Sensory nerves regulate mesenchymal stromal cell lineage commitment by tuning sympathetic tones

Abstract

The sensory nerve was recently identified as being involved in regulation of bone mass accrual. We previously discovered that prostaglandin E2 (PGE2) secreted by osteoblasts could activate sensory nerve EP4 receptor to promote bone formation by inhibiting sympathetic activity. However, the fundamental units of bone formation are active osteoblasts, which originate from mesenchymal stromal/stem cells (MSCs). Here, we found that after sensory denervation, knockout of the EP4 receptor in sensory nerves, or knockout of COX-2 in osteoblasts, could significantly promote adipogenesis and inhibit osteogenesis in adult mice. Furthermore, injection of SW033291 (a small molecule that locally increases the PGE2 level) or propranolol (a beta blocker) significantly promoted osteogenesis and inhibited adipogenesis. This effect of SW033291, but not propranolol, was abolished in conditional EP4-KO mice under normal conditions or in the bone repair process. We conclude that the PGE2/EP4 sensory nerve axis could regulate MSC differentiation in bone marrow of adult mice.

Keywords: Adult stem cells; Bone Biology; Bone marrow differentiation; Stem cells.

Figure 1. Sensory nerve denervation induces adipogenesis of MSCs at the expense of osteogenesis.

(A–C) Representative μCT images of femurs from 1- and 3-month-old male TrkA^WT and TrkA_Avil^–/– mice. The thin yellow lines indicate the area where the cross-section images were captured (0.5 mm proximal from the growth plate). Quantitative analysis of Tb.BV/TV and Tb.Th. Scale bar: 1 mm. (D–F) Representative μCT-detected OsO₄-stained images of decalcified femurs and quantitative analysis of Ad.N and Ad.V/Ma.V in distal femurs from 3-month-old male TrkA^WT and TrkA_Avil^–/– mice. Scale bar: 500 μm. (G–I) Representative images of immunofluorescence staining and quantitative analysis of the perilipin- (red) and OCN-stained (green) femurs from 3-month-old male TrkA^WT and TrkA_Avil^–/– mice. Scale bar: 50 μm. n ≥ 6 per group; *P < 0.05 (Student’s t test). N.Ob./B.Pm, number of osteoblasts per trabecular bone surface.

Figure 2. MSCs derived from sensory denervation mice that had undergone adipogenesis.

(A–D) Representative images of crystal violet–stained CFU-F, oil red O–stained CFU-AD, and alizarin red S–stained CFU-OB. Quantitative analysis of CFU-F, CFU-AD, and CFU-OB MSCs isolated from 3-month-old male TrkA^WT and TrkA_Avil^–/– mice. (E–H) Representative dot plots of flow cytometry and quantitative analysis of CD45^–CD31^–Sca-1⁺CD24^– APCs, CD45^–CD31^–Sca-1^–PDGFRα⁺ (Pα⁺) OPCs, and CD45^–CD31^–Sca-1⁺CD24⁺ MSCs of live cells isolated from femurs of 3-month-old male TrkA^WT and TrkA_Avil^–/– mice. n ≥ 6 per group; *P < 0.05 (Student’s t test).

Figure 3. Loss of sensory nerves potentiate adipogenesis.

(A–C) Representative μCT-detected OsO₄-stained images of decalcified femurs and quantitative analysis of Ad.N and Ad.V/Ma.V in distal femurs of 3-month-old male iDTR_Avil^+/– mice injected with 1 μg/kg/d vehicle or DTX 3 times a week for 4 consecutive weeks. Scale bar: 500 μm. (D–G) Representative dot plot images of flow cytometry and quantitative analysis of CD45^–CD31^–Sca-1⁺CD24^– APCs, CD45^–CD31^–Sca-1^–PDGFRα⁺ (Pα⁺) OPCs, and CD45^–CD31^–Sca-1⁺CD24⁺ MSCs of live cells isolated from femurs of 3-month-old iDTR_Avil^+/– mice injected with vehicle or DTX for 4 weeks. (H–J) Representative images of immunofluorescence staining and quantitative analysis of perilipin (red) and OCN (green) in femurs from 3-month-old male iDTR_Avil^+/– mice injected with vehicle (Veh) or DTX for 4 weeks. Scale bar: 50 μm. n ≥ 6 per group: *P < 0.05 (Student’s t test).

Figure 4. Deletion of the EP4 receptor in sensory nerves promotes adipogenesis and inhibits osteogenesis.

