Lethal Phenotype-Based Database Screening Identifies Ceramide as a Negative Regulator of Primitive Streak Formation.

In early embryogenesis, the primitive streak (PrS) generates the mesendoderm and is essential for organogenesis. However, because the PrS is a minute and transient tissue, elucidating the mechanism of its formation has been challenging. We performed comprehensive screening of 2 knockout mouse databases based on the fact that failure of PrS formation is lethal. We identified 812 genes involved in various cellular functions and responses that might be linked to PrS formation, with the category of greatest abundance being "Metabolism." In this study, we focused on genes of sphingolipid metabolism and investigated their roles in PrS formation using an in vitro mouse ES cell differentiation system. We show here that elevated intracellular ceramide negatively regulates gene expression essential for PrS formation and instead induces neurogenesis. In addition, sphingosine-1-phosphate (a ceramide derivative) positively regulates neural maturation. Our results indicate that ceramide regulates both PrS formation and the induction of neural differentiation.

Stem cell challenges and opportunities.

Hepatocyte-like cells (HLCs) generated from human pluripotent stem cells (PSCs) exhibit hepatocytic properties in vitro; however, their engraftment and functionality in vivo remain unsatisfactory. Despite optimization of differentiation protocols, HLCs did not engraft in a mouse model of liver injury. In contrast, organ-derived hepatocytes reproducibly formed colonies in the liver injury mouse model. As an extension of the phenomenon observed in hematopoietic stem cells giving rise to colonies within the spleen, commonly referred to as "colony-forming units in spleen (CFU-s)", we hypothesize that "colony-forming units in liver (CFU-L)" serves as a reliable indicator of stemness, engraftment, and functionality of hepatocytes. The uniform expression of the randomly inactivated gene in a single colony, as reported by Sugahara et al. 2022, suggests that the colonies generated by isolated hepatocytes likely originate from a single cell. We, therefore, propose that CFU-L can be used to quantify the number of "hepatocytes that engraft and proliferate in vivo" as a quantitative assay for stem cells that utilize colony-forming ability, similar to that observed in hematopoietic stem cells.

Development of small fluorescent probes for the analysis of autophagy kinetics.

Autophagy is a dynamic process that degrades subcellular constituents, and its activity is measured by autophagic flux. The tandem proteins RFP-GFP-LC3 and GFP-LC3-RFP-LC3ΔG, which enable the visualization of autophagic vacuoles of different stages by differences in their fluorescent color, are useful tools to monitor autophagic flux, but they require plasmid transfection. In this study, we hence aimed to develop a new method to monitor autophagic flux using small cell-permeable fluorescent probes. We previously developed two green-fluorescent probes, DALGreen and DAPGreen, which detect autolysosomes and multistep autophagic vacuoles, respectively. We here developed a red-fluorescent autophagic probe, named DAPRed, which recognizes various autophagic vacuoles. By the combinatorial use of these green- and red-fluorescent probes, we were able to readily detect autophagic flux. Furthermore, these probes were useful not only for the visualization of canonical autophagy but also for alternative autophagy. DAPRed was also applicable for the detection of autophagy in living organisms.

Integrator complex subunit 15 controls mRNA splicing and is critical for eye development.

The eye and brain are composed of elaborately organized tissues, development of which is supported by spatiotemporally precise expression of a number of transcription factors and developmental regulators. Here we report the molecular and genetic characterization of Integrator complex subunit 15 (INTS15). INTS15 was identified in search for the causative gene(s) for an autosomal-dominant eye disease with variable individual manifestation found in a large pedigree. While homozygous Ints15 knockout mice are embryonic lethal, mutant mice lacking a small C-terminal region of Ints15 show ocular malformations similar to the human patients. INTS15 is highly expressed in the eye and brain during embryogenesis and stably interacts with the Integrator complex to support small nuclear RNA 3' end processing. Its knockdown resulted in missplicing of a large number of genes, probably as a secondary consequence, and substantially affected genes associated with eye and brain development. Moreover, studies using human iPS cells-derived neural progenitor cells showed that INTS15 is critical for axonal outgrowth in retinal ganglion cells. This study suggests a new link between general transcription machinery and a highly specific hereditary disease.

