Runping Liu

Conjugated bile acids promote cholangiocarcinoma cell invasive growth through activation of sphingosine 1-phosphate receptor 2.

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. (Hepatology 2014;60:908–918)

Conjugated bile acid–activated S1P receptor 2 is a key regulator of sphingosine kinase 2 and hepatic gene expression

Bile acids are important hormones during the feed/fast cycle, allowing the liver to coordinately regulate nutrient metabolism. How they accomplish this has not been fully elucidated. Conjugated bile acids activate both the ERK1/2 and AKT signaling pathways via sphingosine 1‐phosphate receptor 2 (S1PR2) in rodent hepatocytes and in vivo. Here, we report that feeding mice a high‐fat diet, infusion of taurocholate into the chronic bile fistula rat, or overexpression of the gene encoding S1PR2 in mouse hepatocytes significantly upregulated hepatic sphingosine kinase 2 (SphK2) but not SphK1. Key genes encoding nuclear receptors/enzymes involved in nutrient metabolism were significantly downregulated in livers of S1PR2–/– and SphK2–/– mice. In contrast, overexpression of the gene encoding S1PR2 in primary mouse hepatocytes differentially increased SphK2, but not SphK1, and mRNA levels of key genes involved in nutrient metabolism. Nuclear levels of sphingosine‐1‐phosphate, an endogenous inhibitor of histone deacetylases 1 and 2, as well as the acetylation of histones H3K9, H4K5, and H2BK12 were significantly decreased in hepatocytes prepared from S1PR2–/– and SphK2–/– mice. Conclusion: Both S1PR2–/– and SphK2–/– mice rapidly developed fatty livers on a high‐fat diet, suggesting the importance of conjugated bile acids, S1PR2, and SphK2 in regulating hepatic lipid metabolism. (Hepatology 2015;61:1216–1226)

Colonic inflammation and secondary bile acids in alcoholic cirrhosis

Alcohol abuse with/without cirrhosis is associated with an impaired gut barrier and inflammation. Gut microbiota can transform primary bile acids (BA) to secondary BAs, which can adversely impact the gut barrier. The purpose of this study was to define the effect of active alcohol intake on fecal BA levels and ileal and colonic inflammation in cirrhosis. Five age-matched groups {two noncirrhotic (control and drinkers) and three cirrhotic [nondrinkers/nonalcoholics (NAlc), abstinent alcoholic for >3 mo (AbsAlc), currently drinking (CurrAlc)]} were included. Fecal and serum BA analysis, serum endotoxin, and stool microbiota using pyrosequencing were performed. A subgroup of controls, NAlc, and CurrAlc underwent ileal and sigmoid colonic biopsies on which mRNA expression of TNF-α, IL-1β, IL-6, and cyclooxygenase-2 (Cox-2) were performed. One hundred three patients (19 healthy, 6 noncirrhotic drinkers, 10 CurrAlc, 38 AbsAlc, and 30 NAlc, age 56 yr, median MELD: 10.5) were included. Five each of healthy, CurrAlc, and NAlc underwent ileal/colonic biopsies. Endotoxin, serum-conjugated DCA and stool total BAs, and secondary-to-primary BA ratios were highest in current drinkers. On biopsies, a significantly higher mRNA expression of TNF-α, IL-1β, IL-6, and Cox-2 in colon but not ileum was seen in CurrAlc compared with NAlc and controls. Active alcohol use in cirrhosis is associated with a significant increase in the secondary BA formation compared with abstinent alcoholic cirrhotics and nonalcoholic cirrhotics. This increase in secondary BAs is associated with a significant increase in expression of inflammatory cytokines in colonic mucosa but not ileal mucosa, which may contribute to alcohol-induced gut barrier injury.

Taurocholate Induces Cyclooxygenase-2 Expression via the Sphingosine 1-phosphate Receptor 2 in a Human Cholangiocarcinoma Cell Line

