1. Medicine
  2. Neuroscience

Aberrant subchondral osteoblastic metabolism modifies NaV1.8 for osteoarthritis

  1. Jianxi Zhu
  2. Gehua Zhen
  3. Senbo An
  4. Xiao Wang
  5. Mei Wan
  6. Yusheng Li
  7. Zhiyong Chen
  8. Yun Guan
  9. Xinzhong Dong
  10. Yihe Hu  Is a corresponding author
  11. Xu Cao  Is a corresponding author
  1. Departments of Orthopaedic Surgery and Biomedical Engineering and Institute of Cell Engineering, The Johns Hopkins University School of Medicine, United States
  2. Department of Orthopaedic Surgery, Xiangya Hospital, Central South University, China
  3. Department of Anesthesiology and Critical Care Medicine, The Johns Hopkins University School of Medicine, United States
  4. Department of Neuroscience, Neurosurgery, and Dermatology, Center of Sensory Biology, The Johns Hopkins University School of Medicine, Howard Hughes Medical Institute, United States
https://doi.org/10.7554/eLife.57656
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Cite this article
  1. Jianxi Zhu
  2. Gehua Zhen
  3. Senbo An
  4. Xiao Wang
  5. Mei Wan
  6. Yusheng Li
  7. Zhiyong Chen
  8. Yun Guan
  9. Xinzhong Dong
  10. Yihe Hu
  11. Xu Cao
(2020)
Aberrant subchondral osteoblastic metabolism modifies NaV1.8 for osteoarthritis
eLife 9:e57656.
https://doi.org/10.7554/eLife.57656
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8 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
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Nav1.8 modification after mice model of OA.

(a) Heatmap of relative expression levels of NaV channels in ipsilateral L3-L5 DRGs after sham or ACLT surgery. (b, c) Immunostaining of NaV1.8+ (green) nerve fibers (b) and statistical analysis (c) in mouse tibial subchondral bone after sham or ACLT surgery at 1 m and 2 m. Scale bars, 20 μm, n = 6 per group. (d, e) Immunostaining of NaV1.8+ (green) nerve fibers (d) and statistical analysis (e) in ipsilateral sciatic nerve after sham or ACLT surgery at 1 m. Scale bars, 40 μm, n = 6 per group. (f) Western blots of NaV1.8 in mouse ipsilateral L3-5 DRGs 1 month post sham or ACLT surgery. the experiment was repeated three times and a representative result was chosen. (g, h) Retrograde tracing of Nav1.8 (green) and DiI (red) and DAPI (blue) double-labeled neurons (g) and percentage of double labeled neurons (h) in ipsilateral L4 DRG of rat after sham or ACLT surgery in 3 m. Scale bar, 80 μm. n = 6 per group. **p<0.01, ***p<0.001, ****p<0.0001 compared with the sham-operated group at different time points. Statistical significance was determined by multifactorial ANOVA WITH BONFERRONI POST HOC TEST (c, k, l), unpaired Student’s t test (e an i) and all data are shown as scattered plots with means ± standard deviations. (h, i) Representative photomicrographs (h) and statistically analysis of activated neurons (i) in ipsilateral L4 DRG using in vivo Pirt-GCaMP3 imaging treated before or after A803467 1 month post sham or ACLT surgery. n = 6 per group. (j–l) Representative traces of Aps (j upper), maximal current density (j lower), statistical analysis of AP numbers (k) and NaV1.8 currents (l) of DRG 1 month post sham or ACLT. **p<0.01, ***p<0.001, ****p<0.0001 compared with the sham-operated group at different time points. Statistical significance was determined by multifactorial ANOVA WITH BONFERRONI POST HOC TEST (c, k, l), unpaired Student’s t test (e an i) and all data are shown as scattered plots with means ± standard deviations.

