Question: 

Upon endoplasmic reticulum (ER) stress induction, cells endeavor to survive by engaging the adaptive stress response known as the unfolded protein response. Chronic ER stress culminates in apoptotic cell death, which involves induction of the pro-apoptotic protein, CHOP. Here, we show that bestrophin-3 (Best-3), a protein previously associated with Ca²⁺-activated Cl^- channel activity, is upregulated by the ER stressors, thapsigargin (TG, 3mM), tunicamycin (TUN, 6mM) and the toxic metal Cd²⁺ (25mM).

Methods: 

Expression levels were determined by immunoblotting, immunofluorescence and PCR. MTT assay and nuclear staining measured cell death. Cell cycle and cytosolic Ca²⁺ were analysed by flow cytometry. Oxidative stress was determined using carboxy-dichlorodihydrofluorescein and Amplex Red.

Results: 

In cultured rat kidney proximal tubule cells, ER stress by Cd²⁺, TG and TUN after 6h was attenuated by the Ca²⁺ chelator BAPTA-AM (10mM) or the antioxidant a-tocopherol (100mM). Best-3 is expressed in the plasma membrane, nuclei and intracellular compartments, though not in the ER, in both cultured cells and rat kidney cortex sections. Best-3 mRNA was augmented by ER stress and signaled through increased Ca²⁺, oxidative stress and ERK1/2 phosphorylation, which was confirmed by a-tocopherol, BAPTA-AM, calmodulin kinase inhibitor calmidazolium (40mM), ERK1/2 inhibitor U0126 (10mM), and RNAi. Knockdown of Best-3 resulted in decreased cell number consequentially of PARP-1 cleavage and cell death. Furthermore, Best-3 overexpression reduced cell death by Cd²⁺, TG and TUN, by diminishing CHOP.

Conclusions: 

From our novel observations, we conclude that ERK1/2-dependent Best-3 activation regulates cell fate decisions during ER stress by suppressing CHOP induction and cell death.

(Funded by DFG TH345/10-1)