Alcohol dehydrogenase 1 is a tubular mitophagy-dependent apoptosis inhibitor against septic acute kidney injury.

Alcohol dehydrogenase 1 (ADH1) is an alcohol-oxidizing enzyme with poorlydefined biology. Here we report that ADH1 is highly expressed in kidneys of mice with lethal endotoxemia and is transcriptionally upregulated in tubular cells by lipopolysaccharide (LPS) stimuli through TLR4/NF-κB cascade. The Adh1 knockout (Adh1KO) mice with lethal endotoxemia displayed increased susceptibility to acute kidney injury (AKI) but not systemic inflammatory response. Adh1KO mice develop more severe tubular cell apoptosis in comparison to Adh1 wild-type (Adh1WT) mice during course of lethal endotoxemia. ADH1 deficiency facilitates the LPS-induced tubular cell apoptosis in a caspase-dependent manner. Mechanistically, ADH1 deficiency dampens tubular mitophagy that relies on PINK1-Parkin pathway characterized by the reduced membrane potential, reactive oxygen species (ROS) and release of fragmented mtDNA to cytosol. Kidney-specific overexpression of PINK1 and Parkin by adeno-associated viral vector 9 (AAV9) delivery ameliorates AKI exacerbation in Adh1KO mice with lethal endotoxemia. Our study supports the notion that ADH1 is critical for blockade of tubular apoptosis mediated by mitophagy, allowing the rapid identification and targeting of alcohol-metabolic route applicable to septic AKI.

Ascorbate uptake enables tubular mitophagy to prevent septic AKI by PINK1-PARK2 axis.

Ascorbate (Vitamin C) has been proposed as a promising therapeutic agent against sepsis in clinical trials, but there is little experimental evidence on its anti-septic efficacy. We report that Toll-like receptor 4 (TLR4) activation by LPS stimuli augments ascorbate uptake in murine and human tubular cells through upregulation of two ascorbate transporters SVCT-1 and -2 mediated by Fn14/SCFFbxw7α cascade. Ascorbate restriction, or knockout of SVCT-1 and -2, the circumstance reminiscent to blockade of ascorbate uptake, endows tubular cells more vulnerable to the LPS-inducible apoptosis, whereas exogenous administration of ascorbate overrides the ruin execution, for which the PINK1-PARK2, rather than BNIP3-NIX axis is required. Ascorbate increases, while SVCT-1 and -2 knockout or ascorbate restriction dampens tubular mitophagy upon LPS stimuli. Treatment of endotoxemic mice with high-dose ascorbate confers mitophagy and substantial protection against mortality and septic acute kidney injury (AKI). Our work provides a rationale for clinical management of septic AKI with high doses of ascorbate.

Productive transcription of miR-124-3p by RelA and RNA polymerase II directs RIP1 ubiquitination-dependent apoptosis resistance during hypoxia.

The K63-linked ubiquitination of RIP1 coordinates survival/death homeostasis by driving transcription of genes downstream of RelA. Previously, we demonstrated that EGF-dependent RelA transactivation overcomes hypoxia-initiated apoptosis, yet the underlying mechanisms remain mysterious. We report here that UBXN1 deficiency empowers apoptosis resistance against hypoxia through triggering IκBα degradation, for which K63-linked ubiquitination of RIP1 is required. MiR-124-3p is a bona fide inhibitor upstream of UBXN1, thereby antagonizing the hypoxia-initiated apoptosis. UBXN1 repression by miR-124-3p restores the K63-linked ubiquitination of RIP1, IKKß phosphorylation, IκBα-RelA disassembly, RelA nuclear localization and transactivation of EGF gene as well as EGF secretion under hypoxia. Reconstitution of wild-type UBXN1, but not a truncated UBXN1ΔUBA mutant, or pharmacological inhibition of RelA transactivation in miR-124-3p-replete cells compromises the apoptosis-resistant phenotypes of miR-124-3p. Hypoxia transcriptionally downregulates miR-124-3p by disassociating RelA and RNAP II from its promoter. EGFR activation renders the K63-linked ubiquitination of RIP1 and hypoxic tolerance in conjunction with miR-124-3p. Our findings identify a pivotal role of miR-124-3p in ubiquitin conjugation of RIP1 against hypoxic damage and underscore that productive transcription of miR-124-3p by RelA and RNAP II might be a switching mechanism for this process.

Regulation of Fn14 stability by SCFFbxw7α during septic acute kidney injury.