Figure 1—source data 1

Raw data of Navs QPCR, subchondral Nav1.8 fiber density, Retrograde tracing, Von Frey tests, GcAMP3 imaging, and electrophysiological recordings.

https://cdn.elifesciences.org/articles/57656/elife-57656-fig1-data1-v2.xlsx
Figure 1—source data 2

Full scan of western blots in Figure 1f.

https://cdn.elifesciences.org/articles/57656/elife-57656-fig1-data2-v2.pdf
Figure 1—figure supplement 1
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NaV1.8 upregulation and colocalization in different subsets of sensory neurons in vivo and in vitro.

(a–f) Representative photographs showing NaV1.8 colocalization with PGP9.5 (a), CGRP (b), NF200 (c), P2 × 3 (d), PIEZO2 (e) and statistical analysis (f) in tibial subchondral bone in mice 4 weeks after ACLT or sham surgery. scale bar, 20 μm, n = 6 per group. (g–h) Representative photographs of NaV1.8 expression in DRGs (g) and synovial tissue (h) in mice 4 weeks after ACLT or sham surgery. scale bar, 50 μm (g), 20 μm (h). (i–j) Statistical analysis data of NaV1.8 expression in DRGs (i) and synovial tissue (j) in mice 4 weeks after ACLT or sham surgery. (k–i) Representative photographs of Aps (k) and NaV1.8 (green), PGP9.5 (red) and DAPI (blue) colocalization (l) in cultured DRG neurons treated with PGE2 (1 μM) or PBS. scale bar, 20 μm (left), 5 μm (right), experiments were repeated three times. *p<0.01, ***p<0.001, ****p<0.0001 compared with the sham-operated group or healthy donors at different time points. Statistical significance was determined by unpaired Student’s t test (f, i and j), and all data are shown as scattered plots with means ± standard deviations.

Figure 2 with 1 supplement
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Decreased NaV1.8 expression and ameliorated OA progression in Cox2:OCN cKO ACLT mice.

(a,b) Representative pictures (a) and statistical analysis (b) of OCN and Cox2 co-stained cells of murine tibial subchondral bone after sham or ACLT surgery and 1 m. Scale bars, 50 μm (left) and 10 μm (right), n = 6 per group. (c) Relative concentration of subchondral PGE2 compared with total protein concentration before and after ACLT. (e–g) Nav1.8 (green) immunostaining in subchondral bone (d), NeuN (red), NaV1.8 (green) and DAPI (blue) co-immunostaining in ipsilateral L4 DRG (e), Activated neurons in ipsilateral L4 DRG using in vivo Pirt-GCaMP3 imaging (f) and AP traces and NaV currents (g) after sham or ACLT surgery at 1 m. Scale bars, 20 μm (h), 100 μm (e, f). (h–o) Statistical analysis of subchondral PGE2 concentration (h), NaV1.8 immunofluorescence signal in subchondral bone (i), number of NeuN, Nav1.8 co-immunostained neurons in ipsilateral L4 DRG (j), number of activated neurons in ipsilateral L4 DRG using in vivo Pirt-GCaMP3 imaging (k), AP traces (l) and NaV currents (m), Catwalk gait analysis (n) and left HPWT (o) after sham or ACLT surgery. n = 6 per group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared with the sham-operated group at different time points. Statistical significance was determined by multifactorial ANOVA WITH BONFERRONI POST HOC TEST (h) or unpaired Student’s t test (b, c, h–m and o), and all data are shown as scattered plots with means ± standard deviations.

Figure 2—source data 1

Raw data of OCN Cox2 costaining, subchondral PGE2, GcAMP3 imaging, NeuN Nav1.8 merged neurons, electrophysiological recordings, Von Frey tests and catwalk analysis.

https://cdn.elifesciences.org/articles/57656/elife-57656-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
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Subchondral bone remodeling in Cox2:OCN cKO and EP4:Avil cKO mice after ACLT.

