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• W2155256555 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 10−8-10−6M PGE2, sulprostone, M&B 28767, and misoprostol but not with 10−6M 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. 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 10−8-10−6M PGE2, sulprostone, M&B 28767, and misoprostol but not with 10−6M 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. INTRODUCTIONProstaglandin E2 (PGE2) 1The abbreviations used are: PGE2prostaglandin E2RsreceptorsMMPmatrix metalloproteinaseHBSSHanks', balanced salt solutionG proteinheterotrimeric guanine nucleotide-binding proteinIP3inositol triphosphatePTpertussis toxinTGthapsigargin. 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 (1Coleman, R. A., Kennedy, I., Humphrey, P. P. A., Bunce, K., Lumley, P., Emmet, J. C., (eds) (1990) Comprehensive Medicinal Chemistry, Vol 3, p. 643, Pergammon Press, Oxford.Google Scholar, 2Coleman R.A. Smith W.L. Narumiya S. Pharmacol. Rev. 1994; 46: 205-229PubMed Google Scholar). Cell surface expression of multiple functionally distinct subtypes of PGE2 receptors (Rs) is a principal determinant of the diversity and specificity of cellular effects of PGE2. PGE2 is recognized and transduces cellular effects specifically by interacting with PGE2 Rs of at least four subtypes, designated the EP1, EP2, EP3, and EP4 Rs. These subtypes of PGE2 Rs differ in structure, ligand-binding properties, tissue distribution, and coupling to signal transduction pathways (2Coleman R.A. Smith W.L. Narumiya S. Pharmacol. Rev. 1994; 46: 205-229PubMed Google Scholar). All subtypes of PGE2 Rs have recently been cloned and shown to be members of the G protein-coupled seven-transmembrane domain superfamily (1Coleman, R. A., Kennedy, I., Humphrey, P. P. A., Bunce, K., Lumley, P., Emmet, J. C., (eds) (1990) Comprehensive Medicinal Chemistry, Vol 3, p. 643, Pergammon Press, Oxford.Google Scholar, 2Coleman R.A. Smith W.L. Narumiya S. Pharmacol. Rev. 1994; 46: 205-229PubMed Google Scholar). EP1 Rs mediate increases in the intracellular concentration of calcium ([Ca2+]i) (3Watabe A. Sugimoto Y. Honda A. Irie A. Namba T. Negishi M. Ito S. Narumiya S. Ichikawa A. J. Biol. Chem. 1993; 268: 20175-20178Abstract Full Text PDF PubMed Google Scholar). EP2 and EP4 Rs activate adenylyl cyclase via Gs and stimulate increases in the intracellular concentration of cAMP ([cAMP]i) (4Regan J.W. Barley T.J. Pepperl D.J. Pierce K.L. Bogardus A.M. Donello J.E. Fairbairn C.E. Kedzie K.M. Woodward D.F. Gil D.W. Mol. Pharmacol. 1994; 46: 213-220PubMed Google Scholar, 5An S. Yang J. Xia M. Goetzl E.J. Biochem. Biophys. Res. Commun. 1993; 197: 263-270Crossref PubMed Scopus (132) Google Scholar, 6Honda A. Sugimoto Y. Namba T. Watabe A. Irie A. Negishi M. Narumiya S. Ichikawa A. J. Biol. Chem. 1993; 268: 7759-7762Abstract Full Text PDF PubMed Google Scholar). Multiple isoforms of EP3 Rs not only inhibit adenylyl cyclase, resulting in a decrease in [cAMP]i elevated by forskolin or other agonists via Gi, but also stimulate increases in [Ca2+]i via Gi/Go or Gq (1Coleman, R. A., Kennedy, I., Humphrey, P. P. A., Bunce, K., Lumley, P., Emmet, J. C., (eds) (1990) Comprehensive Medicinal Chemistry, Vol 3, p. 643, Pergammon Press, Oxford.Google Scholar, 7An S. Yang J. So S.W. Zeng L. Goetzl E.J. Biochemistry. 1994; 33: 14496-14502Crossref PubMed Scopus (106) Google Scholar, 8Namba T. Sugimoto Y. Negishi M. Irie A. Ushikubi F. Kakizuka A. Ito S. Ichikawa A. Narumiya S. Nature. 