Regulation of expression of matrix metalloproteinase-9 in early human T cells of the HSB.2 cultured line by the EP3 subtype of prostaglandin E2 receptor.

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 PGF2α, 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.

Prostaglandin E 2 (PGE 2 ) 1 is a product of the cyclooxygenation of arachidonic acid released from cellular phospholipids that potently mediates many biological functions in the cardiovascular, pulmonary, renal, endocrine, gastrointestinal, neural, reproductive, and immune systems (1,2). Cell surface expression of multiple functionally distinct subtypes of PGE 2 receptors (Rs) is a principal determinant of the diversity and specificity of cellular effects of PGE 2 . PGE 2 is recognized and transduces cellular effects specifically by interacting with PGE 2 Rs of at least four subtypes, designated the EP 1 , EP 2 , EP 3 , and EP 4 Rs. These subtypes of PGE 2 Rs differ in structure, ligand-binding properties, tissue distribution, and coupling to signal transduction pathways (2). All subtypes of PGE 2 Rs have recently been cloned and shown to be members of the G protein-coupled seven-transmembrane domain superfamily (1,2). EP 1 Rs mediate increases in the intracellular concentration of calcium ([Ca 2ϩ ] i ) (3). EP 2 and EP 4 Rs activate adenylyl cyclase via Gs and stimulate increases in the intracellular concentration of cAMP ([cAMP] i ) (4 -6). Multiple isoforms of EP 3 Rs not only inhibit adenylyl cyclase, resulting in a decrease in [cAMP] i elevated by forskolin or other agonists via G i , but also stimulate increases in [Ca 2ϩ ] i via G i /G o or G q (1,(7)(8)(9). Certain isoforms of the EP 3 R in nonhuman species also activate adenylyl cyclase via G s and transduce increases in [cAMP] i (8,9). PGE 2 potently mediates and modulates cellular and humoral immune responses by stimulating or inhibiting the functions of many different types of immune cells (10). At physiological concentrations, PGE 2 enhances elements of macrophage differentiation but inhibits functional activation and enhances B cell production of IgG1 and IgE while inhibiting that of IgM (11). Of central importance in most host defense and autoimmune responses is that PGE 2 inhibits T cell proliferation, differentiation, expression of membrane Rs, secretion of diverse cytokines, cytotoxicity, and other specific effector functions in cellular immune reactions (10). Some PGE 2 effects on T cells appear to be subset-selective, as for stimulation of the proliferative responses of a suppressor subset of T cells and concurrent suppression of the responses of a subset of helper T cells (10). PGE 2 effects on T cells have been attributed almost exclusively to increases in [cAMP] i , which are transduced by EP 2 and/or EP 4 Rs. Expression of the EP 3 subtype of PGE 2 Rs by T cells at a level capable of altering T cell functions has not been described nor have the functional consequences of EP 3 R-mediated signaling of T cells. The cultured line of human leukemic T cells, termed HSB.2, is an early "double negative" thymocyte bearing CD2 and CD7 but not CD3, CD4, or CD8 (12,13). It is now shown that HSB.2 T cells express predominantly the EP 3 subtype of PGE 2 R and that PGE 2 stimulates increases in HSB.2 cellular content and secretion of matrix metalloproteinase (MMP)-9 by EP 3 R-mediated and [Ca 2ϩ ] i -dependent enhancement of transcription of mRNA encoding MMP-9.
Culture of HSB.2 Cells-Human leukemic T cells of the HSB.2 line (13) (obtained from American Type Culture Collection) were cultured in RPMI 1640 medium (University of California, San Francisco, Cell Culture Facility) with 15 mM HEPES, 10% (v/v) fetal bovine serum (Hyclone Laboratories, Inc., Logan, UT), 100 units/ml of penicillin and 100 g/ml of streptomycin (complete RPMI medium). Cultures were maintained at 37°C in a humidified atmosphere of 5% CO 2 /95% air at a density of 0.4 -1.8 ϫ 10 6 /ml by changing medium every 1-3 days.