Acute kidney injury (AKI) initiated by sepsis remains a thorny problem despite recent advancements in its clinical management. Having been found to be activated during AKI, fibroblast growth factor-inducible molecule 14 (Fn14) may be a potential therapeutic target because of its involvement in the molecular basis of injury. Here, we report that LPS induces apoptosis of mouse cortical tubule cells mediated by Fn14, for which simultaneous Toll-like receptor (TLR)4 activation is required. Mechanistically, TLR4 activation by lipopolysaccharide, through disassociating E3 ligase SCFFbxw7α from Fn14, dismantles Lys48-linked polyubiquitination of Fn14 and stabilizes it. Pharmacological deactivation of Fn14 with monoclonal antibody ITEM-2 provides effective protection against lethal sepsis and AKI in mice. Our study underscores an adaptive mechanism whereby TLR4 regulates SCFFbxw7α-dependent Fn14 stabilization during inflammatory tubular damage and further supports investigation of targeting Fn14 in clinical trials of patients with septic AKI.

MicroRNA-130b transcriptionally regulated by histone H3 deacetylation renders Akt ubiquitination and apoptosis resistance to 6-OHDA.

Apoptosis of DA neurons is a contributing cause of disability and death for Parkinson's disease (PD). Akt may become a potential therapeutic target for PD since Akt has been deactivated during DA neuron apoptosis. We previously demonstrated that Akt confers apoptosis resistance against 6-OHDA in DA neuron-like PC12 cells, yet the underlying mechanisms accounted for this are not fully understood. Here we report that microRNA-130b (miR-130b)-dependent and cylindromatosis (CYLD) repression-mediated Akt ubiquitination renders apoptosis resistance of PC12 cells to 6-OHDA, which elicits histone H3 deacetylation-induced transcriptional downregulation of miR-130b vice versa. CYLD deficiency ubiquitinates Akt at Lys63, thereby phosphorylating Akt and antagonizing 6-OHDA-initiated apoptosis. MiR-130b targetedly represses CYLD and increases apoptosis resistance to 6-OHDA. CYLD repression by miR-130b restores Akt ubiquitination and activation, GSK3ß and FoxO3a phosphorylation, FoxO3a removal from Bim promoter as well as Bim downregulation during 6-OHDA administration. CYLD deficiency-mediated Akt activation is instrumental for the apoptosis-resistant phenotypes of miR-130b. In addition, 6-OHDA transcriptionally downregulates miR-130b through recruitment of HDAC3 at the promoter. Furthermore, EPO potentiates the ability of miR-130b to activate Akt and augment apoptosis resistance. Our findings identify the apoptosis-resistant function of miR-130b and suggest that histone H3 deacetylation plays a pivotal role in regulating miR-130b transcription in response to 6-OHDA.

NF-κB-dependent transcriptional upregulation of cyclin D1 exerts cytoprotection against hypoxic injury upon EGFR activation.

Apoptosis of neural cells is one of the main pathological features in hypoxic/ischemic brain injury. Nuclear factor-κB (NF-κB) might be a potential therapeutic target for hypoxic/ischemic brain injury since NF-κB has been found to be inactivated after hypoxia exposure, yet the underlying molecular mechanisms of NF-κB inactivation are largely unknown. Here we report that epidermal growth factor receptor (EGFR) activation prevents neuron-like PC12 cells apoptosis in response to hypoxia via restoring NF-κB-dependent transcriptional upregulation of cyclin D1. Functionally, EGFR activation by EGF stimulation mitigates hypoxia-induced PC12 cells apoptosis in both dose- and time-dependent manner. Of note, EGFR activation elevates IKKß phosphorylation, increases IκBα ubiquitination, promotes P65 nuclear translocation and recruitment at cyclin D1 gene promoter as well as upregulates cyclin D1 expression. EGFR activation also abrogates the decrease of IKKß phosphorylation, reduction of IκBα ubiquitination, blockade of P65 nuclear translocation and recruitment at cyclin D1 gene promoter as well as downregulation of cyclin D1 expression induced by hypoxia. Furthermore, NF-κB-dependent upregulation of cyclin D1 is instrumental for the EGFR-mediated cytoprotection against hypoxic apoptosis. In addition, the dephosphorylation of EGFR induced by either EGF siRNA transfection or anti-HB-EGF neutralization antibody treatment enhances hypoxic cytotoxicity, which are attenuated by EGF administration. Our results highlight the essential role of NF-κB-dependent transcriptional upregulation of cyclin D1 in EGFR-mediated cytoprotective effects under hypoxic preconditioning and support further investigation of EGF in clinical trials of patients with hypoxic/ischemic brain injury.

