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The mechanosensitive Piezo1 channel is required for bone formation

  1. Weijia Sun
  2. Shaopeng Chi
  3. Yuheng Li
  4. Shukuan Ling
  5. Yingjun Tan
  6. Youjia Xu
  7. Fan Jiang
  8. Jianwei Li
  9. Caizhi Liu
  10. Guohui Zhong
  11. Dengchao Cao
  12. Xiaoyan Jin
  13. Dingsheng Zhao
  14. Xingcheng Gao
  15. Zizhong Liu
  16. Bailong Xiao  Is a corresponding author
  17. Yingxian Li  Is a corresponding author
  1. China Astronaut Research and Training Center, China
  2. Tsinghua University, China
  3. The Second Affiliated Hospital of Soochow University, China
Research Article
Cite this article as: eLife 2019;8:e47454 doi: 10.7554/eLife.47454
7 figures, 1 table and 3 additional files

Figures

Figure 1 with 1 supplement
The expression and function of Piezo1 in the pre-osteoblast cell line MC3T3-E1.

(a) Representative traces of poking-induced inward currents recorded at −60 mV in the indicated cell lines. (b) Scatter plots of the maximal poking-induced currents in the indicated cell lines. (c) QRT-PCR analysis of Piezo1 mRNA level in the indicated cell lines. (d) Representative traces of poking-induced inward currents recorded at −60 mV in MC3T3-E1 under the indicated conditions. 'siRNA' indicates the siRNA-mediated knockdown of Piezo1. NC, Piezo1 negative-control siRNA. GsMTX4 is a relatively specific blocker of the Piezo channel family. (e) Scatter plots of the maximal poking-induced currents in MC3T3-E1 cells under the indicated conditions. (f, g) QRT-PCR analysis of Piezo1 mRNA level (f) and western blot analysis of Piezo1 protein level (g) in MC3T3-E1 cells transfected with control or Piezo1 siRNA for 48 hr. (h) QRT-PCR analysis of Alp, Bglap, and Col1α1 mRNA levels in MC3T3-E1 cells transfected with control or Piezo1 siRNA for 48 hr. (i) Representative images of Alp staining in MC3T3-E1 cells transfected with control or Piezo1 siRNA for 48 hr. Scale bar, 5 mm. *, p<0.05; **, p<0.01; ***, p<0.001.

https://doi.org/10.7554/eLife.47454.003
Figure 1—source data 1

The expression and function of Piezo1 in pre-osteoblast cell line MC3T3-E1.

https://doi.org/10.7554/eLife.47454.005
Figure 1—figure supplement 1
Osteogenic-induced expression of Piezo1 in the pre-osteoblast cell line MC3T3-E1.

QRT-PCR analysis of Piezo1 mRNA level. (a) QRT-PCR analysis of Piezo1 mRNA level and (b) western blot analysis of Piezo1 protein level in the osteoblasts cultured with osteogenic medium for 1 day, 3 days and 5 days. All data are the mean ± s.e.m. from three independent experiments. *, p<0.05; **, p<0.01.

https://doi.org/10.7554/eLife.47454.004
Figure 2 with 2 supplements
Expression and function of Piezo1 in primary osteoblasts isolated from Piezo1fl/fl and Piezo1Ocn/Ocn mice.