Collagen type I-mediated mechanotransduction controls epithelial cell fate conversion during intestinal inflammation.

BACKGROUND: The emerging concepts of fetal-like reprogramming following tissue injury have been well recognized as an important cue for resolving regenerative mechanisms of intestinal epithelium during inflammation. We previously revealed that the remodeling of mesenchyme with collagen fibril induces YAP/TAZ-dependent fate conversion of intestinal/colonic epithelial cells covering the wound bed towards fetal-like progenitors. To fully elucidate the mechanisms underlying the link between extracellular matrix (ECM) remodeling of mesenchyme and fetal-like reprogramming of epithelial cells, it is critical to understand how collagen type I influence the phenotype of epithelial cells. In this study, we utilize collagen sphere, which is the epithelial organoids cultured in purified collagen type I, to understand the mechanisms of the inflammatory associated reprogramming. Resolving the entire landscape of regulatory networks of the collagen sphere is useful to dissect the reprogrammed signature of the intestinal epithelium. METHODS: We performed microarray, RNA-seq, and ATAC-seq analyses of the murine collagen sphere in comparison with Matrigel organoid and fetal enterosphere (FEnS). We subsequently cultured human colon epithelium in collagen type I and performed RNA-seq analysis. The enriched genes were validated by gene expression comparison between published gene sets and immunofluorescence in pathological specimens of ulcerative colitis (UC). RESULTS: The murine collagen sphere was confirmed to have inflammatory and regenerative signatures from RNA-seq analysis. ATAC-seq analysis confirmed that the YAP/TAZ-TEAD axis plays a central role in the induction of the distinctive signature. Among them, TAZ has implied its relevant role in the process of reprogramming and the ATAC-based motif analysis demonstrated not only Tead proteins, but also Fra1 and Runx2, which are highly enriched in the collagen sphere. Additionally, the human collagen sphere also showed a highly significant enrichment of both inflammatory and fetal-like signatures. Immunofluorescence staining confirmed that the representative genes in the human collagen sphere were highly expressed in the inflammatory region of ulcerative colitis. CONCLUSIONS: Collagen type I showed a significant influence in the acquisition of the reprogrammed inflammatory signature in both mice and humans. Dissection of the cell fate conversion and its mechanisms shown in this study can enhance our understanding of how the epithelial signature of inflammation is influenced by the ECM niche.

HBx and YAP expression could promote tumor development and progression in HBV-related hepatocellular carcinoma.

Background: Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) accounts for 10%-20% of the total HCC numbers. Its clinical features include the occurrence in the younger generation, large tumors, and poor prognosis. The contribution of hepatitis B virus X (HBx) protein in hepatocytes during activation of various oncogenic pathways has been reported. We aimed to assess the possible association between HBx and Yes-associated protein (YAP) expression in the liver tissue and the clinical features of HBV-related HCC. Methods: The relationship between HBx and YAP expression was examined in vivo using HCC tumor and peritumor tissues (n = 55). The clinical information including tumor size, marker, and the prognosis was assessed with protein expressions. The in vitro gene expression analyses were conducted using HBx- and YAP-overexpressing HCC cell lines. Results: Among 19 cases of HBV-related, 17 cases of hepatitis C virus (HCV)-related, and 19 cases of nonviral-related HCC, the HBV-related tumor showed the largest size. The HBx-stained area in the tumor and peritumor tissue showed a significant correlation with tumor size and serum α-fetoprotein level. YAP expression was higher in HBV-related tumor tissue than in the peritumor tissue and HCV-related tumor. Additionally, HBx and YAP protein expressions are correlated and both expressions in the tumor contributed to the poor prognosis. An in vitro study demonstrated that HBx and YAP overexpression in the hepatocytes activate the various oncogenic signaling pathways. Conclusions: Our study demonstrated that YAP expression in the liver of HBV-infected patients might be the key factor in HBV-related HCC development and control of tumor-related features.

Hippo-TAZ signaling is the master regulator of the onset of triple-negative basal-like breast cancers.