1993; 365: 166-170Crossref PubMed Scopus (515) Google Scholar, 9Irie A. Sugimoto Y. Namba T. Harazono A. Honda A. Watabe A. Negishi M. Narumiya S. Ichikawa A. Eur. J. Biochem. 1993; 217: 313-318Crossref PubMed Scopus (164) Google Scholar). Certain isoforms of the EP3 R in nonhuman species also activate adenylyl cyclase via Gs and transduce increases in [cAMP]i (8Namba T. Sugimoto Y. Negishi M. Irie A. Ushikubi F. Kakizuka A. Ito S. Ichikawa A. Narumiya S. Nature. 1993; 365: 166-170Crossref PubMed Scopus (515) Google Scholar, 9Irie A. Sugimoto Y. Namba T. Harazono A. Honda A. Watabe A. Negishi M. Narumiya S. Ichikawa A. Eur. J. Biochem. 1993; 217: 313-318Crossref PubMed Scopus (164) Google Scholar).PGE2 potently mediates and modulates cellular and humoral immune responses by stimulating or inhibiting the functions of many different types of immune cells (10Goetzl E.J. An S. Smith W.C. FASEB J. 1995; 9: 1051-1058Crossref PubMed Scopus (246) Google Scholar). At physiological concentrations, PGE2 enhances elements of macrophage differentiation but inhibits functional activation and enhances B cell production of IgG1 and IgE while inhibiting that of IgM (11Roper R.L. Phipps R.P. J. Immunol. 1992; 49: 2984-2991Google Scholar). Of central importance in most host defense and autoimmune responses is that PGE2 inhibits T cell proliferation, differentiation, expression of membrane Rs, secretion of diverse cytokines, cytotoxicity, and other specific effector functions in cellular immune reactions (10Goetzl E.J. An S. Smith W.C. FASEB J. 1995; 9: 1051-1058Crossref PubMed Scopus (246) Google Scholar). Some PGE2 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 (10Goetzl E.J. An S. Smith W.C. FASEB J. 1995; 9: 1051-1058Crossref PubMed Scopus (246) Google Scholar). PGE2 effects on T cells have been attributed almost exclusively to increases in [cAMP]i, which are transduced by EP2 and/or EP4 Rs. Expression of the EP3 subtype of PGE2 Rs by T cells at a level capable of altering T cell functions has not been described nor have the functional consequences of EP3 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 (12Adams R.A. Pothier L. Flowers A. Lazarus H. Farber S. Foley G.E. Exp. Cell Res. 1970; 62: 5-10Crossref PubMed Scopus (51) Google Scholar, 13Hara J. Benedict S.H. Champagne E. Mak T.W. Minden M. Gelfand E.W. J. Clin. Invest. 1988; 81: 989-996Crossref PubMed Scopus (24) Google Scholar). It is now shown that HSB.2 T cells express predominantly the EP3 subtype of PGE2 R and that PGE2 stimulates increases in HSB.2 cellular content and secretion of matrix metalloproteinase (MMP)-9 by EP3 R-mediated and [Ca2+]i-dependent enhancement of transcription of mRNA encoding MMP-9.DISCUSSIONThe expression by T cells of multiple functionally distinct subtypes of PGE2 Rs, designated EP1, EP2, EP3, and EP4 Rs, is a principal determinant of specificity and diversity of the immune effects of PGE2 (1Coleman, R. A., Kennedy, I., Humphrey, P. P. A., Bunce, K., Lumley, P., Emmet, J. C., (eds) (1990) Comprehensive Medicinal Chemistry, Vol 3, p. 643, Pergammon Press, Oxford.Google Scholar, 2Coleman R.A. Smith W.L. Narumiya S. Pharmacol. Rev. 1994; 46: 205-229PubMed Google Scholar). Regulation of T cell functions was not attributed previously to EP3 Rs. We now show that 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. HSB.2 T cells