PADI4通过BMPs通路调控类风关成纤维样滑膜细胞成骨-成脂分化的功能和机制研究

Impaired osteogenesis is one of the causes of bone loss in the synovium of rheumatoid arthritis (RA). Bone morphogenetic proteins (BMPs) plays a paramount role in the regulation of bone formation. However, how the mechanism works in bone loss of RA is poorly understood. Peptide arginine deiminase IV (PADI4), as a co-transcriptional regulator, mediates the proliferation and differentiation of multiple stem cells by converting arginine residues at histone and other associated proteins to citrulline. Our previous studies found increasing expression of PADI4 promotes cell proliferation and inhibits apoptotsis. Pre-experimental results showed that adipogenesis differentiation of RA-FLS increased at the expense of osteogenesis differentiation under hypoxia. Hypoxia provoked PADI4 expression which in turn involved in the osteo-adipo imbalance and associated with BMPs pathway. On the basis of this, the role and function of PADI4 responsible to osteogenesis and adipogensis through BMPs pathway will be investigated in the RA synovium. Bioinformatics on RA synovium have been used to explore the molecules with obvious changes in BMP pathway, then we will screen out and verify in which the potential PADI4-targeted substrates. We will mimic hypoxia, use gain-lose of PADI4 function and interfere the expression of PADI4-targeted substrates in vitro and in vivo to tease out the mechanism of osteogenesis and adipogenesis regulated by PADI4. Our study will explore the therapeutic strategies with targeted inhibition of PADI4 on RA rats and provide more evidences to the clinic.

类风湿关节炎(RA)受累关节滑膜中骨生成减低是导致骨质流失的原因之一，骨形态蛋白(BMPs)通路在骨生成调节中起主导作用，但在RA骨质损伤中具体机制不明。肽精氨酸脱亚氨基酶Ⅳ(PADI4)作为共转录调节子通过蛋白瓜氨酸化介导干细胞的增殖和分化。我们前期证实在RA成纤维滑膜细胞（RA-FLS）中PADI4表达增加，促进细胞增殖并抑制凋亡，预实验结果发现RA-FLS在低氧中成骨分化减弱伴成脂分化增强，PADI4参与调控该表象并与BMPs通路具有相关性。本课题在此基础上深入探究PADI4经BMPs通路控制RA-FLS成骨成脂分化的作用机制。从通过生信分析得到RA滑膜中BMP通路差异明显的分子中筛寻受PADI4调控的靶分子，并模拟RA低氧环境、干扰PADI4和靶分子的表达，验证PADI4诱使成骨成脂失衡的调控机理，最后在RA大鼠模型中探索靶向抑制RA-FLS中PADI4表达的策略对RA的防治效果。

RA成纤维细胞样滑膜细胞（RA derived Fibroblast-Like Synoviocytes, RA-FLSs）是造成RA滑膜增生及迁延不愈的主要病变细胞，我们前期发现他具有间充质干细胞（Mesenchymal Stem cells, MSCs）的表型和特点，表达MSC类似的表面分子标志，并能够分化成成骨细胞和脂肪细胞。在这项研究中，我们旨在研究缺氧在 RA-FLS 的成骨或脂肪形成中的作用。我们发现缺氧维持RA-FLS的MSC样表型，并造成RA-FLS更易分化为脂肪细胞，同时分化为成骨细胞的能力减弱。在缺氧条件下使用肽精氨酸脱亚氨基酶（PAD4）抑制剂或者RNAi干预PAD4的表达，可以部分恢复RA-FLS成骨分化的能力。另一方面PAD4表达增加减弱RA-FLS向成骨分化，增强RA-FLS成脂分化能力。生物信息学分析显示瘦素在缺氧后显著上调，抑制瘦素可以减弱PAD4的表达，抑制瘦素的表达同样可以扭转缺氧诱导的RA-FLS成骨能力下降。但我们并未发现PAD4与瘦素的相互作用。在大鼠关节炎模型中，使用负载siRNA-PAD4的纳米载体治疗胶原诱导的RA大鼠，可以有效缓解RA的症状。因此，我们的结果在体内外证实靶向PAD4在RA治疗中具有良好的应用前景。