Research ArticleMechanotransduction

Microtubules tune mechanotransduction through NOX2 and TRPV4 to decrease sclerostin abundance in osteocytes

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Science Signaling  21 Nov 2017:
Vol. 10, Issue 506, eaan5748
DOI: 10.1126/scisignal.aan5748
  • Fig. 1 The FSS-induced Ca2+ response is required for CaMKII phosphorylation and reduction in sclerostin.

    (A) Ca2+ imaging of Ocy454 cells exposed to 4 dynes/cm2 FSS. Pseudocolored images are shown. n = 5 independent experiments. Scale bars, 100 μm. (B) Ca2+ responses in Ocy454 cells exposed to 4 dynes/cm2 FSS. Trace indicates Fluo-4 fluorescence changes over time. Average trace of all cells (>200 cells in n = 3 independent experiments) shown in bold. Representative individual cell traces are shown in gray. “% Cells responding” indicates the number of cells with >25% increase in fluorescence. (C) Untreated and BAPTA AM ester–loaded Ocy454 cells were subjected to 4 dynes/cm2 FSS with Ca2+-containing or Ca2+-free flow buffer, respectively. Immunoblotting was performed for phosphorylated (p) CaMKII, total CaMKII, sclerostin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (n = 3 independent experiments). The sclerostin/GAPDH and p-CaMKII/total CaMKII ratios are shown. (D) Control and KN-93–treated Ocy454 cells were subjected to 4 dynes/cm2 FSS and immunoblotted for sclerostin and GAPDH (n = 3 independent experiments). The sclerostin/GAPDH ratios are indicated. (E) Ocy454 cells transfected with green fluorescent protein (GFP) control or CaMKII T286A constructs were subjected to 4 dynes/cm2 FSS and immunoblotted for sclerostin and GAPDH (n = 3 independent experiments). The sclerostin/GAPDH ratios are shown. Graphs depict means ± SEM. **P < 0.001, ***P < 0.0001 compared to control by Kruskal-Wallis test. ns, not significantly different.

  • Fig. 2 An intact MT network is required for FSS-induced Ca2+ influx, CaMKII phosphorylation, and decreased sclerostin abundance.

    (A) Ocy454 cells stained for α-tubulin (red), phalloidin (actin, green), and 4′,6-diamidino-2-phenylindole (DAPI) (nuclei, blue). Red arrows in inset depict α-tubulin in osteocyte cell process and primary cilia. Scale bars, 10 μm. n = 3 independent experiments. (B) Murine femurs stained with SiR-tubulin. White arrows indicate MTs in the osteocyte cell processes in situ. Scale bar, 20 μm. n = 3 mice. (C and D) Ca2+ response of Ocy454 cells treated with colchicine and subjected to 4 (C) or 16 (D) dynes/cm2 FSS. Trace indicates average Fluo-4 fluorescence changes over time (>200 cells per treatment, n = 3 independent experiments). “% Cells responding” indicates number of cells with >25% increase in fluorescence; “Peak (ΔF/F)” indicates peak magnitude of Ca2+ response. The Ca2+ data for control 4 dynes/cm2 FSS are the same trace shown in Fig. 1B, because these were run in parallel with colchicine interventions. (E) Ocy454 cells treated with colchicine were subjected to 4 dynes/cm2 FSS and immunoblotted for the indicated proteins. Sclerostin/GAPDH and p-CaMKII/total CaMKII ratios are shown (n = 3 independent experiments). Images are from a single exposure of a contiguous membrane. Dotted lines indicate the removal of irrelevant lanes. (F) Immunostaining for α-tubulin in control and colchicine-treated Ocy454 cells. Scale bars, 10 μm. n = 3 independent experiments. Graphs depict means ± SEM. **P < 0.001, ***P < 0.0001 compared to control by two-tailed Mann-Whitney test (C and D) or Kruskal-Wallis test (E).

  • Fig. 3 Taxol blunts the FSS-induced Ca2+ response, phosphorylation of CaMKII, and decrease in sclerostin abundance, effects that are overcome by increased FSS.

