Alternating Magnetic Fields Drive Stimulation of Gene Expression via Generation of Reactive Oxygen Species

Magnetogenetics represents a method for remote control of cellular function. Despite successful use of magnetic fields to control gene expression in vitro and in vivo, the mechanism underlying magnetogenetics is largely unknown, thereby hindering further development and applications. Previous work suggests a chemical-based mechanism involving the generation of reactive oxygen species (ROS) as a platform initiator and downstream signaling molecule. Herein, a chemical biology approach was used to elucidate further the mechanism of radio frequency-alternating magnetic field (RF-AMF) stimulation of a TRPV1-ferritin magnetogenetics platform that leads to Ca2+ gating. RF-AMF stimulation of HEK 293T cells expressing TRPV1-Ferritin resulted in a [~]30% and [~]140% increase in intra- and extracellular ROS levels, respectively. Mutations to specific cysteine residues in TRPV1 responsible for ROS sensitivity eliminated RF-AMF driven Ca2+-dependent transcription of secreted embryonic alkaline phosphatase (SEAP). Using a non-tethered (to TRPV1) ferritin also eliminated RF-AMF driven SEAP production. These results suggest ferritin-dependent ROS activation of TRPV1 plays a key role in the initiation of magnetogenetics. Furthermore, inhibition of IP3R-based endoplasmic reticulum (ER) Ca2+ release with Xestospongin C, inhibition of protein kinase C (PKC) activity with Go 6983, or inhibition of NADPH oxidase (NOX) isoforms 1/2 gp91phoxcytochrome with GSK 2795039 eliminated short-term RF-AMF potentiation of SEAP production with capsaicin. Similarly, Go 6983 significantly reduced long-term RF-AMF capsaicin potentiation of SEAP production. These results suggest that ROS-activated TRPV1 signaling to increase intracellular Ca2+ includes pathways involving PKC, NOX, and the ER. Together, these findings fill in current knowledge gaps in the mechanism of magnetogenetics, which may lead to translational applications in medicine and biotechnology.