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Inhibition of Thrombopoietin/Mpl Signaling in Adult Hematopoiesis Identifies New Candidates for Hematopoietic Stem Cell Maintenance.

Kohlscheen S, Wintterle S, Schwarzer A, Kamp C, Brugman MH, Breuer DC, Büsche G, Baum C, Modlich U - PLoS ONE (2015)

Bottom Line: Functional analysis of the truncated Mpl in vitro and in vivo demonstrated no internalization after Thpo binding and the inhibition of Thpo/Mpl-signaling in wildtype cells due to dominant-negative (dn) effects by receptor competition with wildtype Mpl for Thpo binding.The gene expression profile supported the exhaustion of HSC due to increased cell cycle progression and identified new and known downstream effectors of Thpo/Mpl-signaling in HSC (namely TIE2, ESAM1 and EPCR detected on the HSC-enriched LSK cell population).We further compared gene expression profiles in LSK cells of dnMpl mice with human CD34+ cells of aplastic anemia patients and identified similar deregulations of important stemness genes in both cell populations.

View Article: PubMed Central - PubMed

Affiliation: Research Group for Gene Modification in Stem Cells, LOEWE Center for Cell and Gene Therapy Frankfurt/Main and the Paul-Ehrlich-Institute, Langen, Germany; Institute of Experimental Hematology; Hannover Medical School, Hannover, Germany.

ABSTRACT
Thrombopoietin (Thpo) signals via its receptor Mpl and regulates megakaryopoiesis, hematopoietic stem cell (HSC) maintenance and post-transplant expansion. Mpl expression is tightly controlled and deregulation of Thpo/Mpl-signaling is linked to hematological disorders. Here, we constructed an intracellular-truncated, signaling-deficient Mpl protein which is presented on the cell surface (dnMpl). The transplantation of bone marrow cells retrovirally transduced to express dnMpl into wildtype mice induced thrombocytopenia, and a progressive loss of HSC. The aplastic BM allowed the engraftment of a second BM transplant without further conditioning. Functional analysis of the truncated Mpl in vitro and in vivo demonstrated no internalization after Thpo binding and the inhibition of Thpo/Mpl-signaling in wildtype cells due to dominant-negative (dn) effects by receptor competition with wildtype Mpl for Thpo binding. Intracellular inhibition of Mpl could be excluded as the major mechanism by the use of a constitutive-dimerized dnMpl. To further elucidate the molecular changes induced by Thpo/Mpl-inhibition on the HSC-enriched cell population in the BM, we performed gene expression analysis of Lin-Sca1+cKit+ (LSK) cells isolated from mice transplanted with dnMpl transduced BM cells. The gene expression profile supported the exhaustion of HSC due to increased cell cycle progression and identified new and known downstream effectors of Thpo/Mpl-signaling in HSC (namely TIE2, ESAM1 and EPCR detected on the HSC-enriched LSK cell population). We further compared gene expression profiles in LSK cells of dnMpl mice with human CD34+ cells of aplastic anemia patients and identified similar deregulations of important stemness genes in both cell populations. In summary, we established a novel way of Thpo/Mpl inhibition in the adult mouse and performed in depth analysis of the phenotype including gene expression profiling.

No MeSH data available.


Related in: MedlinePlus

dnMpl does not transmit Thpo induced signals.(A) The gammaretroviral LTR vector encoded the full length or the intracellular truncated, dominant-negative (dn)Mpl cDNA. For detection of the Mpl proteins a hemagglutinin (HA)Tag was added at the N-terminus between the signal peptide and the ECD. The vector also co-expressed GFP using an internal ribosomal entry site (IRES). As control the retroviral vector only containing IRES.GFP or a truncated form of human CD34 was used. (LTR: long terminal repeat, ψ: packaging signal, SD: splice donor, SA: splice acceptor, wPRE: Woodchuck hepatitis virus posttranscriptional regulatory element, SP: signal peptide, ECD: extracellular domain, TMD: transmembrane domain, ICD: intracellular domain). (B) Western blot analysis of Mpl downstream signaling proteins in 32D cells that were transduced with wtMpl, dnMpl or GFP as a control. Transduced cells were stimulated with mThpo (20ng/mL), IL-3 (5ng/ml) or fixed without stimulation. Activation of STAT3 and STAT5 was analyzed by EMSA. No phosphorylation of ERK1/2, AKT and STATS was detected in dnMpl expressing 32D cells after Thpo stimulation similar to the GFP control transduced cells.
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pone.0131866.g001: dnMpl does not transmit Thpo induced signals.(A) The gammaretroviral LTR vector encoded the full length or the intracellular truncated, dominant-negative (dn)Mpl cDNA. For detection of the Mpl proteins a hemagglutinin (HA)Tag was added at the N-terminus between the signal peptide and the ECD. The vector also co-expressed GFP using an internal ribosomal entry site (IRES). As control the retroviral vector only containing IRES.GFP or a truncated form of human CD34 was used. (LTR: long terminal repeat, ψ: packaging signal, SD: splice donor, SA: splice acceptor, wPRE: Woodchuck hepatitis virus posttranscriptional regulatory element, SP: signal peptide, ECD: extracellular domain, TMD: transmembrane domain, ICD: intracellular domain). (B) Western blot analysis of Mpl downstream signaling proteins in 32D cells that were transduced with wtMpl, dnMpl or GFP as a control. Transduced cells were stimulated with mThpo (20ng/mL), IL-3 (5ng/ml) or fixed without stimulation. Activation of STAT3 and STAT5 was analyzed by EMSA. No phosphorylation of ERK1/2, AKT and STATS was detected in dnMpl expressing 32D cells after Thpo stimulation similar to the GFP control transduced cells.

