25-Hydroxycholesterol regulates lysosome AMP kinase activation and metabolic reprogramming to educate immunosuppressive macrophages.

Macrophages are critical to turn noninflamed "cold tumors" into inflamed "hot tumors". Emerging evidence indicates abnormal cholesterol metabolites in the tumor microenvironment (TME) with unclear function. Here, we uncovered the inducible expression of cholesterol-25-hydroxylase (Ch25h) by interleukin-4 (IL-4) and interleukin-13 (IL-13) via the transcription factor STAT6, causing 25-hydroxycholesterol (25HC) accumulation. scRNA-seq analysis confirmed that CH25Hhi subsets were enriched in immunosuppressive macrophage subsets and correlated to lower survival rates in pan-cancers. Targeting CH25H abrogated macrophage immunosuppressive function to enhance infiltrating T cell numbers and activation, which synergized with anti-PD-1 to improve anti-tumor efficacy. Mechanically, lysosome-accumulated 25HC competed with cholesterol for GPR155 binding to inhibit the kinase mTORC1, leading to AMPKα activation and metabolic reprogramming. AMPKα also phosphorylated STAT6 Ser564 to enhance STAT6 activation and ARG1 production. Together, we propose CH25H as an immunometabolic checkpoint, which manipulates macrophage fate to reshape CD8+ T cell surveillance and anti-tumor response.

ZNT1 and Zn 2+ control TLR4 and PD-L1 endocytosis in macrophages to improve chemotherapy efficacy against liver tumor.

BACKGROUND AND AIMS: HCC is closely associated with inflammation and immune modulation, and combined chemotherapy with other strategies is under extensive investigation to achieve better efficacy. HCC is accompanied by zinc (Zn) deficiency. This study aims to understand how Zn could affect macrophage function and its application for HCC therapy. APPROACH AND RESULTS: Zn 2+ and the Zn transporter 1 (ZNT1, solute carrier family 30 member 1) were markedly reduced in intrahepatic macrophages from patients with HCC and from mouse liver tumors. Lower ZNT1 expression was associated with higher IL-6 production and shorter survival time in patients with HCC. Critically, ZNT1 regulated endosomal Zn 2+ levels for endocytosis of toll-like receptor 4 and programmed cell death ligand 1, thereby decreasing macrophage-induced inflammation and immunosuppression to protect from liver tumors. Myeloid-specific deletion of ZNT1 in mice increased chronic inflammation, liver fibrosis, tumor numbers, and size. Notably, Zn supplementation could reduce inflammation and surface programmed cell death ligand 1 expression in macrophages with the increased CD8 + T cell cytotoxicity, which synergized the antitumor efficacy of Sorafenib/Lenvatinib. CONCLUSIONS: Our study proposes a new concept that ZNT1 and Zn regulate endosome endocytosis to maintain surface receptors, and Zn supplements might be synergized with chemotherapy to treat inflammation-associated tumors, especially those containing programmed cell death ligand 1 + myeloid cells.

Dedifferentiation-associated inflammatory factors of long-term expanded human hepatocytes exacerbate their elimination by macrophages during liver engraftment.

BACKGROUND AND AIMS: Hepatocyte transplantation has been demonstrated to be effective to treat liver metabolic disease and acute liver failure. Nevertheless, the shortage of donor hepatocytes restrained its application in clinics. To expand human hepatocytes at a large scale, several dedifferentiation-based protocols have been established, including proliferating human hepatocytes (ProliHH). However, the decreased transplantation efficiency of these cells after long-term expansion largely impedes their application. APPROACH AND RESULTS: We found that accompanied with dedifferentiation, long-term cultured ProliHH (lc-ProliHH) up-regulated a panel of chemokines and cytokines related to innate immunity, which were referred to as dedifferentiation-associated inflammatory factors (DAIF). DAIF elicited excessive macrophage responses, accounting for the elimination of lc-ProliHH specifically during engraftment. Two possible strategies to increase ProliHH transplantation were then characterized. Blockage of innate immune response by dexamethasone reverted the engraftment and repopulation of lc-ProliHH to a level comparable to primary hepatocytes, resulting in improved liver function and a better survival of fumarylacetoacetate hydrolase-deficient mice. Alternatively, rematuration of lc-ProliHH as organoids reduced the expression of DAIF and led to markedly improved engraftment. CONCLUSIONS: These results revealed that lc-ProliHH triggers exacerbated macrophage activation by DAIF and provided potential solutions for clinical transplantation of lc-ProliHH.

