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GPBAR1/TGR5 mediates bile acid-induced cytokine expression in murine Kupffer cells

PLoS ONE (2014) 9(4)
DOI: 10.1371/journal.pone.0093567
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Abstract

GPBAR1/TGR5 is a novel plasma membrane-bound G protein-coupled bile acid (BA) receptor. BAs are known to induce the expression of inflammatory cytokines in the liver with unknown mechanism. Here we show that without other external stimuli, TGR5 activation alone induced the expression of interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α) in murine macrophage cell line RAW264.7 or murine Kupffer cells. The TGR5-mediated increase of pro-inflammatory cytokine expression was suppressed by JNK inhibition. Moreover, the induced pro-inflammatory cytokine expression in mouse liver by 1% cholic acid (CA) diet was blunted in JNK-/- mice. TGR5 activation by its ligands enhanced the phosphorylation levels, DNA-binding and trans-activities of c-Jun and ATF2 transcription factors. Finally, the induced pro-inflammatory cytokine expression in Kupffer cells by TGR5 activation correlated with the suppression of Cholesterol 7α-hydroxylase (Cyp7a1) expression in murine hepatocytes. These results suggest that TGR5 mediates the BA-induced pro-inflammatory cytokine production in murine Kupffer cells through JNK-dependent pathway. This novel role of TGR5 may correlate to the suppression of Cyp7a1 expression in hepatocytes and contribute to the delicate BA feedback regulation. © 2014 Lou et al.

Figures

  • Figure 2. JNK mediates cytokine expression after TGR5 activation. A. RAW264.7 cells were pretreated with 10 mM H89 for 30 min prior to treatment with 10 mM OA for 24 h. B. RAW264.7 cells were treated with different dose of 8CPT-2Me-cAMP for 24 h. 10 mM BFA (C) or SP (D) was incubated with RAW264.7 cells for 30 min before the treatment of 10 mM OA for 24 h. RNA from each treatment was extracted and subjected to RTPCR analysis. E&F. RAW264.7 cells (E) or Kupffer cells (F) were incubated with 10 mM OA for the indicated time points. G. RAW264.7 cells were incubated with 20 mM 23(s)-MeCDCA for the indicated time points. H. Peritoneal macrophages isolated from mice were incubated with 10 mM OA or 23(s)-MeCDCA for 30 min. The cell lysate was subjected to western blotting analysis for phosphorylation of JNK. Representative western blots are shown. I. Wild-type and JNK2/2 mice were fed with normal diet or 1% CA diet for 3 days. Total RNAs from mouse livers (n = 5) were extracted and subjected to RT-PCR analysis. Three independent experiments were performed. Data are expressed as the mean6SEM.*P,0.05 or **P,0.01 compared to the controls; #P,0.05 or ##P,0.01 compared to the OA as determined by two-way ANOVA. P values of panel B were calculated by one-way ANOVA. doi:10.1371/journal.pone.0093567.g002
  • Figure 3. TGR5 activation increases the activities of ATF2 and c-Jun. A. HEK293 cells transfected with pCRE-luc or pC/EBP-luc were treated with 23(s)-MeCDCA (10 mM) for 6 h. Luciferase and Renilla activities were measured and compared. *P,0.05 compared to the controls. B. RAW246.7 cells pretreated with 10 mM SP were treated with 23(s)-MeCDCA (10 mM) for 45 min. The cell lysate was subjected to western blotting analysis. C&D. Kupffer cells (C) or peritoneal macrophages (D) were incubated with 10 mM 23(s)-MeCDCA, OA for 1 h. The cell lysate was subjected to western blotting analysis. E. RAW246.7 cells were transfected with siRNAs then treated with OA. Total RNAs were subjected to RT-PCR analysis. Three independent experiments were performed. Data are expressed as the mean6SEM. *P,0.05, ***P,0.001 compared to the controls as determined by two-way ANOVA. doi:10.1371/journal.pone.0093567.g003
  • Figure 4. TGR5 activation enhances TNF-a promoter activity. A. pmTNF-a-luc reporter or control plasmid are transfected into cells, then treated with 10 mM 23(s)-MeCDCA or OA for 12 h. Data are expressed as mean6SEM, *P,0.05, **P,0.01 compared to the controls. B. RAW264.7 cells were transfected with pmTNF-a-luc or pmTNF-a-mut-luc reporter constructs, then treated with 23(s)-MeCDCA (10 mM) or OA (10 mM). Cell extracts were prepared and analyzed for luciferase and renilla activities. C. RAW264.7 were treated with 10 mM OA, 20 mM 23(s)-MeCDCA or vehicle control for 1 h. ChIP experiment analysis was performed to measure the binding activity of c-Jun or ATF to the TNF-a promoter. Values are given as fold differences relative to the IgG controls. Two independent experiments were performed. Data are expressed as the mean6SEM. *P,0.05, **P,0.01, ***P,0.001 compared to the controls as determined by two-way ANOVA. doi:10.1371/journal.pone.0093567.g004
  • Figure 5. The culture media of RAW264.7 cells treated with OA repress Cyp7a1 expression in hepatocytes. A. Hepatocytes were treated with the IL-1b (1 ng/ml) or TNF-a (10 ng/ml) for 24 h, RNA was prepared and subjected to RT-PCR analysis. M36b4 mRNA was used as a control. ***P,0.001 compared to the controls as determined by one-way ANOVA. B&C. Hepatocytes were treated with DMSO or OA for 12 h (B), or the cultured medium of hepatocytes was changed to conditional culture medium from RAW264.7 cells, which were pre-incubated for 12 h with DMSO or OA (C). D. RAW264.7 cells were treated with 10 mmol/L OA for 12 h. TNF-a protein concentration in culture medium was detected by ELISA. Three independent experiments were performed. Data are expressed as the mean6SEM. **P,0.01,***P,0.001 compared to the controls as determined by Student’s t-test. doi:10.1371/journal.pone.0093567.g005

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Lou, G., Ma, X., Fu, X., Meng, Z., Zhang, W., Wang, Y. D., … Huang, W. (2014). GPBAR1/TGR5 mediates bile acid-induced cytokine expression in murine Kupffer cells. PLoS ONE, 9(4). https://doi.org/10.1371/journal.pone.0093567

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