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. 2015 Jan 2;290(1):118-26.
doi: 10.1074/jbc.M114.596247. Epub 2014 Nov 11.

Histone deacetylase 7 (Hdac7) suppresses chondrocyte proliferation and β-catenin activity during endochondral ossification

Elizabeth W Bradley  1 , Lomeli R Carpio  2 , Eric N Olson  3 , Jennifer J Westendorf  4
Affiliations

Histone deacetylase 7 (Hdac7) suppresses chondrocyte proliferation and β-catenin activity during endochondral ossification

Elizabeth W Bradley et al. J Biol Chem. .

Abstract

Histone deacetylases (Hdacs) regulate endochondral ossification by suppressing gene transcription and modulating cellular responses to growth factors and cytokines. We previously showed that Hdac7 suppresses Runx2 activity and osteoblast differentiation. In this study, we examined the role of Hdac7 in postnatal chondrocytes. Hdac7 was highly expressed in proliferating cells within the growth plate. Postnatal tissue-specific ablation of Hdac7 with a tamoxifen-inducible collagen type 2a1-driven Cre recombinase increased proliferation and β-catenin levels in growth plate chondrocytes and expanded the proliferative zone. Similar results were obtained in primary chondrocyte cultures where Hdac7 was deleted with adenoviral-Cre. Hdac7 bound β-catenin in proliferating chondrocytes, but stimulation of chondrocyte maturation promoted the translocation of Hdac7 to the cytoplasm where it was degraded by the proteasome. As a result, β-catenin levels and transcription activity increased in the nucleus. These data demonstrate that Hdac7 suppresses proliferation and β-catenin activity in chondrocytes. Reducing Hdac7 levels in early chondrocytes may promote the expansion and regeneration of cartilage tissues.

Keywords: ATDC5 Cells; Beta-catenin (β-catenin); Cartilage; Growth Plate; Histone Deacetylase (HDAC); Histone Deacetylase 7 (Hdac7); Insulin.

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Figures

FIGURE 1.
FIGURE 1.
Hdac7 deletion expands the growth plate proliferative zone and increases chondrocyte numbers. A, 4-week-old Hdac7Col2ERT mice were injected with vehicle (n = 4) or tamoxifen (n = 5). Paraffin-embedded sections of tibias mice were incubated with an IgG isotype control antibody (upper row) or an anti-Hdac7 antibody (lower row). Shown are representative 20× images of the growth plates. The proliferative (P) and hypertrophic (H) zones are designated in select panels. B–F, 9-day-old Hdac7Col2ERT mice injected with vehicle (n = 4) or tamoxifen (n = 5) and BrdU. B, paraffin-embedded sections (10×) of tibias from BrdU-injected Hdac7Col2ERT mice that were incubated with an IgG isotype control antibody or an anti-BrdU antibody. C, in situ hybridization using DIG-labeled type X collagen-specific probes was performed on paraffin-embedded tibia sections. Probes were detected using an anti-DIG antibody or corresponding IgG control. Shown are representative 40× images. D, average total (E) proliferative and hypertrophic zone depths within the growth plate were determined using image J software. F, average number of proliferative cells per tissue area was determined using Image J software. *, p < 0.05 compared with control. G, average femur lengths of 4 week-old vehicle or tamoxifen-injected animals.
FIGURE 2.
FIGURE 2.
Cartilage-specific deletion of Hdac7 decreases bone accrual. A and B, representative three-dimensional reconstructions of trabecular bone from the distal femur of Hdac7Col2ERT mice treated with vehicle or tamoxifen. C, bone volume per total tissue volume (BV/TV), D, trabecular thickness (Tb. Th.), E, trabecular number (Tb. N.), and F, trabecular spacing (Tb. Sp.) from the distal femur are also shown (n = 5 per group). G, cortical bone density was measure at the femoral diaphysis. *, p < 0.05 compared with control.
FIGURE 3.
FIGURE 3.
Hdac7 deficiency increases active β-catenin levels and chondrocyte proliferation. A–N, immature mouse articular chondrocytes from Hdac7Col2ERT mice were cultured in micromass and infected with Ad-GFP or Ad-Cre on day 0. A, Western blotting for the indicated proteins was performed on day 3. This experiment was repeated three times, and data from a representative experiment are shown. B, micromasses were fixed on day 3 and stained with Alcian blue. Relative expression levels of (C) Hdac7, (D) Sox9, (E) Type 2a1 collagen, (F) Ihh, (G) Runx2, (H) Vegf, (I) Aggrecan, (J) Type X collagen, (K) Cyclin D3, (L) p21, (M) Lef1, and (N) Axin2 were assayed by qPCR. *, p < 0.05 compared with control. These data are the average of three independent experiments. O and P, 4-week-old Hdac7Col2ERT mice were injected with vehicle (n = 4) or tamoxifen (n = 5). O, paraffin-embedded sections of tibias were incubated with an IgG isotype control antibody (left column) or an antibody that recognizes p21 (right column). Shown are representative 40× images of the growth plate. P, paraffin-embedded sections of tibias were incubated with an IgG isotype control antibody (left column) or an antibody that recognizes unphosphorylated (active) β-catenin (right column). Shown are representative 40× images of the growth plate.
FIGURE 4.
FIGURE 4.
Chondrogenic stimuli promote Hdac7 degradation and β-catenin activity. A, co-immunoprecipitations of Hdac7 and β-catenin endogenous to ATDC5 cells. B, ATDC5 cells were transfected with Flag-tagged Hdac7 and Myc-tagged β-catenin. Co-immunoprecipitations were performed as shown. C and D, ATDC5 cells were serum starved, treated with 1× ITS for 24 h, and then biochemically fractionated into cytosolic and nuclear extracts. C, Western blotting for the indicated proteins was performed and D, the expression levels of Hdac7 in each compartment were quantified, n = 3. E and F, ATDC5 cells were serum starved, treated with 10 ng/ml Igf1 for 24 h, and then biochemically fractionated into cytosolic and nuclear extracts. E, Western blotting for the indicated proteins was performed and F, the expression levels of Hdac7 in each compartment were quantified, n = 3. G and H, ATDC5 cells were serum starved in the presence of 5 nm MG132 or vehicle control. Cells were then treated with 1× ITS for 24 h. G, Western blotting was then performed as indicated and H, the expression levels of Hdac7 were quantified, n = 3. I, ATDC5 cells were transfected with the TOPFLASH reporter, a constitutively active form of β-catenin and pcDNA3 or pcDNA3-Hdac7 expression constructs as indicated. After transfection, cells were exposed to 1× ITS for 48 h. *, p < 0.05 compared with control, n = 3.
FIGURE 5.
FIGURE 5.
Working model. Igf1/insulin-dependent signaling induces Hdac7 degradation and β-catenin stability.
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