Fatty Acids Activate the Transcriptional Coactivator YAP1 to Promote Liver Fibrosis via p38 Mitogen-Activated Protein Kinase.

BACKGROUND & AIMS: Patients with simple steatosis (SS) and nonalcoholic steatohepatitis can develop progressive liver fibrosis, which is associated with liver-related mortality. The mechanisms contributing to liver fibrosis development in SS, however, are poorly understood. SS is characterized by hepatocellular free fatty acid (FFA) accumulation without lobular inflammation seen in nonalcoholic steatohepatitis. Because the Hippo signaling transcriptional coactivator YAP1 (YAP) has previously been linked with nonalcoholic fatty liver disease (NAFLD)-related fibrosis, we sought to explore how hepatocyte FFAs activate a YAP-mediated profibrogenic program. METHODS: We analyzed RNA sequencing data from a GEO DataSet (accession: GSE162694) consisting of 143 patients with NAFLD. We also performed immunohistochemical, immunofluorescence, immunoblot, and quantitative reverse-transcription polymerase chain reaction analyses (qRT-PCR) in liver specimens from NAFLD subjects, from a murine dietary NAFLD model, and in FFA-treated hepatic spheroids and hepatocytes. RESULTS: YAP-target gene expression correlated with increasing fibrosis stage in NAFLD patients and was associated with fibrosis in mice fed a NAFLD-inducing diet. Hepatocyte-specific YAP deletion in the murine NAFLD model attenuated diet-induced fibrosis, suggesting a causative role of YAP in NAFLD-related fibrosis. Likewise, in hepatic spheroids composed of Huh7 hepatoma cells and primary human hepatic stellate cells, Huh7 YAP silencing reduced FFA-induced fibrogenic gene expression. Notably, inhibition of p38 mitogen-activated protein kinase could block YAP activation in FFA-treated Huh7 cells. CONCLUSIONS: These studies provide further evidence for the pathological role of YAP in NAFLD-associated fibrosis and that YAP activation in NAFLD may be driven by FFA-induced p38 MAPK activation.

Hepatic gap junctions amplify alcohol liver injury by propagating cGAS-mediated IRF3 activation.

Alcohol-related liver disease (ALD) accounts for the majority of cirrhosis and liver-related deaths worldwide. Activation of IFN-regulatory factor (IRF3) initiates alcohol-induced hepatocyte apoptosis, which fuels a robust secondary inflammatory response that drives ALD. The dominant molecular mechanism by which alcohol activates IRF3 and the pathways that amplify inflammatory signals in ALD remains unknown. Here we show that cytoplasmic sensor cyclic guanosine monophosphate-adenosine monophosphate (AMP) synthase (cGAS) drives IRF3 activation in both alcohol-injured hepatocytes and the neighboring parenchyma via a gap junction intercellular communication pathway. Hepatic RNA-seq analysis of patients with a wide spectrum of ALD revealed that expression of the cGAS-IRF3 pathway correlated positively with disease severity. Alcohol-fed mice demonstrated increased hepatic expression of the cGAS-IRF3 pathway. Mice genetically deficient in cGAS and IRF3 were protected against ALD. Ablation of cGAS in hepatocytes only phenocopied this hepatoprotection, highlighting the critical role of hepatocytes in fueling the cGAS-IRF3 response to alcohol. We identified connexin 32 (Cx32), the predominant hepatic gap junction, as a critical regulator of spreading cGAS-driven IRF3 activation through the liver parenchyma. Disruption of Cx32 in ALD impaired IRF3-stimulated gene expression, resulting in decreased hepatic injury despite an increase in hepatic steatosis. Taken together, these results identify cGAS and Cx32 as key factors in ALD pathogenesis and as potential therapeutic targets for hepatoprotection.

Gαs signaling controls intramembranous ossification during cranial bone development by regulating both Hedgehog and Wnt/ß-catenin signaling.

