Institut National de la Santé et de la Recherche Médicale Unité Mixte de Recherche_S U1185, Faculty of Medicine at Université Paris-Sud, University Paris-Sud, Université Paris-Saclay, Le Kremlin Bicêtre, France.
Service d'Endocrinologie-Diabète-Nutrition, Hôpital Robert Debré, Centre Hospitalier Universitaire de Reims, Reims, France.
J Clin Endocrinol Metab. 2019 Nov 1;104(11):5205-5216. doi: 10.1210/jc.2019-00800.
Six patients carrying heterozygous loss-of-function mutations of glucocorticoid (GC) receptor (GR) presented with hypercortisolism, associated with low kalemia, low plasma renin, and aldosterone levels, with or without hypertension, suggesting a pseudohypermineralocorticism whose mechanisms remain unclear. We hypothesize that an impaired activity of the 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2; encoded by the HSD11B2 gene), catalyzing cortisol (F) inactivation, may account for an inappropriate activation of a renal mineralocorticoid signaling pathway in these GC-resistant patients.
We aim at studying the GR-mediated regulation of HSD11B2.
The HSD11B2 promoter was subcloned and luciferase reporter assays evaluated GR-dependent HSD11B2 regulation, and 11β-HSD2 expression/activity was studied in human breast cancer MCF7 cells, endogenously expressing this enzyme.
Transfection assays revealed that GR transactivated the long (2.1-kbp) HSD11B2 promoter construct, whereas a defective 501H GR mutant was unable to stimulate luciferase activity. GR-mediated transactivation of the HSD11B2 gene was inhibited by the GR antagonist RU486. A threefold increase in HSD11B2 mRNA levels was observed after dexamethasone (DXM) treatment of MCF7 cells, inhibited by RU486 or by actinomycin, supporting a GR-dependent transcription. Chromatin immunoprecipitation further demonstrated a DXM-dependent GR recruitment onto the HSD11B2 promoter. 11β-HSD2 activity, evaluated by the cortisone/F ratio, quantified by liquid chromatography/tandem mass spectrometry, was 10-fold higher in the supernatant of DXM-treated cells than controls, consistent with a GR-dependent stimulation of 11β-HSD2 catalytic activity.
Collectively, we demonstrate that 11β-HSD2 expression and activity are transcriptionally regulated by GR. In the context of GR haploinsufficiency, these findings provide evidence that defective GR signaling may account for apparent mineralocorticoid excess in GC-resistant patients.
六位携带糖皮质激素（GC）受体（GR）杂合功能丧失突变的患者表现为皮质醇增多症，伴有低血钾、低血浆肾素和醛固酮水平，伴有或不伴有高血压，提示假性盐皮质激素过多症，其机制尚不清楚。我们假设，11β-羟类固醇脱氢酶 2（11β-HSD2；由 HSD11B2 基因编码）的活性受损，该酶可催化皮质醇（F）失活，可能导致这些 GC 抵抗患者的肾脏盐皮质激素信号通路的不适当激活。
我们旨在研究 GR 介导的 HSD11B2 调节。
亚克隆 HSD11B2 启动子并进行荧光素酶报告基因检测，评估 GR 依赖性 HSD11B2 调节，并用内源性表达该酶的人乳腺癌 MCF7 细胞研究 11β-HSD2 的表达/活性。
转染实验表明，GR 反式激活长（2.1-kbp）HSD11B2 启动子构建体，而缺陷 501H GR 突变体不能刺激荧光素酶活性。GR 拮抗剂 RU486 抑制 GR 介导的 HSD11B2 基因反式激活。MCF7 细胞用地塞米松（DXM）处理后，HSD11B2 mRNA 水平增加了三倍，RU486 或放线菌素处理可抑制其增加，支持 GR 依赖性转录。染色质免疫沉淀进一步表明，DEXM 依赖性 GR 募集到 HSD11B2 启动子上。通过液相色谱/串联质谱定量的可的松/F 比值评估，11β-HSD2 活性在 DXM 处理细胞的上清液中比对照高 10 倍，这与 11β-HSD2 催化活性的 GR 依赖性刺激一致。
总之，我们证明了 11β-HSD2 的表达和活性受 GR 转录调控。在 GR 杂合不足的情况下，这些发现为 GR 信号传导缺陷可能导致 GC 抵抗患者出现明显的盐皮质激素过多症提供了证据。