Targeting SLC22A5 fosters mitophagy inhibition-mediated macrophage immunity against septic acute kidney injury upon CD47-SIRPα axis blockade.

Efferocytosis of apoptotic neutrophils (PMNs) by macrophages is helpful for inflammation resolution and injury repair, but the role of efferocytosis in intrinsic nature of macrophages during septic acute kidney injury (AKI) remains unknown. Here we report that CD47 and signal regulatory protein alpha (SIRPα)-the anti-efferocytotic 'don't eat me' signals-are highly expressed in peripheral blood mononuclear cells (PBMCs) from patients with septic AKI and kidney samples from mice with polymicrobial sepsis and endotoxin shock. Conditional knockout (CKO) of SIRPA in macrophages ameliorates AKI and systemic inflammation response in septic mice, accompanied by an escalation in mitophagy inhibition of macrophages. Ablation of SIRPA transcriptionally downregulates solute carrier family 22 member 5 (SLC22A5) in the lipopolysaccharide (LPS)-stimulated macrophages that efferocytose apoptotic neutrophils (PMNs). Targeting SLC22A5 renders mitophagy inhibition of macrophages in response to LPS stimuli, improves survival and deters development of septic AKI. Our study supports further clinical investigation of CD47-SIRPα signalling in sepsis and proposes that SLC22A5 might be a promising immunotherapeutic target for septic AKI.

Pellino1 orchestrates gut-kidney axis to perpetuate septic acute kidney injury through activation of STING pathway and NLRP3 inflammasome.

AIMS: Intestinal barrier dysfunction is the initial and propagable factor of sepsis in which acute kidney injury (AKI) has been considered as a common life-threatening complication. Our recent study identifies the regulatory role of Pellino1 in tubular death under inflammatory conditions in vitro. The objective of our current study is to explore the impact of Pellino1 on gut-kidney axis during septic AKI and uncover the molecular mechanism (s) underlying this process. MATERIALS AND METHODS: Immunohistochemistry (IHC) was conducted to evaluate Pellino1 and NOD-like receptor thermal protein domain associated protein 3 (NLRP3) levels in renal biopsies from critically ill patients with a clinical diagnosis of sepsis. Functional and mechanistic studies were characterized in septic models of the Peli-knockout (Peli1-/-) mice by histopathological staining, enzyme-linked immunosorbent assay (ELISA), flow cytometry, immunofluorescence, biochemical detection, CRISPR/Cas9-mediated gene editing and intestinal organoid. KEY FINDINGS: Pellino1, together with NLRP3, are highly expressed in renal biopsies from critically ill patients diagnosed with sepsis and kidney tissues of septic mice. The Peli1-/- mice with sepsis become less prone to develop AKI and have markedly compromised NLRP3 activation in kidney. Loss of Peli1 endows septic mice refractory to intestinal inflammation, barrier permeability and enterocyte apoptosis that requires stimulator of interferons genes (STING) pathway. Administration of STING agonist DMXAA deteriorates AKI and mortality of septic Peli1-/- mice in the presence of kidney-specific NLRP3 reconstitution. SIGNIFICANCE: Our studies suggest that Pellino1 has a principal role in orchestrating gut homeostasis towards renal pathophysiology, thus providing a potential therapeutic target for septic AKI.

Tripartite motif containing 69 elicits ERK2-dependent EYA4 turnover to impart pancreatic tumorigenesis.