(a, b) Representative photos of knee joint Safranin Orange and fast green staining (a) and statistical analysis (b) in Bglap-Cre::Cox2fl/fl or Cox2fl/f mice 4 weeks after sham or ACLT surgery at 1 m. Scale bars, 500 μm. (c, d) Statistical analysis of BV/TV (c) and Tb. Pf (d) in Bglap-Cre::Cox2fl/f mice 4 weeks after sham or ACLT surgery at 1 m. n = 6 per group. (e, f) Representative photos of knee joint Safranin Orange and fast green staining (e) and statistical analysis (f) in Avil-Cre::Ptger4fl/fl or Ptger4fl/fl mice 4 weeks after sham or ACLT surgery at 1 m. Scale bars, 500 μm. (g, h) Statistical analysis of BV/TV (g) and Tb. Pf (h) in Ptger4Avi-/- mice 4 weeks after sham or ACLT surgery at 1 m. n = 6 per group. (i) western blot of EPs knockdown on the effect of Nav1.8 upregulation after PGE2 stimulation in DRG. *p<0.01, ***p<0.001, ****p<0.0001 compared with the sham-operated group or healthy donors at different time points. Statistical significance was determined by unpaired Student’s t test (b, c, d, f, g and h), and all data are shown as scattered plots with means ± standard deviations.

Figure 2—figure supplement 1—source data 1

Full scan of western blots in Figure 2—figure supplement 1i.

https://cdn.elifesciences.org/articles/57656/elife-57656-fig2-figsupp1-data1-v2.pdf
Figure 3
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Decreased NaV1.8 expression and ameliorated mechanical allodynia in Avil-Cre::Ptger4fl/fl ACLT mice.

(a, b) NaV1.8 immunostaining in subchondral bone (a), Activated neurons in ipsilateral L4 DRG using in vivo Pirt-GCaMP3 imaging (b) after sham or ACLT surgery at 1 m. Scale bars, 20 μm (a), 100 μm (b). (c) Representative traces of action potentials (upper) and Nav1.8 currents (lower) of ipsilateral L3-5 DRG neurons after sham or ACLT surgery at 1 m. (d–i) Statistical analysis of Nav1.8 immunofluorescence signal in subchondral bone (d), number of activated neurons in ipsilateral L4 DRG using in vivo Pirt-GCaMP3 imaging (e), AP traces (f), max Nav1.8 current density (g), catwalk gait analysis (h) and left hindpaw PWT (i) after sham or ACLT surgery. n = 6 per group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared with the sham-operated group at different time points. Statistical significance was determined by multifactorial ANOVA WITH BONFERRONI POST HOC TEST (i) or unpaired Student’s t test (d–h), and all data are shown as scattered plots with means ± standard deviations.

Figure 3—source data 1

Raw data of subchondral Nav1.8 fiber density, Von Frey tests, catwalk analysis, GcAMP3 imaging, and electrophysiological recordings.

https://cdn.elifesciences.org/articles/57656/elife-57656-fig3-data1-v2.xlsx
Figure 4
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PGE2 upregulates NaV1.8 through PKA signaling.

(a) RT-QPCR analysis of Nav1.8 in cultured lumbar DRG neurons treated with PGE2 (1 μM) for 2–12 hr. n = 6 per group. (b) Representative of western blots of PKA-c, CREB and p-CREB in cultured lumbar DRG neurons treated with PGE2 (1 μM) for 40–160 min. (c) Representative of western blots of the Nav1.8 in cultured lumbar DRG neurons treated with PGE2 (1 μM), forskolin (10 μM), or 666–15 (1 μM) for 6 hr. Representative traces of action potentials (d, upper) and Nav1.8 currents (d, lower) and statistical analysis of maximal Nav1.8 current density (f) of cultured lumbar DRG neurons after sham or ACLT surgery at 1 m. n = 6 per group. (e, f) Co-immunostaining of NeuN and Nav1.8 (g) and statistical analysis of merged cell numbers (h) in ipsilateral L4 DRGs after sham or ACLT surgery at 1 m. n = 6 per group.(i–k) ChIP experiment showing putative primers (i), PCR (j) and gel running results (k) of NaV1.8 promoter, the experiments were repeated three times. n.s, non significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared with the sham-operated group at different time points. Statistical significance was determined by multifactorial ANOVA WITH BONFERRONI POST HOC TEST (a, e and f), unpaired Student’s t test (h), all data are shown as scattered plots with means ± standard deviations.

