Phosphorylation of EIF2S1 (eukaryotic translation initiation factor 2 subunit alpha) is indispensable for nuclear translocation of TFEB and TFE3 during ER stress.

There are diverse links between macroautophagy/autophagy pathways and unfolded protein response (UPR) pathways under endoplasmic reticulum (ER) stress conditions to restore ER homeostasis. Phosphorylation of EIF2S1/eIF2α is an important mechanism that can regulate all three UPR pathways through transcriptional and translational reprogramming to maintain cellular homeostasis and overcome cellular stresses. In this study, to investigate the roles of EIF2S1 phosphorylation in regulation of autophagy during ER stress, we used EIF2S1 phosphorylation-deficient (A/A) cells in which residue 51 was mutated from serine to alanine. A/A cells exhibited defects in several steps of autophagic processes (such as autophagosome and autolysosome formation) that are regulated by the transcriptional activities of the autophagy master transcription factors TFEB and TFE3 under ER stress conditions. EIF2S1 phosphorylation was required for nuclear translocation of TFEB and TFE3 during ER stress. In addition, EIF2AK3/PERK, PPP3/calcineurin-mediated dephosphorylation of TFEB and TFE3, and YWHA/14-3-3 dissociation were required for their nuclear translocation, but were insufficient to induce their nuclear retention during ER stress. Overexpression of the activated ATF6/ATF6α form, XBP1s, and ATF4 differentially rescued defects of TFEB and TFE3 nuclear translocation in A/A cells during ER stress. Consequently, overexpression of the activated ATF6 or TFEB form more efficiently rescued autophagic defects, although XBP1s and ATF4 also displayed an ability to restore autophagy in A/A cells during ER stress. Our results suggest that EIF2S1 phosphorylation is important for autophagy and UPR pathways, to restore ER homeostasis and reveal how EIF2S1 phosphorylation connects UPR pathways to autophagy.Abbreviations: A/A: EIF2S1 phosphorylation-deficient; ACTB: actin beta; Ad-: adenovirus-; ATF6: activating transcription factor 6; ATZ: SERPINA1/α1-antitrypsin with an E342K (Z) mutation; Baf A1: bafilomycin A1; BSA: bovine serum albumin; CDK4: cyclin dependent kinase 4; CDK6: cyclin dependent kinase 6; CHX: cycloheximide; CLEAR: coordinated lysosomal expression and regulation; Co-IP: coimmunoprecipitation; CTSB: cathepsin B; CTSD: cathepsin D; CTSL: cathepsin L; DAPI: 4',6-diamidino-2-phenylindole dihydrochloride; DMEM: Dulbecco's modified Eagle's medium; DMSO: dimethyl sulfoxide; DTT: dithiothreitol; EBSS: Earle's Balanced Salt Solution; EGFP: enhanced green fluorescent protein; EIF2S1/eIF2α: eukaryotic translation initiation factor 2 subunit alpha; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; ERAD: endoplasmic reticulum-associated degradation; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; FBS: fetal bovine serum; gRNA: guide RNA; GSK3B/GSK3ß: glycogen synthase kinase 3 beta; HA: hemagglutinin; Hep: immortalized hepatocyte; IF: immunofluorescence; IRES: internal ribosome entry site; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LMB: leptomycin B; LPS: lipopolysaccharide; MAP1LC3A/B/LC3A/B: microtubule associated protein 1 light chain 3 alpha/beta; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEFs: mouse embryonic fibroblasts; MFI: mean fluorescence intensity; MTORC1: mechanistic target of rapamycin kinase complex 1; NES: nuclear export signal; NFE2L2/NRF2: NFE2 like bZIP transcription factor 2; OE: overexpression; PBS: phosphate-buffered saline; PLA: proximity ligation assay; PPP3/calcineurin: protein phosphatase 3; PTM: post-translational modification; SDS: sodium dodecyl sulfate; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEM: standard error of the mean; TEM: transmission electron microscopy; TFE3: transcription factor E3; TFEB: transcription factor EB; TFs: transcription factors; Tg: thapsigargin; Tm: tunicamycin; UPR: unfolded protein response; WB: western blot; WT: wild-type; Xbp1s: spliced Xbp1; XPO1/CRM1: exportin 1.

Systemic TM4SF5 overexpression in ApcMin/+ mice promotes hepatic portal hypertension associated with fibrosis.

