Per- and polyfluoroalkyl substances activate UPR pathway, induce steatosis and fibrosis in liver cells.

Per- and polyfluoroalkyl substances (PFAS), which include perfluorooctanoic acid (PFOA), heptafluorobutyric acid (HFBA), and perfluorotetradecanoic acid (PFTA), are commonly occurring organic pollutants. Exposure to PFAS affects the immune system, thyroid and kidney function, lipid metabolism, and insulin signaling and is also involved in the development of fatty liver disease and cancer. The molecular mechanisms by which PFAS cause fatty liver disease are not understood in detail. In the current study, we investigated the effect of low physiologically relevant concentrations of PFOA, HFBA, and PFTA on cell survival, steatosis, and fibrogenic signaling in liver cell models. Exposure of PFOA and HFBA (10 to 1000 nM) specifically promoted cell survival in HepaRG and HepG2 cells. PFAS increased the expression of TNFα and IL6 inflammatory markers, increased endogenous reactive oxygen species (ROS) production, and activated unfolded protein response (UPR). Furthermore, PFAS enhanced cell steatosis and fibrosis in HepaRG and HepG2 cells which were accompanied by upregulation of steatosis (SCD1, ACC, SRBP1, and FASN), and fibrosis (TIMP2, p21, TGFß) biomarkers expression, respectively. RNA-seq data suggested that chronic exposures to PFOA modulated the expression of fatty acid/lipid metabolic genes that are involved in the development of NFALD and fatty liver disease. Collectively our data suggest that acute/chronic physiologically relevant concentrations of PFAS enhance liver cell steatosis and fibrosis by the activation of the UPR pathway and by modulation of NFALD-related gene expression.

Cadmium modulates steatosis, fibrosis, and oncogenic signaling in liver cancer cells by activating notch and AKT/mTOR pathways.

Cadmium (Cd) is an environmental pollutant that increases hepatotoxicity and the risk of liver diseases. In the current study, we investigated the effect of a physiologically relevant, low concentration of Cd on the regulation of liver cancer cell proliferation, steatosis, and fibrogenic/oncogenic signaling. Exposure to low concentrations of Cd increased endogenous reactive oxygen species (ROS) production and enhanced cell proliferation in a human bipotent progenitor cell line HepaRG and hepatocellular carcinoma (HCC) cell lines. Acute exposure of Cd increased Jagged-1 expression and activated Notch signaling in HepaRG and HCC cells HepG2 and SK-Hep1. Cd activated AKT/mTOR signaling by increasing phosphorylation of AKT-S473 and mTOR-S-4448 residues. Moreover, a low concentration of Cd also promoted cell steatosis and induced fibrogenic signaling in HCC cells. Chronic exposure to low concentrations of Cd-activated Notch and AKT/mTOR signaling induced the expression of pro-inflammatory cytokines tumor necrosis factor-alpha (TNFα) and its downstream target TNF-α-Induced Protein 8 (TNFAIP8). RNA-Seq data revealed that chronic exposure to low concentrations of Cd modulated the expression of several fatty liver disease-related genes involved in cell steatosis/fibrosis in HepaRG and HepG2 cells. Collectively, our data suggest that low concentrations of Cd modulate steatosis along with fibrogenic and oncogenic signaling in HCC cells by activating Notch and AKT/mTOR pathways.

MicroRNA-99b-5p targets mTOR/AR axis, induces autophagy and inhibits prostate cancer cell proliferation.