Background: Cyclooxygenase-2 (COX-2) and sphingosine 1-phosphate receptor 2 (S1PR2) are highly expressed in human cholangiocarcinoma (CCA) and taurocholate (TCA) promotes CCA cell growth via S1PR2. Results: TCA-mediated activation of S1PR2 contributed to COX-2 expression and CCA cell growth. Conclusion: S1PR2 plays a critical role in TCA-induced COX-2 expression and CCA growth. Significance: S1PR2 represents a novel therapeutic target for CCA. Cholangiocarcinoma (CCA) is a rare, but highly malignant primary hepatobiliary cancer with a very poor prognosis and limited treatment options. Our recent studies reported that conjugated bile acids (CBAs) promote the invasive growth of CCA via activation of sphingosine 1-phosphate receptor 2 (S1PR2). Cyclooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) is the most abundant prostaglandin in various human malignancies including CCA. Previous studies have indicated that COX-2 was highly expressed in CCA tissues, and the survival rate of CCA patients was negatively associated with high COX-2 expression levels. It has also been reported that CBAs induce COX-2 expression, whereas free bile acids inhibit COX-2 expression in CCA mouse models. However, the underlying cellular mechanisms and connection between S1PR2 and COX-2 expression in CCA cells have still not been fully elucidated. In the current study, we examined the role of S1PR2 in conjugated bile acid (taurocholate, (TCA))-induced COX-2 expression in a human HuCCT1 CCA cell line and further identified the potential underlying cellular mechanisms. The results indicated that TCA-induced invasive growth of human CCA cells was correlated with S1PR2-medated up-regulation of COX-2 expression and PGE2 production. Inhibition of S1PR2 activation with chemical antagonist (JTE-013) or down-regulation of S1PR2 expression with gene-specific shRNA not only reduced COX-2 expression, but also inhibited TCA-induced activation of EGFR and the ERK1/2/Akt-NF-κB signaling cascade. In conclusion, S1PR2 plays a critical role in TCA-induced COX-2 expression and CCA growth and may represent a novel therapeutic target for CCA.

The role of sphingosine 1-phosphate receptor 2 in bile-acid–induced cholangiocyte proliferation and cholestasis-induced liver injury in mice

Bile duct obstruction is a potent stimulus for cholangiocyte proliferation, especially for large cholangiocytes. Our previous studies reported that conjugated bile acids (CBAs) activate the protein kinase B (AKT) and extracellular signal‐regulated kinase 1 and 2 (ERK1/2) signaling pathways through sphingosine 1‐phosphate receptor (S1PR) 2 in hepatocytes and cholangiocarcinoma cells. It also has been reported that taurocholate (TCA) promotes large cholangiocyte proliferation and protects cholangiocytes from bile duct ligation (BDL)‐induced apoptosis. However, the role of S1PR2 in bile‐acid–mediated cholangiocyte proliferation and cholestatic liver injury has not been elucidated. Here, we report that S1PR2 is the predominant S1PR expressed in cholangiocytes. Both TCA‐ and sphingosine‐1‐phosphate (S1P)‐induced activation of ERK1/2 and AKT were inhibited by JTE‐013, a specific antagonist of S1PR2, in cholangiocytes. In addition, TCA‐ and S1P‐induced cell proliferation and migration were inhibited by JTE‐013 and a specific short hairpin RNA of S1PR2, as well as chemical inhibitors of ERK1/2 and AKT in mouse cholangiocytes. In BDL mice, expression of S1PR2 was up‐regulated in whole liver and cholangiocytes. S1PR2 deficiency significantly reduced BDL‐induced cholangiocyte proliferation and cholestatic injury, as indicated by significant reductions in inflammation and liver fibrosis in S1PR2 knockout mice. Treatment of BDL mice with JTE‐013 significantly reduced total bile acid levels in serum and cholestatic liver injury. Conclusion: This study suggests that CBA‐induced activation of S1PR2‐mediated signaling pathways plays a critical role in obstructive cholestasis and may represent a novel therapeutic target for cholestatic liver diseases. (Hepatology 2017;65:2005‐2018).

The role of long noncoding RNA H19 in gender disparity of cholestatic liver injury in multidrug resistance 2 gene knockout mice

