应力致HDAC4核外积累及降解在软骨退变中的作用及机制研究

Mechanical loading plays a critical role in cartilage degeneration and osteoarthritis (OA), but it is unclear in mechanism. Our novel findings indicate that histone deacetylase 4 (HDAC4) nuclear export and degradation may play an important role based on recent findings: a) HDAC4 is a critical negative regulator of chondrocyte hypertrophy, and it regulates chondrocyte hypertrophy by nuclear shuttle and suppression of Runx2; b) Over-loading leads to HDAC4 nuclear export and degradation, thus results in chondrocyte hypertrophic differentiation; c) Over-loading activates CaMKⅡδ which regulates HDAC4 nuclear export by phosphorylation of HDAC4. HDAC4 nuclear export The level of HDAC4 was decreased, and the N-terminal of HDAC4, a fragment of HDAC4 degenerated by Caspase-3, was increased dramatically following over-loading; c) The level of HDAC4 was decreased because of degeneration by Caspase-3 which was increased dramatically following over-loading. d) Our pilot study further demonstrated that cartilage degeneration induced by over-loading were attenuated by Caspase inhibitor BAF and HDAC4 nuclear relocation after over-expression of HDAC4 S246/467/632A triple mutant in which the site S246, S467, and S632 of HDAC4 were replaced with the nonphosphorylable residue alanine abolished 14-3-3 binding and nuclear export. On these grounds, we put forth the hypothesis that the progression of OA could attenuate via inhibiting degeneration of HDAC4 and nuclear export of HDAC4. In order to test the hypothesis we will using culturation of chondrocyte and non-invasive loading model of murine osteoarthritis. The hypothesis will also be tested using conditional knockout HDAC4 transgenic mice. This study will facilitate our understanding how the inappropriate mechanical loading, namely overloading, result in OA. If successful in demonstrating our hypotheses, it could yield an entirely new therapeuty.

应力导致软骨细胞肥大，软骨退变和骨关节炎（OA），但机制不清。前期研究发现，应力激活CaMKⅡδ，致组蛋白脱乙酰酶4 (HDAC4）磷酸化，与胞浆蛋白14-3-3结合，核外积累；并激活Capase，降解HDAC4。HDAC4是软骨细胞肥大及基质破坏的重要负性调控因子，实现功能需进入细胞核。在核内HDAC4压制Runx2，抑制软骨细胞肥大，促进增殖和合成代谢。因此我们假设：增加细胞核内HDAC4，并抑制其降解，能逆转应力导致的软骨退变及OA病理。该项目拟转染三个磷酸化位点突变的HDAC4（因不能被磷酸化，进入细胞核），增加细胞核内HDAC4；联合Caspase抑制剂BAF，抑制HDAC4降解。应用Flexcell细胞力学加载系统和小鼠膝关节应力OA模型分别进行体内外实验。并应用条件性HDAC4基因敲除鼠，确证丧失HDAC4引起软骨细胞肥大和OA病变。以揭示OA发病机制，探索OA治疗新方法。

高应力导致软骨退变和骨关节炎（OA）的发生，但发病机制不清。HDAC4是软骨细胞肥大及基质破坏的重要负性调控因子，进入细胞核后，将组蛋白去乙酰化，可调节软骨细胞的增殖和基因表达。前期研究中我们还发现，HDAC4-N末端蛋白过表达可引起软骨细胞凋亡，并呈剂量依赖性，而细胞凋亡是OA的主要病理表现之一。前期研究中我们还发现，生理性应力可促进HDAC4进入细胞核，HDAC4进入细胞核后可抑制Runx2等肥大基因表达，并促进软骨基质蛋白表达和分泌，促进细胞增殖。在这些研究的基础上，本项目研究中，我们应用Flexcell® FX-5000™力学加载系统，对体外培养的软骨细胞施加高应力——每天施加循环拉伸应力（10%，0.5Hz）12小时，同时应用非侵入压应力致小鼠膝关节OA模型进行体内实验验证，发现：①高应力引起HDAC4在细胞核外积累，并发生降解，导致软骨细胞核内HDAC4异常减少，肥大转录因子Runx2失去HDAC4的压制，转录活性增强，促进软骨细胞肥大分化，增加Ⅹ型胶原、MMP-13、ADAMTS-5和Ihh表达，导致软骨降解，关节软骨退变；②高应力通过激活CaMKⅡδ，磷酸化HDAC4，导致HDAC4在细胞核外积累。③高应力导致软骨细胞肥大分化后，增加软骨中Caspase-3酶活性，Caspase-3酶反过来又裂解HDAC4，导致HDAC4含量降低，进一步加剧软骨细胞肥大和终末分化，最终诱发或促进OA的病理发展。证实：①应力导致HDAC4核外积累及降解，引发软骨细胞肥大分化，参与软骨退变和OA病理。②应力增加CaMKIIδ活性，引起HDAC4核外积累；应力增加Caspase-3活性，降解HDAC4；增加细胞核内HDAC4，并抑制其降解，可阻断应力引起的软骨细胞肥大分化和软骨退变。通过项目研究证实了本课题最初的假设：应力增加CaMKⅡδ活性，引起HDAC4细胞核外积累，增加Caspase活性，降解HDAC4；以上作用导致软骨细胞核内HDAC4异常减少，肥大转录因子Runx2失去HDAC4的压制，转录活性增强，促进软骨细胞肥大分化，增加Ⅹ型胶原、MMP-13、ADAMTS-5和Ihh表达，导致软骨降解、关节软骨退变和OA发生。针对关节软骨中HDAC4进行调控：增加细胞核内HDAC4，并抑制其降解，可阻断应力导致的软骨细胞肥大分化和OA病理。