Eyes absent homologue 4 (EYA4) is silenced in pancreatic ductal adenocarcinoma (PDAC) and functions as a tumor suppressor to restrain PDAC development, albeit the molecular mechanism underlying its downregulation remains enigmatic. Methods: Functional studies were determined by immunohistochemistry of PDAC samples from patients and Pdx1-Cre; LSL-KrasG12D/+; Trp53fl/+ (KPC) mice, three-dimensional spheroid culture, flow cytometry, MTT and subcutaneous xenograft experiments. Mechanistical studies were examined by cellular ubiquitination, cycloheximide (CHX) pulse-chase, co-immunoprecipitation, chromatin immunoprecipitation, GST-pulldown, in vitro protein kinase assay, immunofluorescence and luciferase reporter assays. Results: We screen E3 ligase that is negatively correlated with EYA4 and uncover a mutually exclusive interaction of tripartite motif containing 69 (TRIM69) with EYA4 in human PDAC. TRIM69 elicits EYA4 polyubiquitylation and turnover independent of P53 and impedes the EYA4-driven deactivation of ß-catenin/ID2 cascade, fueling PDAC cell proliferation in vitro and tumor development in mice. Expression of TRIM69 is upregulated in PDAC samples from independent cohorts of patients and the Pdx1-Cre; LSL-KrasG12D/+; Trp53fl/+ (KPC) mice, and associated with unfavorable prognosis. Depleting TRIM69 preferentially induces lethality in the EYA4-deficient PDAC cells. We further unearth that ERK2 directly binds to the D-site of mitogen-activated protein kinase (MAPK) docking groove in EYA4 Leu512/514 and phosphorylates EYA4 at Ser37, which is instrumental for EYA4 polyubiquitylation and turnover by TRIM69. Conclusion: Our results define a previously unappreciated role of TRIM69-EYA4 axis in pancreatic tumorigenesis and underscore that targeting TRIM69 might be an effective therapeutic approach for PDAC harboring EYA4 deficiency.

Auto- and paracrine rewiring of NIX-mediated mitophagy by insulin-like growth factor-binding protein 7 in septic AKI escalates inflammation-coupling tubular damage.

AIMS: Inflammation-coupling tubular damage (ICTD) contributes to pathogenesis of septic acute kidney injury (AKI), in which insulin-like growth factor-binding protein 7 (IGFBP-7) serves as a biomarker for risk stratification. The current study aims to discern how IGFBP-7 signalling influences ICTD, the mechanisms that underlie this process and whether blockade of the IGFBP-7-dependent ICTD might have therapeutic value for septic AKI. MATERIALS AND METHODS: In vivo characterization was carried out in B6/JGpt-Igfbp7em1Cd1165/Gpt mice subjected to cecal ligation and puncture (CLP). Transmission electron microscopy, immunofluorescence, flow cytometry, immunoblotting, ELISA, RT-qPCR and dual-luciferase reporter assays were used to determine mitochondrial functions, cell apoptosis, cytokine secretion and gene transcription. KEY FINDINGS: ICTD augments the transcriptional activity and protein secretion of tubular IGFBP-7, which enables an auto- and paracrine signalling via deactivation of IGF-1 receptor (IGF-1R). Genetic knockout (KO) of IGFBP-7 provides renal protection, improves survival and resolves inflammation in murine models of cecal ligation and puncture (CLP), while administering recombinant IGFBP-7 aggravates ICTD and inflammatory invasion. IGFBP-7 perpetuates ICTD in a NIX/BNIP3-indispensable fashion through dampening mitophagy that restricts redox robustness and preserves mitochondrial clearance programs. Adeno-associated viral vector 9 (AAV9)-NIX short hairpin RNA (shRNA) delivery ameliorates the anti-septic AKI phenotypes of IGFBP-7 KO. Activation of BNIP3-mediated mitophagy by mitochonic acid-5 (MA-5) effectively attenuates the IGFBP-7-dependent ICTD and septic AKI in CLP mice. SIGNIFICANCE: Our findings identify IGFBP-7 is an auto- and paracrine manipulator of NIX-mediated mitophagy for ICTD escalation and propose that targeting the IGFBP-7-dependent ICTD represents a novel therapeutic strategy against septic AKI.

Tubule-mitophagic secretion of SerpinG1 reprograms macrophages to instruct anti-septic acute kidney injury efficacy of high-dose ascorbate mediated by NRF2 transactivation.