The multidrug resistance 2 knockout (Mdr2–/–) mouse is a well‐established model of cholestatic cholangiopathies. Female Mdr2–/– mice develop more severe hepatobiliary damage than male Mdr2–/– mice, which is correlated with a higher proportion of taurocholate in the bile. Although estrogen has been identified as an important player in intrahepatic cholestasis, the underlying molecular mechanisms of gender‐based disparity of cholestatic injury remain unclear. The long noncoding RNA H19 is an imprinted, maternally expressed, and estrogen‐targeted gene, which is significantly induced in human fibrotic/cirrhotic liver and bile duct–ligated mouse liver. However, whether aberrant expression of H19 accounts for gender‐based disparity of cholestatic injury in Mdr2–/– mice remains unknown. The current study demonstrated that H19 was markedly induced (∼200‐fold) in the livers of female Mdr2–/– mice at advanced stages of cholestasis (100 days old) but not in age‐matched male Mdr2–/– mice. During the early stages of cholestasis, H19 expression was minimal. We further determined that hepatic H19 was mainly expressed in cholangiocytes, not hepatocytes. Both taurocholate and estrogen significantly activated the extracellular signal–regulated kinase 1/2 signaling pathway and induced H19 expression in cholangiocytes. Knocking down H19 not only significantly reduced taurocholate/estrogen‐induced expression of fibrotic genes and sphingosine 1‐phosphate receptor 2 in cholangiocytes but also markedly reduced cholestatic injury in female Mdr2–/– mice. Furthermore, expression of small heterodimer partner was substantially inhibited at advanced stages of liver fibrosis, which was reversed by H19 short hairpin RNA in female Mdr2–/– mice. Similar findings were obtained in human primary sclerosing cholangitis liver samples. Conclusion: H19 plays a critical role in the disease progression of cholestasis and represents a key factor that causes the gender disparity of cholestatic liver injury in Mdr2–/– mice. (Hepatology 2017;66:869–884).

Gut microbiota drive the development of neuroinflammatory response in cirrhosis in mice

The mechanisms behind the development of hepatic encephalopathy (HE) are unclear, although hyperammonemia and systemic inflammation through gut dysbiosis have been proposed. The aim of this work was to define the individual contribution of hyperammonemia and systemic inflammation on neuroinflammation in cirrhosis using germ‐free (GF) and conventional mice. GF and conventional C57BL/6 mice were made cirrhotic using CCl4 gavage. These were compared to their noncirrhotic counterparts. Intestinal microbiota, systemic and neuroinflammation (including microglial and glial activation), serum ammonia, intestinal glutaminase activity, and cecal glutamine content were compared between groups. GF cirrhotic mice developed similar cirrhotic changes to conventional mice after 4 extra weeks (16 vs. 12 weeks) of CCl4 gavage. GF cirrhotic mice exhibited higher ammonia, compared to GF controls, but this was not associated with systemic or neuroinflammation. Ammonia was generated through increased small intestinal glutaminase activity with concomitantly reduced intestinal glutamine levels. However, conventional cirrhotic mice had intestinal dysbiosis as well as systemic inflammation, associated with increased serum ammonia, compared to conventional controls. This was associated with neuroinflammation and glial/microglial activation. Correlation network analysis in conventional mice showed significant linkages between systemic/neuroinflammation, intestinal microbiota, and ammonia. Specifically beneficial, autochthonous taxa were negatively linked with brain and systemic inflammation, ammonia, and with Staphylococcaceae, Lactobacillaceae, and Streptococcaceae. Enterobacteriaceae were positively linked with serum inflammatory cytokines. Conclusion: Gut microbiota changes drive development of neuroinflammatory and systemic inflammatory responses in cirrhotic animals. (Hepatology 2016;64:1232‐1248)

Continued Alcohol Misuse in Human Cirrhosis is Associated with an Impaired Gut-Liver Axis

BACKGROUND Cirrhosis and alcohol can independently affect the gut-liver axis with systemic inflammation. However, their concurrent impact in humans is unclear. METHODS Our aim was to determine the effect of continued alcohol misuse on the gut-liver axis in cirrhotic patients. Age- and MELD-balanced cirrhotic patients who were currently drinking (Alc) or abstinent (NAlc) and healthy controls underwent serum and stool collection. A subset underwent upper endoscopy and colonoscopy for biopsies and duodenal fluid collection. The groups were compared regarding (i) inflammation/intestinal barrier: systemic tumor necrosis factor levels, intestinal inflammatory cytokine (duodenum, ileum, sigmoid), and ileal antimicrobial peptide expression; (ii) microbiota composition: 16SrRNA sequencing of duodenal, ileal, and colonic mucosal and fecal microbiota; and (iii) microbial functionality: duodenal fluid and fecal bile acid (BA) profile (conjugation and dehydroxylation status), intestinal BA transporter (ASBT, FXR, FGF-19, SHP) expression, and stool metabolomics using gas chromatography/mass spectrometry. RESULTS Alc patients demonstrated a significant duodenal, ileal, and colonic mucosal and fecal dysbiosis, compared to NAlc and controls with lower autochthonous bacterial taxa. BA profile skewed toward a potentially toxic profile (higher secondary and glycine-conjugated BAs) in duodenal fluid and stool in Alc patients. Duodenal fluid demonstrated conjugated secondary BAs only in the Alc group. There was a greater expression of all ileal BA transporters in Alc patients. This group also showed higher endotoxemia, systemic and ileal inflammatory expression, and lower amino acid and bioenergetic-associated metabolites, without change in antimicrobial peptide expression. CONCLUSIONS Despite cirrhosis, continued alcohol misuse predisposes patients to widespread dysbiosis with alterations in microbial functionality such as a toxic BA profile, which can lead to intestinal and systemic inflammation.