(a) QRT-PCR analysis of Piezo1 mRNA level in bone and other tissues from WT mice. (b) QRT-PCR analysis of Piezo1 mRNA level in different tissues from Piezo1fl/fl or Piezo1Ocn/Ocn mice. The Piezo1 mRNA level in all tissues from Piezo1Ocn/Ocn mice was normalized to that in Piezo1fl/fl mice. (c) Western blot analysis of Piezo1 protein level in bone tissues from Piezo1fl/fl and Piezo1Ocn/Ocn mice. (d) QRT-PCR analysis of Piezo1 mRNA level in primary osteoblasts isolated from Piezo1fl/fl or Piezo1Ocn/Ocn mice and cultured with osteogenic medium for 5 days. (e) Western blot analysis of Piezo1 protein level in primary osteoblasts cultured with osteogenic medium for 5 days. (f) Representative traces of poking-induced inward currents recorded at −60 mV in primary osteoblasts isolated from Piezo1fl/fl or Piezo1Ocn/Ocn mice. (g, h) Scatter plots of the maximal poking-induced currents (g) and inactivation tau (h) in primary osteoblasts isolated from Piezo1fl/fl or Piezo1Ocn/Ocn mice. (i) Average single-cell Ca2+ imaging traces of Piezo1fl/fl or Piezo1Ocn/Ocn osteoblasts showing the 340/380 ratio of the Ca2+-sensitive Fura-2 dye in response to the application of 30 μM Yoda1. (j) Scatter plot of baseline and Yoda1-induced Fura-2 amplitude changes in Piezo1fl/fl or Piezo1Ocn/Ocn osteoblasts. (k) Western blot analysis of p-CaMKII, CaMKII, p-Creb, Creb, Runx2 and Atf4 proteins in primary osteoblasts cultured with osteogenic medium for 5 days. (l) QRT-PCR analysis of Alp, Bglap and Col1α1 mRNA levels in primary osteoblasts cultured with osteogenic medium for 5 days. (m) Representative images of Alp staining in primary osteoblasts cultured with osteogenic medium for 5 days. Scale bar, 5 mm. The staining data were confirmed by three repeated tests. All data are the mean ± s.e.m. from three independent experiments. **, p<0.01; ***, p<0.001.

https://doi.org/10.7554/eLife.47454.006
Figure 2—source data 1

The expression and function of Piezo1 in primary osteoblasts isolated from Piezo1fl/fl and Piezo1Ocn/Ocn mice.

https://doi.org/10.7554/eLife.47454.009
Figure 2—figure supplement 1
Characterizations of primary osteoblasts derived from Piezo1fl/fl mice with or without ectopic expression of the Cre recombinase.

(a, b) QRT-PCR analysis of Piezo1 mRNA level (a) and western blot analysis of Piezo1 protein level (b) in osteoblasts derived from Piezo1fl/fl mice after transfection with pIRES-EGFP (Ctrl) or pCAG-Cre-IRES2-GFP (Cre) plasmid. (c) Representative traces of poking-induced inward currents recorded at −60 mV in an osteoblast from Piezo1fl/fl mice after transfection with Ctrl or Cre plasmid. Cells with green fluorescence were used to record current. (d) Scatter plots of the maximal poking-induced currents in osteoblasts from Piezo1fl/fl mice after transfection with Ctrl or Cre plasmid. (e) Western blot analysis of p-CaMKII, CaMKII, p-Creb, Creb, Runx2 and Atf4 proteins in osteoblasts from Piezo1fl/fl mice after transfection with Ctrl or Cre. (f) QRT-PCR analysis of Alp, Bglap and Col1α1 mRNA levels in osteoblasts from Piezo1fl/fl mice after transfection with Ctrl or Cre plasmid. (g) Representative images of Alp staining in osteoblasts from Piezo1fl/fl mice after transfection with Ctrl or Cre plasmid in osteogenic medium for 5 days. Scale bar, 5 mm. (h) Staining of calcium deposition by Alizarin red in osteoblasts derived from Piezo1fl/fl mice after transfection with Ctrl or Cre plasmid in osteogenic medium for 14 days. Scale bar, 5 mm. All data are the mean ± s.e.m. from three independent experiments. *, p<0.05; ***, p<0.001.

https://doi.org/10.7554/eLife.47454.007
Figure 2—figure supplement 2
Expression and function of Piezo1 in osteocytes isolated from Piezo1fl/fl and Piezo1Ocn/Ocn mice.

