of RARα receptor protein, whereas the levels of RXR receptor remained unchanged. The blots were stripped and re-probed with α-actin Ab. E. Reciprocal IP of proteins from EML and BaF3 cells with α-FLRF, α-RARα and α-RXR Abs has shown that endogenous FLRF protein associates with endogenous RARα and RXR receptor proteins in EML and BaF3 cells.

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