differ from blood and lymphoid tissue T cells in expressing predominantly EP3 Rs and lower levels of EP2 Rs and EP4 Rs. The EP3/EP1 R-selective agonist sulprostone and EP3/EP2/EP4 R-selective agonists M&B 28767 and misoprostol competitively inhibited the binding of [3H]PGE2 to HSB.2 cells (Fig. 1). In contrast, the EP2 R-selective agonist butaprost inhibited [3H]PGE2 binding much less at only the highest concentration of 10−6M, and the EP1 R-selective antagonist SC-19220 did not alter [3H]PGE2 binding (Fig. 1). These results indicated predominant expression of EP3 Rs, fewer EP4 Rs, a much lower level of EP2 Rs, and no detectable EP1 Rs in HSB.2 cells.Assessment of adenylyl cyclase signaling revealed that PGE2, misoprostol, and a higher concentration of butaprost evoked increases in [cAMP]i in HSB.2 cells (Fig. 2), confirming expression of functional EP4 Rs and EP2 Rs. The isoforms of EP3 Rs in some nonhuman species that transduce increases in [cAMP]i (8Namba T. Sugimoto Y. Negishi M. Irie A. Ushikubi F. Kakizuka A. Ito S. Ichikawa A. Narumiya S. Nature. 1993; 365: 166-170Crossref PubMed Scopus (515) Google Scholar, 9Irie A. Sugimoto Y. Namba T. Harazono A. Honda A. Watabe A. Negishi M. Narumiya S. Ichikawa A. Eur. J. Biochem. 1993; 217: 313-318Crossref PubMed Scopus (164) Google Scholar) were not detected in HSB.2 cells, because the EP3 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 EP3 R isoforms mediated an increase in [cAMP]i (7An S. Yang J. So S.W. Zeng L. Goetzl E.J. Biochemistry. 1994; 33: 14496-14502Crossref PubMed Scopus (106) Google Scholar). The increases in [Ca2+]i by PGE2 and the EP3/EP1 R-selective agonist sulprostone and the failure of a maximal concentration of the EP1 R-selective antagonist SC-19220 to dampen increases in [Ca2+]i evoked by sulprostone (Table I) argue strongly against the presence of EP1 Rs in HSB.2 cells and confirm the presence of functionally relevant EP3 Rs. EP1 Rs mediate increases in [Ca2+]i by stimulating the entry of extracellular Ca2+ (3Watabe A. Sugimoto Y. Honda A. Irie A. Namba T. Negishi M. Ito S. Narumiya S. Ichikawa A. J. Biol. Chem. 1993; 268: 20175-20178Abstract Full Text PDF PubMed Google Scholar); thus our finding that the elevations of [Ca2+]i by PGE2 and sulprostone were not changed by elimination of extracellular Ca2+ further confirms the absence of EP1 Rs in HSB.2 cells. EP3 Rs are therefore the exclusive transducers of increases in [Ca2+]i evoked by PGE2 in HSB.2 cells.A family of MMPs is the principal physiological system that degrades diverse components of extracellular matrix (21Birkedal-Hansen H. Moore W.G.I. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Crit. Rev. Oral Biol. Med. 1993; 4: 197-250Crossref PubMed Scopus (2630) Google Scholar). In human blood T cells and some human T lymphoblastoma cells, PGE2 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 (15Leppert D. Hauser S.L. Kishiyama J.L. An S. Zeng L. Goetzl E.J. FASEB J. 1995; 9: 1473-1481Crossref PubMed Scopus (111) Google Scholar, 22Goetzl E.J. Banda M.J. Leppert D. J. Immunol. 1996; 156: 1-4PubMed Google Scholar). Using HSB.2 cells as a T cell model, we thus examined the possibility that EP3 Rs mediate regulation of MMPs in T cells responding to PGE2 and the biochemical signaling mechanisms by which EP3 Rs transduce PGE2-evoked increases in MMPs. We demonstrated that stimulation of MMP-9 in HSB.2 cells by PGE2 is attributable to [Ca2+]i-dependent EP3 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 (23Zhou H. Bernhard E.J. Fox F.E. Billings P.C. Biochem. Biophys. Acta. 1993; 1179: 174-178Crossref Scopus (46) Google Scholar). 