Figure 4—source data 1

Raw data of Navs QPCR, subchondral Nav1.8 fiber density, Retrograde tracing, Von Frey tests, GcAMP3 imaging, and electrophysiological recordings.

https://cdn.elifesciences.org/articles/57656/elife-57656-fig4-data1-v2.xlsx
Figure 4—source data 2

Full scan of western blots in Figure 4b.

https://cdn.elifesciences.org/articles/57656/elife-57656-fig4-data2-v2.pdf
Figure 4—source data 3

Full scan of western blots in Figure 4c.

https://cdn.elifesciences.org/articles/57656/elife-57656-fig4-data3-v2.pdf
Figure 4—source data 4

Full scan of western blots in Figure 4k.

https://cdn.elifesciences.org/articles/57656/elife-57656-fig4-data4-v2.pdf
Figure 5
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Mechanical allodynia is reduced in Scn10a-Cre::Rosa26iDTRfl/fl ACLT mice.

(a–d) Representative photos of knee joint Safranin Orange and fast green staining (a), Nav1.8 (green) and DAPI (blue) immunofluorescence in subchondral bone (b) and NeuN (red, Nav1.8 (green) and DAPI (blue) co-immunostaining of ipsilateral L4 DRGs (c) and APs (d) after sham or ACLT surgery at 1 m. Scale bars, 500 μm (a), 20 μm (b) and 100 μm (c), n = 6 per group. (e–j) Statistical analysis of OARSI score (e), Nav1.8 immunofluorescence signal in subchondral bone (f), number of NeuN, Nav1.8 co-immunostained neurons in ipsilateral L4 DRG (g), number of AP (h), catwalk gait analysis (i) and left hindpaw PWT (j) after sham or ACLT surgery. n = 6 per group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared with the sham-operated group at different time points. Statistical significance was determined by multifactorial ANOVA WITH BONFERRONI POST HOC TEST (e–h and j) or unpaired Student’s t test (i), and all data are shown as scattered plots with means ± standard deviations.

Figure 5—source data 1

Raw data of OARSI, subchondral Nav1.8 fiber density, NeuN nav1.8 costaining, Von Frey tests, catwalk analysis, GcAMP3 imaging, and electrophysiological recordings.

https://cdn.elifesciences.org/articles/57656/elife-57656-fig5-data1-v2.xlsx
Figure 6 with 3 supplements
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Targeting aberrant subchondral bone remodeling reduces NaV1.8+ innervation and ameliorates OA pain.

(a–f) Representative photos of Safranin Orange and fast green staining (a), μCT 3D reconstruction (b), pSMAD2/3 (red) and DAPI (blue) immunostaining (c) Osterix immunostaining (d) Nav1.8 (green) and DAPI (blue) immunostaining (e) and T2 weighted fat suppression μMRI image showing bone marrow lesion (yellow arrows) (f) of murine tibial subchondral bone after sham or ACLT surgery at 1 m. Scale bars, 500 μm (a), 2 mm (b), 10 μm (c, d), 20 μm (e), and 5 mm (f), n = 6 per group. (g–l) Quantitative analysis of OARSI score (g), BV/TV (h), number of pSMAD2/3+ cells per mm2 (i) and number of Osterix+ cells per mm2 (j), relative pixel of Nav1.8 immunofluorescence signal (k) and subchondral PGE2 concentrations (l), after sham or ACLT surgery at 1 m. (m) Representative western blots of NaV1.8 and GAPDH of ipsilateral L3-5 DRG lysate, experiments were repeated three times. (n–p) Representative traces of Aps (n upper), maximal current density (n lower), statistical analysis of AP numbers (o) and NaV1.8 currents (p) of DRG 1 month post sham or ACLT. (q, r), left HPWT (q) and catwalk gait analysis (r) n = 6 per group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared with the sham-operated group at different time points. Statistical significance was determined by multifactorial ANOVA WITH BONFERRONI POST HOC TEST (j, k, L, m, n, o, q and r) or unpaired Student’s t test (c), and all data are shown as scattered plots with means ± standard deviations.

