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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

Systemic and cell intrinsic effects of dnMpl expression.(A) wtMpl expressing 32D cells (aHA-PE positive) were transduced with dnMpl.IRES.GFP, constitutive dimerized (cd)-dnMpl.IRES.GFP or GFP encoding vectors to establish cultures with single and double positive cells. Cells were starved for any cytokine stimuli for 16 hrs and stimulated with 2.5 or 5 ng/mL mThpo for 15 minutes the next day. Unstimulated (negative control) and stimulated cells were fixed and permeabelized to allow intracellular staining of phosphorylated signaling molecules. Anti-phosphoERK1/2 or phosphoSTAT5 antibodies conjugated to Alexa Fluor 647 (BD Biosciences) were used. Shown are histogram overlays of pERK1/2 and pSTAT5 activation from wtMpl/GFP negative cells and wtMpl/GFP, wtMpl/dnMpl, wtMpl/cd-dnMpl double positive cells. (Dashed line—activation border; solid line—mean fluorescence intensity of wtMpl/GFP double positive cells). (B) Human erythroid leukemia (HEL) cells were transduced with retroviral vectors encoding dnMpl, cd-dnMpl or GFP as control. wtMpl expressing 32D cells (100% positive) were mixed with untransduced, GFP control, dnMpl or cd-dnMpl expressing HEL cells in different ratios (1:1, 1:2, 2:1). Cells were co-cultured in murine Thpo supplemented medium (6ng/mL). The percentage of 32D cells three and six days after co-culture was measured. In the absence of dnMpl or cd-dnMpl wtMpl/32D cells grew faster than HEL cells. But in the presence of dnMpl (1:1, 1:2 and 2:1) or cd-dnMpl (1:2) wtMpl/32D cells stopped proliferating and were diminished over time in the culture. (C) Blood counts of mice transplanted with dnMpl, cd-dnMpl or GFP control transduced wildtype Lin- BM cells six and twelve weeks after transplantation (*p<0.05, ***p<0.005 Student’s t-test, n = 4–5). One of the cd-dnMpl mice had to be killed at 12 weeks due to the severe anemia.
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pone.0131866.g003: Systemic and cell intrinsic effects of dnMpl expression.(A) wtMpl expressing 32D cells (aHA-PE positive) were transduced with dnMpl.IRES.GFP, constitutive dimerized (cd)-dnMpl.IRES.GFP or GFP encoding vectors to establish cultures with single and double positive cells. Cells were starved for any cytokine stimuli for 16 hrs and stimulated with 2.5 or 5 ng/mL mThpo for 15 minutes the next day. Unstimulated (negative control) and stimulated cells were fixed and permeabelized to allow intracellular staining of phosphorylated signaling molecules. Anti-phosphoERK1/2 or phosphoSTAT5 antibodies conjugated to Alexa Fluor 647 (BD Biosciences) were used. Shown are histogram overlays of pERK1/2 and pSTAT5 activation from wtMpl/GFP negative cells and wtMpl/GFP, wtMpl/dnMpl, wtMpl/cd-dnMpl double positive cells. (Dashed line—activation border; solid line—mean fluorescence intensity of wtMpl/GFP double positive cells). (B) Human erythroid leukemia (HEL) cells were transduced with retroviral vectors encoding dnMpl, cd-dnMpl or GFP as control. wtMpl expressing 32D cells (100% positive) were mixed with untransduced, GFP control, dnMpl or cd-dnMpl expressing HEL cells in different ratios (1:1, 1:2, 2:1). Cells were co-cultured in murine Thpo supplemented medium (6ng/mL). The percentage of 32D cells three and six days after co-culture was measured. In the absence of dnMpl or cd-dnMpl wtMpl/32D cells grew faster than HEL cells. But in the presence of dnMpl (1:1, 1:2 and 2:1) or cd-dnMpl (1:2) wtMpl/32D cells stopped proliferating and were diminished over time in the culture. (C) Blood counts of mice transplanted with dnMpl, cd-dnMpl or GFP control transduced wildtype Lin- BM cells six and twelve weeks after transplantation (*p<0.05, ***p<0.005 Student’s t-test, n = 4–5). One of the cd-dnMpl mice had to be killed at 12 weeks due to the severe anemia.