(a, b) QRT-PCR analysis of Piezo1 mRNA level (a) and western blot analysis of Piezo1 protein level (b) in osteocytes from Piezo1fl/fl and Piezo1Ocn/Ocn mice. (c) Representative traces of poking-induced inward currents recorded at −60 mV in osteocytes from Piezo1fl/fl and Piezo1Ocn/Ocn mice. (d) Scatter plots of the maximal poking-induced currents in osteocytes from Piezo1fl/fl and Piezo1Ocn/Ocn mice. (e) QRT-PCR analysis of Sost mRNA levels in bone tissues from Piezo1fl/fl and Piezo1Ocn/Ocn mice. All data are the mean ± s.e.m. from three independent experiments. *, p<0.05; **, p<0.01.

https://doi.org/10.7554/eLife.47454.008
Figure 3 with 2 supplements
Piezo1Ocn/Ocn mice show severely impaired bone formation.

(a) Alcian blue and Alizarin red staining of the skeletons of Piezo1fl/fl or Piezo1Ocn/Ocn mice at 7 days old. (b, c) Representative images showing the three-dimensional cortical bone and trabecular architecture as shown by micro-CT reconstruction at the distal femurs from Piezo1fl/fl or Piezo1Ocn/Ocn mice at 2 months old. Scale bars: (b) 1.0 mm, (c) 0.5 mm. (d) Micro-CT measurements for bone mineral density (BMD), trabecular bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) and cortical thickness (Cort.Th) at the distal femurs from Piezo1fl/fl (n = 6) or Piezo1Ocn/Ocn (n = 7) mice. (e) Relative maximal (max.) load at failure determined by three-point bending of femurs from Piezo1fl/fl (n = 6) and Piezo1Ocn/Ocn mice (n = 7). (f) Representative images showing new bone formation assessed by double calcein labeling in Piezo1fl/fl mice (n = 6) and Piezo1Ocn/Ocn mice (n = 7). MAR, mineral apposition rate. BFR/BS, bone formation rate/bone surface. Scale bar, 100 μm. (g) Histology images for Col1α1 and Ocn staining of the proximal tibia from Piezo1fl/fl and Piezo1Ocn/Ocn mice. Scale bar, 25 μm. (h) ELISA analysis of the levels of PINP and Ocn protein levels in the serum from Piezo1fl/fl (n = 8) and Piezo1Ocn/Ocn mice (n = 9). (i) QRT-PCR analysis of Alp, Bglap and Col1α1 mRNA levels in bone tissues collected from Piezo1fl/fl (n = 6) and Piezo1Ocn/Ocn mice (n = 7). All data are the mean ± s.e.m. *, p<0.05; **, p<0.01.

https://doi.org/10.7554/eLife.47454.010
Figure 3—source data 1

The data and statistical analysis of Piezo1Ocn/Ocn mice show severely impaired bone formation.

https://doi.org/10.7554/eLife.47454.013
Figure 3—figure supplement 1
The phenotypes of Piezo1fl/fl and Piezo1Ocn/Ocn mice.

(a) The appearance and body length of 2-month-old Piezo1fl/fl and Piezo1Ocn/Ocn mice. (b) The body weight of Piezo1fl/fl (n = 11) and Piezo1Ocn/Ocn mice (n = 11). (c) The appearance and the length of femurs and tibias from Piezo1fl/fl (n = 7) and Piezo1Ocn/Ocn mice (n = 7). (d) Representative images of Trap staining in bone tissue from Piezo1fl/fl and Piezo1Ocn/Ocn mice. N.Oc/B.Pm, osteoclast number/bone perimeter. (e) ELISA analysis of the levels of CTX-1 protein level in the serum from Piezo1fl/fl (n = 10) and Piezo1Ocn/Ocn mice (n = 9). (f) QRT-PCR analysis of Nfatc1, Acp5, Ctsk and Mmp9 mRNA levels in bone tissue from Piezo1fl/fl (n = 6) and Piezo1Ocn/Ocn mice (n = 8). All data are the mean ± s.e.m. **, p<0.01.

https://doi.org/10.7554/eLife.47454.011
Figure 3—figure supplement 2
The phenotypes of female Piezo1fl/fl and Piezo1Ocn/Ocn mice.