Figure 6—source data 1

Raw data of OARSI, microCT data, Osx, TRAP, pSMAD2/3, subchondral Nav1.8 fiber density, Retrograde tracing, Von Frey tests, GcAMP3 imaging, and electrophysiological recordings.

https://cdn.elifesciences.org/articles/57656/elife-57656-fig6-data1-v2.xlsx
Figure 6—source data 2

Full scan of western blots in Figure 6m.

https://cdn.elifesciences.org/articles/57656/elife-57656-fig6-data2-v2.pdf
Figure 6—figure supplement 1
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Aberrant subchondral bone remodeling after ACLT.

(a–g) Parameters of subchondral bone remodeling in OA progression. Safranin Orange and fast green staining (a), H and E staining (b), μCT 3D reconstruction (c), representatives of T2 weighted fat suppression μMRI image showing bone marrow lesion (yellow arrows) (d), pSMAD2/3 (green) immunostaining (e) and Osterix immunostaining (f) and TRAP staining (g) of murine tibial subchondral bone after sham or ACLT surgery at 1 m and 2 m. Scale bars, 500 μm (a, b), 2 mm (c), 5 mm (d), 10 μm (e–g), n = 6 per time point. (h–n) Quantitative analysis of OARSI score (h), number of pSMAD2/3+ cells per mm2 (i), number of Osterix+ cells per mm2 (j), number of TRAP+ cells per mm2 (k), TV (l), BV/TV (g) and Tb Pf (n) after sham or ACLT in 1 m, n = 6 per time point. *p<0.01, ***p<0.001, ****p<0.0001 compared with the sham-operated group or healthy donors at different time points. Statistical significance was determined by multifactorial ANOVA WITH BONFERRONI POST HOC TEST (h–n) and all data are shown as scattered plots with means ± standard deviations.

Figure 6—figure supplement 2
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Parameters of aberrant subchondral bone remodeling in human and mice osteoarthritis.

(a, b) Representative photographs (a) and statistical analysis (b) of TRAP staining in mice 1 month and 2 month after ACLT of sham surgery. scale bar, 50 μm, n = 6 per group. (c, d) μCT 3D analysis data: TV (c) and Tb. Pf (d) of mice knee subchondral bone 4 w after ACLT or sham surgery, n = 6 per group. (e, f) H and E staining of cartilage and subchondral bone of mice ACLT or sham surgery (e) and human OA or healthy samples (f). scale bar, 50 μm (e), 200 μm (f). (g) Demographic data of human patients. *p<0.01, ***p<0.001, ****p<0.0001 compared with the sham-operated group or healthy donors at different time points. Statistical significance was determined by unpaired Student’s t test (b, c and d), and all data are shown as scattered plots with means ± standard deviations.

Figure 6—figure supplement 3
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Alendronate-TβR1I inhibitor conjugate attenuates aberrant subchondral bone remodeling after ACLT.

(a) De novo synthesis of Alendronate-TβR1I inhibitor conjugate. (b, c) Representative photos (b) and statistical analysis (c) of pSMAD signal in immunostaining of human MSCs, 20 μm. (d, e) Representative photos of knee joint H and E (d) and TRAP (e) staining in mice 4 weeks after sham or ACLT or surgery or treatment at 1 m. Scale bars, 50 μm (b), 20 μm (c). (f, g) Statistical analysis of Tb. Pf (f) and TRAP+ cells (g) in WT mice 4 weeks after sham or ACLT surgery or treatment at 1 m. n = 6 per group. *p<0.01, ***p<0.001, ****p<0.0001 compared with the sham-operated group or healthy donors at different time points. Statistical significance was determined by multifactorial ANOVA WITH BONFERRONI POST HOC TEST (f, g), unpaired Student’s t test (c) and all data are shown as scattered plots with means ± standard deviations.

