MMP-13 is one of the critical mediators of the effect of HDAC4 deletion on the skeleton

Abstract

Histone deacetylase 4 (Hdac4) regulates chondrocyte hypertrophy. Hdac4(-/-) mice are runted in size and do not survive to weaning. This phenotype is primarily due to the acceleration of onset of chondrocyte hypertrophy and, as a consequence, inappropriate endochondral mineralization. Previously, we reported that Hdac4 is a repressor of matrix metalloproteinase-13 (Mmp13) transcription, and the absence of Hdac4 leads to increased expression of MMP-13 both in vitro (osteoblastic cells) and in vivo (hypertrophic chondrocytes and trabecular osteoblasts). MMP-13 is thought to be involved in endochondral ossification and bone remodeling. To identify whether the phenotype of Hdac4(-/-) mice is due to up-regulation of MMP-13, we generated Hdac4/Mmp13 double knockout mice and determined the ability of deletion of MMP-13 to rescue the Hdac4(-/-) mouse phenotype. Mmp13(-/-) mice have normal body size. Hdac4(-/-)/Mmp13(-/-) double knockout mice are significantly heavier and larger than Hdac4(-/-) mice, they survive longer, and they recover the thickness of their growth plate zones. In Hdac4(-/-)/Mmp13(-/-) double knockout mice, alkaline phosphatase (ALP) expression and TRAP-positive osteoclasts were restored (together with an increase in Mmp9 expression) but osteocalcin (OCN) was not. Micro-CT analysis of the tibiae revealed that Hdac4(-/-) mice have significantly decreased cortical bone area compared with the wild type mice. In addition, the bone architectural parameter, bone porosity, was significantly decreased in Hdac4(-/-) mice. Hdac4(-/-)/Mmp13(-/-) double knockout mice recover these cortical parameters. Likewise, Hdac4(-/-) mice exhibit significantly increased Tb.Th and bone mineral density (BMD) while the Hdac4(-/-)/Mmp13(-/-) mice significantly recovered these parameters toward normal for this age. Taken together, our findings indicate that the phenotype seen in the Hdac4(-/-) mice is partially derived from elevation in MMP-13 and may be due to a bone remodeling disorder caused by overexpression of this enzyme.

Keywords: Cortical bone; Hdac4; Hdac4(−/−)/Mmp13(−/−) double knockout; Mmp13.

Fig. 1. Images, body weights and survival of Hdac4^−/− mice compared with Hdac4^−/−/Mmp-13^−/−

A, Postnatal day 8 (P8) wild type (WT, n=12), Mmp-13 knockout (Mmp-13^−/−, n=15), Hdac4 knockout (Hdac4^−/−, n=17) and Hdac4^−/−/Mmp-13^−/− (DKO, n=11) male mice are shown. B, Body weights of male and female genotypes. Data shown are mean ± SE. Different letters indicate p<0.05 vs. one another. C, Survival of Hdac4^−/− and Hdac4^−/−/Mmp-13^−/− (DKO) male and female mice. ^*p<0.05 vs. Hdac4^−/−.

Fig. 2. Histological examination of all four genotypes

A, Hematoxylin and eosin stained tibiae at postnatal day 8 (P8) WT, Mmp-13^−/−, Hdac4^−/− and Hdac4^−/−/Mmp-13^−/− male mice. 4x. B, Morphometric analysis of the length of the prehypertrophic (ph), and hypertrophic (h) zones in P8 WT (n=3), Mmp-13^−/− (n=5), Hdac4^−/− (n=6) and DKO (n=5) female and male mice. Data shown are mean ± SE. Different letters indicate p<0.05 vs. one another. The images were quantified using Image J software.

Fig. 3. Immunohistochemistry of all four genotypes

Tibiae from female mice of postnatal day 8 (P8) WT (n=2), Mmp-13^−/− (n=3), Hdac4^−/− (n=4)and Hdac4^−/−/Mmp-13^−/− (n=3)were used. 10x

Fig. 4. Gene expression of all four genotypes

Total RNA was extracted from distal femurs of 5-day postnatal male and female WT (n=6), Mmp-13^−/− (n=6), Hdac4^−/− (n=5), and DKO mice (n=4). RNAs were measured using real time RT-PCR. The relative levels of mRNAs were normalized to β-actin. Data are shown as –fold change to WT. Data shown are mean ± SE. Different letters indicate p<0.05 vs. one another.

Fig. 5. Gene expression of all four genotypes

Total RNA was extracted from distal femurs of 5-day postnatal male and female WT (n=6), Mmp-13^−/− (n=6), Hdac4^−/− (n=5), and DKO mice (n=4). RNAs were measured using real time RT-PCR. The relative levels of mRNAs were normalized to β-actin. Data are shown as –fold change to WT. Data shown are mean ± SE. Different letters indicate p<0.05 vs. one another.

Fig. 6. TRAP staining of all four genotypes

A, Tibiae of postnatal day 8 (P8) WT, Mmp-13^−/−, Hdac4^−/− and Hdac4^−/−/Mmp-13^−/− female mice are shown. 20x. B, Quantification of osteoclast number in postnatal day 8 (P8) WT, Mmp-13^−/−, Hdac4^−/−, and DKO mice. Data shown are mean ± SE. Different letters indicate p<0.05 vs. one another, n = 4 in each group.

Fig. 7. Bone images obtained by μCT analysis

Tibiae from mice at postnatal day 8 (P8) of WT, Mmp-13^−/−, Hdac4^−/− and Hdac4^−/−/Mmp-13^−/− were used. n = 7–8 in each group

Fig. 8. Hdac4^−/− mice have decreased cortical bone area and structure which were restored in Hdac4^−/−/Mmp-13^−/− mice

Tibiae from male and female mice of postnatal day 8 (P8) WT, Mmp-13^−/−, Hdac4^−/− and Hdac4^−/−/Mmp-13^−/− were used. Different letters indicate p<0.05 vs. one another, n = 7–8 in each group.

Fig. 9. The trabecular thickness, BMD, and bone surface were normalized in Hdac4^−/−/Mmp-13^−/− mice

Tibiae from mice at postnatal day 8 (P8) of WT, Mmp-13^−/−, Hdac4^−/− and Hdac4^−/−/Mmp-13^−/− were used. Different letters indicate p<0.05 vs. one another, n = 7–8 in each group.