The Deletion of Hdac4 in Mouse Osteoblasts Influences Both Catabolic and Anabolic Effects in Bone

Abstract

Histone deacetylase 4 (Hdac4) is known to control chondrocyte hypertrophy and bone formation. We have previously shown that parathyroid hormone (PTH) regulates many aspects of Hdac4 function in osteoblastic cells in vitro; however, in vivo confirmation was previously precluded by preweaning lethality of the Hdac4-deficient mice. To analyze the function of Hdac4 in bone in mature animals, we generated mice with osteoblast lineage-specific knockout of Hdac4 (Hdac4^ob-/- ) by crossing transgenic mice expressing Cre recombinase under the control of a 2.3-kb fragment of the Col1a1 promoter with mice bearing loxP-Hdac4. The Hdac4^ob-/- mice survive to adulthood and developed a mild skeletal phenotype. At age 12 weeks, they had short, irregularly shaped and stiff tails due to smaller tail vertebrae, with almost no growth plates. The tibial growth plate zone was also thinned, and Mmp13 and Sost mRNAs were increased in the distal femurs of Hdac4^ob-/- mice. Immunohistochemistry showed that sclerostin was elevated in Hdac4^ob-/- mice, suggesting that Hdac4 inhibits its gene and protein expression. To determine the effect of PTH in these mice, hPTH (1-34) or saline were delivered for 14 days with subcutaneously implanted devices in 8-week-old female Hdac4^ob-/- and wild-type (Hdac4^fl/fl ) mice. Serum CTX, a marker of bone resorption, was increased in Hdac4^ob-/- mice with or without PTH treatment. Tibial cortical bone volume/total volume (BV/TV), cortical thickness (Ct.Th), and relative cortical area (RCA) were decreased in Hdac4^ob-/- mice, but PTH caused no further decrease in Hdac4^ob-/- mice. Tibial trabecular BV/TV and thickness were not changed significantly in Hdac4^ob-/- mice but decreased with PTH treatment. These results indicate that Hdac4 inhibits bone resorption and has anabolic effects via inhibiting Mmp13 and Sost/sclerostin expression. Hdac4 influences cortical bone mass and thickness and knockout of Hdac4 prevents the catabolic effect of PTH in cortical bone. © 2018 American Society for Bone and Mineral Research.

Keywords: CATABOLIC EFFECTS OF PTH; CORTICAL BONE; HDAC4; OSTEOBLASTS; SCLEROSTIN.

Figure:1

(A) Detection of the deletion allele of Hdac4 by PCR in genomic DNA isolated from different tissues using primers arm1 and arm3. (B) Genotyping of Hdac4 mice using primers arm1 and arm2. Analysis of the expression of HDAC4 protein and Hdac4 mRNA in the heart. β-actin was used as a loading control. (C) Hdac4, and (D) Mmp13 levels relative to β-actin were determined in distal femur RNA from 10 week-old Hdac4^fl/fl (n = 6) and Hdac4^ob−/− mice (n = 8). Data are shown as fold changes compared with levels in Hdac4^fl/fl mice. Data shown are means ± SE. *, p< 0.05 by Student’s t test.

Figure. 2.. Phenotype of Hdac4^ob−/− compared with Hdac4^fl/fl mice Twelve-week-old

(A) Male, (B) Female Hdac4 ^fl/fl and Hdac4^ob−/− mice are shown. Fat percentage was obtained by DXA scan analyses. Hdac4 ^fl/fl (n=12) and Hdac4^ob−/− mice (n=17).

Figure 3.. Hdac4^ob−/−mice have decreased caudal bone mass and length compared with Hdac4^fl/fl mice

(A) Representative uCT images of the tails are shown. (B) Caudal vertebrae (caudal 17) from 12-week-old female Hdac4^fl/fl (n = 6) and Hdac4^ob−/− mice (n = 8) were used for µCT analysis. Data shown are means ± SE. *, p< 0.05, **, p< 0.01, ***, p< 0.001 by Student’s t test. Representative images of hematoxylin and eosin stained caudal (C) and lumbar vertebrae (D) are shown. (E) Lumbar vertebrae (L4) from 12-week-old female Hdac4^fl/fl (n = 10) and Hdac4^ob−/− mice (n = 10) were used for µCT analysis. Data shown are as means ± SE. *, p< 0.05, ***, p< 0.001 by Student’s t test.

Figure 4.. Body weight and growth plate histological examination of Hdac4^fl/fl and Hdac4^ob−/−mice after continuous PTH treatment

(A) Body weight and (B) Fat % by DXA in 10-week-old female Hdac4^fl/fl and Hdac4^ob−/− mice treated with continuous PTH or vehicle, (Hdac4 ^fl/fl; n=12, Hdac4 ^fl/fl with PTH; n=13, Hdac4^ob−/−; n=11, Hdac4^ob−/−; with PTH; n=12). (C) Representative hematoxylin and eosin stained sections of the growth plate of the tibiae are shown. (D) Morphometric analysis of the length of the prehypertrophic (ph), and hypertrophic (h) zones in female mice. Hdac4 ^fl/fl; n=5, Hdac4 ^fl/fl with PTH; n=5, Hdac4^ob−/−; n=6, Hdac4^ob−/−; with PTH; n=6. Data shown are mean ± SE. Different letters indicate p<0.05 vs. one another. The images were quantified using Image J software.

