Chloride channel 4 is required for nerve growth factor-induced TrkA signaling and neurite outgrowth in PC12 cells and cortical neurons

Abstract

We have previously shown that chloride ion flux plays an important role in receptor tyrosine kinase A (TrkA)-mediated signaling pathway during nerve growth factor (NGF)-induced differentiation in pheochromocytoma (PC12) cells. Here we found out that chloride channel 4 (CLC-4) is responsible for the NGF-induced neurite outgrowth in neuronal cells. Using a patch-clamp technique, we found that NGF treatment increased anionic conductance in PC12 cells, an effect which was blocked by transfection of siRNA of CLC-4. Also, the NGF-induced TrkA phosphorylation and subsequent Akt/moesin phosphorylation was suppressed in the CLC-4 knock down cells. Moreover, CLC-4 knock down also suppressed the neurite outgrowth in response to long-term treatment of NGF in PC12 cells and in primary cortical neurons. In summary, our results suggest that CLC-4 is an important mediator of the TrkA-mediated signaling pathway and thus, the NGF-induced differentiation of neuronal cells.

Keywords: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid; 5-Nitro-2-(3-phenylpropylamino) benzoic acid; CLC-4; EDTA; EGF; EGTA; FBS; HEPES; HS; MAPK; MTT; NGF; NPPB; PC; PC12 cells; PI3K; PLC-γ; PS; RT-PCR; S/ER; SDS; SITS; Trk; TrkA receptor; chloride channel 4; epidermal growth factor; ethylene glycol tetraacetic acid; ethylenediaminetetraacetic acid; fetal bovine serum; horse serum; hydroxyethyl piperazineethanesulfonic acid; mitogen-activated kinase; nerve growth factor; neurite outgrowth; neurons; penicillin–streptomycin; pheochromocytoma; phosphatidylinositol-3-kinase; phospholipase-C-γ; receptor tyrosine kinase; reverse transcription polymerase chain reaction; sarco/endoplasmic reticulum; sodium dodecyl sulfate.