How osteoblast cells are induced is a central question for understanding skeletal formation. Abnormal osteoblast differentiation leads to a broad range of devastating craniofacial diseases. Here we have investigated intramembranous ossification during cranial bone development in mouse models of skeletal genetic diseases that exhibit craniofacial bone defects. The GNAS gene encodes Gαs that transduces GPCR signaling. GNAS activation or loss-of-function mutations in humans cause fibrous dysplasia (FD) or progressive osseous heteroplasia (POH) that shows craniofacial hyperostosis or craniosynostosis, respectively. We find here that, while Hh ligand-dependent Hh signaling is essential for endochondral ossification, it is dispensable for intramembranous ossification, where Gαs regulates Hh signaling in a ligand-independent manner. We further show that Gαs controls intramembranous ossification by regulating both Hh and Wnt/ß-catenin signaling. In addition, Gαs activation in the developing cranial bone leads to reduced ossification but increased cartilage presence due to reduced cartilage dissolution, not cell fate switch. Small molecule inhibitors of Hh and Wnt signaling can effectively ameliorate cranial bone phenotypes in mice caused by loss or gain of Gnas function mutations, respectively. Our work shows that studies of genetic diseases provide invaluable insights in both pathological bone defects and normal bone development, understanding both leads to better diagnosis and therapeutic treatment of bone diseases.

Induced GnasR201H expression from the endogenous Gnas locus causes fibrous dysplasia by up-regulating Wnt/ß-catenin signaling.

Fibrous dysplasia (FD; Online Mendelian Inheritance in Man no. 174800) is a crippling skeletal disease caused by activating mutations of the GNAS gene, which encodes the stimulatory G protein Gαs FD can lead to severe adverse conditions such as bone deformity, fracture, and severe pain, leading to functional impairment and wheelchair confinement. So far there is no cure, as the underlying molecular and cellular mechanisms remain largely unknown and the lack of appropriate animal models has severely hampered FD research. Here we have investigated the cellular and molecular mechanisms underlying FD and tested its potential treatment by establishing a mouse model in which the human FD mutation (R201H) has been conditionally knocked into the corresponding mouse Gnas locus. We found that the germ-line FD mutant was embryonic lethal, and Cre-induced Gnas FD mutant expression in early osteochondral progenitors, osteoblast cells, or bone marrow stromal cells (BMSCs) recapitulated FD features. In addition, mosaic expression of FD mutant Gαs in BMSCs induced bone marrow fibrosis both cell autonomously and non-cell autonomously. Furthermore, Wnt/ß-catenin signaling was up-regulated in FD mutant mouse bone and BMSCs undergoing osteogenic differentiation, as we have found in FD human tissue previously. Reduction of Wnt/ß-catenin signaling by removing one Lrp6 copy in an FD mutant line significantly rescued the phenotypes. We demonstrate that induced expression of the FD Gαs mutant from the mouse endogenous Gnas locus exhibits human FD phenotypes in vivo, and that inhibitors of Wnt/ß-catenin signaling may be repurposed for treating FD and other bone diseases caused by Gαs activation.

Hepatic Hippo signaling inhibits protumoural microenvironment to suppress hepatocellular carcinoma.

OBJECTIVE: Hippo signalling is a recently identified major oncosuppressive pathway that plays critical roles in inhibiting hepatocyte proliferation, survival and hepatocellular carcinoma (HCC) formation. Hippo kinase (Mst1 and Mst2) inhibits HCC proliferation by suppressing Yap/Taz transcription activities. As human HCC is mainly driven by chronic liver inflammation, it is not clear whether Hippo signalling inhibits HCC by shaping its inflammatory microenvironment. DESIGN: We have established a genetic HCC model by deleting Mst1 and Mst2 in hepatocytes. Functions of inflammatory responses in this model were characterised by molecular, cellular and FACS analysis, immunohistochemistry and genetic deletion of monocyte chemoattractant protein-1 (Mcp1) or Yap. Human HCC databases and human HCC samples were analysed by immunohistochemistry. RESULTS: Genetic deletion of Mst1 and Mst2 in hepatocytes (DKO) led to HCC development, highly upregulated Mcp1 expression and massive infiltration of macrophages with mixed M1 and M2 phenotypes. Macrophage ablation or deletion of Mcp1 in DKO mice markedly reduced hepatic inflammation and HCC development. Moreover, Yap removal abolished induction of Mcp1 expression and restored normal liver growth in the Mst1/Mst2 DKO mice. Finally, we showed that MCP1 is a direct transcription target of YAP in hepatocytes and identified a strong gene expression correlation between YAP targets and MCP-1 in human HCCs. CONCLUSIONS: Hippo signalling in hepatocytes maintains normal liver growth by suppressing macrophage infiltration during protumoural microenvironment formation through the inhibition of Yap-dependent Mcp1 expression, providing new targets and strategies to treat HCCs.

