Endoplasmic reticulum stress and type 2 diabetes.

Given the functional importance of the endoplasmic reticulum (ER), an organelle that performs folding, modification, and trafficking of secretory and membrane proteins to the Golgi compartment, the maintenance of ER homeostasis in insulin-secreting ß-cells is very important. When ER homeostasis is disrupted, the ER generates adaptive signaling pathways, called the unfolded protein response (UPR), to maintain homeostasis of this organelle. However, if homeostasis fails to be restored, the ER initiates death signaling pathways. New observations suggest that both chronic hyperglycemia and hyperlipidemia, known as important causative factors of type 2 diabetes (T2D), disrupt ER homeostasis to induce unresolvable UPR activation and ß-cell death. This review examines how the UPR pathways, induced by high glucose and free fatty acids (FFAs), interact to disrupt ER function and cause ß-cell dysfunction and death.

Sphingomyelin synthase 1 mediates hepatocyte pyroptosis to trigger non-alcoholic steatohepatitis.

OBJECTIVE: Lipotoxic hepatocyte injury is a primary event in non-alcoholic steatohepatitis (NASH), but the mechanisms of lipotoxicity are not fully defined. Sphingolipids and free cholesterol (FC) mediate hepatocyte injury, but their link in NASH has not been explored. We examined the role of free cholesterol and sphingomyelin synthases (SMSs) that generate sphingomyelin (SM) and diacylglycerol (DAG) in hepatocyte pyroptosis, a specific form of programmed cell death associated with inflammasome activation, and NASH. DESIGN: Wild-type C57BL/6J mice were fed a high fat and high cholesterol diet (HFHCD) to induce NASH. Hepatic SMS1 and SMS2 expressions were examined in various mouse models including HFHCD-fed mice and patients with NASH. Pyroptosis was estimated by the generation of the gasdermin-D N-terminal fragment. NASH susceptibility and pyroptosis were examined following knockdown of SMS1, protein kinase Cδ (PKCδ), or the NLR family CARD domain-containing protein 4 (NLRC4). RESULTS: HFHCD increased the hepatic levels of SM and DAG while decreasing the level of phosphatidylcholine. Hepatic expression of Sms1 but not Sms2 was higher in mouse models and patients with NASH. FC in hepatocytes induced Sms1 expression, and Sms1 knockdown prevented HFHCD-induced NASH. DAG produced by SMS1 activated PKCδ and NLRC4 inflammasome to induce hepatocyte pyroptosis. Depletion of Nlrc4 prevented hepatocyte pyroptosis and the development of NASH. Conditioned media from pyroptotic hepatocytes activated the NOD-like receptor family pyrin domain containing 3 inflammasome (NLRP3) in Kupffer cells, but Nlrp3 knockout mice were not protected against HFHCD-induced hepatocyte pyroptosis. CONCLUSION: SMS1 mediates hepatocyte pyroptosis through a novel DAG-PKCδ-NLRC4 axis and holds promise as a therapeutic target for NASH.

Establishment of a reporter system for monitoring activation of the ER stress transducer ATF6ß.

ATF6 has two isoforms, ATF6α and ATF6ß, which are ubiquitously expressed type II transmembrane glycoproteins in the endoplasmic reticulum (ER). While the regulatory mechanisms and transcriptional roles of ATF6α in response to ER stress have been well-studied, those of its paralogue ATF6ß are less understood. Moreover, there is no specific cell-based reporter assay to monitor ATF6ß activation. Here, we developed a new cell-based reporter system that can monitor activation of endogenous ATF6ß. This system expresses a chimeric protein containing a synthetic transcription factor followed by the transmembrane domain and C-terminal luminal domain of ATF6ß. Under ER stress conditions, the chimeric protein was cleaved by regulated intramembrane proteolysis (RIP) to liberate the N-terminal synthetic transcription factor, which induced luciferase expression in the HeLa Luciferase Reporter cell line. This new stable reporter cell line will be an innovative tool to investigate RIP of ATF6ß.

Translation Inhibitors Activate Autophagy Master Regulators TFEB and TFE3.

