Mouse epiblast stem cells (mEpiSCs) and human embryonic stem cells (hESCs) are primed pluripotent stem cells whose self-renewal can be maintained through cytoplasmic stabilization and retention of β-catenin. The underlying mechanism, however, remains largely unknown. Here, we show that cytoplasmic β-catenin interacts with and retains TAZ, a Hippo pathway effector, in the cytoplasm. Cytoplasmic retention of TAZ promotes mEpiSC self-renewal in the absence of nuclear β-catenin, whereas nuclear translocation of TAZ induces mEpiSC differentiation. TAZ is dispensable for naive mouse embryonic stem cell (mESC) self-renewal but required for the proper conversion of mESCs to mEpiSCs. The self-renewal of hESCs, like that of mEpiSCs, can also be maintained through the cytoplasmic retention of β-catenin and TAZ. Our study indicates that how TAZ regulates cell fate depends on not only the cell type but also its subcellular localization.

Liver injury after experimental acetaminophen treatment is mediated both by direct hepatocyte injury through a P450-generated toxic metabolite and indirectly by activated liver Kupffer cells and neutrophils. This study was designed to investigate the role of Notch signaling in the regulation of innate immune responses in acetaminophen (APAP)-induced liver injury. Using a mouse model of APAP-induced liver injury, wild-type (WT) and toll-like receptor 4 knockout (TLR4 KO) mice were injected intraperitoneally with APAP or PBS. Some animals were injected with γ-secretase inhibitor DAPT or DMSO vehicle. For the in vitro study, bone marrow-derived macrophages (BMMs) were transfected with Notch1 siRNA, TLR4 siRNA, and non-specific (NS) siRNA and stimulated with LPS. Indeed, paracetamol/acetaminophen-induced liver damage was worse after Notch blockade with DAPT in wild-type mice, which was accompanied by significantly increased ALT levels, diminished hairy and enhancer of split-1 (Hes1), and phosphorylated Stat3 and Akt but enhanced high mobility group box 1 (HMGB1), TLR4, NF-κB, and NLRP3 activation after APAP challenge. Mice receiving DAPT increased macrophage and neutrophil accumulation and hepatocellular apoptosis. However, TLR4 KO mice that received DAPT reduced APAP-induced liver damage and NF-κB, NLRP3, and cleaved caspase-1 activation. BMMs transfected with Notch1 siRNA reduced Hes1 and phosphorylated Stat3 and Akt but augmented HMGB1, TLR4, NF-κB, and NLRP3. Furthermore, TLR4 siRNA knockdown resulted in decreased NF-κB and NLRP3 and cleaved caspase-1 and IL-1β levels following LPS stimulation. These results demonstrate that Notch signaling regulates innate NLRP3 inflammasome activation through regulation of HMGB1/TLR4/NF-κB activation in APAP-induced liver injury. Our novel findings underscore the critical role of the Notch1-Hes1 signaling cascade in the regulation of innate immunity in APAP-triggered liver inflammation. This might imply a novel therapeutic potential for the drug-induced damage-associated lethal hepatitis.

UNLABELLED: Notch signaling plays an emerging role in the regulation of immune cell development and function during inflammatory response. Activation of the ras homolog gene family member A/Rho-associated protein kinase (ROCK) pathway promotes leukocyte accumulation in tissue injury. However, it remains unknown whether Notch signaling regulates ras homolog gene family member A/ROCK-mediated immune responses in liver ischemia and reperfusion (IR) injury. This study investigated intracellular signaling pathways regulated by Notch receptors in the IR-stressed liver and in vitro. In a mouse model of IR-induced liver inflammatory injury, we found that mice with myeloid-specific Notch1 knockout showed aggravated hepatocellular damage, with increased serum alanine aminotransferase levels, hepatocellular apoptosis, macrophage/neutrophil trafficking, and proinflammatory mediators compared to Notch1-proficient controls. Unlike in the controls, myeloid Notch1 ablation diminished hairy and enhancer of split-1 (Hes1) and augmented c-Jun N-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), JNK, ROCK1, and phosphatase and tensin homolog (PTEN) activation in ischemic livers. Disruption of JSAP1 in myeloid-specific Notch1 knockout livers improved hepatocellular function and reduced JNK, ROCK1, PTEN, and toll-like receptor 4 activation. Moreover, ROCK1 knockdown inhibited PTEN and promoted Akt, leading to depressed toll-like receptor 4. In parallel in vitro studies, transfection of lentivirus-expressing Notch1 intracellular domain promoted Hes1 and inhibited JSAP1 in lipopolysaccharide-stimulated bone marrow-derived macrophages. Hes1 deletion enhanced JSAP1/JNK activation, whereas clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9-mediated JSAP1 knockout diminished ROCK1/PTEN and toll-like receptor 4 signaling. CONCLUSION: Myeloid Notch1 deficiency activates the ras homolog gene family member A/ROCK pathway and exacerbates hepatocellular injury by inhibiting transcriptional repressor Hes1 and inducing scaffold protein JSAP1 in IR-triggered liver inflammation; our findings underscore the crucial role of the Notch-Hes1 axis as a novel regulator of innate immunity-mediated inflammation and imply the therapeutic potential for the management of organ IR injury in transplant recipients. (Hepatology 2018;67:1041-1055).

