Calcineurin-dependent TNF production via calcium sensing receptor (CaR) activation in medullary thick ascending limb (mTAL) cells

Primary cultures of rat mTAL cells express the G-protein coupled CaR, sensing changes in extracellular Ca2+ concentration that are transduced into several intracellular signals. Activation of this receptor increases expression and activity of cyclooxygenase-2 in a TNF-dependent manner, an interaction that regulates ion transport mechanisms in the TAL. TNF production has been linked to a calcineurin-dependent signaling pathway; however, activation of this pathway via CaR has not been described. Exposure of mTAL cells to Ca2+o (1.2mM) for 6 hr increased TNF production (control: 0.49±0.02; Ca2+: 2.26±0.06 pg TNF/μg protein). The CaR-selective agonist neomycin (100 μM) also increased TNF production. Transient transfection of mTAL cells with a dominant negative CaR (R796W) abolished Ca2+o- and neomycin-mediated TNF production in a concentration-dependent manner. These data indicate that the effects of extracellular Ca2+ and neomycin were dependent on signaling pathways initiated after stimulation of CaR. Additional evidence of TNF production via CaR-dependent signaling was provided, by demonstrating that inhibition of phospholipase C with U73122 (0.5 μM) or calmodulin kinase II with KN-62 (150 μM) abolished TNF production. Subsequently, CaR-mediated TNF production was inhibited by the calcineurin inhibitor cyclosporin A (CsA; 0.1 ng/ml) (control: 0.94±0.27; Ca2+: 2.25±0.21; CsA: 0.77±0.2; Ca2++CsA: 0.83±0.28 pg TNF/μg protein; p<0.001, n=4), implicating a role of downstream effectors of calcineurin in TNF gene transcription. Indeed, a peptide inhibitor (20 μM) of nuclear factor of activated T-cells (NF-AT) activation prevented CaR-mediated TNF production. Collectively, these data are the first to suggest that CaR-mediated TNF gene transcription in the mTAL is initiated via a Gq/11a signaling pathway that is calcineurin- and NF-AT-dependent. Characterization of the signaling pathways for CaR-dependent TNF production is crucial for understanding natriuretic/diuretic responses and metabolic disorders linked to CaR dysfunction.