Bile acids have been shown to be important regulatory molecules for cells in the liver and gastrointestinal tract. They can activate various cell signaling pathways including the extracellular regulated kinase (ERK)1/2 and AKT as well as the G-protein coupled receptor (GPCR), TGR5/M-BAR. Activation of the ERK1/2 and AKT signaling pathways by conjugated bile acids has been reported to be pertussis toxin (PTX) and dominant negative Gαi sensitive in primary rodent hepatocytes. However, the GPCRs responsible for activation of these pathways have not been identified. Screening GPCRs in the lipid activated phylogenetic family, expressed in HEK293 cells, identified sphingosine 1-phosphate receptor 2 (S1P2) as being activated by taurocholate (TCA). TCA, taurodeoxycholic acid (TDCA), tauroursodeoxycholic acid (TUDCA), glycocholic acid (GCA), glycodeoxycholic acid (GDCA), and S1P-induced activation of ERK1/2 and AKT were significantly inhibited by JTE-013, a S1P2 antagonist, in primary rat hepatocytes. JTE-013 significantly inhibited hepatic ERK1/2 and AKT activation as well as short heterodimeric partner (SHP) mRNA induction by TCA in the chronic bile fistula rat. Knock down of the expression of S1P2 by a recombinant lentivirus encoding S1P2 shRNA, markedly inhibited the activation of ERK1/2 and AKT by TCA and S1P in rat primary hepatocytes. Primary hepatocytes prepared from S1P2 knock out (S1P2−/−) mice were significantly blunted in the activation of the ERK1/2 and AKT pathways by TCA. Structural modeling of the S1P receptors indicated that only S1P2 can accommodate TCA binding. In summary, all these data support the hypothesis that conjugated bile acids activate the ERK1/2 and AKT signaling pathways primarily via S1P2 in primary rodent hepatocytes.

Cholangiocarcinoma (CCA) is an often fatal primary malignancy of the intra- and extrahepatic biliary tract that is commonly associated with chronic cholestasis and significantly elevated levels of primary and conjugated bile acids (CBAs), which are correlated with bile duct obstruction (BDO). BDO has also recently been shown to promote CCA progression. However, whereas there is increasing evidence linking chronic cholestasis and abnormal bile acid profiles to CCA development and progression, the specific mechanisms by which bile acids may be acting to promote cholangiocarcinogenesis and invasive biliary tumor growth have not been fully established. Recent studies have shown that CBAs, but not free bile acids, stimulate CCA cell growth, and that an imbalance in the ratio of free to CBAs may play an important role in the tumorigenesis of CCA. Also, CBAs are able to activate extracellular signal-regulated kinase (ERK)1/2- and phosphatidylinositol-3-kinase/protein kinase B (AKT)-signaling pathways through sphingosine 1-phosphate receptor 2 (S1PR2) in rodent hepatocytes. In the current study, we demonstrate S1PR2 to be highly expressed in rat and human CCA cells, as well as in human CCA tissues. We further show that CBAs activate the ERK1/2- and AKT-signaling pathways and significantly stimulate CCA cell growth and invasion in vitro. Taurocholate (TCA)-mediated CCA cell proliferation, migration, and invasion were significantly inhibited by JTE-013, a chemical antagonist of S1PR2, or by lentiviral short hairpin RNA silencing of S1PR2. In a novel organotypic rat CCA coculture model, TCA was further found to significantly increase the growth of CCA cell spheroidal/“duct-like” structures, which was blocked by treatment with JTE-013. Conclusion: Our collective data support the hypothesis that CBAs promote CCA cell-invasive growth through S1PR2.

