Functional Characterization of a Human Choroid Plexus Cell Line and TRPV4 Stimulated Transepithelial Ion Flux

Abstract

The choroid plexus epithelium (CPe) establishes the blood‐cerebrospinal fluid barrier (BCSFB) and is the primary producer of cerebrospinal fluid (CSF). The CPe has a unique, polarized structure with distinct transporters and channels localized to the apical (CSF‐facing) or basolateral (blood‐facing) membrane. These proteins tightly regulate the composition of CSF by controlling transport of electrolytes and water from the blood into the intraventricular space. Significantly, CSF volume and composition varies on a diurnal basis, as well as in response to pathophysiological stresses. Developing an in vitromodel of the CPe that represents its unique epithelial structure and barrier function is thus key for studying the role of CSF in health and disease. The human choroid plexus papilloma (HIBCPP) cell line is a continuous culture model originally developed by Schwerk et al (2012). The current studies provide characterization of this line to determine its potential as a culture model for electrophysiological studies investigating the regulation of electrogenic ion transport. The HIBCPP cells expresses several CP markers via RT‐PCR, including E2f‐5, transthyretin, forkhead box protein J1 and orthodenticle homeobox 2, confirming lineage of the model. Characteristic of a barrier epithelium, the cells express appropriate junctional complex proteins, zonula occludens‐1, occludin, claudin 1 and claudin 2, via both RT‐PCR and immunofluorescence (IF). When cultured on 30mm Transwell filters, these cells predominately grow as a monolayer and have a transepithelial electrical resistance of ∼400 Ω*cm2. The cells are ciliated, determined by expression of ciliary markers Arl13B and acetylated tubulin. By RT‐PCR, the HIBCPP cells express numerous transporters previously implicated in regulating CSF production in the native CPe, including the Na‐K‐ATPase α1 and β2 subunits, transient receptor potential vanilloid 4 (TRPV4), Na‐K‐Cl cotransporter 1, Na2+ HCO3‐ cotransporter 2, Na2+ dependent Cl‐/ HCO3‐ exchanger, anion exchange protein 2 (AE2), K+ Cl‐ cotransporter 3, volume regulated ion channel, and aquaporin 1. Correct polarization of the N‐K‐ATPase pump, TRPV4 and AE2 suggests transporters in this in vitro model localize to the correct membrane. IF and 3D imaging was used to confirm apical localization of the pump and TRPV4, versus basolateral localization of AE2, consistent with expression patterns in the native tissue. Previous studies have implicated TRPV4 in the regulation of CSF production. In the HIBCPP model, addition of a TRPV4 agonist, GSK1016790A, stimulates a change in net transepithelial ion flux and increase in conductance that is inhibited by a TRPV4 specific antagonist, RN1734. These initial studies support the idea that TRPV4 contributes to CSF secretion by controlling solute movement across the CPe. Overall, this cell line holds great promise as an in vitro model for BCSFB‐based studies.