Wang Xianghu, Wang Yuanguo, Bai Bing, Shaha Aurpita, Bao Wenming, He Lianping, Wang Tian, Kitange Gaspar J, Kang Ningling
Tumor Microenvironment and Metastasis, The Hormel Institute, University of Minnesota, Austin, Minnesota.
School of Medicine, Taizhou University, Taizhou, Zhejiang, P. R. China.
Cell Mol Gastroenterol Hepatol. 2025;19(3):101429. doi: 10.1016/j.jcmgh.2024.101429. Epub 2024 Nov 13.
BACKGROUND & AIMS: Transforming growth factor (TGF)β1 induces plasma membrane (PM) accumulation of glucose transporter 1 (Glut1) required for glycolysis of hepatic stellate cells (HSCs) and HSC activation. This study aimed to understand how Glut1 is anchored/docked onto the PM of HSCs.
HSC expression of protein kinase M zeta isoform (PKMζ) was detected by reverse transcription polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescence. PKMζ level was manipulated by short hairpin RNA (shRNA) or overexpression; HSC activation was assessed by cell expression of activation markers; PM Glut1, glucose uptake, and glycolysis of HSCs were analyzed by biotinylation, 2-NBDG-based assay, and Seahorse Glycolysis Stress Test. Phospho-mutants of vasodilator-stimulated phosphorylated protein (VASP) were created by site-directed mutagenesis. TGFβ transcriptome was obtained by RNA sequencing. Single-cell RNA sequencing datasets and immunofluorescence were leveraged to analyze PKMζ expression in cancer-associated fibroblasts (CAFs) of colorectal liver metastases. Function of HSC PKMζ was determined by tumor/HSC co-implantation study.
Primary human and murine HSCs express PKMζ, but not full-length PKCζ. PKMζ knockdown suppresses, whereas PKMζ overexpression potentiates PM accumulation of Glut1, glycolysis, and HSC activation induced by TGFβ1. Mechanistically, PKMζ binds to and induces VASP phosphorylation at serines 157 and 239 facilitating anchoring/docking of Glut1 onto the PM of HSCs. PKMζ expression is increased in the CAFs of murine and patient colorectal liver metastases compared with quiescent HSCs. Targeting PKMζ suppresses transcriptome, CAF activation of HSCs, and colorectal tumor growth in mice.
Because HSCs are also a major contributor of liver fibrosis, our data highlight PKMζ and VASP as targets to inhibit metabolic reprogramming, HSC activation, liver fibrosis, and the pro-metastatic microenvironment of the liver.
转化生长因子（TGF）β1可诱导肝星状细胞（HSC）糖酵解及HSC激活所需的葡萄糖转运蛋白1（Glut1）在质膜（PM）上积聚。本研究旨在了解Glut1如何锚定/停靠在HSC的质膜上。
通过逆转录聚合酶链反应（RT-PCR）、蛋白质印迹法和免疫荧光法检测HSC中蛋白激酶Mζ亚型（PKMζ）的表达。通过短发夹RNA（shRNA）或过表达来调控PKMζ水平；通过激活标志物的细胞表达评估HSC激活情况；通过生物素化、基于2-NBDG的检测方法和海马糖酵解应激试验分析HSC的质膜Glut1、葡萄糖摄取和糖酵解。通过定点诱变构建血管舒张刺激磷蛋白（VASP）的磷酸化突变体。通过RNA测序获得TGFβ转录组。利用单细胞RNA测序数据集和免疫荧光分析结直肠癌肝转移癌相关成纤维细胞（CAF）中PKMζ的表达。通过肿瘤/HSC共植入研究确定HSC中PKMζ的功能。
原代人源和鼠源HSC表达PKMζ，但不表达全长PKCζ。PKMζ敲低可抑制，而PKMζ过表达可增强TGFβ1诱导的Glut1质膜积聚、糖酵解和HSC激活。机制上，PKMζ与VASP结合并诱导其丝氨酸157和239位点磷酸化，促进Glut1锚定/停靠在HSC的质膜上。与静止的HSC相比，鼠源和患者结直肠癌肝转移灶的CAF中PKMζ表达增加。靶向PKMζ可抑制转录组、CAF对HSC的激活以及小鼠结直肠癌肿瘤生长。
由于HSC也是肝纤维化的主要促成因素，我们的数据突出了PKMζ和VASP作为抑制代谢重编程、HSC激活、肝纤维化及肝脏促转移微环境的靶点。