The autophagy-lysosome pathway is a major protein degradation pathway stimulated by multiple cellular stresses, including nutrient or growth factor deprivation, hypoxia, misfolded proteins, damaged organelles, and intracellular pathogens. Recent studies have revealed that transcription factor EB (TFEB) and transcription factor E3 (TFE3) play a pivotal role in the biogenesis and functions of autophagosome and lysosome. Here we report that three translation inhibitors (cycloheximide, lactimidomycin, and rocaglamide A) can facilitate the nuclear translocation of TFEB/TFE3 via dephosphorylation and 14-3-3 dissociation. In addition, the inhibitor-mediated TFEB/TFE3 nuclear translocation significantly increases the transcriptional expression of their downstream genes involved in the biogenesis and function of autophagosome and lysosome. Furthermore, we demonstrated that translation inhibition increased autophagosome biogenesis but impaired the degradative autolysosome formation because of lysosomal dysfunction. These results highlight the previously unrecognized function of the translation inhibitors as activators of TFEB/TFE3, suggesting a novel biological role of translation inhibition in autophagy regulation.

Roles of the Translation Initiation Factor eIF2α Phosphorylation in Cell Structure and Function.

It is often assumed that α-subunit phosphorylation of the eukaryotic translation initiation factor 2 (eIF2) complex is just a mechanism to control protein synthesis. However, eIF2α phosphorylation induced by multiple kinases can recognize various intracellular and extracellular stress conditions, and it is involved in various other cellular processes beyond protein synthesis. This review introduces the roles of eIF2α phosphorylation in translational regulation, the generation of reactive oxygen species, changes in mitochondria structure and shape, and mitochondrial retrograde signaling pathways in response to diverse stress conditions.Key words: eIF2α phosphorylation, Translation, Unfolded Protein Response, Reactive Oxygen Species, Mitochondria.

Quantitative structural characterization of phosphatidylinositol phosphates from biological samples.

The aspects of cellular metabolism controlled by phosphatidylinositol phosphates (PtdInsPs) have been broadly expanded, and these phospholipids have drawn tremendous attention as pleiotropic signaling molecules. PtdInsPs analysis using LC/MS/MS has remained challenging due to the strong hydrophilicity of these lipids. Multiple reaction monitoring (MRM) or a neutral loss scan has been performed to quantitatively measure PtdInsPs after chemical derivatization on the phosphate groups of inositol moieties. Only predefined PtdInsPs can be measured in MRM mode, and fatty acyl compositions of sn-1 and sn-2 positions of PtdInsPs cannot be obtained from a neutral loss scan. In our present study, we developed a simple LC/MS/MS method for structural identification of sn-1 and sn-2 fatty acids of PtdInsPs and their relative quantitation. Precursor ion scans of sn-1 monoacylglycerols (MAGs) of PtdInsPs provided structural information about the lipids, and ammonium adduction enhanced signal intensities of PtdInsPs. The relative amount of observed PtdInsPs in biological samples could be compared using chromatographic peak areas from the neutral loss scans. Using precursor ion scans of sn-1 MAG and neutral loss scans of headgroups, major PtdInsPs in cells and tissues were successfully identified with structural information of sn-1 and sn-2 fatty acids, and their relative amounts in different samples were compared.

The novel resveratrol derivative 3,5-diethoxy-3',4'-dihydroxy-trans-stilbene induces mitochondrial ROS-mediated ER stress and cell death in human hepatoma cells in vitro.

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a well-known polyphenol that is present in grapes, peanuts, pine seeds, and several other plants. Resveratrol exerts deleterious effects on various types of human cancer cells. Here, we analyzed the cell death-inducing mechanisms of resveratrol-006 (Res-006), a novel resveratrol derivative in human liver cancer cells in vitro. Res-006 was more effectively suppressed the viability of HepG2 human hepatoma cells than resveratrol (the IC50 values were 67.2 and 354.8 µmol/L, respectively). Co-treatment with the ER stress regulator 4-phenylbutyrate (0.5 mmol/L) or the ROS inhibitor N-acetyl-L-cysteine (NAC, 1 mmol/L) significantly attenuated Res-006-induced HepG2 cell death, suggesting that pro-apoptotic ER stress and/or ROS may govern the Res-006-induced HepG2 cell death. We further revealed that treatment of HepG2 cells with Res-006 (65 µmol/L) immediately elicited the dysregulation of mitochondrial dynamics and the accumulation of mitochondrial ROS. It also collapsed the mitochondrial membrane potential and further induced ER stress and cell death. These events, except for the change in mitochondrial morphology, were prevented by the exposure of the HepG2 cells to the mitochondrial ROS scavenger, Mito-TEMPO (300-1000 µmol/L). The results suggest that Res-006 may kill HepG2 cells through cell death pathways, including the ER stress initiated by mitochondrial ROS accumulation. The cell death induced by this novel resveratrol derivative involves crosstalk between the mitochondria and ER stress mechanisms.