Mutation of the gene for adenomatous polyposis coli (APC), as seen in ApcMin/+ mice, leads to intestinal adenomas and carcinomas via stabilization of ß-catenin. Transmembrane 4 L six family member 5 (TM4SF5) is involved in the development of non-alcoholic fatty liver disease, fibrosis, and cancer. However, the functional linkage between TM4SF5 and APC or ß-catenin has not been investigated for pathological outcomes. After interbreeding ApcMin/+ with TM4SF5-overexpressing transgenic (TgTM4SF5) mice, we explored pathological outcomes in the intestines and livers of the offspring. The intestines of 26-week-old dual-transgenic mice (ApcMin/+:TgTM4SF5) had intramucosal adenocarcinomas beyond the single-crypt adenomas in ApcMin/+ mice. Additional TM4SF5 overexpression increased the stabilization of ß-catenin via reduced glycogen synthase kinase 3ß (GSK3ß) phosphorylation on Ser9. Additionally, the livers of the dualtransgenic mice showed distinct sinusoidal dilatation and features of hepatic portal hypertension associated with fibrosis, more than did the relatively normal livers in ApcMin/+ mice. Interestingly, TM4SF5 overexpression in the liver was positively linked to increased GSK3ß phosphorylation (opposite to that seen in the colon), ß-catenin level, and extracellular matrix (ECM) protein expression, indicating fibrotic phenotypes. Consistent with these results, 78-week-old TgTM4SF5 mice similarly had sinusoidal dilatation, immune cell infiltration, and fibrosis. Altogether, systemic overexpression of TM4SF5 aggravates pathological abnormalities in both the colon and the liver. [BMB Reports 2022; 55(12): 609-614].

O-GlcNAcylation: An Emerging Protein Modification Regulating the Hippo Pathway.

The balance between cellular proliferation and apoptosis and the regulation of cell differentiation must be established to maintain tissue homeostasis. These cellular responses involve the kinase cascade-mediated Hippo pathway as a crucial regulator. Hence, Hippo pathway dysregulation is implicated in diverse diseases, including cancer. O-GlcNAcylation is a non-canonical glycosylation that affects multiple signaling pathways through its interplay with phosphorylation in the nucleus and cytoplasm. An abnormal increase in the O-GlcNAcylation levels in various cancer cells is a potent factor in Hippo pathway dysregulation. Intriguingly, Hippo pathway dysregulation also disrupts O-GlcNAc homeostasis, leading to a persistent elevation of O-GlcNAcylation levels, which is potentially pathogenic in several diseases. Therefore, O-GlcNAcylation is gaining attention as a protein modification that regulates the Hippo pathway. This review presents a framework on how O-GlcNAcylation regulates the Hippo pathway and forms a self-perpetuating cycle with it. The pathological significance of this self-perpetuating cycle and clinical strategies for targeting O-GlcNAcylation that causes Hippo pathway dysregulation are also discussed.

Past, present, and future perspectives of transcription factor EB (TFEB): mechanisms of regulation and association with disease.

Transcription factor EB (TFEB), a member of the MiT/TFE family of basic helix-loop-helix leucine zipper transcription factors, is an established central regulator of the autophagy/lysosomal-to-nucleus signaling pathway. Originally described as an oncogene, TFEB is now widely known as a regulator of various processes, such as energy homeostasis, stress response, metabolism, and autophagy-lysosomal biogenesis because of its extensive involvement in various signaling pathways, such as mTORC1, Wnt, calcium, and AKT signaling pathways. TFEB is also implicated in various human diseases, such as lysosomal storage disorders, neurodegenerative diseases, cancers, and metabolic disorders. In this review, we present an overview of the major advances in TFEB research over the past 30 years, since its description in 1990. This review also discusses the recently discovered regulatory mechanisms of TFEB and their implications for human diseases. We also summarize the moonlighting functions of TFEB and discuss future research directions and unanswered questions in the field. Overall, this review provides insight into our understanding of TFEB as a major molecular player in human health, which will take us one step closer to promoting TFEB from basic research into clinical and regenerative applications.

Regulation of Hippo signaling by metabolic pathways in cancer.