(a) QRT-PCR analysis of Piezo1 mRNA level in bone tissue from Piezo1fl/fl or Piezo1Ocn/Ocn female mice at 2 months. (b) Western blot analysis of Piezo1 protein level in bone tissue from Piezo1fl/fl and Piezo1Ocn/Ocn female mice at 2 months. (c, d) Representative images showing three-dimensional cortical bone and trabecular architecture as determined by micro-CT reconstruction at the distal femurs of Piezo1fl/fl or Piezo1 Ocn/Ocn female mice at 2 months old. Scale bars: 1.0 mm (c), 0.5 mm (d). (e) Micro-CT measurements for BMD, BV/TV, Tb.N, Tb.Th, Tb.Sp and Cort.Th in the distal femurs of Piezo1fl/fl or Piezo1Ocn/Ocn female mice. (f) QRT-PCR analysis of Alp, Bglap and Col1α1 mRNA levels in bone tissues collected from Piezo1fl/fl and Piezo1Ocn/Ocn female mice. Each group, n = 3. All data are the mean ± s.e.m. *, p<0.05; **, p<0.01.

https://doi.org/10.7554/eLife.47454.012
The effect of mechanical unloading on bone remodeling and osteoblast function in Piezo1fl/fl and Piezo1Ocn/Ocn mice.

(a) Representative images showing three-dimensional trabecular architecture as determined by micro-CT reconstruction of the distal femurs from the groups of mice indicated. Scale bar, 0.5 mm. (b) Micro-CT measurements for BMD, BV/TV, Tb.N and Tb.Th in the distal femurs from the groups of mice indicated. n = 6 in each group. (c) Relative maximal (max.) load at failure determined by three-point bending of femurs from the groups of mice indicated. Ctrl-Piezo1fl/fl group, n = 9; HS-Piezo1fl/fl group, n = 7; Ctrl- Piezo1Ocn/Ocn group, n = 7; HS-Piezo1Ocn/Ocn group, n = 8. (d) Histology images for Col1α1 and Ocn staining of the proximal tibia from the groups of mice indicated. Scale bar: 25 μm. (e) ELISA analysis of the levels of PINP and Ocn proteins in serum from the groups of mice indicated. Ctrl- Piezo1fl/fl group, n = 9; HS-Piezo1fl/fl group, n = 7; Ctrl-Piezo1Ocn/Ocn group, n = 7; HS-Piezo1Ocn/Ocn group, n = 10. (f) QRT-PCR analysis of Alp, Bglap and Col1α1 mRNA levels in bone tissues collected from the groups of mice indicated. n = 6 in each group. All data are the mean ± s.e.m. *, p<0.05; **, p<0.01; ***, p<0.001.

https://doi.org/10.7554/eLife.47454.014
Figure 4—source data 1

The data and statistical analysis of the effect of mechanical unloading on bone remodeling and osteoblast function in Piezo1fl/fl  and Piezo1Ocn/Ocn mice.

https://doi.org/10.7554/eLife.47454.015
Mechanical unloading suppresses Piezo1 expression in Piezo1Ocn/Ocn mice and osteoblasts.

(a, b) QRT-PCR analysis of Piezo1 mRNA level (a) and western blot analysis of Piezo1 protein level (b) in bone tissues from the groups of mice with the indicated treatment conditions. (c, d) QRT-PCR analysis of Piezo1 mRNA level and western blot analysis of Piezo1 protein level in osteoblasts under control (Ctrl) and simulated microgravity (MG) conditions. (e) Representative traces of poking-induced inward currents recorded at −60 mV in osteoblasts under Ctrl and MG conditions. (f) Scatter plots of the maximal poking-induced currents in osteoblasts under Ctrl and MG conditions. (g) QRT-PCR analysis of Alp, Bglap and Col1α1 mRNA levels in primary osteoblasts isolated from Piezo1fl/fl and Piezo1Ocn/Ocn mice under Ctrl and MG conditions. (h) Representative images of Alp staining in osteoblasts isolated from the indicated mice under Ctrl and MG conditions. Scale bar: 5 mm. All data are the mean ± s.e.m. from three independent experiments. **, p<0.01; ***, p<0.001.

https://doi.org/10.7554/eLife.47454.016
Figure 5—source data 1

The data and statistical analysis of the effect of mechanical unloading on Piezo1 expression in osteoblasts.

https://doi.org/10.7554/eLife.47454.017
Mechanical loading promotes Piezo1 expression in osteoblasts.