Quantification of HSB.2 Cell Binding of [ 3 H]PGE 2 and cAMP
Responses to PGE 2 -Replicate suspensions of 10 6 HSB.2 cells in 100 l of binding buffer composed of 100 mM NaCl-10 mM KH 2 PO 4 (pH 6.0) with 1 mg/ml of recrystallized ovalbumin were incubated with 3 nM [ 3 H]PGE 2 without and with 10 Ϫ10 -10 Ϫ6 M nonradioactive PGE 2 , other synthetic prostanoids and agonists for 2 h at room temperature. The binding mixtures then were applied directly to GF/C filters (Whatman) pretreated with 0.3% polyethylenimine for rapid vacuum filtration (Hoeffer Scientific, San Francisco, CA). The filters then were washed with 12 ml of cold binding buffer and assessed for radioactivity in an LC5801 scintillation counter (Beckman). Binding data were analyzed by the LIGAND computer program (14).
Suspensions of 10 6 HSB.2 cells in 100 l of Hanks' balanced salt solution (HBSS) with 1 mg/ml of ovalbumin and 20 mM HEPES (pH 7.4) were preincubated with 1 mM 3-isobutyl-1-methylxanthine, a cAMP phosphodiesterase inhibitor, at 37°C for 10 min. 10 Ϫ10 -10 Ϫ6 M PGE 2 and other agonists then were added, and the incubation was continued at 37°C for 10 min. The reaction was stopped, and the cells were lysed by the addition of 250 l of ice-cold 100% ethanol at Ϫ20°C. cAMP in 15,000 ϫ g supernatants of ethanol solutions was quantified by radioimmunoassay, according to the manufacturer's protocol.

Measurement of Changes in [Ca 2ϩ ] i in HSB.2 Cells Induced by PGE 2 -
The calcium-sensitive fluorescent dye fura-2 was used to quantify [Ca 2ϩ ] i , as described in a similar system (7). HSB.2 cells were incubated in the dark at 37°C for 30 min at 10 7 /3 ml of Ca 2ϩ -and Mg 2ϩ -free HBSS containing 0.1% ovalbumin, 25 mM HEPES, and 2.5 M fura-2 acetoxymethylester. The fura-2 acetoxymethylester-loaded HSB.2 cells were washed two times with Ca 2ϩ -and Mg 2ϩ -free HBSS and resuspended in normal HBSS containing 1 mM Ca 2ϩ or Ca 2ϩ -and Mg 2ϩ -free HBSS with 1 mM EGTA at a concentration of 10 6 HSB.2 cells/ml. Replicate 2-ml aliquots of the suspension were placed in a cuvette with a stirring bar and warmed to 37°C for 5 min before addition of PGE 2 , other agonists/antagonist, ionomycin, and thapsigargin. Fura-2 fluorescence was recorded with a Perkin-Elmer model LS-50B luminometer using excitation and emission wavelengths of 340/380 nm and 510 nm, respectively. Maximum fluorescence (R max ) and minimum fluorescence (R min ), respectively, were determined after addition of 100 l of 2% (v/v) Triton X-100 in distilled H 2 O and subsequent addition of 100 l of 250 mM Tris buffer (pH 10) with 250 mM EGTA. A computer program calculated [Ca 2ϩ ] i from the ratio of fluorescence intensity (340/380 nm).
