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. 2017 Feb;46:83-95.e6.
doi: 10.1016/j.exphem.2016.10.002. Epub 2016 Oct 15.

HIF1α-induced PDGFRβ Signaling Promotes Developmental HSC Production via IL-6 Activation

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HIF1α-induced PDGFRβ Signaling Promotes Developmental HSC Production via IL-6 Activation

Sung-Eun Lim et al. Exp Hematol. .
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Abstract

Hematopoietic stem cells (HSCs) have the ability to both self-renew and differentiate each of the mature blood cell lineages and thereby reconstitute the entire blood system. Therefore, HSCs are therapeutically valuable for treatment of hematological malignances and bone marrow failure. We showed recently that transient glucose elevation elicited dose-dependent effects on HSCs through elevated metabolic activity and subsequent reactive oxygen species-mediated induction of Hypoxia-Inducible Factor 1α (Hif1α). Platelet-Derived Growth Factor B (pdgfb), a Hif1α-target, and its receptor, pdgfrb, were significantly upregulated in response to metabolic stimulation. Although the function of PDGF signaling is well established in vascular development, its role in hematopoiesis is less understood. Exposure to either a pan-PDGF inhibitor or a PDGFRβ-selective antagonist in the context of Hif1α stimulation blocked elevations in hematopoietic stem and progenitor cell (HSPC) formation as determined by runx1;cmyb whole-mount in situ hybridization (WISH) and HSPC-reporter flow cytometry analysis. Similar results were observed for morpholino (MO) knockdown of pdgfrb or dominant-negative pdgfrb expression, indicating that PDGFRβ signaling is a key downstream mediator of Hif1α-mediated induction of HSPCs. Notably, overexpression of Pdgfb ligand enhanced HSPC numbers in the aorta-gonado-mesonephros (AGM) at 36 hours postfertilization (hpf) and in the caudal hematopoietic tissue at 48 hpf. A survey of known PDGF-B/PDGFRβ regulatory targets by expression analysis revealed a significant increase in inflammatory intermediates, including Interleukin 6 (IL-6) and its receptor (IL-6R). MO-mediated knockdown of il6 or chemical inhibition of IL-6R antagonized the effect of Pdgfb overexpression. Furthermore, epistatic analysis of IL-6/IL-6R function confirmed activity downstream of Hif1α. Together, these findings define a Hif1α-regulated signaling axis mediated through PBFGB/PDGFRβ and IL-6/IL-6R that acts to control embryonic HSPC production.