Hippo signaling is known to maintain balance between cell proliferation and apoptosis via tight regulation of factors, such as metabolic cues, cell-cell contact, and mechanical cues. Cells directly recognize glucose, lipids, and other metabolic cues and integrate multiple signaling pathways, including Hippo signaling, to adjust their proliferation and apoptosis depending on nutrient conditions. Therefore, the dysregulation of the Hippo signaling pathway can promote tumor initiation and progression. Alteration in metabolic cues is considered a major factor affecting the risk of cancer formation and progression. It has recently been shown that the dysregulation of the Hippo signaling pathway, through diverse routes activated by metabolic cues, can lead to cancer with a poor prognosis. In addition, unique crosstalk between metabolic pathways and Hippo signaling pathways can inhibit the effect of anticancer drugs and promote drug resistance. In this review, we describe an integrated perspective of the relationship between the Hippo signaling pathway and metabolic signals in the context of cancer. We also characterize the mechanisms involved in changes in metabolism that are linked to the Hippo signaling pathway in the cancer microenvironment and propose several novel targets for anticancer drug treatment.

Role of the Hippo pathway and mechanisms for controlling cellular localization of YAP/TAZ.

The Hippo pathway is a crucial signaling mechanism that inhibits the growth of cells and organs during development and in disease. When the Hippo pathway is activated, YAP/TAZ transcriptional coactivators are phosphorylated by upstream kinases, preventing nuclear localization of YAP/TAZ. However, when the Hippo pathway is inhibited, YAP/TAZ localize mainly in the nucleus and induce the expression of target genes related to cell proliferation. Abnormal proliferation of cells is one of the hallmarks of cancer initiation, and activation of Hippo pathway dampens such cell proliferation. Various types of diseases including cancer can occur due to the dysregulation of the Hippo pathway. Therefore, a better understanding of the Hippo pathway signaling mechanisms, and in particular how YAP/TAZ exist in the nucleus, may lead to the identification of new therapeutic targets for treating cancer and other diseases. In this review, we summarize the overall Hippo pathway and discuss mechanisms related to nuclear localization of YAP/TAZ.

Hypermethylation of Mest promoter causes aberrant Wnt signaling in patients with Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that leads to dementia and behavioral changes. Extracellular deposition of amyloid plaques (Aß) and intracellular deposition of neurofibrillary tangles in neurons are the major pathogenicities of AD. However, drugs targeting these therapeutic targets are not effective. Therefore, novel targets for the treatment of AD urgently need to be identified. Expression of the mesoderm-specific transcript (Mest) is regulated by genomic imprinting, where only the paternal allele is active for transcription. We identified hypermethylation on the Mest promoter, which led to a reduction in Mest mRNA levels and activation of Wnt signaling in brain tissues of AD patients. Mest knockout (KO) using the CRIPSR/Cas9 system in mouse embryonic stem cells and P19 embryonic carcinoma cells leads to neuronal differentiation arrest. Depletion of Mest in primary hippocampal neurons via lentivirus expressing shMest or inducible KO system causes neurodegeneration. Notably, depletion of Mest in primary cortical neurons of rats leads to tau phosphorylation at the S199 and T231 sites. Overall, our data suggest that hypermethylation of the Mest promoter may cause or facilitate the progression of AD.

Regulation of the Low-Density Lipoprotein Receptor-Related Protein LRP6 and Its Association With Disease: Wnt/ß-Catenin Signaling and Beyond.

Wnt signaling plays crucial roles in development and tissue homeostasis, and its dysregulation leads to various diseases, notably cancer. Wnt/ß-catenin signaling is initiated when the glycoprotein Wnt binds to and forms a ternary complex with the Frizzled and low-density lipoprotein receptor-related protein 5/6 (LRP5/6). Despite being identified as a Wnt co-receptor over 20 years ago, the molecular mechanisms governing how LRP6 senses Wnt and transduces downstream signaling cascades are still being deciphered. Due to its role as one of the main Wnt signaling components, the dysregulation or mutation of LRP6 is implicated in several diseases such as cancer, neurodegeneration, metabolic syndrome and skeletal disease. Herein, we will review how LRP6 is activated by Wnt stimulation and explore the various regulatory mechanisms involved. The participation of LRP6 in other signaling pathways will also be discussed. Finally, the relationship between LRP6 dysregulation and disease will be examined in detail.

TFEB regulates pluripotency transcriptional network in mouse embryonic stem cells independent of autophagy-lysosomal biogenesis.