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 10−7-10−6M PGE2. Parallel Western blot analyses showed that PGE2 stimulated MMP-9 activity in part by increasing both MMP-9 protein expression and secretion (Fig. 3). PGE1 but not PGF2α, PGD2, or PGI2 enhanced MMP-9 activity of HSB.2 cells to the same extent as PGE2, reflecting PGE2 R specificity on MMP-9 and the similar preference of PGE2 Rs for PGE2 and PGE1, which was observed in other immune cells (24Zeng L. An S. Goetzl E.J. Immunology. 1995; 86: 620-628PubMed Google Scholar). That the EP3 R-directed agonists sulprostone, M&B 28767, and misoprostol enhanced MMP-9 activity of HSB.2 cells to the same extent as PGE2 or higher, whereas the EP2 R-selective agonist butaprost did not (Fig. 4), supported the critical involvement of EP3 Rs in stimulation of HSB.2 T cell MMP-9 by PGE2.The potent effects of PGE2 on MMP-9 have been attributed principally to increases in [cAMP]i (25Mertz P.M. DeWitt D.L. Stetler-Stevenson W.G. Wahl L.M. J. Biol. Chem. 1994; 269: 21322-21329Abstract Full Text PDF PubMed Google Scholar, 26Corcoran M.L. Stetler-Stevenson W.G. Brown P.D. Wahl L.M. J. Biol. Chem. 1992; 267: 515-519Abstract Full Text PDF PubMed Google Scholar, 27Corcoran M.L. Stetler-Stevenson W.G. DeWitt D.L. Wahl L.M. Arch. Biochem. Biophys. 1994; 310: 481-488Crossref PubMed Scopus (76) Google Scholar, 28McCarthy J.B. Wahl S.B. Rees J.C. Olsen C.E. Sandberg A.L. Whal L.M. J. Immunol. 1980; 124: 2405-2409PubMed Google Scholar), which are presumably transduced by EP2 and/or EP4 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 PGE2 on MMP-9 expression in HSB.2 T cells. This is consistent with the result that the EP2 R-selective agonist butaprost did not affect MMP-9 activity of HSB.2 cells. Thus the effect of PGE2 on MMP-9 of HSB.2 cells is not mediated by EP2 Rs or EP4 Rs.The finding that PGE2 stimulation of MMP-9 was not changed by elimination of extracellular Ca2+ but was suppressed by pretreatment with the Ca2+-ATPase inhibitor thapsigargin, which depletes intracellular Ca2+ (18Gouy H. Cefai D. Christensen S.B. Debre P. Bismuth G. Eur. J. Immunol. 1990; 20: 2269-2275Crossref PubMed Scopus (88) Google Scholar, 19Missiaen L. De Smedt H. Droogmans G. Casteels R. J. Biol. Chem. 1992; 267: 22961-22966Abstract Full Text PDF PubMed Google Scholar, 20Premack B.A. McDonald T.V. Gardner P. J. Immunol. 1994; 152: 5226-5240PubMed Google Scholar) (Fig. 4), indicated the critical role of release of Ca2+ from intracellular stores in EP3 R mediation of MMP-9 secretion. Increases in [Ca2+]i appear to be necessary but insufficient for the effect of PGE2 on MMP-9 in HSB.2 cells, because incubation of the cells with 10−9-10−6M ionomycin in medium containing 1 mM Ca2+ did not mimic a PGE2 effect on MMP-9, although ionomycin evoked increases in [Ca2+]i of HSB.2 cells by a maximal 100-fold enhancement at 10−6M. Other unidentified signals that were simultaneously evoked by PGE2 binding to EP3 Rs along with the increases of [Ca2+]i released from intracellular stores also may be required for the PGE2 effect. IP3 formation in HSB.2 cells was not changed significantly by up to 10−6M PGE2 (data not shown), suggesting either that EP3 Rs transduced production of small amounts of IP3 that could not be detected in whole-cells assays but was capable of evoking increases in [Ca2+]i in HSB.2 cells or that the increases in [Ca2+]i from intracellular stores result from an IP3-independent mechanism. It was shown that TG releases stored Ca2+ from intracellular pools without production of IP3 (20Premack B.A. McDonald T.V. Gardner P. J. Immunol. 