Research ArticleBONE DEVELOPMENT

Histone deacetylase 3 supports endochondral bone formation by controlling cytokine signaling and matrix remodeling

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Science Signaling  09 Aug 2016:
Vol. 9, Issue 440, pp. ra79
DOI: 10.1126/scisignal.aaf3273
  • Fig. 1 Hdac3 ablation in chondrocytes during postnatal growth delays skeletal development.

    (A) Immunohistochemistry for HDAC3 on tibial growth plates of 4-week-old Hdac3-CKOCol2ERT mice that had been injected with tamoxifen or vehicle at P5. Scale bars, 200 μm. (B) qPCR for Hdac3 in the xiphoid processes of 8-week-old mice described in (A) (n = 3 mice per group). (C) Weights of male mice at indicated ages (n = 5 mice per group). (D) Femur lengths of 8-week-old male animals described in (A) were measured in micro-CT (computed tomography) scans (n = 5 mice per group). (E) Alcian blue/eosin-stained tibial sections isolated at the indicated ages from mice described in (A). Scale bars, 100 μm. (F) Quantification of SOC area to total tissue area of epiphyses from 2-week-old Hdac3-CKOCol2ERT or C57BL/6 mice injected at P5 with tamoxifen or vehicle (n = 5 mice per group). N.S., not significant. (G) Quantification of total growth plate (GP) depth across the indicated regions in 4-week-old animals described in (A) (n = 5 mice per group). (H) Equilibrium partitioning of an ionic contrast agent (EPIC)–CT scans of femoral condyles from 4-week-old mice described in (A) (top). The yellow arrow points to residual cartilage along the growth plate in the Hdac3-CKOCol2ERT mice. Three-dimensional reconstructions of the femoral condyles show the growth plate (solid gray) within the bone architecture (semitransparent blue). A, anterior; D, dorsal; L, lateral; M, medial; P, posterior; V, ventral axes. (I) Immunohistochemistry for phosphorylated γH2A.X on tibiae of 2- and 4-week-old mice described in (A). Scale bars, 100 μm. (J) Immunohistochemistry for PECAM-1 on tibiae of P9 pups described in (A). Scale bars, 100 μm. *P ≤ 0.05, #P ≤ 0.075, Student’s t test.

  • Fig. 2 Hdac3 deletion alters global gene expression and histone acetylation in chondrocytes.

    (A) Western blotting for the indicated proteins performed 48 hours after adenoviral transduction in primary IMCs isolated from 1-week-old Hdac3fl/fl;Cre pups, plated in micromass cultures, and transduced with Ad-GFP or Ad-Cre on day 3 of culture. (B) Micromasses described in (A) were fixed 48 hours after adenoviral transduction and stained with Alcian blue. (C and D) qPCR to validate RNA-seq results from Hdac3-depleted IMCs described in (A). Differential expression of representative genes with decreased (C) or increased (D) expression in HDAC3-deficient micromasses (Ad-Cre) is shown relative to control (Ad-GFP) IMCs, which was set to 1 (n = 3). (E to I) ChIP-seq was performed for H3K27ac in HDAC3-deficient micromasses described in (A). Average binding profiles of H3K27ac 5 kb upstream or downstream of the transcriptional start site (TSS) in control (blue) or HDAC3-deficient (green) micromasses: all genes (E), genes with increased expression after Hdac3 depletion (F), and genes with decreased expression after Hdac3 depletion (H). Representative distribution of H3K27ac in select genes with corresponding increased (G) or decreased (I) expression. *P ≤ 0.05, Student’s t test.

  • Fig. 3 HDAC3 deficiency in chondrocytes increases proinflammatory response signals through increased IL-6–STAT3 signaling and is rescued with JAK inhibition.

    (A, B, G, and J to O) Primary IMCs were harvested from 1-week-old Hdac3fl/fl pups, plated in micromasses, and transduced with Ad-GFP or Ad-Cre on day 3. (A and B) Conditioned media (CM) from micromasses were analyzed with an ELISA to measure mouse IL-6 (A) or a fluorometric MMP enzymatic assay (B) (n = 3). (C to F) ATDC5 cells in monolayer (C and E) or micromass (D and F) were treated with the broad HDAC inhibitor SAHA (1 μM) or the HDAC3-specific inhibitor RGFP966 (10 μM) for 24 hours. qPCR for expression of Il-6 (C and D) and Mmp13 (E and F) (n = 3). (G to I) Western blotting for the indicated proteins in HDAC3-deficient chondrocytes in micromass (G), ATDC5 cells in micromass and treated with RGFP966 (H), or wild-type (WT) IMCs treated with RGFP966 (I) (10 μM) for 24 hours. (J to N) HDAC3-deficient chondrocytes in micromasses were treated with JAK Inhibitor I (1 μM) for 24 hours, at which point Western blotting (J) for the indicated proteins and qPCR (K to N) for the indicated mRNA transcripts were performed (n = 3). *P ≤ 0.05, #P = 0.06, Student’s t test.

