Counteracting effect of TRPC1-associated CA²⁺ influx on TNF-α-induced COX-2-dependent prostaglandin E² production in human colonic myofibroblasts

TNF-α-NF-κB signaling plays a central role in inflammation, apoptosis, and neoplasia. One major consequence of this signaling in the gut is increased production of prostaglandin E₂ (PGE₂) via cyclooxygenase-2 (COX-2) induction in myofibroblasts, which has been reported to be dependent on Ca²⁺. In this study, we explored a potential role of canonical transient receptor potential (TRPC) proteins in this Ca²⁺-mediated signaling using a human colonic myofibroblast cell line CCD-18Co. In CCD-18Co cell, treatment with TNF-α greatly enhanced Ca²⁺ influx induced by store depletion along with increased cell-surface expression of TRPC1 protein (but not of the other TRPC isoforms) and induction of a Gd³⁺-sensitive nonselective cationic conductance. Selective inhibition of TRPC1 expression by small interfering RNA (siRNA) or functionally effective TRPC1 antibody targeting the near-pore region of TRPC1 (T1E3) antagonized the enhancement of store-dependent Ca²⁺ influx by TNF-α, whereas potentiated TNF-α induced PGE2 production. Overexpression of TRPC1 in CCD-18Co produced opposite consequences. Inhibitors of NF-κB (curcumin, SN-50) attenuated TNF-α-induced enhancement of TRPC1 expression, store-dependent Ca²⁺ influx, and COX-2-dependent PGE₂ production. In contrast, inhibition of calcineurin-nuclear factor of activated T-cell proteins (NFAT) signaling by FK506 or NFAT Activation Inhibitor III enhanced the PGE₂ production without affecting TRPC1 expression and the Ca²⁺ influx. Finally, the suppression of store-dependent Ca²⁺ influx by T1E3 antibody or siRNA knockdown significantly facilitated TNF-α-induced NF-κB nuclear translocation. In aggregate, these results strongly suggest that, in colonic myofibroblasts, NF-κB and NFAT serve as important positive and negative transcriptional regulators of TNF-α-induced COX-2-dependent PGE₂ production, respectively, at the downstream of TRPC1-associated Ca²⁺ influx.