OBJECTIVES: MicroRNAs (miRNAs) are the small non-coding regulatory RNA molecules involved in gene regulation via base-pairing with complementary sequences in mRNAs. The dysregulation of specific miRNAs, such as miR-99b-5p (miR-99b), is associated with prostate cancer (PCa) progression. However, the mechanistic role of miR-99b in PCa remains to be determined. In this study, we aimed to investigate the functional and clinical significance of miR-99b in PCa. STUDY DESIGN: The expression of miR-99b and its downstream targets mTOR/AR in the PCa samples were analyzed by RT/qPCR. The effects of miR-99b overexpression/inhibition on PCa cell survival/proliferation, spheroid formation, and cell migration were examined by specific assays. Luciferase reporter assays were performed to determine the binding of miR-99b to 3' untranslated region (UTR) of the mTOR gene. The effects of miR-99b on the expression of mTOR, AR, and PSA proteins, as well as on AKT/mTOR signaling, autophagy, and neuroendocrine differentiation markers were analyzed by western blotting. The expression of miR-99b, mTOR, AR, PSA in AR-negative PC3 and AR-positive LNCaP cells was analyzed by RT/qPCR. The effect of miR-99b on global gene expression in PC3 cells was analyzed by RNA-seq. RESULTS: The expression of miR-99b was downregulated in tumor samples from PCa patients, whereas the expression of mTOR and AR was upregulated. In PCa cell lines, overexpression of miR-99b inhibited cell proliferation and cell colony/spheroid formation; induced apoptosis, and increased sensitivity towards docetaxel (DTX). In contrast, inhibition of miR-99b by miR-99b inhibitor resulted in increased cell growth in PCa cells. Mechanistically, miR-99b inhibited the expression of the mammalian target of the rapamycin (mTOR) gene by binding to its 3' UTR and induced autophagy. Furthermore, miR-99b inhibited androgen receptor (AR) activity in LNCaP cells and induced apoptosis. Activation of AR signaling by dihydrotestosterone (DHT) downregulated miR-99b expression and promoted cell PCa cell growth/survival, whereas inactivation of mTOR by rapamycin or AR by enzalutamide decreased miR-99b mediated PCa cell growth. CONCLUSION: Our data suggest that miR-99b functions as a tumor suppressor by targeting the mTOR/AR axis in PCa cells, implicating miR-99b as a novel biomarker and therapeutic target for PCa management.

Serotonin induced hepatic steatosis is associated with modulation of autophagy and notch signaling pathway.

BACKGROUND: Besides its neurotransmitter and vasoconstriction functions, serotonin is an important mediator of numerous biological processes in peripheral tissues including cell proliferation, steatosis, and fibrogenesis. Recent reports indicate that serotonin may promote tumor growth in liver cancer, however, the molecular mechanisms remain elusive. n this study, we investigated the role and molecular signaling mechanisms mediated by serotonin in liver cancer cell survival, drug resistance, and steatosis. METHODS: Effect of serotonin on modulation of cell survival/proliferation was determined by MTT/WST1 assay. Effect of serotonin on the regulation of autophagy biomarkers and lipid/fatty acid proteins expression, AKT/mTOR and Notch signaling was evaluated by immunoblotting. The role of serotonin in normal human hepatocytes and liver cancer cell steatosis was analyzed by Oil Red O staining. The mRNA expression levels of lipid/fatty acid proteins and serotonin receptors were validated by qRT-PCR. The important roles of autophagy, Notch signaling, serotonin receptors and serotonin re-uptake proteins on serotonin-mediated cell steatosis were investigated by using selective inhibitors or antagonists. The association of peripheral serotonin, autophagy, and hepatic steatosis was also investigated using chronic EtOH fed mouse model. RESULTS: Exposure of liver cancer cells to serotonin induced Notch signaling and autophagy, independent of AKT/mTOR pathway. Also, serotonin enhanced cancer cell proliferation/survival and drug resistance. Furthermore, serotonin treatment up-regulated the expression of lipogenic proteins and increased steatosis in liver cancer cells. Inhibition of autophagy or Notch signaling reduced serotonin-mediated cell steatosis. Treatment with serotonin receptor antagonists 5-HTr1B and 5-HTr2B reduced serotonin-mediated cell steatosis; in contrast, treatment with selective serotonin reuptake inhibitors (SSRIs) increased steatosis. In addition, mice fed with chronic EtOH resulted in increased serum serotonin levels which were associated with the induction of hepatic steatosis and autophagy. CONCLUSIONS: Serotonin regulates liver cancer cell steatosis, cells survival, and may promote liver carcinogenesis by activation of Notch signaling and autophagy.

Oncogene PKCε controls INrf2-Nrf2 interaction in normal and cancer cells through phosphorylation of INrf2.

