Flynn J M, Dimitrijevich S D, Younes M, Skliris G, Murphy L C, Cammarata P R
Department of Cell Biology, University of North Texas Health Science Center, Fort Worth, TX, USA.
Am J Physiol Endocrinol Metab. 2008 Sep;295(3):E637-47. doi: 10.1152/ajpendo.90407.2008. Epub 2008 Jun 24.
The influence of sexual category as a modifier of cellular function is underinvestigated. Whether sex differences affect estrogen-mediated mitochondrial cytoprotection was determined using cell cultures of normal human lens epithelia (nHLE) from postmortem male and female donors. Experimental indicators assessed included differences in estrogen receptor-beta (ERbeta) isoform expression, receptor localization in mitochondria, and estrogen-mediated prevention of loss of mitochondrial membrane potential using the potentiometric fluorescent compound JC-1 after nHLE were exposed to peroxide. The impact of wild-type ERbeta (wtERbeta1) was also assessed using wtERbeta1 siRNA to suppress expression. A triple-primer PCR assay was employed to determine the proportional distribution of the receptor isoforms (wtERbeta1, -beta2, and -beta5) from the total ERbeta message pool in male and female cell cultures. Irrespective of sex, nHLE express wtERbeta1 and the ERbeta2 and ERbeta5 splice variants in similar ratios. Confocal microscopy and immunofluorescence revealed localization of the wild-type receptor in peripheral mitochondrial arrays and perinuclear mitochondria as well as nuclear staining in both cell populations. The ERbeta2 and ERbeta5 isoforms were distributed primarily in the nucleus and cytosol, respectively; no association with the mitochondria was detected. Both male and female nHLE treated with E(2) (1 muM) displayed similar levels of protection against peroxide-induced oxidative stress. In conjunction with acute oxidative insult, RNA suppression of wtERbeta1 elicited the collapse of mitochondrial membrane potential and markedly diminished the otherwise protective effects of E(2). Thus, whereas the estrogen-mediated prevention of mitochondrial membrane permeability transition is sex independent, the mechanism of estrogen-induced mitochondrial cytoprotection is wtERbeta1 dependent.
作为细胞功能调节因素的性别类别影响尚未得到充分研究。我们使用来自男性和女性死后捐赠者的正常人晶状体上皮细胞（nHLE）培养物，确定性别差异是否影响雌激素介导的线粒体细胞保护作用。评估的实验指标包括雌激素受体-β（ERβ）亚型表达的差异、受体在线粒体中的定位，以及nHLE暴露于过氧化物后使用电位荧光化合物JC-1检测雌激素介导的线粒体膜电位丧失的预防情况。还使用野生型ERβ（wtERβ1）小干扰RNA（siRNA）抑制表达来评估野生型ERβ（wtERβ1）的影响。采用三引物聚合酶链反应（PCR）测定法确定男性和女性细胞培养物中总ERβ信息库中受体亚型（wtERβ1、-β2和-β5）的比例分布。无论性别如何，nHLE以相似比例表达wtERβ1以及ERβ2和ERβ5剪接变体。共聚焦显微镜和免疫荧光显示野生型受体定位于外周线粒体阵列和核周线粒体以及两个细胞群体中的核染色。ERβ2和ERβ5亚型分别主要分布在细胞核和细胞质中；未检测到与线粒体的关联。用E（2）（1μM）处理的男性和女性nHLE对过氧化物诱导的氧化应激均表现出相似水平的保护作用。与急性氧化损伤相结合，wtERβ1的RNA抑制导致线粒体膜电位崩溃，并显著减弱E（2）的保护作用。因此，虽然雌激素介导的线粒体膜通透性转换预防与性别无关，但雌激素诱导的线粒体细胞保护机制依赖于wtERβ
