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The potential protective effect of basic fibroblast growth factor (BFGF) on the cardiovascular system has been proposed previously, however, its effect on calcific aortic valve disease (CAVD) and underlying mechanisms have not been elucidated. The valvular interstitial cell (VIC) were isolated from porcine aortic valve leaflets. To investigate the effect of BFGF on osteogenic differentiation of VIC, the osteogenic induced medium (OIM) and BFGF were added. The protein expression level was detected by Western blot, and apoptosis was determined by flow cytometry. The effect of BFGF on CAVD process in vivo was assessed by a rat CAVD model, which was identified by echocardiography and Alizarin red staining. The expression level of BFGF in the aortic valve and serum were significantly upregulated in CAVD patients compared to control group. In addition, exogenous BFGF injection attenuates CAVD process in vivo. The protein markers of osteogenic differentiation, endoplasmic reticulum stress (ERS), and apoptosis were significantly upregulated by culture with OIM. On the contrary, the aforementioned proteins were suppressed after adding 100 ng/mL of BFGF. Inhibition of PI3K/Akt and ERK1/2 pathways by specific inhibitors abolished the protective effect of BFGF. In conclusion, BFGF could alleviate the VIC calcification by inhibiting ERS-mediated apoptosis, which is partly regulated by activation of the PI3K/Akt and ERK1/2 signaling pathways. BFGF may provide a potential avenue for CAVD therapy. [ABSTRACT FROM AUTHOR]

Organelles are dynamic entities whose functions are essential for the optimum functioning of cells. It is now known that the juxtaposition of organellar membranes is essential for the exchange of metabolites and their communication. These functional apposition sites are termed membrane contact sites. Dynamic membrane contact sites between various sub-cellular structures such as mitochondria, endoplasmic reticulum, peroxisomes, Golgi apparatus, lysosomes, lipid droplets, plasma membrane, endosomes, etc. have been reported in various model systems. The burgeoning area of research on membrane contact sites has witnessed several manuscripts in recent years that identified the contact sites and components involved. Several methods have been developed to identify, measure and analyze the membrane contact sites. In this manuscript, we aim to discuss important methods developed to date that are used to study membrane contact sites. • Organelles communicate with their surrounding organelles via regions of close apposition called membrane contact sites. • Membrane contact sites are dynamic in nature and may have unique protein and lipid composition. • Their role in optimum functioning of cells and in human disease conditions is now established. • Modern microscopy methods combined with proximity dependent assays have significantly enhanced our understanding of various membrane contact sites. [ABSTRACT FROM AUTHOR]

In this study, we investigated the effect of doxorubicin and tunicamycin treatment alone or in combination on MDM-, Cul9-and prion protein (PrP)-mediated subcellular regulation of p53 in the context of apoptosis and autophagy. MTT analysis was performed to determine the cytotoxic effect of the agents. Apoptosis was monitorized by ELISA, flow cytometry and JC-1 assay. Monodansylcadaverine assay was performed for autophagy. Western blotting and immunofluorescence were performed to determine p53, MDM2, CUL9 and PrP levels. Doxorubicin increased p53, MDM2 and CUL9 levels in a dose-dependent manner. Expression of p53 and MDM2 was higher at the 0.25 μM concentration of tunicamycin compared to the control, but it decreased at 0.5 μM and 1 μM concentrations. CUL9 expression was significantly decreased only after treatment of tunicamycin at 0.25 μM. According to its glycosylation status, the upper band of PrP increased only in combination treatment. In combination treatment, p53 expression was higher than control, whereas MDM2 and CUL9 expressions were decreased. Combination treatments may make MCF-7 cells more susceptible to apoptosis rather than autophagy. In conclusion, PrP may be important in determining the fate of cell death through crosstalk between proteins such as p53 and MDM2 under endoplasmic reticulum (ER) stress conditions. Further studies are needed to obtain in-depth information on these potential molecular networks. • p53 accumulation in blebs and nuclei induced by doxorubicin treatment. • Tunicamycin induced endoplasmic reticulum stress causes p53 accumulation only in blebs. • Doxorubicin treatment induces MDM2 expression only in apoptotic cells. • CUL9 moderately decreased by doxorubicin and tunicamycin treatments. • Doxorubicin decrease PrP level in a dose dependent manner. [ABSTRACT FROM AUTHOR]

Abstract Oxidized low-density lipoprotein (ox-LDL)-induced endothelial cell (EC) apoptosis is the initial step of atherogenesis and associated with Ca2+ overload. Mitochondria-associated endoplasmic reticulum (ER) membrane (MAM), regulated by tethering proteins such as phosphofurin acidic cluster sorting protein 2 (PACS2), is essential for mitochondrial Ca2+ overload by mediating ER-mitochondria Ca2+ transfer. In our study, we aimed to investigate the role of PACS2 in ox-LDL-induced apoptosis in human umbilical vein endothelial cells (HUVECs) and the underlying mechanisms. Ox-LDL dose- and time-dependently increased cell apoptosis concomitant with mitochondrial Ca2+ elevation, mitochondrial membrane potential (MMP) loss, reactive oxygen species (ROS) production, and cytochrome c release. Silencing PACS2 significantly inhibited ox-LDL-induced cell apoptosis at 24 h in addition to the effects of ox-LDL on mitochondrial Ca2+, MMP, and ROS at 2 h. Besides, ox-LDL promoted PACS2 localization at mitochondria as well as ER-mitochondria contacts at 2 h. Not only that, ox-LDL upregulated PACS2 expression at 24 h. Furthermore, silencing PACS2 inhibited ox-LDL-induced mitochondrial localization of PACS2 and MAM formation at 24 h. Altogether, our findings suggest that PACS2 plays an important role in ox-LDL-induced EC apoptosis by regulating MAM formation and mitochondrial Ca2+ elevation, implicating that PACS2 may be a promising therapeutic target for atherosclerosis. Graphical abstract Schematic illustration of how PACS2 mediates ox-LDL-increased ER-mitochondria Ca 2+ transfer and subsequent EC apoptosis. In resting ECs, there are a few ER-mitochondria contacts (MAMs) mediating ER-mitochondria Ca2+ transfer. However, ox-LDL significantly increases mitochondrial localization of PACS2, leading to elevated MAM formation to facilitate ER-mitochondria Ca2+ transfer. Subsequently, ox-LDL-induced mitochondrial Ca2+ elevation caused MMP loss and ROS production, resulting in cytochrome c release and EC apoptosis. Image 1 Highlights • PACS2 is required for oxidized low-density lipoprotein (oxLDL)-induced endothelial cell apoptosis. • Mitochondrial Ca2+ elevation is critical for oxLDL-induced mitochondrial dysfunction and endothelial cell apoptosis. • PACS2 regulates mitochondrial Ca2+ elevation by promoting MAM formation following oxLDL treatments. [ABSTRACT FROM AUTHOR]

