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In vivo regulation of erythropoiesis by chemically inducible dimerization of the erythropoietin receptor intracellular domain.

Suzuki N, Mukai HY, Yamamoto M - PLoS ONE (2015)

Bottom Line: This conformational change is sufficient for the initiation of Epo-EpoR signal transduction.Because transgene expression is limited to hematopoietic cells by the Gata1 gene regulatory region, the effect of the CID is limited to erythropoiesis without adverse effects.Additionally, we show that the 160 amino acid sequence is the minimal essential domain of EpoR for intracellular signaling of chemically inducible erythropoiesis in vivo.

View Article: PubMed Central - PubMed

Affiliation: Division of Interdisciplinary Medical Science, Center for Oxygen Medicine, United Centers for Advanced Research and Translational Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan.

ABSTRACT
Erythropoietin (Epo) and its receptor (EpoR) are required for the regulation of erythropoiesis. Epo binds to the EpoR homodimer on the surface of erythroid progenitors and erythroblasts, and positions the intracellular domains of the homodimer to be in close proximity with each other. This conformational change is sufficient for the initiation of Epo-EpoR signal transduction. Here, we established a system of chemically regulated erythropoiesis in transgenic mice expressing a modified EpoR intracellular domain (amino acids 247-406) in which dimerization is induced using a specific compound (chemical inducer of dimerization, CID). Erythropoiesis is reversibly induced by oral administration of the CID to the transgenic mice. Because transgene expression is limited to hematopoietic cells by the Gata1 gene regulatory region, the effect of the CID is limited to erythropoiesis without adverse effects. Additionally, we show that the 160 amino acid sequence is the minimal essential domain of EpoR for intracellular signaling of chemically inducible erythropoiesis in vivo. We propose that the CID-dependent dimerization system combined with the EpoR intracellular domain and the Gata1 gene regulatory region generates a novel peroral strategy for the treatment of anemia.

No MeSH data available.


Related in: MedlinePlus

CID Dose- and Transgene Expression Level-dependent Colony Formation of G1HRD-idEpoRic Bone Marrow Cells.(A) Increased CFU-E—derived colony formation in response to incremental doses of CID or rHuEPO. Bone marrow MNCs from line A transgenic mice were used. (B) The bone marrow MNCs of 3 independent transgenic mouse lines were cultured in methylcellulose medium supplemented with CID (1.0 nmol/mL) or rHuEPO (2.0 U/mL) for 3 days, and the numbers of CFU-E—derived colonies were counted. Note that the numbers of CFU-E—derived colonies correlate tightly with the expression levels of the transgene (line A > line B > line C, see Fig. 1C). These assays were performed in triplicate and repeated 3 times. The results are shown as the mean ± standard deviations.
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pone.0119442.g002: CID Dose- and Transgene Expression Level-dependent Colony Formation of G1HRD-idEpoRic Bone Marrow Cells.(A) Increased CFU-E—derived colony formation in response to incremental doses of CID or rHuEPO. Bone marrow MNCs from line A transgenic mice were used. (B) The bone marrow MNCs of 3 independent transgenic mouse lines were cultured in methylcellulose medium supplemented with CID (1.0 nmol/mL) or rHuEPO (2.0 U/mL) for 3 days, and the numbers of CFU-E—derived colonies were counted. Note that the numbers of CFU-E—derived colonies correlate tightly with the expression levels of the transgene (line A > line B > line C, see Fig. 1C). These assays were performed in triplicate and repeated 3 times. The results are shown as the mean ± standard deviations.

Mentions: To test whether the CID-idEpoRic system recapitulates the Epo-EpoR system, a conventional erythroid colony-formation assay was performed with various concentrations of CID or rHuEPO. Increasing CID as well as rHuEPO doses resulted in an increased number of CFU-E−derived colonies in the line A bone marrow cells, and the colony counts reached maximum levels (approximately 200 colonies in 1.0 × 105 cells) when the cells were incubated with 0.5 nmol/mL CID or 0.5 U/mL rHuEPO (Fig. 2A). These data show that the CID-idEpoRic system mimics the Epo-EpoR system in colony-forming assays and that the erythroid progenitors of the transgenic mice respond to CID in a dose-dependent manner.


