PTHrP targets HDAC4 and HDAC5 to repress chondrocyte hypertrophy
Shigeki Nishimori, Forest Lai, Mieno Shiraishi, Tatsuya Kobayashi, Elena Kozhemyakina, Tso-Pang Yao, Andrew B. Lassar, Henry M. Kronenberg
Shigeki Nishimori, Forest Lai, Mieno Shiraishi, Tatsuya Kobayashi, Elena Kozhemyakina, Tso-Pang Yao, Andrew B. Lassar, Henry M. Kronenberg
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Research Article Bone biology Development

PTHrP targets HDAC4 and HDAC5 to repress chondrocyte hypertrophy

Abstract

During endochondral bone formation, chondrocyte hypertrophy represents a crucial turning point from chondrocyte differentiation to bone formation. Both parathyroid hormone-related protein (PTHrP) and histone deacetylase 4 (HDAC4) inhibit chondrocyte hypertrophy. Using multiple mouse genetics models, we demonstrate in vivo that HDAC4 is required for the effects of PTHrP on chondrocyte differentiation. We further show in vivo that PTHrP leads to reduced HDAC4 phosphorylation at the 14-3-3–binding sites and subsequent HDAC4 nuclear translocation. The Hdac4-KO mouse shares a similar but milder phenotype with the Pthrp-KO mouse, indicating the possible existence of other mediators of PTHrP action. We identify HDAC5 as an additional mediator of PTHrP signaling. While the Hdac5-KO mouse has no growth plate phenotype at birth, the KO of Hdac5 in addition to the KO of Hdac4 is required to block fully PTHrP action on chondrocyte differentiation at birth in vivo. Finally, we show that PTHrP suppresses myocyte enhancer factor 2 (Mef2) action that allows runt-related transcription factor 2 (Runx2) mRNA expression needed for chondrocyte hypertrophy. Our results demonstrate that PTHrP inhibits chondrocyte hypertrophy and subsequent bone formation in vivo by allowing HDAC4 and HDAC5 to block the Mef2/Runx2 signaling cascade. These results explain the phenotypes of several genetic abnormalities in humans.

Authors

Shigeki Nishimori, Forest Lai, Mieno Shiraishi, Tatsuya Kobayashi, Elena Kozhemyakina, Tso-Pang Yao, Andrew B. Lassar, Henry M. Kronenberg

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Figure 3

HDAC4 dephosphorylation and nuclear translocation by PTHrP signaling in vivo.

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HDAC4 dephosphorylation and nuclear translocation by PTHrP signaling in ...
(A and C) Western blots for the 14-3-3–binding sites (see complete unedited blots in the supplemental material). Whole cell lysate from microdissected proliferating chondrocyte regions in the proximal tibial growth plates at birth. Different sets of animals were used (A, n = 3, biological triplicates; C, n = 2, biological duplicates). Tg, Pthrp-Tg/+; KO, Pthrp-KO. WT1 or WT2 and WT3 are derived from Tg litter or KO litter, respectively (A). Tg1 and Tg2 are littermates. The KO pups are derived from different litters. (B and D) Relative band intensities were calculated from the bands in A (n = 3) or C (n = 2) as well as Supplemental Figure 4A (n = 2). HDAC4 was normalized to β-actin. Phosphorylated HDACs were normalized to HDAC4. S245+S250, the sum of phospho HDAC4-Ser245 and phospho HDAC5-Ser250; S465, phospho HDAC4-Ser465; S629, phospho HDAC4-Ser629. *P = 0.03, **P = 0.002, ***P = 1 × 10–5, ****P = 4 × 10–4, *****P = 5 × 10–5 by the 2-tailed Student’s t test. (E) Representative IHC images by confocal microscopy (original magnification, ×620). Round chondrocytes in the proximal tibial growth plates at birth: green (HDAC4) and blue (DAPI, nuclear stain). (F) Average ratio of total HDAC4 intensities in nuclei to those in whole cells (mean ± SEM, n = 5, biological replicates). The ratio for Pthrp-Tg/+ cells was 50.7% ± 2.4% and for Pthrp-KO cells was 38.4% ± 1.3%. The detailed calculation is shown in Supplemental Figure 4B and Supplemental Table 1. *P < 0.0001 by random intercept linear mixed-effects model (SAS Institute). A P value of less than 0.05 was considered significant (B, D, and F).