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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 Ca2+ (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 Ca2+ 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 TNFmore » 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.« less

Authors:
; ; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
944776
Report Number(s):
PNNL-SA-51497
TRN: US200902%%944
DOE Contract Number:
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: American Journal of Physiology. Renal Physiology, 290(5):F1110-7; Journal Volume: 290; Journal Issue: 5
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; AUTOMOBILES; CYCLOSPORINE; GENES; HOMEOSTASIS; LUCIFERASE; PEPTIDES; PHOSPHATASES; PRODUCTION; PROMOTERS; TRANSCRIPTION; WATER; tumor necrosis factor; calcium-sensing receptor; loop of Henle; cyclosporine A

Citation Formats

abdullah, huda ismail, Pedraza, Paulina L., Hao, Shoujin, Rodland, Karin D., McGiff, John C., and Ferreri, Nicholas R. NFAT regulates calcium-sensing receptor-mediated TNF production. United States: N. p., 2006. Web. doi:10.1152/ajprenal.00223.2005.
abdullah, huda ismail, Pedraza, Paulina L., Hao, Shoujin, Rodland, Karin D., McGiff, John C., & Ferreri, Nicholas R. NFAT regulates calcium-sensing receptor-mediated TNF production. United States. doi:10.1152/ajprenal.00223.2005.
abdullah, huda ismail, Pedraza, Paulina L., Hao, Shoujin, Rodland, Karin D., McGiff, John C., and Ferreri, Nicholas R. Mon . "NFAT regulates calcium-sensing receptor-mediated TNF production". United States. doi:10.1152/ajprenal.00223.2005.
@article{osti_944776,
title = {NFAT regulates calcium-sensing receptor-mediated TNF production},
author = {abdullah, huda ismail and Pedraza, Paulina L. and Hao, Shoujin and Rodland, Karin D. and McGiff, John C. and Ferreri, Nicholas R.},
abstractNote = {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 Ca2+ (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 Ca2+ 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.},
doi = {10.1152/ajprenal.00223.2005},
journal = {American Journal of Physiology. Renal Physiology, 290(5):F1110-7},
number = 5,
volume = 290,
place = {United States},
year = {Mon May 01 00:00:00 EDT 2006},
month = {Mon May 01 00:00:00 EDT 2006}
}
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