UNLABELLED: Heat shock transcription factor 1 (HSF1) has been implicated in the differential regulation of cell stress and disease states. β-catenin activation is essential for immune homeostasis. However, little is known about the role of macrophage HSF1-β-catenin signaling in the regulation of NLRP3 inflammasome activation during ischemia/reperfusion (I/R) injury (IRI) in the liver. This study investigated the functions and molecular mechanisms by which HSF1-β-catenin signaling influenced NLRP3-mediated innate immune response in vivo and in vitro. Using a mouse model of IR-induced liver inflammatory injury, we found that mice with a myeloid-specific HSF1 knockout (HSF1M-KO ) displayed exacerbated liver damage based on their increased serum alanine aminotransferase levels, intrahepatic macrophage/neutrophil trafficking, and proinflammatory interleukin (IL)-1β levels compared to the HSF1-proficient (HSF1FL/FL ) controls. Disruption of myeloid HSF1 markedly increased transcription factor X-box-binding protein (XBP1), NLR family, pyrin domain-containing 3 (NLRP3), and cleaved caspase-1 expression, which was accompanied by reduced β-catenin activity. Knockdown of XBP1 in HSF1-deficient livers using a XBP1 small interfering RNA ameliorated hepatocellular functions and reduced NLRP3/cleaved caspase-1 and IL-1β protein levels. In parallel in vitro studies, HSF1 overexpression increased β-catenin (Ser552) phosphorylation and decreased reactive oxygen species (ROS) production in bone-marrow-derived macrophages. However, myeloid HSF1 ablation inhibited β-catenin, but promoted XBP1. Furthermore, myeloid β-catenin deletion increased XBP1 messenger RNA splicing, whereas a CRISPR/CRISPR-associated protein 9-mediated XBP1 knockout diminished NLRP3/caspase-1. CONCLUSION: The myeloid HSF1-β-catenin axis controlled NLRP3 activation by modulating the XBP1 signaling pathway. HSF1 activation promoted β-catenin, which, in turn, inhibited XBP1, leading to NLRP3 inactivation and reduced I/R-induced liver injury. These findings demonstrated that HSF1/β-catenin signaling is a novel regulator of innate immunity in liver inflammatory injury and implied the therapeutic potential for management of sterile liver inflammation in transplant recipients. (Hepatology 2016;64:1683-1698).

Notch signaling plays an emerging role in the regulation of immune cell development and function during inflammatory response. Activation of the ras homolog gene family member A/Rho-associated protein kinase (ROCK) pathway promotes leukocyte accumulation in tissue injury. However, it remains unknown whether Notch signaling regulates ras homolog gene family member A/ROCK-mediated immune responses in liver ischemia and reperfusion (IR) injury. This study investigated intracellular signaling pathways regulated by Notch receptors in the IR-stressed liver and in vitro. In a mouse model of IR-induced liver inflammatory injury, we found that mice with myeloid-specific Notch1 knockout showed aggravated hepatocellular damage, with increased serum alanine aminotransferase levels, hepatocellular apoptosis, macrophage/neutrophil trafficking, and proinflammatory mediators compared to Notch1-proficient controls. Unlike in the controls, myeloid Notch1 ablation diminished hairy and enhancer of split-1 (Hes1) and augmented c-Jun N-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), JNK, ROCK1, and phosphatase and tensin homolog (PTEN) activation in ischemic livers. Disruption of JSAP1 in myeloid-specific Notch1 knockout livers improved hepatocellular function and reduced JNK, ROCK1, PTEN, and toll-like receptor 4 activation. Moreover, ROCK1 knockdown inhibited PTEN and promoted Akt, leading to depressed toll-like receptor 4. In parallel in vitro studies, transfection of lentivirus-expressing Notch1 intracellular domain promoted Hes1 and inhibited JSAP1 in lipopolysaccharide-stimulated bone marrow-derived macrophages. Hes1 deletion enhanced JSAP1/JNK activation, whereas clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9-mediated JSAP1 knockout diminished ROCK1/PTEN and toll-like receptor 4 signaling.

