METHODS: Senescence was induced HSCs by serum withdrawal or progressive passage. DNA-protein interaction was evaluated by chromatin immunoprecipitation (ChIP).
RESULTS: SRF expression was down-regulated in HSCs entering into senescence. Coincidently, SRF depletion by RNAi accelerated HSC senescence. Of note, treatment of an anti-oxidant (N-acetylcysteine or NAC) blocked HSC senescence by SRF deficiency suggesting that SRF may antagonize HSC senescence by eliminating excessive reactive oxygen species (ROS). PCR-array based screening identified peroxidasin (PXDN) as a potential target for SRF in HSCs. PXDN expression was inversely correlated with HSC senescence whereas PXDN knockdown accelerated HSC senescence. Further analysis reveals that SRF directly bound to the PXDN promoter and activated PXDN transcription. Consistently, PXDN over-expression protected whereas PXDN depletion amplified HSC senescence. Finally, PXDN knockout mice displayed diminished liver fibrosis compared to wild type mice when subjected to bile duct ligation (BDL).
CONCLUSIONS: Our data suggest that SRF, via its downstream target PXDN, plays a key role in regulating HSC senescence.
方法:通过血清停药或逐步传代诱导HSC衰老。通过染色质免疫沉淀(ChIP)评估DNA-蛋白质相互作用。
结果:在进入衰老的HSC中,SRF表达下调。巧合的是,通过RNAi消除SRF加速HSC衰老。值得注意的是,抗氧化剂(N-乙酰半胱氨酸或NAC)的治疗可通过SRF缺乏症阻断HSC衰老,这表明SRF可能通过消除过多的活性氧(ROS)来拮抗HSC衰老。基于PCR阵列的筛选将过氧化物酶素(PXDN)鉴定为HSC中SRF的潜在靶标。PXDN表达与HSC衰老呈负相关,而PXDN敲低可加速HSC衰老。进一步分析揭示SRF直接结合PXDN启动子并激活PXDN转录。始终如一,PXDN过表达受保护,而PXDN耗竭扩增HSC衰老。最后,当经受胆管结扎(BDL)时,与野生型小鼠相比,PXDN敲除小鼠显示出减少的肝纤维化。
结论:我们的数据表明SRF,通过其下游目标PXDN,在调节HSC衰老中起关键作用。