High-dose ascorbate confers tubular mitophagy responsible for septic acute kidney injury (AKI) amelioration, yet its biological roles in immune regulation remain poorly understood. Methods: The role of tubular mitophagy in macrophage polarization upon high-dose ascorbate treatment was assessed by fluorescence-activated cell sorter analysis (FACS) in vitro and by immunofluorescence in AKI models of LPS-induced endotoxemia (LIE) from Pax8-cre; Atg7 flox/flox mice. The underlying mechanisms were revealed by RNA-sequencing, gene set enrichment analysis (GSEA), luciferase reporter, chromatin immunoprecipitation (ChIP) and adeno-associated viral vector serotype 9 (AAV9) delivery assays. Results: High-dose ascorbate enables conversion of macrophages from a pro-inflammatory M1 subtype to an anti-inflammatory M2 subtype in murine AKI models of LIE, leading to decreased renal IL-1ß and IL-18 production, reduced mortality and alleviated tubulotoxicity. Blockade of tubular mitophagy abrogates anti-inflammatory macrophages polarization under the high-dose ascorbate-exposed coculture systems. Similar abrogations are verified in LIE mice with tubular epithelium-specific ablation of Atg7, where the high-dose ascorbate-inducible renal protection and survival improvement are substantially weaker than their control littermates. Mechanistically, high-dose ascorbate stimulates tubular secretion of serpin family G member 1 (SerpinG1) through maintenance of mitophagy, for which nuclear factor-erythroid 2 related factor 2 (NRF2) transactivation is required. SerpinG1 perpetuates anti-inflammatory macrophages to prevent septic AKI, while kidney-specific disruption of SerpinG1 by adeno-associated viral vector serotype 9 (AAV9)-short hairpin RNA (shRNA) delivery thwarts the anti-inflammatory macrophages polarization and anti-septic AKI efficacy of high-dose ascorbate. Conclusion: Our study identifies SerpinG1 as an intermediate of tubular mitophagy-orchestrated myeloid function during septic AKI and reveals a novel rationale for ascorbate-based therapy.

Crosstalk between hypoxia-sensing ULK1/2 and YAP-driven glycolysis fuels pancreatic ductal adenocarcinoma development.

Autophagy and glycolysis are two catabolic processes that manipulate pancreatic ductal adenocarcinoma (PDAC) development in response to hypoxia sensing, yet the underlying mechanism of how they are interlinked remain elusive. Methods: The functional roles of Unc-51 like kinase 1 and 2 (ULK1/2) in pyruvate kinase M2 (PKM2) transcription and glycolysis under hypoxia were assessed by chromatin immunoprecipitation, luciferase reporter, glucose consumption and lactate production assay. Co-immunoprecipitation, cellular ubiquitination, His-pulldown, in vitro protein kinase assay, immunofluorescence, immunohistochemistry, CRISPR technology, in silico studies were adopted to determine the molecular mechanism. Correlation analyses were performed in KPC (Pdx1-Cre; LSL-KrasG12D/+; Trp53fl/+) mice and clinical samples from PDAC patients. Therapeutic potential of ULK1/2 inhibitor and 2-deoxyglucose (2-DG) or 3-bromopyruvate (3-BP) was evaluated in cell-derived xenograft (CDX) and the patient-derived xenograft (PDX) models of nude mice. Results: ULK1/2, but not ULK3, augments hypoxic glycolysis in PDAC cells mediated by PKM2 independent of BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3). Mechanistically, hypoxia stimulates ULK1 to translocate into nucleus, where it interacts with and phosphorylates yes-associated protein (YAP) at Ser227, resulting in YAP stabilization through blockade of ubiquitin-proteasome system (UPS), which in turn facilitates PKM2 transcription, glycolysis, cell proliferation in vitro as well as PDAC growth in mice. ULK1/2 is positively correlated with YAP and PKM2 in tumor tissues from KPC mice and clinical samples from PDAC patients. Pharmacological deactivation of ULK1/2 potentiates the antineoplastic efficacy of 2-DG and 3-BP in CDX and PDX models. Conclusion: Our findings underscore the Ser227 autophosphorylation-dependent nuclear YAP stabilization as a central node that couples ULK1/2-initiated autophagy to hypoxic glycolysis during PDAC development and propose that targeting ULK1/2 combined with 2-DG or 3-BP might be a feasible therapeutic strategy against PDAC.

Interruption of neutrophil extracellular traps formation dictates host defense and tubular HOXA5 stability to augment efficacy of anti-Fn14 therapy against septic AKI.