  • Fig. 4 HDAC3 depletion in chondrocytes increases NF-κB acetylation.

    (A to D) Primary IMCs were harvested from 1-week-old Hdac3fl/fl pups, plated in micromass, and treated with Ad-GFP or Ad-Cre on day 3 of culture. (A) Western blotting was performed after 48 hours. (B) Western blotting for the indicated proteins in cytosolic and nuclear fractions collected from micromasses. (C) Quantification of the percentage of nuclear NF-κB relative to the total amount of NF-κB present from n = 3 experiments. (D) Primary immature mouse chondrocytes from 1-week-old Hdac3fl/fl mice were plated in monolayer and transduced with Ad-GFP or Ad-Cre. Immunofluorescence for total NF-κB/p65 and Cre recombinase was performed 48 hours after viral transduction. The yellow arrows indicate cells expressing Cre, and white arrowheads indicate control cells (not expressing Cre). Scale bars, 20 μm. (E to J) Micromass cultures were simultaneously transduced with Ad-GFP or Ad-Cre and either Ad-GFP or Ad-dnIKK2. (E) Western blotting for the indicated proteins was performed on the cellular fractions 48 hours after transduction. (F) Quantification of nuclear NF-κB relative to the total amount of NF-κB present (n = 3). (G to J) qPCR for the indicated mRNAs in HDAC3-deficient IMCs transduced with Ad-dnIKK2: Il-6 (G), Mmp13 (H), Saa3 (I), and Col2a1 (J) (n = 3). (K to N) HDAC3-deficient micromasses were treated with JQ-1 (10 μM) for 24 hours, and qPCR was performed for the indicated transcripts: Il-6 (K), Mmp13 (L), Saa3 (M), and Col2a1 (N) (n = 3). *P ≤ 0.05, Student’s t test.

  • Fig. 5 Chondrocytic HDAC3 deficiency increases osteoclastogenesis in vitro.

    (A to G) Osteoclast precursors were collected from 8-week-old WT mice and incubated overnight with 15% CM from HDAC3-deficient or control chondrocytes. Nonadherent cells were plated in normal osteoclast differentiation medium. (A) Day 4 TRAP-stained mature osteoclasts. Scale bars, 100 μm. (B) Quantification of TRAP+ mature osteoclasts (n = 3). No. Ocl, total number of osteoclasts. (C) RNA was isolated from mature osteoclast cultures, and qPCR was performed for the indicated genes. Relative fold changes were normalized to osteoclast cultures treated with CM from Ad-GFP–treated chondrocytes, indicated by the dotted line set to 1 (n = 3). (D and E) CM were collected from HDAC3-deficient chondrocytes treated with 1 μM JAK inhibitor for 24 hours. WT osteoclast precursors were preincubated with the CM from JAK inhibitor–treated HDAC3-deficient chondrocytes. (D) Day 4 TRAP-stained mature osteoclasts. Scale bars, 100 μm. (E) Quantification of TRAP+ osteoclasts (n = 3). (F and G) CM from HDAC3-deficient chondrocytes was pretreated with a neutralizing antibody recognizing IL-6 or an immunoglobulin G (IgG) control antibody (Ab). WT osteoclast precursors were collected and primed with the neutralized CM. (F) Day 4 TRAP-stained mature osteoclasts. (G) Quantification of TRAP+ osteoclasts (n = 3). *P ≤ 0.05, #P = 0.08, Student’s t test. Black arrowheads indicate multinucleated osteoclasts.

  • Fig. 6 HDAC3-deficient chondrocytes increase inflammatory responses and osteoclast proliferation in vivo.

    (A) ELISAs for IL-6 on sera collected from 2-week-old Hdac3f/.fl;Cre or Col2ERT-Cre+ mice. (B) Immunohistochemistry for IL-6 on sections from 4-week-old tibiae. Scale bars, 100 μm. (C) Immunohistochemistry for MMP13 on tibia sections from P9 Hdac3-CKOCol2ERT or control mice. (D) Tibiae from 4-week-old Hdac3-CKOCol2ERT and control animals were stained for TRAP. Shown are representative images of the primary spongiosa. Scale bars, 200 μm. (E to G) Histomorphometric quantification of osteoclast parameters from 4-week-old Hdac3-CKOCol2ERT mice: (E) total number of osteoclasts (N. Oc), (F) number of osteoclast per bone perimeter (N. Oc/B. Pm), and (G) osteoclast surface per bone surface (Oc. S/BS) (n = 3 for control animals and n = 4 for CKO animals). (H to L) Micro-CT analysis of bone architecture from 8-week-old Hdac3-CKOCol2 mice. (H) Micro-CT reconstructions of the cancellous bone. (I to L) Quantification of micro-CT data on trabecular bone volume fraction (Tb. BV/TV) (I), trabecular number (Tb. N) (J), trabecular thickness (Tb. Th) (K), and trabecular separation (Tb. Sp) (L) (n = 5 mice for each group). (M) Tibiae from 6-week-old Hdac3-CKOCol2ERT mice were treated with ruxolitinib for 2 weeks and were TRAP-stained. Shown are representative images of the primary spongiosa. Scale bars, 200 μm (n = 3 vehicle/vehicle and tamoxifen/vehicle mice, 5 vehicle/ruxolitinib mice, and 4 tamoxifen/ruxolitinib mice). In (A) to (M), *P ≤ 0.05 and #P = 0.14, Student’s t test. (N) Working model. HDAC3 suppresses production of cytokines and catabolic matrix enzymes in chondrocytes during bone development.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/9/440/ra79/DC1