Transcription factor EB (TFEB), a well-known master regulator of autophagy and lysosomal biogenesis, is a member of the microphthalmia family of transcription factors (MiT family). Over the years, TFEB has been shown to have diverse roles in various physiological processes such as clearance for intracellular pathogenic factors and having developmental functions such as dendritic maturation, as well as osteoclast, and endoderm differentiation. However, in the present study, we propose a novel mechanism for TFEB governing pluripotency of mouse ESCs (mESCs) by regulating the pluripotency transcriptional network (PTN) in these cells. We observed high levels of TFEB mRNA and protein levels in undifferentiated mESCs. Interestingly, we found a reduction of Nanog and Sox2 levels in TFEB knockout (KO) mESCs while pluripotency was maintained as there was an upregulation of TFE3, a potent stem cell maintenance factor. In consistent, double knockout of TFEB/TFE3 (TFEB/3 DKO) reduced mESC pluripotency, as indicated by the loss of ESC morphology, reduction of ESC markers, and the emergence of differentiation markers. We further discovered that Nanog was a TFEB target gene in undifferentiated mESCs. TFEB also promoted sex-determining region Y-box2 (Sox2) transcription by forming a heterodimer with Sox2 in mESCs. Notably, Sox2, Oct4, and Nanog were also binding to the TFEB promoter and thus generating a feed-forward loop in relation to TFEB. Although high levels of nuclear TFEB are expected to enhance autophagy-lysosomal activity, undifferentiated mESC remarkably displayed low basal autophagy-lysosomal activity. Overexpression or knockout of TFEB did not affect the expression of TFEB lysosomal-autophagy target genes and TFEB also had a lesser binding affinity to its own lysosomal promoter-target genes in mESCs compared to differentiated cells. Collectively, these findings define a newly incorporative, moonlighting function for TFEB in regulating PTN, independent of its autophagy-lysosomal biogenesis roles.

PARsylated transcription factor EB (TFEB) regulates the expression of a subset of Wnt target genes by forming a complex with ß-catenin-TCF/LEF1.

Wnt signaling is mainly transduced by ß-catenin via regulation of the ß-catenin destruction complex containing Axin, APC, and GSK3ß. Transcription factor EB (TFEB) is a well-known master regulator of autophagy and lysosomal biogenesis processes. TFEB's nuclear localization and transcriptional activity are also regulated by various upstream signals. In this study, we found that Wnt signaling induces the nuclear localization of TFEB and the expression of Wnt target genes is regulated by TFEB-ß-catenin-TCF/LEF1 as well as ß-catenin-TCF/LEF1 complexes. Our biochemical data revealed that TFEB is a part of the ß-catenin destruction complex, and destabilization of the destruction complex by knockdown of either Axin or APC causes nuclear localization of TFEB. Interestingly, RNA-sequencing analysis revealed that about 27% of Wnt3a-induced genes were TFEB dependent. However, these "TFEB mediated Wnt target genes" were different from TFEB target genes involved in autophagy and lysosomal biogenesis processes. Mechanistically, we found that Tankyrase (TNKS) PARsylates TFEB with Wnt ON signaling, and the nuclear localized PARsylated TFEB forms a complex with ß-catenin-TCF/LEF1 to induce the "TFEB mediated Wnt target genes". Finally, we found that in various types of cancer, the levels of TFEB mediated Wnt target genes exhibit strong correlations with the level of Axin2, which represents the activity of Wnt signaling. Overall, our data suggest that Wnt signaling induces the expression of a subset of genes that are distinct from previously known genes regulated by the ß-catenin-TCF/LEF1 complex or TFEB, by forming a transcription factor complex consisting of PARsylated TFEB and ß-catenin-TCF/LEF1.

LGK974 suppresses lipopolysaccharide-induced endotoxemia in mice by modulating the crosstalk between the Wnt/ß-catenin and NF-κB pathways.

