m6A demethylation of FOSL1 mRNA protects hepatoma cells against necrosis under glucose deprivation.

Stress-adaptive mechanisms enabling cancer cells to survive under glucose deprivation remain elusive. N6-methyladenosine (m6A) modification plays important roles in determining cancer cell fate and cellular stress response to nutrient deficiency. However, whether m6A modification functions in the regulation of cancer cell survival under glucose deprivation is unknown. Here, we found that glucose deprivation reduced m6A modification levels. Increasing m6A modification resulted in increased hepatoma cell necrosis under glucose deprivation, whereas decreasing m6A modification had an opposite effect. Integrated m6A-seq and RNA-seq revealed potential targets of m6A modification under glucose deprivation, including the transcription factor FOSL1; further, glucose deprivation upregulated FOSL1 by inhibiting FOSL1 mRNA decay in an m6A-YTHDF2-dependent manner through reducing m6A modification in its exon1 and 5'-UTR regions. Functionally, FOSL1 protected hepatoma cells against glucose deprivation-induced necrosis in vitro and in vivo. Mechanistically, FOSL1 transcriptionally repressed ATF3 by binding to its promoter. Meanwhile, ATF3 and MAFF interacted via their leucine zipper domains to form a heterodimer, which competed with NRF2 for binding to antioxidant response elements in the promoters of NRF2 target genes, thereby inhibiting their transcription. Consequently, FOSL1 reduced the formation of the ATF3-MAFF heterodimer, thereby enhancing NRF2 transcriptional activity and the antioxidant capacity of glucose-deprived-hepatoma cells. Thus, FOSL1 alleviated the necrosis-inducing effect of glucose deprivation-induced reactive oxygen species accumulation. Collectively, our study uncovers the protective role of m6A-FOSL1-ATF3 axis in hepatoma cell necrosis under glucose deprivation, and may provide new targets for cancer therapy.

Tubular Epithelial Cell HMGB1 Promotes AKI-CKD Transition by Sensitizing Cycling Tubular Cells to Oxidative Stress: A Rationale for Targeting HMGB1 during AKI Recovery.

SIGNIFICANCE STATEMENT: Cells undergoing necrosis release extracellular high mobility group box (HMGB)-1, which triggers sterile inflammation upon AKI in mice. Neither deletion of HMGB1 from tubular epithelial cells, nor HMGB1 antagonism with small molecules, affects initial ischemic tubular necrosis and immediate GFR loss upon unilateral ischemia/reperfusion injury (IRI). On the contrary, tubular cell-specific HMGB1 deficiency, and even late-onset pharmacological HMGB1 inhibition, increased functional and structural recovery from AKI, indicating that intracellular HMGB1 partially counters the effects of extracellular HMGB1. In vitro studies indicate that intracellular HMGB1 decreases resilience of tubular cells from prolonged ischemic stress, as in unilateral IRI. Intracellular HMGB1 is a potential target to enhance kidney regeneration and to improve long-term prognosis in AKI. BACKGROUND: Late diagnosis is a hurdle for treatment of AKI, but targeting AKI-CKD transition may improve outcomes. High mobility group box-1 (HMGB1) is a nuclear regulator of transcription and a driver of necroinflammation in AKI. We hypothesized that HMGB1 would also modulate AKI-CKD transition in other ways. METHODS: We conducted single-cell transcriptome analysis of human and mouse AKI and mouse in vivo and in vitro studies with tubular cell-specific depletion of Hmgb1 and HMGB1 antagonists. RESULTS: HMGB1 was ubiquitously expressed in kidney cells. Preemptive HMGB1 antagonism with glycyrrhizic acid (Gly) and ethyl pyruvate (EP) did not affect postischemic AKI but attenuated AKI-CKD transition in a model of persistent kidney hypoxia. Consistently, tubular Hmgb1 depletion in Pax8 rtTA, TetO Cre, Hmgb1fl/fl mice did not protect from AKI, but from AKI-CKD transition. In vitro studies confirmed that absence of HMGB1 or HMGB1 inhibition with Gly and EP does not affect ischemic necrosis of growth-arrested differentiated tubular cells but increased the resilience of cycling tubular cells that survived the acute injury to oxidative stress. This effect persisted when neutralizing extracellular HMGB1 with 2G7. Consistently, late-onset HMGB1 blockade with EP started after the peak of ischemic AKI in mice prevented AKI-CKD transition, even when 2G7 blocked extracellular HMGB1. CONCLUSION: Treatment of AKI could become feasible when ( 1 ) focusing on long-term outcomes of AKI; ( 2 ) targeting AKI-CKD transition with drugs initiated after the AKI peak; and ( 3 ) targeting with drugs that block HMGB1 in intracellular and extracellular compartments.

