1 Prostaglandin H synthase-2 (PGHS-2), is an inducible enzyme involved in various in¯ammatory responses. We established here that interleukin-1b (IL-1b) but not tumour necrosis factor-a (TNF-a) increased its expression in human pulmonary microvascular endothelial cells (HPMEC). However, associated with IL-1b, TNF-a greatly potentiated this enzyme induction. 2 Although unable to induce PGHS-2 expression by itself, TNF-a promoted a similar transcription nuclear factor-kB (NF-kB) activation to IL-1b. This e ect was more pronounced when cells were coexposed to both cytokines. HPMEC pre-treatment with MG-132, a proteasome inhibitor, prevented NF-kB activation as well as more distal signalling response, indicating that NF-kB activation is required but not su cient for PGHS-2 expression. 3 Both IL-1b and TNF-a failed to activate c-Jun NH2-terminal kinase (JNK). In addition, PD98059, a p42/44 mitogen-activated protein kinase (MAPK) phosphorylation inhibitor, did not decrease PGHS-2 expression. However, SB 203580, a p38 MAPK inhibitor, suppressed PGHS-2 induction by IL-1b alone or combined with TNF-a, demonstrating that p38 MAPK but not p42/44 MAPK or JNK cascades are required for PGHS-2 up-regulation. 4 Finally, TNF-a, unlike IL-1b, was unable to promote p38 MAPK phosphorylation, indicating that the failure of TNF-a to induce PGHS-2 expression is linked, at least in part, to its inability to activate p38 MAPK signalling pathway. Altogether, these data enhanced our understanding of PGHS-2 regulation in HPMEC and emphasize the heterogeneity of cellular responses to proin¯ammatory cytokines.
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-PGF1␣ 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-PGF1␣ 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 ectoprotein 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.
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