Miglionico Rocchina, Ostuni Angela, Armentano Maria Francesca, Milella Luigi, Crescenzi Elvira, Carmosino Monica, Bisaccia Faustino
Department of Sciences, University of Basilicata, Via dell'Ateneo Lucano, 85100 Potenza, Italy.
Institute of Experimental Endocrinology and Oncology (IEOS), National Research Council (CNR), 80131 Naples, Italy.
Cell Mol Biol Lett. 2017 Apr 4;22:7. doi: 10.1186/s11658-017-0036-2. eCollection 2017.
Pseudoxanthoma elasticum (PXE) is characterized by progressive ectopic mineralization of elastic fibers in dermal, ocular and vascular tissues. No effective treatment exists. It is caused by inactivating mutations in the gene encoding for the ATP-binding cassette, sub-family C member 6 transporter (ABCC6), which is mainly expressed in the liver. The ABCC6 substrate (s) and the PXE pathomechanism remain unknown. Recent studies have shown that overexpression of ABCC6 in HEK293 cells results in efflux of ATP, which is rapidly converted into nucleoside monophosphates and pyrophosphate (PPi). Since the latter inhibits mineralization, it was proposed that the absence of circulating PPi in PXE patients results in the characteristic ectopic mineralization. These studies also demonstrated that the presence of ABCC6 modifies cell secretory activity and suggested that ABCC6 can change the cell phenotype.
Stable knockdown HepG2 clones were generated using small hairpin RNA (shRNA) technology. The intracellular glutathione and ROS levels were determined. Experiments using cell cycle analysis, real-time PCR and western blot were performed on genes involved in the senescence phenotype.
To shed light on the physiological role of ABCC6, we focused on the phenotype of HepG2 cells that lack ABCC6 activity. Interestingly, we found that knockdown HepG2 cells show: 1) intracellular reductive stress; 2) cell cycle arrest in G1 phase; 3) upregulation of p21 p53 independent; and 4) downregulation of lamin A/C.
These findings show that the absence of ABCC6 profoundly changes the HepG2 phenotype, suggesting that the PXE syndrome is a complex metabolic disease that is not exclusively related to the absence of pyrophosphate in the bloodstream.
弹性假黄瘤（PXE）的特征是真皮、眼部和血管组织中的弹性纤维进行性异位矿化。目前尚无有效治疗方法。它是由编码ATP结合盒转运体C亚家族成员6（ABCC6）的基因发生失活突变引起的，该基因主要在肝脏中表达。ABCC6的底物和PXE的发病机制仍不清楚。最近的研究表明，ABCC6在HEK293细胞中的过表达导致ATP外流，ATP迅速转化为单磷酸核苷和焦磷酸（PPi）。由于后者抑制矿化，因此有人提出PXE患者循环中PPi的缺乏导致了特征性的异位矿化。这些研究还表明，ABCC6的存在改变了细胞分泌活性，并提示ABCC6可以改变细胞表型。
使用小发夹RNA（shRNA）技术构建稳定敲低HepG2细胞系。测定细胞内谷胱甘肽和活性氧水平。对与衰老表型相关的基因进行细胞周期分析、实时PCR和蛋白质印迹实验。
为了阐明ABCC6的生理作用，我们重点研究了缺乏ABCC6活性的HepG2细胞的表型。有趣的是，我们发现敲低HepG2细胞表现出：1）细胞内还原应激；2）细胞周期停滞于G1期；3）p21非p53依赖性上调；4）核纤层蛋白A/C下调。
这些发现表明，ABCC6的缺失深刻改变了HepG2细胞表型，提示PXE综合征是一种复杂的代谢性疾病，并不完全与血液中焦磷酸的缺乏有关。