The immunosuppressive, inflammatory microenvironment orchestrated by neutrophil extracellular traps (NETs) plays a principal role in pathogenesis of sepsis. Fibroblast growth factor-inducible molecule 14 (Fn14) has been established as a potential target for septic acute kidney injury (AKI), making further therapeutic benefits from combined NETs and Fn14 blockade possible. Methods: The concurrence of NETs and Fn14 in mice and patients with septic AKI were assessed by immunofluorescence, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and in silico studies. Survival, histopathological and biochemical analyses of wild-type and PAD4-deficient CMV-Cre; PAD4 fl/fl mice with septic AKI were applied to evaluate the efficacy of either pharmacological or genetic NETs interruption in combination with Fn14 blockade. Molecular mechanisms underlying such effects were determined by CRISPR technology, fluorescence-activated cell sorter analysis (FACS), cycloheximide (CHX) pulse-chase, luciferase reporter and chromatin immunoprecipitation (ChIP) assay. Results: NETs formation is concurred with Fn14 upregulation in murine AKI models of abdominal, endotoxemic, multidrug-resistant sepsis as well as in serum samples of patients with septic AKI. Pharmacological or genetic interruption of NETs formation synergizes with ITEM-2, a monoclonal antibody (mAb) of Fn14, to prolong mice survival and provide renal protection against abdominal sepsis, the effects that could be abrogated by elimination of macrophages. Interrupting NETs formation predominantly perpetuates infiltration and survival of efferocytic growth arrest-specific protein 6+ (GAS6+) macrophages in combination with ITEM-2 therapy and enhances transcription of tubular cell-intrinsic Fn14 in a DNA methyltransferase 3a (DNMT3a)-independent manner through dismantling the proteasomes-mediated turnover of homeobox protein Hox-A5 (HOXA5) upon abdominal sepsis challenge or LPS stimuli. Pharmacological NETs interruption potentiates the anti-septic AKI efficacy of ITEM-2 in murine models of endotoxemic and multidrug-resistant sepsis. Conclusion: Our preclinical data propose that interrupting NETs formation in combination with Fn14 mAb might be a feasible therapeutic strategy for septic AKI.

MicroRNA-19a Targets Fibroblast Growth Factor-Inducible Molecule 14 and Prevents Tubular Damage in Septic AKI.

Fibroblast growth factor-inducible molecule 14 (Fn14) plays a principal role in triggering tubular damage during septic acute kidney injury (AKI). Here, we explore the mechanism underlying Fn14 deregulation in septic AKI. We identify Fn14 as a bona fide target of miR-19a, which directly binds to 3' UTR of Fn14 for repression independent of cylindromatosis (CYLD), the deubiquitinase (DUB) downstream of miR-19a, and thereby antagonizes the LPS-induced tubular cell apoptosis. Genetic ablation of Fn14, but not of CYLD, abolishes the ability of miR-19a to antagonize the tubular apoptosis by lipopolysaccharide (LPS). In mice, systemic delivery of miR-19a confers protection against septic AKI. Our findings implicate that miR-19a may serve as a promising therapeutic candidate in the prevention of septic AKI.

Redox DAPK1 destabilizes Pellino1 to govern inflammation-coupling tubular damage during septic AKI.

Tubular damage initiated by inflammatory response and ischemic/hypoxic stress is a hallmark of septic acute kidney injury (AKI), albeit the molecular mechanism coupling the two events remains unclear. We investigated the intrinsic nature of tubular damage with respect to inflammatory/hypoxic stress during septic AKI. Methods: The apoptotic response of tubular cells to LPS stimuli was analyzed before and after hypoxia exposure. Cellular ubiquitination, co-immunoprecipitation, GST-pulldown, in vitro protein kinase assay, immunofluorescence and CRISPR technology were adopted to determine the molecular mechanism underlying this process. In vivo characterization was performed in wild-type and DAPK1-/- mice models of cecal ligation and puncture (CLP). Results: We found that the MyD88-dependent inflammatory response couples to tubular damage during LPS stimuli under hypoxia in a Fn14/SCFFbxw7α-dispensable manner via recruitment of caspase-8 with TRIF-RIP1 signalosome mediated by DAPK1, which directly binds to and phosphorylates Pellino1 at Ser39, leading to Pellino1 poly-ubiquitination and turnover. Either pharmacological deactivation or genetic ablation of DAPK1 makes tubular cells refractory to the LPS-induced damage in the context of hypoxia, while kinase activity of DAPK1 is essential for ruin execution. Targeting DAPK1 effectively protects mice against septic AKI and potentiates the efficacy of a MyD88 homodimerization inhibitor, ST2825. Conclusion: Our findings provide a rationale for the mechanism whereby inflammation intersects with hypoxic tubular damage during septic AKI through a previously unappreciated role of DAPK1-inducible Ser39 phosphorylation in Pellino1 turnover and underscore that combined targeting DAPK1 and MyD88 might be a feasible strategy for septic AKI management.

Phosphorylation of 14-3-3ζ links YAP transcriptional activation to hypoxic glycolysis for tumorigenesis.

