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Flow-induced protein kinase A-CREB pathway acts via BMP signaling to promote HSC emergence.

Kim PG, Nakano H, Das PP, Chen MJ, Rowe RG, Chou SS, Ross SJ, Sakamoto KM, Zon LI, Schlaeger TM, Orkin SH, Nakano A, Daley GQ - J. Exp. Med. (2015)

Bottom Line: By chemical modulation of the PKA-CREB and BMP pathways in isolated AGM VE-cadherin(+) cells from mid-gestation embryos, we demonstrate that PKA-CREB regulates hematopoietic engraftment and clonogenicity of hematopoietic progenitors, and is dependent on secreted BMP ligands through the type I BMP receptor.Finally, we observed blunting of this signaling axis using Ncx1- embryos, which lack a heartbeat and intravascular flow.Collectively, we have identified a novel PKA-CREB-BMP signaling pathway downstream of shear stress that regulates HSC emergence in the AGM via the endothelial-to-hematopoietic transition.

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Affiliation: Division of Pediatric Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute; Howard Hughes Medical Institute, Harvard Stem Cell Institute; Stem Cell Transplantation Program, Division of Pediatric Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute; and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115 Division of Pediatric Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute; Howard Hughes Medical Institute, Harvard Stem Cell Institute; Stem Cell Transplantation Program, Division of Pediatric Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute; and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115 Division of Pediatric Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute; Howard Hughes Medical Institute, Harvard Stem Cell Institute; Stem Cell Transplantation Program, Division of Pediatric Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute; and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.

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BMP4 rescues hematopoiesis from PKA–CREB inhibition. (A) Immunofluorescence section of the E11.5 AGM showing the co-localization of phospho-CREB and Bmp4. ao, dorsal aorta; nc, notochord; nt, neural tube. (B) Immunofluorescence of adjacent section to A showing the localization of phospho-Smad1/5/8. (C) Immunofluorescence of adjacent section to A showing the localization of phospho-Smad1/5. (D) Zoomed view of the ventral mesenchyme indicated by an arrow in A. (E) Experimental scheme for working with AGM-derived VE-cadherin+ cells. (F) Normalized high-content immunofluorescence of the phospho-CREB dynamics upon KT5720 treatment over 8 h. n = 2. (G) CFU of sorted VE-cadherin+ cells from E11.5 per embryo equivalent (e.e) treated with various compounds for 8 h. Representative multipotent CFUs at 12 d after plating shown on right. n = 3—5. (H) CFU assay of sorted VE-cadherin+ cells from E10.5 per e.e treated with various compounds for 8 h. n = 3. (I) CFU assay of sorted VE-cadherin− cells from E10.5 per e.e treated with various compounds for 8 h. n = 3. (J) Example of peripheral blood analysis for donor chimerism for myeloid and lymphoid cell contribution using flow cytometry. (K) Donor chimerism levels at 12-wk after transplantation. 2.5 e.e. were used per recipient. Averages are shown in red. One-sided Wilcoxon rank-sum test was performed on log-transformed values. Bars: (A–C) 100 µm.
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fig3: BMP4 rescues hematopoiesis from PKA–CREB inhibition. (A) Immunofluorescence section of the E11.5 AGM showing the co-localization of phospho-CREB and Bmp4. ao, dorsal aorta; nc, notochord; nt, neural tube. (B) Immunofluorescence of adjacent section to A showing the localization of phospho-Smad1/5/8. (C) Immunofluorescence of adjacent section to A showing the localization of phospho-Smad1/5. (D) Zoomed view of the ventral mesenchyme indicated by an arrow in A. (E) Experimental scheme for working with AGM-derived VE-cadherin+ cells. (F) Normalized high-content immunofluorescence of the phospho-CREB dynamics upon KT5720 treatment over 8 h. n = 2. (G) CFU of sorted VE-cadherin+ cells from E11.5 per embryo equivalent (e.e) treated with various compounds for 8 h. Representative multipotent CFUs at 12 d after plating shown on right. n = 3—5. (H) CFU assay of sorted VE-cadherin+ cells from E10.5 per e.e treated with various compounds for 8 h. n = 3. (I) CFU assay of sorted VE-cadherin− cells from E10.5 per e.e treated with various compounds for 8 h. n = 3. (J) Example of peripheral blood analysis for donor chimerism for myeloid and lymphoid cell contribution using flow cytometry. (K) Donor chimerism levels at 12-wk after transplantation. 2.5 e.e. were used per recipient. Averages are shown in red. One-sided Wilcoxon rank-sum test was performed on log-transformed values. Bars: (A–C) 100 µm.

Mentions: We investigated the interaction between the PKA–CREB and BMP pathways during AGM hematopoiesis, as predicted by bioinformatics, by examining the localization of the BMP ligand Bmp4 in the E11.5 AGM. Similar to the distribution of phospho-CREB (Fig. 1, C and D), Bmp4 was localized in cells lining the dorsal aorta and, more strongly, in the ventral than the dorsal mesenchyme, as previously reported (Fig. 3 A; Durand et al., 2007). In immediately adjacent sections, the transcriptionally active BMP target protein complexes phospho-Smad1/5 and Smad1/5/8 showed a similar distribution (Fig. 3, B and C). Upon closer examination of the ventral floor of the dorsal aorta, we observed cells with phospho-CREB in close proximity to Bmp4 (Fig. 3 D).