Fibroblast growth factor 21 participates in adaptation to endoplasmic reticulum stress and attenuates obesity-induced hepatic metabolic stress.

AIMS/HYPOTHESIS: Fibroblast growth factor 21 (FGF21) is an endocrine hormone that exhibits anti-diabetic and anti-obesity activity. FGF21 expression is increased in patients with and mouse models of obesity or nonalcoholic fatty liver disease (NAFLD). However, the functional role and molecular mechanism of FGF21 induction in obesity or NAFLD are not clear. As endoplasmic reticulum (ER) stress is triggered in obesity and NAFLD, we investigated whether ER stress affects FGF21 expression or whether FGF21 induction acts as a mechanism of the unfolded protein response (UPR) adaptation to ER stress induced by chemical stressors or obesity. METHODS: Hepatocytes or mouse embryonic fibroblasts deficient in UPR signalling pathways and liver-specific eIF2α mutant mice were employed to investigate the in vitro and in vivo effects of ER stress on FGF21 expression, respectively. The in vivo importance of FGF21 induction by ER stress and obesity was determined using inducible Fgf21-transgenic mice and Fgf21-null mice with or without leptin deficiency. RESULTS: We found that ER stressors induced FGF21 expression, which was dependent on a PKR-like ER kinase-eukaryotic translation factor 2α-activating transcription factor 4 pathway both in vitro and in vivo. Fgf21-null mice exhibited increased expression of ER stress marker genes and augmented hepatic lipid accumulation after tunicamycin treatment. However, these changes were attenuated in inducible Fgf21-transgenic mice. We also observed that Fgf21-null mice with leptin deficiency displayed increased hepatic ER stress response and liver injury, accompanied by deteriorated metabolic variables. CONCLUSIONS/INTERPRETATION: Our results suggest that FGF21 plays an important role in the adaptive response to ER stress- or obesity-induced hepatic metabolic stress.

The unfolded protein response transducer IRE1α prevents ER stress-induced hepatic steatosis.

The endoplasmic reticulum (ER) is the cellular organelle responsible for protein folding and assembly, lipid and sterol biosynthesis, and calcium storage. The unfolded protein response (UPR) is an adaptive intracellular stress response to accumulation of unfolded or misfolded proteins in the ER. In this study, we show that the most conserved UPR sensor inositol-requiring enzyme 1 α (IRE1α), an ER transmembrane protein kinase/endoribonuclease, is required to maintain hepatic lipid homeostasis under ER stress conditions through repressing hepatic lipid accumulation and maintaining lipoprotein secretion. To elucidate physiological roles of IRE1α-mediated signalling in the liver, we generated hepatocyte-specific Ire1α-null mice by utilizing an albumin promoter-controlled Cre recombinase-mediated deletion. Deletion of Ire1α caused defective induction of genes encoding functions in ER-to-Golgi protein transport, oxidative protein folding, and ER-associated degradation (ERAD) of misfolded proteins, and led to selective induction of pro-apoptotic UPR trans-activators. We show that IRE1α is required to maintain the secretion efficiency of selective proteins. In the absence of ER stress, mice with hepatocyte-specific Ire1α deletion displayed modest hepatosteatosis that became profound after induction of ER stress. Further investigation revealed that IRE1α represses expression of key metabolic transcriptional regulators, including CCAAT/enhancer-binding protein (C/EBP) ß, C/EBPδ, peroxisome proliferator-activated receptor γ (PPARγ), and enzymes involved in triglyceride biosynthesis. IRE1α was also found to be required for efficient secretion of apolipoproteins upon disruption of ER homeostasis. Consistent with a role for IRE1α in preventing intracellular lipid accumulation, mice with hepatocyte-specific deletion of Ire1α developed severe hepatic steatosis after treatment with an ER stress-inducing anti-cancer drug Bortezomib, upon expression of a misfolding-prone human blood clotting factor VIII, or after partial hepatectomy. The identification of IRE1α as a key regulator to prevent hepatic steatosis provides novel insights into ER stress mechanisms in fatty liver diseases associated with toxic liver injuries.