Figure 7
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The working model of osteoblastic PGE2 induces OA progression by NaV1.8 modification.
Author response image 1
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Tables

Key resources table
Reagent type
(species) or resource		DesignationSource or referenceIdentifiersAdditional information
Strain, Strain backgroud
(Mus musculus)		Ptger4floxed(Chen et al., 2019)N/AC57BL/6 background
Strain, Strain background
(Mus musculus)		Bglap-Cre(Tomlinson et al., 2016)N/AC57BL/6 background
Strain, Strain background
(Mus musculus)		Pirt-GcaMP3 floxed(Kim et al., 2008)N/AC57BL/6 background
Strain, Strain background
(Mus musculus)		Advillin-Cre(Avil-Cre)(Zurborg et al., 2011)N/AC57BL/6 background
Strain, Strain background
(Mus musculus)		Rosa26iDTRfloxedJackson LaboratoryC57BL/6-Gt(ROSA)26Sortm1(HBEGF)Awai/J Stock No: 007900C57BL/6 background
Strain, Stran background Rattus norvegicusSprague Dawley (SD)Charles RiverN/A
Strain, Strain backgroud
(Mus musculus)		Scn10a-Cre(Duan et al., 2018)N/AC57BL/6 background
Strain, Strain backgroud
(Mus musculus)		Cox2 floxedHarvey HerschmanN/AC57BL/6 background
Sequence- based reagentScn10a-Cre forwardPCR Primer5′-TGTAGATGGACTGCAGAGGATGGA-3′
Sequence- based reagentScn10a-Cre reversePCR Primer5′-AAATGTTGCTGGATAGTTTTTACTGCC-3′
Sequence- based reagentPirt-GCaMP3 fl primer 1PCR Primer5′-TCCCCTCTACTGAGAGCCAG-3′
Sequence- based reagentPirt-GCaMP3fl primer 2PCR Primer5′-GGCCCTATCATCCTGAGCAC-3′
Sequence- based reagentPirt-GCaMP3fl primer 3PCR Primer5′-ATAGCTCTGACTGCGTGACC-3′
Sequence- based reagentAvil-Cre: forwardPCR Primer5′-CCCTGTTCACTGTGAGTAGG-3′
Sequence- based reagentAvil-Cre: reversePCR Primer5′-GCGATCCCTGAACATGTCCATC-3′
Sequence- based reagentAvil-Cre: wildtypePCR Primer5′-AGTATCTGGTAGGTGCTTCCAG-3′
Sequence- based reagentBglap-Cre: forwardPCR Primer5′-CAAATAGCCCTGGCAGATTC-3′
Sequence- based reagentBglap-Cre: reversePCR PrimerReverse: 5′-TGATACAAGGGACATCTTCC-3′
Sequence- based reagentCox2 loxP allele forward:PCR Primer5′-AATTACTGCTGAAGCCCACC-3
Sequence- based reagentCox2 loxP allele reversePCR Primer5′-GAATCTCCTAGAACTGACTGG-3′
Sequence- based reagentPtger4 loxP allele forwardPCR Primer5′-TCTGTGAAGCGAGTCCTTAGGCT-3′
Sequence- based reagentPtger4 loxP allele reversePCR Primer5′-CGCACTCTCTCTCTCCCAAGGAA-3′
Sequence- based reagentRosa26iDTRfloxed forwardPCR Primer5′-GCGAAGAGTTTGTCCTCAACC-3′
Sequence- based reagentRosa26iDTRfloxed reversePCR Primer5′-AAAGTCGCTCTGAGTTGTTAT-3′
