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 Ca 2+ levels. The calcium ionophore A23187 or ionomycin was used to increase intracellular Ca 2+ levels in Schwann cell cultures that had been treated with dibutyryl cAMP to induce Krox‐20. Increase in Ca 2+ levels significantly potentiated Krox‐20 induction, determined by Krox‐20 immunolabeling of individual cells and Western blotting. Levels of the myelin proteins periaxin and P 0 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.