Abstract
The expression by T lymphocytes (T cells) of more than one of the functionally distinct subtypes of prostaglandin E2 (PGE2) receptors (Rs), designated EP1, EP2, EP3, and EP4 Rs, is a principal determinant of specificity and diversity of the immune effects of PGE2. The cultured line of human leukemic T cells, termed HSB.2, co-expresses a total of 7282 +/- 1805 EP3, EP4, and EP2 Rs per cell with a Kd of 3.7 +/- 1.4 nM (mean +/- S.E., n = 9). The EP3/EP1 R-selective agonist sulprostone, EP3/EP2/EP4 R-selective agonists M&B 28767 and misoprostol, and EP2 R-selective agonist butaprost but not the EP1 R-selective antagonist SC-19220 competitively inhibited the binding of [3H]PGE2 to HSB.2 cells. Stimulation of increases in the intracellular concentration of cyclic AMP ([cAMP]i) by PGE2, misoprostol, and butaprost and of increases in the intracellular concentration of calcium ([Ca2+]i) by PGE2 and sulprostone demonstrated the respective involvement of EP2/EP4 Rs and EP3 Rs in transduction of biochemical signals. Matrix metalloproteinase (MMP)-9 was identified by zymography and Western blots as the principal MMP secreted by HSB.2 cells. The cytosolic level and secretion of MMP-9 were increased maximally after 24 h of incubation of HSB.2 cells with 10(-8)-10(-6) M PGE2, sulprostone, M&B 28767, and misoprostol but not with 10(-6) M PGF2alpha, PGD2, PGI2, or butaprost, suggesting a principal dependence on EP3 Rs. That stimulation of MMP-9 secretion by PGE2 was not diminished in Ca2+-free medium but was suppressed significantly and dose-dependently by thapsigargin, an inhibitor of endomembrane Ca2+-ATPase, suggested that MMP-9 expression by HSB.2 cells is mediated by increases in [Ca2+]i attributable to release of Ca2+ from intracellular stores. The lack of effect of dibutyryl cAMP, forskolin, and SQ 22536, an adenylyl cyclase inhibitor, on MMP-9 secretion by HSB.2 cells argued against any role for cAMP-dependent mechanisms linked to EP2/EP4 Rs. Cycloheximide and actinomycin D, which respectively inhibited protein and RNA synthesis, suppressed basal and PGE2 induction of MMP-9 production by HSB.2 cells. Northern analysis indicated that PGE2 and sulprostone time-dependently increased expression of MMP-9 mRNA. Thus, stimulation of MMP-9 in HSB.2 T cells by PGE2 is attributable to [Ca2+]i-dependent EP3 R-mediation of increases in message transcription.
Highlights
prostaglandin E2 (PGE2) potently mediates and modulates cellular and humoral immune responses by stimulating or inhibiting the functions of many different types of immune cells [10]
The binding of [3H]PGE2 to HSB.2 cells was inhibited only by the highest concentration of the EP2 R-selective agonist butaprost (IC50 Ͼ 1 M) and not at all by the EP1 R-selective antagonist SC-19220 (Fig. 1B). These results suggest that HSB.2 cells express EP3 and EP4 Rs, fewer EP2 Rs, and no EP1 Rs
Gelatin zymographic analyses of the secretions of HSB.2 cells revealed that a range of concentrations of TG did not change basal levels of matrix metalloproteinase (MMP)-9 activities but dose-dependently suppressed the PGE2 stimulation of increases in MMP-9 activities (Fig. 4C). These results suggest that TG and PGE2 mobilize Ca2ϩ from common intracellular pools and that the PGE2 effects on MMP-9 in HSB.2 cells are mediated by changes in [Ca2ϩ]i attributable to mobilization of Ca2ϩ from intracellular stores
Summary
Materials—[5,6,8,11,12,14,15-3H]PGE2 (153 Ci/mmol, DuPont NEN), M&B 28767 (Rhone-Poulenc Rorer Research, Dagenham, Essex), sulprostone (Schering Pharmaceuticals, Berlin), PGE1, PGD2, PGF2␣, PGI2 (Upjohn Co., Kalamazoo, MI), SC 19220 and misoprostol (Searle, Skokie, IL), cAMP radioimmunoassay kit (DuPont NEN), mouse monoclonal IgG antibodies specific for human MMP-9, MMP-1, MMP-2, and MMP-3 (Oncogene Science, Cambridge, MA), 3-isobutyl-1-methylxanthine, 9-tetrahydro-2-furyl-adenosine (SQ 22536), H-89 and KT5720. Cells by Zymographic and Western Blot Analyses—Replicate pellets of 107 HSB. cells were washed three times with 40 ml of protein-free RPMI 1640 and incubated in 2 ml of protein-free Iscove’s/RPMI 1640 medium (1:1, v/v) containing 1 mM CaCl2 without and with 10Ϫ9–10Ϫ6 M PGE2, other prostanoids, or synthetic agonists at 37 °C in 5% CO2/95% air for up to 24 h. These conditions have been shown not to significantly change the total number of cells and the amounts of proteins secreted by HSB. cells. MMP-9 mRNA was identified by hybridization with the 1.1-kilobase pairs 32P-labeled MMP-9 cDNA probe in a Northern analysis under high stringency conditions
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