Inhibition by extracellular cAMP of phorbol 12-myristate 13-acetate-induced prostaglandin H synthase-2 expression in human pulmonary microvascular endothelial cells. Involvement of an ecto-protein kinase A activity.
Exposure of human pulmonary microvascular endothelial cells (HPMECs) to phorbol 12-myristate 13-acetate (PMA) leads to the increase of prostaglandin H synthase (PGHS)-2 protein levels. Under same conditions and according to its constitutive nature, no significant variation of PGHS-1 protein was noted. The elevation of the intracellular cAMP rate is known to enhance PGHS-2 levels through a protein kinase A pathway in various cells. To determine whether the extracellular cAMP also regulates the inducible expression of PGHS, cultured HPMECs were exposed to cAMP alone or in combination with PMA. The PMA-induced PGHS-2 protein was attenuated by the extracellular cAMP. In addition, PGHS-2 activity evaluated through 6-keto-PGF1alpha generation, which was enhanced by PMA was inhibited by extracellular cAMP. Furthermore, in HPMEC medium, PMA-induced PGHS-2 expression was accompanied by the generation of a transferable activity (TA) able to abolish platelet aggregation. This resulting TA was dependent from PGHS-2 pathway, because NS-398, a selective inhibitor of PGHS-2, suppressed its production. The inhibitory TA released by treated HPMECs was also prevented by extracellular cAMP. The specific protein kinase A (PKA) inhibitor blocked the extracellular cAMP effect on both PMA-induced 6-keto-PGF1alpha synthesis and inhibitory TA generation, suggesting the involvement of PKA signaling at the outer surface of HPMECs. Accordingly, we established, in phosphorylation experiments, the presence of an endothelial ecto-protein kinase activity, able to phosphorylate the synthetic substrate kemptide in a cAMP-dependent mode. Reverse transcription-polymerase chain reaction analysis showed that PMA-induced PGHS-2 mRNA was markedly reduced by extracellular cAMP. Together, these findings provide the first experimental evidence that extracellular cAMP is able to reduce HPMEC PGHS-2 expression in terms of mRNA, protein, and enzyme activity through an ecto-PKA pathway. In addition, they outline the potential role of endothelial PGHS-2 in the limitation of platelet activation during inflammatory processes.
Elalamy
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I
Unité de Pharmacologie Cellulaire, Unité Associée Institut Pasteur-INSERM U 485, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France.
Said
F A
FA
Singer
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Couetil
J P
JP
Hatmi
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M
D002273:Carcinogens / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism* / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D051546:Cyclooxygenase 2; D004347:Drug Interactions; D004730:Endothelium, Vascular / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis*; D008168:Lung / Q000098:blood supply; D008565:Membrane Proteins; D008833:Microcirculation; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D002273:Carcinogens; D007527:Isoenzymes; D008565:Membrane Proteins; D000242:Cyclic AMP; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D017868:Cyclic AMP-Dependent Protein Kinases; D013755:Tetradecanoylphorbol Acetate