Mentions: We constructed gammaretroviral vectors encoding a truncated Mpl receptor lacking the intracellular domain leaving only seven amino acids C-terminal of the transmembrane domain (termed dominant-negative (dn)Mpl, Fig 1A). As neutral controls, retroviral vectors were generated that expressed GFP or the human truncated (tr)CD34 as marker proteins and as positive control for in vitro experiments we expressed the wildtype (wt)Mpl (Fig 1A). For detection of the transgenic Mpl or truncated CD34, an HA tag was added at the extracellular N-terminus between the signal peptide and the ECD. HA-trCD34 was used in experiment 2 to allow equal staining procedures to the HA-Mpl on the target cell populations. To test the functionality of the expressed Mpl, 32D cells were transduced with the respective vectors and stimulated with Thpo. 32D cells transduced with dnMpl did not transmit Thpo mediated signals, while Thpo stimulation of wtMpl-expressing 32D cells induced ERK, AKT, STAT3 and STAT5 phosphorylation at similar or higher levels than after stimulation with IL3 (Fig 1B) indicating that Thpo binding to the transgenic receptor was possible. In addition, transduced 32D cells were stained for the presence of the HA-tagged protein on the cell surface showing equal protein expression (S1A Fig).


Inhibition of Thrombopoietin/Mpl Signaling in Adult Hematopoiesis Identifies New Candidates for Hematopoietic Stem Cell Maintenance.

Kohlscheen S, Wintterle S, Schwarzer A, Kamp C, Brugman MH, Breuer DC, Büsche G, Baum C, Modlich U - PLoS ONE (2015)

dnMpl does not transmit Thpo induced signals.(A) The gammaretroviral LTR vector encoded the full length or the intracellular truncated, dominant-negative (dn)Mpl cDNA. For detection of the Mpl proteins a hemagglutinin (HA)Tag was added at the N-terminus between the signal peptide and the ECD. The vector also co-expressed GFP using an internal ribosomal entry site (IRES). As control the retroviral vector only containing IRES.GFP or a truncated form of human CD34 was used. (LTR: long terminal repeat, ψ: packaging signal, SD: splice donor, SA: splice acceptor, wPRE: Woodchuck hepatitis virus posttranscriptional regulatory element, SP: signal peptide, ECD: extracellular domain, TMD: transmembrane domain, ICD: intracellular domain). (B) Western blot analysis of Mpl downstream signaling proteins in 32D cells that were transduced with wtMpl, dnMpl or GFP as a control. Transduced cells were stimulated with mThpo (20ng/mL), IL-3 (5ng/ml) or fixed without stimulation. Activation of STAT3 and STAT5 was analyzed by EMSA. No phosphorylation of ERK1/2, AKT and STATS was detected in dnMpl expressing 32D cells after Thpo stimulation similar to the GFP control transduced cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493002&req=5