Hypoxic microenvironment deregulates metabolic homeostasis in cancer cells albeit the underlying mechanisms involved in this process remain hitherto enigmatic. 14-3-3ζ/Yes-associated protein (YAP) axis plays a principal role in malignant transformation and tumor development. Here, we report that hypoxia disassembles 14-3-3ζ from YAP and thereby promotes YAP nuclear localization mediated by ERK2, which directly binds to the D-site of mitogen-activated protein kinase (MAPK) docking domain in 14-3-3ζ Leu98/100 and phosphorylates 14-3-3ζ at Ser37. When localizing in nucleus, YAP recruits at pyruvate kinase M2 (PKM2) gene promoter with hypoxia-inducible factor 1α (HIF-1α), for which PKM2 transcription is required. 14-3-3ζ Ser37 phosphorylation is instrumental for the hypoxia-induced glucose uptake, lactate production, and clonogenicity of pancreatic ductal adenocarcinoma (PDAC) cells, as well as tumorigenesis in mice. The 14-3-3ζ Ser37 phosphorylation positively correlates with p-ERK1/2 activity and HIF-1α expression in clinical samples from patients with PDAC and predicts unfavorable prognosis. Our findings underscore an appreciable linkage between YAP transcriptional activation and hypoxic glycolysis governed by ERK2-dependent 14-3-3ζ Ser37 phosphorylation for malignant progression of PDAC.

SAA1 increases NOX4/ROS production to promote LPS-induced inflammation in vascular smooth muscle cells through activating p38MAPK/NF-κB pathway.

BACKGROUND: To investigate the effects of serum amyloid A1 (SAA1) on lipopolysaccharide (LPS) -induced inflammation in vascular smooth muscle cells (VSMCs). SAA1 expression was detected in LPS induced VSMCs at different concentrations for different time by using Western blotting. After pre-incubation with recombinant SAA1 protein, VSMCs were treated with 1 µg/ml LPS for 24 h. The VSMCs were then divided into Control, SAA1 siRNA, Nox4 siRNA, LPS, LPS + SAA1 siRNA, LPS + Nox4 siRNA and LPS + SAA1 siRNA + Nox4 groups. MTT was performed to observe the toxicity of VSMCs. Lucigenin-enhanced chemiluminescence method was used to detect superoxide anion (O2-) production and NADPH oxidase activity. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine expressions of inflammatory factors. Western blotting was used to determine expressions of NOX-4 and p38MAPK/NF-κB pathway related proteins. RESULTS: LPS promoted SAA1 protein expression in a concentration-/time-dependent manner. Recombinant SAA1 protein could increase NOX4/ROS production and promote the release of inflammatory factors (IL-1ß, IL-6, IL-8, IL-17, TNF-α and MCP-1) in LPS (1 µg/ml) - induced VSMCs. Besides, both SAA1 siRNA and NOX-4 siRNA could not only enhance the O2- production and NADPH oxidase activity, but also up-regulate the protein expression of NOX4, the release of inflammatory factors, and the levels of p-p38 and p-NF-κB p65 in LPS-induced VSMCs. However, no significant differences in each index were observed between LPS group and LPS + SAA1 siRNA + Nox4 group. CONCLUSION: SAA1-mediated NOX4/ROS pathway could activate p38MAPK/NF-κB pathway, thereby contributing to the release of inflammatory factors in LPS-induced VSMCs.

EYA4 inhibits hepatocellular carcinoma growth and invasion by suppressing NF-κB-dependent RAP1 transactivation.

