Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response.

NRF2 is a transcription factor responsible for antioxidant stress responses that is usually regulated in a redox-dependent manner. p62 bodies formed by liquid-liquid phase separation contain Ser349-phosphorylated p62, which participates in the redox-independent activation of NRF2. However, the regulatory mechanism and physiological significance of p62 phosphorylation remain unclear. Here, we identify ULK1 as a kinase responsible for the phosphorylation of p62. ULK1 colocalizes with p62 bodies, directly interacting with p62. ULK1-dependent phosphorylation of p62 allows KEAP1 to be retained within p62 bodies, thus activating NRF2. p62S351E/+ mice are phosphomimetic knock-in mice in which Ser351, corresponding to human Ser349, is replaced by Glu. These mice, but not their phosphodefective p62S351A/S351A counterparts, exhibit NRF2 hyperactivation and growth retardation. This retardation is caused by malnutrition and dehydration due to obstruction of the esophagus and forestomach secondary to hyperkeratosis, a phenotype also observed in systemic Keap1-knockout mice. Our results expand our understanding of the physiological importance of the redox-independent NRF2 activation pathway and provide new insights into the role of phase separation in this process.

Lactic acidosis induces metabolic and phenotypic reprogramming in cholangiocarcinoma cells via the upregulation of thrombospondin-1.

The high glycolytic activity of cancer cells leads to lactic acidosis (LA) in the tumor microenvironment. LA is not merely a consequence of metabolic activities but also has functional roles in metabolic reprogramming and cancer progression. Cholangiocarcinoma (CCA) cells exhibit a high dependency on glycolysis for survival and growth, but the specific effects of LA on cellular characteristics remain unknown. Here, we demonstrate that long-term LA (LLA) reprograms the metabolic phenotype of CCA cells from glycolytic to oxidative and enhances their migratory activity. In CCA cell culture, short-term LA (24 h) showed a growth inhibitory effect, while extended LA exposure for more than 2 weeks (LLA) led to enhanced cell motility. Coincidentally, LLA enhanced the respiratory capacity with an increase in mitochondrial mass. Inhibition of mitochondrial function abolished LLA-induced cell motility, suggesting that metabolic remodeling affects the phenotypic outcomes. RNA-sequencing analysis revealed that LLA upregulated genes associated with cell migration and epithelial-mesenchymal transition (EMT), including thrombospondin-1 (THBS1), which encodes a pro-EMT-secreted protein. Inhibition of THBS1 resulted in the suppression of both LLA-induced cell motility and respiratory capacity. Moreover, high THBS1 expression was associated with poor survival in patients with CCA. Collectively, our study suggests that the increased expression of THBS1 by LLA promotes phenotypic alterations, leading to CCA progression.

High glucose-ROS conditions enhance the progression in cholangiocarcinoma via upregulation of MAN2A2 and CHD8.

Diabetes is a major risk factor in the development and progression of several cancers including cholangiocarcinoma (CCA). However, the molecular mechanism by which hyperglycemia potentiates progression of CCA is not clearly understood. Here, we showed that a high glucose condition significantly increased reactive oxygen species (ROS) production and promoted aggressive phenotypes of CCA cells, including proliferation and migration activities. Mannosidase alpha class 2a member 2 (MAN2A2), was upregulated at both mRNA and protein levels in a high glucose- and ROS-dependent manner. In addition, cell proliferation and migration were significantly reduced by MAN2A2 knockdown. Based on our proteome and in silico analyses, we further found that chromodomain helicase DNA-binding protein 8 (CHD8) was induced by ROS signaling and regulated MAN2A2 expression. Overexpression of CHD8 increased MAN2A2 expression, while CHD8 knockdown dramatically reduced proliferation and migration as well as MAN2A2 expression in CCA cells. Moreover, both MAN2A2 and CHD8 were highly expressed with positive correlation in CCA tumor tissues. Collectively, these data suggested that high glucose conditions promote CCA progression through ROS-mediated upregulation of MAN2A2 and CHD8. Thus, glucose metabolism is a promising therapeutic target to control tumor progression in patients with CCA and diabetes.