Intracellular protein crystallization occurs under various physiological and pathological settings, yet the underlying cellular processes remain enigmatic. After validating individual crystallization events using cellular proteins that readily crystallize in the ER (NEU1), cytosol (crystallin-γD mutant) or nucleus (CLC protein), I demonstrate three independent crystallization events can take place concurrently in different subcellular compartments of a single cell without compromising cell viability. By co-expressing NEU1 and previously reported two human monoclonal antibodies that undergo crystallization and liquid-liquid phase separation in the ER, I additionally demonstrate two independent phase separation events can be simultaneously induced in the ER lumen of a single cell without mixing or interfering each other's phase separation behaviors. Intracellular protein crystallization thus takes place in a crowded physiological cellular environment and does not require high protein purity. Furthermore, I report a simple method to increase the yield of intracellular protein crystals by treating the cells with a topoisomerase II inhibitor that blocks cell division without preventing cell size growth. This study not only presents accessible model tools for studying cryptic in vivo protein crystallization events, but also paves a way toward establishing the intracellular protein crystallization as a novel platform for recombinant protein expression and purification. • Two protein phase separation events can occur simultaneously in the same compartment. • Three independent protein crystallization events can take place concurrently in a single cell. • Multiple intracellular crystal formation proceeds without interfering each other's events. • Intracellular protein crystallization does not require high protein purity. • Cell enlargement is accompanied by marked intracellular crystal growth in size or number. [ABSTRACT FROM AUTHOR]

Abstract Protein kinase RNA-like Endoplasmic Reticulum Kinase (PERK) is an endoplasmic reticulum stress sensor that possesses pro-survival capability and contributes to cell homeostasis and survival. Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) has been recognized as a stem cell marker in intestinal epithelial cells. To determine whether PERK modulates the proliferation of intestinal stem cells, we investigated the effects of PERK knock-down on intestinal Lgr5-positive stem cells in mice. Lgr5-EGFP knock-in mice were fed with lentivirus-PERK shRNA twice a day for three days. Isolated intestinal Lgr5-positive stem cells were treated with lentivirus-PERK shRNA. The number of Lgr5-positive cells, the proliferation and apoptotic indices, several biomarkers for proliferation and differentiation, and Akt expression in intestinal stem cells were detected in vivo, in vitro and in two intestinal epithelial injury models caused by radiotherapy and sepsis. PERK knock-down could significantly diminish the number and proliferation of Lgr5-positive cells, induce the low expression of several proliferation markers and the high expression of several differentiation markers in Lgr5-positive cells, enhance the apoptotic Lgr5-positive cells, and reduce the Akt expression in intestinal Lgr5-positive stem cells. Similar results were observed in radiotherapy- and sepsis-induced intestinal injuries. Moreover, PERK inhibition markedly decreased the survival of mice in response to radiation and sepsis. These results suggest a critical role for PERK in the proliferation and survival of intestinal stem cells in mice. [ABSTRACT FROM AUTHOR]

STEAP2 is a member of the Six-Transmembrane Epithelial Antigen of the Prostate (STEAP) protein family that is proposed to function as metalloreductase. While STEAP2 shows a complex subcellular distribution pattern localizing to both secretory and endocytic pathway organelles, how such broad steady-state distribution is maintained is unknown. Similarly, whether STEAP2 undergoes any compartment-specific modulation during intracellular trafficking has not been reported. Leveraging a newly-identified monoclonal antibody that recognizes a conformation-sensitive epitope nested in the second extracellular loop of STEAP2, we demonstrate that the epitope formation was dependent on the cholesterol content of the membrane in which STEAP2 was embedded. Monitoring the STEAP2-dependent internalization of this antibody uncovered STEAP2's rapid internalization from the cell surface and their subsequence trafficking to the Golgi region and endosome-like puncta. Acute inhibition of endocytosis also increased the detectable amount of STEAP2 at the plasma membrane. Collectively, these experiments demonstrate that an intricate balance of membrane flux between the secretory and endocytic pathways underlies the characteristic broad subcellular localization of STEAP2. By using a cell-based assay that detects the metalloreductase functions of cell surface-localizing STEAP4, STEAP2's metalloreductase activities were not detectable, suggesting that its enzymatic function is suppressed at the plasma membrane. The conformational modulation of STEAP2 by the local membrane cholesterol content can therefore serve as a potential mechanism to modulate STEAP2 function in a compartment-restricted manner, by coupling a pre-existing difference in cholesterol content among different cellular membranes to a dynamic trafficking process leading to broad subcellular distribution. [ABSTRACT FROM AUTHOR]

Orosomucoid 1-like protein 3 (ORMDL3) is an asthma candidate gene associated with virus-triggered recurrent wheeze. Stimulator of interferon gene (STING) controls TLR-independent cytosolic responses to viruses. However, the association of STING with ORMDL3 is unclear. Here, we have shown that ORMDL3 expression shows a linear correlation with STING in recurrent wheeze patients. In elucidating the molecular mechanisms of the ORMDL3-STING relationship, we found that STING promoted the transcriptional activity of ORMDL3, which was significantly associated with increased levels of interferon regulatory factor 3 (IRF3) and signal transducer and activator of transcription 6 (STAT6). Further study showed that via activation of TANK binding kinase 1 (TBK1), STING enhanced the phosphorylation and binding of IRF3 and STAT6, which upregulated ORMDL3 by binding to the promoter. Our results showed that STING positively regulated ORMDL3 through the TBK1-IRF3-STAT6 complex. [ABSTRACT FROM AUTHOR]

Oxysterol-binding protein (OSBP)-related proteins (ORPs) are conserved lipid binding proteins found in organisms ranging from yeast to mammals. Recent findings have indicated that these proteins mainly localize to contact sites of 2 different membranous organelles. ORP6, a member of the ORP subfamily III, is one of the least studied ORPs. Using approaches in molecular cell biology, we attempted to study the characteristics of ORP6 and found that ORP6 is abundantly expressed in mouse cultured neurons. Deconvolution microscopy of cultured cerebellar granular cells revealed that ORP6 is localized to the endoplasmic reticulum (ER) and ER–plasma membrane (PM) contact sites, where it co-localized with extended synaptotagmin2 (E-Syt2), a well-known ER–PM contact site marker. E-Syt2 also co-localized with ORP3, another subfamily III member, and ORP5, a subfamily IV member. However, ORP5 does not distribute to the same ER-PM contact sites as subfamily III members. Also, the co-expression of ORP3 but not ORP5 altered the distribution of ORP6 into the processes of cerebellar neurons. Immunoprecipitation demonstrated binding between the intermediate region of ORP6 and ORP3 or ORP6 itself. Additionally, the localization of ORP6 in the PM decreased when co-expressed with the intermediate region of ORP6, in which the pleckstrin homology (PH) domain and OSBP-related ligand binding domain (ORD) are deleted. Over-expression of this intermediate region shifted the location of a phophtidylinositol-4-phosphate (PI4P) marker from the Golgi to the PM. Knockdown of ORP6 resulted in the same shift of the PI4P marker. Collectively, our data suggests that the recruitment of ORP6 to ER–PM contact sites is involved in the turnover of PI4P in cerebellar granular neurons. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER) stress signaling is activated in retinal degeneration disease. Activating transcription factor 4 (ATF4), an important mediator of the unfolded protein response (UPR), is a key element that maintains cell survival and proliferation in hypoxic conditions. Our previous studies showed that a small ribosomal protein L41 (RPL41) inhibits ATF4 by inducing its phosphorylation and degradation. In the present study, the effects of mini-peptide RPL41 on retinal neovascularization (RNV) in oxygen-induced retinopathy (OIR) mice was investigated. We induced OIR in C57BL/6 mice and obtained retinas from normoxia, OIR, OIR control (treated with PBS), and OIR treated (treated with RPL41) mice. Our results showed that ER stress signaling was activated and ATF4 was overexpressed in the retinas of OIR mice. After intravitreal injection of RPL41, the size of RNV and vaso-obliteration, and the number of preretinal neovascular cell nuclei in the retinas of OIR mice were significantly decreased. Western blot analysis and quantitative real-time polymerase chain reaction (qPCR) showed ATF4 and VEGF expression decreased after intravitreal injection of RPL41. Furthermore, the expression levels of inflammatory genes including TNF-α, IL-1β, and IL-6 were significantly decreased compared with the OIR control mice. In conclusion, RPL41 prevented pathologic neovascularization and exerted anti-inflammatory effects by degrading the important ER stress factor ATF4, thus, RPL41 could be a promising therapeutic agent for the treatment of neovascular eye diseases, especially retinopathy of prematurity (ROP). [ABSTRACT FROM AUTHOR]