The INrf2 (Keap1)-Nrf2 cell sensor complex has a crucial role in protection against chemical- and radiation-induced oxidative stress and cellular transformation. INrf2, in association with Cul3-Rbx1, ubiquitylates and degrades Nrf2. Exposure to stressors leads to stabilization of Nrf2 and the coordinated activation of cytoprotective proteins and cellular protection. However, the molecular signal(s) that regulate control of Nrf2 by INrf2 remain elusive. In this report, we demonstrate that phosphorylation of INrf2 at Ser599 and Ser602 by the oncoprotein PKCε is essential for INrf2-Nrf2 interaction, and the subsequent ubiquitylation and degradation of Nrf2. Inhibition of PKCε, knockdown of PKCε and the INrf2S602A mutant all failed to phosphorylate INrf2, leading to loss of the INrf2-Nrf2 interaction, Nrf2 degradation and enhanced cytoprotection and drug resistance. Molecular modeling analyses revealed that phosphorylation of S599 exposes the deeply buried S602 for phosphorylation and enhanced INrf2-Nrf2 interaction. Analysis of human lung and liver tumor protein arrays showed lower PKCε and higher Nrf2 levels, which presumably promoted cancer cell survival and drug resistance. In conclusion, phosphorylation of INrf2 by PKCε leads to regulation of Nrf2, with significant implications for the survival of cancer cells, which often express lower levels of PKCε.

Antioxidant-induced INrf2 (Keap1) tyrosine 85 phosphorylation controls the nuclear export and degradation of the INrf2-Cul3-Rbx1 complex to allow normal Nrf2 activation and repression.

INrf2 (Keap1) serves as a negative regulator of the cytoprotective transcription factor Nrf2. At basal levels, INrf2 functions as a substrate adaptor to sequester Nrf2 into the Cul3-Rbx1 E3 ligase complex for ubiquitylation and proteasomal degradation. In response to antioxidants, Nrf2 is released from the INrf2-Cul3-Rbx1 complex and translocates into the nucleus, where it activates ARE-mediated cytoprotective gene expression. The present studies demonstrate that INrf2, Cul3 and Rbx1 export out of the nucleus and are degraded during the early or pre-induction response to antioxidants. Mutation of Tyr85 in INrf2 stymied the nuclear export of INrf2, suggesting that tyrosine phosphorylation controls the pre-induction nuclear export and degradation in response to antioxidants. The nuclear export of Cul3-Rbx1 were also blocked when INrf2Tyr85 was mutated, suggesting that INrf2-Cul3-Rbx1 undergo nuclear export as a complex. INrf2 siRNA also inhibited the nuclear export of Cul3-Rbx1, confirming that Cul3-Rbx1 requires INrf2 for nuclear export. Newly synthesized INrf2-Cul3-Rbx1 is imported back into the nucleus during the post-induction period to ubiquitylate and degrade Nrf2. Mutation of INrf2Tyr85 had no effect on activation of Nrf2 but led to nuclear accumulation of Nrf2 during the post-induction period owing to reduced export and degradation of Nrf2. Our results also showed that nuclear export and degradation followed by the new synthesis of INrf2-Cul3-Rbx1 controls the cellular abundance of the proteins during different phases of antioxidant responses. In conclusion, the early or pre-induction nuclear export of INrf2 in response to antioxidants is controlled by tyrosine phosphorylation, whereas the nuclear export of Cul3 and Rbx1 is controlled by INrf2, allowing normal activation or repression of Nrf2.

Clemastine and hyperthermia enhance sensitization of osteosarcoma cells for apoptosis.

Clemastine is an antagonist of histamine H1 receptor may provide benefits in the treatment of osteosarcoma (OS). In the current study, we used hyperthermia approach to sensitize OS cells to clemastine-mediated cell death. Osteosarcoma U-2 OS and Saos-2 cells were treated with clemastine at 37°C, followed by 42°C for 2 h, and released at 37°C for 6 h. The impact of clemastine and hyperthermia on OS cell survival and autophagy-mediated cell death was investigated. Exposure of U-2 OS and Saos-2 cells to clemastine and hyperthermia (42°C) inhibited dose-dependent clemastine-mediated cell survival by increasing cell apoptosis. Hyperthermia and clemastine exposure modulated inflammatory and unfolded protein response (UPR) signaling differentially in U-2 OS and Saos-2 cells. Exposure of U-2 OS and Saos-2 cells to hyperthermia and clemastine inhibited AKT/mTOR and induced expression of the autophagy biomarkers LC3B II and LC3-positive puncta formation. The inhibition of autophagy by 3-methyladenine blocked hyperthermia and clemastine-mediated induction of LC3B II, LC3-positive puncta formation, and OS cell apoptosis. These results indicate that clemastine and hyperthermia sensitize OS cell lines by inducing increased autophagic cell death. Collectively, our data suggest that hyperthermia along with antihistamine therapy may provide an improved approach for the treatment of OS.