Endotoxemia, a type of sepsis caused by gram-negative bacterial endotoxin [i.e., lipopolysaccharide (LPS)], is associated with manifestations such as cytokine storm; failure of multiple organs, including the liver; and a high mortality rate. We investigated the effect and mechanism of action of LGK974, a Wnt signaling inhibitor, in mice with LPS-induced endotoxemia, an animal model of sepsis. LGK974 significantly and dose-dependently increased the survival rate and reduced plasma cytokine levels in mice with LPS-induced endotoxemia. Transcriptome analysis of liver tissues revealed significant changes in the expression of genes associated with the Wnt pathway as well as cytokine and NF-κB signaling during endotoxemia. LGK974 treatment suppressed the activation of NF-κB signaling and cytokine expression as well as the Wnt/ß-catenin pathway in the livers of endotoxemic mice. Coimmunoprecipitation of phospho-IκB and ß-transducin repeat-containing protein (ß-TrCP) was increased in the livers of endotoxemic mice but was reduced by LGK974 treatment. Moreover, LGK974 treatment decreased the coimmunoprecipitation and colocalization of ß-catenin and NF-κB, which were elevated in the livers of endotoxemic mice. Our results reveal crosstalk between the Wnt/ß-catenin and NF-κB pathways via interactions between ß-TrCP and phospho-IκB and between ß-catenin and NF-κB during endotoxemia. The results of this study strongly suggest that the crosstalk between the Wnt/ß-catenin and NF-κB pathways contributes to the mutual activation of these two pathways during endotoxemia, which results in amplified cytokine production, liver damage and death, and that LGK974 suppresses this vicious amplification cycle by reducing the crosstalk between these two pathways.

SGK1 inhibition in glia ameliorates pathologies and symptoms in Parkinson disease animal models.

Astrocytes and microglia are brain-resident glia that can establish harmful inflammatory environments in disease contexts and thereby contribute to the progression of neuronal loss in neurodegenerative disorders. Correcting the diseased properties of glia is therefore an appealing strategy for treating brain diseases. Previous studies have shown that serum/ glucocorticoid related kinase 1 (SGK1) is upregulated in the brains of patients with various neurodegenerative disorders, suggesting its involvement in the pathogenesis of those diseases. In this study, we show that inhibiting glial SGK1 corrects the pro-inflammatory properties of glia by suppressing the intracellular NFκB-, NLRP3-inflammasome-, and CGAS-STING-mediated inflammatory pathways. Furthermore, SGK1 inhibition potentiated glial activity to scavenge glutamate toxicity and prevented glial cell senescence and mitochondrial damage, which have recently been reported as critical pathologic features of and therapeutic targets in Parkinson disease (PD) and Alzheimer disease (AD). Along with those anti-inflammatory/neurotrophic functions, silencing and pharmacological inhibition of SGK1 protected midbrain dopamine neurons from degeneration and cured pathologic synuclein alpha (SNCA) aggregation and PD-associated behavioral deficits in multiple in vitro and in vivo PD models. Collectively, these findings suggest that SGK1 inhibition could be a useful strategy for treating PD and other neurodegenerative disorders that share the common pathology of glia-mediated neuroinflammation.

TAZ/Wnt-ß-catenin/c-MYC axis regulates cystogenesis in polycystic kidney disease.

Autosomal-dominant polycystic kidney disease (ADPKD) is the most common genetic renal disease, primarily caused by germline mutation of PKD1 or PKD2, leading to end-stage renal disease. The Hippo signaling pathway regulates organ growth and cell proliferation. Herein, we demonstrate the regulatory mechanism of cystogenesis in ADPKD by transcriptional coactivator with PDZ-binding motif (TAZ), a Hippo signaling effector. TAZ was highly expressed around the renal cyst-lining epithelial cells of Pkd1-deficient mice. Loss of Taz in Pkd1-deficient mice reduced cyst formation. In wild type, TAZ interacted with PKD1, which inactivated ß-catenin. In contrast, in PKD1-deficient cells, TAZ interacted with AXIN1, thus increasing ß-catenin activity. Interaction of TAZ with AXIN1 in PKD1-deficient cells resulted in nuclear accumulation of TAZ together with ß-catenin, which up-regulated c-MYC expression. Our findings suggest that the PKD1-TAZ-Wnt-ß-catenin-c-MYC signaling axis plays a critical role in cystogenesis and might be a potential therapeutic target against ADPKD.

LDL receptor-related protein LRP6 senses nutrient levels and regulates Hippo signaling.

