NFAT regulates calcium-sensing receptor-mediated TNF production.
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.
Abdullah
Huda Ismail
HI
Dept. of Pharmacology, New York Medical College, Valhalla, NY 10595, USA.
Pedraza
Paulina L
PL
Hao
Shoujin
S
Rodland
Karin D
KD
McGiff
John C
JC
Ferreri
Nicholas R
NR
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*
D050778:NFATC Transcription Factors; D044169:Receptors, Calcium-Sensing; D014409:Tumor Necrosis Factor-alpha; D019703:Calcineurin