Sequence- based reagentGapdh forwardRT-PCR Primer5′-TCCATGACAACTTTGGCATTG-3′
Sequence- based reagentGapdh reverseRT-PCR Primer5′-CAGTCTTCTGGGTGGCAGTGA-3′
Sequence- based reagentScn1a forwardRT-PCR Primer5′-AACAAGCTTGATTCACATACAATAAG-3′
Sequence- based reagentScn1a reverseRT-PCR Primer5′-AGGAGGGCGGACAAGCTG-3′
Sequence- based reagentScn2a forwardRT-PCR Primer5′-GGGAACGCCCATCAAAGAAG-3′
Sequence- based reagentScn2a reverseRT-PCR Primer5′-ACGCTATCGTAGGAAGGTGG-3′
Sequence- based reagentScn3a forwardRT-PCR Primer5′-AGGCATGAGGGTGGTTGTGAACG-3′
Sequence- based reagentScn3a reverseRT-PCR Primer5′-CAGAAGATGAGGCACACCAGTAGC-3′
Sequence- based reagentScn8a forwardRT-PCR Primer5′-AGTAACCCTCCAGAATGGTCCAA-3′
Sequence- based reagentScn8a reverseRT-PCR Primer5′-GTCTAACCAGTTCCACGGGTCT-3′
Sequence- based reagentScn9a forwardRT-PCR Primer5′-TCCTTTATTCATAATCCCAGCCTCAC-3′
Sequence- based reagentScn9a reverseRT-PCR Primer5′-GATCGGTTCCGTCTCTCTTTGC-3′
Sequence- based reagentScn10a forwardRT-PCR Primer5′-ACCGACAATCAGAGCGAGGAG-3′
Sequence- based reagentScn10a reverseRT-PCR Primer5′-ACAGACTAGAAATGGACAGAATCACC-3′
Sequence- based reagentScn11a forwardRT-PCR Primer5′-TGAGGCAACACTACTTCACCAATG-3′
Sequence- based reagentScn11a reverseRT-PCR Primer5′-AGCCAGAAACCAAGGTACTAATGATG-3′
Sequence- based reagentCreb1
forward		ChIP-PCR
Primer 1		5′-AGTATGGTCCTTCGTGGAATACCAG-3′
Sequence- based reagentCreb1reverseChIP-PCR
Primer 1		5′-GCTATACTGCAGGAAACTGGCGA-3′
Sequence- based reagentCreb1forwardChIP-PCR
Primer 2		5′-AGCTCCCTTCTCAGCTCTCAC-3′
Sequence- based reagentCreb1reverseChIP-PCR
Primer 2		5′-CAATCTACCCAGTCTCCCTCTTTGG-3′
Sequence- based reagentCreb1forwardChIP-PCR
Primer 3		5′-GAGCACCATCCAGCAAGCAG-3′
Sequence- based reagentCreb1reverseChIP-PCR
Primer 3		5′-CCAGCTCTGCGAAACTTACACT-3′
AntibodyRabbit polyclonal Anti-Nav1.8Alomone LabsASC-016,1:50
AntibodyRabbit polyclonal Anti-pSmad2/3Santa Cruz Biosc-117691:50,
AntibodyRabbit polyclonal Anti-OsterixAbcamab225521:300
AntibodyRabbit polyclonal Anti- OsteocalcinTakara bio Inc,M1731:200
AntibodyRabbit polyclonal Anti- Cox2Abcamab151911:100
AntibodyMouse monocloncal anti-PKA-cAbcamab759911:200
SoftwareGraphpad 8.0Statistical Analysisgraph preparation, statistical analysis

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  1. Jianxi Zhu
  2. Gehua Zhen
  3. Senbo An
  4. Xiao Wang
  5. Mei Wan
  6. Yusheng Li
  7. Zhiyong Chen
  8. Yun Guan
  9. Xinzhong Dong
  10. Yihe Hu
  11. Xu Cao
(2020)
Aberrant subchondral osteoblastic metabolism modifies NaV1.8 for osteoarthritis
eLife 9:e57656.
https://doi.org/10.7554/eLife.57656