Flow-induced protein kinase A-CREB pathway acts via BMP signaling to promote HSC emergence.

Kim PG, Nakano H, Das PP, Chen MJ, Rowe RG, Chou SS, Ross SJ, Sakamoto KM, Zon LI, Schlaeger TM, Orkin SH, Nakano A, Daley GQ - J. Exp. Med. (2015)

BMP4 rescues hematopoiesis from PKA–CREB inhibition. (A) Immunofluorescence section of the E11.5 AGM showing the co-localization of phospho-CREB and Bmp4. ao, dorsal aorta; nc, notochord; nt, neural tube. (B) Immunofluorescence of adjacent section to A showing the localization of phospho-Smad1/5/8. (C) Immunofluorescence of adjacent section to A showing the localization of phospho-Smad1/5. (D) Zoomed view of the ventral mesenchyme indicated by an arrow in A. (E) Experimental scheme for working with AGM-derived VE-cadherin+ cells. (F) Normalized high-content immunofluorescence of the phospho-CREB dynamics upon KT5720 treatment over 8 h. n = 2. (G) CFU of sorted VE-cadherin+ cells from E11.5 per embryo equivalent (e.e) treated with various compounds for 8 h. Representative multipotent CFUs at 12 d after plating shown on right. n = 3—5. (H) CFU assay of sorted VE-cadherin+ cells from E10.5 per e.e treated with various compounds for 8 h. n = 3. (I) CFU assay of sorted VE-cadherin− cells from E10.5 per e.e treated with various compounds for 8 h. n = 3. (J) Example of peripheral blood analysis for donor chimerism for myeloid and lymphoid cell contribution using flow cytometry. (K) Donor chimerism levels at 12-wk after transplantation. 2.5 e.e. were used per recipient. Averages are shown in red. One-sided Wilcoxon rank-sum test was performed on log-transformed values. Bars: (A–C) 100 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4419355&req=5

fig3: BMP4 rescues hematopoiesis from PKA–CREB inhibition. (A) Immunofluorescence section of the E11.5 AGM showing the co-localization of phospho-CREB and Bmp4. ao, dorsal aorta; nc, notochord; nt, neural tube. (B) Immunofluorescence of adjacent section to A showing the localization of phospho-Smad1/5/8. (C) Immunofluorescence of adjacent section to A showing the localization of phospho-Smad1/5. (D) Zoomed view of the ventral mesenchyme indicated by an arrow in A. (E) Experimental scheme for working with AGM-derived VE-cadherin+ cells. (F) Normalized high-content immunofluorescence of the phospho-CREB dynamics upon KT5720 treatment over 8 h. n = 2. (G) CFU of sorted VE-cadherin+ cells from E11.5 per embryo equivalent (e.e) treated with various compounds for 8 h. Representative multipotent CFUs at 12 d after plating shown on right. n = 3—5. (H) CFU assay of sorted VE-cadherin+ cells from E10.5 per e.e treated with various compounds for 8 h. n = 3. (I) CFU assay of sorted VE-cadherin− cells from E10.5 per e.e treated with various compounds for 8 h. n = 3. (J) Example of peripheral blood analysis for donor chimerism for myeloid and lymphoid cell contribution using flow cytometry. (K) Donor chimerism levels at 12-wk after transplantation. 2.5 e.e. were used per recipient. Averages are shown in red. One-sided Wilcoxon rank-sum test was performed on log-transformed values. Bars: (A–C) 100 µm.
Mentions: We investigated the interaction between the PKA–CREB and BMP pathways during AGM hematopoiesis, as predicted by bioinformatics, by examining the localization of the BMP ligand Bmp4 in the E11.5 AGM. Similar to the distribution of phospho-CREB (Fig. 1, C and D), Bmp4 was localized in cells lining the dorsal aorta and, more strongly, in the ventral than the dorsal mesenchyme, as previously reported (Fig. 3 A; Durand et al., 2007). In immediately adjacent sections, the transcriptionally active BMP target protein complexes phospho-Smad1/5 and Smad1/5/8 showed a similar distribution (Fig. 3, B and C). Upon closer examination of the ventral floor of the dorsal aorta, we observed cells with phospho-CREB in close proximity to Bmp4 (Fig. 3 D).

Bottom Line: By chemical modulation of the PKA-CREB and BMP pathways in isolated AGM VE-cadherin(+) cells from mid-gestation embryos, we demonstrate that PKA-CREB regulates hematopoietic engraftment and clonogenicity of hematopoietic progenitors, and is dependent on secreted BMP ligands through the type I BMP receptor.Finally, we observed blunting of this signaling axis using Ncx1- embryos, which lack a heartbeat and intravascular flow.Collectively, we have identified a novel PKA-CREB-BMP signaling pathway downstream of shear stress that regulates HSC emergence in the AGM via the endothelial-to-hematopoietic transition.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Pediatric Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute; Howard Hughes Medical Institute, Harvard Stem Cell Institute; Stem Cell Transplantation Program, Division of Pediatric Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute; and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115 Division of Pediatric Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute; Howard Hughes Medical Institute, Harvard Stem Cell Institute; Stem Cell Transplantation Program, Division of Pediatric Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute; and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115 Division of Pediatric Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute; Howard Hughes Medical Institute, Harvard Stem Cell Institute; Stem Cell Transplantation Program, Division of Pediatric Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute; and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.

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