Disruption of Notch signaling aggravates irradiation-induced bone marrow injury, which is ameliorated by a soluble Dll1 ligand through Csf2rb2 upregulation

Abstract

Physical and chemical insult-induced bone marrow (BM) damage often leads to lethality resulting from the depletion of hematopoietic stem and progenitor cells (HSPCs) and/or a deteriorated BM stroma. Notch signaling plays an important role in hematopoiesis, but whether it is involved in BM damage remains unclear. In this study, we found that conditional disruption of RBP-J, the transcription factor of canonical Notch signaling, increased irradiation sensitivity in mice. Activation of Notch signaling with the endothelial cell (EC)-targeted soluble Dll1 Notch ligand mD1R promoted BM recovery after irradiation. mD1R treatment resulted in a significant increase in myeloid progenitors and monocytes in the BM, spleen and peripheral blood after irradiation. mD1R also enhanced hematopoiesis in mice treated with cyclophosphamide, a chemotherapeutic drug that induces BM suppression. Mechanistically, mD1R increased the proliferation and reduced the apoptosis of myeloid cells in the BM after irradiation. The β chain cytokine receptor Csf2rb2 was identified as a downstream molecule of Notch signaling in hematopoietic cells. mD1R improved hematopoietic recovery through up-regulation of the hematopoietic expression of Csf2rb2. Our findings reveal the role of Notch signaling in irradiation- and drug-induced BM suppression and establish a new potential therapy of BM- and myelo-suppression induced by radiotherapy and chemotherapy.

Figure 1. RBP-J disruption led to increased sensitivity to TBI-induced mortality and myelo-suppression.

(A) RBP-J cKO and control mice (MxCre-RBP-J^f/f and MxCre-RBP-J^f/+ mice induced with poly(I)-poly(C)) were subjected to TBI with 600 cGy of γ-radiation. The survival of mice was plotted for 14 days. (B) The number of nucleated BM cells was counted and compared between RBP-J cKO and control mice after TBI. (C) The number of MNCs in peripheral blood was counted and compared between RBP-J cKO and control mice after TBI. (D) FACS analysis of hematopoietic cells from RBP-J cKO and control mice after TBI. (E) The numbers of KSL (c-Kit⁺Sca-1⁺Lin⁻) and myeloid progenitors (c-Kit⁺Sca-1⁻Lin⁻) were compared between RBP-J cKO and control mice. (F) The number of Ly6G⁺CD11b⁺ monocytes in the BM, spleen (SP) and peripheral blood (PB) was compared between RBP-J cKO and control mice. Bars = means ± SD (n = 6). *P < 0.05; **P < 0.01.

Figure 2. Activation of Notch signaling by mD1R improved radiation tolerance and accelerated myeloid recovery.

(A) Eight-week-old C57BL/6 mice subjected to sublethal TBI were injected i.p. with mD1R (4 mg/kg) or PBS every day for 14 days. The survival of the mice was plotted. (B,C) The total numbers of nucleated cells in the BM (B) and peripheral blood (C) were determined every two days. (D) Cells from the BM and peripheral blood (PB) were stained with Giemsa-Wright staining. Bottom, femurs were subjected to H&E staining. (E) BM cells from mice on day 7 in (A) were subjected to colony-forming assays. The numbers of total CFU, GEMM-CFU, GM-CFU, G-CFU, and M-CFU were compared. The inset pictures show typical colonies in the two groups (n = 4). (F,G) Single-cell suspensions were prepared from the BM, spleen (SP) and peripheral blood (PB) of the mice in (A) on day 7 and day 14 and were analyzed by FACS (F). The numbers of Ly6G⁺CD11b⁺ cells in the BM, SP and PB were compared (G). Bars = means ± SD (n = 6). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3. Activation of Notch signaling by mD1R protected HSPCs after TBI.

(A,B) Eight-week-old C57BL/6 mice subjected to sublethal TBI were injected i.p. with mD1R (4 mg/kg) or PBS every day for 7 or 14 days. Single-cell suspensions were prepared from the BM and analyzed by FACS (A). The numbers of KSL cells (B, left) and c-Kit⁺Sca-1⁻Lin⁻ progenitor cells (B, right) were compared. (C,D) BM cells from mice in (A, D14) were subjected to FACS analyses. The numbers of LT-HSC (CD150⁺CD48^- KSL), ST-HSC (CD150⁺CD48⁺KSL), MPP (CD150^-CD48⁺KSL), GMP (Lin⁻Sca-1⁻c-Kit⁺CD34⁺FcγRII/III^high), CMP (Lin⁻Sca-1⁻c-Kit⁺CD34⁺FcγRII/III^low), and MEP (Lin⁻Sca-1⁻c-Kit⁺CD34^-FcγRII/III^low) were determined and compared. (E) GFP⁺C57BL/6 mice were subjected to sublethal TBI and treated with mD1R or PBS for 7 days. BM cells were isolated, mixed with equivalent numbers of normal BM cells, and transplanted into lethally irradiated (900 cGy) congenic mice. The numbers of GFP⁺ KSL cells in the BM and spleen (SP) of the recipient mice were determined with FACS 8 weeks after BM transplantation. Bars = means ± SD (n = 6). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4. Activation of Notch signaling by mD1R protected against hematopoiesis in mice after chemotherapy.