Characterization of MMPs of HSB.2 Cells by Zymographic and Western Blot Analyses-Replicate pellets of 10 7 HSB.2 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 CaCl 2 without and with 10 Ϫ9 -10 Ϫ6 M PGE 2 , other prostanoids, or synthetic agonists at 37°C in 5% CO 2 /95% air for up to 24 h. These conditions have been shown not to significantly change the total num-ber of cells and the amounts of proteins secreted by HSB.2 cells. The suspensions then were centrifuged at 15,000 ϫ g for 5 min. The supernatants were collected, and the cell pellets were lysed with 0.5% Triton X-100 in distilled H 2 O. Replicate 3-g protein aliquots of the 15,000 ϫ g supernatants that represented the secretions from about 1.5 ϫ 10 5 HSB.2 cells, and 25-g protein aliquots of the Triton extracts were electrophoresed to resolve secreted and intracellular MMPs, respectively. Quantification of enzymatic activity by zymography was performed in a nonreducing 10% SDS-polyacrylamide gel, which had been co-polymerized with 1 mg/ml of type A porcine skin-derived gelatin as described (15). The gels then were incubated in 2.5% Triton X-100 and then in 50 mM Tris-HCl/50 mM NaCl/5 mM CaCl 2 for 24 h at 37°C to permit digestion of gelatin by MMPs. After staining undigested protein with Coomassie Blue, the decrease in staining of each band that reflected protease activity was determined by densitometry with a Scan-Jet IIC and quantified by NIH Image 1.41 software. All results were expressed as relative percentages of untreated controls.
Replicate 5-g protein aliquots of the secreted and cytosolic supernatants were subjected to 10% SDS-polyacrylamide gel electrophoresis for quantification by immunoreactivity, and the proteins resolved were then transferred by electroblotting to a 0.45-m pore nitrocellulose membrane (Hybond, Amersham Corp.). The blots were developed with 1 g/ml of mouse monoclonal IgG antibodies specific for human MMP-9, MMP-2, MMP-1, and MMP-3, and then a 1/2,000 dilution of horse radish peroxidase-labeled sheep anti-mouse IgG (Amersham Corp.). Luminescence analysis was performed according to the manufacturer's protocol (ECL, Amersham Corp.), and the MMP-9 was quantified by densitometry.
Reverse Transcription-Polymerase Chain Reaction and Northern Blot Analyses-Total RNA of HSB.2 cells was isolated by TRIzol Reagent kit (Life Technologies, Inc.). First strand cDNAs were synthesized from HSB.2 cell total RNA with random hexamer primers and Superscript II reverse transcriptase (Life Technologies, Inc.) and were used as templates for polymerase chain reaction with 36 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min. The MMP-9 primers were 5Ј-AGACCTGAGAACCAATCT (upstream) and 5Ј-GGCACTGAGGAAT-GATCTAA (downstream) (16). The resultant reverse transcription-polymerase chain reaction products were analyzed by electrophoresis in a 2% agarose gel, and the product of 1.1 kilobase pairs of MMP-9 cDNA was extracted and purified from agarose gels and 32 P-labeled for Northern blot analyses.
Replicate pellets of 10 8 HSB.2 cells were washed and incubated in 20 ml of protein-free Iscove's/RPMI 1640 medium (1:1, v/v) at 37°C for 24 h. 10 Ϫ6 M PGE 2 or sulprostone was added to HSB.2 cell incubation at 24, 12, 4, and 1 h before harvesting the cells. Poly(A ϩ ) RNA then was isolated from the control and treated HSB.2 cells by the Fast Track kit (Invitrogen, San Diego, CA). Replicate aliquots of 7 g of poly(A ϩ ) RNA were electrophoresed and transferred to a nylon membrane as described (17). MMP-9 mRNA was identified by hybridization with the 1.1-kilobase pairs 32 P-labeled MMP-9 cDNA probe in a Northern analysis under high stringency conditions.

