ABSTRACT
Renal endothelin-converting enzyme (ECE)-1 is induced in experimental diabetes and following radiocontrast administration, conditions characterized by renal hypoxia, hypoxia-inducible factor (HIF) stabilization, and enhanced endothelin synthesis. Here we tested whether ECE-1 might be a HIF-target gene in vitro and in vivo. ECE-1 transcription and expression increased in cultured vascular endothelial and proximal tubular cell lines, subject to hypoxia, to mimosine or cobalt chloride. These interventions are known to stabilize HIF signaling by inhibition of HIF-prolyl hydroxylases. In rats, HIF-prolyl-hydroxylase inhibition by mimosine or FG-4497 increased HIF-1α immunostaining in renal tubules, principally in distal nephron segments. This was associated with markedly enhanced ECE-1 protein expression, predominantly in the renal medulla. A progressive and dramatic increase in ECE-1 immunostaining over time, in parallel with enhanced HIF expression, was also noted in conditional von Hippel-Lindau knockout mice. Since HIF and STAT3 are cross-stimulated, we triggered HIF expression by STAT3 activation in mice, transfected by or injected with a chimeric IL-6/IL-6-receptor protein, and found a similar pattern of enhanced ECE-1 expression. Chromatin immunoprecipitation sequence (ChIP-seq) and PCR analysis in hypoxic endothelial cells identified HIF binding at the ECE-1 promoter and intron regions. Thus, our findings suggest that ECE-1 may be a novel HIF-target gene.
Subject(s)
Aspartic Acid Endopeptidases/genetics , Aspartic Acid Endopeptidases/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Metalloendopeptidases/genetics , Metalloendopeptidases/metabolism , Animals , Cell Hypoxia/physiology , Cells, Cultured , Cobalt/pharmacology , Dioxygenases/antagonists & inhibitors , Endothelin-Converting Enzymes , Human Umbilical Vein Endothelial Cells , Humans , Introns , Kidney Tubules, Proximal/cytology , Kidney Tubules, Proximal/metabolism , Male , Mice , Mice, Inbred BALB C , Mice, Knockout , Mimosine/pharmacology , Oligonucleotide Array Sequence Analysis , Prolyl-Hydroxylase Inhibitors/pharmacology , Promoter Regions, Genetic , Rats , Rats, Sprague-Dawley , STAT3 Transcription Factor/metabolism , Signal Transduction/drug effects , Signal Transduction/physiology , Transcription, Genetic , von Hippel-Lindau Disease/genetics , von Hippel-Lindau Disease/metabolismABSTRACT
The multinucleate syncytiotrophoblast of the human placenta is formed by fusion of the underlying cytotrophoblast progenitor cells. The large surface area of the syncytiotrophoblast is necessary for transport functions while it also serves as the site of synthesis of hormones and steroids. Studies of syncytiotrophoblast transcription are puzzling, demonstrating that many of the nuclei in the multinucleated syncytium are transcriptionally inactive. To further elucidate RNA activity in the syncytiotrophoblast, we investigated expression of snRNAs involved in RNA splicing. Using RNA in situ hybridization, we observed that snRNAs were markedly reduced in the syncytium throughout the course of pregnancy. Recapitulating these results in primary trophoblasts and in trophoblast cell lines in vitro, we found, using qRT-PCR and RNA in situ hybridization, that snRNA expression is reduced in trophoblasts cultured under fusion conditions. Our finding that snRNA is markedly reduced in the syncytiotrophoblast suggests that the placenta has evolved a balance between the large surface area essential for its transport function and the need to regulate protein production in the multinucleated syncytium.
Subject(s)
Placenta/metabolism , Pregnancy Proteins/genetics , RNA, Small Nuclear/genetics , Trophoblasts/metabolism , Cadherins/genetics , Cadherins/metabolism , Cell Fusion , Cells, Cultured , Down-Regulation/genetics , Female , Humans , In Situ Hybridization, Fluorescence , Pregnancy , Pregnancy Proteins/metabolism , RNA, Small Nuclear/metabolismABSTRACT
BACKGROUND: Exogenous hyperisulinemia causes pregnancy, induced hypertension and intrauterine growth restriction (IUGR) in pregnant rats. Hyperinsulinemia may increase production of endothelin-1 (ET-1), produced by sequential proteolysis of the big endothelin by the endothelin-converting enzyme (ECE)-1, the expression of which is examined here in the placenta, kidney, heart, and liver. METHODS: Rats were rendered hyperinsulinemic by subcutaneous insulin pellet, mated and followed to the twenty-first day of pregnancy. They were then killed, and their fetuses and placentas were examined. RESULTS: Hyperinsulinemic dams (HD) had higher blood pressure (BP) (130 ± 17 mm Hg in HD vs. 115 ± 16 mm Hg in normal pregnant dams (NPD), P < 0.05), lower placenta weight (0.44 ± 0.08 g in HD vs. 0.47 ± 0.08 NPD, P < 0.05) and lower fetus weight: males 4.9 ± 0.4 g in HD vs. 5.5 ± 0.4 g in NPD, P < 0.0001; females 4.7 ± 0.4 g in HD vs. 5.2 ± 0.4 g in NPD (P < 0.0001). ECE-1 expression as determined by western blot was significantly increased in the placenta and its implantation site, i.e., the mesometrial triangle (MT) of HD by 46 and 48%, respectively. In the kidney and heart of HD ECE-1, protein expression was increased by 230 and 220%, respectively, but its level in the liver was similar in both groups. Immunohistochemical staining revealed ECE-1 expression in endothelial cells and trophoblastic cells of the placenta and MT. Endothelin receptor A (ET-A), a mediator of vasoconstriction by ET-1, was also expressed in the endothelium and in trophoblasts of the placenta and MT. The expression of both ECE-1 and ET-A, as measured by automated image analysis, was generally stronger in placentas of HD. CONCLUSIONS: ECE-1 and ET-A are expressed in the trophoblastic cells of the placenta and MT. This may affect local endothelin levels, BP and IUGR.