Transcriptional cross talk between orphan nuclear receptor ERRγ and transmembrane transcription factor ATF6α coordinates endoplasmic reticulum stress response.

Orphan nuclear receptor ERRγ is a member of nuclear receptor superfamily that regulates several important cellular processes including hepatic glucose and alcohol metabolism. However, mechanistic understanding of transcriptional regulation of the ERRγ gene remains to be elucidated. Here, we report that activating transcription factor 6α (ATF6α), an endoplasmic reticulum (ER)-membrane-bound basic leucine zipper (bZip) transcription factor, directly regulates ERRγ gene expression in response to ER stress. ATF6α binds to ATF6α responsive element in the ERRγ promoter. The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) is required for this transactivation. Chromatin immunoprecipitation (ChIP) assay confirmed the binding of both ATF6α and PGC1α on the ERRγ promoter. ChIP assay demonstrated histone H3 and H4 acetylation occurs at the ATF6α and PGC1α binding site. Of interest, ERRγ along with PGC1α induce ATF6α gene transcription upon ER stress. ERRγ binds to an ERRγ responsive element in the ATF6α promoter. ChIP assay confirmed that both ERRγ and PGC1α bind to a site in the ATF6α promoter that exhibits histone H3 and H4 acetylation. Overall, for the first time our data show a novel pathway of cross talk between nuclear receptors and ER-membrane-bound transcription factors and suggest a positive feed-forward loop regulates ERRγ and ATF6α gene transcription.

Endoplasmic reticulum stress induces hepatic steatosis via increased expression of the hepatic very low-density lipoprotein receptor.

UNLABELLED: Recent evidence suggests that obese animals exhibit increased endoplasmic reticulum (ER) stress in the liver and adipose tissue. Although ER stress is closely associated with lipid homeostasis, it is largely unknown how ER stress contributes to hepatic steatosis. In this study, we demonstrate that the induction of ER stress stimulates hepatic steatosis through increased expression of the hepatic very low-density lipoprotein receptor (VLDLR). Among the unfolded protein response sensors, the protein kinase RNA-like ER kinase-activating transcription factor 4 signaling pathway was required for hepatic VLDLR up-regulation. In primary hepatocytes, ER stress-dependent VLDLR expression induced intracellular triglyceride accumulation in the presence of very low-density lipoprotein. Moreover, ER stress-dependent hepatic steatosis was diminished in the livers of VLDLR-deficient and apolipoprotein E-deficient mice compared with wild-type mice. In addition, the VLDLR-deficient mice exhibited decreased hepatic steatosis upon high-fat diet feeding. CONCLUSION: These data suggest that ER stress-dependent expression of hepatic VLDLR leads to hepatic steatosis by increasing lipoprotein delivery to the liver, which might be a novel mechanism explaining ER stress-induced hepatic steatosis.

Quantitative determination of major platelet activating factors from human plasma.

Platelet activating factor (PAF) is a potent lipid mediator that is involved in many important biological functions, including platelet aggregation and neuronal differentiation. Although an ELISA assay has been used to measure PAF levels, it cannot distinguish between its isoforms. To achieve this, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been used instead. However, isobaric lysophosphatidylcholine (lyso PC), which is often present in large amounts in complex biological samples and has similar retention times in many LC conditions, can affect the accurate measurement of PAF. The present study examined the fragmentation behavior of major PAF and lyso PC during various MS/MS conditions. Fragment ions at m/z 184 and at m/z 104 were abundantly observed from MS/MS of lyso PCs. PAF provided a dominant fragment ion at m/z 184, but a fragment ion at m/z 104 was almost never produced, regardless of the collision energy. Thus, the two fragment ions at m/z 184 and m/z 104 were used to accurately measure PAF levels. First, the fragment ion at m/z 184 and the retention time of PAF in LC-MS/MS were used to identify and quantitate PAF. However, if there were small retention time shifts, which are common in multiple sample runs, and lipid composition in a sample is very complicated, the fragment ion at m/z 104 was used to confirm whether the fragment ion at m/z 184 belonged to PAF. This novel method accurately determined the major PAF (C16:0 PAF, C18:0 PAF, and C18:1 PAF) levels in human plasma.

