Abstract
We investigated proteinase-activated receptor-2 (PAR2)-triggered signal transduction pathways causing increased prostaglandin E2 (PGE2) formation in human lung-derived A549 epithelial cells. The PAR2 agonist, SLIGRL-NH2 (Ser-Leu-Ile-Gly-Arg-Leu-amide), evoked immediate cytosolic Ca2+ mobilization and delayed (0.5-3 h) PGE2 formation. The PAR2-triggered PGE2 formation was attenuated by inhibition of the following signal pathway enzymes: cyclooxygenases 1 and 2 (COX-1 and COX-2, respectively), cytosolic Ca2+-dependent phospholipase A2 (cPLA2), the mitogen-activated protein kinases (MAPKs), mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) and p38 MAPK, Src family tyrosine kinase, epidermal growth factor (EGF) receptor tyrosine kinase (EGFRK), and protein kinase C (PKC), but not by inhibition of matrix metalloproteinases. SLIGRL-NH2 caused prompt (5 min) and transient ERK phosphorylation, blocked in part by inhibitors of PKC and tyrosine kinases but not by an EGFRK inhibitor. SLIGRL-NH2 also evoked a relatively delayed (15 min) and persistent (30 min) phosphorylation of p38 MAPK, blocked by inhibitors of Src and EGFRK but not by inhibitors of COX-1 or COX-2. SLIGRL-NH2 elicited a Src inhibitor-blocked prompt (5 min) and transient phosphorylation of the EGFRK. SLIGRL-NH2 up-regulated COX-2 protein and/or mRNA levels that were blocked by inhibition of p38 MAPK, EGFRK, Src, and COX-2 but not MEK-ERK. SLIGRL-NH2 also caused COX-1-dependent up-regulation of microsomal PGE synthase-1 (mPGES-1). We conclude that PAR2-triggered PGE2 formation in A549 cells involves a coordinated up-regulation of COX-2 and mPGES-1 involving cPLA2, increased cytosolic Ca2+, PKC, Src, MEK-ERK, p38 MAPK, Src-mediated EGF receptor trans-activation, and also metabolic products of both COX-1 and COX-2.
Footnotes
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This work was supported by Kinki University Research Grant.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.105.089490.
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ABBREVIATIONS: PAR, proteinase-activated receptor; MAPK, mitogen-activated protein kinase; EGF, epidermal growth factor; MMP, matrix metalloproteinase; TGF-α, transforming growth factor-α; PGE2, prostaglandin E2; mPGES-1, microsomal prostaglandin E synthase-1; ERK, extracellular signal-regulated kinase; EGFRK, EGF receptor tyrosine kinase; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; COX, cyclooxygenase; HRP, horseradish peroxidase; PCR, polymerase chain reaction; HB-EGF; heparin-binding EGF; Igepal CA-630, (octylphenoxy)-polyethoxyethanol; iPLA2, Ca2+-independent phospholipase A2; cPLA2, cytosolic Ca2+-dependent phospholipase A2; BEL, bromoenol lactone; PI3-kinase, phosphatidylinositol 3-kinase; AACOCF3, arachidonyl trifluoromethyl ketone; MEK, MAPK/ERK kinase; DMSO, dimethyl sulfoxide; SLIGRL-NH2, Ser-Leu-Ile-Gly-Arg-Leu-amide; TFLLR-NH2, Thr-Phe-Leu-Leu-Arg-amide; U0126, 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene; SC-560, 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole); NS-398, N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide); PD98059, 2-(2′-amino-3′-methoxyphenyl)-oxanaphthalen-4-one); SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5(4-pyridyl)imidazole); GF109203X, 3-(1-(3-(dimethylamino)propyl)-1H-indol-3-yl)-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride; PP2 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; PD153035, 4-(3-bromoanilino)-6,7-dimethoxyquinazoline; GM6001, N-[2(R)-2-(hydroxamido carbonylmethyl)-4-methylpentanoyl]-l-tryptophane methylamide; BAPTA-AM, 1,2-bis(2-aminophenoxyethane)-N,N,N′,N′-tetraacetic acid/acetomethoxy ester; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide.
- Received May 13, 2005.
- Accepted August 23, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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