Progress in patient-derived liver cancer cell models: a step forward for precision medicine.

The development of effective precision treatments for liver cancers has been hindered by the scarcity of preclinical models that accurately reflect the heterogeneity of this disease. Recent progress in developing patient-derived liver cancer cell lines and organoids has paved the way for precision medicine research. These expandable resources of liver cancer cell models enable a full spectrum of pharmacogenomic analysis for liver cancers. Moreover, patient-derived and short-term cultured two-dimensional tumor cells or three-dimensional organoids can serve as patient avatars, allowing for the prediction of patients' response to drugs and facilitating personalized treatment for liver cancer patients. Furthermore, the current novel techniques have expanded the scope of cancer research, including innovative organoid culture, gene editing and bioengineering. In this review, we provide an overview of the progress in patient-derived liver cancer cell models, focusing on their applications in precision and personalized medicine research. We also discuss the challenges and future perspectives in this field.

RNA helicase DDX5 enables STAT1 mRNA translation and interferon signalling in hepatitis B virus replicating hepatocytes.

OBJECTIVE: RNA helicase DDX5 is downregulated during HBV replication and poor prognosis HBV-related hepatocellular carcinoma (HCC). The objective of this study is to investigate the role of DDX5 in interferon (IFN) signalling. We provide evidence of a novel mechanism involving DDX5 that enables translation of transcription factor STAT1 mediating the IFN response. DESIGN AND RESULTS: Molecular, pharmacological and biophysical assays were used together with cellular models of HBV replication, HCC cell lines and liver tumours. We demonstrate that DDX5 regulates STAT1 mRNA translation by resolving a G-quadruplex (rG4) RNA structure, proximal to the 5' end of STAT1 5'UTR. We employed luciferase reporter assays comparing wild type (WT) versus mutant rG4 sequence, rG4-stabilising compounds, CRISPR/Cas9 editing of the STAT1-rG4 sequence and circular dichroism determination of the rG4 structure. STAT1-rG4 edited cell lines were resistant to the effect of rG4-stabilising compounds in response to IFN-α, while HCC cell lines expressing low DDX5 exhibited reduced IFN response. Ribonucleoprotein and electrophoretic mobility assays demonstrated direct and selective binding of RNA helicase-active DDX5 to the WT STAT1-rG4 sequence. Immunohistochemistry of normal liver and liver tumours demonstrated that absence of DDX5 corresponded to absence of STAT1. Significantly, knockdown of DDX5 in HBV infected HepaRG cells reduced the anti-viral effect of IFN-α. CONCLUSION: RNA helicase DDX5 resolves a G-quadruplex structure in 5'UTR of STAT1 mRNA, enabling STAT1 translation. We propose that DDX5 is a key regulator of the dynamic range of IFN response during innate immunity and adjuvant IFN-α therapy.

Fatty acid synthesis is critical for stem cell pluripotency via promoting mitochondrial fission.

Pluripotent stem cells are known to display distinct metabolic phenotypes than their somatic counterparts. While accumulating studies are focused on the roles of glucose and amino acid metabolism in facilitating pluripotency, little is known regarding the role of lipid metabolism in regulation of stem cell activities. Here, we show that fatty acid (FA) synthesis activation is critical for stem cell pluripotency. Our initial observations demonstrated enhanced lipogenesis in pluripotent cells and during cellular reprogramming. Further analysis indicated that de novo FA synthesis controls cellular reprogramming and embryonic stem cell pluripotency through mitochondrial fission. Mechanistically, we found that de novo FA synthesis regulated by the lipogenic enzyme ACC1 leads to the enhanced mitochondrial fission via (i) consumption of AcCoA which affects acetylation-mediated FIS1 ubiquitin-proteasome degradation and (ii) generation of lipid products that drive the mitochondrial dynamic equilibrium toward fission. Moreover, we demonstrated that the effect of Acc1 on cellular reprogramming via mitochondrial fission also exists in human iPSC induction. In summary, our study reveals a critical involvement of the FA synthesis pathway in promoting ESC pluripotency and iPSC formation via regulating mitochondrial fission.

