Ca(2+) is a Regulator of the WNK/OSR1/NKCC Pathway in a Human Salivary Gland Cell Line.

Wnk kinase maintains cell volume, regulating various transporters such as sodium-chloride cotransporter, potassium-chloride cotransporter, and sodium-potassium-chloride cotransporter 1 (NKCC1) through the phosphorylation of oxidative stress responsive kinase 1 (OSR1) and STE20/SPS1-related proline/alanine-rich kinase (SPAK). However, the activating mechanism of Wnk kinase in specific tissues and specific conditions is broadly unclear. In the present study, we used a human salivary gland (HSG) cell line as a model and showed that Ca(2+) may have a role in regulating Wnk kinase in the HSG cell line. Through this study, we found that the HSG cell line expressed molecules participating in the WNK-OSR1-NKCC pathway, such as Wnk1, Wnk4, OSR1, SPAK, and NKCC1. The HSG cell line showed an intracellular Ca(2+) concentration ([Ca(2+)]i) increase in response to hypotonic stimulation, and the response was synchronized with the phosphorylation of OSR1. Interestingly, when we inhibited the hypotonically induced [Ca(2+)]i increase with nonspecific Ca(2+) channel blockers such as 2-aminoethoxydiphenyl borate, gadolinium, and lanthanum, the phosphorylated OSR1 level was also diminished. Moreover, a cyclopiazonic acid-induced passive [Ca(2+)]i elevation was evoked by the phosphorylation of OSR1, and the amount of phosphorylated OSR1 decreased when the cells were treated with BAPTA, a Ca(2+) chelator. Finally, through that process, NKCC1 activity also decreased to maintain the cell volume in the HSG cell line. These results indicate that Ca(2+) may regulate the WNK-OSR1 pathway and NKCC1 activity in the HSG cell line. This is the first demonstration that indicates upstream Ca(2+) regulation of the WNK-OSR1 pathway in intact cells.

p62/SQSTM1 is required for the protection against endoplasmic reticulum stress-induced apoptotic cell death.

Endoplasmic reticulum (ER) stress is triggered by various cellular stresses that disturb protein folding or calcium homeostasis in the ER. To cope with these stresses, ER stress activates the unfolded protein response (UPR) pathway, but unresolved ER stress induces reactive oxygen species (ROS) accumulation leading to apoptotic cell death. However, the mechanisms that underlie protection from ER stress-induced cell death are not clearly defined. The nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway plays a crucial role in the protection of cells against ROS-mediated oxidative damage. Keap1 acts as a negative regulator of Nrf2 activation. In this study, we investigated the role of the Nrf2-Keap1 pathway in protection from ER stress-induced cell death using tunicamycin (TM) as an ER stress inducer. We found that Nrf2 is an essential protein for the prevention from TM-induced apoptotic cell death and its activation is driven by autophagic Keap1 degradation. Furthermore, ablation of p62, an adapter protein in the autophagy process, attenuates the Keap1 degradation and Nrf2 activation that was induced by TM treatment, and thereby increases susceptibility to apoptotic cell death. Conversely, reinforcement of p62 alleviated TM-induced cell death in p62-deficient cells. Taken together, these results demonstrate that p62 plays an important role in protecting cells from TM-induced cell death through Nrf2 activation.

Ezetimibe, an NPC1L1 inhibitor, is a potent Nrf2 activator that protects mice from diet-induced nonalcoholic steatohepatitis.

Oxidative stress is important for the pathogenesis of nonalcoholic fatty liver disease (NAFLD), a chronic disease that ranges from hepatic steatosis to nonalcoholic steatohepatitis (NASH). The nuclear factor erythroid 2-related factor 2-Kelch-like ECH associated protein 1 (Nrf2-Keap1) pathway is essential for cytoprotection against oxidative stress. In this study, we found that oxidative stress or inflammatory biomarkers and TUNEL positive cells were markedly increased in NASH patients compared to normal or simple steatosis. In addition, we identified that the hepatic mRNA levels of Nrf2 target genes such as Nqo-1 and GSTA-1 were significantly increased in NASH patients. Ezetimibe, a drug approved by the Food and Drug Administration for the treatment of hypercholesterolemia, improves NAFLD and alleviates oxidative stress. However, the precise mechanism of its antioxidant function remains largely unknown. We now demonstrate that ezetimibe activates Nrf2-Keap1 pathway which was dependent of autophagy adaptor protein p62, without causing cytotoxicity. Ezetimibe activates AMP-activated protein kinase (AMPK), which in turn phosphorylates p62 (p-S351) via their direct interaction. Correspondingly, Ezetimibe protected liver cells from saturated fatty acid-induced apoptotic cell death through p62-dependent Nrf2 activation. Furthermore, its role as an Nrf2 activator was supported by methione- and choline- deficient (MCD) diet-induced NASH mouse model, showing that ezetimibe decreased the susceptibility of the liver to oxidative injury. These data demonstrate that the molecular mechanisms underlying ezetimibe's antioxidant role in the pathogenesis of NASH.