    Fig. S1. Expression pattern of Hdac3 during embryonic and postnatal long bone development.

    Fig. S2. Hdac3 knockout in chondrocytes during embryogenesis is lethal.

    Fig. S3. Tamoxifen does not affect growth plate maturation in wild-type mice.

    Fig. S4. Top 30 annotation clusters of genes with decreased gene expression in Hdac3-depleted IMCs.

    Fig. S5. Top 30 annotation clusters of genes with increased gene expression in Hdac3-depleted IMCs.

    Fig. S6. Gene lists associated with top annotation clusters from DAVID pathway analysis.

    Fig. S7. GSEA on differentially expressed genes in Hdac3-depleted IMCs.

    Fig. S8. JAK Inhibitor I reduces STAT3 phosphorylation and Il-6 and Mmp13 gene expression in a concentration-dependent manner in Hdac3-depleted chondrocytes.

    Fig. S9. JAK-STAT pathway inhibitors reduce STAT3 phosphorylation and Il-6 and Mmp13 gene expression in Hdac3-depleted chondrocytes.

    Fig. S10. Combining JAK-STAT pathway and NF-κB inhibitors does not further reduce Il-6 and Mmp13 gene expression in Hdac3-depleted chondrocytes.

    Fig. S11. The Col2a1ERT-Cre driver is expressed in chondrocytes and osteoblasts but does not increase IL-6 abundance in osteoblasts.

    Fig. S12. JAK inhibition partially restores the bone phenotype of Hdac3-CKOCol2ERT mice.

    Table S1. Primer sequences.

  • Supplementary Materials for:

    Histone deacetylase 3 supports endochondral bone formation by controlling cytokine signaling and matrix remodeling

    Lomeli R. Carpio, Elizabeth W. Bradley, Meghan E. McGee-Lawrence, Megan M. Weivoda, Daniel D. Poston, Amel Dudakovic, Ming Xu, Tamar Tchkonia, James L. Kirkland, Andre J. van Wijnen, Merry Jo Oursler, Jennifer J. Westendorf*

    *Corresponding author. Email: westendorf.jennifer{at}mayo.edu

    This PDF file includes:

    • Fig. S1. Expression pattern of Hdac3 during embryonic and postnatal long bone development.
    • Fig. S2. Hdac3 knockout in chondrocytes during embryogenesis is lethal.
    • Fig. S3. Tamoxifen does not affect growth plate maturation in wild-type mice.
    • Fig. S4. Top 30 annotation clusters of genes with decreased gene expression in Hdac3-depleted IMCs.
    • Fig. S5. Top 30 annotation clusters of genes with increased gene expression in Hdac3-depleted IMCs.
    • Fig. S6. Gene lists associated with top annotation clusters from DAVID pathway analysis.
    • Fig. S7. GSEA on differentially expressed genes in Hdac3-depleted IMCs.
    • Fig. S8. JAK Inhibitor I reduces STAT3 phosphorylation and Il-6 and Mmp13 gene expression in a concentration-dependent manner in Hdac3-depleted chondrocytes.
    • Fig. S9. JAK-STAT pathway inhibitors reduce STAT3 phosphorylation and Il-6 and Mmp13 gene expression in Hdac3-depleted chondrocytes.
    • Fig. S10. Combining JAK-STAT pathway and NF-κB inhibitors does not further reduce Il-6 and Mmp13 gene expression in Hdac3-depleted chondrocytes.
    • Fig. S11. The Col2a1ERT-Cre driver is expressed in chondrocytes and osteoblasts but does not increase IL-6 abundance in osteoblasts.
    • Fig. S12. JAK inhibition partially restores the bone phenotype of Hdac3-CKOCol2ERT mice.
    • Table S1. Primer sequences.

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    Citation: L. R. Carpio, E. W. Bradley, M. E. McGee-Lawrence, M. M. Weivoda, D. D. Poston, A. Dudakovic, M. Xu, T. Tchkonia, J. L. Kirkland, A. J. van Wijnen, M. J. Oursler, J. J. Westendorf, Histone deacetylase 3 supports endochondral bone formation by controlling cytokine signaling and matrix remodeling. Sci. Signal. 9, ra79 (2016).

    © 2016 American Association for the Advancement of Science

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