Coexpression of EP 3 , EP 4 , and EP 2 Rs by HSB.2 Cells-
LIGAND program analysis of the results of studies of competitive inhibition of specific binding of [ 3 H]PGE 2 to HSB.2 cells by unlabeled PGE 2 revealed a K d of 3.7 Ϯ 1.4 nM (mean Ϯ S.E., n ϭ 9) and a B max of 12.1 Ϯ 3.0 fmol/10 6 cells (7282 Ϯ 1805 PGE 2 Rs/cell). The specific binding of [ 3 H]PGE 2 to HSB.2 cells was inhibited competitively by nonradioactive synthetic prostanoids with a rank order of potency of PGE 2 ϭ PGE 1 Ͼ Ͼ PGF 2␣ ϭ PGD 2 Ͼ PGI 2 (Fig. 1A). The specific binding of [ 3 H]PGE 2 also was inhibited by the EP 3 /EP 1 R-selective agonist sulprostone (IC 50 ϭ 2.3 Ϯ 0.6 nM) (mean Ϯ S.E., n ϭ 6), the EP 3 /EP 2 /EP 4 R-selective agonists M&B 28767 (IC 50 ϭ 4.0 Ϯ 0.4 nM) and misoprostol (IC 50 ϭ 44 Ϯ 1.3 nM) (Fig. 1B). The binding of [ 3 H]PGE 2 to HSB.2 cells was inhibited only by the highest concentration of the EP 2 R-selective agonist butaprost (IC 50 Ͼ 1 M) and not at all by the EP 1 R-selective antagonist SC-19220 (Fig. 1B) (Table I). Furthermore, the elevations of [Ca 2ϩ ] i in response to PGE 2 and sulprostone were not changed by elimination of extracellular Ca 2ϩ (Table I). However, preincubation for 10 min of HSB.2 cells with 10 Ϫ7 M thapsigargin, an inhibitor of endomembrane Ca 2ϩ -ATPase that depletes intracellular storage of Ca 2ϩ (18 -20), elevated the basal level of [Ca 2ϩ ] i but suppressed the increases of [Ca 2ϩ ] i in response to 10 Ϫ7 M PGE 2 (Table I). These results suggest that PGE 2 -elicited increases in [Ca 2ϩ ] i are attributable to release of Ca 2ϩ from intracellular stores linked to EP 3 R signals.
Enhancement by PGE 2 of the Cytosolic Level and Secretion of MMP-9 by HSB.2 Cells-Gelatin zymographic analyses of the secretion and extracts of HSB.2 cells revealed one vastly predominant gelatinolytic activity of 92 kDa (Fig. 3, A and B) that corresponds to the molecular mass of MMP-9 (21). Neither the related MMP-2 (72 kDa) gelatinase nor other MMPs, such as MMP-1 and MMP-3 (55-59 kDa), were detected. Secreted MMP-9 activity was detected as early as 4 h, reached a maximal level at 24 h, and persisted through 48 h (data not shown). At 24 h, 10 Ϫ9 , 10 Ϫ8 , 10 Ϫ7 , and 10 Ϫ6 M PGE 2 increased MMP-9 activities in a concentration-dependent manner ranging from means of 1.1-3.0-fold higher than unstimulated control cells (Fig. 3A). Cytosolic MMP-9 activities also were augmented by PGE 2 (Fig. 3B) with a pattern similar to that of the secreted MMP-9, suggesting that PGE 2 increases both the synthesis and secretion of MMP-9 protein. Western blot analyses of the secreted (Fig. 3C) and cytosolic (data not shown) MMP from HSB.2 cells confirmed the identity of MMP-9 and the stimulatory effect of PGE 2 on the level of MMP-9 protein. When the same amount of total protein was used for each Western blot analysis, the amount of MMP-9 immunoreactive protein was increased by PGE 2 dose-dependently, as compared with the control in buffer alone (Fig. 3C), implying that PGE 2 stimulated the levels of MMP-9 activity in part by increasing the cytosolic and secreted amounts of MMP-9 (Fig. 3, A and B). Moreover, indomethacin at 10 Ϫ5 M, which suppresses endogenous PGE 2 synthesis, did not change the basal MMP-9 level, suggesting either that HSB.2 cells do not produce relevant amounts of PGE 2 or that the basal MMP-9 activity of HSB.2 cells is independent of endogenous PGE 2 .

Mediation of PGE 2 Effect on MMP-9 by an EP 3 R-dependent Mechanism Involving Increases in [Ca 2ϩ ] i from Intracellular
Stores-PGE 1 and the EP 3 R-directed agonists sulprostone, M&B 28767, and misoprostol enhanced MMP-9 activity of HSB.2 cells to the same extent as PGE 2 or higher at optimal concentrations, whereas there was no effect with the other synthetic prostanoids PGF 2␣ , PGD 2 , and PGI 2 nor the EP 2 R-selective agonist butaprost (Fig. 4A). The dependence of MMP-9 enhancement on EP 3 R-directed effects of both natural and pharmacological agonists confirms the specificity of coupling of MMP-9 responses to the EP 3 R subtype. Neither dibutyryl-cAMP nor the adenylyl cyclase stimulator forskolin or inhibitor SQ 22536 changed significantly the activity of MMP-9 (data not shown), diminishing the possibility that the effect of PGE 2 is through a cAMP-dependent mechanism.