Joint contracture is a common complication for people with joint immobility that involves fibrosis structural alteration in the joint capsule. Considering that endoplasmic reticulum (ER) stress plays a prominent role in the promotion of tissue fibrosis, we investigated whether the unfolded protein response (UPR) contributes to the fibrotic development in immobilization-induced knee joint contractures. Using a non-traumatic rat knee joint contracture model, twelve female Sprague-Dawley rats received knee joint immobilization for a period of 8 weeks. We found that fibrosis protein markers (type I collagen, α-SMA) and UPR (GRP78, ATF6α, XBP1s) markers were parallelly upregulated in rat primary cultured synovial myofibroblasts. In the same cell types, pre-treatment with an ER stress inhibitor, 4-phenylbutyric acid (4-PBA), not only abrogated cytokine TGFβ1 stimulation but also reduced the protein level of UPR. Additionally, high reactive oxygen species (ROS) generation was detected in synovial myofibroblasts through flow cytometry, as expected. Notably, TGFβ1-induced UPR was significantly reduced through the inhibition of ROS with antioxidants. These data suggest that ER stress act as a pro-fibrotic stimulus through the overexpression of ROS in synovial fibroblasts. Interestingly, immunohistochemical results showed an increase in the UPR protein levels both in human acquired joint contractures capsule tissue and in animal knee joint contracture tissue. Together, our findings suggest that ER stress contributes to synovial myofibroblastic differentiation in joint capsule fibrosis and may also serve as a potential therapeutic target in joint contractures. [ABSTRACT FROM AUTHOR]

ER-localized proteins have been reported function in endoplasmic reticulum, unfolded protein degradation and destruction of misfolded proteins by the ER-associated protein degradation (ERAD) system, but their function in the chemotaxis of macrophage cells remained un-addressed. Here, we showed that ER protein with ubiquitin like domain 1(Herpud1) was upregulated in IL-4-treated M2 macrophage cells and its expression pattern was similar with macrophage polarization markers, such as Arg1, Mrc1 and Fizz1. Inhibition of Herpud1 by using specific target shRNA decreased these marker's expression at mRNA and protein level in IL-4-treated or -untreated M2 macrophage cells. IL-4 treatment promoted M2 macrophage cell migration and polarization, but this promotion was weakened by Herpud1 depletion and we got similar results by inhibition of ER stress response with chemical molecule 4-phenylbutyric acid (4-PBA) in IL-4-treated or untreated-M2 macrophage cells with Herpud1 overexpression. These results indicated that depending on ER-associated protein degradation (ERAD) to help unfolded protein degradation or destruction is not the only function of Herpud1 and acting as a mediator of IL-4 induced macrophage activation and polarization maybe another unrevealed function, elucidating the role of Herpud1-associated M2 macrophage cell polarization and activation are helpful for exploration the function of macrophage cells in immune response. [ABSTRACT FROM AUTHOR]

The unfolded protein response (UPR) is widely activated in cancers. The mammalian UPR encompasses three signaling branches, namely inositol-requiring enzyme-1α (IRE1α), protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) and activating transcription factor 6α (ATF6α). The functional significance of each branch in tumorigenesis is incompletely understood, especially in cancer stem cells (CSCs). Here, we report that inhibition and silencing of the three UPR sensors has differential effects on breast cancer growth and the CSC population. The levels of PERK and ATF6α strongly correlate with the expression of sex determining region Y (SRY)-box 2 (SOX2), a pluripotency regulator, in human breast cancer tissues. UPR activation is also elevated in the CSC-enriched mammospheres. Inhibition of the UPR sensors or excess ER stress markedly reduces the formation and maintenance of mammospheres, suggesting that an appropriate level of UPR activation is critical for the CSC survival. Mechanistically, transcription factors from UPR and pluripotency pathways interact and reciprocally influence each other. A transcription modulator, CCAAT-enhancer-binding protein delta (C/EBPδ), interacts with pluripotency regulator, SOX2, and UPR transcription factors, thus likely serving as a link to coordinate UPR and pluripotency maintenance in CSCs. Our findings demonstrate that UPR is critical for both cancer growth and pluripotency, and highlight the differential role and complexity of the three UPR branches in tumorigenesis. [ABSTRACT FROM AUTHOR]

Insect nephrocytes provide a valuable model for kidney disease, as they are structurally and functionally homologous to mammalian kidney podocytes. They possess an exceptional macromolecular assembly, the nephrocyte diaphragm (ND), which serves as a filtration barrier and helps maintain tissue homeostasis by filtering out wastes and toxic products. However, the elements that maintain nephrocyte architecture and the ND are not understood. We show that Drosophila nephrocytes have a unique cytoplasmic cluster of F-actin, which is maintained by the microtubule cytoskeleton and Rho-GTPases. A balance of Rac1 and Cdc42 activity as well as proper microtubule organization and endoplasmic reticulum structure, are required to position the actin cluster. Further, ND proteins Sns and Duf also localize to this cluster and regulate organization of the actin and microtubule cytoskeleton. Perturbation of any of these inter-dependent components impairs nephrocyte ultrafiltration. Thus cytoskeletal components, Rho-GTPases and ND proteins work in concert to maintain the specialized nephrocyte architecture and function. [ABSTRACT FROM AUTHOR]