pone.0131866.g001: dnMpl does not transmit Thpo induced signals.(A) The gammaretroviral LTR vector encoded the full length or the intracellular truncated, dominant-negative (dn)Mpl cDNA. For detection of the Mpl proteins a hemagglutinin (HA)Tag was added at the N-terminus between the signal peptide and the ECD. The vector also co-expressed GFP using an internal ribosomal entry site (IRES). As control the retroviral vector only containing IRES.GFP or a truncated form of human CD34 was used. (LTR: long terminal repeat, ψ: packaging signal, SD: splice donor, SA: splice acceptor, wPRE: Woodchuck hepatitis virus posttranscriptional regulatory element, SP: signal peptide, ECD: extracellular domain, TMD: transmembrane domain, ICD: intracellular domain). (B) Western blot analysis of Mpl downstream signaling proteins in 32D cells that were transduced with wtMpl, dnMpl or GFP as a control. Transduced cells were stimulated with mThpo (20ng/mL), IL-3 (5ng/ml) or fixed without stimulation. Activation of STAT3 and STAT5 was analyzed by EMSA. No phosphorylation of ERK1/2, AKT and STATS was detected in dnMpl expressing 32D cells after Thpo stimulation similar to the GFP control transduced cells.
Mentions: We constructed gammaretroviral vectors encoding a truncated Mpl receptor lacking the intracellular domain leaving only seven amino acids C-terminal of the transmembrane domain (termed dominant-negative (dn)Mpl, Fig 1A). As neutral controls, retroviral vectors were generated that expressed GFP or the human truncated (tr)CD34 as marker proteins and as positive control for in vitro experiments we expressed the wildtype (wt)Mpl (Fig 1A). For detection of the transgenic Mpl or truncated CD34, an HA tag was added at the extracellular N-terminus between the signal peptide and the ECD. HA-trCD34 was used in experiment 2 to allow equal staining procedures to the HA-Mpl on the target cell populations. To test the functionality of the expressed Mpl, 32D cells were transduced with the respective vectors and stimulated with Thpo. 32D cells transduced with dnMpl did not transmit Thpo mediated signals, while Thpo stimulation of wtMpl-expressing 32D cells induced ERK, AKT, STAT3 and STAT5 phosphorylation at similar or higher levels than after stimulation with IL3 (Fig 1B) indicating that Thpo binding to the transgenic receptor was possible. In addition, transduced 32D cells were stained for the presence of the HA-tagged protein on the cell surface showing equal protein expression (S1A Fig).

Bottom Line: Functional analysis of the truncated Mpl in vitro and in vivo demonstrated no internalization after Thpo binding and the inhibition of Thpo/Mpl-signaling in wildtype cells due to dominant-negative (dn) effects by receptor competition with wildtype Mpl for Thpo binding.The gene expression profile supported the exhaustion of HSC due to increased cell cycle progression and identified new and known downstream effectors of Thpo/Mpl-signaling in HSC (namely TIE2, ESAM1 and EPCR detected on the HSC-enriched LSK cell population).We further compared gene expression profiles in LSK cells of dnMpl mice with human CD34+ cells of aplastic anemia patients and identified similar deregulations of important stemness genes in both cell populations.

View Article: PubMed Central - PubMed

Affiliation: Research Group for Gene Modification in Stem Cells, LOEWE Center for Cell and Gene Therapy Frankfurt/Main and the Paul-Ehrlich-Institute, Langen, Germany; Institute of Experimental Hematology; Hannover Medical School, Hannover, Germany.

ABSTRACT
Thrombopoietin (Thpo) signals via its receptor Mpl and regulates megakaryopoiesis, hematopoietic stem cell (HSC) maintenance and post-transplant expansion. Mpl expression is tightly controlled and deregulation of Thpo/Mpl-signaling is linked to hematological disorders. Here, we constructed an intracellular-truncated, signaling-deficient Mpl protein which is presented on the cell surface (dnMpl). The transplantation of bone marrow cells retrovirally transduced to express dnMpl into wildtype mice induced thrombocytopenia, and a progressive loss of HSC. The aplastic BM allowed the engraftment of a second BM transplant without further conditioning. Functional analysis of the truncated Mpl in vitro and in vivo demonstrated no internalization after Thpo binding and the inhibition of Thpo/Mpl-signaling in wildtype cells due to dominant-negative (dn) effects by receptor competition with wildtype Mpl for Thpo binding. Intracellular inhibition of Mpl could be excluded as the major mechanism by the use of a constitutive-dimerized dnMpl. To further elucidate the molecular changes induced by Thpo/Mpl-inhibition on the HSC-enriched cell population in the BM, we performed gene expression analysis of Lin-Sca1+cKit+ (LSK) cells isolated from mice transplanted with dnMpl transduced BM cells. The gene expression profile supported the exhaustion of HSC due to increased cell cycle progression and identified new and known downstream effectors of Thpo/Mpl-signaling in HSC (namely TIE2, ESAM1 and EPCR detected on the HSC-enriched LSK cell population). We further compared gene expression profiles in LSK cells of dnMpl mice with human CD34+ cells of aplastic anemia patients and identified similar deregulations of important stemness genes in both cell populations. In summary, we established a novel way of Thpo/Mpl inhibition in the adult mouse and performed in depth analysis of the phenotype including gene expression profiling.

No MeSH data available.


Related in: MedlinePlus