Interacting network of Hippo, Wnt/ß-catenin and Notch signaling represses liver tumor formation.

Acquiring a selective growth advantage by breaking the proliferation barrier established by gatekeeper genes is a centrally important event in tumor formation. Removal of the mammalian Hippo kinase Mst1 and Mst2 in hepatocytes leads to rapid hepatocellular carcinoma (HCC) formation, indicating that the Hippo signaling pathway is a critical gatekeeper that restrains abnormal growth in hepatocytes. By rigorous genetic approaches, we identified an interacting network of the Hippo, Wnt/ß-catenin and Notch signaling pathways that control organ size and HCC development. We found that in hepatocytes, the loss of Mst1/2 leads to the activation of Notch signaling, which forms a positive feedback loop with Yap/Taz (transcription factors controlled by Mst1/2). This positive feedback loop results in severe liver enlargement and rapid HCC formation. Blocking the Yap/Taz-Notch positive feedback loop by Notch inhibition in vivo significantly reduced the Yap/Taz activities, hepatocyte proliferation and tumor formation. Furthermore, we uncovered a surprising inhibitory role of Wnt/ß-catenin signaling to Yap/Taz activities, which are important in tumor initiation. Genetic removal of ß-catenin in the liver of the Mst1/2 mutants significantly accelerates tumoriogenesis. Therefore, Wnt/ß-catenin signaling, known for its oncogenic property, exerts an unexpected function in restricting Yap/Taz and Notch activities in HCC initiation. The molecular interplay between the three signaling pathways identified in our study provides new insights in developing novel therapeutic strategies to treat liver tumors. [BMB Reports 2017; 50(1): 1-2].

Hippo signaling interactions with Wnt/ß-catenin and Notch signaling repress liver tumorigenesis.

Malignant tumors develop through multiple steps of initiation and progression, and tumor initiation is of singular importance in tumor prevention, diagnosis, and treatment. However, the molecular mechanism whereby a signaling network of interacting pathways restrains proliferation in normal cells and prevents tumor initiation is still poorly understood. Here, we have reported that the Hippo, Wnt/ß-catenin, and Notch pathways form an interacting network to maintain liver size and suppress hepatocellular carcinoma (HCC). Ablation of the mammalian Hippo kinases Mst1 and Mst2 in liver led to rapid HCC formation and activated Yes-associated protein/WW domain containing transcription regulator 1 (YAP/TAZ), STAT3, Wnt/ß-catenin, and Notch signaling. Previous work has shown that abnormal activation of these downstream pathways can lead to HCC. Rigorous genetic experiments revealed that Notch signaling forms a positive feedback loop with the Hippo signaling effector YAP/TAZ to promote severe hepatomegaly and rapid HCC initiation and progression. Surprisingly, we found that Wnt/ß-catenin signaling activation suppressed HCC formation by inhibiting the positive feedback loop between YAP/TAZ and Notch signaling. Furthermore, we found that STAT3 in hepatocytes is dispensable for HCC formation when mammalian sterile 20-like kinase 1 and 2 (Mst1 and Mst2) were removed. The molecular network we have identified provides insights into HCC molecular classifications and therapeutic developments for the treatment of liver tumors caused by distinct genetic mutations.

Cryptochrome 1 regulates the circadian clock through dynamic interactions with the BMAL1 C terminus.