1994; 152: 5226-5240PubMed Google Scholar, 18Gouy H. Cefai D. Christensen S.B. Debre P. Bismuth G. Eur. J. Immunol. 1990; 20: 2269-2275Crossref PubMed Scopus (88) Google Scholar), but there is a large degree of overlap between the IP3- and TG-sensitive Ca2+ pools (20Premack B.A. McDonald T.V. Gardner P. J. Immunol. 1994; 152: 5226-5240PubMed Google Scholar). We suggest in this study that PGE2 and TG mobilize Ca2+ from common intracellular pools. Thus, the PGE2-mediated increase in [Ca2+]i via EP3 Rs in HSB.2 cells may be from both TG-sensitive and IP3-sensitive pools.All subtypes of PGE2 Rs are members of the G protein-coupled seven-transmembrane domain superfamily. We have shown previously that elevation of [Ca2+]i mediated by all human EP3 R isoforms was partially blocked by pertussis toxin treatment (7An S. Yang J. So S.W. Zeng L. Goetzl E.J. Biochemistry. 1994; 33: 14496-14502Crossref PubMed Scopus (106) Google Scholar). The PGE2-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 (29Xie B. Dong Z. Fidler I. J. Immunol. 1994; 152: 3637-3644PubMed Google Scholar). src-related protein-tyrosine kinase plays a role in MMP-9 transcriptional activation (30Sato H. Kita M. Seiki M. J. Biol. Chem. 1993; 268: 23460-23468Abstract Full Text PDF PubMed Google Scholar). 12-O-Tetradecanoylphorbol 13-acetate-enhanced MMP-9 activity in HSB cells also is mediated through activation of protein kinase C (23Zhou H. Bernhard E.J. Fox F.E. Billings P.C. Biochem. Biophys. Acta. 1993; 1179: 174-178Crossref Scopus (46) Google Scholar). However, the PGE2 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 10−10-10−7M, with protein-tyrosine kinase inhibitors genistein and tyrphostin at concentrations of 10−7-10−4M, or with protein kinase A/protein kinase G inhibitors H-89 and KT5720 at concentrations of 10−9-10−6M did not change the basal and PGE2 elevated level of MMP-9 (data not shown). Further work is needed to elucidate biochemical signal transduction mechanisms by which PGE2 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 α, epidermal growth factor, platelet-derived growth factor, nerve growth factor, interleukin-1, transforming growth factor-β, progesterone, and corticosteroids (21Birkedal-Hansen H. Moore W.G.I. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Crit. Rev. Oral Biol. Med. 1993; 4: 197-250Crossref PubMed Scopus (2630) Google Scholar, 22Goetzl E.J. Banda M.J. Leppert D. J. Immunol. 1996; 156: 1-4PubMed Google Scholar). We now show that PGE2 enhances MMP-9 expression by increasing transcription as well. Prevention of PGE2-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 PGE2 treatment (Fig. 5). The ability of the EP3 R-directed agonist sulprostone to mimic a PGE2 stimulatory effect on MMP-9 mRNA (Fig. 5) supports the hypothesis that EP3 Rs signal predominantly at the transcriptional level. 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.The HSB.2 T cell (12Adams R.A. Pothier L. Flowers A. Lazarus H. Farber S. Foley G.E. Exp. Cell Res. 1970; 62: 5-10Crossref PubMed Scopus (51) Google Scholar) is at an early thymocyte stage (13Hara J. Benedict S.H. Champagne E. Mak T.W. Minden M. Gelfand E.W. J. Clin. Invest. 