Psychological stress disturbs bone metabolism via miR-335-3p/Fos signaling in osteoclast
- Shanghai Engineering Research Center of Tooth Restoration and Regeneration & Tongji Research Institute of Stomatology & Department of Prosthodontics, Shanghai Tongji Stomatological Hospital and Dental School, Tongji University, China
- Department of Neurology and Neurological Rehabilitation, Shanghai Disabled Persons' Federation Key Laboratory of Intelligent Rehabilitation Assistive Devices and Technologies, Yangzhi Rehabilitation Hospital (Shanghai Sunshine Rehabilitation Center), School of Medicine, Tongji University, China
Figures
Figure 1
Effect of chronic unpredictable mild stress (CUMS) on body weight, behavioral performance, and serum metabolism.
(A) Schematic of the procedure used to establish a CUMS model in mice. (B) Body weight (n = 9). (C) Distance moved and (D) time spent in the central area of open-field test (OFT) (n = 9). (E) Total distance moved in OFT (n = 9). (F) Frequency and amplitude of body movements during struggle. (G) Immobility in tail suspension test (TST) (n = 9). (H) Sucrose preference index (n = 9). (I) Serum corticosterone (CORT) levels (n = 6). (J) Serum norepinephrine (NE) levels (n = 5). All data are presented as means ± SD; ns, p>0.05, *p<0.05, **p<0.01, ***p<0.001, by unpaired Student’s t-test.
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Figure 1—source data 1
Full dataset for Figure 1.
- https://cdn.elifesciences.org/articles/95944/elife-95944-fig1-data1-v1.xlsx
Figure 2
Bone mass of the femur decreased in the chronic unpredictable mild stress (CUMS) model.
(A) Three-dimensional reconstruction images of femurs from control and CUMS mice. (B) Representative X-ray images of femurs from control and CUMS mice. Quantitative micro-CT analysis of femur (C) trabecular bone and (D) cortical bone. (E) H&E staining, scale bar, 200 um. All data are presented as mean ± SD, n = 7; *p<0.05, **p<0.01, ***p<0.001, by unpaired Student’s t-test.
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Figure 2—source data 1
Full dataset for Figure 2.
- https://cdn.elifesciences.org/articles/95944/elife-95944-fig2-data1-v1.xlsx
Figure 3 with 2 supplements
Osteogenic and osteoclastic metabolism of mouse femur under psychological stress.
(A) Masson staining. (B, C) Representative IHC images for OSX, OCN expression in the femur. (D) Calcein double labeling of cortical bone with quantification of MAR. (E) qRT-PCR quantification analysis of osteogenic markers including Osx, Ocn, Opn, Dmp1, Runx2, and Alpl. (F) Representative tartrate-resistant acid phosphatase (TRAP) staining images of the femur with quantitative analysis of (G) osteoclast surface per bone surface (Oc.S/BS) and (H) number per bone surface (N.OC/B). (I) qRT-PCR quantification analysis of osteogenic markers including Ca2, Mmp9, Nfatc1, and Acp5. (J) Representative IHC images for MMP9 in the femur. All data are presented as mean ± SD, n = 3; *p<0.05, **p<0.01, ***p<0.001, by unpaired Student’s t-test. Scale bar, 100 um.
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Figure 3—source data 1
Full dataset for Figure 3.
- https://cdn.elifesciences.org/articles/95944/elife-95944-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Detection of osteogenic and osteoclastic markers in serum.
(A) Serum ELISA assay of osteogenic and osteoclastic markers. (B) Serum biochemical assay of osteogenic and osteoclastic markers. All data are presented as mean ± SD, n = 4; ns, p>0.05, *p<0.05, **p<0.01, by unpaired Student’s t-test.
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Figure 3—figure supplement 1—source data 1
Full dataset for Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/95944/elife-95944-fig3-figsupp1-data1-v1.xlsx
Figure 3—figure supplement 2
qRT-PCR quantification analysis of osteoclastic markers, including Oscar, Dastamp, Calar, Ctsk, Rank/Opg, and Clcn7.
All data are presented as mean ± SD, n = 3; ns, p>0.05, by unpaired Student’s t-test.
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Figure 3—figure supplement 2—source data 1
Full dataset for Figure 3—figure supplement 2.
- https://cdn.elifesciences.org/articles/95944/elife-95944-fig3-figsupp2-data1-v1.xlsx
Figure 4 with 1 supplement
Screening of key miRNAs.
(A) The heatmap and (B) volcano plot for visualization of differentially expressed miRNAs in the femur proximal to the distal femoral growth plate (p<0.05). (C, D) Gene Ontology (GO) terms associated with targeted mRNA of differentially expressed miRNAs between groups obtained by database (miRDB and miRTarBase) prediction. (E) Validation by real-time expression analysis of miRNAs with statistically significant differences (Con, n = 3; chronic unpredictable mild stress [CUMS], n = 5). (F) Real-time expression of miR-335-3p in five stress-related brain regions (n = 4) and (G) serum (n = 3). All data are presented as mean ± SD, n = 5; *p<0.05, **p<0.01, ***p<0.001, by unpaired Student’s t-test.