Previously, we reported that vitamin A deficiency resulted in the reduction of stearoyl-CoA desaturase 1 (SCD1) and monounsaturated fatty acid (MUFA) levels, which corroborated with attenuation of high fructose-induced hepatic steatosis. Here, we aimed at assessing the effect of vitamin A deficiency on SCD1, MUFA levels and their impact on pancreas’ structure and functions. Male weanling Wistar rats fed one of the four diets, namely control (Con), vitamin A-deficient (VAD), highfructose (HFr) and vitamin A-deficient diet with highfructose (VADHFr) for 16 weeks period. Compared to the control, feeding of VAD diet (alone or with HFr) resulted in pancreatic intra-islet vessel dilation and reduced plasma insulin, glucagon and C-peptide levels, however, glucose levels decreased only in VADHFr group. In line with plasma levels, VAD diet-fed animals displayed lower immunostaining for insulin and glucagon, which corroborated with increased apoptotic staining observed in the islet regions, possibly due to increased cellular stress, as indicated by high immunostaining for endothelial nitric oxide synthase (eNOS) and CCAAT/Enhancer-binding protein homologues protein (CHOP). On the other hand, it significantly decreased the SCD1 protein, which corroborated with reduced MUFA levels, particularly, oleic acid (C18:1), when compared to the control and HFr groups. In conclusion, chronic vitamin A deficiency altered the structure and functions of pancreas by diminishing the islet cells, possibly by inducing cellular stress-mediated apoptosis and decreasing SCD1-mediated oleic acid (C18:1) synthesis. Thus, the data suggest that unlike liver, the reduction in SCD1 and MUFA levels in the pancreas exerts deleterious effects on its functions and perturb the overall cellular metabolism. [ABSTRACT FROM AUTHOR]

The X-box binding protein 1 (XBP1) is a pivotal transcription factor in the endoplasmic reticulum stress response. Our previous studies have proven that XBP1 is involved in vascular endothelial growth factor (VEGF)-mediated endothelial cell (EC) proliferation and angiogenesis. In this study, we used EC monolayer wound healing, tube formation and transwell migration models to explore the role of XBP1 splicing in EC migration. We found that scratching on EC monolayer triggered XBP1 splicing, which was attenuated by the presence of SU5416and LY294002, suggesting that VEGF signalling pathways may be involved. Over-expression of the spliced XBP1 (XBP1s) via Ad- XBP1s gene transfer increased while knockdown of IRE1α or XBP1 by ShRNA lentivirus suppressed EC migration. Over-expression of XBP1s up-regulated the nitric oxide synthase 3 ( NOS3 )mRNA through the 3’UTR-mediated stabilisation and increased eNOS protein translation. Further experiments demonstrated that miR-24 participated in the XBP1s-induced eNOSup-regulation and EC migration. Further co-IP and immunofluorescence staining assays revealed that protein kinase B (Akt), eNOS andXBP1s form a complex, resulting in Akt and eNOS nucleus relocation. These results suggest that XBP1 splicing can regulate eNOS expression and cellular location, leading to EC migration and therefore contributing to wound healing and angiogenesis. [ABSTRACT FROM AUTHOR]

Coronary artery spasm (CAS) is a pathophysiological phenomenon that may cause myocardial infarction and lead to circulatory collapse and death. Aberrant endoplasmic reticulum (ER) stress causes accumulation of misfolding proteins and has been reported to be involved in a variety of vascular diseases. The present study investigated the role of ER stress in the development of CAS and explored the possible molecular mechanisms. Initially, it was found that ER stress markers were elevated in response to drug-induced vascular smooth muscle cells (VSMCs) contraction. Pharmacologic activation of ER stress using Tunicamycin (Tm) persistently induced CAS and significantly promoted Pituitrin-induced CAS in mice as well as in a collagen gel contraction assay. On the contrary, pharmacologic inhibition of ER stress using 4-phenylacetic acid (4-PBA) completely blunted Pituitrin-induced CAS development in mice. Moreover, during the drug-induced VSMCs contraction, expression of ER stress markers were increased in parallel to those of myosin light chain kinase (MLCK) and phosphor-MLC2 (p-MLC2, at Ser19). After inhibiting MLCK activity by using its specific inhibitor ML-7, the ER stress activator Tm failed to activate the MLCK/MLC2 pathway and could neither trigger CAS in mice nor induce VSMCs contraction in vitro . Our results suggested that aberrant ER stress mediated CAS via regulating the MLCK/MLC2 pathway. ER stress activators might be more robust than the common drugs (Pituitrin or acetylcholine) as to induce vasocontraction and thus may serve as potential therapeutics against chronic bleeding, while its inhibitor might be useful for treatment of severe CAS caused by other medication. [ABSTRACT FROM AUTHOR]

Cytoplasmic lipid droplets (CLD) are considered as neutral lipid reservoirs, which protect cells from lipotoxicity. It became clear that these fascinating dynamic organelles play a role not only in energy storage and metabolism, but also in cellular lipid and protein handling, inter-organelle communication, and signaling among diverse functions. Their dysregulation is associated with multiple disorders, including obesity, liver steatosis and cardiovascular diseases. The central aim of this review is to highlight the link between intra-enterocyte CLD dynamics and the formation of chylomicrons, the main intestinal dietary lipid vehicle, after overviewing the morphology, molecular composition, biogenesis and functions of CLD. [ABSTRACT FROM AUTHOR]

Never in mitosis A-related kinase 2A (Nek2A), a centrosomal serine/threonine kinase, is involved in mitotic progression by regulating the centrosome cycle. Particularly, Nek2A is necessary for dissolution of the intercentriole linkage between the duplicated centrosomes prior to mitosis. Nek2A activity roughly parallels its cell cycle-dependent expression levels, but the precise mechanism regulating its activity remains unclear. In this study, we found that γ-taxilin co-localized with Nek2A at the centrosome during interphase and interacted with Nek2A in yeast two-hybrid and pull-down assays and that γ-taxilin regulated centrosome disjunction in a Nek2A-dependent manner. γ-Taxilin depletion increased the number of cells with striking splitting of centrosomes. The precocious splitting of centrosomes induced by γ-taxilin depletion was attenuated by Nek2A depletion, suggesting that γ-taxilin depletion induces the Nek2A-mediated dissolution of the intercentriole linkage between the duplicated centrosomes nevertheless mitosis does not yet begin. Taken together with the result that γ-taxilin protein expression levels were decreased at the onset of mitosis, we propose that γ-taxilin participates in Nek2A-mediated centrosome disjunction as a negative regulator through its interaction with Nek2A. [ABSTRACT FROM AUTHOR]