Metabolic benefits of inhibition of p38α in white adipose tissue in obesity.

p38 has long been known as a central mediator of protein kinase A (PKA) signaling in brown adipocytes, which positively regulate the transcription of uncoupling protein 1 (UCP-1). However, the physiological role of p38 in adipose tissues, especially the white adipose tissue (WAT), is largely unknown. Here, we show that mice lacking p38α in adipose tissues display a lean phenotype, improved metabolism, and resistance to diet-induced obesity. Surprisingly, ablation of p38α causes minimal effects on brown adipose tissue (BAT) in adult mice, as evident from undetectable changes in UCP-1 expression, mitochondrial function, body temperature (BT), and energy expenditure. In contrast, genetic ablation of p38α in adipose tissues not only markedly facilitates the browning in WAT upon cold stress but also prevents diet-induced obesity. Consistently, pharmaceutical inhibition of p38α remarkably enhances the browning of WAT and has metabolic benefits. Furthermore, our data suggest that p38α deficiency promotes white-to-beige adipocyte reprogramming in a cell-autonomous manner. Mechanistically, inhibition of p38α stimulates the UCP-1 transcription through PKA and its downstream cAMP-response element binding protein (CREB), which form a positive feedback loop that functions to reinforce the white-to-beige phenotypic switch during cold exposure. Together, our study reveals that inhibition of p38α is able to promote WAT browning and confer metabolic benefits. Our study also indicates that p38α in WAT represents an exciting pharmacological target to combat obesity and metabolic diseases.

The evidence of a macrophage barrier in the xenotransplantation of human hematopoietic stem cells to severely immunodeficient rats.

BACKGROUND: The human-to-rat hematopoietic stem cell transplantation (HSCT) model is rare, unlike its human-to-mouse counterpart. The rat models are desired, especially in areas of physiology, toxicology, and pharmacology. In addition to lymphocytes, macrophages are also considered to be important for xenotransplantation. We generated a rat xenotransplantation model to prove the role of macrophages as a xenotransplantation barrier. METHODS: Immunodeficiency in SRG rats, which are Sprague-Dawley (SD) rats lacking Rag2 and Il2rg, was confirmed by flow cytometry and spleen immunostaining. Human umbilical cord blood was collected after scheduled cesarean section at the University of Tsukuba Hospital. Cord blood mononuclear cells (CB-MNCs) were transplanted into the SRG rats administered several injections of clodronate liposome (CL), which cause macrophage depletion. Survival of human cells was observed by flow cytometry. Rat macrophage phagocytosis assay was performed to check the species-specific effects of rat macrophages on injected human/rat blood cells. RESULTS: SRG rats were deficient in T/B/NK cells. Without CL pretreatment, human CB-MNCs were removed from SRG rats within 7 hours after transplantation. The rats pretreated with CL could survive after transplantation. Prolonged survival for more than 4 weeks was observed only following a one-time CL injection. Rat macrophages had a species-specific potential for the phagocytosis of human blood cells in vivo. CONCLUSION: In human-to-rat HSCT, the short period of early macrophage control, leading to macrophage immunotolerance, is important for engraftment. The generated model can be useful for the creation of future xenotransplantation models or other clinical research.

ECM1 Prevents Activation of Transforming Growth Factor ß, Hepatic Stellate Cells, and Fibrogenesis in Mice.