When HSB.2 cells were washed and incubated with proteinand Ca 2ϩ -free medium containing 1 mM EGTA, we observed that HSB.2 cell growth was inhibited by 62 Ϯ 12% (mean Ϯ S.E., n ϭ 3) after 24 h when compared with that in medium containing 1 mM Ca 2ϩ . Consistently, the basal and PGE 2 -stimulated levels of MMP-9 detected in Ca 2ϩ -free medium were lower than that in medium with 1 mM Ca 2ϩ (Fig. 4). However, PGE 2 still significantly stimulated MMP-9 secretion in Ca 2ϩfree medium when compared with the control level without PGE 2 (Fig. 4B), suggesting that PGE 2 enhancement of secretion of MMP-9 is independent of extracellular Ca 2ϩ . Western blots also showed that PGE 2 dose-dependently increased intracellular expression of MMP-9 by HSB.2 cells in Ca 2ϩ -free me-dium (data not shown). Ionomycin, an ionophore that increases the permeability of plasma membrane to divalent cations, such as Ca 2ϩ , evoked increases in [Ca 2ϩ ] i of HSB.2 cells (in buffer with 1 mM Ca 2ϩ ) that attained mean maxima (n ϭ 4) of 1.1-, 6-, and 100-fold at respective concentrations of 10 Ϫ8 , 10 Ϫ7 , and 10 Ϫ6 M. However, when HSB.2 cells were incubated with 10 Ϫ9 -10 Ϫ6 M ionomycin in medium containing 1 mM Ca 2ϩ , neither basal nor PGE 2 -stimulated increases in MMP-9 secretion was significantly affected (data not shown), ruling out the possibility that increases in Ca 2ϩ influx mediate the effect of PGE 2 on MMP-9.
To investigate further whether EP 3 R-dependent increases in cytosolic and secreted MMP-9 are mediated by increases in [Ca 2ϩ ] i from intracellular stores, we preincubated HSB.2 cells with thapsigargin (TG), an inhibitor of endomembrane Ca 2ϩ -ATPase that depletes intracellular storage of Ca 2ϩ (18 -20) (Table I). Gelatin zymographic analyses of the secretions of HSB.2 cells also revealed that a range of concentrations of TG did not change basal levels of MMP-9 activities but dose-dependently suppressed the PGE 2 stimulation of increases in MMP-9 activities (Fig. 4C). These results suggest that TG and PGE 2 mobilize Ca 2ϩ from common intracellular pools and that the PGE 2 effects on MMP-9 in HSB.2 cells are mediated by changes in [Ca 2ϩ ] i attributable to mobilization of Ca 2ϩ from intracellular stores. PGE 2 Enhancement of Transcription of MMP-9 mRNA-The increases in cytosolic level and secretion of MMP-9 induced by PGE 2 were completely blocked by co-treatment of HSB.2 cells with 5-50 g/ml of the protein synthesis inhibitor cycloheximide (data not shown), indicating that PGE 2 effect requires de novo MMP-9 protein synthesis by HSB.2 cells. When HSB.2 cells were incubated for 24 h with both PGE 2 and 0.1-1 g/ml of actinomycin D, an inhibitor of cellular RNA synthesis, previously observed increases in cytosolic and secreted MMP-9 were completely prevented (data not shown), suggesting a requirement for mRNA synthesis in the PGE 2 effect on MMP-9. This transcriptional regulatory mechanism was confirmed by the increase of MMP-9 mRNA level by PGE 2 treatment. Northern analysis of HSB.2 cell poly(A ϩ ) RNA, by hybridization with a cDNA probe specific for human MMP-9 showed one predominant transcript of 2.8 kilobase pairs that corresponds in size to mRNA encoding MMP-9 (Fig. 5A). MMP-9 mRNA was expressed constitutively at a low level in HSB.2 cells, and the content was significantly increased by PGE 2 . PGE 2 increased the MMP-9 mRNA level with a time dependence characterized by slight (1.8-fold) enhancement at 4 h and a mean maximal increase of 3.8-fold at 12 h that persisted for up to 24 h (Fig.  5A). The enhancement by PGE 2 of MMP-9 mRNA was mimicked by the EP 3 R-directed agonist sulprostone (Fig. 5A), which confirms an EP 3 R specificity of the effect of PGE 2 on MMP-9 mRNA. Northern analysis of the same blot with a probe specific for glyceraldehyde-3-phosphate dehydrogenase, a constitutively expressed mRNA, exhibited a similar intensity for all of the samples from different times of incubation of HSB.2 cells with PGE 2 , sulprostone, or buffer alone (Fig. 5B).