Blood-brain barrier (BBB) breakdown has been determined to play a critical role in the pathogenesis of Alzheimer's disease (AD). However, the underlying mechanisms of BBB disruption in AD remain unclear. Our previous study suggested that the receptor for advanced glycation end-products (RAGE) functioned as a signal transduction receptor in Aβ 1–42 -induced damage in endothelial cells. In our present study, we revealed that RAGE-mediated endoplasmic reticulum stress (ERS) is essential for Aβ-induced endothelial cell damage. Here, we found that Aβ 1–42 activated ERS by upregulation of Grp78, xbp-1 and CHOP in endothelial cells and that Aβ 1–42 -resulted lesions, including the upregulations of caspase-12 and caspase-3, the augment of bax/bcl-2 ratio, and the downregulations of ZO-1 and Occludin in bEnd.3 cells, were ameliorated by the pretreatment of salubrinal, an ERS inhibitor. Furthermore, the expressions of Grp78, xbp-1 and CHOP induced by Aβ 1–42 were blocked by transfection of RAGE small interfering RNA (siRNA), which indicated that Aβ 1–42 activated ERS in a RAGE-dependent manner. Additionally, bEnd.3 cells transfected with RAGE siRNA showed lower expressions of caspase-12 and caspase-3, decreased bax/bcl-2 ratio, and higher expressions of ZO-1 and Occludin following Aβ1-42 treatment, comparing to control cells. In conclusion, our data demonstrated that Aβ 1–42 induced endothelial cells damage via activation of ERS in a RAGE-dependent manner. [ABSTRACT FROM AUTHOR]

Ca 2+ signaling is essential for bone metabolism. Fluid shear stress (FSS), which can induce a rapid release of calcium from endoplasmic reticulum (ER) to produce calcium transients, plays a significant role in osteoblast proliferation and differentiation. However, it is still unclear of how calcium transients induced by FSS activating a number of downstream signals which subsequently regulate cell functions. In this study, we performed a group of Ca 2+ transients models, which were induced by FSS to investigate the effects of different magnitudes of Ca 2+ transients in osteoblast proliferation. Further, we performed a global proteomic profile of MC3T3-E1 cells in different Ca 2+ transients models stimulated by FSS. GO enrichment and KEGG pathway analysis revealed that the TCA cycle was activated in the proliferating process. The activation of TCA needed mitochondrial Ca 2+ uptake which were influenced by the amplitude of Ca 2+ transients induced by FSS. Our work elucidate that osteoblast proliferation induced by FSS was related to the magnitude of calcium transients, which further activated energetic metabolism signaling pathway. This work revealed further understanding the mechanism of osteoblast proliferation induced by mechanic loading and help us to design new methods for osteoporosis therapy. [ABSTRACT FROM AUTHOR]

Retinal pigment epithelium (RPE) has been implicated as key source of cholesterol-rich deposits at Bruch's membrane (BrM) and in drusen in aging human eye. We have shown that serum-deprivation of confluent RPE cells is associated with upregulation of cholesterol synthesis and accumulation of unesterified cholesterol (UC). Here we investigate the cellular processes involved in this response. We compared the distribution and localization of UC and esterified cholesterol (EC); the age-related macular degeneration (AMD) associated EFEMP1/Fibulin3 (Fib3); and levels of acyl-coenzyme A (CoA): cholesterol acyltransferases (ACAT) ACAT1, ACAT2 and Apolipoprotein B (ApoB) in ARPE-19 cells cultured in serum-supplemented and serum-free media. The results were compared with distributions of these lipids and proteins in human donor eyes with AMD. Serum deprivation of ARPE-19 was associated with increased formation of FM dye-positive membrane vesicles, many of which co-labeled for UC. Additionally, UC colocalized with Fib3 in distinct granules. By day 5, serum-deprived cells grown on transwells secreted Fib3 basally into the matrix. While mRNA and protein levels of ACTA1 were constant over several days of serum-deprivation, ACAT2 levels increased significantly after serum-deprivation, suggesting increased formation of EC. The lower levels of intracellular EC observed under serum-deprivation were associated with increased formation and secretion of ApoB. The responses to serum-deprivation in RPE-derived cells: accumulation and secretion of lipids, lipoproteins, and Fib3 are very similar to patterns seen in human donor eyes with AMD and suggest that this model mimics processes relevant to disease progression. [ABSTRACT FROM AUTHOR]

Diabetic encephalopathy (DE) is one of the most common complications of diabetes. The major pathological variations include neurofibrillary tangles (NFTs), which are caused by tau hyperphosphorylation, and senile plaques (SPs) consisting of amyloid β- protein(Aβ) deposits. In recent years, DE research studies have focused on exploring the activation of the inflammatory signaling pathway in immune cells. Toll-like receptor 9 (TLR9) is well known to regulate the inflammatory reactions in immune processes. During the tau hyperphosphorylation process, TLR9 in microglia plays bidirectional roles. However, no studies have clearly demonstrated the relationship between TLR9 and tau hyperphosphorylation in neurons. Based on our experiments, we found significant increase in TLR9 expression in neurons and an increase in tau hyperphosphorylation in high-glucose media. However, these alterations can be reversed by TLR9 inhibitor. Furthermore, we specifically inhibited the activation of P38mitogenactivated protein kinase(P38MAPK) and found an effective decrease in tau hyperphosphorylation. This effect is likely related to Unc93b1. Meanwhile, High glucose levels can induce neuronal apoptosis via the TLR9 signaling pathway. Our studies are the first to reveal that high glucose can regulate tau hyperphosphorylation and neuronal apoptosis via TLR9-P38MAPK signaling pathway. These findings provide a new method for studying the mechanism underlying DE. [ABSTRACT FROM AUTHOR]

The mitochondria-associated ER membrane (MAM) is a specialized subdomain of ER that physically connects with mitochondria. Although disruption of inter-organellar crosstalk via the MAM impairs cellular homeostasis, its pathological significance in insulin resistance in type 2 diabetes mellitus remains unclear. Here, we reveal the importance of reduced MAM formation in the induction of fatty acid–evoked insulin resistance in hepatocytes. Palmitic acid (PA) repressed insulin-stimulated Akt phosphorylation in HepG2 cells within 12 h. Treatment with an inhibitor of the ER stress response failed to restore PA-mediated suppression of Akt activation. Mitochondrial reactive oxygen species (ROS) production did not increase in PA-treated cells. Even short-term exposure (3 h) to PA reduced the calcium flux from ER to mitochondria, followed by a significant decrease in MAM contact area, suggesting that PA suppressed the functional interaction between ER and mitochondria. Forced expression of mitofusin-2, a critical component of the MAM, partially restored MAM contact area and ameliorated the PA-elicited suppression of insulin sensitivity with Ser473 phosphorylation of Akt selectively improved. These results suggest that loss of proximity between ER and mitochondria, but not perturbation of homeostasis in the two organelles individually, plays crucial roles in PA-evoked Akt inactivation in hepatic insulin resistance. [ABSTRACT FROM AUTHOR]