BACKGROUND & AIMS: Activation of TGFB (transforming growth factor ß) promotes liver fibrosis by activating hepatic stellate cells (HSCs), but the mechanisms of TGFB activation are not clear. We investigated the role of ECM1 (extracellular matrix protein 1), which interacts with extracellular and structural proteins, in TGFB activation in mouse livers. METHODS: We performed studies with C57BL/6J mice (controls), ECM1-knockout (ECM1-KO) mice, and mice with hepatocyte-specific knockout of EMC1 (ECM1Δhep). ECM1 or soluble TGFBR2 (TGFB receptor 2) were expressed in livers of mice after injection of an adeno-associated virus vector. Liver fibrosis was induced by carbon tetrachloride (CCl4) administration. Livers were collected from mice and analyzed by histology, immunohistochemistry, in situ hybridization, and immunofluorescence analyses. Hepatocytes and HSCs were isolated from livers of mice and incubated with ECM1; production of cytokines and activation of reporter genes were quantified. Liver tissues from patients with viral or alcohol-induced hepatitis (with different stages of fibrosis) and individuals with healthy livers were analyzed by immunohistochemistry and in situ hybridization. RESULTS: ECM1-KO mice spontaneously developed liver fibrosis and died by 2 months of age without significant hepatocyte damage or inflammation. In liver tissues of mice, we found that ECM1 stabilized extracellular matrix-deposited TGFB in its inactive form by interacting with αv integrins to prevent activation of HSCs. In liver tissues from patients and in mice with CCl4-induced liver fibrosis, we found an inverse correlation between level of ECM1 and severity of fibrosis. CCl4-induced liver fibrosis was accelerated in ECM1Δhep mice compared with control mice. Hepatocytes produced the highest levels of ECM1 in livers of mice. Ectopic expression of ECM1 or soluble TGFBR2 in liver prevented fibrogenesis in ECM1-KO mice and prolonged their survival. Ectopic expression of ECM1 in liver also reduced the severity of CCl4-induced fibrosis in mice. CONCLUSIONS: ECM1, produced by hepatocytes, inhibits activation of TGFB and its activation of HSCs to prevent fibrogenesis in mouse liver. Strategies to increase levels of ECM1 in liver might be developed for treatment of fibrosis.

Cas9-nickase-mediated genome editing corrects hereditary tyrosinemia in rats.

Hereditary tyrosinemia type I (HTI) is a metabolic genetic disorder caused by mutation of fumarylacetoacetate hydrolase (FAH). Because of the accumulation of toxic metabolites, HTI causes severe liver cirrhosis, liver failure, and even hepatocellular carcinoma. HTI is an ideal model for gene therapy, and several strategies have been shown to ameliorate HTI symptoms in animal models. Although CRISPR/Cas9-mediated genome editing is able to correct the Fah mutation in mouse models, WT Cas9 induces numerous undesired mutations that have raised safety concerns for clinical applications. To develop a new method for gene correction with high fidelity, we generated a Fah mutant rat model to investigate whether Cas9 nickase (Cas9n)-mediated genome editing can efficiently correct the Fah First, we confirmed that Cas9n rarely induces indels in both on-target and off-target sites in cell lines. Using WT Cas9 as a positive control, we delivered Cas9n and the repair donor template/single guide (sg)RNA through adenoviral vectors into HTI rats. Analyses of the initial genome editing efficiency indicated that only WT Cas9 but not Cas9n causes indels at the on-target site in the liver tissue. After receiving either Cas9n or WT Cas9-mediated gene correction therapy, HTI rats gained weight steadily and survived. Fah-expressing hepatocytes occupied over 95% of the liver tissue 9 months after the treatment. Moreover, CRISPR/Cas9-mediated gene therapy prevented the progression of liver cirrhosis, a phenotype that could not be recapitulated in the HTI mouse model. These results strongly suggest that Cas9n-mediated genome editing is a valuable and safe gene therapy strategy for this genetic disease.

Bacterial effector NleL promotes enterohemorrhagic E. coli-induced attaching and effacing lesions by ubiquitylating and inactivating JNK.

As a major diarrheagenic human pathogen, enterohemorrhagic Escherichia coli (EHEC) produce attaching and effacing (A/E) lesions, characterized by the formation of actin pedestals, on mammalian cells. A bacterial T3SS effector NleL from EHEC O157:H7 was recently shown to be a HECT-like E3 ligase in vitro, but its biological functions and host targets remain elusive. Here, we report that NleL is required to effectively promote EHEC-induced A/E lesions and bacterial infection. Furthermore, human c-Jun NH2-terminal kinases (JNKs) were identified as primary substrates of NleL. NleL-induced JNK ubiquitylation, particularly mono-ubiquitylation at the Lys 68 residue of JNK, impairs JNK's interaction with an upstream kinase MKK7, thus disrupting JNK phosphorylation and activation. This subsequently suppresses the transcriptional activity of activator protein-1 (AP-1), which modulates the formation of the EHEC-induced actin pedestals. Moreover, JNK knockdown or inhibition in host cells complements NleL deficiency in EHEC infection. Thus, we demonstrate that the effector protein NleL enhances the ability of EHEC to infect host cells by targeting host JNK, and elucidate an inhibitory role of ubiquitylation in regulating JNK phosphorylation.