(A–C) Representative μCT images of femurs from 1- and 3-month-old male EP4^WT and EP4_Avil^–/– mice. The thin yellow lines indicate the area where the cross-section images were captured (0.5 mm proximal from the growth plate). Quantitative analysis of Tb.BV/TV and Tb.Th. Scale bar: 1 mm. (D–F) Representative μCT-detected OsO₄-stained images of decalcified femurs and quantitative analysis of Ad.N and Ad.V/Ma.V in distal femurs from 3-month-old male EP4^WT and EP4_Avil^–/– mice. Scale bar: 500 μm. (G–I) Immunohistochemical staining of perilipin (red) and OCN (green) in femurs from 3-month-old male EP4^WT and EP4_Avil^–/– mice treated with SW033291 (10 mg/kg/d) or vehicle for 1 month. Quantitative analysis of the density of perilipin and OCN in femurs from 3-month-old male EP4^WT and EP4_Avil^–/– mice treated with SW033291 or vehicle for 1 month. Scale bar: 50 μm. (J–M) Representative images of crystal violet–stained CFU-F, oil red O–stained CFU-AD, and alizarin red S–stained CFU-OB. Quantitative analysis of CFU-F, CFU-AD, and CFU-OB MSCs isolated from 3-month-old male EP4^WT and EP4_Avil^–/– mice. (N–Q) Representative dot plot images of flow cytometry and quantitative analysis of CD45^–CD31^–Sca-1⁺CD24^– APCs, CD45^–CD31^–Sca-1^–Pα⁺ OPCs, and CD45^–CD31^–Sca-1⁺CD24⁺ MSCs of live cells isolated from femurs of 3-month-old male EP4^WT and EP4_Avil^–/– mice. n ≥ 6 per group; *P < 0.05 (Student’s t test; 2-way ANOVA for H and I).

Figure 5. PGE2 derived from osteoblasts regulates the differentiation of MSCs through EP4 in sensory nerves.

(A–C) Representative μCT images of femurs from 1- and 3-month-old male COX2^WT and COX2_OCN^–/– mice. The thin yellow lines indicate the area where the cross-section images were captured (0.5 mm proximal from the growth plate). Quantitative analysis of Tb.BV/TV and Tb.Th. Scale bar: 1 mm. (D–F) Representative μCT-detected OsO₄-stained images of decalcified femurs and quantitative analysis of Ad.N and Ad.V/Ma.V in distal femurs of 3-month-old male EP4^WT and EP4_Avil^–/– mice. Scale bar: 500 μm. (G–I) Representative images of immunohistochemical staining of perilipin (red) and OCN (green) in femurs of 3-month-old male COX2^WT and COX2_OCN^–/– mice treated with SW033291 (10 mg/kg/d) or vehicle for 1 month. Quantitative analysis of density of perilipin and OCN in femurs of 3-month-old male COX2^WT and COX2_OCN^–/– mice treated with SW033291 or vehicle for 1 month. Scale bar: 50 μm. (J–M) Representative images of crystal violet–stained CFU-F, oil red O–stained CFU-AD, and alizarin red S–stained CFU-OB. Quantitative analysis of CFU-F, CFU-AD, and CFU-OB MSCs isolated from 3-month-old male COX2^WT and COX2_OCN^–/– mice. (N–Q) Representative dot plots of flow cytometry and quantitative analysis of CD45^–CD31^–Sca-1⁺CD24^– APCs, CD45^–CD31^–Sca-1^–PDGFRα⁺ (Pα⁺) OPCs, and CD45^–CD31^–Sca-1⁺CD24⁺ MSCs of live cells isolated from femurs of 3-month-old male COX2^WT and COX2_OCN^–/– mice. n ≥ 6 per group; *P < 0.05 (Student’s t test for B, C, E, F, K–M, and O–Q; 2-way ANOVA for H and I).

Figure 6. Capsaicin-induced sensory nerve denervation promotes adipogenesis and inhibits osteogenesis.

(A–C) Representative μCT images and quantitative analysis of Tb. BV/TV and Tb.Th of femurs from 3-month-old male LepR-Cre;YFP mice injected with capsaicin (Cap; 30 mg/kg/d) or vehicle for 1 week and euthanized after another 2 weeks. The yellow lines indicated the area where the cross-section images were captured (0.5 mm proximal from the growth plate). Scale bar: 1 mm. (D–F) Representative μCT-detected OsO₄-stained images of decalcified femurs and quantitative analysis of Ad.N and Ad.V/Ma.V in distal femurs of 3-month-old male LepR-Cre;YFP mice injected with capsaicin or vehicle for 1 week and euthanized after another 2 weeks. Scale bar: 500 μm. (G–I) Representative images of immunohistochemical staining of perilipin (red) and OCN (green) in femurs of 3-month-old male LepR-Cre;YFP mice injected with capsaicin or vehicle for 1 week and euthanized after another 2 weeks. Scale bar: 50 μm. n ≥ 6 per group; *P < 0.05 (Student’s t test).

Figure 7. LepR⁺ MSCs are the major source of the increase in adipocytes and are regulated by the PGE2/EP4 sensory nerve axis.