Conclusion

Myeloid Notch1 deficiency activates the ras homolog gene family member A/ROCK pathway and exacerbates hepatocellular injury by inhibiting transcriptional repressor Hes1 and inducing scaffold protein JSAP1 in IR-triggered liver inflammation; our findings underscore the crucial role of the Notch-Hes1 axis as a novel regulator of innate immunity-mediated inflammation and imply the therapeutic potential for the management of organ IR injury in transplant recipients. (Hepatology 2018;67:1041-1055).

Heat shock transcription factor 1 (HSF1) has been implicated in the differential regulation of cell stress and disease states. β-catenin activation is essential for immune homeostasis. However, little is known about the role of macrophage HSF1-β-catenin signaling in the regulation of NLRP3 inflammasome activation during ischemia/reperfusion (I/R) injury (IRI) in the liver. This study investigated the functions and molecular mechanisms by which HSF1-β-catenin signaling influenced NLRP3-mediated innate immune response in vivo and in vitro. Using a mouse model of IR-induced liver inflammatory injury, we found that mice with a myeloid-specific HSF1 knockout (HSF1^M-KO ) displayed exacerbated liver damage based on their increased serum alanine aminotransferase levels, intrahepatic macrophage/neutrophil trafficking, and proinflammatory interleukin (IL)-1β levels compared to the HSF1-proficient (HSF1^FL/FL ) controls. Disruption of myeloid HSF1 markedly increased transcription factor X-box-binding protein (XBP1), NLR family, pyrin domain-containing 3 (NLRP3), and cleaved caspase-1 expression, which was accompanied by reduced β-catenin activity. Knockdown of XBP1 in HSF1-deficient livers using a XBP1 small interfering RNA ameliorated hepatocellular functions and reduced NLRP3/cleaved caspase-1 and IL-1β protein levels. In parallel in vitro studies, HSF1 overexpression increased β-catenin (Ser552) phosphorylation and decreased reactive oxygen species (ROS) production in bone-marrow-derived macrophages. However, myeloid HSF1 ablation inhibited β-catenin, but promoted XBP1. Furthermore, myeloid β-catenin deletion increased XBP1 messenger RNA splicing, whereas a CRISPR/CRISPR-associated protein 9-mediated XBP1 knockout diminished NLRP3/caspase-1.

Conclusion

The myeloid HSF1-β-catenin axis controlled NLRP3 activation by modulating the XBP1 signaling pathway. HSF1 activation promoted β-catenin, which, in turn, inhibited XBP1, leading to NLRP3 inactivation and reduced I/R-induced liver injury. These findings demonstrated that HSF1/β-catenin signaling is a novel regulator of innate immunity in liver inflammatory injury and implied the therapeutic potential for management of sterile liver inflammation in transplant recipients. (Hepatology 2016;64:1683-1698).

Glycogen synthase kinase 3 (GSK3) plays a central role in diverse cellular processes. GSK3 has two mammalian isozymes, GSK3α and GSK3β, whose functions remain ill-defined because of a lack of inhibitors that can distinguish between the two highly homologous isozymes. Here, we show that GSK3α and GSK3β can be selectively inhibited in mouse embryonic stem cells (ESCs) using a chemical-genetic approach. Selective inhibition of GSK3β is sufficient to maintain mouse ESC self-renewal, whereas GSK3α inhibition promotes mouse ESC differentiation toward neural lineages. Genome-wide transcriptional analysis reveals that GSK3α and GSK3β have distinct sets of downstream targets. Furthermore, selective inhibition of individual GSK3 isozymes yields distinct phenotypes from gene deletion, highlighting the power of the chemical-genetic approach in dissecting kinase catalytic functions from the proteins scaffolding functions. Our study opens new avenues for defining GSK3 isozyme-specific functions in various cellular processes.

Wildlife biodiversity is essential for healthy, resilient and sustainable ecosystems. For biologists, this diversity also represents a treasure trove of genetic, molecular and developmental mechanisms that deepen our understanding of the origins and rules of life. However, the rapid decline in biodiversity reported recently foreshadows a potentially catastrophic collapse of many important ecosystems and the associated irreversible loss of many forms of life on our planet. Immediate action by conservationists of all stripes is required to avert this disaster. In this Spotlight, we draw together insights and proposals discussed at a recent workshop hosted by Revive & Restore, which gathered experts to discuss how stem cell technologies can support traditional conservation techniques and help protect animal biodiversity. We discuss reprogramming, in vitro gametogenesis, disease modelling and embryo modelling, and we highlight the prospects for leveraging stem cell technologies beyond mammalian species.