Arid1a regulates response to anti-angiogenic therapy in advanced hepatocellular carcinoma.

BACKGROUND & AIMS: AT-rich interaction domain 1a (Arid1a), a component of the chromatin remodeling complex, has emerged as a tumor suppressor gene. It is frequently mutated in hepatocellular carcinoma (HCC). However, it remains unknown how Arid1a suppresses HCC development and whether Arid1a deficiency could be exploited for therapy, we aimed to explore these questions. METHODS: The expression of Arid1a in human and mouse HCCs was determined by immunohistochemical (IHC) staining. Gene expression was determined by quantitative PCR, ELISA or western blotting. Arid1a knockdown HCC cell lines were established by lentiviral-based shRNA. Tumor angiogenesis was quantified based on vessel density. The regulation of angiopoietin (Ang2) expression by Arid1a was identified by chromatin immunoprecipitation (ChIP) assay. The tumor promoting function of Arid1a loss was studied with a xenograft model in nude mice and diethylnitrosamine (DEN)-induced HCC in Arid1a conditional knockout mice. The therapeutic values of Ang2 antibody and sorafenib treatment were evaluated both in vitro and in vivo. RESULTS: We demonstrate that Arid1a deficiency, occurring in advanced human HCCs, is associated with increased vessel density. Mechanistically, loss of Arid1a causes aberrant histone H3K27ac deposition at the angiopoietin-2 (Ang2) enhancer and promoter, which eventually leads to ectopic expression of Ang2 and promotes HCC development. Ang2 blockade in Arid1a-deficient HCCs significantly reduces vessel density and tumor progression. Importantly, sorafenib treatment, which suppresses H3K27 acetylation and Ang2 expression, profoundly halts the progression of Arid1a-deficient HCCs. CONCLUSIONS: Arid1a-deficiency activates Ang2-dependent angiogenesis and promotes HCC progression. Loss of Arid1a in HCCs confers sensitivity to Ang2 blockade and sorafenib treatment. LAY SUMMARY: AT-rich interaction domain 1a (Arid1a), is a tumor suppressor gene. Arid1a-deficiency promotes Ang2-dependent angiogenesis leading to hepatocellular carcinoma progression. Arid1a-deficiency also sensitizes tumors to Ang2 blockade by sorafenib treatment.

Hepatic Loss of Borealin Impairs Postnatal Liver Development, Regeneration, and Hepatocarcinogenesis.

Borealin, a member of the chromosomal passenger complex, plays a key regulatory role at centromeres and the central spindle during mitosis. Loss of Borealin leads to defective cell proliferation and early embryonic lethality. The in vivo functions of Borealin in mammalian postnatal development, tissue homeostasis, and tumorigenesis remain elusive. We specifically analyzed the role of Borealin in regulating postnatal liver development, damage-induced liver regeneration, and liver carcinogenesis using mice carrying conditional Borealin alleles. Perinatal loss of Borealin caused increased genome ploidy and enlarged cell size in hepatocytes, likely due to the impaired function of the chromosomal passenger complex in mitosis. Borealin deletion also showed attenuated expansion of Sox9+HNF4α+ progenitor-like cells in liver regeneration during 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet-induced liver injury. Moreover, ΔN90-ß-Catenin and c-Met-induced hepatocarcinogenesis development was largely impeded by Borealin deletion. These findings indicate that Borealin plays a key role in liver development, regeneration, and tumorigenesis and suggests that Borealin could be a potential target for related liver diseases.

High-throughput screening for Survivin and Borealin interaction inhibitors in hepatocellular carcinoma.