(a, b) QRT-PCR analysis of Piezo1 mRNA level (a) and Western blotting analysis of Piezo1 protein level (b) in bone tissues from the groups of mice with the indicated treatment conditions. Ex, Exercise. Ctrl-Piezo1fl/fl group, n = 4; Ex-Piezo1fl/fl group, n = 5; Ctrl-Piezo1Ocn/Ocn group, n = 3; Ex-Piezo1Ocn/Ocn group, n = 4. (c) QRT-PCR analysis of Alp, Bglap and Col1α1 mRNA levels in bone tissues collected from the groups of mice as indicated above. (d, e) QRT-PCR analysis of Piezo1 mRNA level (d) and Western blotting analysis of Piezo1 protein level (e) in osteoblasts isolated from Piezo1fl/fl and Piezo1Ocn/Ocn mice under control (Ctrl) and fluid shear stress (FSS) conditions. (f) QRT-PCR analysis of Alp, Bglap and Col1α1 mRNA levels in osteoblasts isolated from the indicated mice under Ctrl and FSS conditions. (g) Representative images of Alp staining in osteoblasts isolated from the indicated mice under Ctrl and FSS conditions. Scale bar: 1 cm. All data are the mean ± s.e.m. from three independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001.

https://doi.org/10.7554/eLife.47454.018
Figure 6—source data 1

The data and statistical analysis of the effect of mechanicalloading on Piezo1 expression in osteoblasts.

https://doi.org/10.7554/eLife.47454.019
Osteoporosis bone specimens have decreased Piezo1 expression and correlated defective osteoblast function.

(a) QRT-PCR analysis of Piezo1 mRNA level in bone specimens from two T-score groups. T > −2.5 group, n = 10, and T ≤ −2.5 group, n = 10. (b) Western blot analysis of Piezo1 protein level in bone specimens from two T-score groups. T > −2.5 group, n = 7, and T≤- 2.5 group, n = 7. Quantification of Piezo1 protein level was normalized to GAPDH. (c) Correlation analysis between Piezo1 level and the levels of ALP, BGLAP or COL1α1. T > −2.5 group, n = 10, and T ≤ −2.5 group, n = 10. (d) Correlation analysis between Piezo1 levels and the levels of CTSK (cathepsin K), ACP5 (acid phosphatase 5) or MMP9 (matrix metallopeptidase 9). T > −2.5 group, n = 10, and T ≤ −2.5 group, n = 10. (e) Correlation analysis between Piezo1 levels and the level of DMP1 (dentin matrix acidic phosphoprotein 1 ) or SOST (sclerostin). All data are the mean ± s.e.m. *, p<0.05; **, p<0.01; ***, p<0.001.

https://doi.org/10.7554/eLife.47454.020
Figure 7—source data 1

The data and statistical analysis of the relationship between Piezo1 expression levels and bone formation in human specimens.