Epigenetic silencing of GCH1promotes hepatocellular carcinoma growth by activating superoxide anion-mediated ASK1/p38 signaling via inhibiting tetrahydrobiopterin de novo biosynthesis.

Metabolic reprogramming is a hallmark of cancer, including hepatocellular carcinoma (HCC). However, its role in HCC remains to be elucidated. Herein, we identified GTP cyclohydrolase 1 (GCH1), the first rate-limiting enzyme in tetrahydrobiopterin (BH4) de novo biosynthesis, as a novel metabolic regulator of HCC. GCH1 was frequently down-regulated in HCC tissues and cell lines by promoter methylation. Low GCH1 expression was associated with larger tumor size, increased tumor number, and worse prognosis in two independent cohorts of HCC patients. Functionally, GCH1 silencing promoted HCC growth in vitro and in vivo, while GCH1 overexpression exerted an opposite effect. The metabolite BH4 inhibited HCC growth in vitro and in vivo. GCH1 silencing exerted its growth-promoting effect through directly inhibiting BH4 de novo biosynthesis. Mechanistically, GCH1 silencing activated ASK1/p38 signaling; pharmacological or genetic inhibition of ASK1 or p38 abolished GCH1 silencing-induced growth-promoting effect. Further mechanistic studies found that GCH1 silencing-induced BH4 reduction resulted in an increase of intracellular superoxide anion levels in a dose-dependent manner, which mediated the activation of ASK1/p38 signaling. Collectively, our study reveals that epigenetic silencing of GCH1 promotes HCC growth by activating superoxide anion-mediated ASK1/p38 signaling via inhibiting BH4 de novo biosynthesis, suggesting that targeting GCH1/BH4 pathway may be a promising therapeutic strategy to combat HCC.

Electric cell-substrate impedance sensing in kidney research.

Electric cell-substrate impedance sensing (ECIS) is a quantitative, label-free, non-invasive analytical method allowing continuous monitoring of the behaviour of adherent cells by online recording of transcellular impedance. ECIS offers a wide range of practical applications to study cell proliferation, migration, differentiation, toxicity and monolayer barrier integrity. All of these applications are relevant for basic kidney research, e.g. on endothelial cells, tubular and glomerular epithelial cells. This review gives an overview on the fundamental principles of the ECIS technology. We name strengths and remaining hurdles for practical applications, present an ECIS array reuse protocol, and review its past, present and potential future contributions to preclinical kidney research.

Overexpression of Rab40b Promotes Hepatocellular Carcinoma Cell Proliferation and Metastasis via PI3K/AKT Signaling Pathway.

BACKGROUND: Rab40b is an evolutionarily conserved Rab GTPase that plays an important role in intracellular trafficking and is closely related to cancer progression. However, the role and potential molecular mechanism of Rab40b in hepatocellular carcinoma (HCC) have not yet been elucidated. MATERIALS AND METHODS: The expression of Rab40b in HCC tissues and peritumour tissues was tested by qRT-PCR, Western blotting and histological analysis. A Kaplan-Meier survival curve was generated based on the expression of Rab40b in the HCC samples. Cell proliferation assays, wound healing assays, and transwell assays are used to examine the effect of Rab40b on HCC cell growth in vitro. The effect of Rab40b on cell cycle was examined by flow cytometry. A xenograft implantation model was used to assess the effect of Rab40b on the development of HCC cells in vivo. RESULTS: Rab40b protein expression is upregulated in HCC tissues, and this upregulation is associated with high pathological stage and poor prognosis in HCC patients. Rab40b overexpression promotes the proliferation and metastasis of HCC cells by upregulating cyclin D1, cyclin E1 and matrix metalloproteinase 2 (MMP2) through the PI3K/AKT signalling pathway. Conversely, Rab40b inhibitors can significantly inhibit the proliferation and metastasis of HCC cell lines and induce G0/G1 cell cycle arrest and apoptosis. Studies of a nude mouse xenograft model demonstrated that Rab40b knockdown can significantly inhibit the proliferation and progression of HCC tumours in vivo. CONCLUSION: Overall, this study demonstrates that Rab40b is an important oncoprotein that promotes HCC progression by regulating the expression of cyclin D1, cyclin E1, p21 and MMP2 through the PI3K/AKT signalling pathway.