The molecular circadian clock in mammals is generated from transcriptional activation by the bHLH-PAS transcription factor CLOCK-BMAL1 and subsequent repression by PERIOD and CRYPTOCHROME (CRY). The mechanism by which CRYs repress CLOCK-BMAL1 to close the negative feedback loop and generate 24-h timing is not known. Here we show that, in mouse fibroblasts, CRY1 competes for binding with coactivators to the intrinsically unstructured C-terminal transactivation domain (TAD) of BMAL1 to establish a functional switch between activation and repression of CLOCK-BMAL1. TAD mutations that alter affinities for co-regulators affect the balance of repression and activation to consequently change the intrinsic circadian period or eliminate cycling altogether. Our results suggest that CRY1 fulfills its role as an essential circadian repressor by sequestering the TAD from coactivators, and they highlight regulation of the BMAL1 TAD as a critical mechanism for establishing circadian timing.

Cell type-specific functions of period genes revealed by novel adipocyte and hepatocyte circadian clock models.

In animals, circadian rhythms in physiology and behavior result from coherent rhythmic interactions between clocks in the brain and those throughout the body. Despite the many tissue specific clocks, most understanding of the molecular core clock mechanism comes from studies of the suprachiasmatic nuclei (SCN) of the hypothalamus and a few other cell types. Here we report establishment and genetic characterization of three cell-autonomous mouse clock models: 3T3 fibroblasts, 3T3-L1 adipocytes, and MMH-D3 hepatocytes. Each model is genetically tractable and has an integrated luciferase reporter that allows for longitudinal luminescence recording of rhythmic clock gene expression using an inexpensive off-the-shelf microplate reader. To test these cellular models, we generated a library of short hairpin RNAs (shRNAs) against a panel of known clock genes and evaluated their impact on circadian rhythms. Knockdown of Bmal1, Clock, Cry1, and Cry2 each resulted in similar phenotypes in all three models, consistent with previous studies. However, we observed cell type-specific knockdown phenotypes for the Period and Rev-Erb families of clock genes. In particular, Per1 and Per2, which have strong behavioral effects in knockout mice, appear to play different roles in regulating period length and amplitude in these peripheral systems. Per3, which has relatively modest behavioral effects in knockout mice, substantially affects period length in the three cellular models and in dissociated SCN neurons. In summary, this study establishes new cell-autonomous clock models that are of particular relevance to metabolism and suitable for screening for clock modifiers, and reveals previously under-appreciated cell type-specific functions of clock genes.

Monitoring cell-autonomous circadian clock rhythms of gene expression using luciferase bioluminescence reporters.

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere. Individual cells are the functional units for generation and maintenance of circadian rhythms, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms. Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection or stable transduction. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Identification of a novel cryptochrome differentiating domain required for feedback repression in circadian clock function.

Circadian clocks in mammals are based on a negative feedback loop in which transcriptional repression by the cryptochromes, CRY1 and CRY2, lies at the heart of the mechanism. Despite similarities in sequence, domain structure, and biochemical activity, they play distinct roles in clock function. However, detailed biochemical studies have not been straightforward and Cry function has not been examined in real clock cells using kinetic measurements. In this study, we demonstrate, through cell-based genetic complementation and real-time molecular recording, that Cry1 alone is able to maintain cell-autonomous circadian rhythms, whereas Cry2 cannot. Using this novel functional assay, we identify a cryptochrome differentiating α-helical domain within the photolyase homology region (PHR) of CRY1, designated as CRY1-PHR(313-426), that is required for clock function and distinguishes CRY1 from CRY2. Contrary to speculation, the divergent carboxyl-terminal tail domain (CTD) is dispensable, but serves to modulate rhythm amplitude and period length. Finally, we identify the biochemical basis of their distinct function; CRY1 is a much more potent transcriptional repressor than CRY2, and the strength of repression by various forms of CRY proteins significantly correlates with rhythm amplitude. Taken together, our results demonstrate that CRY1-PHR(313-426), not the divergent CTD, is critical for clock function. These findings provide novel insights into the evolution of the diverse functions of the photolyase/cryptochrome family of flavoproteins and offer new opportunities for mechanistic studies of CRY function.