Leukotriene A4 hydrolase deficiency protects mice from diet-induced obesity by increasing energy expenditure through neuroendocrine axis.

Obesity is a health problem worldwide, and brown adipose tissue (BAT) is important for energy expenditure. Here, we explored the role of leukotriene A4 hydrolase (LTA4 H), a key enzyme in the synthesis of the lipid mediator leukotriene B4 (LTB4 ), in diet-induced obesity. LTA4 H-deficient (LTA4 H-KO) mice fed a high-fat diet (HFD) showed a lean phenotype, and bone-marrow transplantation studies revealed that LTA4 H-deficiency in non-hematopoietic cells was responsible for this lean phenotype. LTA4 H-KO mice exhibited greater energy expenditure, but similar food intake and fecal energy loss. LTA4 H-KO BAT showed higher expression of thermogenesis-related genes. In addition, the plasma thyroid-stimulating hormone and thyroid hormone concentrations, as well as HFD-induced catecholamine secretion, were higher in LTA4 H-KO mice. In contrast, LTB4 receptor (BLT1)-deficient mice did not show a lean phenotype, implying that the phenotype of LTA4 H-KO mice is independent of the LTB4 /BLT1 axis. These results indicate that LTA4 H mediates the diet-induced obesity by reducing catecholamine and thyroid hormone secretion.

Leukotriene B4 receptor 1 exacerbates inflammation following myocardial infarction.

Leukotriene B4 receptor 1 (BLT1), a high-affinity G-protein-coupled receptor for leukotriene B4 (LTB4 ), is expressed on various inflammatory cells and plays critical roles in several inflammatory diseases. In myocardial infarction (MI), various inflammatory cells are known to be recruited to the infarcted area, but the function of BLT1 in MI is poorly understood. Here, we investigated the role of BLT1 in MI and the therapeutic effect of a BLT1 antagonist, ONO-4057, on MI. Mice with infarcted hearts showed increased BLT1 expression and LTB4 levels. BLT1-knockout mice with infarcted hearts exhibited attenuated leukocyte infiltration, proinflammatory cytokine production, and cell death, which led to reduced mortality and improved cardiac function after MI. Bone-marrow transplantation studies showed that BLT1 expressed on bone marrow-derived cells was responsible for the exacerbation of inflammation in infarcted hearts. Furthermore, ONO-4057 administration attenuated the inflammatory responses in hearts surgically treated for MI, which resulted in reduced mortality and improved cardiac function after MI. Our study demonstrated that BLT1 contributes to excessive inflammation after MI and could represent a new therapeutic target for MI.

STT3B-dependent posttranslational N-glycosylation as a surveillance system for secretory protein.

Nascent secretory proteins are extensively scrutinized at the endoplasmic reticulum (ER). Various signatures of client proteins, including exposure of hydrophobic patches or unpaired sulfhydryls, are coordinately utilized to reduce nonnative proteins in the ER. We report here the cryptic N-glycosylation site as a recognition signal for unfolding of a natively nonglycosylated protein, transthyretin (TTR), involved in familial amyloidosis. Folding and ER-associated degradation (ERAD) perturbation analyses revealed that prolonged TTR unfolding induces externalization of cryptic N-glycosylation site and triggers STT3B-dependent posttranslational N-glycosylation. Inhibition of posttranslational N-glycosylation increases detergent-insoluble TTR aggregates and decreases cell proliferation of mutant TTR-expressing cells. Moreover, this modification provides an alternative pathway for degradation, which is EDEM3-mediated N-glycan-dependent ERAD, distinct from the major pathway of Herp-mediated N-glycan-independent ERAD. Hence we postulate that STT3B-dependent posttranslational N-glycosylation is part of a triage-salvage system recognizing cryptic N-glycosylation sites of secretory proteins to preserve protein homeostasis.

Inhibition of Local Macrophage Growth Ameliorates Focal Inflammation and Suppresses Atherosclerosis.

