Phosphoproteomic Insights into TRPV4-AMPK Signaling Axis and Barrier Regulation in the Choroid Plexus Epithelium

Hydrocephalus is characterized by the abnormal accumulation of cerebrospinal fluid (CSF) due to disrupted secretion, circulation, or reabsorption. CSF homeostasis is regulated by ion and water channels on choroid plexus epithelial (CPe) cells, including the mechanosensitive cation channel, which is transient receptor potential vanilloid 4 (TRPV4). Although TRPV4 antagonism prevents hydrocephalus progression in rats, how TRPV4 activity modulates CSF production remains unclear. Because TRPV4 function is phosphorylation-dependent, we examined its activation (GSK1016790A) and inhibition (RN1734) and the downstream signaling alterations in the human choroid plexus papilloma cell line (HIBCPP) using phosphoproteomic mass spectrometry. Our phosphoproteomic analysis revealed significant changes in kinases and tight junction (TJ) proteins regulating epithelial barrier integrity. TRPV4 activation altered the phosphorylation of TJ proteins, including Zonula Occludens-1 (ZO-1) and Claudin-7 (CLDN7), as well as AMP-activated protein kinase (AMPK), with a notable increase in the phosphorylation of the AMPK inhibitory site (Ser496). Using electrophysiological techniques, we showed that AMPK had a more profound effect on epithelial barrier function than on net transepithelial electrolyte ion flux. The inhibition of AMPK-associated inhibitory phosphorylation prior to TRPV4 activation increased barrier tightness, and these permeability changes were mirrored by changes in ZO-1 continuity within junctional complexes. Furthermore, the pharmacological modulation of AMPK-associated signaling altered TRPV4-stimulated epithelial conductance, while metformin treatment also reduced TRPV4-stimulated conductance under the experimental conditions used. These findings suggest an interaction between TRPV4 signaling, AMPK phosphorylation, and epithelial barrier regulation that warrants further mechanistic investigation. Overall, these findings provide insight into signaling pathways associated with the TRPV4-dependent regulation of choroid plexus epithelial barrier properties and provide a basis for the further investigation of their relevance to CSF homeostasis and hydrocephalus.