Lenz Bernd, Bleich Stefan, Beutler Sonja, Schlierf Beate, Schwager Katja, Reulbach Udo, Kornhuber Johannes, Bönsch Dominikus
Department of Psychiatry and Psychotherapy, Friedrich-Alexander-University of Erlangen-Nuremberg, Germany.
Exp Cell Res. 2006 Dec 10;312(20):4049-55. doi: 10.1016/j.yexcr.2006.09.004. Epub 2006 Sep 9.
Herp (homocysteine-induced endoplasmic reticulum protein) is an ER-resident membrane protein, which has a ubiquitin-like domain at its N-terminus. Expression of Herp protein is up-regulated in response to ER stress, including homocysteine. Herp stabilizes neuronal Ca(2+) homeostasis and is involved in improving the balance of the folding capacity and protein loading in the ER. In patients with alcoholism, we observed a significant decrease in Herp mRNA expression, and an increase of Herp promoter DNA methylation, which was associated with elevated homocysteine levels. Therefore, we studied the mechanism of Herp CpG islands regulation by luciferase assays and mRNA analysis in neuronal SH-SY5Y (human neuroblastoma cell line) and HEK 293T (human embryonic kidney 293T) cells. Acute homocysteine treatment caused transient demethylation of the Herp promoter and an increase in Herp mRNA level. Global DNA methylation was increased over the following 48 h period. We identified the transcription factor binding site AARE (amino acid response element) by mutational analysis involved in Herp induction in SH-SY5Y cells, and the more significant role of the CREB binding site (cyclic AMP response element-binding protein) compared to AARE in HEK 293T cells. Stimulation with SAM (S-adenosyl methionine) and homocysteine led to an increase in Herp promoter methylation, which correlated to an acute decrease in luciferase expression in SAM, but not in homocysteine stimulated cells. Complete methylation of the CpG islands resulted in suppressed gene expression.
Herp（同型半胱氨酸诱导的内质网蛋白）是一种内质网驻留膜蛋白，其N端有一个泛素样结构域。Herp蛋白的表达在对内质网应激（包括同型半胱氨酸）的反应中上调。Herp可稳定神经元钙离子稳态，并参与改善内质网中折叠能力与蛋白质负载的平衡。在酒精中毒患者中，我们观察到Herp mRNA表达显著降低，且Herp启动子DNA甲基化增加，这与同型半胱氨酸水平升高有关。因此，我们通过荧光素酶测定和mRNA分析，在神经元SH-SY5Y（人神经母细胞瘤细胞系）和HEK 293T（人胚肾293T）细胞中研究了Herp CpG岛的调控机制。急性同型半胱氨酸处理导致Herp启动子短暂去甲基化，并使Herp mRNA水平升高。在接下来的48小时内，整体DNA甲基化增加。我们通过突变分析确定了参与SH-SY5Y细胞中Herp诱导的转录因子结合位点AARE（氨基酸反应元件），以及在HEK 293T细胞中与AARE相比，CREB结合位点（环磷酸腺苷反应元件结合蛋白）发挥的更重要作用。用S-腺苷甲硫氨酸（SAM）和同型半胱氨酸刺激导致Herp启动子甲基化增加，这与SAM刺激的细胞中荧光素酶表达急性降低相关，但同型半胱氨酸刺激的细胞中未出现这种情况。CpG岛的完全甲基化导致基因表达受抑制。
