Calcineurin–nuclear factor of activated t cells regulation of Krox-20 expression in Schwann cells requires elevation of intracellular cyclic AMP

The transcription factor Krox-20 (Egr2) is a master regulator of Schwann cell myelination. In mice from which calcineurin B had been excised in cells of the neural crest lineage, calcineurin–nuclear factor of activated T cells (NFAT) signaling was required for neuregulin-related Schwann cell myelination (Kao et al. [2009] Immunity 12:359–372). Whether NFAT signaling required simultaneous elevation of intracellular cAMP levels was not explored. In vivo, Krox-20 expression requires continuous axon–Schwann cell signaling that in Schwann cell cultures can be mimicked by elevation of intracellular cAMP. We have investigated the role of the calcineurin–NFAT pathway in Krox-20 induction in purified rat Schwann cell cultures. Activation of this pathway requires elevation of intracellular Ca2+ levels. The calcium ionophore A23187 or ionomycin was used to increase intracellular Ca2+ levels in Schwann cell cultures that had been treated with dibutyryl cAMP to induce Krox-20. Increase in Ca2+ levels significantly potentiated Krox-20 induction, determined by Krox-20 immunolabeling of individual cells and Western blotting. Levels of the myelin proteins periaxin and P0 were also elevated. The potentiating effect was blocked by cyclosporin A, a specific blocker of the calcineurin–NFAT pathway. We found that, in the absence of cAMP elevation, treatment with A23187 alone failed to induce Krox-20 expression, indicating that NFAT upregulation of Krox-20 requires elevation of cAMP levels in Schwann cells. P-VIVIT, another specific inhibitor of calcineurin–NFAT interaction, blocked Krox-20 induction in response to dibutyryl cAMP and ionophore. HA-NFAT1 (1–460)-GFP translocated to the nucleus on treatment with dibutyryl cAMP with or without added ionophore. NFAT isoforms 1–4 were detected in purified Schwann cells by quantitative RT-PCR. © 2012 Wiley Periodicals, Inc.