Breast cancer is the most frequent malignancy in women worldwide. Basal-like breast cancer (BLBC) is the most aggressive form of this disease, and patients have a poor prognosis. Here, we present data suggesting that the Hippo-transcriptional coactivator with PDZ-binding motif (TAZ) pathway is a key driver of BLBC onset and progression. Deletion of Mob1a/b in mouse mammary luminal epithelium induced rapid and highly reproducible mammary tumorigenesis that was dependent on TAZ but not yes-associated protein 1 (YAP1). In situ early-stage BLBC-like malignancies developed in mutant animals by 2 wk of age, and invasive BLBC appeared by 4 wk. In a human estrogen receptor+ luminal breast cancer cell line, TAZ hyperactivation skewed the features of these luminal cells to the basal phenotype, consistent with the aberrant TAZ activation frequently observed in human precancerous BLBC lesions. TP53 mutation is rare in human precancerous BLBC but frequent in invasive BLBC. Addition of Trp53 deficiency to our Mob1a/b-deficient mouse model enhanced tumor grade and accelerated cancer progression. Our work justifies targeting the Hippo-TAZ pathway as a therapy for human BLBC, and our mouse model represents a powerful tool for evaluating candidate agents.

Abnormal male reproduction and embryonic development induced by downregulation of a phospholipid fatty acid-introducing enzyme Lpgat1 in zebrafish.

Phospholipids in the membrane consist of diverse pairs of fatty acids bound to a glycerol backbone. The biological significance of the diversity, however, remains mostly unclear. Part of this diversity is due to lysophospholipid acyltransferases (LPLATs), which introduce a fatty acid into lysophospholipids. The human genome has 14 LPLATs and most of them are highly conserved in vertebrates. Here, we analyzed the function of one of these enzymes, lysophosphatidylglycerol acyltransferase 1 (Lpgat1), in zebrafish. We found that the reproduction of heterozygous (lpgat1+/-) male mutants was abnormal. Crosses between heterozygous males and wild-type females produced many eggs with no obvious cleavage, whereas eggs produced by crosses between heterozygous females and wild-type males cleaved normally. Consistent with this, spermatozoa from heterozygous males had reduced motility and abnormal morphology. We also found that the occurrence of lpgat1 homozygous (lpgat1-/-) mutants was far lower than expected. In addition, downregulation of lpgat1 by morpholino antisense oligonucleotides resulted in severe developmental defects. Lipidomic analysis revealed that selective phospholipid species with stearic acid and docosahexaenoic acid were reduced in homozygous larvae and spermatozoa from heterozygotes. These results suggest that the specific phospholipid molecular species produced by Lpgat1 have an essential role in sperm fertilization and in embryonic development.

Physiological and pathological roles of the Hippo-YAP/TAZ signaling pathway in liver formation, homeostasis, and tumorigenesis.

The liver plays central homeostatic roles in metabolism and detoxification, and has a remarkable capacity to fully recover from injuries caused by the various insults to which it is constantly exposed. To fulfill these functions, the liver must maintain a specific size and so must regulate its cell numbers. It must also remove senescent, transformed, and/or injured cells that impair liver function and can lead to diseases such as cirrhosis and liver cancer. Despite their importance, however, the mechanisms governing liver size control and homeostasis have resisted delineation. The discovery of the Hippo intracellular signaling pathway and its downstream effectors, the transcriptional coactivators Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), has provided partial elucidation of these mechanisms. The Hippo-YAP/TAZ pathway is considered to be a cell's sensor of its immediate microenvironment and the cells that surround it, in that this pathway responds to changes in elements such as the ECM, cell-cell tension, and cell adhesion. Once triggered, Hippo signaling negatively regulates the binding of YAP/TAZ to transcription factors such as TEAD and Smad, controlling their ability to drive gene expression needed for cellular responses such as proliferation, survival, and stemness. Numerous KO mouse strains lacking YAP/TAZ, as well as transgenic mice showing YAP/TAZ hyperactivation, have been generated, and the effects of these mutations on liver development, size, regeneration, homeostasis, and tumorigenesis have been reported. In this review, I summarize the components and regulation of Hippo-YAP/TAZ signaling, and discuss this pathway in the context of liver physiology and pathology.

Protein kinase Cα activation switches YAP1 from TEAD-mediated signaling to p73-mediated signaling.