Structure-activity relationship studies of novel pyrazole and imidazole carboxamides as cannabinoid-1 (CB1) antagonists.

The synthesis and biological evaluation of novel pyrazole and imidazole carboxamides as CB1 antagonists are described. As a part of eastern amide SAR, various chemically diverse motifs were introduced on rimonabant template. The central pyrazole core was also replaced with its conformationally constrained motif and imidazole moieties. In general, a range of modifications were well tolerated. Several molecules with low- and sub-nanomolar potencies were identified as potent CB1 receptor antagonists. The in vivo proof of principle for weight loss is demonstrated with a lead compound in DIO mice model.

Circadian regulation of ATP release in astrocytes.

Circadian clocks sustain daily oscillations in gene expression, physiology, and behavior, relying on transcription-translation feedback loops of clock genes for rhythm generation. Cultured astrocytes display daily oscillations of extracellular ATP, suggesting that ATP release is a circadian output. We hypothesized that the circadian clock modulates ATP release via mechanisms that regulate acute ATP release from glia. To test the molecular basis for circadian ATP release, we developed methods to measure in real-time ATP release and Bmal1::dLuc circadian reporter expression in cortical astrocyte cultures from mice of different genotypes. Daily rhythms of gene expression required functional Clock and Bmal1, both Per1 and Per2, and both Cry1 and Cry2 genes. Similarly, high-level, circadian ATP release also required a functional clock mechanism. Whereas blocking IP(3) signaling significantly disrupted ATP rhythms with no effect on Bmal1::dLuc cycling, blocking vesicular release did not alter circadian ATP release or gene expression. We conclude that astrocytes depend on circadian clock genes and IP(3) signaling to express daily rhythms in ATP release.

Novel pyrazole-3-carboxamide derivatives as cannabinoid-1 (CB1) antagonists: journey from non-polar to polar amides.

The synthesis and biological evaluation of novel pyrazole-3-carboxamide derivatives as CB1 antagonists are described. As a part of eastern amide SAR, various chemically diverse motifs were introduced. In general, a range of modifications were well tolerated. Several molecules with high polar surface area were also identified as potent CB1 receptor antagonists. The in vivo proof of principle for weight loss is exemplified with a lead compound from this series.

Novel astaxanthin prodrug (CDX-085) attenuates thrombosis in a mouse model.

BACKGROUND: Cardiovascular disease remains the leading cause of morbidity and premature mortality in most industrialized countries as well as in developing nations. A pro-oxidative state appears to promote and/or exacerbate vascular disease complications. Furthermore, a state of low-grade chronic inflammation can promote increased oxidative stress and lead to endothelial cell and platelet dysfunction ultimately contributing to thrombogenesis. OBJECTIVES: In this study, the effect of a proprietary astaxanthin prodrug (CDX-085) on thrombus formation was investigated using a mouse model of arterial thrombosis. The influence of free astaxanthin, the active drug of CDX-085, on human endothelial cells and rat platelets was evaluated to investigate potential mechanisms of action. METHODS AND RESULTS: Oral administration of CDX-085 (0.4% in chow, approximately 500 mg/kg/day) to 6-8 week old C57BL/6 male mice for 14 days resulted in significant levels of free astaxanthin in the plasma, liver, heart and platelets. When compared to control mice, the CDX-085 fed group exhibited significant increases in basal arterial blood flow and significant delays in occlusive thrombus formation following the onset of vascular endothelial injury. Primary human umbilical vein endothelial cells (HUVECs) and platelets isolated from Wistar-Kyoto rats treated with free astaxanthin demonstrated significantly increased levels of released nitric oxide (NO) and significantly decreased peroxynitrite (ONOO-) levels. CONCLUSION: Observations of increased NO and decreased ONOO- levels in endothelial cells and platelets support a potential mechanism of action for astaxanthin (CDX-085 active drug). These studies support the potential of CDX-085 and its metabolite astaxanthin in the treatment or prevention of thrombotic cardiovascular complications.