In vivo regulation of erythropoiesis by chemically inducible dimerization of the erythropoietin receptor intracellular domain.

Suzuki N, Mukai HY, Yamamoto M - PLoS ONE (2015)

CID Dose- and Transgene Expression Level-dependent Colony Formation of G1HRD-idEpoRic Bone Marrow Cells.(A) Increased CFU-E—derived colony formation in response to incremental doses of CID or rHuEPO. Bone marrow MNCs from line A transgenic mice were used. (B) The bone marrow MNCs of 3 independent transgenic mouse lines were cultured in methylcellulose medium supplemented with CID (1.0 nmol/mL) or rHuEPO (2.0 U/mL) for 3 days, and the numbers of CFU-E—derived colonies were counted. Note that the numbers of CFU-E—derived colonies correlate tightly with the expression levels of the transgene (line A > line B > line C, see Fig. 1C). These assays were performed in triplicate and repeated 3 times. The results are shown as the mean ± standard deviations.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4366189&req=5

pone.0119442.g002: CID Dose- and Transgene Expression Level-dependent Colony Formation of G1HRD-idEpoRic Bone Marrow Cells.(A) Increased CFU-E—derived colony formation in response to incremental doses of CID or rHuEPO. Bone marrow MNCs from line A transgenic mice were used. (B) The bone marrow MNCs of 3 independent transgenic mouse lines were cultured in methylcellulose medium supplemented with CID (1.0 nmol/mL) or rHuEPO (2.0 U/mL) for 3 days, and the numbers of CFU-E—derived colonies were counted. Note that the numbers of CFU-E—derived colonies correlate tightly with the expression levels of the transgene (line A > line B > line C, see Fig. 1C). These assays were performed in triplicate and repeated 3 times. The results are shown as the mean ± standard deviations.
Mentions: To test whether the CID-idEpoRic system recapitulates the Epo-EpoR system, a conventional erythroid colony-formation assay was performed with various concentrations of CID or rHuEPO. Increasing CID as well as rHuEPO doses resulted in an increased number of CFU-E−derived colonies in the line A bone marrow cells, and the colony counts reached maximum levels (approximately 200 colonies in 1.0 × 105 cells) when the cells were incubated with 0.5 nmol/mL CID or 0.5 U/mL rHuEPO (Fig. 2A). These data show that the CID-idEpoRic system mimics the Epo-EpoR system in colony-forming assays and that the erythroid progenitors of the transgenic mice respond to CID in a dose-dependent manner.

Bottom Line: This conformational change is sufficient for the initiation of Epo-EpoR signal transduction.Because transgene expression is limited to hematopoietic cells by the Gata1 gene regulatory region, the effect of the CID is limited to erythropoiesis without adverse effects.Additionally, we show that the 160 amino acid sequence is the minimal essential domain of EpoR for intracellular signaling of chemically inducible erythropoiesis in vivo.

View Article: PubMed Central - PubMed

Affiliation: Division of Interdisciplinary Medical Science, Center for Oxygen Medicine, United Centers for Advanced Research and Translational Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan.

ABSTRACT
Erythropoietin (Epo) and its receptor (EpoR) are required for the regulation of erythropoiesis. Epo binds to the EpoR homodimer on the surface of erythroid progenitors and erythroblasts, and positions the intracellular domains of the homodimer to be in close proximity with each other. This conformational change is sufficient for the initiation of Epo-EpoR signal transduction. Here, we established a system of chemically regulated erythropoiesis in transgenic mice expressing a modified EpoR intracellular domain (amino acids 247-406) in which dimerization is induced using a specific compound (chemical inducer of dimerization, CID). Erythropoiesis is reversibly induced by oral administration of the CID to the transgenic mice. Because transgene expression is limited to hematopoietic cells by the Gata1 gene regulatory region, the effect of the CID is limited to erythropoiesis without adverse effects. Additionally, we show that the 160 amino acid sequence is the minimal essential domain of EpoR for intracellular signaling of chemically inducible erythropoiesis in vivo. We propose that the CID-dependent dimerization system combined with the EpoR intracellular domain and the Gata1 gene regulatory region generates a novel peroral strategy for the treatment of anemia.

No MeSH data available.


Related in: MedlinePlus