Sensing endoplasmic reticulum stress by protein kinase RNA-like endoplasmic reticulum kinase promotes adaptive mitochondrial DNA biogenesis and cell survival via heme oxygenase-1/carbon monoxide activity.

Endoplasmic reticulum (ER) stress activates the adaptive unfolded protein response, allowing cells to recover folding capacity in the organelle. However, the overwhelming response to severe damage results in apoptotic cell death. Because of the physical proximity between ER and mitochondria, a functional interrelationship between these two organelles, including mitochondrial ATP production and apoptosis, has been suggested. The adaptive response to ER stress includes the maintenance of cellular energetics, which eventually determines cell fate. We previously demonstrated that heme oxygenase-1 (HO-1) activity protects cells against ER stress in a protein kinase RNA-like endoplasmic reticulum kinase (PERK)-dependent pathway. Here, we provide evidence that PERK-mediated induction of HO-1 in murine macrophages, RAW264.7, relays ER stress to mitochondrial DNA (mtDNA) replication and function. ER stress induced by thapsigargin treatments (10-100 nM) resulted in a 2-fold increase in mtDNA contents compared with that in the untreated control. HO-1 activity on ER stress is proven to be critical for mitochondrial integrity because chemical inhibition (zinc protoporphyrin, 5-20 µM) and genetic depletion of HO-1 by small interference RNA transfection suppress the activation of transcription factors for mitochondrial biogenesis. Carbon monoxide (CO), an enzymatic by-product of HO-1 activity is responsible for the function of HO-1. Limited bioavailability of CO by hemoglobin treatment triggers cell death with a concomitant decline in ATP production. Approximately 78.1% of RAW264.7 cells were damaged in the presence of hemoglobin compared with the percentage of injured cells (26.9%) under ER stress alone. Mitochondrial generation of ATP levels significantly declined when CO availability was limited under prolonged ER stress. Taken together, these results suggest that the cellular HO-1/CO system conveys ER stress to cell survival signals from mitochondria via both the activation of transcriptional factors and functional integrity of mtDNA.

Double-layered blood vessels over 3 mm in diameter extruded by the inverse-gravity technique.

One of the most promising techniques for treating severe peripheral artery disease is the use of cellular tissue-engineered vascular grafts (TEVGs). This study proposes an inverse-gravity (IG) extrusion technique for creating long double-layered cellular TEVGs with diameters over 3 mm. A three-layered coaxial laminar hydrogel flow in an 8 mm-diameter pipe was realised simply by changing the extrusion direction of the hydrogel from being aligned with the direction of gravity to against it. This technique produced an extruded mixture of human aortic smooth muscle cells (HASMCs) and type-I collagen as a tubular structure with an inner diameter of 3.5 mm. After a 21 day maturation period, the maximal burst pressure, longitudinal breaking force, and circumferential breaking force of the HASMC TEVG were 416 mmHg, 0.69 N, and 0.89 N, respectively. The HASMC TEVG was endothelialised with human umbilical vein endothelial cells to form a tunica intima that simulated human vessels. Besides subcutaneous implantability on mice, the double-layered blood vessels showed a considerably lower adherence of platelets and red blood cells once exposed to heparinised mouse blood and were considered nonhaemolytic. The proposed IG extrusion technique can be applied in various fields requiring multilayered materials with large diameters.

Bridging Energy Need and Feeding Behavior: The Impact of eIF2α Phosphorylation in AgRP Neurons.