https://doi.org/10.7554/eLife.47454.021

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or referenceIdentifiersAdditional
information
Genetic reagent (M. musculus)Ocn-CrePMID:27966526RRID:MGI:5514364Dr. Xiaochun Bai(Southern Medical University, Guangzhou, China)
Genetic reagent (M. musculus)Piezo1fl/flPMID:26001274RRID:MGI:3603204Dr. Ardem Patapoutian(The Scripps Research Institute, La Jolla, United States)
Cell line (M. musculus)MC3T3-E1China Infrastructure of Cell Line ResourceRRID:CVCL_0409
Cell line (M. musculus)RAW264.7China Infrastructure of Cell Line ResourceRRID:CVCL_0493
AntibodyRabbit anti-Piezo1ProteintechCat#:15939–1-APWB (1:1000)
AntibodyMouse anti-GapdhZSGB-BIOCat#:TA-08; RRID:AB_2747414WB (1:1000)
AntibodyRabbit anti-p-CamkIICell Signaling TechnologyCat#:12716 sWB (1:1000)
AntibodyRabbit anti-CamkIICell Signaling TechnologyCat#:4436 sWB (1:1000)
AntibodyRabbit anti-p-CrebCell Signaling TechnologyCat#:9198 sWB (1:1000)
AntibodyRabbit anti-CrebCell Signaling TechnologyCat#:9197 sWB (1:1000)
AntibodyRabbit anti-Runx2Cell Signaling TechnologyCat#:12556 sWB (1:1000)
AntibodyRabbit anti-Atf4Cell Signaling TechnologyCat#:11815 sWB (1:1000)
AntibodyRabbit anti-Col1α1AbcamCat#:ab64883IHC (1:400)
AntibodyRabbit anti-OcnProteintechCat#:23418–1-APIHC (1:200)
Recombinant DNA reagentpCAG-Cre-IRES2-GFPShanghai Biological Technology Co., Ltd. enzyme researchCat#:MY8104
Recombinant DNA reagentpIRES-EGFPAddgeneAddgene plasmid Cat#:45567
Sequence-based reagentRT-qPCR primersThis paperSee Supplementary file 2
Sequence-based reagentsiRNAThis paperSee 'Materials and methods'
Peptide, recombinant proteinRecombinant mouse RANKL proteinR and D462-TR-010
Commercial assay or kitPrimeScript RT reagent KitTAKARARR037A
Commercial assay or kitTB Green Premix Ex Taq II (Tli RNaseH Plus)TAKARARR820A
Commercial assay or kitVector Blue Substrate kitVector LaboratoriesSK-5300
Commercial assay or kitDAB kitZSGB-BIOZLI-9017
Commercial assay or kitMouse PINP ELISA KitImmunowayKE1744
Commercial assay or kitMouse Ocn ELISA KitNOVUSNBP2-68151
Commercial assay or kitMouse CTX-I ELISA KitSangon BiotechD720090
Chemical compound, drugDulbecco's Modified Eagle Medium (DMEM)Gibco/Thermo FisherGibco/Thermo Fisher:11965118
Chemical compound, drugMinimum Essential Medium (MEM) αHyCloneSH30265.01B
Chemical compound, drugOpti-MEM I Reduced Serum MediumThermo FisherCat#:31985070
Chemical compound, drugGibco Fetal Bovine SerumGibco/Thermo FisherGibco/Thermo Fisher:10099141C
Chemical compound, drugPenicillin/streptomycinThermo FisherCat#:15140122
Chemical compound, drugDexamethasoneSigmaSigma D1796
Chemical compound, drugAscorbic acidSigmaSigma A4544
Chemical compound, drugβ-glycerophosphateSigmaSigma G9422
Chemical compound, drugCollagenase type 2WorthingtonCat#:LS004176
Chemical compound, drugEDTAAmrescoCat#:0322
Chemical compound, drugLipofectamine RNAiMAXThermo FisherCat#:13778150
Chemical compound, drugLipofectamine 3000Thermo FisherCat#:L3000015
Chemical compound, drugAlcian BlueSigmaCat#:A5268
Chemical compound, drugAlizarin redSigmaCat#:A5533
Chemical compound, drugCalcein greenSigmaCat#:C0875
Software, algorithmGraphPad PrismGraphPad Prism (https://www.graphpad.com)RRID:SCR_015807Version 6
Software, algorithmOsteomeasure Analysis SystemOM-HRDVS
Software, algorithmAdobe IllustratorAdobe Illustrator (http://www.adobe.com)RRID:SCR_010279
Software, algorithmImageJImageJ (http://imagej.nih.gov/ij/)RRID:SCR_003070

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

Additional files

Supplementary file 1

Clinical features of fracture patients involved in bone specimens analysis.

https://doi.org/10.7554/eLife.47454.022
Supplementary file 2

The sequences of mRNA primers.

https://doi.org/10.7554/eLife.47454.023
Transparent reporting form
https://doi.org/10.7554/eLife.47454.024

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