SIRT4 silencing in tumor-associated macrophages promotes HCC development via PPARδ signalling-mediated alternative activation of macrophages.

BACKGROUND: The activation of tumour-associated macrophages (TAMs) contributes to the progression of hepatocellular carcinoma (HCC). SIRT4 acts as a tumour suppressor of tumour growth by regulating cell metabolism, inflammation, and anti-tumourigenesis. However, the involvement of SIRT4 in the activation of TAMs is unknown. Based on previous findings, the expression of SIRT4 in distinct groups of TAMs as well as the effect of SIRT4 silencing on macrophage polarization was investigated. METHODS: The expression of SIRT4 in HCC tissues and peritumour tissues was tested by qRT-PCR, western blotting and histological analysis. A Kaplan-Meier survival curve was generated based on the expression of SIRT4 in the HCC samples. Next, immunofluorescence staining was used to evaluate distinct groups of TAMs in human HCC samples, and the expression of SIRT4 in M1 and M2 TAMs was examined by flow cytometry. A homograft mouse model was used to assess the effect of SIRT4 silencing in TAMs on the development of HCC cells. RESULTS: SIRT4 was significantly downregulated in HCC tumour tissues, and the expression of SIRT4 in peritumour tissues was positively associated with survival in patients. We further found that downregulation of SIRT4 was associated with increased macrophage infiltration and a high ratio of M2/M1 macrophages in HCC peritumour tissues. Using gene interference, we found that SIRT4 silencing in TAMs significantly modulated the alternative activation of macrophages and promoted in vitro and in vivo HCC cell growth. Mechanistically, we revealed that HCM restricted the expression of SIRT4 in macrophages and promoted alternative activation of macrophages via the FAO-PPARδ-STAT3 axis. Furthermore, we also revealed that elevated MCP-1 expression induced by SIRT4 downregulation was responsible for increased TAM infiltration in peritumour tissues. CONCLUSIONS: Overall, our results demonstrate that downregulation of SIRT4 in TAMs modulates the alternative activation of macrophages and promotes HCC development via the FAO-PPARδ-STAT3 axis. These results could provide a new therapeutic target for the treatment of HCC.

Mitochondria Permeability Transition versus Necroptosis in Oxalate-Induced AKI.

BACKGROUND: Serum oxalate levels suddenly increase with certain dietary exposures or ethylene glycol poisoning and are a well known cause of AKI. Established contributors to oxalate crystal-induced renal necroinflammation include the NACHT, LRR and PYD domains-containing protein-3 (NLRP3) inflammasome and mixed lineage kinase domain-like (MLKL) protein-dependent tubule necroptosis. These studies examined the role of a novel form of necrosis triggered by altered mitochondrial function. METHODS: To better understand the molecular pathophysiology of oxalate-induced AIK, we conducted in vitro studies in mouse and human kidney cells and in vivo studies in mice, including wild-type mice and knockout mice deficient in peptidylprolyl isomerase F (Ppif) or deficient in both Ppif and Mlkl. RESULTS: Crystals of calcium oxalate, monosodium urate, or calcium pyrophosphate dihydrate, as well as silica microparticles, triggered cell necrosis involving PPIF-dependent mitochondrial permeability transition. This process involves crystal phagocytosis, lysosomal cathepsin leakage, and increased release of reactive oxygen species. Mice with acute oxalosis displayed calcium oxalate crystals inside distal tubular epithelial cells associated with mitochondrial changes characteristic of mitochondrial permeability transition. Mice lacking Ppif or Mlkl or given an inhibitor of mitochondrial permeability transition displayed attenuated oxalate-induced AKI. Dual genetic deletion of Ppif and Mlkl or pharmaceutical inhibition of necroptosis was partially redundant, implying interlinked roles of these two pathways of regulated necrosis in acute oxalosis. Similarly, inhibition of mitochondrial permeability transition suppressed crystal-induced cell death in primary human tubular epithelial cells. PPIF and phosphorylated MLKL localized to injured tubules in diagnostic human kidney biopsies of oxalosis-related AKI. CONCLUSIONS: Mitochondrial permeability transition-related regulated necrosis and necroptosis both contribute to oxalate-induced AKI, identifying PPIF as a potential molecular target for renoprotective intervention.

Protist communities are more sensitive to nitrogen fertilization than other microorganisms in diverse agricultural soils.