DISCUSSION
The expression by T cells of multiple functionally distinct subtypes of PGE 2 Rs, designated EP 1 , EP 2 , EP 3 , and EP 4 Rs, is a principal determinant of specificity and diversity of the immune effects of PGE 2 (1,2). Regulation of T cell functions was not attributed previously to EP 3 Rs. We now show that the cultured line of human leukemic T cells, termed HSB.2, coexpresses a total of 7282 Ϯ 1805 EP 3 , EP 4 , and EP 2 Rs per cell with a K d of 3.7 Ϯ 1.4 nM. HSB.2 T cells differ from blood and lymphoid tissue T cells in expressing predominantly EP 3 Rs and lower levels of EP 2 Rs and EP 4 Rs. The EP 3 /EP 1 R-selective agonist sulprostone and EP 3 /EP 2 /EP 4 R-selective agonists M&B 28767 and misoprostol competitively inhibited the binding of [ 3 H]PGE 2 to HSB.2 cells (Fig. 1). In contrast, the EP 2 R-selective agonist butaprost inhibited [ 3 H]PGE 2 binding much less at only the highest concentration of 10 Ϫ6 M, and the EP 1 R-selective antagonist SC-19220 did not alter [ 3 H]PGE 2 binding (Fig. 1). These results indicated predominant expression of EP 3 Rs, fewer EP 4 Rs, a much lower level of EP 2 Rs, and no detectable EP 1 Rs in HSB.2 cells.
Assessment of adenylyl cyclase signaling revealed that PGE 2 , misoprostol, and a higher concentration of butaprost evoked increases in [cAMP] i in HSB.2 cells (Fig. 2), confirming expression of functional EP 4 Rs and EP 2 Rs. The isoforms of EP 3 Rs in some nonhuman species that transduce increases in [cAMP] i (8,9) were not detected in HSB.2 cells, because the EP 3 R-selective agonist sulprostone did not stimulate increases in [cAMP] i (Fig. 2). This is consistent with our previous finding that none of the human EP 3 R isoforms mediated an increase in [cAMP] i (7). The increases in [Ca 2ϩ ] i by PGE 2 and the EP 3 /EP 1 R-selective agonist sulprostone and the failure of a maximal concentration of the EP 1 R-selective antagonist SC-19220 to dampen increases in [Ca 2ϩ ] i evoked by sulprostone (Table I)  A family of MMPs is the principal physiological system that degrades diverse components of extracellular matrix (21). In human blood T cells and some human T lymphoblastoma cells, PGE 2 stimulates surface expression and secretion of MMPs-2, -3, and -9, which create channels in the basement membrane required to admit migrating blood T cells and Tsup-1 cells (15,22). Using HSB.2 cells as a T cell model, we thus examined the possibility that EP 3 Rs mediate regulation of MMPs in T cells responding to PGE 2 and the biochemical signaling mechanisms by which EP 3 Rs transduce PGE 2 -evoked increases in MMPs. We demonstrated that stimulation of MMP-9 in HSB.2 cells by PGE 2 is attributable to [Ca 2ϩ ] i -dependent EP 3 R mediation of increases in message transcription.
