Division of Gene Function in Animals, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
Exp Eye Res. 2013 Jul;112:79-92. doi: 10.1016/j.exer.2013.04.013. Epub 2013 Apr 24.
Genetic predisposition and senescence of retinal pigment epithelium induced by oxidative stress are major contributors to age-related macular degeneration (AMD). Single-nucleotide polymorphisms in HTRA1 are strongly linked to the onset of AMD. In this study, we examine the role of HtrA1 in premature senescence and cell death induced by oxidative stress. HtrA1 mRNA and protein were up-regulated during premature senescence induced by H2O2 in both mouse embryonic fibroblasts (MEFs) and ARPE-19 cells. Expression of the senescence markers p21(CIP1/WAF1) and p16(INK4a), and SA-β-galactosidase activity, were higher in HtrA1+/- MEFs than in HtrA1-/- MEFs. HtrA1+/+ and HtrA1+/- MEFs were more resistant than HtrA1-/- MEFs to H2O2-induced cell death. Activation of p38 MAPK by oxidative stress was quicker in HtrA1+/- MEFs than in HtrA1-/- MEFs. The effects of excess HtrA1 were examined by transient transfection of cells with HtrA1 expression vectors or by addition of recombinant proteins. Excess wild type HtrA1 accelerated premature senescence of MEFs and ARPE-19 cells, while the protease-inactive HtrA1 S328A did not. HtrA1-induced senescence was abrogated by inhibition of p38 MAPK. We conclude that HtrA1 is induced by oxidative stress and promotes premature cell senescence through p38 MAPK in a protease activity-dependent manner.
氧化应激引起的视网膜色素上皮的遗传易感性和衰老，是导致年龄相关性黄斑变性（AMD）的主要原因。HTRA1 中的单核苷酸多态性与 AMD 的发病密切相关。在这项研究中，我们研究了 HtrA1 在氧化应激诱导的细胞过早衰老和死亡中的作用。在 H2O2 诱导的小鼠胚胎成纤维细胞（MEFs）和 ARPE-19 细胞的过早衰老过程中，HtrA1 mRNA 和蛋白表达上调。衰老标志物 p21（CIP1/WAF1）和 p16（INK4a）的表达，以及 SA-β-半乳糖苷酶的活性，在 HtrA1+/- MEFs 中均高于 HtrA1-/- MEFs。与 HtrA1-/- MEFs 相比，HtrA1+/+ 和 HtrA1+/- MEFs 对 H2O2 诱导的细胞死亡更具抗性。氧化应激激活的 p38 MAPK 在 HtrA1+/- MEFs 中比 HtrA1-/- MEFs 更快。通过用 HtrA1 表达载体瞬时转染细胞或添加重组蛋白来研究过量 HtrA1 的作用。过量的野生型 HtrA1 加速了 MEFs 和 ARPE-19 细胞的过早衰老，而无蛋白酶活性的 HtrA1 S328A 则没有。HtrA1 诱导的衰老可被 p38 MAPK 的抑制所阻断。我们的结论是，HtrA1 被氧化应激诱导，并通过依赖蛋白酶活性的 p38 MAPK 促进细胞过早衰老。
