Abstract
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-1β or CCL4. ET-1-induced MIP-1β 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-1β expression involved hypoxia-inducible factor-1α (HIF-1α), independent of hypoxia, as demonstrated by silencing with HIF-1α small interfering RNA, EMSA, and chromatin immunoprecipitation analysis. ET-1-induced MIP-1β promoter luciferase activity was attenuated when any of the five hypoxia-response elements, AP-1, or NF-κB binding motifs in the proximal MIP-1β 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-1β mRNA. Moreover, ET-1-induced MIP-1β 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-1β expression. Taken together, these studies showed that ET-1-mediated MIP-1β gene expression is regulated via hypoxia-response elements, AP-1, and NF-κB cis-binding elements in its promoter and negatively regulated by microRNA-195, which targets the 3' untranslated region of MIP-1β RNA. These studies provide what we believe are new avenues, based on targets of HIF-1α and microRNAs, for ameliorating inflammation in SCD.
Highlights
Patients with sickle cell disease (SCD) exhibit a chronic inflammatory state manifested by leukocytosis and increased circulating levels of proinflammatory cytochemokines
Abbreviations used in this paper: Chromatin immunoprecipitation (ChIP), chromatin immunoprecipitation; CRE, cAMP response element; Ct, threshold cycle; Diphenyleneiodonium chloride (DPI), diphenyleneiodonium chloride; ET-1, endothelin-1; ET-AR, endothelin receptor-A; ET-BR, endothelin receptor-B; HIF1a, hypoxia-inducible factor-1a; hypoxia response element (HRE), hypoxia-response element; MKK, MAPK kinase; PBM, peripheral blood monocyte; PHD-2, prolylhydroxylase-2; PlGF, placental growth factor; qRT-PCR, quantitative real-time PCR; Relative quantification (RQ), relative quantification; SCD, sickle cell disease; scRNA, scrambled small interfering RNA; SDM, sitedirected mutagenesis; Small interfering RNA (siRNA), small interfering RNA; UTR, untranslated region
We show that ET-1 activates monocytes, both primary PBMs and THP-1 monocytic cells, resulting in the upregulation of chemokine MIP-1b mRNA and protein expression
Summary
Patients with sickle cell disease (SCD) exhibit a chronic inflammatory state manifested by leukocytosis and increased circulating levels of proinflammatory cytochemokines. Transfection of monocytes with anti–microRNA195a oligonucleotide augmented several-fold ET-1–induced MIP-1b expression Taken together, these studies showed that ET-1– mediated MIP-1b gene expression is regulated via hypoxia-response elements, AP-1, and NF-kB cis-binding elements in its promoter and negatively regulated by microRNA-195, which targets the 39 untranslated region of MIP-1b RNA. Abbreviations used in this paper: ChIP, chromatin immunoprecipitation; CRE, cAMP response element; Ct, threshold cycle; DPI, diphenyleneiodonium chloride; ET-1, endothelin-1; ET-AR, endothelin receptor-A; ET-BR, endothelin receptor-B; HIF1a, hypoxia-inducible factor-1a; HRE, hypoxia-response element; MKK, MAPK kinase; PBM, peripheral blood monocyte; PHD-2, prolylhydroxylase-2; PlGF, placental growth factor; qRT-PCR, quantitative real-time PCR; RQ, relative quantification; SCD, sickle cell disease; scRNA, scrambled small interfering RNA; SDM, sitedirected mutagenesis; siRNA, small interfering RNA; UTR, untranslated region
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