Parathyroid Hormone Receptor-1 Signaling Aggravates Hepatic Fibrosis through Upregulating cAMP Response Element Binding Protein-Like 2 (human). Parathyroid Hormone Receptor-1 Signaling Aggravates Hepatic Fibrosis through Upregulating cAMP Response Element Binding Protein-Like 2 (human)

Background & Aims Parathyroid hormone receptor-1 (PTH1R) is a class B G protein-coupled receptor central to skeletal development, bone turnover, and calcium homeostasis. However, the role of PTH1R signaling in liver fibrosis is largely unknown. Here, the role of PTH1R signaling in the activation of hepatic stellate cells (HSCs) and hepatic fibrosis was examined. Approach & Results PTH1R was highly expressed in activated HSCs and fibrotic liver by using human liver specimens or carbon tetrachloride (CCl4)-treated or methionine and choline-deficient diet (MCD)-fed C57/BL6 mice. The mRNA level of hepatic PTH1R was positively correlated to α-smooth muscle actin (α-SMA) in patients with liver cirrhosis. Mice with HSCs-specific PTH1R deletion were protected from CCl4, MCD, or western diet plus low-dose CCl4-induced liver fibrosis. Conversely, parathyroid hormone (PTH) aggravated liver fibrosis in CCl4-treated mice. Mouse primary HSCs and LX2 cell lines were used for in vitro experiments. Molecular analyses by luciferase reporter assays and chromatin-immunoprecipitation assays in combination with mRNA sequencing in HSCs revealed that cAMP response element-binding protein-like 2 (Crebl2), a novel regulator in HSCs treated by PTH that interacted with Mothers against decapentaplegic homolog 3 (SMAD3) and increased the transcription of transforming growth factor β (TGFβ) in activating HSCs and collagen deposition. In agreement, HSCs-specific Crebl2 deletion ameliorated PTH-induced liver fibrosis in CCl4-treated mice. Conclusions In both mouse and human models, we found that PTH1R was highly expressed in activated HSCs and fibrotic liver. PTH1R signaling regulated collagen production in the HSCs via Crebl2/SMAD3/TGFβ regulatory circuits. Blockade of PTH1R signaling in HSCs might help mitigate the development of liver fibrosis. Overall design: Comparative gene expression profiling analysis of RNA-seq data for mice with or without PTH

研究背景与目的：甲状旁腺激素受体1 (Parathyroid hormone receptor-1, PTH1R) 属于B类G蛋白偶联受体，在骨骼发育、骨转换及钙稳态调控中发挥核心作用。然而，目前学界对PTH1R信号通路在肝纤维化中的作用仍不甚明晰。本研究旨在探讨PTH1R信号通路在肝星状细胞 (hepatic stellate cells, HSCs) 活化及肝纤维化进程中的功能。
研究方法与结果：通过人类肝脏标本，或经四氯化碳 (carbon tetrachloride, CCl4) 处理、饲喂蛋氨酸胆碱缺乏饮食 (methionine and choline-deficient diet, MCD) 的C57/BL6小鼠实验，发现PTH1R在活化的HSCs及纤维化肝脏中呈高表达状态。肝硬化患者的肝脏PTH1R mRNA水平与α-平滑肌肌动蛋白 (α-smooth muscle actin, α-SMA) 呈正相关。构建HSC特异性PTH1R敲除小鼠后，该小鼠可抵御CCl4、MCD，或高脂饮食联合低剂量CCl4诱导的肝纤维化。反之，甲状旁腺激素 (parathyroid hormone, PTH) 会加重CCl4处理小鼠的肝纤维化程度。体外实验采用小鼠原代HSCs及LX2细胞系开展。通过荧光素酶报告基因实验、染色质免疫共沉淀实验结合HSCs的mRNA测序进行分子分析，结果显示cAMP反应元件结合蛋白样2 (cAMP response element-binding protein-like 2, Crebl2) 是PTH处理HSCs后的新型调控因子，该因子可与母抗decapentaplegic同源物3 (Mothers against decapentaplegic homolog 3, SMAD3) 结合，并在活化HSCs及胶原沉积过程中上调转化生长因子β (transforming growth factor β, TGFβ) 的转录水平。与之相符的是，HSC特异性Crebl2敲除可改善CCl4处理小鼠中PTH诱导的肝纤维化。
研究结论：在小鼠及人类模型中，我们证实PTH1R在活化的HSCs及纤维化肝脏中高表达。PTH1R信号通路可通过Crebl2/SMAD3/TGFβ调控环路调节HSCs的胶原产生。靶向抑制HSCs中的PTH1R信号通路或可缓解肝纤维化的进展。
实验整体设计：对接受或未接受PTH处理的小鼠的RNA测序数据开展比较基因表达谱分析。