Figure 5.. Hdac4^ob−/− mice have decreased cortical bone but not trabecular bone and PTH is unable to cause a catabolic effect in cortical bone while this is retained in trabecular bone

(A) Tibiae from female 10-week-old female Hdac4 ^fl/fl and Hdac4^ob−/− mice treated with continuous PTH for 14 days or vehicle were used for cortical or trabecular (B) µCT analysis. (Hdac4 ^fl/fl; n=9, Hdac4 ^fl/fl with PTH; n=11, Hdac4^ob−/−; n=9, Hdac4^ob−/−; with PTH; n=9). Data shown are means ± SE. Different letters indicate p<0.05 vs. one another.

Figure 6.. Osteoclast activity is increased in Hdac4^ob−/− mice

(A) TRAP staining of tibiae of female 10-week-old Hdac4^fl/fl and Hdac4^ob−/− mice treated with PTH or vehicle is shown. 20X. (B) Quantification of osteoclast number and (C) osteoclast surface % in the animals in (A). Data shown as means ± SE. Different letters indicate p<0.05 vs. one another. (Hdac4 ^fl/fl; n=4, Hdac4 ^fl/fl with PTH; n=5, Hdac4^ob−/−; n=−5, Hdac4^ob−/−; with PTH; n=4). (D) Serum CTX levels (Hdac4 ^fl/fl; n=9, Hdac4 ^fl/fl with PTH; n=10, Hdac4^ob−/−; n=10, Hdac4^ob−/− with PTH; n=10), data shown as means ± SE and different letters indicate p<0.05 vs. one another. (E) The relative levels of Mmp13 mRNA normalized to β-actin (Hdac4 ^fl/fl; n=7, Hdac4 ^fl/fl with PTH; n=7, Hdac4^ob−/−; n=6, Hdac4^ob−/− with PTH; n=8). Total RNA was extracted from distal femurs of 10-week-old females and measured using real time RT-PCR. Data are shown as fold change to vehicle-treated Hdac4^fl/fl and as means ± SE. Different letters indicate p<0.05 vs. one another.

Figure 7.. Bone formation markers were increased by PTH in Hdac4 ^fl/fl and Hdac4^ob−/− mice while Runx2 decreased

Ten-week-old female Hdac4 ^fl/fl and Hdac4 ^ob−/− mice were examined after treatment with PTH or vehicle. (A) Serum P1NP levels. n=10 in all groups. Data are shown as are means ± SE. (B) Type I collagen mRNA, (Hdac4 ^fl/fl; n=10, Hdac4 ^fl/fl with PTH; n=10, Hdac4^ob−/−; n=10, Hdac4^ob−/− with PTH; n=11), (C) Runx2 mRNA(Hdac4^fl/fl; n=8, Hdac4^fl/fl with PTH; n=10, Hdac4^ob−/−; n=10, Hdac4^ob−/− with PTH; n=11), (D) Osx mRNA(Hdac4^fl/fl; n=8, Hdac4^fl/fl with PTH; n=10, Hdac4^ob−/−; n=10, Hdac4^ob−/− with PTH; n=11), were measured using real time RT-PCR. The relative levels of mRNAs were normalized to β-actin. Data are shown as fold change to vehicle-treated Hdac4^fl/fl and as means ± SE.

Figure 8.. Increased expression of Sost/sclerostin in Hdac4^ob−/− mice Sost

mRNA (A) was measured using real time RT-PCR. The relative levels of mRNAs were normalized to β-actin. Data are shown as means ± SE for –fold change to vehicle-treated Hdac4^fl/fl. (Hdac4 ^fl/fl; n= 9, Hdac4 ^fl/fl with PTH; n= 9, Hdac4^ob−/−; n= 9, Hdac4^ob−/− with PTH; n=11). (B) Immunohistochemistry for sclerostin. Representative longitudinal sections of the cortical region of the tibiae. (C) Ratio of number of sclerostin-ositive osteocytes to total number of osteocytes were quantified in lacunae of tibial cortical bones using image J (Hdac4 ^fl/fl; n=4, Hdac4 ^fl/fl with PTH; n=4, Hdac4^ob−/−; n=8, Hdac4^ob−/− with PTH; n=5). Data shown as means ± SE. Different letters indicate p<0.05 vs. one another. Hdac5 mRNA (D) was measured using real time RT-PCR (Hdac4 ^fl/fl; n= 8, Hdac4 ^fl/fl with PTH; n= 8, Hdac4^ob−/−; n= 8, Hdac4^ob−/−; with PTH; n=7). Data are shown as fold change to vehicle-treated Hdac4^fl/fl and as means ± SE.

Figure 9.. Proposed model depicting the functions of HDAC4 in the mature osteoblast and osteocyte

Under basal conditions, HDAC4 in mature osteoblasts and osteocytes inhibits bone resorption and also has an anabolic effect via inhibiting Sost gene and sclerostin protein expression. After continuous PTH treatment, HDAC4 is necessary for maintaining Runx2 expression. Since Runx2 contributes to Sost gene transcription, continuous PTH decreases Sost and sclerostin in the absence of HDAC4. This leads to a combined increase in bone formation and no resorptive effect of PTH in the absence of HDAC4. These actions indicate the function of HDAC4 in maintaining cortical bone mass and its role in the catabolic effects of PTH on cortical bone.