Eukaryotic translation initiation factor 2α (eIF2α) is a key mediator of the endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR). In mammals, eIF2α is phosphorylated by overnutrition-induced ER stress and is related to the development of obesity. Here, we studied the function of phosphorylated eIF2α (p-eIF2α) in agouti-related peptide (AgRP) neurons using a mouse model (AgRPeIF2αA/A) with an AgRP neuron-specific substitution from Ser 51 to Ala in eIF2α, which impairs eIF2α phosphorylation in AgRP neurons. These AgRPeIF2αA/A mice had decreases in starvation-induced AgRP neuronal activity and food intake and an increased responsiveness to leptin. Intriguingly, impairment of eIF2α phosphorylation produced decreases in the starvation-induced expression of UPR and autophagy genes in AgRP neurons. Collectively, these findings suggest that eIF2α phosphorylation regulates AgRP neuronal activity by affecting intracellular responses such as the UPR and autophagy during starvation, thereby participating in the homeostatic control of whole-body energy metabolism. ARTICLE HIGHLIGHTS: This study examines the impact of eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, triggered by an energy deficit, on hypothalamic AgRP neurons and its subsequent influence on whole-body energy homeostasis. Impaired eIF2α phosphorylation diminishes the unfolded protein response and autophagy, both of which are crucial for energy deficit-induced activation of AgRP neurons. This study highlights the significance of eIF2α phosphorylation as a cellular marker indicating the availability of energy in AgRP neurons and as a molecular switch that regulates homeostatic feeding behavior.

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

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

Curcumin differentially regulates endoplasmic reticulum stress through transcriptional corepressor SMILE (small heterodimer partner-interacting leucine zipper protein)-mediated inhibition of CREBH (cAMP responsive element-binding protein H).

Curcumin (diferuloylmethane), a major active component of turmeric (Curcuma longa), is a natural polyphenolic compound. Herein the effect of curcumin on endoplasmic reticulum (ER) stress responsive gene expression was investigated. We report that curcumin induces transcriptional corepressor small heterodimer partner-interacting leucine zipper protein (SMILE) gene expression through liver kinase B1 (LKB1)/adenosine monophosphate-activated kinase (AMPK) signaling pathway and represses ER stress-responsive gene transcription in an ER-bound transcription factor specific manner. cAMP responsive element-binding protein H (CREBH) and activating transcription factor 6 (ATF6) are both ER-bound bZIP family transcription factors that are activated upon ER stress. Of interest, we observed that both curcumin treatment and SMILE overexpression only represses CREBH-mediated transactivation of the target gene but not ATF6-mediated transactivation. Knockdown of endogenous SMILE significantly releases the inhibitory effect of curcumin on CREBH transactivation. Intrinsic repressive activity of SMILE is observed in the Gal4 fusion system, and the intrinsic repressive domain is mapped to the C terminus of SMILE spanning amino acid residues 203-269, corresponding to the basic region leucine zipper (bZIP) domain. In vivo interaction assay revealed that through its bZIP domain, SMILE interacts with CREBH and inhibits its transcriptional activity. Interestingly, we observed that SMILE does not interact with ATF6. Furthermore, competition between SMILE and the coactivator peroxisome proliferator-activated receptor α (PGC-1α) on CREBH transactivation has been demonstrated in vitro and in vivo. Finally, chromatin immunoprecipitation assays revealed that curcumin decreases the binding of PGC-1α and CREBH on target gene promoter in a SMILE-dependent manner. Overall, for the first time we suggest a novel phenomenon that the curcumin/LKB1/AMPK/SMILE/PGC1α pathway differentially regulates ER stress-mediated gene transcription.

TMBIM6 (transmembrane BAX inhibitor motif containing 6) enhances autophagy through regulation of lysosomal calcium.