OBJECTIVE: Macrophages play a central role in various stages of atherosclerotic plaque formation and progression. The local macrophages reportedly proliferate during atherosclerosis, but the pathophysiological significance of macrophage proliferation in this context remains unclear. Here, we investigated the involvement of local macrophage proliferation during atherosclerosis formation and progression using transgenic mice, in which macrophage proliferation was specifically suppressed. APPROACH AND RESULTS: Inhibition of macrophage proliferation was achieved by inducing the expression of cyclin-dependent kinase inhibitor 1B, also known as p27kip, under the regulation of a scavenger receptor promoter/enhancer. The macrophage-specific human p27kip Tg mice were subsequently crossed with apolipoprotein E-deficient mice for the atherosclerotic plaque study. Results showed that a reduced number of local macrophages resulted in marked suppression of atherosclerotic plaque formation and inflammatory response in the plaque. Moreover, fewer local macrophages in macrophage-specific human p27kip Tg mice helped stabilize the plaque, as evidenced by a reduced necrotic core area, increased collagenous extracellular matrix, and thickened fibrous cap. CONCLUSIONS: These results provide direct evidence of the involvement of local macrophage proliferation in formation and progression of atherosclerotic plaques and plaque stability. Thus, control of macrophage proliferation might represent a therapeutic target for treating atherosclerotic diseases.

Profiling of bioactive lipids in different dendritic cell subsets using an improved multiplex quantitative LC-MS/MS method.

Lipids are an energy source and key components of the cell membrane; however, they are also bioactive mediators of physiological and pathophysiological phenomena. Quantification of bioactive lipids is not easy because they have diverse chemical properties and are present in trace amounts. Here, we improved a multiplex method of quantifying bioactive lipids, thereby enabling measurement of 90 compounds simultaneously. We then used this system to quantify bioactive lipids produced by two subsets of dendritic cells (DCs): all-trans retinoic acid-treated DCs (RA-DCs) (a type of tolerogenic DC (tDC)) and conventional DCs (cDCs). We found that cDCs produced inflammatory lipid mediators such as leukotrienes, whereas RA-DCs produced anti-inflammatory lipid mediators such as prostaglandin I2. Consistent with this, cDCs expressed larger amounts of mRNA encoding 5-lipoxygenase and LTA4 hydrolase (both responsible for leukotriene biosynthesis) and RA-DCs produced larger amounts of mRNA encoding prostaglandin I2 synthase. Taken together, the results suggest that the method is useful for clarifying the roles of bioactive lipids during immune responses.

Generation and characterization of a human-mouse chimeric high-affinity antibody that detects the DYKDDDDK FLAG peptide.

DYKDDDDK peptide (FLAG) is a useful tool for investigating the function and localization of proteins whose antibodies (Abs) are not available. We recently established a high-affinity monoclonal antibody (mAb) for FLAG (clone 2H8). The 2H8 Ab is highly sensitive for detecting FLAG-tagged proteins by flowcytometry and immunoprecipitation, but it can yield nonspecific signals in immunohistochemistry of mouse tissues because it is of mouse origin. In this study, we reduced nonspecific signals by generating a chimeric 2H8 Ab with Fc fragments derived from human immunoglobulin. We fused a 5' terminal cDNA fragments for the Fab region of 2H8 mAb with 3' terminal cDNA fragments for Fc region of human IgG1. We transfected both chimeric plasmids and purified the resulting human-mouse chimeric 2H8. The chimeric 2H8 Ab successfully detected FLAG-tagged proteins in flowcytometry with anti-human IgG secondary Ab with comparable sensitivity to 2H8 mAb. Importantly, chimeric 2H8 detected specific FLAG peptide signals without nonspecific signals in immunohistochemical analysis with mouse tissues. This human-mouse chimeric high-affinity anti-FLAG Ab will prove useful for future immunohistochemical analysis of mouse tissues.

Mild electrical stimulation with heat shock guides differentiation of embryonic stem cells into Pdx1-expressing cells within the definitive endoderm.