Introduction Pancreatic pathologies are characterized by a progressive fibrosis process. Pancreatic stellate cells (PSC) play a crucial role in pancreatic fibrogenesis. Endoplasmic reticulum (ER) stress emerges as an important determinant of fibrotic remodeling. Overload of fatty acids (FA), typical to obesity, may lead to lipotoxic state and cellular stress. Aim To study the effect of different lipolytic challenges on pancreatic ER stress and PSC activation. Methods Primary PSCs were exposed to different FAs, palmitate (pal) and oleate (ole), at pathophysiological concentrations typical to obese state, and in acute caerulein-induced stress (cer). PSC activation and differentiation were analyzed by measuring fat accumulation (oil-red staining and quantitation), proliferation (cells count) and migration (wound- healing assay). PSC differentiation markers (α-sma, fibronectin, tgf-β and collagen secretion), ER stress unfolded protein response and immune indicators (Xbp1, CHOP, TNF-α, IL-6) were analyzed at the transcript and protein expression levels (quantitative RT-PCR and western blotting). Results PSC exposure to pal and ole FAs (500 µM) increased significantly fat accumulation. Proliferation and migration analysis demonstrated that ole FA retained PSC activation, while exposure to pal FA significantly halted proliferation rate and delayed migration. Cer significantly augmented PSC differentiation markers α- sma, fibronectin and collagen, and ER stress and inflammation markers including Xbp1, CHOP, TNF-α and IL-6. The ole FA treatment significantly elevated PSC differentiation markers α-sma, fibronectin and collagen secretion. PSC ER stress was demonstrated following pal treatment with significant elevation of Xbp1 splicing and CHOP levels. Conclusion Exposure to pal FA halted PSC activation and differentiation and elevated ER stress markers, while cer and ole exposure significantly induced activation, differentiation and fibrosis. Thus, dietary FA composition should be considered and optimized to regulate PSC activation and differentiation in pancreatic pathologies. [ABSTRACT FROM AUTHOR]

Annexin A6 (AnxA6) has been implicated in the regulation of endo-/exocytic pathways, cholesterol transport, and the formation of multifactorial signaling complexes in many different cell types. More recently, AnxA6 has also been linked to triglyceride storage in adipocytes. Here we investigated the potential role of AnxA6 in fatty acid (FA) – induced lipid droplet (LD) formation in hepatocytes. AnxA6 was associated with LD from rat liver and HuH7 hepatocytes. In oleic acid (OA) -loaded HuH7 cells, substantial amounts of AnxA6 bound to LD in a Ca 2+ -independent manner. Remarkably, stable or transient AnxA6 overexpression in HuH7 cells led to elevated LD numbers/size and neutral lipid staining under control conditions as well as after OA loading compared to controls. In contrast, overexpression of AnxA1, AnxA2 and AnxA8 did not impact on OA-induced lipid accumulation. On the other hand, incubation of AnxA6-depleted HuH7 cells or primary hepatocytes from AnxA6 KO-mice with OA led to reduced FA accumulation and LD numbers. Furthermore, morphological analysis of liver sections from A6-KO mice revealed significantly lower LD numbers compared to wildtype animals. Interestingly, pharmacological inhibition of cytoplasmic phospholipase A2α (cPLA 2 α)-dependent LD formation was ineffective in AnxA6-depleted HuH7 cells. We conclude that cPLA 2 α-dependent pathways contribute to the novel regulatory role of hepatic AnxA6 in LD formation. [ABSTRACT FROM AUTHOR]

The Piezo1 protein ion channel is a novel mechanical stretch-activated ion channel (SAC) closely related to mechanical signals. Mechanotransduction plays a crucial role in organ development and homeostasis. Previous studies identified Piezo1 and demonstrated that it is distinct from other ion channels with well-established roles in lower organisms. Mechanical stretch-activated ion channels from other organisms are not conserved in mammals or do not act as mechanically activated channels in mammals. In the current study, we explored the role of the Piezo1 ion channel in human nucleus pulposus cell (NP cell) apoptosis through mechanical force-induced mitochondrial dysfunction and endoplasmic reticulum stress. Reverse Transcription Polymerase chain reaction (RT-PCR), immunofluorescence, immunohistochemistry and Annexin V binding and propidium iodide analyses revealed that the Piezo1 protein ion channel was highly expressed in human NP cells, which are the primary cells that comprise the intervertebral disc. In patients with intervertebral disc degeneration (IVDD), the Piezo1 protein may play a crucial role in human NP cell apoptosis through mitochondrial dysfunction and endoplasmic reticulum stress under abnormal loading conditions. This study also verified that human NP cells have an intimate connection with the cytoskeleton upon treatment of the cells with the Piezo1 blocking peptide GsMTx4 from tarantula venom. In summary, Piezo1 functions in human NP cell apoptosis, which may be one underlying mechanism of apoptosis induced by abnormal loading in IVDD patients. [ABSTRACT FROM AUTHOR]

Inflammatory bowel disease (IBD) is a chronic inflammatory disease involving the digestive tract, characterized by abdominal pain, diarrhea, rectal bleeding, and so on, which can make patients physically weakened and live difficultly. Although IBD has been recognized for many years, the pathogenesis of IBD has not yet been established and damage to intestinal barrier is thought to be closely associated with IBD. Intestinal barrier is an innate barrier that maintains the homeostasis of the intestinal environment and impedes pathogenic bacteria and toxins, and the endoplasmic reticulum (ER) has recently been found to be involved in maintaining the integrity of intestinal barrier. Endoplasmic reticulum stress (ERS) is a status of endoplasmic reticulum damaged when unfolded or misfolded proteins accumulate in excess of the degradation systematic clearance limit of the misfolded proteins. The regulation of ERS on protein folding synthesis and maintenance of cellular homeostasis is an important factor in influencing the integrity of the intestinal barrier. This paper mainly discusses the relationship between ERS and the intestinal barrier, aiming to understand the regulatory role of ERS on the intestinal barrier and the mechanism and to improve new solutions and notions for the treatment or prevention of IBD. [ABSTRACT FROM AUTHOR]

Gastric cancer is the most common type of tumor in developing countries and the fourth most frequently diagnosed cancer worldwide. Here, we demonstrated the apoptotic effects of GL-V9 on several human gastric cancer cells and selected MGC-803 cells to uncover the underlying mechanism. GL-V9 elevated Bax/Bcl-2 ratio, abated mitochondrial membrane potential and triggered the onset of apoptotic execution in MGC-803 cells. Our research revealed that CHOP silencing could not inhibit apoptosis, neither could it block Ca 2+ release, suggesting that GL-V9-induced apoptosis was independent of CHOP. Furthermore, GL-V9 increased mitochondrial Ca 2+ uptake through 1,4,5-triphosphate (IP3) receptor via the activation of phospholipase C-γ1 and the translocation of phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase from nucleus to endoplasmic reticulum. Moreover, in-vivo studies indicated that GL-V9 exhibited significant MGC-803 xenografts regression in nude mice with low systemic toxicity. In conclusion, GL-V9 could induce apoptosis in gastric cancer cells, and would be a promising therapeutical agent against gastric cancer. [ABSTRACT FROM AUTHOR]

