(The FASEB Journal. 2007;21:937.30)
© 2007 FASEB
Expression and function of NFAT5 in medullary thick ascending limb (mTAL) cells
shoujin hao,
Huda Abdullah,
John C. McGiff and
Nicholas R. Ferreri
pharmacology, New York Medical College, grasslands reservation, valhalla, NY, 10595
ABSTRACT
The distribution of distinct NFAT isoforms and their potential contribution to the regulation of tumor necrosis factor-alpha (TNF) gene expression in mTAL cells is unclear. RT-PCR analysis was performed on primary cultures of mouse mTAL cells and freshly isolated mTAL tubules to determine which NFAT isoforms are present in this nephron segment. Primer pairs were designed, based on published sequences for mouse NFAT1-5, to produce fragments of approximately 200 bp. Analysis of PCR products by gel electrophoresis and subsequent DNA sequencing indicated that cells and tubules contained mRNA for all five NFAT isoforms. The relative expression of NFAT isoforms was then determined using quantitative real-time RT-PCR. The data indicate that NFAT isoforms 1 and 5 are the predominant isoforms present in mTAL cells and tubules. Overexpression of NFAT5 in mTAL cells increased calcium sensing receptor (CaR)-mediated TNF production (n=3; p< 0.05). The functional implications of inhibiting NFAT activity in mTAL cells also were evaluated. Preincubation for 30 min with either cyclosporine A or the peptide inhibitor, VIVIT, attenuated the CaR- and TNF-dependent inhibitory effects on ouabain-sensitive O2 consumption in mTAL cells. Collectively, these data suggest that activation of NFAT5 is part of a TNF-dependent pathway that inhibits ion transport mechanisms in the mTAL after activation of CaR.