Yes-associated protein 1 (YAP1) interacts with TEAD transcription factor in the nucleus and upregulates TEAD-target genes. YAP1 is phosphorylated by large tumor suppressor (LATS) kinases, the core kinases of the Hippo pathway, at 5 serine residues and is sequestered and degraded in the cytoplasm. In human cancers with the dysfunction of the Hippo pathway, YAP1 becomes hyperactive and confers malignant properties to cancer cells. We have observed that cold shock induces protein kinase C (PKC)-mediated phosphorylation of YAP1. PKC phosphorylates YAP1 at 3 serine residues among LATS-mediate phosphorylation sites. Importantly, PKC activation recruits YAP1 to the cytoplasm even in LATS-depleted cancer cells and reduces the cooperation with TEAD. PKC activation induces promyelocytic leukemia protein-mediated SUMOylation of YAP1. SUMOylated YAP1 remains in the nucleus, binds to p73, and promotes p73-target gene transcription. Bryostatin, a natural anti-neoplastic reagent that activates PKC, induces YAP1/p73-mediated apoptosis in cancer cells. Bryostatin reverses malignant transformation caused by the depletion of LATS kinases. Therefore, bryostatin and other reagents that activate PKC are expected to control cancers with the dysfunction of the Hippo pathway.

Puromycin-based purification of cells with high expression of the cytochrome P450 CYP3A4 gene from a patient with drug-induced liver injury (DILI).

BACKGROUND: Many drugs have the potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in hepatocytes. Hepatocytes can be accurately evaluated for drug-mediated CYP3A4 induction; this is the gold standard for in vitro hepatic toxicology testing. However, the variation from lot to lot is an issue that needs to be addressed. Only a limited number of immortalized hepatocyte cell lines have been reported. In this study, immortalized cells expressing CYP3A4 were generated from a patient with drug-induced liver injury (DILI). METHODS: To generate DILI-derived cells with high expression of CYP3A4, a three-step approach was employed: (1) Differentiation of DILI-induced pluripotent stem cells (DILI-iPSCs); (2) Immortalization of the differentiated cells; (3) Selection of the cells by puromycin. It was hypothesized that cells with high cytochrome P450 gene expression would be able to survive exposure to cytotoxic antibiotics because of their increased drug-metabolizing activity. Puromycin, a cytotoxic antibiotic, was used in this study because of its rapid cytocidal effect at low concentrations. RESULTS: The hepatocyte-like cells differentiated from DILI-iPSCs were purified by exposure to puromycin. The puromycin-selected cells (HepaSM or SI cells) constitutively expressed the CYP3A4 gene at extremely high levels and exhibited hepatocytic features over time. However, unlike primary hepatocytes, the established cells did not produce bile or accumulate glycogen. CONCLUSIONS: iPSC-derived hepatocyte-like cells with intrinsic drug-metabolizing enzymes can be purified from non-hepatocytes and undifferentiated iPSCs using the cytocidal antibiotic puromycin. The puromycin-selected hepatocyte-like cells exhibited characteristics of hepatocytes after immortalization and may serve as another useful source for in vitro hepatotoxicity testing of low molecular weight drugs.

DNA Damage Triggers the Nuclear Accumulation of RASSF6 Tumor Suppressor Protein via CDK9 and BAF53 To Regulate p53 Target Gene Transcription.

RASSF6, a member of the tumor suppressor Ras-association domain family (RASSF) proteins, regulates cell cycle arrest and apoptosis via p53 and plays a tumor suppressor role. We previously reported that RASSF6 blocks MDM2-mediated p53 degradation and enhances p53 expression. In this study, we demonstrated that RASSF6 has nuclear localization and nuclear export signals and that DNA damage triggers the nuclear accumulation of RASSF6. We found that RASSF6 directly binds to BAF53, the component of SWI/SNF complex. DNA damage induces CDK9-mediated phosphorylation of BAF53, which enhances the interaction with RASSF6 and increases the amount of RASSF6 in the nucleus. Subsequently, RASSF6 augments the interaction between BAF53 and BAF60a, another component of the SWI/SNF complex, and further promotes the interaction of BAF53 and BAF60a with p53. BAF53 silencing or BAF60a silencing attenuates RASSF6-mediated p53 target gene transcription and apoptosis. Thus, RASSF6 is involved in the regulation of DNA damage-induced complex formation, including BAF53, BAF60a, and p53.

