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.
Department of Veterinary Anatomy, Sokoine University of Agriculture, Chuo Kikuu, Morogoro, Tanzania.
Maulilio J
MJ
Kipanyula
Ashwin
A
Woodhoo
Mary
M
Rahman
Donna
D
Payne
Kristján R
KR
Jessen
Rhona
R
Mirsky
D000818:Animals; D015153:Blotting, Western; D000242:Cyclic AMP / Q000378:metabolism*; D051767:Early Growth Response Protein 2 / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology*; D007150:Immunohistochemistry; D050778:NFATC Transcription Factors / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012583:Schwann Cells / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection; D015854:Up-Regulation
D051767:Early Growth Response Protein 2; D050778:NFATC Transcription Factors; D000242:Cyclic AMP