Nrf2 protein up-regulates antiapoptotic protein Bcl-2 and prevents cellular apoptosis.

Nuclear transcription factor Nrf2 regulates the expression and coordinated induction of a battery of genes encoding cytoprotective and drug transporter proteins in response to chemical and radiation stress. This leads to reduced apoptosis, enhanced cell survival, and increased drug resistance. In this study, we investigated the role of Nrf2 in up-regulation of antiapoptotic protein Bcl-2 and its contribution to stress-induced apoptosis and cell survival. Exposure of mouse hepatoma (Hepa-1) and human hepatoblastoma (HepG2) cells to antioxidant tert-butylhydroquinone led to induction of Bcl-2. Mutagenesis and transfection assays identified an antioxidant response element between nucleotides -3148 and -3140 on the reverse strand of the Bcl-2 gene promoter that was essential for activation of Bcl-2 gene expression. Band/supershift and ChIP assays demonstrated binding of Nrf2 to Bcl-2 antioxidant response element. Alterations in Nrf2 led to altered Bcl-2 induction and cellular apoptosis. Moreover, dysfunctional/mutant inhibitor of Nrf2 (INrf2) in human lung cancer cells failed to degrade Nrf2, resulting in an increased Bcl-2 level and decreased etoposide- and UV/γ radiation-mediated DNA fragmentation. In addition, siRNA-mediated down-regulation of Nrf2 also led to decreased apoptosis and increased cell survival. Furthermore, the specific knockdown of Bcl-2 in Nrf2-activated tumor cells led to increased etoposide-induced apoptosis and decreased cell survival and growth/proliferation. These data provide the first evidence of Nrf2 in control of Bcl-2 expression and apoptotic cell death with implications in antioxidant protection, survival of cancer cells, and drug resistance.

Deficiency of spns1 exacerbates per- and polyfluoroalkyl substances mediated hepatic toxicity and steatosis in zebrafish (Danio rerio).

Per- and polyfluoroalkyl substances (PFAS) are man-made long-lasting chemical compounds that are found in everyday household items. Today they occur in the environment as a major group of pollutants. These compounds are broadly used in commercial product preparation such as, for food packaging, nonstick coatings, and firefighting foam. In humans, PFAS can cause immune disorders, impaired fetal development, abnormal skeletal tissue development, osteoarthritis, thyroid dysfunctions, cholesterol changes, affect insulin regulation and lipid metabolism, and are also involved in the development of fatty liver disease. In the current study, we investigated the effect of low, but physiologically relevant, concentrations of perfluorooctanoic acid (PFOA), heptafluorobutyric acid (HFBA), and perfluorotetradecanoic acid (PFTA) on gene expression markers of an inflammatory response (tnfa, il-1b, il-6, rplp0, edem1, and dnajc3a), unfolded protein response (UPR) (bip, atf4a, atf6, xbp1, and ddit3), senescence (p21, pai1, smp30, mdm2, and baxa), lipogenesis (scd1, acc, srebp1, pparγ, and fasn) and autophagy (p62, atg3, atg7, rab7, lc3b, and becn1) in AB wild-type (+/+), spns1-wt sibling (+/+), (+/-) and spns1 homozygous mutant (-/-) zebrafish embryos. Exposure to PFOA and HFBA (50 and 100 nM) specifically modulated inflammatory, UPR, senescence, lipogenic, and autophagy signaling in spns1-wt (+/+), (+/-), and spns1-mutant (-/-) zebrafish embryos. Furthermore, PFOA, but not HFBA, upregulated lipogenic-related gene expression and enhanced hepatic steatosis in spns1-wt (+/+), (+/-) zebrafish embryos. Combined exposure to PFOA, HFBA, and PFTA differentially expressed inflammatory, senescence, lipogenic, and autophagy-associated gene expression in spns1-mutant (-/-) zebrafish embryos compared with spns1-wt (+/+), (+/-) and AB-wt (+/+) zebrafish embryos. In addition, chronic exposure (∼2 months) to PFOA (120-600 nM) upregulated the expression of hepatic lipogenic and steatosis biomarkers in AB-wt (+/+) zebrafish. Collectively, our data suggest that acute/chronic physiologically relevant concentrations of PFOA upregulate inflammatory, UPR, senescence, and lipogenic signaling in spns1-wt (+/+), (+/-) and spns1-mutant (-/-) zebrafish embryos as well as in two-month-old AB-wt zebrafish, by targeting autophagy and hence induces toxicity that could promote nonalcoholic fatty liver disease.