Characterization of mouse embryonic fibroblasts derived from Rassf6 knockout mice shows the implication of Rassf6 in the regulation of NF-κB signaling.

RASSF6 is a member of the tumor suppressor Ras association domain family (RASSF) proteins. We have reported using human cancer cell lines that RASSF6 induces apoptosis and cell cycle arrest via p53 and plays tumor suppressive roles. In this study, we generated Rassf6 knockout mice by CRISPR/Cas technology. Contrary to our expectation, Rassf6 knockout mice were apparently healthy. However, Rassf6-null mouse embryonic fibroblasts (MEF) were resistant against ultraviolet (UV)-induced apoptosis/cell cycle arrest and senescence. UV-induced p53-target gene expression was compromised, and DNA repair was delayed in Rassf6-null MEF. More importantly, KRAS active mutant promoted the colony formation of Rassf6-null MEF but not the wild-type MEF. RNA sequencing analysis showed that NF-κB signaling was enhanced in Rassf6-null MEF. Consistently, 7,12-dimethylbenz(a)anthracene (DMBA) induced skin inflammation in Rassf6 knockout mice more remarkably than in the wild-type mice. Hence, Rassf6 deficiency not only compromises p53 function but also enhances NF-κB signaling to lead to oncogenesis.

YAP drives cell competition by activating choline metabolism.

Cell competition is a phenomenon that eliminates unfit cells from cell society, a function vital for maintaining cellular and organismal homeostasis. We previously showed that Madin-Darby canine kidney (MDCK) epithelial cells expressing the active form of the transcriptional coactivator Yes-associated protein (YAP) are apically extruded when surrounded by normal MDCK cells. Although we demonstrated that the arachidonic acid (AA) cascade is involved in YAP-dependent apical extrusion, the metabolic events leading to this outcome remained unclear. Here, we present the results of metabolomic analysis that identified phosphatidylcholine (PC) biosynthesis as the most significant player in this process. Removal of the PC biosynthetic components choline and methionine from culture medium inhibited YAP-dependent apical extrusion. Inhibition of either choline uptake or metabolic cycles involving choline or methionine also decreased YAP-dependent apical extrusion. At the molecular level, active YAP induced expression of the genes encoding glycerophosphocholine phosphodiesterase 1 (GPCPD1) and lecithin-cholesterol acyltransferase (LCAT), which are involved in choline metabolism. Our results indicate that YAP-dependent cell competition depends on YAP-mediated activation of the choline metabolic cycle.

The Light-Inducible Genes Per2, Cry1a, and Cry2a Regulate Oxidative Status in Zebrafish.

The circadian clock is a highly conserved 24 h biological oscillation mechanism and is affected by environmental stimuli such as light, food and temperature. Disruption of the circadian clock results in disorders of diverse biological processes, including the sleep-wake cycle and metabolism. Although we previously identified several components of the circadian clock in zebrafish, our understanding of the relationship between light-inducible clock genes and metabolism remains incomplete. To investigate how light-inducible clock genes regulate metabolism, we performed transcriptomic and metabolomic analyses of the light-inducible clock genes zPer2, zCry1a, and zCry2a in zebrafish. Transcriptomic analysis of zPer2/zCry1a double knockout (DKO) and zPer2/zCry1a/zCry2a triple knockout (TKO) mutants showed that their gene expression profiles differed from that of wild type (WT) zebrafish. In particular, mRNA levels of zKeap1a, which encodes an oxidative stress sensor, were increased in DKO and TKO mutants. Metabolomic analysis showed genotype-dependent alteration of metabolomic profiles. Principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) showed the alteration of cysteine/methionine metabolism and glutathione metabolism. Specifically, cysteine and glutathione were decreased but methionine sulfoxide was increased in TKO zebrafish. These results indicate that the light-inducible genes zPer2, zCry1a, and zCry2a are involved in regulating the oxidative status of zebrafish.

