Endothelin-1-Induced Macrophage Inflammatory Protein-1β Expression in Monocytic Cells Involves Hypoxia-Inducible Factor-1α and AP-1 and Is Negatively Regulated by microRNA-195

Patients with sickle cell disease (SCD) exhibit a chronic inflammatory state manifested by leukocytosis and increased circulating levels of proinflammatory cytochemokines. Our studies show that placenta growth factor levels are high in SCD, and placental growth factor induces the release of the vasoconstrictor endothelin-1 (ET-1) from pulmonary microvascular endothelial cells. In this study, we observed that ET-1 increased the expression of the chemokines MIP-1 beta or CCL4. ET-1-induced MIP-1 beta mRNA expression in THP-1 cells and human peripheral blood monocytes occurred via the activation of PI3K, NADPH oxidase, p38 MAPK, and JNK-1 but not JNK-2. ET-1-induced MIP-1 beta expression involved hypoxia-inducible factor-1 alpha (HIF-1 alpha), independent of hypoxia, as demonstrated by silencing with HIF-1 alpha small interfering RNA, EMSA, and chromatin immunoprecipitation analysis. ET-1-induced MIP-1 beta promoter luciferase activity was attenuated when any of the five hypoxia-response elements, AP-1, or NF-kappa B binding motifs in the proximal MIP-1 beta promoter (-1053/+43 bp) were mutated. Furthermore, ET-1 significantly downregulated the expression of a key microRNA, microRNA-195a, which showed a complementary binding site in the 3' untranslated region of MIP-1 beta mRNA. Moreover, ET-1-induced MIP-1 beta mRNA expression in either THP-1 cells or peripheral blood monocytes was reduced upon expression of microRNA-195a. Conversely, transfection of monocytes with anti-microRNA-195a oligonucleotide augmented several-fold ET-1-induced MIP-1 beta expression. Taken together, these studies showed that ET-1-mediated MIP-1 beta gene expression is regulated via hypoxia-response elements, AP-1, and NF-kappa B cis-binding elements in its promoter and negatively regulated by microRNA-195, which targets the 3' untranslated region of MIP-1 beta RNA. These studies provide what we believe are new avenues, based on targets of HIF-1 alpha and microRNAs, for ameliorating inflammation in SCD. The Journal of Immunology, 2010, 185: 6253-6264.