We have previously shown that palmitate causes ER stress in primary cardiomyocytes and this was associated with a diffuse lipid staining histology. In contrast, oleate, which was non-toxic, led to the formation of abundant, clearly delineated lipid droplets. The aberrant lipid histology in palmitate treated cells led us to hypothesize that perhaps there was an impairment in lipid droplet formation, which could lead to accumulation of lipids in the ER and consequent ER stress. To test this hypothesis we treated H9C2s (a cardiomyoblast cell line) with either 300 µM oleate or palmitate for 8 h. We found that palmitate resulted in significantly less lipid droplet abundance despite elevated intracellular lipid accumulation. Next we showed that palmitate was packaged primarily as diacylglycerol (DAG), in contrast oleate formed primarily triacylglycerol (TAG). Furthermore, the palmitate induced DAG accumulated mostly in the ER, while oleate treatment resulted in accumulation of TAG primarily in lipid droplets. The palmitate-induced accumulation of lipid in the ER was associated with a strong ER stress response. Interestingly, we found that ER stress induced by either palmitate, tunicamycin, or thapsigargin led to the degradation of Plin2, an important lipid droplet binding protein. In contrast palmitate had little effect on either Plin3 or Plin5. Furthermore, we found that acute MG132 administration significantly attenuated palmitate mediated ER stress and cell death. This protection was associated with a moderate attenuation of Plin2 degradation. [ABSTRACT FROM AUTHOR]

Mammalian or mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth, metabolism, and cell differentiation. Recent studies have revealed that the recruitment of mTORC1 to lysosomes is essential for its activation. The ceramide analogue 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), a well known glycosphingolipid synthesis inhibitor, also affects the structures and functions of various organelles, including lysosomes and endoplasmic reticulum (ER). We investigated whether PDMP regulates the mTORC1 activity through its effects on organellar behavior. PDMP induced the translocation of mTORC1 from late endosomes/lysosomes, leading to the dissociation of mTORC1 from its activator Rheb in MC3T3-E1 cells. Surprisingly, we found mTORC1 translocation to the ER upon PDMP treatment. This effect of PDMP was independent of its action as the inhibitor, since two stereoisomers of PDMP, with and without the inhibitor activity, showed essentially the same effect. We confirmed that PDMP inhibits the mTORC1 activity based on the decrease in the phosphorylation of ribosomal S6 kinase, a downstream target of mTORC1, and the increase in LC3 puncta, reflecting autophagosome formation. Furthermore, PDMP inhibited the mTORC1-dependent osteoblastic cell proliferation and differentiation of MC3T3-E1 cells. Accordingly, the present results reveal a novel mechanism of PDMP, which inhibits the mTORC1 activity by inducing the translocation of mTOR from lysosomes to the ER. [ABSTRACT FROM AUTHOR]

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) undergoes numerous post-translational modifications, which impart new function and influence intracellular location. For example, atypical PKC ι/λ phosphorylates GAPDH that locates to vesicular tubular clusters and is required for retrograde membrane trafficking in the early secretory pathway. GAPDH is also required in the endocytic pathway; substitution of Pro 234 to Ser (Pro 234 Ser) rendered CHO cells defective in endocytosis. To determine if GAPDH (Pro 234 Ser) could inhibit endoplasmic reticulum to Golgi trafficking, we introduced the recombinant mutant enzyme into several biochemical and morphological transport assays. The mutant protein efficiently blocked vesicular stomatitis virus-G protein transport. Because GAPDH binds to microtubules (MTs), we evaluated MT binding and MT intracellular distribution in the presence of the mutant. Although these properties were not changed relative to wild-type, GAPDH (Pro 234 Ser) altered Golgi complex morphology. We determined that the GAPDH point mutation disrupted association between the enzyme and the serine/threonine kinase Akt. Interestingly Rab1, which functions in anterograde-directed trafficking, stimulates GAPDH-Akt association with membranes in a quantitative binding assay. In contrast, Rab2 does not stimulate GAPDH-Akt membrane binding but instead recruits GAPDH-aPKC. We propose a mechanism whereby the association of GAPDH with Akt or with aPKC serves as a switch to discriminate between anterograde directed cargo and recycling cargo retrieved back to the ER, respectively. [ABSTRACT FROM AUTHOR]

Heat stress induces intracellular protein denaturation and endoplasmic reticulum (ER) stress, which elicits unfolded protein response (UPR) in cells. UPR involves three ER-localized sensor proteins: the inositol-requiring protein 1α (IRE1α), the dsRNA-activated protein kinase-like ER kinase (PERK), and activating transcription factor-6 (ATF6). However, the precise mechanism by which cells deal with heat stress remains to be elucidated. We report herein that heat stress effectively activates all branches of the UPR. While splicing of the XBP-1 mRNA is usually triggered by activated IRE1α, the level of this protein was found to be decreased immediately after the occurrence of heat stress and the down-regulation gradually recovered thereafter. The protein levels of other UPR sensors or ER luminal proteins were unaffected. The down-regulation of IRE1α was independent of cellular viability. It thus appears that the heat-stress induced down-regulation of the IRE1α protein may lead to the termination of the IRE1α signaling pathway in an attempt to avoid excessive responses. We investigated the machinery causing IRE1α down-regulation and found that heat stress effectively promoted the activation of autophagy. Importantly, the inhibition of either proteasomes or autophagic flux failed to rescue the loss of IRE1α following heat stress. These collective results suggest that heat stress simultaneously activates both the UPR and autophagy, followed by the activation of a negative feedback system in UPR by modulating the responses related to the IRE1α–XBP-1 axis. [ABSTRACT FROM AUTHOR]

Atlastins are large, membrane-bound GTPases that participate in the fusion of endoplasmic reticulum (ER) tubules to generate the polygonal ER network in eukaryotes. They also regulate lipid droplet size and inhibit bone morphogenetic protein (BMP) signaling, though mechanisms remain unclear. Humans have three atlastins (ATL1, ATL2, and ATL3), and ATL1 and ATL3 are mutated in autosomal dominant hereditary spastic paraplegia and hereditary sensory neuropathies. Cellular investigations of atlastin orthologs in most yeast, plants, flies and worms are facilitated by the presence of a single or predominant isoform, but loss-of-function studies in mammalian cells are complicated by multiple, broadly-expressed paralogs. We have generated mouse NIH-3T3 cells lacking all three mammalian atlastins (Atl1/2/3) using CRISPR/Cas9-mediated gene knockout (KO). ER morphology is markedly disrupted in these triple KO cells, with prominent impairment in formation of three-way ER tubule junctions. This phenotype can be rescued by expression of distant orthologs from Saccharomyces cerevisiae (Sey1p) and Arabidopsis (ROOT HAIR DEFECTIVE3) as well as any one of the three human atlastins. Minimal, if any, changes are observed in the morphology of mitochondria and the Golgi apparatus. Alterations in BMP signaling and increased sensitivity to ER stress are also noted, though effects appear more modest. Finally, atlastins appear required for the proper differentiation of NIH-3T3 cells into an adipocyte-like phenotype. These findings have important implications for the pathogenesis of hereditary spastic paraplegias and sensory neuropathies associated with atlastin mutations. [ABSTRACT FROM AUTHOR]

