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. 2015 Feb;1853(2):370-376.
doi: 10.1016/j.bbamcr.2014.11.003. Epub 2014 Nov 13.

Mitogen-activated protein kinase p38 induces HDAC4 degradation in hypertrophic chondrocytes

Jingming Zhou  1 , Pengcui Li #  1   2 , Qian Chen  1 , Xiaochun Wei  2 , Ting Zhao  3 , Zhengke Wang  4 , Lei Wei  1   2
Affiliations

Mitogen-activated protein kinase p38 induces HDAC4 degradation in hypertrophic chondrocytes

Jingming Zhou et al. Biochim Biophys Acta. 2015 Feb.

Abstract

Histone deacetylase 4 (HDAC4) is a critical negative regulator for chondrocyte hypertrophy by binding to and inhibiting Runx2, a critical transcription factor for chondrocyte hypertrophy. It is unclear how HDAC4 expression and stability are regulated during growth plate development. We report here that inhibition of mitogen-activated protein kinase (MAPK) p38 by dominant negative p38 or p38 inhibitor prevents HDAC4 degradation. Mutation of a potential caspase-2 and 3 cleavage site Asp289 stabilizes HDAC4 in chondrocytes. In contrast, constitutively active MAPK kinase 6 (constitutive activator of p38) transgenic mice exhibit decreased HDAC4 content in vivo. We also observed that p38 stimulates caspase-3 activity in chondrocytes. Inhibition of p38 or caspases reduced HDAC4 degradation. HDAC4 inhibited Runx2 promoter activity in a dose-dependent manner and caspase inhibitors further enhanced this inhibition by preventing HDAC4 degradation. Overall, these results demonstrate that p38 promotes HDAC4 degradation by increasing caspase-mediated cleavage, which releases Runx2 from a repressive influence of HDAC4 and promotes the chondrocyte hypertrophy and bone formation.

Keywords: Caspase; Degradation; HDAC4; Inhibition; p38.

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Figures

Figure 1
Figure 1. Inhibition of p38 MAPK activity reduces HDAC4 degradation
(A) Degraded HDAC4 band (34 kDa) was not detected in DN p38 treated cells, even after overexposure (B). (C) Intensity of the full length HDAC4/34 kDa degradation fragment was calculated and presented. *, #, p<0.05. (D) Western blot showed phosphorylation of p38 (p-p38) was induced by CAMKK but not DN p38. (E) Chicken chondrocytes were treated with p38 inhibitor SB203580 (15 μM) for 48 hours. Western blot showed that the degraded band (34 kDa) was decreased. (F) The intensity of full length HDAC4/degradation fragment was presented. *, p<0.05. SB203580 (15 μM) effectively inhibited p38 phosphorylation (E).
Figure 2
Figure 2. Mutation of Asp-289 stabilized HDAC4 and CaMKK6 induces capase-3 activity in chondrocytes
(A) Schematic structure of HDAC4 revealed Asp 289 as the target of mutation to remove capase-2 and 3 cleavage site, and HDAC4 antibody detects both N-termimal degradation fragment and full length HDAC4. (B) Western blot shows that HDAC4 is cleaved in WT but the mutant D289 is resistant to degradation of HDAC4. The result indicates that the degradation of HDAC4 is related to ASP-289. (C) Intensity of full length HDAC4/degradation fragment was presented. *, p<0.05. (D) Caspase activity assays showed that over-activation of p38 by CAMKK6 stimulates caspase-3 activity while inhibition of p38 by DN p38 has the opposite effect when comparing to the empty RCAS control. Data are the means of three independent experiments, with S.D. indicated. *, p< 0.05 compared with control. #, p< 0.05 compared with DN p38.
Figure 3
Figure 3. The expression of HDAC4 is dramatically decreased in the growth plate of CAMKK6 mice
(A) Immunofluorescent histochemistry shows that in WT mice, HDAC4 is highly expressed in the pre-hypertrophic zone (red), but the expression is reduced in the hypertrophic zone. In contrast, CAMKK6 homozygote mice (CAMKK6-TG) exhibit a weaker HDAC4 staining in the pre-hypertrophic zone in comparison to the strong signal in WT mice. Magnified view of the pre-hypertrophic and hypertrophic chondrocytes from WT and CAMKK6-TG mice correspond to the boxed areas. Blue: Nucleus stained by DAPI. Red: HDAC4 stained by Rhoda mine. Purple: overlap of blue and red. Multiple animals were examined and consistent results were obtained (N=4). Safranin-O staining was used to show PG staining on the growth plate. (B) Intensity of the HDAC4 signal was quantified. CAMKK6-TG mice had much lower signal than WT controls. * p<0.05.
Figure 4
Figure 4. HDAC4 is stabilized by caspase-2 or 3 inhibiters and by DN p38 MAPK
(A) Full length HDAC4 is stabilized by inhibition of caspase-2 or 3 as shown by western. (B) Ratio of the full length HDAC4/degradation fragment was presented. *, p<0.05. (C) HDAC4 is stabilized by caspase-2 and caspase-3 inhibiters or by DN p38, and cycloheximide treatment excludes the variation of full length HDAC4 content due to protein neosynthesis. (D) Ratio of the full length HDAC4/degradation fragment was presented. *, #, p<0.05. (E) Realtime PCR excludes the possibility that caspase-2, 3 inhibitors may induce HDAC4 expression at the transcription level.
Figure 5
Figure 5. Caspases inhibitors further suppress Runx2 promoter activity by inhibiting degradation of HDAC4
(A) Promoter assay showed that inhibition of Runx2 promoter activity presents an HDAC4-dose-dependent manner in chondrocytes. (B) HDAC4 inhibition of Runx2 promoter could be enhanced by caspases inhibitors via suppressing degradation of HDAC4.
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References

    1. Arnold MA, Kim Y, Czubryt MP, Phan D, McAnally J, Qi X, Shelton JM, Richardson JA, Bassel-Duby R, Olson EN. MEF2C transcription factor controls chondrocyte hypertrophy and bone development. Developmental Cell. 2007;12:377–389. - PubMed
    1. Backs J, Worst BC, Lehmann LH, Patrick DM, Jebessa Z, Kreusser MM, Sun Q, Chen L, Heft C, Katus HA, Olson EN. Selective repression of MEF2 activity by PKA-dependent proteolysis of HDAC4. The Journal of cell biology. 2011;195:403–415. - PMC - PubMed
    1. Chen Q, Johnson DM, Haudenschild DR, Goetinck PF. Progression and recapitulation of the chondrocyte differentiation program: cartilage matrix protein is a marker for cartilage maturation. Dev Biol. 1995;172:293–306. - PubMed
    1. Chrysis D, Nilsson O, Ritzen EM, Savendahl L. Apoptosis is developmentally regulated in rat growth plate. Endocrine. 2002;18:271–278. - PubMed
    1. Guan Y-J, Yang X, Wei L, Chen Q. MiR-365: a mechanosensitive microRNA stimulates chondrocyte differentiation through targeting histone deacetylase 4. FASEB Journal. 2011;25:4457–4466. - PMC - PubMed

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