Sidhu Jaspreet S, Liu Fei, Omiecinski Curtis J
Department of Environmental Health, University of Washington, Seattle, WA 98105, USA.
Exp Cell Res. 2004 Jan 15;292(2):252-64. doi: 10.1016/j.yexcr.2003.09.001.
We used a serum-free, highly defined primary hepatocyte culture model to investigate the mechanisms whereby dexamethasone (Dex) and extracellular matrix (ECM) coordinate cell differentiation and transcriptional responsiveness to the inducer, phenobarbital (PB). Low nanomolar levels of Dex and dilute concentrations of ECM overlay were essential in the maintenance of normal hepatocyte physiology, as assessed by cell morphology, LDH release, expression of the hepatic nuclear factors C/EBPalpha, -beta, -gamma, HNF-1alpha, -1beta, -4alpha, and RXRalpha, expression of prototypical hepatic marker genes, including albumin and transferrin, and ultimately, cellular capacity to respond to PB. The loss of hepatocyte integrity produced by deficiency of these components correlated with the activation of several stress signaling pathways including the MAPK, SAPK/JNK, and c-Jun signaling pathways, with resulting nuclear recruitment of the activated protein-1 (AP-1) complex. In Dex-deficient cultures, normal cellular function, including the PB induction response, was largely restored in a dose-dependent manner by reintroduction of nanomolar additions of the hormone, in the presence of ECM. Our results demonstrate critical and cooperative roles for Dex and ECM in establishing hepatocyte integrity and in the coordination of an array of liver-specific functions. These studies further establish the PB gene induction response as an exceptionally sensitive indicator of hepatocyte differentiation status.
我们使用无血清、高度明确的原代肝细胞培养模型，来研究地塞米松（Dex）和细胞外基质（ECM）协调细胞分化以及对诱导剂苯巴比妥（PB）转录反应的机制。通过细胞形态、乳酸脱氢酶（LDH）释放、肝核因子C/EBPα、-β、-γ、肝细胞核因子-1α、-1β、-4α和视黄酸X受体α（RXRα）的表达、包括白蛋白和转铁蛋白在内的典型肝脏标志物基因的表达，以及最终细胞对PB的反应能力评估，低纳摩尔水平的Dex和稀释浓度的ECM覆盖对于维持正常肝细胞生理至关重要。这些成分缺乏导致的肝细胞完整性丧失与包括丝裂原活化蛋白激酶（MAPK）、应激激活蛋白激酶/应激活化蛋白激酶（SAPK/JNK）和c-Jun信号通路在内的几种应激信号通路的激活相关，从而导致活化蛋白-1（AP-1）复合物的核募集。在缺乏Dex的培养物中，在存在ECM的情况下，通过重新添加纳摩尔剂量的激素，正常细胞功能（包括PB诱导反应）在很大程度上以剂量依赖的方式得以恢复。我们的结果表明，Dex和ECM在建立肝细胞完整性以及协调一系列肝脏特异性功能方面发挥着关键且协同的作用。这些研究进一步确立了PB基因诱导反应作为肝细胞分化状态的一个极其敏感的指标。