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Brain inflammation has a critical role in the pathophysiology of brain diseases. Microglia, the resident immune cells in the brain, play an important role in brain inflammation, while brain mast cells are the "first responder" in the injury rather than microglia. Functional aspects of mast cell-microglia interactions remain poorly understood. Our results demonstrated that site-directed injection of the "mast cell degranulator" compound 48/80 (C48/80) in the hypothalamus induced mast cell degranulation, microglial activation, and inflammatory factor production, which initiated the acute brain inflammatory response. "Mast cell stabilizer" disodium cromoglycate (cromolyn) inhibited this effect, including decrease of inflammatory cytokines, reduced microglial activation, inhibition of MAPK and AKT pathways, and repression of protein expression of histamine receptor 1 (HR), histamine receptor 4 (HR), protease-activated receptor 2 (PAR2), and toll-like receptor 4 (TLR4) in microglia. We also demonstrated that C48/80 had no effect on microglial activation in mast cell-deficient Kit mice. These results implicate that activated brain mast cells trigger microglial activation and stabilization of mast cell inhibits microglial activation-induced central nervous system (CNS) inflammation. Interactions between mast cells and microglia could constitute a new and unique therapeutic target for CNS immune inflammation-related diseases.

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Author contributions: Wen-Ming Chu conceived and directed the project and performed a large body of experiments.

Inflammation plays a pivotal role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Berberine (BBR), an isoquinoline alkaloid isolated from many medicinal herbs, has been used to treat various diseases including liver diseases for hundreds of years. Our previous studies have shown that BBR inhibits high fat‐diet‐induced steatosis and inflammation in rat model. However, the underlying molecular mechanisms remain to be identified. This study was to identify the potential mechanism by which BBR inhibits free fatty acid (FFA) and LPS‐induced inflammatory response in mouse macrophages. Methods Mouse RAW2674.1 macrophages were treated with palmitic acid (PA) or LPS or PA+ LPS with or without different concentrations of BBR (0–10 μM) for different periods (0–24h). The mRNA and protein levels of IL‐1β, IL‐6, TNF‐α, MCP‐1, COX‐2, and ER stress genes (CHOP, ATF4, XBP‐1) were detected by real time RT‐PCR, Western blot and ELISA, respectively. Results BBR significantly inhibited PA‐ and LPS‐induced activation of ER stress and expression of proinflammatory cytokines (IL‐1β, IL‐6) and COX‐2. PA/LPS‐mediated activation of ERK1/2 was inhibited by BBR in a dose‐dependent manner. Conclusion BBR inhibits PA/LPS‐induced inflammatory responses through modulating ER stress‐mediated ERK1/2 activation in macrophages. Support or Funding Information This work was supported by VA Merit Award I01BX004033 and 1I01BX001390, VA Career Scientist Award IK6BX004477 and National Institutes of Health Grant R01 DK104893 and R01DK‐057543 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Background: Hypoxia is a risk factor for non-alcoholic fatty liver diseases, leading to permanent imbalance of liver lipid homeostasis and steatohepatitis. The current study examined the effect of HIF-2α, an oxygen-sensitive heterodimeric transcription factor, on hypoxia-induced dysregulation of lipid metabolism in HepG2 cells. Methods: Studies were conducted in C57BL/6 male mice and human HepG2 cells under hypoxic conditions, transfected with HIF-2α-targeted shRNA. The mRNA and protein expressions of key genes relevant to lipid metabolism were determined via RT-qPCR and western blot, respectively. Intracellular lipid accumulation was determined by Nile red, filipin staining and quantitative assay kits. Results: HIF-2α protein was quantified in both HepG2 cells and C57BL/6 mice under hypoxic conditions. Intracellular lipid accumulation and increased lipid levels induced by hypoxia were significantly reduced by silence of HIF-2α expression, associated with reversed expression of ABCA1 and ADRP, key genes in involved cholesterol excretion and fatty acid uptake respectively. However, HIF-2α had no effect on enzymatic activity and expression of key genes involved in fatty acid β-oxidation or cholesterol metabolism. Conclusion: Inhibition of HIF-2α protein reversed lipid metabolism dysregulation induced by acute hypoxia in HepG2 cells, which suggested that HIF-2α signaling may be relevant to oxygen-dependent lipid homeostasis in the liver.