REGULATION OF EXPRESSION OF MATRIX METALLOPROTEINASE-9 IN EARLY HUMANT-CELLS OF THE HSB.2 CULTURED LINE BY THE EP(3) SUBTYPE OF PROSTAGLANDIN E(2) RECEPTOR

The expression by T lymphocytes (T cells) of more than one of the func tionally distinct subtypes of prostaglandin E(2) (PGE(2)) receptors (R s), designated EP(1), EP(2), EP(3), and EP(4) RS, is a principal deter minant of specificity and diversity of the immune effects of PGE(2). T he cultured line of human leukemic T cells, termed HSB.2, co-expresses a total of 7282 +/- 1805 EP(3), EP(4), and EP(2) Rs per cell with a K -d of 3.7 +/- 1.4 nM (mean +/- S.E., n = 9), The EP(3)/EP(1) R-selecti ve agonist sulprostone, EP(3)/EP(2)/EP(4) R-selective agonists M&B 287 67 and misoprostol, and EP(2) R-selective agonist butaprost but not th e EP(1) R-selective antagonist SC-19220 competitively inhibited the bi nding of [H-3]PGE(2) to HSB.2 cells, Stimulation of increases in the i ntracellular concentration of cyclic AMP ([cAMP](i)) by PGE(2), misopr ostol, and butaprost and of increases in the intracellular concentrati on of calcium ([Ca2+](i)) by PGE(2) and sulprostone demonstrated the r espective involvement of EP(2)/EP(4) Rs and EP(3) Rs in transduction o f 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 max imally after 24 h of incubation of HSB.2 cells with 10(-8)-10(-6) M PG E(2), sulprostone, M&B 28767, and misoprostol but not with 10(-6) M PG F(2 alpha), PGD(2), PGI(2), or butaprost, suggesting a principal depen dence on EP(3) Rs. That stimulation of MMP-9 secretion by PGE(2) was n ot diminished in Ca2+-free medium but was suppressed significantly and dose-dependently by thapsigargin, an inhibitor of endomembrane Ca2+-A TPase, suggested that MMP-9 expression by HSB.2 cells is mediated by i ncreases in [Ca2+](i) attributable to release of Ca2+ from intracellul ar stores, The lack of effect of dibutyryl cAMP, forskolin, and SQ 225 36, an adenylyl cyclase inhibitor, on MMP-9 secretion by HSB.2 cells a rgued against any role for cAMP-dependent mechanisms linked to EP(2)/E P(4) Rs, Cycloheximide and actinomycin D, which respectively inhibited protein and RNA synthesis, suppressed basal and PGE(2) induction of M MP-9 production by HSB.2 cells, Northern analysis indicated that PGE(2 ) and sulprostone time-dependently increased expression of MMP-9 mRNA, Thus, stimulation of MMP-9 in HSB.2 T cells by PGE(2) is attributable to [Ca2+](i)-dependent EP(3) R-mediation of increases in message tran scription.