Role of toll-like receptors in changes in gene expression and NF-kappa B activation in mouse hepatocytes stimulated with lipopolysaccharide

Abstract

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-kappa B 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.

FIG. 1.

Expression of TLR, MyD88, and MD-2 in human hepatocytes by RT-PCR. (Top panel) Total RNA from both cultured human primary hepatocytes and HepG2 cells were reverse transcribed with an oligo(dT) primer, followed by PCR with primers specific to TLR1 to TLR9, MyD88, or β-actin (as a loading control). (Bottom panel) Total RNA from freshly isolated hepatocytes from saline- or LPS-injected (2.5 mg/kg of body weight) (intraperitoneally [i.p.] administered at 24 h) C3H/HeN or C3H/HeJ mice were subjected to RT-PCR and analysis of MD-2 and β-actin.

FIG. 2.

LPS activates ERK1/2 in mouse hepatocytes. (A) Total cell lysates from LPS (500 ng/ml)-stimulated hepatocytes, treated for the indicated time periods, were subjected to Western blotting analysis with anti-phospho-ERK1/2 antibody. The blot was then stripped and immunostained with anti-total ERK1/2 antibody. (B) The films were scanned (HP ScanJet 6100C scanner), and band intensity was analyzed by using the digitized imaging analysis software UN-SCAN-IT gel (Silk Scientific). Multiple exposures were performed to ascertain that the films used for scanning were within linear-exposure range. Data (means ± SE [error bars]) are combined results from nine independent experiments (∗, P < 0.05 [for C57BL/10SnJ versus C57BL/10ScN at 5, 30, and 60 min]).

FIG. 3.

Effect of LPS on NF-κB and AP-1 binding and transcriptional activation in hepatocytes. (A) Hepatocytes from C3H/HeN, C3H/HeJ mice were treated with the indicated concentrations of LPS for 60 min or for the indicated time periods with LPS at 500 ng/ml. Nuclear extracts were prepared and the binding of nuclear proteins to a ³²P-labeled NF-κB consensus oligonucleotide was analyzed by EMSA. Data are representative of at least three independent experiments. (B) Hepatocytes from C57BL/10SnJ, C57BL/10ScN mice were treated with the indicated concentrations of LPS for 60 min or for the indicated time periods with LPS at 500 ng/ml. Binding of nuclear proteins to a ³²P-labeled NF-κB or AP-1 consensus oligonucleotide was analyzed by EMSA. Data are representative of at least three independent experiments. (C and D) Hepatocytes from C3H/HeN or C3H/HeJ mice on six-well plates were transiently transfected with an NF-κB (pELAM, 0.1 μg/well) or AP-1 (AP-1-luc, 0.2 μg/well) luciferase reporter vector and cotransfected with a β-galactosidase reporter vector (pIEPlacZ, 0.5 μg/well). The data (means ± SE [error bars]) shown here are a representative of six independent experiments with similar results (∗, P < 0.05 [for C3H/HeN versus C3H/HeJ]).

FIG. 4.

NF-κB activation by LPS in mouse hepatocytes infected with Ad.mTLR4. (Top panel) Hepatocytes from C3H/HeN or C3H/HeJ mice were infected with Ad.mTLR4 (multiplicity of infection = 10) or Ad.LacZ for 3 h and recovered overnight. At 24 h after the start of infection, cells were treated with the indicated concentrations of LPS for 60 min. Nuclear extracts were prepared, and the binding of nuclear proteins to NF-κB consensus oligonucleotide was analyzed by EMSA. Data are representative of three independent experiments. (Bottom panel) Total RNA (10 μg per sample) was subjected to Northern blot analysis and hybridized with a ³²P-labeled rat TLR4 cDNA.