BACKGROUND: Because of the increasing number of diabetic patients, it is important to generate pancreatic and duodenal homeobox gene 1 (Pdx1)-expressing cells, which are capable of differentiating into pancreatic endocrine ß cells. Mild electrical stimulation was reported to modulate the differentiation of ES cells into ectoderm-derived neuronal cells or mesoderm-derived cardiac cells. RESULTS: In this study, we report that mild electrical stimulation with heat shock (MET) potentiates the differentiation of ES cells into definitive endoderm-derived Pdx1-expressing cells. MET has no effect when applied to early definitive endoderm on differentiation day 5. A 1.87-fold increase in the proportion of Pdx1-expressing cells was observed when stimulation was applied to the late definitive endoderm one day prior to the immergence of Pdx1/GFP-expressing cells on differentiation day 7. Pdx1 mRNA was also up-regulated by MET. The potentiating effect of MET synergized with activin and basic fibroblast growth factor into Pdx1-expressing cells. Moreover, MET stimulation on late definitive endoderm up-regulated heat shock protein 72 and activated various kinases including Akt, extracellular signal-regulated kinase, p38, and c-jun NH2-terminal kinase in ES cells. CONCLUSIONS: Our findings indicate that MET induces the differentiation of Pdx1-expressing cells within the definitive endoderm in a time-dependent manner, and suggest useful application for regenerative medicine.

Inhibition of Leukotriene B4 Action Mitigates Intracerebral Hemorrhage-Associated Pathological Events in Mice.

Infiltration of neutrophils has been suggested to play an important role in the pathogenesis of intracerebral hemorrhage (ICH) for which effective therapeutic interventions remain unavailable. In the present study we focused on leukotriene B4 (LTB4) as a potent chemotactic factor for neutrophils in order to address its contribution to the pathologic events associated with ICH. ICH with hematoma expansion into the internal capsule that resulted in severe sensorimotor dysfunction was induced by injection of collagenase in mouse striatum. We found that LTB4 as well as mRNAs of 5-lipoxygenase (5-LOX) and 5-LOX-activating protein were increased in the brain after ICH. Daily treatment with a 5-LOX inhibitor zileuton (3 or 10 mg/kg, i.v.) prevented ICH-induced increase in LTB4, attenuated neutrophil infiltration into the hematoma, and ameliorated sensorimotor dysfunction. In addition, mice deficient in LTB4 receptor BLT1 exhibited a lower number of infiltrating neutrophils in the hematoma and lower levels of sensorimotor dysfunction after ICH than did wild-type mice. Similarly, daily treatment of mice with BLT antagonist ONO-4057 (30 or 100 mg/kg, by mouth) from 3 hours after induction of ICH inhibited neutrophil infiltration and ameliorated sensorimotor dysfunction. ONO-4057 also attenuated inflammatory responses of microglia/macrophages in the perihematoma region and axon injury in the internal capsule. These results identify LTB4 as a critical factor that plays a major role in the pathogenic events in ICH, and BLT1 is proposed as a promising target for ICH therapy.

An aromatic amino acid within intracellular loop 2 of the prostaglandin EP2 receptor is a prerequisite for selective association and activation of Gαs.

We previously demonstrated that the aromatic moiety of Tyr143 within the intracellular loop 2 (ICL2) region of the prostaglandin EP2 receptor plays a crucial role in Gs coupling. Here we investigated whether the ICL2 of the EP2 receptor directly binds to Gαs and whether an aromatic moiety affects this interaction. In Chinese hamster ovary cells, mutations of Tyr143 reduced the ability of the EP2 receptor to interact with G proteins as demonstrated by GTPγS sensitivity, as well as the ability of agonist-induced cAMP formation, with the rank order of Phe>Tyr (wild-type)=Trp>Leu>Ala (=0). We found that the wild-type ICL2 peptide (i2Y) and its mutant with Phe at Tyr143 (i2F) inhibited receptor-G protein complex formation of wild-type EP2 in membranes, whereas the Ala-substituted mutant (i2A) did not. Specific interactions between these peptides and the Gαs protein were detected by surface plasmon resonance, but Gαs showed different association rates, with a rank order of i2F>i2Y≫i2A, with similar dissociation rates. Moreover, i2F and i2Y, but not i2A activated membrane adenylyl cyclase. These results indicate that the ICL2 region of the EP2 receptor is its potential interaction site with Gαs, and that the aromatic side chain moiety at position 143 is a determinant for the accessibility of the ICL2 to the Gαs protein.

