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. Comparative gene expression profiling analysis of RNA-seq data for mice with or without PTH

背景与目的
甲状旁腺激素受体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，PTH1R信号通路在肝纤维化中的作用尚不明确。本研究旨在探究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作用。

方法与结果
通过人类肝脏标本、四氯化碳（CCl4）诱导造模的小鼠及蛋氨酸胆碱缺乏饮食（methionine and choline-deficient diet, MCD）喂养的C57/BL6小鼠的检测，证实PTH1R在活化的肝星状细胞及纤维化肝脏中高表达。肝硬化患者肝脏组织中PTH1R的mRNA水平与α-平滑肌肌动蛋白（α-SMA）呈正相关。肝星状细胞特异性敲除PTH1R的小鼠，可免受四氯化碳、蛋氨酸胆碱缺乏饮食或高脂饮食联合低剂量四氯化碳诱导的肝纤维化损伤。反之，甲状旁腺激素（PTH）可加重四氯化碳造模小鼠的肝纤维化程度。本研究采用小鼠原代肝星状细胞及LX2细胞系开展体外实验。通过荧光素酶报告基因实验、染色质免疫共沉淀实验结合肝星状细胞转录组测序的分子分析显示，cAMP应答元件结合蛋白样2（Crebl2）是PTH处理肝星状细胞后的新型调控因子，其可与母抗decapentaplegic同源物3（SMAD3）相互结合，在活化肝星状细胞及胶原沉积过程中促进转化生长因子β（TGFβ）的转录。与此一致，肝星状细胞特异性敲除Crebl2可改善四氯化碳造模小鼠中PTH诱导的肝纤维化。

结论
在小鼠及人类模型中，本研究均证实PTH1R在活化的肝星状细胞及纤维化肝脏中高表达。PTH1R信号通路可通过Crebl2/SMAD3/TGFβ调控轴调控肝星状细胞的胶原生成。靶向抑制肝星状细胞的PTH1R信号通路，或可延缓肝纤维化的进展。对接受/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