Role of Toll-Like Receptors in Changes in Gene Expression and NF-κB Activation in Mouse Hepatocytes Stimulated with Lipopolysaccharide

The liver is an important site of host-microbe interaction. Although hepatocytes have been reported to be responsive to lipopolysaccharide (LPS), the global gene expression changes by LPS and mechanism(s) by which LPS stimulates cultured hepatocytes remain uncertain. Cultures of primary mouse hepatocytes were incubated with LPS to assess its effects on the global gene expression, hepatic transcription factors, and mitogen-activated protein (MAP) kinase activation. DNA microarray analysis indicated that LPS modulates the selective expression of more than 80 genes and expressed sequence tags. We have shown previously that hepatocytes express CD14, which is required both for uptake and responsiveness to LPS. In other cells, responsiveness to microbial products requires expression of Toll-like receptors (TLR) and their associated accessory molecules. Hepatocytes expressed TLR1 through TLR9 as well as MyD88 and MD-2 transcripts, as shown by reverse transcriptase PCR analysis, indicating that hepatocytes express all known microbe recognition molecules. The MAP kinase extracellular signal-regulated kinase 1/2 was phosphorylated in response to LPS in mouse hepatocytes, and the levels of phosphorylation were lower in hepatocytes from TLR4-null mice. NF-κB activation was reduced in TLR4-mutant or -null hepatocytes compared to control hepatocytes, and this defect was partially restored by adenoviral transduction of mouse TLR4. Thus, hepatocytes respond to nanogram concentrations of LPS through a TLR4 response pathway.

肝脏是宿主与微生物互作的关键位点。尽管已有研究证实肝细胞可对脂多糖（LPS）产生应答，但脂多糖所引发的全基因表达改变，以及脂多糖刺激培养肝细胞的具体分子机制，目前仍未明确。我们将原代小鼠肝细胞培养物与脂多糖共孵育，以评估其对全基因表达、肝转录因子以及丝裂原活化蛋白（MAP）激酶激活的影响。DNA微阵列分析结果显示，脂多糖可调控超过80个基因及表达序列标签的选择性表达。我们此前已证实，肝细胞表达CD14，该分子是脂多糖摄取与应答所必需的。在其他细胞中，对微生物产物的应答依赖于Toll样受体（TLR）及其相关辅助分子的表达。通过逆转录聚合酶链式反应分析证实，肝细胞表达TLR1至TLR9，以及MyD88与MD-2转录本，表明肝细胞表达所有已知的微生物识别分子。小鼠肝细胞在脂多糖刺激下可发生细胞外信号调节激酶1/2的磷酸化，而TLR4敲除小鼠的肝细胞中该磷酸化水平更低。与对照肝细胞相比，TLR4突变或敲除的肝细胞中核因子κB（NF-κB）的激活水平有所降低，而通过腺病毒转导小鼠TLR4可部分修复该缺陷。综上，肝细胞可通过TLR4应答通路对纳克级浓度的脂多糖产生应答。