Leukotriene B4 receptor type 2 (BLT2) enhances skin barrier function by regulating tight junction proteins.

GPCRs are involved in numerous physiologic functions and are important drug targets. Although the epithelial barrier is important for protection from invading pathogens, the correlation between GPCRs and epithelial barrier function remains unknown. Leukotriene B4 (LTB4) receptor type 2 (BLT2), mainly expressed in epithelial cells, is a GPCR for 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) and LTB4. In our study, BLT2 localized at the lateral membrane in BLT2-overexpressing Madin-Darby canine kidney (MDCK) II cells and in the small intestine of BLT2-transgenic mice. BLT2-deficient mice exhibited higher transepidermal water loss and were more sensitive to epicutaneous sensitization. MDCK-BLT2 cells recovered transepithelial electrical resistance (TER) after a calcium switch faster than did MDCK-Mock cells, and 12-HHT stimulation accelerated TER recovery only in MDCK-BLT2 cells. Quantitative PCR and immunoblot analyses revealed that the 12-HHT/BLT2 axis up-regulated claudin-4 (CLDN4) expression in MDCK-BLT2 cells and human primary keratinocytes, and CLDN4 knockdown abolished 12-HHT-dependent TER recovery. Acceleration of TER recovery and induction of CLDN4 expression by 12-HHT stimulation were abolished by inhibition of Gαi protein or p38 MAPK. These results show that 12-HHT/BLT2 enhances epithelial barrier function by increasing CLDN4 expression via the Gαi protein-p38 MAPK pathway.

Modulation of leukotriene B4 receptor 1 signaling by receptor for advanced glycation end products (RAGE).

Leukotriene B4 (LTB4) receptor 1 (BLT1), a high-affinity GPCR for LTB4, plays important roles in acute and chronic inflammatory diseases. Although the LTB4-BLT1 axis is known to promote inflammation, no studies have defined the binding proteins that modulate LTB4-BLT1 signaling. In this study, the receptor for advanced glycation end products (RAGE) interacted with BLT1 in human cervical epithelial HeLa cells. RAGE increased LTB4-BLT1-dependent ERK phosphorylation and inhibited LTB4-BLT1-dependent activation of NF-κB and up-regulation of proinflammatory cytokines and chemokines. RAGE-dependent inhibition of NF-κB was blunted by treatment with an MEK inhibitor, suggesting that RAGE suppresses LTB4-BLT1-dependent NF-κB signaling by enhancing the MEK-ERK pathway. Meanwhile, in a chemotaxis assay of mouse bone marrow-derived neutrophils, the velocity of LTB4-dependent neutrophil migration was attenuated by soluble RAGE, which is an inhibitory decoy protein for RAGE signaling, in a dose-dependent manner (0.2-5 µg/ml), or by RAGE deficiency. Furthermore, both LTB4-dependent ERK phosphorylation in neutrophils and LTB4-dependent neutrophil accumulation in a murine peritonitis model were significantly attenuated in RAGE-deficient mice compared with C57BL/6J wild-type mice, indicating that RAGE potentiates LTB4-dependent neutrophil migration by enhancing ERK phosphorylation. Our results demonstrate that RAGE interacts with BLT1 and modulates LTB4-BLT1 signaling through potentiation of the MEK-ERK pathway.-Ichiki, T., Koga, T., Okuno, T., Saeki K., Yamamoto, Y., Yamamoto, H., Sakaguchi, M., Yokomizo, T. Modulation of leukotriene B4 receptor 1 signaling by receptor for advanced glycation end products (RAGE).

Podocyte p53 Limits the Severity of Experimental Alport Syndrome.