1988; 81: 989-996Crossref PubMed Scopus (24) Google Scholar) and expresses predominantly EP3 and EP4 Rs, of which the former is the principal transducer of PGE2 effects on MMP-9. However, mature human blood CD4+ and CD8+ T cells express principally EP4 Rs and only the CD8+ subset bears a prominent number of EP3 Rs. 2L. Zeng, S. An, and E. J. Goetzl, unpublished data. In human blood T cells of mixed CD4+ and CD8+ composition, PGE2 stimulates surface expression and secretion of MMP-2 and −3, as well as MMP-9 (15Leppert D. Hauser S.L. Kishiyama J.L. An S. Zeng L. Goetzl E.J. FASEB J. 1995; 9: 1473-1481Crossref PubMed Scopus (111) Google Scholar), but the PGE2 R subtype dependence has not been defined. Whether HSB.2 cell is representative of human normal early thymocytes and whether the EP3 Rs of CD8+ T cells transduce increases in MMP-9 content and secretion remain to be evaluated in the corresponding population of T cells. INTRODUCTIONProstaglandin E2 (PGE2) 1The abbreviations used are: PGE2prostaglandin E2RsreceptorsMMPmatrix metalloproteinaseHBSSHanks', balanced salt solutionG proteinheterotrimeric guanine nucleotide-binding proteinIP3inositol triphosphatePTpertussis toxinTGthapsigargin. 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 (1Coleman, R. A., Kennedy, I., Humphrey, P. P. A., Bunce, K., Lumley, P., Emmet, J. C., (eds) (1990) Comprehensive Medicinal Chemistry, Vol 3, p. 643, Pergammon Press, Oxford.Google Scholar, 2Coleman R.A. Smith W.L. Narumiya S. Pharmacol. Rev. 1994; 46: 205-229PubMed Google Scholar). Cell surface expression of multiple functionally distinct subtypes of PGE2 receptors (Rs) is a principal determinant of the diversity and specificity of cellular effects of PGE2. PGE2 is recognized and transduces cellular effects specifically by interacting with PGE2 Rs of at least four subtypes, designated the EP1, EP2, EP3, and EP4 Rs. These subtypes of PGE2 Rs differ in structure, ligand-binding properties, tissue distribution, and coupling to signal transduction pathways (2Coleman R.A. Smith W.L. Narumiya S. Pharmacol. Rev. 1994; 46: 205-229PubMed Google Scholar). All subtypes of PGE2 Rs have recently been cloned and shown to be members of the G protein-coupled seven-transmembrane domain superfamily (1Coleman, R. A., Kennedy, I., Humphrey, P. P. A., Bunce, K., Lumley, P., Emmet, J. C., (eds) (1990) Comprehensive Medicinal Chemistry, Vol 3, p. 643, Pergammon Press, Oxford.Google Scholar, 2Coleman R.A. Smith W.L. Narumiya S. Pharmacol. Rev. 1994; 46: 205-229PubMed Google Scholar). EP1 Rs mediate increases in the intracellular concentration of calcium ([Ca2+]i) (3Watabe A. Sugimoto Y. Honda A. Irie A. Namba T. Negishi M. Ito S. Narumiya S. Ichikawa A. J. Biol. Chem. 1993; 268: 20175-20178Abstract Full Text PDF PubMed Google Scholar). EP2 and EP4 Rs activate adenylyl cyclase via Gs and stimulate increases in the intracellular concentration of cAMP ([cAMP]i) (4Regan J.W. Barley T.J. Pepperl D.J. Pierce K.L. Bogardus A.M. Donello J.E. Fairbairn C.E. Kedzie K.M. Woodward D.F. Gil D.W. Mol. Pharmacol. 1994; 46: 213-220PubMed Google Scholar, 5An S. Yang J. Xia M. Goetzl E.J. Biochem. Biophys. Res. Commun. 