(A) C57BL/6 mice were injected i.p. once with CTX (150 mg/kg), followed by PBS or mD1R (4 mg/kg) every day for 7 days. The ratios of spleen weight/body weight (left) and liver weight/body weight (right) were measured. (B) The numbers of nucleated cells in the BM (left), spleen (SP, middle), and peripheral blood MNCs (PB, right) were determined. (C) Peripheral blood (PB) and BM were collected and subjected to Giemsa and H&E staining, respectively. (D–F) Single-cell suspensions were prepared from the BM and peripheral blood (PB) of mice in (A) and were analyzed by FACS (D). Monocytes (Ly6G⁺CD11b⁺) in the BM and peripheral blood were calculated and compared (E). In (F), the numbers of KSL cells and c-Kit⁺Sca-1⁻Lin⁻ progenitor cells were compared. Bars = means ± SD (n = 6). *P < 0.05; **P < 0.01.

Figure 5. Notch signaling regulated the proliferation and apoptosis of hematopoietic cells after irradiation in vivo.

(A,B) C57BL/6 mice subjected to sublethal TBI were injected i.p. with mD1R (4 mg/kg) or PBS every day for 14 days. Meanwhile, the mice were injected i.p. with BrdU (100 mg/kg) every two days and were maintained with drinking water containing BrdU (1 mg/ml) until the analysis of BM cells by FACS (A). The percentages of BrdU⁺ HSPCs (Sca-1⁺Lin⁻) and myeloid cells (Ly6G⁺CD11b⁺) in the BM were compared (B). (C,D) KSL cells in the BM from mice in (A) were analyzed by FACS after staining with Annexin V and PI (C). The apoptosis of KSL and Lin⁻Sca-1⁻c-Kit⁺ cells in the BM was compared on day 7 after irradiation. (E) Lin⁻ cells in the BM from mice in (A) were sorted by FACS. The levels of P-STAT3, STAT3, P-Erk1/2, Erk1/2, and Bcl-2 were determined by using Western blotting. The gels had been run under the same experimental conditions, and cropped gels are presented to show the targeted bands. Bars = means ± SD (n = 6). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6. Csf2rb2 was downstream of Notch signaling.

(A) Lin⁻ cells were isolated from the mouse BM and co-cultured with HUVECs + m5GF in the presence of PBS or mD1R. Floating hematopoietic cells were collected 48 h after the start of the culture. The expression levels of Hes1, Notch1, IL-6, Lif, Csf2rb2 and Csf1 were determined by using qRT-PCR. (B) Mice treated with mD1R or PBS were subjected to sublethal TBI. Lin⁻ cells were isolated on day 7, and Csf2rb2 mRNA expression was determined by using qRT-PCR. (C) RBP-J cKO and control mice were subjected to sublethal TBI. Lin⁻ cells were isolated on day 7, and Csf2rb2 mRNA expression was determined by using qRT-PCR. (D) Reporter assay. Upper, schematic representation of the reporter constructs driven by the mouse Csf2rb2 gene promoter and its derivatives. Red blocks represent putative RBP-J-binding sites. Lower, luciferase assay. HeLa cells were co-transfected with increasing amounts of pEF-BOS-NICD and the reporter constructs (Reporter 1~6) as described above. Cells were lysed 24 h after transfection, and luciferase activities were evaluated. (E,F) ChIP assay. BM cells were treated with 1% formaldehyde to crosslink chromatin and subjected to immunoprecipitation using anti-RBP-J (E) or anti-NICD (F), with preimmune IgG as a control. Precipitated chromatin fragments were further analyzed by qPCR using primers spanning the most distal putative RBP-J-binding sites (Fig. 6D, upper) in the Csf2rb2 promoter. Bars = means ± SD (n = 6). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 7. Activation of Notch signaling with mD1R protected hematopoietic cells through Csf2rb2.

(A) C57BL/6 mice subjected to lethal TBI (900 cGy) were injected i.p. with mD1R (4 mg/kg) or PBS every other day for 30 days. At 2 h post irradiation, the mice were injected with lentiviral particles expressing Csf2rb2-shRNA-EGFP (shCsf2rb2) or control-shRNA-EGFP (shCtrl) through the caudal veins. The survival of the mice was plotted for 30 days (n = 20). (B) The percentage of EGFP⁺ cells in the BM, spleen (SP) or peripheral blood (PB) of the mice in (A) was determined and compared. (C,D) BM and spleen (SP) cells were analyzed by FACS, and EGFP⁺ KSL cells were compared. Bars = means ± SD (n = 10). *P < 0.05; **P < 0.01.