The effect of ROS-YAP crosstalk on osteoimmune response orchestrating osteogenesis

Abstract

Bone defect repair is a common medical concern. In spite of various existing treatments, its management still requires improvement. Here we show that YAP, a downstream signaling of Hippo pathway, might interplay with redox oxygen species (ROS) and modulate osteoimmunology, which refers to the interaction between immune and skeletal system during bone defect repair. We modulated the ROS level of RAW264.7 cells and found YAP level was reversely regulated. Meanwhile, we detected the feedback of YAP on oxidation level. The results demonstrated that the antioxidant enzyme expression was in proportion to the YAP level of RAW264.7 cells. Additionally, indirect coculture system was applied and it indicated that RAW264.7 cells under oxidative stress could impede proliferation and migration ability of MC3T3-E1 pre-osteoblasts. Consistently, in vivo experiment verified high oxidant level slowed down mice osteogenesis during bone defect repair, while antioxidant and upregulation of YAP accelerated this process. Additionally, we established a mouse model with YAP conditional knockout in macrophages. The results identified that deficiency of YAP in macrophages negatively affected bone defect repair in vivo. In summary, our study indicated that ROS and YAP could jointly modulate osteogenesis via their effect on osteoimmunology.ABBREVIATIONS: GPX4, glutathione peroxidase 4; NAC, N-Acetyl-L-cysteine; qRT-PCR, real-time quantitative PCR; ROS, reactive oxygen species; Tb.N, trabecular number; Tb.Sp, trabecular separation.

Keywords: Redox oxygen species; YAP; osteogenesis; osteoimmunology; oxidative stress.

Figure 1.

Upregulation of intracellular ROS level abrogates expression of antioxidant gene in RAW264.7 cells. RAW264.7 was untreated, treated with rotenone or NAC, respectively. a, DCFH-DA fluorescent probe experiment indicated the ROS level of RAW264.7 was significantly improved by rotenone treatment but it was not significantly changed by NAC treatment. qRT-PCR experiment results suggested significant downregulation of GPX4 (b) and YAP(c) when dealing with rotenone, and robust upregulation of GPX4 (b) and YAP(c) when dealing with NAC. Additionally, rotenone induced RAW264.7 cell apoptosis while NAC treatment made insignificant difference (d). Scale bar=250 μm. Representative data of three independent experiments are used for statistical analysis. Data are presented as dot plots with the mean ± SEM, and significant differences are marked by asterisks (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n.S., not significant). GPX4, glutathione peroxidase 4; NAC, N-Acetyl-L-cysteine.

Figure 2.

The GPX4 expression was positively related to the level of YAP. RAW264.7 was transfected with YAP-overexpressed lenti-virus. a, qRT-PCR test was used to verify the efficiency of the lenti-virus. It showed that the mRNA expression of YAP was significantly enhanced under MOI 10. b, qRT-PCR test further revealed that YAP upregulation had a significantly positive effect on GPX4 expression, and its inhibition diminished GPX4 expression. Representative data of three independent experiments are used for statistical analysis. Data are presented as dot plots with the mean ± SEM, and significant differences are marked by asterisks (*p < 0.05; ***p < 0.001; ****p < 0.0001). LV, lenti-virus; VP, verteporfin.

Figure 3.

Upregulated macrophage ROS level inhibited proliferation and migration of MC3T3-E1. RAW264.7 was untreated, treated with rotenone or NAC, respectively. the conditioned medium (i.e. the supernatant) of RAW264.7 was collected to culture MC3T3-E1. a, CCK-8 assay was performed. Proliferation was significantly suppressed by rotenone treated RAW264.7 supernatant while not influenced by NAC treated RAW264.7 supernatant. Data are presented as dot plots with the mean ± SEM, and significant differences are marked by asterisks (*p < 0.05; ***p < 0.001; ****p < 0.0001). b, wound healing assay was performed and uncovered that the migration ability of MC3T3-E1 could be blocked by rotenone-treated RAW264.7 while promoted by NAC-treated RAW264.7. Scale bar=200 μm. c, morphologic alteration was observed in MC3T3-E1 cultured with rotenone-treated RAW264.7 supernatant after 3 days, but not in those cultured with NAC-treated RAW264.7 supernatant. Scale bar=100μm. Representative data of three independent experiments are used for statistical analysis.