1993; 197: 263-270Crossref PubMed Scopus (132) Google Scholar, 6Honda A. Sugimoto Y. Namba T. Watabe A. Irie A. Negishi M. Narumiya S. Ichikawa A. J. Biol. Chem. 1993; 268: 7759-7762Abstract Full Text PDF PubMed Google Scholar). Multiple isoforms of EP3 Rs not only inhibit adenylyl cyclase, resulting in a decrease in [cAMP]i elevated by forskolin or other agonists via Gi, but also stimulate increases in [Ca2+]i via Gi/Go or Gq (1Coleman, R. A., Kennedy, I., Humphrey, P. P. A., Bunce, K., Lumley, P., Emmet, J. C., (eds) (1990) Comprehensive Medicinal Chemistry, Vol 3, p. 643, Pergammon Press, Oxford.Google Scholar, 7An S. Yang J. So S.W. Zeng L. Goetzl E.J. Biochemistry. 1994; 33: 14496-14502Crossref PubMed Scopus (106) Google Scholar, 8Namba T. Sugimoto Y. Negishi M. Irie A. Ushikubi F. Kakizuka A. Ito S. Ichikawa A. Narumiya S. Nature. 1993; 365: 166-170Crossref PubMed Scopus (515) Google Scholar, 9Irie A. Sugimoto Y. Namba T. Harazono A. Honda A. Watabe A. Negishi M. Narumiya S. Ichikawa A. Eur. J. Biochem. 1993; 217: 313-318Crossref PubMed Scopus (164) Google Scholar). Certain isoforms of the EP3 R in nonhuman species also activate adenylyl cyclase via Gs and transduce increases in [cAMP]i (8Namba T. Sugimoto Y. Negishi M. Irie A. Ushikubi F. Kakizuka A. Ito S. Ichikawa A. Narumiya S. Nature. 1993; 365: 166-170Crossref PubMed Scopus (515) Google Scholar, 9Irie A. Sugimoto Y. Namba T. Harazono A. Honda A. Watabe A. Negishi M. Narumiya S. Ichikawa A. Eur. J. Biochem. 1993; 217: 313-318Crossref PubMed Scopus (164) Google Scholar).PGE2 potently mediates and modulates cellular and humoral immune responses by stimulating or inhibiting the functions of many different types of immune cells (10Goetzl E.J. An S. Smith W.C. FASEB J. 1995; 9: 1051-1058Crossref PubMed Scopus (246) Google Scholar). At physiological concentrations, PGE2 enhances elements of macrophage differentiation but inhibits functional activation and enhances B cell production of IgG1 and IgE while inhibiting that of IgM (11Roper R.L. Phipps R.P. J. Immunol. 1992; 49: 2984-2991Google Scholar). Of central importance in most host defense and autoimmune responses is that PGE2 inhibits T cell proliferation, differentiation, expression of membrane Rs, secretion of diverse cytokines, cytotoxicity, and other specific effector functions in cellular immune reactions (10Goetzl E.J. An S. Smith W.C. FASEB J. 1995; 9: 1051-1058Crossref PubMed Scopus (246) Google Scholar). Some PGE2 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 (10Goetzl E.J. An S. Smith W.C. FASEB J. 1995; 9: 1051-1058Crossref PubMed Scopus (246) Google Scholar). PGE2 effects on T cells have been attributed almost exclusively to increases in [cAMP]i, which are transduced by EP2 and/or EP4 Rs. Expression of the EP3 subtype of PGE2 Rs by T cells at a level capable of altering T cell functions has not been described nor have the functional consequences of EP3 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 (12Adams R.A. Pothier L. Flowers A. Lazarus H. Farber S. Foley G.E. Exp. Cell Res. 1970; 62: 5-10Crossref PubMed Scopus (51) Google Scholar, 13Hara J. Benedict S.H. Champagne E. Mak T.W. Minden M. Gelfand E.W. J. Clin. Invest. 1988; 81: 989-996Crossref PubMed Scopus (24) Google Scholar). It is now shown that HSB.2 T cells express predominantly the EP3 subtype of PGE2 R and that PGE2 stimulates increases in HSB.2 cellular content and secretion of matrix metalloproteinase (MMP)-9 by EP3 R-mediated and [Ca2+]i-dependent enhancement of transcription of mRNA encoding MMP-9." @default.
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