Alport syndrome (AS) is one of the most common types of inherited nephritis caused by mutation in one of the glomerular basement membrane components. AS is characterized by proteinuria at early stage of the disease and glomerular hyperplastic phenotype and renal fibrosis at late stage. Here, we show that global deficiency of tumor suppressor p53 significantly accelerated AS progression in X-linked AS mice and decreased the lifespan of these mice. p53 protein expression was detected in 21-week-old wild-type mice but not in age-matched AS mice. Expression of proinflammatory cytokines and profibrotic genes was higher in p53(+/-) AS mice than in p53(+/+) AS mice. In vitro experiments revealed that p53 modulates podocyte migration and positively regulates the expression of podocyte-specific genes. We established podocyte-specific p53 (pod-p53)-deficient AS mice, and determined that pod-p53 deficiency enhanced the AS-induced renal dysfunction, foot process effacement, and alteration of gene-expression pattern in glomeruli. These results reveal a protective role of p53 in the progression of AS and in maintaining glomerular homeostasis by modulating the hyperplastic phenotype of podocytes in AS.

Endoplasmic Reticulum (ER) Stress Induces Sirtuin 1 (SIRT1) Expression via the PI3K-Akt-GSK3ß Signaling Pathway and Promotes Hepatocellular Injury.

Sirtuin 1 (SIRT1), an NAD(+)-dependent histone deacetylase, plays crucial roles in various biological processes including longevity, stress response, and cell survival. Endoplasmic reticulum (ER) stress is caused by dysfunction of ER homeostasis and exacerbates various diseases including diabetes, fatty liver, and chronic obstructive pulmonary disease. Although several reports have shown that SIRT1 negatively regulates ER stress and ER stress-induced responses in vitro and in vivo, the effect of ER stress on SIRT1 is less explored. In this study, we showed that ER stress induced SIRT1 expression in vitro and in vivo. We further determined the molecular mechanisms of how ER stress induces SIRT1 expression. Surprisingly, the conventional ER stress-activated transcription factors XBP1, ATF4, and ATF6 seem to be dispensable for SIRT1 induction. Based on inhibitor screening experiments with SIRT1 promoter, we found that the PI3K-Akt-GSK3ß signaling pathway is required for SIRT1 induction by ER stress. Moreover, we showed that pharmacological inhibition of SIRT1 by EX527 inhibited the ER stress-induced cellular death in vitro and severe hepatocellular injury in vivo, indicating a detrimental role of SIRT1 in ER stress-induced damage responses. Collectively, these data suggest that SIRT1 expression is up-regulated by ER stress and contributes to ER stress-induced cellular damage.

Citrate metabolism controls the senescent microenvironment via the remodeling of pro-inflammatory enhancers.

The senescent microenvironment and aged cells per se contribute to tissue remodeling, chronic inflammation, and age-associated dysfunction. However, the metabolic and epigenomic bases of the senescence-associated secretory phenotype (SASP) remain largely unknown. Here, we show that ATP-citrate lyase (ACLY), a key enzyme in acetyl-coenzyme A (CoA) synthesis, is essential for the pro-inflammatory SASP, independent of persistent growth arrest in senescent cells. Citrate-derived acetyl-CoA facilitates the action of SASP gene enhancers. ACLY-dependent de novo enhancers augment the recruitment of the chromatin reader BRD4, which causes SASP activation. Consistently, specific inhibitions of the ACLY-BRD4 axis suppress the STAT1-mediated interferon response, creating the pro-inflammatory microenvironment in senescent cells and tissues. Our results demonstrate that ACLY-dependent citrate metabolism represents a selective target for controlling SASP designed to promote healthy aging.

Insulin receptor activation through its accumulation in lipid rafts by mild electrical stress.