Reduction of the HIV protease inhibitor-induced ER stress and inflammatory response by raltegravir in macrophages.

Background HIV protease inhibitor (PI), the core component of highly active antiretroviral treatment (HAART) for HIV infection, has been implicated in HAART-associated cardiovascular complications. Our previous studies have demonstrated that activation of endoplasmic reticulum (ER) stress is linked to HIV PI-induced inflammation and foam cell formation in macrophages. Raltegravir is a first-in-its-class HIV integrase inhibitor, the newest class of anti-HIV agents. We have recently reported that raltegravir has less hepatic toxicity and could prevent HIV PI-induced dysregulation of hepatic lipid metabolism by inhibiting ER stress. However, little information is available as to whether raltegravir would also prevent HIV PI-induced inflammatory response and foam cell formation in macrophages. Methodology and Principal Findings In this study, we examined the effect of raltegravir on ER stress activation and lipid accumulation in cultured mouse macrophages (J774A.1), primary mouse macrophages, and human THP-1-derived macrophages, and further determined whether the combination of raltegravir with existing HIV PIs would potentially exacerbate or prevent the previously observed activation of inflammatory response and foam cell formation. The results indicated that raltegravir did not induce ER stress and inflammatory response in macrophages. Even more interestingly, HIV PI-induced ER stress, oxidative stress, inflammatory response and foam cell formation were significantly reduced by raltegravir. High performance liquid chromatography (HPLC) analysis further demonstrated that raltegravir did not affect the uptake of HIV PIs in macrophages. Conclusion and Significance Raltegravir could prevent HIV PI-induced inflammatory response and foam cell formation by inhibiting ER stress. These results suggest that incorporation of this HIV integrase inhibitor may reduce the cardiovascular complications associated with current HAART.

HIV Protease Inhibitors Sensitize Human Head and Neck Squamous Carcinoma Cells to Radiation by Activating Endoplasmic Reticulum Stress

Background Human head and neck squamous cell carcinoma (HNSCC) is the sixth most malignant cancer worldwide. Despite significant advances in the delivery of treatment and surgical reconstruction, there is no significant improvement of mortality rates for this disease in the past decades. Radiotherapy is the core component of the clinical combinational therapies for HNSCC. However, the tumor cells have a tendency to develop radiation resistance, which is a major barrier to effective treatment. HIV protease inhibitors (HIV PIs) have been reported with radiosensitizing activities in HNSCC cells, but the underlying cellular/molecular mechanisms remain unclear. Our previous study has shown that HIV PIs induce cell apoptosis via activation of endoplasmic reticulum (ER) stress. The aim of this study was to examine the role of ER stress in HIV PI-induced radiosensitivity in human HNSCC. Methodology and Principal Findings HNSCC cell lines, SQ20B and FaDu, and the most commonly used HIV PIs, lopinavir and ritonavir (L/R), were used in this study. Clonogenic assay was used to assess the radiosensitivity. Cell viability, apoptosis and cell cycle were analyzed using Cellometer Vision CBA. The mRNA and protein levels of ER stress-related genes (eIF2α, CHOP, ATF-4, and XBP-1), as well as cell cycle related protein, cyclin D1, were detected by real time RT-PCR and Western blot analysis, respectively. The results demonstrated that L/R dose-dependently sensitized HNSCC cells to irradiation and inhibited cell growth. L/R-induced activation of ER stress was correlated to down-regulation of cyclin D1 expression and cell cycle arrest under G0/G1 phase. Conclusion and Significance HIV PIs sensitize HNSCC cells to radiotherapy by activation of ER stress and induction of cell cycle arrest. Our results provided evidence that HIV PIs can be potentially used in combination with radiation in the treatment of HNSCC.