Mentions: To test whether dnMpl exerts its effects by a cell intrinsic or extrinsic mechanism, we performed a set of in vitro experiments using cell line models. First, we investigated whether dnMpl expression interfered with wtMpl receptor signaling when co-expressed on the same cell. The myeloid cell line 32D was engineered to grow Thpo dependent by retroviral expression of wtMpl from the phosphoglycerate kinase (PGK) promoter. wtMpl/32D cells were transduced with the SF91.dnMpl.IRES.GFP retroviral vector to obtain a mixture of wtMpl single and wtMpl/dnMpl double positive cells. dnMpl expression from the SFFV promoter was stronger than the expression of wtMpl from the PGK promoter which mimics the in vivo situation. In an attempt to exclude heterodimerization of dnMpl with the wtMpl and, therefore, prevent the possibility of dnMpl monomers to block the activity of endogenous wtMpl, we also expressed a constitutive-dimerized (cd) form of dnMpl. To achieve covalent dimerization, we introduced an S to C mutation at amino acid 368 of the dnMpl extracellular domain. This mutation results in a stable dimerized conformation via a disulfide bridge of two Mpl receptor chains [32]. wtMpl/dnMpl and wtMpl/cd-dnMpl cells were then stimulated by Thpo and analyzed for STAT5 and ERK1/2 phosphorylation by flow cytometry. Strikingly, the expression of dnMpl but not cd-dnMpl inhibited wtMpl-induced STAT5 phosphorylation at low Thpo concentrations (2.5 and 5 ng/ml) and inhibition declined with higher doses (20 ng/ml and 50ng/ml; S4 Fig). This suggests that expression of dnMpl intrinsically inhibits wtMpl signaling at physiological Thpo concentrations by heterodimerization. However, at high Thpo doses this effect was abrogated (Fig 3A).


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)

Systemic and cell intrinsic effects of dnMpl expression.(A) wtMpl expressing 32D cells (aHA-PE positive) were transduced with dnMpl.IRES.GFP, constitutive dimerized (cd)-dnMpl.IRES.GFP or GFP encoding vectors to establish cultures with single and double positive cells. Cells were starved for any cytokine stimuli for 16 hrs and stimulated with 2.5 or 5 ng/mL mThpo for 15 minutes the next day. Unstimulated (negative control) and stimulated cells were fixed and permeabelized to allow intracellular staining of phosphorylated signaling molecules. Anti-phosphoERK1/2 or phosphoSTAT5 antibodies conjugated to Alexa Fluor 647 (BD Biosciences) were used. Shown are histogram overlays of pERK1/2 and pSTAT5 activation from wtMpl/GFP negative cells and wtMpl/GFP, wtMpl/dnMpl, wtMpl/cd-dnMpl double positive cells. (Dashed line—activation border; solid line—mean fluorescence intensity of wtMpl/GFP double positive cells). (B) Human erythroid leukemia (HEL) cells were transduced with retroviral vectors encoding dnMpl, cd-dnMpl or GFP as control. wtMpl expressing 32D cells (100% positive) were mixed with untransduced, GFP control, dnMpl or cd-dnMpl expressing HEL cells in different ratios (1:1, 1:2, 2:1). Cells were co-cultured in murine Thpo supplemented medium (6ng/mL). The percentage of 32D cells three and six days after co-culture was measured. In the absence of dnMpl or cd-dnMpl wtMpl/32D cells grew faster than HEL cells. But in the presence of dnMpl (1:1, 1:2 and 2:1) or cd-dnMpl (1:2) wtMpl/32D cells stopped proliferating and were diminished over time in the culture. (C) Blood counts of mice transplanted with dnMpl, cd-dnMpl or GFP control transduced wildtype Lin- BM cells six and twelve weeks after transplantation (*p<0.05, ***p<0.005 Student’s t-test, n = 4–5). One of the cd-dnMpl mice had to be killed at 12 weeks due to the severe anemia.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4493002&req=5