    (A and B) Ca2+ response of Fluo-4–loaded Ocy454 cells treated with Taxol and subjected to 4 (A) or 16 (B) dynes/cm2 FSS. Trace indicates average Fluo-4 fluorescence changes over time (>200 cells per treatment, n = 3 independent experiments). “% Cells responding” indicates number of cells with >25% increase in fluorescence; “Peak (ΔF/F)” indicates peak magnitude of Ca2+ response. The Ca2+ data for the controls at 4 and 16 dynes/cm2 FSS are the same traces as in Figs. 1B and 2D, because these controls were run in parallel with the Taxol interventions. (C) Control and Taxol-treated Ocy454 cells were subjected to FSS and immunoblotted for the indicated proteins. Sclerostin/GAPDH and p-CaMKII/total CaMKII ratios are indicated (n = 3 independent experiments). (D) Immunostaining for α-tubulin in control and Taxol-treated Ocy454 cells. Scale bars, 10 μm. n = 3 independent experiments. Graphs depict means ± SEM. *P < 0.05, **P < 0.001, ***P < 0.0001 compared to control by two-tailed Mann-Whitney test (A and B) or Kruskal-Wallis test (C).

  • Fig. 4 Loss of detyrosinated tubulin, which is found within mechanically sensitive areas of osteocytes and is increased by Taxol, abrogates FSS-induced mechanosignaling.

    (A) Ocy454 cells immunostained for α-tubulin (red), detyrosinated (deTyr)–tubulin (green), and DAPI (blue). Osteocyte cell process and primary cilia are indicated by the red arrow and red arrowheads. Scale bars, 20 μm. n = 3 independent experiments. (B) Murine long bone sections immunostained for deTyr-tubulin. Red arrows indicate deTyr-tubulin in the osteocyte cell processes in situ. Scale bar, 50 μm. n = 3 mice. (C) Ocy454 cells and ex vivo murine long bone were treated with Taxol and immunoblotted for indicated proteins. DeTyr-tubulin/α-tubulin ratios are indicated (n = 3 independent experiments). The image is from a single exposure of a contiguous membrane. Dotted lines indicate the removal of irrelevant lanes. (D) Immunostaining for α-tubulin (red), deTyr-tubulin (green), and DAPI (blue) in control and Taxol-treated Ocy454 cells. Scale bars, 10 μm. n = 3 independent experiments. (E and F) Ca2+ response of Ocy454 cells treated with parthenolide (PTL) and subjected to 4 (E) or 16 (F) dynes/cm2 FSS. Trace indicates average Fluo-4 fluorescence changes over time (>200 cells per treatment, n = 3 independent experiments). “% Cells responding” indicates number of cells with >25% increase in fluorescence; “Peak (ΔF/F)” indicates peak magnitude of Ca2+ response. The Ca2+ data for the controls at 4 and 16 dynes/cm2 FSS are the same traces as in Figs. 1B and 2D, respectively, because these controls were run in parallel with the PTL interventions. (G) Control and PTL-treated Ocy454 cells were subjected to FSS and immunoblotted for indicated proteins. Sclerostin/GAPDH and p-CaMKII/total CaMKII ratios are shown (n = 3 independent experiments). (H) Control and PTL-treated Ocy454 cells were immunostained for α-tubulin (red), deTyr-tubulin (green), and DAPI (blue). n = 3 independent experiments. Scale bars, 10 μm. Graphs depict means ± SEM. **P < 0.001, ***P < 0.0001 compared to control by Mann-Whitney test (C, E, and F) or Kruskal-Wallis test (G).

  • Fig. 5 Combination treatment with PTL and Taxol restores mechanosignaling and alters MT-dependent cytoskeletal stiffness.

    (A) Ca2+ response of Fluo-4–loaded Ocy454 cells treated with combination of PTL and Taxol and subjected to 4 dynes/cm2 FSS. Trace indicates average Fluo-4 fluorescence changes over time (>200 cells per treatment, n = 3 independent experiments). “% Cells responding” indicates number of cells with >25% increase in fluorescence; “Peak (ΔF/F)” indicates peak magnitude of Ca2+ response. (B) Control Ocy454 cells and cells treated with combination of PTL and Taxol were subjected to FSS and immunoblotted for indicated proteins. Sclerostin/GAPDH and p-CaMKII/total CaMKII ratios are shown (n = 3 independent experiments). (C) Control Ocy454 cells or Ocy454 cells treated with a combination of Taxol and PTL (PTL/Taxol) were immunostained for α-tubulin (red), deTyr-tubulin (green), and DAPI (blue). Scale bars, 10 μm. n = 4 independent experiments. Graphs depict means ± SEM. **P < 0.001, ***P < 0.0001 compared to control by two-tailed Mann-Whitney test (A) or Kruskal-Wallis test (B). (D) Atomic force microscopy nanoindentation of control Ocy454 cells or cells treated with Taxol, PTL, or PTL/Taxol. Box edges denote 25th and 75th percentile, whiskers denote 10th and 90th percentile, and white lines indicate mean. Data are from three independent experiments, with number of cells per group indicated. (E) Protein extracts from control Ocy454 cells or Ocy454 cells treated with PTL, Taxol, or PTL/Taxol were probed for deTyr-tubulin and α-tubulin. The deTyr-tubulin to α-tubulin ratio (means ± SEM) is shown. (D and E) Statistical significance determined using one-way analysis of variance (ANOVA) with Holm-Sidak’s multiple comparison test. “*” denotes statistical significance between all groups. Exact P values for each comparison are shown in fig. S3.

