The Role of the Primary Cilium in Sensing Extracellular pH

Abstract

Biosensors on the membrane of the vascular endothelium are responsible for sensing mechanical and chemical signals in the blood. Transduction of these stimuli into intracellular signaling cascades regulate cellular processes including ion transport, gene expression, cell proliferation, and/or cell death. The primary cilium is a well-known biosensor of shear stress but its role in sensing extracellular pH change has never been examined. As a cellular extension into the immediate microenvironment, the cilium could be a prospective sensor for changes in pH and regulator of acid response in cells. We aim to test our hypothesis that the primary cilium plays the role of an acid sensor in cells using vascular endothelial and embryonic fibroblast cells as in vitro models. We measure changes in cellular pH using pH-sensitive 2′,7′-biscarboxyethy1-5,6-carboxyfluorescein acetoxy-methylester (BCECF) fluorescence and mitogen-activated protein kinase (MAPK) activity to quantify responses to both extracellular pH (pHo) and intracellular pH (pHi) changes. Our studies show that changes in pHo affect pHi in both wild-type and cilia-less Tg737 cells and that the kinetics of the pHi response are similar in both cells. Acidic pHo or pHi was observed to change the length of primary cilia in wild-type cells while the cilia in Tg737 remained absent. Vascular endothelial cells respond to acidic pH through activation of ERK1/2 and p38-mediated signaling pathways. The cilia-less Tg737 cells exhibit delayed responsiveness to pHo dependent and independent pHi acidification as depicted in the phosphorylation profile of ERK1/2 and p38. Otherwise, intracellular pH homeostatic response to acidic pHo is similar between wild-type and Tg737 cells, indicating that the primary cilia may not be the sole sensor for physiological pH changes. These endothelial cells respond to pH changes with a predominantly K+-dependent pHi recovery mechanism, regardless of ciliary presence or absence.