Korean J Physiol Pharmacol.  2019 Mar;23(2):95-102. 10.4196/kjpp.2019.23.2.95.

Involvement of Orai1 in tunicamycin-induced endothelial dysfunction

• Yang H
• Xue Y
• Kuang S
• Zhang M
• Chen J
• Liu L
• Shan Z
• Lin Q
• Li X
• Yang M
• Zhou H
• Rao F
• Deng C

• KMID: 2438083
• DOI: http://doi.org/10.4196/kjpp.2019.23.2.95

Abstract

Endoplasmic reticulum (ER) stress is mediated by disturbance of Ca²âº homeostasis. The store-operated calcium (SOC) channel is the primary Ca²âº channel in non-excitable cells, but its participation in agent-induced ER stress is not clear. In this study, the effects of tunicamycin on Ca²âº influx in human umbilical vein endothelial cells (HUVECs) were observed with the fluorescent probe Fluo-4 AM. The effect of tunicamycin on the expression of the unfolded protein response (UPR)-related proteins BiP and CHOP was assayed by western blotting with or without inhibition of Orai1. Tunicamycin induced endothelial dysfunction by activating ER stress. Orai1 expression and the influx of extracellular Ca²âº in HUVECs were both upregulated during ER stress. The SOC channel inhibitor SKF96365 reversed tunicamycin-induced endothelial cell dysfunction by inhibiting ER stress. Regulation of tunicamycin-induced ER stress by Orai1 indicates that modification of Orai1 activity may have therapeutic value for conditions with ER stress-induced endothelial dysfunction.

Keyword

Endoplasmic reticulum stress; Endothelial cells; Orai1; Store-operated calcium channel

MeSH Terms

Blotting, Western
Calcium
Endoplasmic Reticulum
Endoplasmic Reticulum Stress
Endothelial Cells
Homeostasis
Human Umbilical Vein Endothelial Cells
Tunicamycin
Unfolded Protein Response
Calcium
Tunicamycin

Figure

• 

  Fig. 1 Tunicamycin induced endothelial dysfunction by ER stress. HUVECs were treated with 0.25, 0.5, 1, 2 µg/ml tunicamycin for 24 h. Cell viability was assayed with Cell Counting Assay Kit-8 (A). Expression of eNOS (B), BiP (C) and CHOP (D) was determined by western blotting. GAPDH was used as an internal control. *p < 0.05 vs. control; n = 4.

• 

  Fig. 2 Alteration of intracellular Ca2+ concentration in HUVECs induced by tunicamycin. HUVECs were treated with 1 µg/ml tunicamycin for 24 h. Cells were incubated in a Ca2+-free buffer containing 2 µM thapsigargin for 8 min, and Ca2+ influx was induced during the Ca2+ loading period (left panel). Ca2+ influx was enhanced by tunicamycin (right panel). **p < 0.01 vs. control; n = 60 cells.

• 

  Fig. 3 Orai1 expression was upregulated by tunicamycin in HUVECs. HUVECs were treated with tunicamycin (0.25, 0.5, 1, 2 µg/ml respectively). Twenty-four hours later, the expression of Orai1 was determined by western blotting. *p < 0.05 vs. control; n = 4.

• 

  Fig. 4 SOC channel inhibitor SKF-96365 reduced tunicamycin-induced ER stress to protect endothelial function. HUVECs were preincubated with 1.25, 2.5, 5 µM SKF96365 before treatment with 1 µg/ml tunicamycin. BiP (A), CHOP (B), and eNOS (C) expression were determined by western blotting. *p < 0.05 vs. control; #p < 0.05 vs. tunicamycin treatment only, n = 4.

• 

  Fig. 5 Effect of Orai1 siRNA on tunicamycin-induced eNOS and UPR markers expression. HUVECs were preincubated with 50, 100, or 200 MOI adenovirus-Orai1siRNA for 24 h; 1 µg/ml tunicamycin was added for another 24 h. (A) eNOS, UPR markers and Orai1 expression were assayed by western blotting. (B-E) Densitometric analysis of the protein levels of eNOS, BiP, Xbp1-s and Orai1 respectively, GAPDH was used as the internal control. *p < 0.05, **p < 0.01 vs. control; #p < 0.05, ##p < 0.01 vs. tunicamycin treatment only, n = 4.

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