It has been reported that treatment of HSB.2 cells with the tumor promoter 12-O-tetradecanoylphorbol 13-acetate elicits secretion of MMP-9 activity (23). We now confirm by zymography and Western blots that MMP-9 is the exclusive MMP secreted constitutively by HSB.2 cells. Zymographic analyses revealed that the cytosolic level and secretion of MMP-9 were increased maximally after 24 h of incubation of HSB.2 cells with 10 Ϫ7 -10 Ϫ6 M PGE 2 . Parallel Western blot analyses showed that PGE 2 stimulated MMP-9 activity in part by increasing both MMP-9 protein expression and secretion (Fig. 3). PGE 1 but not PGF 2␣ , PGD 2 , or PGI 2 enhanced MMP-9 activity of HSB.2 cells to the same extent as PGE 2 , reflecting PGE 2 R specificity on MMP-9 and the similar preference of PGE 2 Rs for PGE 2 and PGE 1 , which was observed in other immune cells  (24). That the EP 3 R-directed agonists sulprostone, M&B 28767, and misoprostol enhanced MMP-9 activity of HSB.2 cells to the same extent as PGE 2 or higher, whereas the EP 2 R-selective agonist butaprost did not (Fig. 4), supported the critical involvement of EP 3 Rs in stimulation of HSB.2 T cell MMP-9 by PGE 2 .
The potent effects of PGE 2 on MMP-9 have been attributed principally to increases in [cAMP] i (25)(26)(27)(28), which are presumably transduced by EP 2 and/or EP 4 Rs. However, neither dibutyryl-cAMP nor the adenylyl cyclase stimulator forskolin and inhibitor SQ 22536 affected the cytosolic or secreted levels of MMP-9 in HSB.2 cells, confirming that [cAMP] i is not the mediator for effect of PGE 2 on MMP-9 expression in HSB.2 T cells. This is consistent with the result that the EP 2 R-selective agonist butaprost did not affect MMP-9 activity of HSB.2 cells. Thus the effect of PGE 2 on MMP-9 of HSB.2 cells is not mediated by EP 2 Rs or EP 4 Rs.
The finding that PGE 2 stimulation of MMP-9 was not changed by elimination of extracellular Ca 2ϩ but was suppressed by pretreatment with the Ca 2ϩ -ATPase inhibitor thapsigargin, which depletes intracellular Ca 2ϩ (18 -20) (Fig. 4), indicated the critical role of release of Ca 2ϩ from intracellular stores in EP 3 R mediation of MMP-9 secretion. Increases in [Ca 2ϩ ] i appear to be necessary but insufficient for the effect of PGE 2 on MMP-9 in HSB.2 cells, because incubation of the cells with 10 Ϫ9 -10 Ϫ6 M ionomycin in medium containing 1 mM Ca 2ϩ did not mimic a PGE 2 effect on MMP-9, although ionomycin evoked increases in [Ca 2ϩ ] i of HSB.2 cells by a maximal 100fold enhancement at 10 Ϫ6 M. Other unidentified signals that were simultaneously evoked by PGE 2 binding to EP 3 Rs along with the increases of [Ca 2ϩ ] i released from intracellular stores also may be required for the PGE 2 effect. IP 3 formation in HSB.2 cells was not changed significantly by up to 10 Ϫ6 M PGE 2 (data not shown), suggesting either that EP 3 Rs transduced production of small amounts of IP 3 that could not be detected in whole-cells assays but was capable of evoking increases in [Ca 2ϩ ] i in HSB.2 cells or that the increases in [Ca 2ϩ ] i from intracellular stores result from an IP 3 -independent mechanism. It was shown that TG releases stored Ca 2ϩ from intracellular pools without production of IP 3 (20,18), but there is a large degree of overlap between the IP 3 -and TG-sensitive Ca 2ϩ pools (20). We suggest in this study that PGE 2 and TG mobilize Ca 2ϩ from common intracellular pools. Thus, the PGE 2 -mediated increase in [Ca 2ϩ ] i via EP 3 Rs in HSB.2 cells may be from both TG-sensitive and IP 3 -sensitive pools.