  • Fig. 6 TRPV4 is necessary and sufficient for the osteocyte FSS-induced Ca2+ response, CaMKII phosphorylation, and decrease in sclerostin.

    (A) Ocy454 cells and sections of murine long bones immunostained with α-tubulin (red), TRPV4 (green), and DAPI (blue). Scale bars, 100 μm. n = 3 independent experiments, n = 3 mice. (B) Immunoblotting of Ocy454 whole-cell lysates and murine long bone extracts for TRPV4 and GAPDH. n = 3 independent experiments. (C and D) Ca2+ response of Ocy454 cells in the presence or absence (control) of the TRPV4 antagonist (Antag.) GSK2193874 (C) or transfected with control or TRPV4 siRNA (D) and subjected to 4 dynes/cm2 FSS. Trace indicates average Fluo-4 fluorescence changes over time (>200 cells per treatment, n = 3 independent experiments). “% Cells responding” indicates number of cells with >25% increase in fluorescence; “Peak (ΔF/F)” indicates peak magnitude of Ca2+ response. (E) Ocy454 cells were treated with or without (control) the TRPV4 antagonist GSK2193874, subjected to 4 dynes/cm2 FSS, and immunoblotted for the indicated proteins. Sclerostin/GAPDH and p-CaMKII/total CaMKII ratios are shown (n = 3 independent experiments). (F) Ocy454 cells transfected with control or TRPV4 siRNA were subjected to 4 dynes/cm2 FSS and immunoblotted for the indicated proteins. Sclerostin/GAPDH, p-CaMKII/total CaMKII, and TRPV4/GAPDH ratios are shown (n = 3 independent experiments). Image is from a single exposure of a contiguous membrane. Dotted lines indicate the removal of irrelevant lanes. (G) Ocy454 cells treated with or without (control) the TRPV4 agonist GSK-1016790A and immunoblotted for indicated proteins. Sclerostin/GAPDH and p-CaMKII/total CaMKII ratios are shown (n = 3 independent experiments). Graphs depict means ± SEM. *P < 0.05, **P < 0.001, ***P < 0.0001 compared to control by two-tailed Mann-Whitney test (C, D, and G) or Kruskal-Wallis test (E and F).

  • Fig. 7 ROS is required for the FSS-induced Ca2+ response, CaMKII phosphorylation, and decrease in sclerostin.

    (A) Ca2+ response in Ocy454 cells treated with α-NAC and subjected to 4 dynes/cm2 FSS. Trace indicates average Fluo-4 fluorescence changes over time (>200 cells per treatment, n = 3 independent experiments). “% Cells responding” indicates number of cells with >25% increase in fluorescence; “Peak (ΔF/F)” indicates peak magnitude of Ca2+ response. The Ca2+ data for the control at 4 dynes/cm2 FSS are the same trace as in Fig. 1B, because these controls were run in parallel with the NAC interventions. (B) Ocy454 cells treated with or without (control) NAC were subjected to FSS and immunoblotted for indicated proteins. Sclerostin/GAPDH and p-CaMKII/total CaMKII ratios are shown (n = 3 independent experiments). (C) Ocy454 cells were treated with H2O2 and immunoblotted for the indicated proteins. Sclerostin/GAPDH and p-CaMKII/total CaMKII ratios are shown (n = 3 independent experiments). Graphs depict means ± SEM. ***P < 0.0001 compared to control by two-tailed Mann-Whitney test (A and C) or Kruskal-Wallis test (B). (D) Ca2+ and ROS response in Ocy454 cells simultaneously loaded with the Ca2+ indicator Fluo-4 and the ROS indicator CellROX and subjected to 4 dynes/cm2 FSS. Ca2+ and ROS traces are aggregated from >200 cells per treatment over n = 3 independent experiments. Graphs depict means ± SEM. Statistical significance was determined using one-way ANOVA with Holm-Sidak’s multiple comparison test. **P < 0.001, ***P < 0.0001 compared to control (line depicts statistical significance between indicated groups).

