Distinct Activation Properties of the Nuclear Factor of Activated T-cells (NFAT) Isoforms NFATc3 and NFATc4 in Neurons

Abstract

Background:Ca 2ϩ /calcineurin-dependent transcription factor NFAT regulates many neuronal functions. Results: Depolarization/Ca 2ϩ influx-induced activation and nuclear translocation are markedly faster and stronger for NFATc3 than NFATc4 in neurons, which is determined in part by SP-containing regions of NFATc3 and NFATc4. Conclusion: Activation of NFATc3 and NFATc4 is distinctly regulated in neurons. Significance: The distinct regulation of NFATc3 and NFATc4 may underlie isoform-specific NFAT functions in neurons.

Nerve Growth Factor (NGF) Regulates Activity of Nuclear Factor of Activated T-cells (NFAT) in Neurons via the Phosphatidylinositol 3-Kinase (PI3K)-Akt-Glycogen Synthase Kinase 3β (GSK3β) Pathway

“…Prior to plating, DRG neurons were transfected using the Amaxa nucleofection system (Program G-013; Lonza, Switzerland) as described previously (7,30,33). Typically, five rat pups or three mice were required to obtain a sufficient number of cells for performing a single transfection.…”

“…The shRNA constructs targeting NFATc3, NFATc4, and GSK3␤ were kindly provided by Dr. Michal Hetman (University of Kentucky) (12). We previously validated all of these constructs (33). A constitutively active form of GSK3␤ (GSK3␤ S9A) (36) was purchased from Addgene (plasmid 14754; Cambridge, MA).…”

“…NFAT Reporter Assays-NFAT reporter expression was assessed using a Dual-Luciferase assay as described previously (7,33). In brief, DRG neurons were co-transfected with the NFAT-luciferase (NFAT-luc) reporter plasmid (which encodes firefly luciferase under the control of three copies of the NFATbinding motif; pNFAT-TA-luciferase; Clontech) and the Renilla reniformis luciferase (TK-luc) reporter (which encodes Renilla luciferase under the control of constitutively active HSV-TK promoter; pRL-TK; Promega, Madison, WI).…”

“…The [Ca 2ϩ ] i changes and EGFP-NFATc3 movement were simultaneously recorded as described previously (7,33). In brief, DRG neurons were loaded with the ratiometric Ca 2ϩ indicator dye Fura-2/AM (2 M) for 30 min.…”

“…They were counterstained with DAPI (Invitrogen) to mark the nucleus and mounted on glass slides using Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL). The distribution of EGFP-NFATc3 was analyzed using a ϫ60 oil immersion objective (numerical aperture ϭ 1.4) on an Olympus BX61 microscope equipped with the Fluoview 300 laser-scanning confocal imaging system as described previously (33). Data were acquired and analyzed by a blinded experimenter using the Fluoview software (Olympus).…”