Estrogen-dependent inhibition of dextrose-induced endoplasmic reticulum stress and superoxide generation in endothelial cells

Increased oxidative stress and endoplasmic reticulum stress (ER stress) have been implicated in atherosclerosis. Estrogens have potent antioxidant activity but their effects on ER stress have not been well studied. Therefore, we studied the effects of estradiol and related sex steroids on dextrose-induced ER stress and superoxide (SO) generation in human umbilical vein endothelial cells (HUVECs). Oxidative stress was measured using hydroethidine fluorescence and MCLA chemiluminescence. ER stress was measured with an ER stress-sensitive secreted alkaline phosphatase (ES-TRAP) assay and by Western blot analysis of the expression of GRP78, JNK1, and phosphorylated JNK1, markers for ER stress. A supraphysiological dextrose concentration (27.5 mM) increased ER stress and SO generation compared to treatment with a physiological concentration (5.5 mM) of dextrose. In the presence of estradiol or testosterone (T), ER stress and SO generation were significantly reduced. In contrast to T-treated cells, dihydrotestosterone and 5-methyltestosterone were ineffective at alleviating ER stress or SO generation. When HUVECs were treated with T and the aromatase inhibitor 4-hydroxy-4-androstene-3,17-dione, T was no longer effective at suppressing ER stress or inhibiting SO generation. Changes in GRP78 expression and JNK activity in HUVECs support the results obtained in the ES-TRAP assay. These results indicate that dextrose-induced endoplasmic reticulum stress and superoxide generation are reversed by estradiol and testosterone; however, the latter requires aromatase-dependent conversion to estradiol.

Graphical AbstractDiagram showing effect of estradiol (E) and testosterone (T) on superoxide (SO) generation and endoplasmic reticulum (ER) stress. Testosterone is converted to estrogen via aromatase activity .Estradiol-mediated inhibition of ER stress was associated with down-regulation of several components of the unfolded protein response (UPR) including glucose-regulated protein 78 (GRP78) expression and c-jun N-terminal kinase (JNK) activity.Figure optionsDownload high-quality image (46 K)Download as PowerPoint slideHighlights
► Dextrose increases superoxide (SO) generation in endothelial cells.
► Dextrose increases endoplasmic reticulum (ER) stress in endothelial cells.
► Estradiol and testosterone inhibit dextrose-induced ER stress and SO generation.
► Dihydrotestosterone and 5-methyltestosterone do not alter ER or oxidative stress.
► Testosterone effects require aromatase-dependent conversion to estradiol.