Cyanotoxins Increase Cytotoxicity and Promote Nonalcoholic Fatty Liver Disease Progression by Enhancing Cell Steatosis.

Freshwater prokaryotic cyanobacteria within harmful algal blooms produce cyanotoxins which are considered major pollutants in the aquatic system. Direct exposure to cyanotoxins through inhalation, skin contact, or ingestion of contaminated drinking water can target the liver and may cause hepatotoxicity. In the current study, we investigated the effect of low concentrations of cyanotoxins on cytotoxicity, inflammation, modulation of unfolded protein response (UPR), steatosis, and fibrosis signaling in human hepatocytes and liver cell models. Exposure to low concentrations of microcystin-LR (MC-LR), microcystin-RR (MC-RR), nodularin (NOD), and cylindrospermopsin (CYN) in human bipotent progenitor cell line HepaRG and hepatocellular carcinoma (HCC) cell lines HepG2 and SK-Hep1 resulted in increased cell toxicity. MC-LR, NOD, and CYN differentially regulated inflammatory signaling, activated UPR signaling and lipogenic gene expression, and induced cellular steatosis and fibrotic signaling in HCC cells. MC-LR, NOD, and CYN also regulated AKT/mTOR signaling and inhibited autophagy. Chronic exposure to MC-LR, NOD, and CYN upregulated the expression of lipogenic and fibrosis biomarkers. Moreover, RNA sequencing (RNA seq) data suggested that exposure of human hepatocytes, HepaRG, and HCC HepG2 cells to MC-LR and CYN modulated expression levels of several genes that regulate non-alcoholic fatty liver disease (NAFLD). Our data suggest that low concentrations of cyanotoxins can cause hepatotoxicity and cell steatosis and promote NAFLD progression.

MicroRNA-483-5p Inhibits Hepatocellular Carcinoma Cell Proliferation, Cell Steatosis, and Fibrosis by Targeting PPARα and TIMP2.

MicroRNAs (miRNAs) are small non-coding RNA molecules that bind with the 3' untranslated regions (UTRs) of genes to regulate expression. Downregulation of miR-483-5p (miR-483) is associated with the progression of hepatocellular carcinoma (HCC). However, the significant roles of miR-483 in nonalcoholic fatty liver disease (NAFLD), alcoholic fatty liver diseases (AFLD), and HCC remain elusive. In the current study, we investigated the biological significance of miR-483 in NAFLD, AFLD, and HCC in vitro and in vivo. The downregulation of miR-483 expression in HCC patients' tumor samples was associated with Notch 3 upregulation. Overexpression of miR-483 in a human bipotent progenitor liver cell line HepaRG and HCC cells dysregulated Notch signaling, inhibited cell proliferation/migration, induced apoptosis, and increased sensitivity towards antineoplastic agents sorafenib/regorafenib. Interestingly, the inactivation of miR-483 upregulated cell steatosis and fibrosis signaling by modulation of lipogenic and fibrosis gene expression. Mechanistically, miR-483 targets PPARα and TIMP2 gene expression, which leads to the suppression of cell steatosis and fibrosis. The downregulation of miR-483 was observed in mice liver fed with a high-fat diet (HFD) or a standard Lieber-Decarli liquid diet containing 5% alcohol, leading to increased hepatic steatosis/fibrosis. Our data suggest that miR-483 inhibits cell steatosis and fibrogenic signaling and functions as a tumor suppressor in HCC. Therefore, miR-483 may be a novel therapeutic target for NAFLD/AFLD/HCC management in patients with fatty liver diseases and HCC.