Insulin resistance is due to the reduced cellular response to insulin in peripheral tissues. The interaction of insulin with its receptor is the first step in insulin action and thus the identified target of insulin resistance. It has been well established that defects or mutations in the insulin receptor (IR) cause insulin resistance. Therefore, an IR activator might be a novel therapeutic approach for insulin resistance. Our previous report showed that mild electrical stress (MES) enhanced the insulin-induced signaling pathway. However, the molecular mechanism of the effect of MES remains unclear. We assessed the effect of MES, which is characterized by low-intensity direct current, on insulin signaling in vitro and in vivo. Here, we showed that MES activated the insulin signaling in an insulin-independent manner and improved insulin resistance in peripheral tissues of high fat-fed mice. Moreover, we found that MES increased the localization of IR in lipid rafts and enhanced the level of phosphorylated Akt in insulin-resistant hepatic cells. Ablation of lipid rafts disrupted the effect of MES on Akt activation. Our findings indicate that MES has potential as an activator of IR in an insulin-independent manner, and might be beneficial for insulin resistance in type 2 diabetes.

MEF/ELF4 transactivation by E2F1 is inhibited by p53.

Myeloid elf-1-like factor (MEF) or Elf4 is an E-twenty-six (ETS)-related transcription factor with strong transcriptional activity that influences cellular senescence by affecting tumor suppressor p53. MEF downregulates p53 expression and inhibits p53-mediated cellular senescence by transcriptionally activating MDM2. However, whether p53 reciprocally opposes MEF remains unexplored. Here, we show that MEF is modulated by p53 in human cells and mice tissues. MEF expression and promoter activity were suppressed by p53. While we found that MEF promoter does not contain p53 response elements, intriguingly, it contains E2F consensus sites. Subsequently, we determined that E2F1 specifically binds to MEF promoter and transactivates MEF. Nevertheless, E2F1 DNA binding and transactivation of MEF promoter was inhibited by p53 through the association between p53 and E2F1. Furthermore, we showed that activation of p53 in doxorubicin-induced senescent cells increased E2F1 and p53 interaction, diminished E2F1 recruitment to MEF promoter and reduced MEF expression. These observations suggest that p53 downregulates MEF by associating with and inhibiting the binding activity of E2F1, a novel transcriptional activator of MEF. Together with previous findings, our present results indicate that a negative regulatory mechanism exists between p53 and MEF.

Lactic acidosis promotes aggressive features of cholangiocarcinoma cells via upregulating ALDH1A3 expression through EGFR axis.

AIMS: Lactic acidosis (LA) generated in tumor microenvironment promotes tumor metastasis and drug resistance. This study aimed to demonstrate the impacts and the mechanisms of LA on aldehyde dehydrogenase1A3 (ALDH1A3) in promoting aggressiveness and gemcitabine resistance in cholangiocarcinoma (CCA) cell lines. The clinical relevance and the molecular pathway related to the upregulation of ALDH1A3 in LA cells will be revealed. MAIN METHODS: ALDH1A3 expression and its clinical significances in CCA tissues were analyzed using the GEO databases. Human CCA cell lines, KKU-213A-LA and KKU-213B-LA maintained in the LA medium were studied and compared with its parental cells cultured in normal medium. Aggressive features-proliferation, colony formation, migration, invasion, and gemcitabine response were determined. Expression of ALDH1A3, EGFR and the downstream effectors were analyzed using real-time PCR and Western blotting. KEY FINDINGS: ALDH1A3 was upregulated in patient CCA tissues and correlated with LDHA and shorter survival of CCA patients. mRNA and protein of ALDH1A3 were increased in LA cells. Attenuation of ALDH1A3 expression by siRNA significantly reduced cell proliferation, colony formation, migration, invasion, and gemcitabine resistance of LA cells, and gemcitabine resistant cells. The EGF/EGFR signaling via Erk and STAT3 was pinned to be involved in the induction of ALDH1A3 expression in LA cells. The transcriptomic analysis from TCGA dataset supported the links between LDHA, EGFR and ALDH1A3 in several tumor tissues. SIGNIFICANCE: Lactic acidosis upregulated EGFR and ALDH1A3 expression, leading to the aggressiveness of CCA cells. The EGFR/ALDH1A3 axis could be a novel therapeutic target to eradicate metastatic CCA.