Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University (TMDU), Tokyo, Japan.
Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University (TMDU), Tokyo, Japan; Department of Liver Disease Control, Tokyo Medical and Dental University (TMDU), Tokyo, Japan.
J Hepatol. 2019 Jul;71(1):143-152. doi: 10.1016/j.jhep.2019.02.024. Epub 2019 Mar 19.
BACKGROUND & AIMS: Congenital hepatic fibrosis (CHF) is a genetic liver disease resulting in abnormal proliferation of cholangiocytes and progressive hepatic fibrosis. CHF is caused by mutations in the PKHD1 gene and the subsequent dysfunction of the protein it encodes, fibrocystin. However, the underlying molecular mechanism of CHF, which is quite different from liver cirrhosis, remains unclear. This study investigated the molecular mechanism of CHF pathophysiology using a genetically engineered human induced pluripotent stem (iPS) cell model to aid the discovery of novel therapeutic agents for CHF.
PKHD1-knockout (PKHD1-KO) and heterozygously mutated PKHD1 iPS clones were established by RNA-guided genome editing using the CRISPR/Cas9 system. The iPS clones were differentiated into cholangiocyte-like cells in cysts (cholangiocytic cysts [CCs]) in a 3D-culture system.
The CCs were composed of a monolayer of cholangiocyte-like cells. The proliferation of PKHD1-KO CCs was significantly increased by interleukin-8 (IL-8) secreted in an autocrine manner. IL-8 production was significantly elevated in PKHD1-KO CCs due to mitogen-activated protein kinase pathway activation caused by fibrocystin deficiency. The production of connective tissue growth factor (CTGF) was also increased in PKHD1-KO CCs in an IL-8-dependent manner. Furthermore, validation analysis demonstrated that both the serum IL-8 level and the expression of IL-8 and CTGF in the liver samples were significantly increased in patients with CHF, consistent with our in vitro human iPS-disease model of CHF.
Loss of fibrocystin function promotes IL-8-dependent proliferation of, and CTGF production by, human cholangiocytes, suggesting that IL-8 and CTGF are essential for the pathogenesis of CHF. IL-8 and CTGF are candidate molecular targets for the treatment of CHF.
Congenital hepatic fibrosis (CHF) is a genetic liver disease caused by mutations of the PKHD1 gene. Dysfunction of the protein it encodes, fibrocystin, is closely associated with CHF pathogenesis. Using an in vitro human induced pluripotent stem cell model and patient samples, we showed that the loss of fibrocystin function promotes proliferation of cholangiocytes and the production of connective tissue growth factor (CTGF) in an interleukin 8 (IL-8)-dependent manner. These results suggest that IL-8 and CTGF are essential for the pathogenesis of CHF.
先天性肝纤维化（CHF）是一种遗传性肝病，导致胆管细胞异常增殖和进行性肝纤维化。CHF 是由 PKHD1 基因突变引起的，其编码的蛋白纤连蛋白功能障碍。然而，与肝硬化有很大不同的 CHF 的潜在分子机制尚不清楚。本研究使用基因工程人诱导多能干细胞（iPS）模型研究 CHF 病理生理学的分子机制，以帮助发现 CHF 的新型治疗药物。
使用 CRISPR/Cas9 系统的 RNA 引导基因组编辑建立 PKHD1 敲除（PKHD1-KO）和杂合突变 PKHD1 iPS 克隆。iPS 克隆在 3D 培养系统中分化为胆管细胞样细胞（胆管囊性细胞 [CCs]）。
CCs 由单层胆管细胞样细胞组成。PKHD1-KO CCs 中自分泌分泌的白细胞介素 8（IL-8）显著增加了增殖。由于纤连蛋白缺乏导致丝裂原活化蛋白激酶途径激活，PKHD1-KO CCs 中 IL-8 的产生显著增加。结缔组织生长因子（CTGF）的产生也以 IL-8 依赖的方式增加在 PKHD1-KO CCs 中。此外，验证分析表明，CHF 患者的血清 IL-8 水平以及肝脏样本中 IL-8 和 CTGF 的表达均显著升高，与我们的 CHF 体外人 iPS 疾病模型一致。
纤连蛋白功能丧失促进人胆管细胞中 IL-8 依赖性增殖和 CTGF 产生，提示 IL-8 和 CTGF 是 CHF 发病机制的关键。IL-8 和 CTGF 是治疗 CHF 的候选分子靶点。
先天性肝纤维化（CHF）是一种由 PKHD1 基因突变引起的遗传性肝病。其编码的蛋白纤连蛋白功能障碍与 CHF 的发病机制密切相关。使用体外人诱导多能干细胞模型和患者样本，我们表明纤连蛋白功能丧失以白细胞介素 8（IL-8）依赖性方式促进胆管细胞增殖和结缔组织生长因子（CTGF）的产生。这些结果表明 IL-8 和 CTGF 是 CHF 发病机制的关键。