Inhibitor of Nrf2 (INrf2 or Keap1) protein degrades Bcl-xL via phosphoglycerate mutase 5 and controls cellular apoptosis.

INrf2 (Keap1) is an adaptor protein that facilitates INrf2-Cul3-Rbx1-mediated ubiquitination/degradation of Nrf2, a master regulator of cytoprotective gene expression. Here, we present evidence that members of the phosphoglycerate mutase family 5 (PGAM5) proteins are involved in the INrf2-mediated ubiquitination/degradation of anti-apoptotic factor Bcl-xL. Mass spectrometry and co-immunoprecipitation assays revealed that INrf2, through its DGR domain, interacts with PGAM5, which in turn interacts with anti-apoptotic Bcl-xL protein. INrf2-Cul3-Rbx1 complex facilitates ubiquitination and degradation of both PGAM5 and Bcl-xL. Overexpression of PGAM5 protein increased INrf2-mediated degradation of Bcl-xL, whereas knocking down PGAM5 by siRNA decreased INrf2 degradation of Bcl-xL, resulting in increased stability of Bcl-xL. Mutation of PGMA5-E79A/S80A abolished INrf2/PGAM5/Bcl-xL interaction. Therefore, PGAM5 protein acts as a bridge between INrf2 and Bcl-xL interaction. Further studies showed that overexpression of INrf2 enhanced degradation of PGAM5-Bcl-xL complex, led to etoposide-mediated accumulation of Bax, increased release of cytochrome c from mitochondria, activated caspase-3/7, and enhanced DNA fragmentation and apoptosis. In addition, antioxidant (tert-butylhydroquinone) treatment destabilized the Nrf2-INrf2-PGAM5-Bcl-xL complex, which resulted in release of Nrf2 in cytosol and mitochondria, release of Bcl-xL in mitochondria, increase in Bcl-xL heterodimerization with Bax in mitochondria, and reduced cellular apoptosis. These data provide the first evidence that INrf2 controls Bcl-xL via PGAM5 and controls cellular apoptosis.

Src subfamily kinases regulate nuclear export and degradation of transcription factor Nrf2 to switch off Nrf2-mediated antioxidant activation of cytoprotective gene expression.

Nrf2 (NF-E2-related factor 2) is a nuclear transcription factor that in response to chemical and radiation stress regulates coordinated induction of a battery of cytoprotective gene expressions leading to cellular protection. In this study, we investigated the role of Src kinases in the regulation of Nrf2 and downstream signaling. siRNA-mediated inhibition of Fyn, Src, Yes, and Fgr, but not Lyn, in mouse hepatoma Hepa-1 cells, led to nuclear accumulation of Nrf2 and up-regulation of Nrf2 downstream gene expression. Mouse embryonic fibroblasts with combined deficiency of Fyn/Src/Yes/Fgr supported results from siRNA. In addition, steady-state overexpression of Fyn, Src, and Yes phosphorylated Nrf2Tyr568 that triggered nuclear export and degradation of Nrf2 and down-regulation of Nrf2 downstream gene expression. Exposure of cells to antioxidant, oxidant, or UV radiation increased nuclear import of Fyn, Src, and Yes kinases, which phosphorylated Nrf2Tyr568 resulting in nuclear export and degradation of Nrf2. Further analysis revealed that stress-activated GSK3ß acted upstream to the Src kinases and phosphorylated the Src kinases, leading to their nuclear localization and Nrf2 phosphorylation. The overexpression of Src kinases in Hepa-1 cells led to decreased Nrf2, increased apoptosis, and decreased cell survival. Mouse embryonic fibroblasts deficient in Src kinases showed nuclear accumulation of Nrf2, induction of Nrf2 and downstream gene expression, reduced apoptosis, and increased cell survival. The studies together demonstrate that Src kinases play a critical role in nuclear export and degradation of Nrf2, thereby providing a negative feedback mechanism to switch off Nrf2 activation and restore normal cellular homeostasis.