Figures

Figure 1
Figure 1. Hif1α stabilization enhances HSPC production via PDGF activity
(A) Embryonic exposure to the pan-PDGFR inhibitor AG1295 (10μM) (during HSC formation (12-36hpf) decreased runx1;cmyb WISH expression in the AGM and blocked the increase in runx1;cmyb expression due to exposure to Hif1α agonist (CoCl2, 500μM). Tx = treatment. (B) Qualitative phenotypic distribution of embryos from panel 1A scored with low, medium or high runx1;cmyb expression in the AGM (n≥20 condition × 3 replicate clutches). (C) FACS analysis confirmed that chemical stabilization of Hif1α enhanced HSCs, while inhibition of PDGFR decreased CD41+gata1 HSPCs at 36hpf in the presence of CoCl2 (*p<0.05, ***p<0.001, one-tailed t-test, n≥13 replicates/condition). (D) RT-qPCR analysis showed that pdgfba and pdgfrb were significantly upregulated over baseline in CoCl2 treated embryos, whereas related family members were not affected (Tx 12-36hpf) (*p<0.05, two-tailed t-test, n≥3). (E) In vhl MO injected embryos RT-qPCR analysis likewise indicated that only pdgfba and pdgfrb were significantly upregulated over matched controls (Tx 12-36hpf) (*p<0.05, **p<0.01, two-tailed t-test, n≥4). (F) WISH analysis of wild-type embryos at 36hpf indicated pdgfrb expression throughout the trunk of the embryo, including enrichment in the region of the VDA; pdgfrb expression was notably increased by CoCl2 exposure. (G) Qualitative phenotypic distribution of embryos from panel 1F scored with low, medium or high pdgfrb expression in the AGM (n≥20 condition × 2 replicate clutches).
Fig 2
Fig 2. PDGFRβ signaling acts downstream of Hif1α to control AGM HSPC production
(A) Morpholino knockdown of pdgfrb attenuated the increase in runx1;cmyb expression in embryos treated with the Hif1α agonist DMOG (75μM). (B) Qualitative phenotypic distribution of embryos from panel 2A (n≥20/condition × 3 replicate clutches). (C) Knockdown of vhl increased runx1;cmyb WISH expression in the AGM at 36hpf, while co-injection with the pdgfrb MO blocked this effect. (D) Qualitative phenotypic distribution of embryos from panel 2D (n≥20/condition × 2 replicate clutches). (E) FACS analysis for CD41+(Gata1) HSPCs following injection of MOs to vhl and pdgfrb alone and combined confirmed a reduced impact for Hif1α stabilization with loss of PDGF signaling (*p<0.05, one-tailed t-test, n≥7). (F) Ectopic activation of a dominant negative pdgfrb transgene, Tg(hsp70:dn-pdgfrb), at 27hpf (37°C for 1 hour) diminished the impact of CoCl2 stimulation on cmyb expression at 48hpf. (G) Qualitative phenotypic distribution of embryos from panel S2G (n≥20/condition × 4 replicate clutches). (H) CD41+ HSPC cell counts at 48hpf confirmed the effect of loss of PDGFRB signaling, mediated by induction of a dominant negative receptor, on Hif1α activation by CoCl2 (*p<0.05, **p<0.01, one-tailed t-test, n≥8).
Fig 3
Fig 3. PDGF-B stimulation increases developmental HSPC numbers
(A) Overexpression of pdgfb by mRNA injection enhanced runx1;cmyb expression in the VDA by WISH at 36hpf. (B) Qualitative phenotypic distribution of embryos from panel 3A (n≥20/condition × 5 replicate clutches). (C) FACS analysis confirmed that pdgfb overexpression increased Flk1:dsRed+cMyb:GFP+ HSPCs at 48hpf (1.54 fold increase, ***p≤0.0001, two-tailed t test, n≥5 replicates/condition). (D) WISH analysis showed enhanced cmyb expression at 48hpf following ectopic activation of PDGFRβ signaling using hsp70:ca-pdgfrb induced at 27hpf (37°C for 1 hour) (E) Qualitative phenotypic distribution of embryos from panel 3D (n≥20/condition × 5 replicate clutches). (F) FACS analysis confirmed a significant increase in Flk1+cMyb+ HSPCs (48hpf) following PDGFRβ activation mediated by induced expression of ca-pdgfrb (1.47 fold increase, *p<0.05, two-tailed t test, n≥5 replicates/condition). (G) Overexpression of pdgfb ligand rescued the reduction in runx1;cmyb expression found in hif1a morphants at 36hpf. (H) Qualitative phenotypic distribution of embryos from panel 3G (n≥20/condition × 3 replicate clutches).
Fig 4
Fig 4. IL-6 acts downstream of PDGFRβ signaling to stimulate AGM HSPC production
(A) qPCR analysis showed il6 and its’ receptor and co-receptor, il6R and gp130, are upregulated in pdgfb mRNA-injected embryos (*p<0.05, **p<0.01, two-tailed t-test, n≥3). (B) Morpholino knockdown of il6 blocked the ability of pdgfb mRNA to increase runx1;cmyb expression in the AGM. (C) Qualitative phenotypic distribution of embryos from panel 4B (n≥20/condition × 3 replicate clutches). (D) Absolute counts of Flk1:dsRed+cMyb:GFP+ HSPCs from embryos overexpressing pdgfb were significantly reduced with MO-mediated loss of il6 (***p<0.0005, *p<0.05, two-tailed t-test, n≥15/condition). (E) FACS analysis for Flk1+cMyb+ HSPCs at 48hpf showed that overexpression of il6 significantly increases HSPCs (1.49-fold vs. control, *p<0.02, two-tailed t-test, n≥5 replicates/condition). (F) Absolute cell counts of phospho histone H3 expressing (pHH3+) cells in the VDA were increased in both pdgfb and il6 mRNA-injected embryos compared to matched sibling controls (*p<0.05, **p<0.01, two-tailed t-test, n ≥10/condition).
Fig 5
Fig 5. IL-6 signaling functions downstream of Hif1α in AGM HSPC regulation
(A) RT-qPCR analysis demonstrated that expression of il6 and its receptor il6R are increased by CoCl2 exposure (*p<0.05, **p<0.01, one-tailed t-test, n≥3). (B) Morpholino knockdown of il6 diminished the ability of CoCl2 to stimulate runx1;cmyb expression in the VDA over that seen in controls as determined by WISH analysis at 36hpf. (C) Qualitative phenotypic distribution of embryos from panel 5B (n≥20/condition × 3 replicate clutches). (D) FACS analysis confirmed that CoCl2 was unable to enhance the number of CD41+ HSPCs in the presence of il6 knockdown (*p<0.05, one-tailed t-test, n≥4). (E) Injection of dnhif1a mRNA decreased runx1;cmyb expression in the VDA at 36hpf, which could be partially ameliorated by overexpression of il6. (F) Qualitative phenotypic distribution of embryos from panel 5E (n≥20/condition × 3 replicate clutches.

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