This study examines the regulation of tumor necrosis factor-alpha (TNF-alpha) promoter activity by prostaglandin F-2alpha (PGF(2alpha)) in HEK cells stably expressing either the FPA or FPB prostanoid receptors. Cells were transiently transfected with a luciferase reporter plasmid under the control of a TNF-alpha promoter and luciferase activity was measured. In the absence of PGF(2alpha) basal TNT-alpha reporter gene activity is elevated in FPB cells as compared with FPA cells. This elevated basal activity is blocked by pretreatment with a Rho inhibitor, but not by pretreatment with an inhibitor of protein kinase C (PKC). TNF-alpha reporter activity in FPB cells is stimulated by PGF(2alpha) and this is decreased by pretreatment with a chelator of intracellular calcium or by a gap junction inhibitor. In FPB cells pretreatment with a Rho inhibitor combined with either a calcium chelator or a gap junction inhibitor decreases both basal and PGF(2alpha) stimulated TNF-alpha reporter activity. Interestingly post-treatment of FPB cells with an inhibitor of PKC decreased PGF(2alpha) stimulated TNF-alpha reporter gene activity even though pretreatment did not. It, therefore, appears that PGF(2alpha) stimulated TNF-alpha reporter activity in FPB cells is amplified by a Rho-dependent mechanism involving calcium, gap junctions, and PKC. These findings may help in understanding the function of the FPB isoform in the corpus luteum. (C) 2004 Elsevier Inc. All rights reserved.