Controlled cell growth and proliferation are essential for tissue homeostasis and development. Wnt and Hippo signaling are well known as positive and negative regulators of cell proliferation, respectively. The regulation of Hippo signaling by the Wnt pathway has been shown, but how and which components of Wnt signaling are involved in the activation of Hippo signaling during nutrient starvation are unknown. Here, we report that a reduction in the level of low-density lipoprotein receptor-related protein 6 (LRP6) during nutrient starvation induces phosphorylation and cytoplasmic localization of YAP, inhibiting YAP-dependent transcription. Phosphorylation of YAP via loss of LRP6 is mediated by large tumor suppressor kinases 1/2 (LATS1/2) and Merlin. We found that O-GlcNAcylation of LRP6 was reduced, and the overall amount of LRP6 was decreased via endocytosis-mediated lysosomal degradation during nutrient starvation. Merlin binds to LRP6; when LRP6 is less O-GlcNAcylated, Merlin dissociates from it and becomes capable of interacting with LATS1 to induce phosphorylation of YAP. Our data suggest that LRP6 has unexpected roles as a nutrient sensor and Hippo signaling regulator.

The Distinct Role of Tcfs and Lef1 in the Self-Renewal or Differentiation of Mouse Embryonic Stem Cells.

BACKGROUND AND OBJECTIVES: Tcfs and Lef1 are DNA-binding transcriptional factors in the canonical Wnt signaling pathway. In the absence of ß-catenin, Tcfs and Lef1 generally act as transcriptional repressors with co-repressor proteins such as Groucho, CtBP, and HIC-5. However, Tcfs and Lef1 turn into transcriptional activators during the interaction with ß-catenin. Therefore, the activity of Tcfs and Lef1 is regulated by ß-catenin. However, the intrinsic role of Tcfs and Lef1 has yet to be examined. The purpose of this study was to determine whether Tcfs and Lef1 play differential roles in the regulation of self-renewal and differentiation of mouse ES cells. METHODS AND RESULTS: Interestingly, the expression of Tcfs and Lef1 was dynamically altered under various differentiation conditions, such as removal of LIF, EB formation and neuronal differentiation in N2B27 media, suggesting that the function of each Tcf and Lef1 may vary in ES cells. Ectopic expression of Tcf1 or the dominant negative form of Lef1 (Lef1-DN) contributes to ES cells to self-renew in the absence of leukemia inhibitory factor (LIF), whereas ectopic expression of Tcf3, Lef1 or Tcf1-DN did not support ES cells to self-renew. Ectopic expression of either Lef1 or Lef1-DN blocked neuronal differentiation, suggesting that the transient induction of Lef1 was necessary for the initiation and progress of differentiation. ChIP analysis shows that Tcf1 bound to Nanog promoter and ectopic expression of Tcf1 enhanced the transcription of Nanog. CONCLUSIONS: The overall data suggest that Tcf1 plays a critical role in the maintenance of stemness whereas Lef1 is involved in the initiation of differentiation.

MAML1/2 promote YAP/TAZ nuclear localization and tumorigenesis.

The Hippo pathway plays a pivotal role in tissue homeostasis and tumor suppression. YAP and TAZ are downstream effectors of the Hippo pathway, and their activities are tightly suppressed by phosphorylation-dependent cytoplasmic retention. However, the molecular mechanisms governing YAP/TAZ nuclear localization have not been fully elucidated. Here, we report that Mastermind-like 1 and 2 (MAML1/2) are indispensable for YAP/TAZ nuclear localization and transcriptional activities. Ectopic expression or depletion of MAML1/2 induces nuclear translocation or cytoplasmic retention of YAP/TAZ, respectively. Additionally, mutation of the MAML nuclear localization signal, as well as its YAP/TAZ interacting region, both abolish nuclear localization and transcriptional activity of YAP/TAZ. Importantly, we demonstrate that the level of MAML1 messenger RNA (mRNA) is regulated by microRNA-30c (miR-30c) in a cell-density-dependent manner. In vivo and clinical results suggest that MAML potentiates YAP/TAZ oncogenic function and positively correlates with YAP/TAZ activation in human cancer patients, suggesting pathological relevance in the context of cancer development. Overall, our study not only provides mechanistic insight into the regulation of YAP/TAZ subcellular localization, but it also strongly suggests that the miR30c-MAML-YAP/TAZ axis is a potential therapeutic target for developing novel cancer treatments.

O-GlcNAcylation on LATS2 disrupts the Hippo pathway by inhibiting its activity.