BACKGROUND: Agricultural food production is at the base of food and fodder, with fertilization having fundamentally and continuously increased crop yield over the last decades. The performance of crops is intimately tied to their microbiome as they together form holobionts. The importance of the microbiome for plant performance is, however, notoriously ignored in agricultural systems as fertilization disconnects the dependency of plants for often plant-beneficial microbial processes. Moreover, we lack a holistic understanding of how fertilization regimes affect the soil microbiome. Here, we examined the effect of a 2-year fertilization regime (no nitrogen fertilization control, nitrogen fertilization, and nitrogen fertilization plus straw amendment) on entire soil microbiomes (bacteria, fungi, and protist) in three common agricultural soil types cropped with maize in two seasons. RESULTS: We found that the application of nitrogen fertilizers more strongly affected protist than bacterial and fungal communities. Nitrogen fertilization indirectly reduced protist diversity through changing abiotic properties and bacterial and fungal communities which differed between soil types and sampling seasons. Nitrogen fertilizer plus straw amendment had greater effects on soil physicochemical properties and microbiome diversity than nitrogen addition alone. Moreover, nitrogen fertilization, even more together with straw, increased soil microbiome network complexity, suggesting that the application of nitrogen fertilizers tightened soil microbiomes interactions. CONCLUSIONS: Together, our results suggest that protists are the most susceptible microbiome component to the application of nitrogen fertilizers. As protist communities also exhibit the strongest seasonal dynamics, they serve as the most sensitive bioindicators of soil changes. Changes in protist communities might have long-term effects if some of the key protist hubs that govern microbiome complexities as top microbiome predators are altered. This study serves as the stepping stone to promote protists as promising agents in targeted microbiome engineering to help in reducing the dependency on exogenous unsustainably high fertilization and pesticide applications.

Liraglutide protects against inflammatory stress in non-alcoholic fatty liver by modulating Kupffer cells M2 polarization via cAMP-PKA-STAT3 signaling pathway.

Nonalcoholic fatty liver disease (NAFLD) is the second major chronic liver disease world-wide and growing. Current medical treatment of NAFLD is not effective, and there is an urgent need to find new effective drugs. Liraglutide is now the first-line treatment for type 2 diabetes mellitus (T2DM) with promise, according to recent reports, to mitigate the fatty degeneration of the liver. The investigators of the current study discern if liraglutide reduces non-alcoholic fatty liver disease (NAFLD) induced by a high-fat diet using mice via modulating Kupffer cells M2 polarization in the liver. The mice underwent four weeks of intraperitoneal injections of liraglutide (0.6 mg/kg body weight). In the NAFLD model used in this study, the liver index, the body weight, and the serum levels of ALT, AST, total cholesterol, and triglycerides were meaningfully improved. In sections using H&E and Oil Red O staining, hepatic steatosis was significantly improved. Liraglutide decreased liver inflammation and the inflammatory properties of Kupffer cells in the NAFLD mouse model and there was a higher ratio of M2/M1 Kupffer cells. In vitro studies found that Liraglutide treatment modulates Kupffer cells to M2-like activation via the cAMP-PKA-STAT3 signaling pathway. The perilous effects of a high-fat diet were alleviated by liraglutide, including hepatic steatosis, by modulating Kupffer cells M2 polarization via the cAMP-PKA-STAT3 signaling pathway. Liraglutide can indeed reverse the negative effects of NAFLD.

Computational determination of binding structures and free energies of glucose 6-phosphate dehydrogenase with novel steroid inhibitors.

Glucose 6-phosphate dehydrogenase (G6PD), the first and the rate-limiting enzyme in the pentose phosphate pathway (PPP), catalyzes the oxidation of G6P to 6-phosphogluconolactone and the reduction of NADP(+) to NADPH. Its key role in cancer promotes the development of a potent and selective inhibitor that might increase cancer cell death when combined with radiotherapy. In the present study, we investigated the detailed binding modes and binding free energies for G6PD interacting with a promising series of recently developed inhibitors, i.e., the steroid derivatives, by performing molecular docking, molecular dynamics (MD) simulations, and binding free energy calculations. The docking indicates that the inhibitors occupy the binding sites of both G6P and NADP(+). The calculated binding free energies on the basis of the MD-simulated enzyme-inhibitor complexes are in good agreement with the experimental activity data for all of the examined inhibitors. The valuable insights into the detailed enzyme-inhibitor binding including the important intermolecular interactions, e.g., the hydrogen bond interaction and the hydrophobic interaction, have been provided. The computational results provide new insights into future rational design of more potent inhibitors of G6PD as a treatment for cancer.