Alantolactone is a natural product that potently inhibits YAP1/TAZ through promotion of reactive oxygen species accumulation.

Yes-associated protein 1 (YAP1) and its paralogue PDZ-binding motif (TAZ) play pivotal roles in cell proliferation, migration, and invasion, and abnormal activation of these TEAD transcriptional coactivators is found in diverse cancers in humans and mice. Targeting YAP1/TAZ signaling is thus a promising therapeutic avenue but, to date, few selective YAP1/TAZ inhibitors have been effective against cancer cells either in vitro or in vivo. We screened chemical libraries for potent YAP1/TAZ inhibitors using a highly sensitive luciferase reporter system to monitor YAP1/TAZ-TEAD transcriptional activity in cells. Among 29 049 low-molecular-weight compounds screened, we obtained nine hits, and the four of these that were the most effective shared a core structure with the natural product alantolactone (ALT). We also tested 16 other structural derivatives of ALT and found that natural ALT was the most efficient at increasing ROS-induced LATS kinase activities and thus YAP1/TAZ phosphorylation. Phosphorylated YAP1/TAZ proteins were subject to nuclear exclusion and proteosomic degradation such that the growth of ALT-treated tumor cells was inhibited both in vitro and in vivo. Our data show for the first time that ALT can be used to target the ROS-YAP pathway driving tumor cell growth and so could be a potent anticancer drug.

CSE1L promotes nuclear accumulation of transcriptional coactivator TAZ and enhances invasiveness of human cancer cells.

The transcriptional coactivator with PDZ-binding motif (TAZ) (WWTR1) induces epithelial-mesenchymal transition and enhances drug resistance in multiple cancers. TAZ has been shown to interact with transcription factors in the nucleus, but when phosphorylated, translocates to the cytoplasm and is degraded through proteasomes. Here, we identified a compound TAZ inhibitor 4 (TI-4) that shifted TAZ localization to the cytoplasm independently of its phosphorylation. We used affinity beads to ascertain a putative target of TI-4, chromosomal segregation 1 like (CSE1L), which is known to be involved in the recycling of importin α and as a biomarker of cancer malignancy. We found that TI-4 suppressed TAZ-mediated transcription in a CSE1L-dependent manner. CSE1L overexpression increased nuclear levels of TAZ, whereas CSE1L silencing delayed its nuclear import. We also found via the in vitro coimmunoprecipitation experiments that TI-4 strengthened the interaction between CSE1L and importin α5 and blocked the binding of importin α5 to TAZ. WWTR1 silencing attenuated CSE1L-promoted colony formation, motility, and invasiveness of human lung cancer and glioblastoma cells. Conversely, CSE1L silencing blocked TAZ-promoted colony formation, motility, and invasiveness in human lung cancer and glioblastoma cells. In human cancer tissues, the expression level of CSE1L was found to correlate with nuclear levels of TAZ. These findings support that CSE1L promotes the nuclear accumulation of TAZ and enhances malignancy in cancer cells.

Heat shock induces the nuclear accumulation of YAP1 via SRC.

Yes-associated protein 1 (YAP1), a co-transcription activator, shuttles between the cytoplasm and the nucleus. Phosphorylation by large tumor suppressor kinases (LATS1/2) is the major determinant of YAP1 subcellular localization. Unphosphorylated YAP1 interacts with transcription factors in the nucleus and regulates gene transcription, while phosphorylated YAP1 is trapped in the cytoplasm and is degraded. We found that when U2OS and HeLa cells are exposed to 42 °C, YAP1 enters the nucleus within 30 min and returns to the cytoplasm at 4 h. SRC and HSP90 are involved in nuclear accumulation and return to the cytoplasm, respectively. Upon heat shock, LATS2 forms aggregates including protein phosphatase 1 and is dephosphorylated and inactivated. SRC activation is necessary for the formation of aggregates, while HSP90 is required for their dissociation. YAP1 is involved in heat shock-induced NF-κB signaling. Mechanistically, YAP1 is implicated in strengthening the interaction between RELA and DPF3, a component of SWI/SNF chromatin remodeling complex, in response to heat shock. Thus, YAP1 plays a role as a thermosensor.