  • Fig. 8 NOX2 generates ROS in response to FSS and is required for FSS-induced Ca2+ response, CaMKII phosphorylation, and decrease in sclerostin.

    (A) Immunoblotting of Ocy454 whole-cell lysates for NOX2 and α-tubulin. n = 3 independent experiments. (B) ROS response in Ocy454 cells loaded with the ROS indicator CellROX and subjected to 4 dynes/cm2 FSS. ROS traces are aggregated data from >200 cells per treatment from n = 3 independent experiments. Graphs depict means ± SEM. Statistical significance was determined using one-way ANOVA with Holm-Sidak’s multiple comparison test. ***P < 0.0001. (C) Ca2+ response of Ocy454 cells treated with GP91ds-TAT and subjected to 4 dynes/cm2 FSS. Ca2+ traces are aggregated from >200 cells per treatment from n = 3 independent experiments. The Ca2+ data for the control at 4 dynes/cm2 FSS are the same trace as in Fig. 5A, because these controls were run in parallel with the GP91ds-TAT interventions. (D) Ocy454 cells treated with or without (control) GP91ds-TAT were subjected to FSS and immunoblotted for the indicated proteins. Sclerostin/GAPDH and p-CaMKII/total CaMKII ratios are shown (n = 3 independent experiments). Graphs depict means ± SEM. **P < 0.001, ***P < 0.0001 compared to control by two-tailed Mann-Whitney test (C) or Kruskal-Wallis test (D). (E) Representation of MT-dependent mechanotransduction pathway showing the interventions used to alter osteocyte mechanoresponse (top). Proposed model of deTyr-tubulin and cytoskeletal stiffness regulation of osteocyte response to mechanical stimuli (bottom), in which cytoskeletal stiffness tunes the mechanoresponsive range of an osteocyte. This responsive range can be influenced not only by the cytoskeletal stiffness but also by altering the amount of FSS applied to the cell.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/506/eaan5748/DC1

    Fig. S1. Control and PTL/Taxol-treated Ocy454 cells show indistinguishable FSS-induced Ca2+ responses at 16 dynes/cm2.

    Fig. S2. Statistical significance of treatment groups in Fig. 6.

    Fig. S3. The Ca2+ channel TRPV4 is abundant at the mRNA level in Ocy454 cells.

    Fig. S4. Increased FSS does not rescue FSS-induced Ca2+ influx in Ocy454 cells treated with α-NAC or GP91ds-TAT, and TRPV4 activation does not affect ROS production.

  • Supplementary Materials for:

    Microtubules tune mechanotransduction through NOX2 and TRPV4 to decrease sclerostin abundance in osteocytes

    James S. Lyons, Humberto C. Joca, Robert A. Law, Katrina M. Williams, Jaclyn P. Kerr, Guoli Shi, Ramzi J. Khairallah, Stuart S. Martin, Konstantinos Konstantopoulos, Christopher W. Ward,* Joseph P. Stains*

    *Corresponding author. Email: jstains{at}som.umaryland.edu (J.P.S.); ward{at}som.umaryland.edu (C.W.W.)

    This PDF file includes:

    • Fig. S1. Control and PTL/Taxol-treated Ocy454 cells show indistinguishable FSS-induced Ca2+ responses at 16 dynes/cm2.
    • Fig. S2. Statistical significance of treatment groups in Fig. 6.
    • Fig. S3. The Ca2+ channel TRPV4 is abundant at the mRNA level in Ocy454 cells.
    • Fig. S4. Increased FSS does not rescue FSS-induced Ca2+ influx in Ocy454 cells treated with α-NAC or GP91ds-TAT, and TRPV4 activation does not affect ROS production.

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    Citation: J. S. Lyons, H. C. Joca, R. A. Law, K. M. Williams, J. P. Kerr, G. Shi, R. J. Khairallah, S. S. Martin, K. Konstantopoulos, C. W. Ward, J. P. Stains, Microtubules tune mechanotransduction through NOX2 and TRPV4 to decrease sclerostin abundance in osteocytes. Sci. Signal. 10, eaan5748 (2017).

    © 2017 American Association for the Advancement of Science

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