pone.0131866.g003: Systemic and cell intrinsic effects of dnMpl expression.(A) wtMpl expressing 32D cells (aHA-PE positive) were transduced with dnMpl.IRES.GFP, constitutive dimerized (cd)-dnMpl.IRES.GFP or GFP encoding vectors to establish cultures with single and double positive cells. Cells were starved for any cytokine stimuli for 16 hrs and stimulated with 2.5 or 5 ng/mL mThpo for 15 minutes the next day. Unstimulated (negative control) and stimulated cells were fixed and permeabelized to allow intracellular staining of phosphorylated signaling molecules. Anti-phosphoERK1/2 or phosphoSTAT5 antibodies conjugated to Alexa Fluor 647 (BD Biosciences) were used. Shown are histogram overlays of pERK1/2 and pSTAT5 activation from wtMpl/GFP negative cells and wtMpl/GFP, wtMpl/dnMpl, wtMpl/cd-dnMpl double positive cells. (Dashed line—activation border; solid line—mean fluorescence intensity of wtMpl/GFP double positive cells). (B) Human erythroid leukemia (HEL) cells were transduced with retroviral vectors encoding dnMpl, cd-dnMpl or GFP as control. wtMpl expressing 32D cells (100% positive) were mixed with untransduced, GFP control, dnMpl or cd-dnMpl expressing HEL cells in different ratios (1:1, 1:2, 2:1). Cells were co-cultured in murine Thpo supplemented medium (6ng/mL). The percentage of 32D cells three and six days after co-culture was measured. In the absence of dnMpl or cd-dnMpl wtMpl/32D cells grew faster than HEL cells. But in the presence of dnMpl (1:1, 1:2 and 2:1) or cd-dnMpl (1:2) wtMpl/32D cells stopped proliferating and were diminished over time in the culture. (C) Blood counts of mice transplanted with dnMpl, cd-dnMpl or GFP control transduced wildtype Lin- BM cells six and twelve weeks after transplantation (*p<0.05, ***p<0.005 Student’s t-test, n = 4–5). One of the cd-dnMpl mice had to be killed at 12 weeks due to the severe anemia.
Mentions: To test whether dnMpl exerts its effects by a cell intrinsic or extrinsic mechanism, we performed a set of in vitro experiments using cell line models. First, we investigated whether dnMpl expression interfered with wtMpl receptor signaling when co-expressed on the same cell. The myeloid cell line 32D was engineered to grow Thpo dependent by retroviral expression of wtMpl from the phosphoglycerate kinase (PGK) promoter. wtMpl/32D cells were transduced with the SF91.dnMpl.IRES.GFP retroviral vector to obtain a mixture of wtMpl single and wtMpl/dnMpl double positive cells. dnMpl expression from the SFFV promoter was stronger than the expression of wtMpl from the PGK promoter which mimics the in vivo situation. In an attempt to exclude heterodimerization of dnMpl with the wtMpl and, therefore, prevent the possibility of dnMpl monomers to block the activity of endogenous wtMpl, we also expressed a constitutive-dimerized (cd) form of dnMpl. To achieve covalent dimerization, we introduced an S to C mutation at amino acid 368 of the dnMpl extracellular domain. This mutation results in a stable dimerized conformation via a disulfide bridge of two Mpl receptor chains [32]. wtMpl/dnMpl and wtMpl/cd-dnMpl cells were then stimulated by Thpo and analyzed for STAT5 and ERK1/2 phosphorylation by flow cytometry. Strikingly, the expression of dnMpl but not cd-dnMpl inhibited wtMpl-induced STAT5 phosphorylation at low Thpo concentrations (2.5 and 5 ng/ml) and inhibition declined with higher doses (20 ng/ml and 50ng/ml; S4 Fig). This suggests that expression of dnMpl intrinsically inhibits wtMpl signaling at physiological Thpo concentrations by heterodimerization. However, at high Thpo doses this effect was abrogated (Fig 3A).

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