All subtypes of PGE 2 Rs are members of the G proteincoupled seven-transmembrane domain superfamily. We have shown previously that elevation of [Ca 2ϩ ] i mediated by all human EP 3 R isoforms was partially blocked by pertussis toxin treatment (7). The PGE 2 -enhanced effect on cytosolic and secreted MMP-9 in HSB.2 cells also was partially blocked by 24 h of co-treatment with 100 ng/ml pertussis toxin without a change in the basal level of MMP-9 (data not shown), implying involvement of both pertussis toxin-sensitive and pertussis toxin-insensitive G proteins in signal transduction. LPS-triggered secretion of MMP-9 in macrophages involves both protein kinase C and protein-tyrosine kinase (29). src-related proteintyrosine kinase plays a role in MMP-9 transcriptional activation (30). 12-O-Tetradecanoylphorbol 13-acetate-enhanced MMP-9 activity in HSB cells also is mediated through activation of protein kinase C (23). However, the PGE 2 effect on MMP-9 in HSB.2 T cells was independent of protein kinase C, protein-tyrosine kinase, protein kinase A, and protein kinase G. Co-treatment of HSB.2 cells with the protein kinase C inhibitors staurosporine or calphostin C at concentrations of 10 Ϫ10 -10 Ϫ7 M, with protein-tyrosine kinase inhibitors genistein and tyrphostin at concentrations of 10 Ϫ7 -10 Ϫ4 M, or with protein kinase A/protein kinase G inhibitors H-89 and KT5720 at concentrations of 10 Ϫ9 -10 Ϫ6 M did not change the basal and PGE 2 elevated level of MMP-9 (data not shown). Further work is needed to elucidate biochemical signal transduction mechanisms by which PGE 2 affects MMP-9 in HSB.2 T cells.
It has been demonstrated that MMP-1, -3, and -9 expression is regulated at the transcriptional level by factors including 12-O-tetradecanoylphorbol 13-acetate, tumor necrosis factor ␣, epidermal growth factor, platelet-derived growth factor, nerve growth factor, interleukin-1, transforming growth factor-␤, progesterone, and corticosteroids (21,22). We now show that PGE 2 enhances MMP-9 expression by increasing transcription as well. Prevention of PGE 2 -induced increases in the cytosolic level and secretion of MMP-9 in HSB.2 cells by both protein synthesis inhibitor cycloheximide and RNA synthesis inhibitor actinomycin D suggested a requirement for de novo protein synthesis and transcription. Northern analyses revealed enhancement of the level of MMP-9 mRNA after 12-24 h of PGE 2 treatment (Fig. 5). The ability of the EP 3 R-directed agonist sulprostone to mimic a PGE 2 stimulatory effect on MMP-9 mRNA (Fig. 5) supports the hypothesis that EP 3 Rs signal predominantly at the transcriptional level. Thus, stimulation of MMP-9 in HSB.2 T cells by PGE 2 is attributable to [Ca 2ϩ ] i -dependent EP 3 R-mediation of increases in message transcription.
The HSB.2 T cell (12) is at an early thymocyte stage (13) and expresses predominantly EP 3 and EP 4 Rs, of which the former is the principal transducer of PGE 2 effects on MMP-9. However, mature human blood CD4 ϩ and CD8 ϩ T cells express principally EP 4 Rs and only the CD8 ϩ subset bears a prominent number of EP 3 Rs. 2 In human blood T cells of mixed CD4 ϩ and CD8 ϩ composition, PGE 2 stimulates surface expression and secretion of MMP-2 and -3, as well as MMP-9 (15), but the PGE 2 R subtype dependence has not been defined. Whether HSB.2 cell is representative of human normal early thymocytes and whether the EP 3 Rs of CD8 ϩ T cells transduce increases in MMP-9 content and secretion remain to be evaluated in the corresponding population of T cells.