Fig 2: HDAC4 and HDAC5 control osteocyte biology in vivo.(a) Endogenous MEF2C was immunoprecipitated from Ocy454 cells, followed by immunoblotting for the indicated proteins. Data shown are representative of n=3 independent experiments. (b) Osteocyte density in cortical bone 3 mm below the growth plate. 4–5, 8 week old male mice per genotype was analysed, *indicates P<0.01 versus WT by student's unpaired two tailed t-test. (c) Representative H+E section demonstrating increased osteocyte density and disorganized cortical bone in DKO (HDAC4f/f; HDAC5−/−;DMP1-Cre) mice. Scale bar, 40 μm. (d) Sections were stained with Sirius Red and analysed under polarized light to view collagen fibre organization. Disorganized collagen fibers are only seen in DKO sections. Scale bar, 40 μm. Error bars indicate s.e.m for all figures.

Fig 3: Model showing PTH signaling via inhibition of SIK2 in osteocytes.In the absence of PTH signaling, SIK2 tonically phosphorylates its substrates HDAC4/5 and CRTC2, leading to their cytoplasmic retention via binding to 14-3-3 chaperones. PTH signaling leads to PKA-mediated phosphorylation of SIK2, which inhibits its cellular activity. This in turn reduces phosphorylation of HDAC4/5 and CRTC2, leading to their dephosphorylation by an unknown phosphatase (ppase), and subsequent nuclear translocation. Small molecule SIK inhibitors (YKL-05-093 and YKL-05-099) mimic the effects of PTH by directly blocking SIK2 kinase activity. In the nucleus, HDAC4/5 block MEF2C-driven SOST expression, while CRTC2 enhances CREB-mediated RANKL gene transcription. PTH-induced reductions in sclerostin contribute to increased bone formation, while PTH-induced increases in RANKL drive increased bone resorption.

Fig 4: Hepatic nuclear HDAC3 protein content immunoprecipitated (IP) with HDAC4/5/7 antibody (A), representative western blot of cytosolic phospho-HDAC4,5, and 7 (B), cytosolic level of phospho-HDAC4 (C) and phospho-HDAC5/7 (D), and nuclear content of acetyl-foxo1 protein (E) in 4 month-old male rat offspring exposed to ethanol (EtOH) or water only during early, mid, or late pregnancy.Control and pair-fed offspring were from dams with free access to chow and dams fed the amount of chow consumed by EtOH dams, respectively. A subgroup of EtOH rats was given TUDCA (T) 3 weeks before the experiments. Data shown as the mean±SE, n = 6. *P<0.05, **P<0.01 EtOH vs. control; #P<0.05, ##P<0.01 EtOH vs. pair-fed.

Fig 5: Overexpression of CKIP-1 inhibits the phosphorylation of HDAC4. (A) Western blotting showing the expression levels of CKIP-1, phosphorylated HDAC4 (S632), phosphorylated HDAC4 (S246), and the total protein expression levels of HDAC4. (B) Quantification of p-HDAC4 (S632), p-HDAC4 (S246), and HDAC4 levels. (C–E) Transcript levels of hypertrophic marker genes (ANP, BNP, and β-MHC) in the hearts of WT and CKIP-1 TG mice after swimming exercise. Values are means ± SEM, n = 4∼6, ∗P < 0.05, ∗∗P < 0.01. Statistical differences among groups were analyzed by two-way analysis of variance (ANOVA) followed by the Bonferroni procedure. CKIP-1, casein kinase-2 interacting protein-1; WT, wild type; TG, transgenic; HDAC4, histone deacetylase 4; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; β-MHC, myosin heavy chain β.