Survivin, a key member of the chromatin passenger complex (CPC), is often highly expressed in human cancers, making it a promising target for cancer treatment. Out of the numerous reported Survivin inhibitors, YM155 is only one entering clinical trial, but was recently failed in the Phase II trial. It is important to develop Survivin inhibitors with new strategies. We recently reported that both Survivin and its binding protein Borealin in the CPC complex are essential for the development of hepatocellular carcinoma, suggesting that disrupting the interaction between Survivin and Borealin would be a promising strategy. Here, we developed a high-throughput screening method based on bimolecular fluorescence complementation (BiFC) technology in cultured cells, which allowed the identification of small chemical inhibitors specifically blocking the Survivin and Borealin interaction. Primary hits from BiFC were further validated in an in vitro AlphaScreen system, which detects the direct interactions of Survivin and Borealin. Etoposide was identified as one of the effective hits. Direct interaction between Survivin and Etoposide was confirmed by surface plasmon resonance assay, and molecular docking analysis suggested the structural information on how Etoposide inhibits the Survivin and Borealin interaction. These results demonstrate a screening system to identify small molecule chemicals inhibiting Survivin and Borealin interaction. In future, an even larger scale screening may lead to identification of better Survivin and Borealin inhibitors.

Hepatocellular carcinoma repression by TNFα-mediated synergistic lethal effect of mitosis defect-induced senescence and cell death sensitization.

UNLABELLED: Hepatocellular carcinoma (HCC) is a cancer lacking effective therapies. Several measures have been proposed to treat HCCs, such as senescence induction, mitotic inhibition, and cell death promotion. However, data from other cancers suggest that single use of these approaches may not be effective. Here, by genetic targeting of Survivin, an inhibitor of apoptosis protein (IAP) that plays dual roles in mitosis and cell survival, we identified a tumor necrosis factor alpha (TNFα)-mediated synergistic lethal effect between senescence and apoptosis sensitization in malignant HCCs. Survivin deficiency results in mitosis defect-associated senescence in HCC cells, which triggers local inflammation and increased TNFα. Survivin inactivation also sensitizes HCC cells to TNFα-triggered cell death, which leads to marked HCC regression. Based on these findings, we designed a combination treatment using mitosis inhibitor and proapoptosis compounds. This treatment recapitulates the therapeutic effect of Survivin deletion and effectively eliminates HCCs, thus representing a potential strategy for HCC therapy. CONCLUSION: Survivin ablation dramatically suppresses human and mouse HCCs by triggering senescence-associated TNFα and sensitizing HCC cells to TNFα-induced cell death. Combined use of mitotic inhibitor and second mitochondrial-derived activator of caspases mimetic can induce senescence-associated TNFα and enhance TNFα-induced cell death and synergistically eliminate HCC. (Hepatology 2016;64:1105-1120).

Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors.

The generation of functional hepatocytes independent of donor liver organs is of great therapeutic interest with regard to regenerative medicine and possible cures for liver disease. Induced hepatic differentiation has been achieved previously using embryonic stem cells or induced pluripotent stem cells. Particularly, hepatocytes generated from a patient's own induced pluripotent stem cells could theoretically avoid immunological rejection. However, the induction of hepatocytes from induced pluripotent stem cells is a complicated process that would probably be replaced with the arrival of improved technology. Overexpression of lineage-specific transcription factors directly converts terminally differentiated cells into some other lineages, including neurons, cardiomyocytes and blood progenitors; however, it remains unclear whether these lineage-converted cells could repair damaged tissues in vivo. Here we demonstrate the direct induction of functional hepatocyte-like (iHep) cells from mouse tail-tip fibroblasts by transduction of Gata4, Hnf1α and Foxa3, and inactivation of p19(Arf). iHep cells show typical epithelial morphology, express hepatic genes and acquire hepatocyte functions. Notably, transplanted iHep cells repopulate the livers of fumarylacetoacetate-hydrolase-deficient (Fah(-/-)) mice and rescue almost half of recipients from death by restoring liver functions. Our study provides a novel strategy to generate functional hepatocyte-like cells for the purpose of liver engineering and regenerative medicine.

Optimized Hepatocyte-Like Cells with Functional Drug Transporters Directly-Reprogrammed from Mouse Fibroblasts and their Potential in Drug Disposition and Toxicology.

