br Conflict of interest br Introduction The

Conflict of interest
Introduction The Piezo proteins, Piezo1/FAM38A and Piezo2/FAM38B, are multi-pass transmembrane proteins that have been identified as mechanically-activated (MA) homomultimeric, pore-forming ion channels [1,2]. Piezo1 is broadly expressed with its highest expression in bladder, colon, kidney, lung, and skin. Piezo2 is highly expressed in bladder, colon, lung, and sensory neurons from dorsal root ganglia. Recent studies have demonstrated that Piezo1, in particular, can mediate MA cationic currents in several different cell types, including endothelial Sulindac sulfone (ECs) [3,4]. Additionally, Piezo1 was shown to be critical for calcium influx into ECs as well as EC alignment in response to fluid shear stress [3,4]. Expression of Piezo1 in HEK 293 cells, which lack endogenous Piezo1 and are typically unresponsive to shear stress, evoked shear stress-induced calcium influx, suggesting that Piezo1 channels may directly function as shear stress sensors [3]. Most recently, Piezo1 channels have been proposed as “exercise sensors” that are activated by increased blood flow during whole body physical activity and cause vascular bed-specific vasoconstriction [5]. There are no known endogenous agonists and/or antagonists of Piezo channels to date, but Piezo channels are known to be activated by mechanical stimuli and also by a recently identified synthetic small molecule called Yoda1. Yoda1 was first discovered using a high-throughput screen to test whether chemical compounds exist that can activate Piezo channels. It was determined to be a selective small molecule agonist of Piezo1 based on its ability to induce a calcium response in HEK293 cells that overexpressed Piezo1, but not in vector-transfected cells or in cells that overexpressed Piezo2 [6]. Yoda1 stimulation has since been utilized as an invaluable tool for investigating the activation and regulation of Piezo1. The concept of a Yoda1-like “exercise pill”, which has the ability to specifically target and enhance Piezo1 activity during exercise, has even been introduced [7]. It has been recently demonstrated that Yoda1 can induce responses similar to those activated by fluid shear stress in ECs, such as increased intracellular calcium and nitrate formation [8]. These endothelial responses were shown to be mediated by Piezo1 by using an siRNA targeting approach. Pharmacological inhibition of Piezo1 is an alternative approach that has been utilized in other studies, including the examination of mechanically-activated channel currents in a mouse neuroblastoma cell line [1], the investigation of shear-induced ATP release and calcium influx in RBCs [9], and the study of shear stress-induced endothelial polarization in mesenteric arteries [5]. Using a similar pharmacological approach, we sought to examine the role of Piezo1 on the simultaneous activations of the Akt and ERK1/2 signaling pathways by Yoda1 in ECs.
Materials and methods
Results
Discussion It is widely accepted that calcium (Ca2+) is an important signaling molecule in cells. In endothelial cells, changes in intracellular Ca2+ can lead to the production and release of vasoactive factors, such as nitric oxide (NO), that affect a wide range of vascular functions, including EC permeability and vascular tone. The Akt and ERK1/2 signaling pathways, which mediate cell proliferation and survival, are also activated by changes in intracellular Ca2+. Membrane ion channels play a critical role in regulating this influx of Ca2+ and the specific activation and opening of Piezo channels, whether through mechanical (i.e. shear stress) or chemical means (i.e. Yoda1), can trigger an influx of Ca2+ causing its various downstream signaling pathways to also be initiated. Our results showed that both Akt and ERK1/2 phosphorylation are increased in endothelial cells in response to Yoda1 treatment, suggesting that Piezo channels have been activated. It has recently been shown that Akt phosphorylation is induced in human umbilical artery endothelial cells (HUAECs) exposed to 1 μM Yoda for 5 min [8]. Moreover, it was shown that Yoda1-induced Akt phosphorylation was strongly reduced by knockdown of Piezo1 with siRNA, which implies that Piezo1 mediates this response in HUAECs. Although our results confirm that Yoda1 can induce Akt activation in ECs regardless of its origin, we could not observe a significant decrease in Akt phosphorylation in cells pre-treated with the Piezo1 antagonist, GsMTx4, prior to exposure to Yoda1. It should be noted here that siRNA knockdown of Piezo1 has been reported to decrease the expression levels of eNOS (∼40% decrease) in ECs [3]. Consequently, decreased eNOS expression may cause a decrease in NO production and release from cells in response to Yoda1 stimulation. Since NO is known to activate PI3K-Akt [13,14], a decrease in phosphorylated Akt in Yoda-induced ECs in which Piezo1 is knocked down via siRNA may be partly attributed to a decrease in eNOS expression.