Winkler Rebecca, Lu Hong
Department of Pharmacology, SUNY Upstate Medical University, Syracuse, NY 13210, USA.
Mediators Inflamm. 2023 Nov 28;2023:4399998. doi: 10.1155/2023/4399998. eCollection 2023.
Literature and data mining found abnormal induction of chemokine (C-X-C motif) ligand 1 (CXCL1) and CXCL8 and down-regulation of CXCL2 in inflammatory liver diseases. This study was performed to understand the glucocorticoid receptor's (GR's) effects on chemokine and acute-phase protein expression in human liver, in settings of bacterial infection (modeled using LPS) or inflammation (modeled using TNF).
Primary human hepatocytes (PHH) were treated with combinations of tumor necrosis factor alpha (TNF), lipopolysaccharide (LPS), and dexamethasone (DEX) for 24 h, following which chemokine mRNA and protein expression were analyzed using qPCR and enzyme-linked immunosorbent assay assays. Dual luciferase assays were performed on transfected cell lines. Mutant CXCL2 promoters were used in dual luciferase assays to identify specific regions of the CXCL2 promoter affected by GR, TNF, or hepatocyte nuclear factor 4 (HNF4, a liver-enriched transcription factor).
In PHH from donor 1, GR strongly inhibited LPS-induced CXCL1 and CXCL8 translation and transcription, whereas CXCL2 transcription tended to increase with DEX treatment. In PHH from donor 2, DEX treatment inhibited protein expression and secretion of CXCL1 and CXCL8 induced by TNF and/or LPS, whereas CXCL2 upregulation was largely unaffected by DEX treatment. In nonliver HEK293T cells GR activity inhibited CXCL2 promoter activity. However, in liver-derived HEPG2 cells, GR induced CXCL2 promoter activity. A 407-base pair region upstream of CXCL2 promoter is necessary for full GR functionality in HEPG2 cells. TNF synergized with HNF4 in inducing CXCL2 promoter activity in HEPG2 cells.
GR's effects on chemokine expression are cell-type specific and chemokine specific. GR down-regulated CXCL1 and CXCL8 in different cell types, whereas the specific activation of CXCL2 in hepatocytes and down-regulation of CXCL2 in nonhepatocytes by GR appears due to cell-specific utilization of CXCL2 promoter. By specifically increasing GR activity in the liver, we may normalize chemokine imbalances and prevent sepsis in inflammatory liver diseases.
文献研究和数据挖掘发现，趋化因子（C-X-C基序）配体1（CXCL1）和CXCL8在炎症性肝病中异常诱导，而CXCL2表达下调。本研究旨在了解在细菌感染（使用脂多糖模拟）或炎症（使用肿瘤坏死因子模拟）情况下，糖皮质激素受体（GR）对人肝脏中趋化因子和急性期蛋白表达的影响。
将原代人肝细胞（PHH）用肿瘤坏死因子α（TNF）、脂多糖（LPS）和地塞米松（DEX）联合处理24小时，然后使用qPCR和酶联免疫吸附测定法分析趋化因子mRNA和蛋白表达。对转染细胞系进行双荧光素酶测定。在双荧光素酶测定中使用突变型CXCL2启动子，以鉴定受GR、TNF或肝细胞核因子4（HNF4，一种肝脏富集转录因子）影响的CXCL2启动子的特定区域。
在供体1的PHH中，GR强烈抑制LPS诱导的CXCL1和CXCL8翻译及转录，而DEX处理后CXCL2转录有增加趋势。在供体2的PHH中，DEX处理抑制TNF和/或LPS诱导的CXCL1和CXCL8蛋白表达及分泌，而CXCL2上调在很大程度上不受DEX处理影响。在非肝脏的HEK293T细胞中，GR活性抑制CXCL2启动子活性。然而，在肝脏来源的HEPG2细胞中，GR诱导CXCL2启动子活性。CXCL2启动子上游407个碱基对区域对于HEPG2细胞中GR的完整功能是必需的。在HEPG2细胞中，TNF与HNF4协同诱导CXCL2启动子活性。
GR对趋化因子表达的影响具有细胞类型特异性和趋化因子特异性。GR在不同细胞类型中下调CXCL1和CXCL8，而GR对肝细胞中CXCL2的特异性激活及对非肝细胞中CXCL2的下调，似乎是由于CXCL2启动子的细胞特异性利用。通过特异性增加肝脏中的GR活性，我们可能使趋化因子失衡正常化，并预防炎症性肝病中的脓毒症。
