The Sur1-Trpm4 channel regulates NOS2 transcription in TLR4-activated microglia. Issue 1 (December 2016)

• Record Type: Journal Article
• Authors: Kurland, David; Gerzanich, Volodymyr; Karimy, Jason; Woo, Seung; Vennekens, Rudi; Freichel, Marc; Nilius, Bernd; Bryan, Joseph; Simard, J.
• Is Part Of: Journal of neuroinflammation. Volume 13:Issue 1(2016)
• Page Start: 1
• Page End: 23
• Publication Date: 2016-12
• Subjects: Sur1-Trpm4 -- KATP -- NO -- NOS2 -- TLR4 -- Microglia; Central nervous system -- Diseases -- Periodicals; Inflammation -- Periodicals; 616.8
• DOI: 10.1186/s12974-016-0599-2
• Languages: English
• ISSNs: 1742-2094

Abstract

Abstract Background Harmful effects of activated microglia are due, in part, to the formation of peroxynitrite radicals, which is attributable to the upregulation of inducible nitric oxide (NO) synthase (NOS2). Because NOS2 expression is determined by Ca2+ -sensitive calcineurin (CN) dephosphorylating nuclear factor of activated T cells (NFAT), and because Sur1-Trpm4 channels are crucial for regulating Ca2+ influx, we hypothesized that, in activated microglia, Sur1-Trpm4 channels play a central role in regulating CN/NFAT and downstream target genes such asNos2 . Methods We studied microglia in vivo and in primary culture from adult rats, and from wild type, Abcc8 −/− andTrpm4 −/− mice, and immortalized N9 microglia, following activation of Toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS), using in situ hybridization, immunohistochemistry, co-immunoprecipitation, immunoblot, qPCR, patch clamp electrophysiology, calcium imaging, the Griess assay, and chromatin immunoprecipitation. Results In microglia in vivo and in vitro, LPS activation of TLR4 led to de novo upregulation of Sur1-Trpm4 channels and CN/NFAT-dependent upregulation ofNos2 mRNA, NOS2 protein, and NO. Pharmacological inhibition of Sur1 (glibenclamide), Trpm4 (9-phenanthrol), or gene silencing ofAbcc8 orTrpm4 reducedNos2 upregulation. Inhibiting Sur1-Trpm4 increased the intracellular calcium concentration ([Ca2+ ]i ), as expected, but also decreased NFAT nuclear translocation. The increase in [Ca2+ ]i induced by inhibiting or silencing Sur1-Trpm4 resulted in phosphorylation of Ca2+ /calmodulin protein kinase II and of CN, consistent with reduced nuclear translocation of NFAT. The regulation of NFAT by Sur1-Trpm4 was confirmed using chromatin immunoprecipitation. Conclusions Sur1-Trpm4 constitutes a novel mechanism by which TLR4-activated microglia regulate pro-inflammatory, Ca2+ -sensitive gene expression, includingNos2 .