(A and B) Representative images of immunofluorescence staining and quantitative analysis of CGRP⁺ sensory nerves (green) in distal femurs of 3-month-old male LepR-Cre;YFP mice injected with capsaicin or vehicle for 1 week and euthanized after another 2 weeks (SB, subchondral bone). Scale bar: 100 μm. (C) Representative images of immunofluorescence staining of colocalization of perilipin (red) and YFP (representing LepR⁺ cells) (green) in femur bone marrow from 3-month-old LepR-Cre;YFP mice treated with capsaicin (30 mg/kg/d for 1 week), SW033291 (10 mg/kg/d for 1 month), or propranolol (0.5 mg/kg/d for 6 weeks). (D) Quantitative analysis of YFP⁺ adipocytes for each of the groups (marrow adipocytes labeled by white arrowheads). Scale bar: 50 μm. n ≥ 6 per group; *P < 0.05 (Student’s t test for B, 2-way ANOVA for C).

Figure 8. Impairment of EP4 sensory nerves promotes adipogenesis and attenuates bone regeneration.

(A and B) Representative μCT images of bone regeneration 7 days after femoral bone marrow ablation in 3-month-old male LepR-Cre;YFP mice treated with capsaicin (30 mg/kg/d) or vehicle. Scale bar: 1 mm. Areas selected for measurement of BV/TV are indicated by yellow squares. (C) Representative SO/FG and Masson’s staining (red, muscle and cytoplasm; blue, bone) images in the regeneration area in 3-month-old male LepR-Cre;YFP mice treated with capsaicin or vehicle 7 days after bone marrow ablation. Red represents cartilage area; green represents woven bone area in SO/FG staining. Scale bars: 100 μm. (D–F) Representative images and analysis of perilipin (red) and OCN (green) staining in the regeneration area from the capsaicin-treated and control groups. Scale bar: 50 μm. (G and H) Representative images of immunofluorescence staining of colocalization of perilipin (red) and YFP (representing LepR⁺ cells; green), and quantitative analysis of density of YFP⁺ adipocytes in the regeneration area from capsaicin-treated and control groups. Scale bar: 100 μm. (I and J) Representative μCT images of bone regeneration after femoral bone marrow ablation in 3-month-old male EP4^WT and EP4_Avil^–/– mice treated with 10 mg/kg/d SW033291 or vehicle 7 days after bone marrow ablation. Scale bar: 1 mm. Selected areas for the measurements of BV/TV are indicated by yellow squares. (K–M) Immunohistochemical staining and quantitative analysis of perilipin (red) and OCN (green) in the regeneration area from 3-month-old male EP4^WT and EP4_Avil^–/– mice treated with 10 mg/kg/d SW033291 or vehicle. Scale bar: 50 μm. n ≥ 6 per group; *P < 0.05 (Student’s t test for B, E, F, and H; 2-way ANOVA for J–L).

Figure 9. Sensory nerve denervation impaired bone fracture healing.

(A–C) Representative μCT images and quantitative analysis of BV/TV and Tb.Th of fracture healing area in 3-month-old male LepR-Cre;YFP mice treated with capsaicin (30 mg/kg/d) or vehicle 2 weeks after bone fracture. Scale bar: 1 mm. (D) Representative SO/FG (red, cartilage; green, bone) and Masson’s staining (red, muscle and cytoplasm; blue, bone) images in the fracture healing area in the capsaicin-treated and control groups. Scale bar: 100 μm. (E–G) Representative images of immunohistochemical staining and quantitative analysis of density of perilipin (red) and OCN (green) in the fracture healing area in the capsaicin-treated and control groups. Scale bar: 50 μm. (H and I) Representative images of immunofluorescence staining of colocalization of perilipin (red) and YFP (representing LepR⁺ cells; green), and quantitative analysis of the density of YFP⁺ adipocytes in fracture healing area in the capsaicin-treated and control groups. Scale bar: 50 μm. n ≥ 5 per group; *P < 0.05 (Student’s t test).

Figure 10. Impairment of EP4 sensory nerves interrupts bone fracture healing.

(A–E) Representative μCT images and quantitative analysis of BV/TV, Tb.Th, Tb.N, and Tb.Sp of bone fracture healing area in 3-month-old male EP4^WT and EP4_Avil^–/– mice treated with 10 mg/kg/d SW033291 or vehicle 2 weeks after bone fracture. Scale bar: 2 mm. (F) Representative images of SO/FG and Masson’s staining and (G–I) immunohistochemical staining and quantitative analysis of perilipin (red) and OCN (green) in the fracture healing area in 3-month-old male EP4^WT and EP4_Avil^–/– mice treated with SW033291 or vehicle 2 weeks after bone fracture. Scale bar: 50 μm. n ≥ 5 per group; *P < 0.05 (2-way ANOVA).