Lysosomal Ca2+ contributes to macroautophagy/autophagy, an intracellular process for the degradation of cytoplasmic material and organelles in the lysosomes to protect cells against stress responses. TMBIM6 (transmembrane BAX inhibitor motif containing 6) is a Ca2+ channel-like protein known to regulate ER stress response and apoptosis. In this study, we examined the as yet unknown role of TMBIM6 in regulating lysosomal Ca2+ levels. The Ca2+ efflux from the ER through TMBIM6 was found to increase the resting lysosomal Ca2+ level, in which ITPR-independent regulation of Ca2+ status was observed. Further, TMBIM6 regulated the local release of Ca2+ through lysosomal MCOLN1/TRPML1 channels under nutrient starvation or MTOR inhibition. The local Ca2+ efflux through MCOLN1 channels was found to activate PPP3/calcineurin, triggering TFEB (transcription factor EB) nuclear translocation, autophagy induction, and lysosome biogenesis. Upon genetic inactivation of TMBIM6, lysosomal Ca2+ and the associated TFEB nuclear translocation were decreased. Furthermore, autophagy flux was significantly enhanced in the liver or kidney from starved Tmbim6+/+ mice compared with that in the counter tmbim6-/- mice. Together, our observations indicated that under stress conditions, TMBIM6 increases lysosomal Ca2+ release, leading to PPP3/calcineurin-mediated TFEB activation and subsequently enhanced autophagy. Thus, TMBIM6, an ER membrane protein, is suggested to be a lysosomal Ca2+ modulator that coordinates with autophagy to alleviate metabolism stress.Abbreviations: AVs: autophagic vacuoles; CEPIA: calcium-measuring organelle-entrapped protein indicator; ER: endoplasmic reticulum; GPN: glycyl-L-phenylalanine-beta-naphthylamide; ITPR/IP3R: inositol 1,4,5-trisphosphate receptor; LAMP1: lysosomal associated membrane protein 1; MCOLN/TRPML: mucolipin; MEF: mouse embryonic fibroblast; ML-SA1: mucolipin synthetic agonist 1; MTORC1: mechanistic target of rapamycin kinase complex 1; RPS6KB1: ribosomal protein S6 kinase B1; SQSTM1: sequestosome 1; TFEB: transcription factor EB; TKO: triple knockout; TMBIM6/BI-1: transmembrane BAX inhibitor motif containing 6.

Butyrate Prevents TGF-ß1-Induced Alveolar Myofibroblast Differentiation and Modulates Energy Metabolism.

Idiopathic pulmonary fibrosis (IPF) is a serious lung disease characterized by excessive collagen matrix deposition and extracellular remodeling. Signaling pathways mediated by fibrotic cytokine transforming growth factor ß1 (TGF-ß1) make important contributions to pulmonary fibrosis, but it remains unclear how TGF-ß1 alters metabolism and modulates the activation and differentiation of pulmonary fibroblasts. We found that TGF-ß1 lowers NADH and NADH/NAD levels, possibly due to changes in the TCA cycle, resulting in reductions in the ATP level and oxidative phosphorylation in pulmonary fibroblasts. In addition, we showed that butyrate (C4), a short chain fatty acid (SCFA), exhibits potent antifibrotic activity by inhibiting expression of fibrosis markers. Butyrate treatment inhibited mitochondrial elongation in TGF-ß1-treated lung fibroblasts and increased the mitochondrial membrane potential (MMP). Consistent with the mitochondrial observations, butyrate significantly increased ADP, ATP, NADH, and NADH/NAD levels in TGF-ß1-treated pulmonary fibroblasts. Collectively, our findings indicate that TGF-ß1 induces changes in mitochondrial dynamics and energy metabolism during myofibroblast differentiation, and that these changes can be modulated by butyrate, which enhances mitochondrial function.

Coaxial printing of double-layered and free-standing blood vessel analogues without ultraviolet illumination for high-volume vascularised tissue.

Human umbilical vein endothelial cells (HUVECs) and human aortic smooth muscle cells (HASMCs) were coaxially and continuously extruded without ultraviolet illumination using a microfluidic-based nozzle. Type I collagen (3 mg ml-1) containing HUVECs and a crosslinking reagent (100 mM CaCl2) were supplied as the core material. A mixture of 3 mg ml-1 of type I collagen (25%) and 1.8% weight volume-1 of sodium alginate (75%) was provided as the shell layer material surrounding the core material. The HUVECs were well proliferated at the core and reshaped into a monolayer formation along the axial direction of the scaffold. The HASMCs showed more than 90% cell viability in the shell layer. Fluorescent beads were passed through the inside channel of the scaffold with the HUVEC core and HASMC shell using an in-house connector. This double-layered scaffold showed higher angiogenesis in growth factor-free medium than the scaffold with only a HUVEC core. The HASMCs in the shell layer affected angiogenesis, extracellular matrix secretion, and outer diameter. The proposed technique could be applied to three-dimensional bioprinting for the production of high-volume vascularised tissue.