Figure 4.

Antioxidants such as NAC promote osteogenesis during bone defect repair. a, a 1mm in depth and 1mm in diameter bone defect was made at 1mm mesial from femur metaphysis. The yellow dotted circle indicated the location of bone defect. b, micro-CT was adopted to analyze the osteogenesis status at the bone defect. It turns out that osteogenesis was significantly inhibited by rotenone treatment but enhanced by NAC treatment. Scale bar=1mm. c, rotenone treatment reduced the trabecular number (Tb.N) while NAC treatment increased it at 1 week and 2 weeks. d, Rot group presented significantly increased trabecular separation (Tb.Sp) at 2 weeks compared with Ctr group, whereas NAC group was not significantly different from Ctr group. e, HE staining results also suggested that osteogenesis was significantly inhibited by rotenone treatment but enhanced by NAC treatment. Black arrows indicated the newborn bone trabeculae. Scale bar=200μm at low magnification. f, Histomorphometry analysis of bone volume versus total tissue volume (BV/TV) was broadly in line with the former results. Scale bar=100μm at high magnification. Data are presented as dot plots with the mean ± SEM, and significant differences are marked by asterisks (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n.S., not significant). NAC, N-Acetyl-L-cysteine.

Figure 5.

Upregulation of YAP level could accelerate osteogenesis during bone defect repair. a, three-dimensional reconstruction of micro-CT unraveled that YAP upregulation could promote osteogenesis during bone defect repair. Scale bar=1mm. b, YAP overexpression increased Tb.N while YAP downregulation made insignificant difference at 1 week and 2 weeks. c, Tb.Sp was significantly reduced in YAP+ group at 1 week and 2 weeks, while there was no significant difference between YAP- group and Ctr group. d, HE staining results also demonstrated that regenerated new bone was denser in YAP+ group at 1 week and 2 weeks, while insignificant difference was observed between YAP- group and Ctr group. Black arrows indicated the newborn bone trabeculae. Scale bar=200μm at low magnification. e, Histomorphometry analysis of bone volume versus total tissue volume (BV/TV) was basically consistent with the former results. Scale bar=100μm at high magnification. Data are presented as dot plots with the mean ± SEM, and significant differences are marked by asterisks (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n.S., not significant).

Figure 6.

Deficiency of YAP in macrophages negatively affected bone defect repair. a, three-dimensional reconstruction of micro-CT indicated that YAP knockout in macrophages could inhibit osteogenesis during bone defect repair. Scale bar=1mm. b & c, YAP knockout in macrophages decreased Tb.N and increased Tb.Sp at 2 week, while the difference was not significant at 1 week. d & e, HE staining and histomorphometry analysis results similarly suggested looser regenerated new bone in LysM-Cre;YAP^fl/fl mice. Black arrows indicated the newborn bone trabeculae. Scale bar=200μm at low magnification. Scale bar=100μm at high magnification. Data are presented as dot plots with the mean ± SEM, and significant differences are marked by asterisks (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n.S., not significant).

Figure 7.

ROS and YAP could interact with each other in osteo-immune response and make a difference in osteogenesis procedure. Cellular ROS level could downregulate the YAP mRNA expression. The expression of GPX4, an important antioxidant enzyme, was dependent on activation of YAP, which indicated the reverse effect of YAP on oxidation level. Additionally, the oxidation level impacted the pre-osteoblast function through osteo-immune response between macrophages and bone cells. Furthermore, osteogenesis process during bone defect repair was also associated with the oxidation level. GPX4, glutathione peroxidase 4; ROS, reactive oxygen species; TFs, transcriptional factors.