The Hippo pathway controls organ size and tissue homeostasis by regulating cell proliferation and apoptosis. The LATS-mediated negative feedback loop prevents excessive activation of the effectors YAP/TAZ, maintaining homeostasis of the Hippo pathway. YAP and TAZ are hyperactivated in various cancer cells which lead to tumor growth. Aberrantly increased O-GlcNAcylation has recently emerged as a cause of hyperactivation of YAP in cancer cells. However, the mechanism, which induces hyperactivation of TAZ and blocks LATS-mediated negative feedback, remains to be elucidated in cancer cells. This study found that in breast cancer cells, abnormally increased O-GlcNAcylation hyperactivates YAP/TAZ and inhibits LATS2, a direct negative regulator of YAP/TAZ. LATS2 is one of the newly identified O-GlcNAcylated components in the MST-LATS kinase cascade. Here, we found that O-GlcNAcylation at LATS2 Thr436 interrupted its interaction with the MOB1 adaptor protein, which connects MST to LATS2, leading to activation of YAP/TAZ by suppressing LATS2 kinase activity. LATS2 is a core component in the LATS-mediated negative feedback loop. Thus, this study suggests that LATS2 O-GlcNAcylation is deeply involved in tumor growth by playing a critical role in dysregulation of the Hippo pathway in cancer cells.

A concise review of human brain methylome during aging and neurodegenerative diseases.

DNA methylation at CpG sites is an essential epigenetic mark that regulates gene expression during mammalian development and diseases. Methylome refers to the entire set of methylation modifications present in the whole genome. Over the last several years, an increasing number of reports on brain DNA methylome reported the association between aberrant methylation and the abnormalities in the expression of critical genes known to have critical roles during aging and neurodegenerative diseases. Consequently, the role of methylation in understanding neurodegenerative diseases has been under focus. This review outlines the current knowledge of the human brain DNA methylomes during aging and neurodegenerative diseases. We describe the differentially methylated genes from fetal stage to old age and their biological functions. Additionally, we summarize the key aspects and methylated genes identified from brain methylome studies on neurodegenerative diseases. The brain methylome studies could provide a basis for studying the functional aspects of neurodegenerative diseases. [BMB Reports 2019; 52(10): 577-588].

Hippo signaling is intrinsically regulated during cell cycle progression by APC/CCdh1.

The Hippo-YAP/TAZ signaling pathway plays a pivotal role in growth control during development and regeneration and its dysregulation is widely implicated in various cancers. To further understand the cellular and molecular mechanisms underlying Hippo signaling regulation, we have found that activities of core Hippo signaling components, large tumor suppressor (LATS) kinases and YAP/TAZ transcription factors, oscillate during mitotic cell cycle. We further identified that the anaphase-promoting complex/cyclosome (APC/C)Cdh1 E3 ubiquitin ligase complex, which plays a key role governing eukaryotic cell cycle progression, intrinsically regulates Hippo signaling activities. CDH1 recognizes LATS kinases to promote their degradation and, hence, YAP/TAZ regulation by LATS phosphorylation is under cell cycle control. As a result, YAP/TAZ activities peak in G1 phase. Furthermore, we show in Drosophila eye and wing development that Cdh1 is required in vivo to regulate the LATS homolog Warts with a conserved mechanism. Cdh1 reduction increased Warts levels, which resulted in reduction of the eye and wing sizes in a Yorkie dependent manner. Therefore, LATS degradation by APC/CCdh1 represents a previously unappreciated and evolutionarily conserved layer of Hippo signaling regulation.

Complementary Wnt Sources Regulate Lymphatic Vascular Development via PROX1-Dependent Wnt/ß-Catenin Signaling.

Wnt/ß-catenin signaling is necessary for lymphatic vascular development. Oscillatory shear stress (OSS) enhances Wnt/ß-catenin signaling in cultured lymphatic endothelial cells (LECs) to induce expression of the lymphedema-associated transcription factors GATA2 and FOXC2. However, the mechanisms by which OSS regulates Wnt/ß-catenin signaling and GATA2 and FOXC2 expression are unknown. We show that OSS activates autocrine Wnt/ß-catenin signaling in LECs in vitro. Tissue-specific deletion of Wntless, which is required for the secretion of Wnt ligands, reveals that LECs and vascular smooth muscle cells are complementary sources of Wnt ligands that regulate lymphatic vascular development in vivo. Further, the LEC master transcription factor PROX1 forms a complex with ß-catenin and the TCF/LEF transcription factor TCF7L1 to enhance Wnt/ß-catenin signaling and promote FOXC2 and GATA2 expression in LECs. Thus, our work defines Wnt sources, reveals that PROX1 directs cell fate by acting as a Wnt signaling component, and dissects the mechanisms of PROX1 and Wnt synergy.