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Figure 4—source data 1
Full dataset for Figure 4A–D.
- https://cdn.elifesciences.org/articles/95944/elife-95944-fig4-data1-v1.xls
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Figure 4—source data 2
Full dataset for Figure 4E–G.
- https://cdn.elifesciences.org/articles/95944/elife-95944-fig4-data2-v1.xlsx
Figure 4—figure supplement 1
Reads per million mapped reads (RPMM) expression of miR-335-3p in different tissues.
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Figure 4—figure supplement 1—source data 1
Full dataset for Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/95944/elife-95944-fig4-figsupp1-data1-v1.csv
Figure 5 with 1 supplement
Initial validation of the function of miR-335-3p.
(A) Representative cytochemical tartrate-resistant acid phosphatase (TRAP) staining images, Scale bar, 200 um. (B) The amounts of osteoclasts (multinucleated TRAP-positive cells), n = 5. (C) The positive TRAP area of osteoclasts, n = 5. (D) Representative resorption pits, Scale bar, 200 um. (E) The area of resorbed surface, n = 6. (F) qRT-PCR quantification analysis of Acp5, Nfatc1, Mmp9 expression of RANKL-induced RAW264.7 transfected with mimic-NC, mimic-miR-335-3p, inhibitor-NC, or inhibitor-miR-335-3p, n = 3. (G) Representative IF images for FOS, NFATC1, and HOCHEST expression, Scale bar, 200 um. (H) Three-dimensional reconstruction images of femurs from agomir NC, agomir-mir-335-3p, antagomir NC, and antagomir-miR-335-3p mice. (I) Quantitative micro-CT analysis of femur trabecular bone, n = 3; scale bar, 1 mm. All data are presented as mean ± SD; *p<0.05, **p<0.01, ***p<0.001, by unpaired Student’s t-test.
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Figure 5—source data 1
Full dataset for Figure 5.
- https://cdn.elifesciences.org/articles/95944/elife-95944-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Cell transfection efficiency.
All data are presented as mean ± SD; *p<0.05, ****p<0.0001, by unpaired Student’s t-test.
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Figure 5—figure supplement 1—source data 1
Full dataset for Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/95944/elife-95944-fig5-figsupp1-data1-v1.xlsx
Figure 6 with 1 supplement
Seek and verification mechanism of miR-335-3p in vivo and in vitro.
(A) The ingenuity pathways analysis (IPA) of miR-335-3p and its predictive target gene. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) terms associated with targeted mRNA of differentially expressed miRNAs between groups obtained by database (miRDB and miRTarBase) prediction. (D) Firefly luciferase activity (n = 3). (C) Sequence alignment of miR-335-3p and its predictive target sites in 3′UTR of Fos. (E) Representative IF images for FOS, CTSK, and HOCHEST expression in the femur (scale bar, 50 um). (F) Representative cytochemical tartrate-resistant acid phosphatase (TRAP) staining images (scale bar, 200 um). (G) The area and (H) the number of osteoclasts (multinucleated TRAP-positive cells) (n = 4). (I) qRT-PCR quantification analysis of Acp5, Nfatc1, Mmp9 expression of RANKL-induced RAW264.7 transfected with inhibitor-NC, miR-335-3p-inhibitor, or miR-335-3p-inhibitor+T5224 (n = 3).All data are presented as mean ± SD; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, by one-way ANOVA with Tukey’s post hoc test.
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Figure 6—source data 1
Full dataset for Figure 6.
- https://cdn.elifesciences.org/articles/95944/elife-95944-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
qRT-PCR quantification analysis of Fos expression in femur of control and chronic unpredictable mild stress (CUMS) mice.
All data are presented as mean ± SD, n = 3; ns, p>0.05, by unpaired Student’s t-test.
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Figure 6—figure supplement 1—source data 1
Full dataset for Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/95944/elife-95944-fig6-figsupp1-data1-v1.xlsx
Figure 7
Schematic model of miR-335-3p as a regulator of osteoclast in psychological stress-induced osteoporosis.
This study demonstrates that miR-335-3p levels in nucleus ambiguous (NAC), serum, and bone tissue are decreased under psychological stress, which reduces its targeting of Fos. These promote Fos translation and binding to NFATC1, increasing osteoclast activity and ultimately leading to bone loss.
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Psychological stress disturbs bone metabolism via miR-335-3p/Fos signaling in osteoclast
eLife 13:RP95944.
https://doi.org/10.7554/eLife.95944.3
