title: NFAT regulates calcium-sensing receptor-mediated TNF production.
abstract: Because nuclear factor of activated T cells (NFAT) has been implicated in TNF production as well as osmoregulation and salt and water homeostasis, we addressed whether calcium-sensing receptor (CaR)-mediated TNF production in medullary thick ascending limb (mTAL) cells was NFAT dependent. TNF production in response to addition of extracellular Ca(2+) (1.2 mM) was abolished in mTAL cells transiently transfected with a dominant-negative CaR construct (R796W) or pretreated with the phosphatidylinositol phospholipase C (PI-PLC) inhibitor U-73122. Cyclosporine A (CsA), an inhibitor of the serine/threonine phosphatase calcineurin, and a peptide ligand, VIVIT, that selectively inhibits calcineurin-NFAT signaling, also prevented CaR-mediated TNF production. Increases in calcineurin activity in cells challenged with Ca(2+) were inhibited after pretreatment with U-73122 and CsA, suggesting that CaR activation increases calcineurin activity in a PI-PLC-dependent manner. Moreover, U-73122, CsA, and VIVIT inhibited CaR-dependent activity of an NFAT construct that drives expression of firefly luciferase in transiently transfected mTAL cells. Collectively, these data verify the role of calcineurin and NFAT in CaR-mediated TNF production by mTAL cells. Activation of the CaR also increased the binding of NFAT to a consensus oligonucleotide, an effect that was blocked by U-73122 and CsA, suggesting that a calcineurin- and NFAT-dependent pathway increases TNF production in mTAL cells. This mechanism likely regulates TNF gene transcription as U-73122, CsA, and VIVIT blocked CaR-dependent activity of a TNF promoter construct. Elucidating CaR-mediated signaling pathways that regulate TNF production in the mTAL will be crucial to understanding mechanisms that regulate extracellular fluid volume and salt balance.
authors.affiliation: Dept. of Pharmacology, New York Medical College, Valhalla, NY 10595, USA.
authors.forename: Huda Ismail
authors.initials: HI
authors.lastname: Abdullah
authors.forename: Paulina L
authors.initials: PL
authors.lastname: Pedraza
authors.forename: Shoujin
authors.initials: S
authors.lastname: Hao
authors.forename: Karin D
authors.initials: KD
authors.lastname: Rodland
authors.forename: John C
authors.initials: JC
authors.lastname: McGiff
authors.forename: Nicholas R
authors.initials: NR
authors.lastname: Ferreri
mesh_terms: D000818:Animals; D019703:Calcineurin / Q000502:physiology; D018929:Cell Culture Techniques; D007679:Kidney Medulla / Q000166:cytology / Q000502:physiology; D008297:Male; D050778:NFATC Transcription Factors / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D044169:Receptors, Calcium-Sensing / Q000502:physiology*; D015398:Signal Transduction; D014158:Transcription, Genetic; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
chemical_list: D050778:NFATC Transcription Factors; D044169:Receptors, Calcium-Sensing; D014409:Tumor Necrosis Factor-alpha; D019703:Calcineurin