BACKGROUND/AIMS: To develop a suitable hepatocyte-like cell model that could be a substitute for primary hepatocytes with essential transporter expression and functions. Induced hepatocyte-like (iHep) cells directly reprogrammed from mice fibroblast cells were fully characterized. METHODS: Naïve iHep cells were transfected with nuclear hepatocyte factor 4 alpha (Hnf4α) and treated with selected small molecules. Sandwich cultured configuration was applied. The mRNA and protein expression of transporters were determined by Real Time PCR and confocal. The functional transporters were estimated by drug biliary excretion measurement. The inhibition of bile acid efflux transporters by cholestatic drugs were assessed. RESULTS: The expression and function of p-glycoprotein (P-gp), bile salt efflux pump (Bsep), multidrug resistance-associated protein 2 (Mrp2), Na+-dependent taurocholate cotransporting polypeptide (Ntcp), and organic anion transporter polypedtides (Oatps) in iHep cells were significantly improved after transfection of hepatocyte nuclear factor 4 alpha (Hnf4α) and treatment with selected inducers. In vitro intrinsic biliary clearances (CLb,int) of optimized iHep cells for rosuvastatin, methotrexate, d8-TCA (deuterium-labeled sodium taurocholate acid) and DPDPE ([D-Pen2,5] enkephalin hydrate) correlated well with that of sandwich-cultured primary mouse hepatocytes (SCMHs) (r2 = 0.984). Cholestatic drugs were evaluated and the results were compared well with primary mice hepatocytes. CONCLUSION: The optimized iHep cells expressed functional drug transporters and were comparable to primary mice hepatocytes. This study suggested direct reprogramming could provide a potential alternative to primary hepatocytes for drug candidate hepatobiliary disposition and hepatotoxicity screening.

Nuclear beta-arrestin1 functions as a scaffold for the dephosphorylation of STAT1 and moderates the antiviral activity of IFN-gamma.

Signal transducers and activators of transcription 1 (STAT1) is activated by tyrosine phosphorylation upon interferon-gamma (IFN-gamma) stimulation. Phosphorylated STAT1 translocates into nucleus to initiate the transcription of IFN-gamma target genes that are important in mediating antiviral, antiproliferative, and immune response. The inactivation of STAT1 is mainly accomplished via tyrosine dephosphorylation by the nuclear isoform of T cell protein tyrosine phosphatase (TC45) in nucleus. Here we show that beta-arrestin1 directly interacts with STAT1 in nucleus after IFN-gamma treatment and accelerates STAT1 tyrosine dephosphorylation by recruiting TC45. Consequently, beta-arrestin1 negatively regulates STAT1 transcription activity as well as the IFN-gamma-induced gene transcription. Application of beta-arrestin1 siRNA significantly enhances IFN-gamma-induced antiviral response in vesicular stomatitis virus (VSV)-infected cells. Our results reveal that nuclear beta-arrestin1, acting as a scaffold for the dephosphorylation of STAT1, is an essential negative regulator of IFN-gamma signaling and participates in the IFN-gamma-induced cellular antiviral response.

p38alpha suppresses normal and cancer cell proliferation by antagonizing the JNK-c-Jun pathway.

The mitogen-activated protein kinase (MAPK) p38alpha controls inflammatory responses and cell proliferation. Using mice carrying conditional Mapk14 (also known as p38alpha) alleles, we investigated its function in postnatal development and tumorigenesis. When we specifically deleted Mapk14 in the mouse embryo, fetuses developed to term but died shortly after birth, probably owing to lung dysfunction. Fetal hematopoietic cells and embryonic fibroblasts deficient in p38alpha showed increased proliferation resulting from sustained activation of the c-Jun N-terminal kinase (JNK)-c-Jun pathway. Notably, in chemical-induced liver cancer development, mice with liver-specific deletion of Mapk14 showed enhanced hepatocyte proliferation and tumor development that correlated with upregulation of the JNK-c-Jun pathway. Furthermore, inactivation of JNK or c-Jun suppressed the increased proliferation of Mapk14-deficient hepatocytes and tumor cells. These results demonstrate a new mechanism whereby p38alpha negatively regulates cell proliferation by antagonizing the JNK-c-Jun pathway in multiple cell types and in liver cancer development.