BACKGROUND: Our previous studies demonstrated that eyes absent homolog 4 (EYA4), a member of the eye development-related EYA family in Drosophila, is frequently methylated and silenced in hepatocellular carcinoma (HCC) specimens and associated with shorter survival. The current work aimed to explore the mechanisms through which EYA4 functions as a tumor suppressor in HCC. METHODS: Stable EYA4-expressing plasmid (pEYA4) transfectants of the human HCC cell lines Huh-7 and PLC/PRF/5 (PLC) were established. Xenografts tumors were established via subcutaneous injection of the stable transfectants into BALB/c nude mice. Tissue samples were obtained from 75 pathologically diagnosed HCC patients. Quantitative real-time polymerase chain reaction, Western blotting and immunohistochemistry were performed to determine the expression of EYA4 in cell lines, xenografts and clinical specimens. The cell proliferation, colony formation, invasiveness and tumor formation of stable transfectants were studied. A gene expression microarray was utilized to screen genes regulated by EYA4 expression. The effect of EYA4 on nuclear factor-κB (NF-κB)/RAS-related protein 1 (RAP1) signaling was demonstrated through the co-transfection of pEYA4 and Flag-tagged RAS-related protein 1A gene-expressing plasmid (Flag-RAP1A), functional studies, chromatin immunoprecipitation, immunofluorescence staining and cellular ubiquitination assay. RESULTS: The restoration of EYA4 expression in HCC cell lines suppressed cell proliferation, inhibited clonogenic outgrowth, reduced cell invasion and restrained xenograft tumor growth, and Flag-RAP1A reversed the suppressive effects of pEYA4 in vitro. Activation of NF-κB with tumor necrosis factor-α (TNF-α) increased the binding of p65 to the RAP1A gene promoter and up-regulated RAP1 protein expression. The inhibition of NF-κB with BAY 11-7085 and p65 siRNA successfully blocked TNF-α-induced RAP1 up-regulation. EYA4 antagonized the TNF-α-induced phosphorylation and ubiquitination of inhibitor of NF-κBα (IκBα) as well as the nuclear translocation and transactivation of p65, resulting in repressed NF-κB activity and RAP1 expression. Blocking the serine/threonine phosphatase activity of EYA4 with calyculin A notably abrogated its suppressive effect on NF-κB activity. In addition, EYA4 expression was inversely correlated with IκBα/RAP1 activity in clinical HCC specimens. CONCLUSION: Our findings provide a functional and mechanistic basis for identifying EYA4 as a bona fide tumor suppressor that disrupts aberrant activation of the NF-κB/RAP1 signaling pathway and thus orchestrates a physiological impediment to HCC growth and invasion.

Transcriptional downregulation of microRNA-19a by ROS production and NF-κB deactivation governs resistance to oxidative stress-initiated apoptosis.

Cell apoptosis is one of the main pathological alterations during oxidative stress (OS) injury. Previously, we corroborated that nuclear factor-κB (NF-κB) transactivation confers apoptosis resistance against OS in mammalian cells, yet the underlying mechanisms remain enigmatic. Here we report that microRNA-19a (miR-19a) transcriptionally regulated by reactive oxygen species (ROS) production and NF-κB deactivation prevents OS-initiated cell apoptosis through cylindromatosis (CYLD) repression. CYLD contributes to OS-initiated cell apoptosis, for which NF-κB deactivation is essential. MiR-19a directly represses CYLD via targeting 3' UTR of CYLD, thereby antagonizing OS-initiated apoptosis. CYLD repression by miR-19a restores the IKKß phosphorylation, RelA disassociation from IκBα, IκBα polyubiquitination and degradation, RelA recruitment at VEGF gene promoter as well as VEGF secretion in the context of OS. Either pharmacological deactivation of NF-κB or genetic upregulation of CYLD compromises the apoptosis-resistant phenotypes of miR-19a. Furthermore, miR-19a is transcriptionally downregulated upon OS in two distinct processes that require ROS production and NF-κB deactivation. VEGF potentiates the ability of miR-19a to activate NF-κB and render apoptosis resistance. Our findings underscore a putative mechanism whereby CYLD repression-mediated and NF-κB transactivation-dependent miR-19a regulatory feedback loop prevents cell apoptosis in response to OS microenvironment.

EYA4 functions as tumor suppressor gene and prognostic marker in pancreatic ductal adenocarcinoma through ß-catenin/ID2 pathway.

Eye absent homolog 4 (EYA4) was initially found as key gene in controlling eye development in Drosophila. We recently found that EYA4 was an independent prognostic factor in hepatocellular carcinoma. Its biological functions in malignancies remained unknown. The present study aimed at investigating its biological functions, molecular mechanisms and prognostic values in pancreatic ductal adenocarcinoma (PDAC). Overexpression of EYA4 in PDAC cells inhibited proliferation and invasion in vitro and tumor growth in vivo. Depletion of EYA4 in PDAC cells enhanced proliferation and invasion in vitro and tumor growth in vivo. Mechanistically, armed with the serine/threonine-specific protein phosphatase activity, EYA4 dephosphorylated ß-catenin at Ser675, blocked ß-catenin nuclear translocation and inhibited ID2 transactivation. Consistently, EYA4 expression inversely correlated with the levels of p-Ser675-ß-catenin and ID2 in tissues. EYA4 expression in PDAC tissues was significantly reduced as compared with adjacent non-tumoral tissues. EYA4 expression was an independent prognostic factor in PDAC, with a lower EYA4 level in association with shorter long-term survival and disease-free time. We showed that EYA4 functioned as tumor suppressor gene in PDAC via repressing ß-catenin/ID2 activation, and was an independent prognostic factor in PDAC.