Angiogenesis in Liver Disease Research Group, August Pi i Sunyer Biomedical Research Institute, Hospital Clinic, University of Barcelona, Barcelona, Spain; Biomedical Research Networking Center on Hepatic and Digestive Disease, Institute of Health Carlos III, Spain.
Angiogenesis in Liver Disease Research Group, August Pi i Sunyer Biomedical Research Institute, Hospital Clinic, University of Barcelona, Barcelona, Spain.
Gastroenterology. 2020 Jul;159(1):273-288. doi: 10.1053/j.gastro.2020.03.008. Epub 2020 Mar 10.
BACKGROUND & AIMS: We investigated mechanisms of hepatic stellate cell (HSC) activation, which contributes to liver fibrogenesis. We aimed to determine whether activated HSCs increase glycolysis, which is regulated by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3), and whether this pathway might serve as a therapeutic target.
We performed studies with primary mouse HSCs, human LX2 HSCs, human cirrhotic liver tissues, rats and mice with liver fibrosis (due to bile duct ligation [BDL] or administration of carbon tetrachloride), and CPEB4-knockout mice. Glycolysis was inhibited in cells and mice by administration of a small molecule antagonist of PFKFB3 (3-[3-pyridinyl]-1-[4-pyridinyl]-2-propen-1-one [3PO]). Cells were transfected with small interfering RNAs that knock down PFKFB3 or CPEB4.
Up-regulation of PFKFB3 protein and increased glycolysis were early and sustained events during HSC activation and accompanied by increased expression of markers of fibrogenesis; incubation of HSCs with 3PO or knockdown of PFKFB3 reduced their activation and proliferation. Mice with liver fibrosis after BDL had increased hepatic PFKFB3; injection of 3PO immediately after the surgery prevented HSC activation and reduced the severity of liver fibrosis compared with mice given vehicle. Levels of PFKFB3 protein were increased in fibrotic liver tissues from patients compared with non-fibrotic liver. Up-regulation of PFKFB3 in activated HSCs did not occur via increased transcription, but instead via binding of CPEB4 to cytoplasmic polyadenylation elements within the 3'-untranslated regions of PFKFB3 messenger RNA. Knockdown of CPEB4 in LX2 HSCs prevented PFKFB3 overexpression and cell activation. Livers from CPEB4-knockout had decreased PFKFB3 and fibrosis after BDL or administration of carbon tetrachloride compared with wild-type mice.
Fibrotic liver tissues from patients and rodents (mice and rats) have increased levels of PFKFB3 and glycolysis, which are essential for activation of HSCs. Increased expression of PFKFB3 is mediated by binding of CPEB4 to its untranslated messenger RNA. Inhibition or knockdown of CPEB4 or PFKFB3 prevents HSC activation and fibrogenesis in livers of mice.
我们研究了肝星状细胞（HSC）激活的机制，这有助于肝纤维化的发生。我们旨在确定激活的 HSC 是否会增加糖酵解，糖酵解受 6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶-3（PFKFB3）调节，以及该途径是否可以作为治疗靶点。
我们对原代小鼠 HSC、人 LX2 HSC、人肝硬化组织、胆管结扎（BDL）或四氯化碳给药致肝纤维化的大鼠和小鼠以及 CPEB4 敲除小鼠进行了研究。通过给予 PFKFB3 的小分子拮抗剂 3-[3-吡啶基]-1-[4-吡啶基]-2-丙烯-1-酮（3PO）来抑制细胞和小鼠中的糖酵解。用敲低 PFKFB3 或 CPEB4 的小干扰 RNA 转染细胞。
PFKFB3 蛋白的上调和糖酵解的增加是 HSC 激活的早期和持续事件，伴随着纤维化标志物表达的增加；HSC 孵育用 3PO 或敲低 PFKFB3 可降低其激活和增殖。BDL 后发生肝纤维化的小鼠肝内 PFKFB3 增加；与给予载体的小鼠相比，手术后立即注射 3PO 可防止 HSC 激活并降低肝纤维化的严重程度。与非纤维化肝组织相比，患者的纤维化肝组织中 PFKFB3 蛋白水平升高。激活的 HSC 中 PFKFB3 的上调不是通过转录增加引起的，而是通过 CPEB4 与 PFKFB3 信使 RNA 的 3'-非翻译区中的细胞质多聚腺苷酸化元件结合引起的。在 LX2 HSC 中敲低 CPEB4 可防止 PFKFB3 过表达和细胞激活。与野生型小鼠相比，BDL 或四氯化碳给药后 CPEB4 敲除小鼠的肝脏中 PFKFB3 和纤维化减少。
患者和啮齿动物（小鼠和大鼠）的纤维化肝组织中 PFKFB3 和糖酵解水平升高，这对于 HSC 的激活是必需的。PFKFB3 的表达增加是由 CPEB4 与其未翻译的信使 RNA 结合介导的。抑制或敲低 CPEB4 或 PFKFB3 可防止小鼠肝脏中 HSC 的激活和纤维化。