We have previously demonstrated the protective action of hydrogen sulfide (H 2 S) in 1-Methy-4-Phenylpyridinium Ion (MPP + )-induced neurotoxicity. However, the exact mechanisms of this protection remain largely unknown. Aldehyde stress and endoplasmic reticulum (ER) stress play significant roles in the neurotoxicity of MPP + . Brain derived neurotrophic factor (BDNF) is an important endogenous neuroprotectant. Therefore, we speculated that the protection of H 2 S against MPP + neurotoxicity results from inhibiting MPP + -induced aldehyde stress and ER stress via upregulation of BDNF. In the present study, we found that NaHS, a donor of H 2 S, inhibited MPP + -induced aldehyde stress (the accumulations of the intracellular 4-HNE and MDA) and ER stress (the increases in the expressions of GRP78 and Cleaved-caspase-12) in PC12 cells and upregulated the BDNF expression in MPP + -exposed PC12 cells. Furthermore, we found that pretreatment of PC12 cells with K252a, an inhibitor of the BDNF receptor TrkB, not only markedly reversed the inhibitiory role of NaHS in MPP + -induced aldehyde stress and ER stress, but also ablated the protection of NaHS against MPP + -induced neurotoxicity. These data demonstrated that the protective role of H 2 S against MPP + -induced neurotoxicity by inhibiting aldehyde stress and ER stress, which is involved in upregulation of BDNF. [ABSTRACT FROM AUTHOR]

Copper is an essential trace element, however, excess copper is harmful to human health. Excess copper-derived oxidants contribute to the progression of Wilson disease, and oxidative stress induces accumulation of abnormal proteins. It is known that the endoplasmic reticulum (ER) plays an important role in proper protein folding, and that accumulation of misfolded proteins disturbs ER homeostasis resulting in ER stress. However, copper-induced ER homeostasis disturbance has not been fully clarified. We treated human hepatoma cell line (Huh7) and immortalized-human hepatocyte cell line (OUMS29) with copper and chemical chaperones, including 4-phenylbutyrate and ursodeoxycholic acid. We examined copper-induced oxidative stress, ER stress and apoptosis by immunofluorescence microscopy and immunoblot analyses. Furthermore, we examined the effects of copper on carcinogenesis. Excess copper induced not only oxidative stress but also ER stress. Furthermore, excess copper induced DNA damage and reduced cell proliferation. Chemical chaperones reduced this copper-induced hepatotoxicity. Excess copper induced hepatotoxicity via ER stress. We also confirmed the abnormality of ultra-structure of the ER of hepatocytes in patients with Wilson disease. These findings show that ER stress plays a pivotal role in Wilson disease, and suggests that chemical chaperones may have beneficial effects in the treatment of Wilson disease. [ABSTRACT FROM AUTHOR]

Whether and how sevoflurane preconditioning (SevoPre) exerts protection against acute myocardial ischemia/reperfusion (MI/R) injury remains elusive. We observed significant myocardial injury, as evidenced by infarct size, cardiomyocyte apoptosis, and circulating troponin-I, at 3 h of MI/R in both wildtype and adiponectin knockout mice. The injury was significantly ameliorated by SevoPre in wildtype mice, but not in adiponectin knockout mice. In wildtype mice, we found that MI/R could increase endoplasmic reticulum stress of cardiomyocytes, and impair association of adiponectin receptor 1 and ceveolin-3, both of which processes were largely restored by SevoPre. In summary, we demonstrated that significant injury had already took place at 3 h of MI/R, which could be ameliorated by SevoPre via promoting affinity of adiponectin receptor 1 and ceveolin-3, and then attenuating endoplasmic reticulum stress of cardiomyocytes. • Previous efforts focused on injury of >24 h of MI/R. • Here we found that significant acute injury had already taken place 3 h after MI/R. • Sevoflurane preconditioning protects against the acute MI/R injury. • The novel time frame of injuring calls for more elaborative management of MI/R. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum (ER) presents unique properties to establishing bacterium symbiosis in eukaryotic cells since it synthesizes and glycosylates essential molecules like proteins and lipids. Tunicamycin (TM) is an antibiotic that inhibits the first step in the N-linked glycosylation in eukaryotes and has been used as an ER stress inducer to activate the Unfolded Protein Response (UPR). Mutualistic symbiosis in trypanosomatids is characterized by structural adaptations and intense metabolic exchanges, thus we investigated the effects of TM in the association between Angomonas deanei and its symbiotic bacterium, through ultrastructural and proteomic approaches. Cells treated with the inhibitor showed a decrease in proliferation, enlargement of the ER and Golgi cisternae and an increased distance between the symbiont and the ER. TM proved to be an important tool to better understand ER stress in trypanosomatids, since changes in protein composition were observed in the host protozoan, especially the expression of the Hsp90 chaperone. Furthermore, data obtained indicates the importance of the ER for the adaptation and maintenance of symbiotic associations between prokaryotes and eukaryotes, considering that this organelle has recognized importance in the biogenesis and division of cell structures. [ABSTRACT FROM AUTHOR]

Diabetic cardiomyopathy increases the risk for the development of heart failure independent of coronary artery disease and hypertension. Either type 1 or type 2 diabetes is often accompanied by varying degrees of hyperglycemia, which has been proven to induce myocardial apoptosis in animal models. Recently, a novel small molecule, ZLN005, has been reported to show antidiabetic efficacy in a mouse model, possibly by induction of PGC-1α expression. In this study, we investigated whether ZLN005 protects cardiomyocytes against high glucose-induced cytotoxicity and the mechanisms involved. Neonatal mouse cardiomyocytes were incubated with media containing 5.5 or 33 mM glucose for 24 h in the presence or absence of ZLN005. ZLN005 treatment led to ameliorated cardiomyocyte oxidative injury, enhanced cell viability, and reduced apoptosis in the high glucose environment. Western blot analysis revealed that high glucose suppressed cardiomyocyte autophagy, whereas ZLN005 increased the expression of autophagy marker proteins ATG5, beclin1, and LC3 II/LC3 I; this increase was accompanied by increased expression of SIRT1. Furthermore, EX527, a SIRT1-specific inhibitor, weakened the protective effects of ZLN005 on cardiomyocytes subjected to high glucose. Taken together, these results suggest that ZLN005 suppresses high glucose-induced cardiomyocyte injury by promoting SIRT1 expression and autophagy. [ABSTRACT FROM AUTHOR]

A mutation in VAPB causes a familial form of Amyotrophic Lateral Sclerosis. The mutant protein (VAPB-P56S) is aggregate prone and blocks retrograde traffic from the endoplasmic reticulum (ER) Golgi intermediate compartment (ERGIC) including trafficking to the nuclear envelope (NE). Here we report a morphological screen where overexpression of oxysterol binding protein-related protein-3 (ORP3) rescued the mutant VAPB phenotype. It resolved the mutant VAPB-induced membrane expansions, restored solubility of the mutant protein in non-ionic detergent, and restored trafficking of Emerin to the NE. Knockdown of ORP3 or VAPB increased the intracellular level of phosphatidylinositol 4-phosphate (PtdIns4 P ). Decreasing PtdIns4 P levels by inhibiting its synthesis reduced the severity of the mutant VAPB-induced membrane expansions and restored Emerin trafficking to the NE. Thus, VAPB and its interacting partners cooperatively regulate protein trafficking through the ERGIC by modulating PtdIns4 P levels. [ABSTRACT FROM AUTHOR]

Lipid droplets (LDs) in steroidogenic tissues have a cholesteryl ester (CE) core surrounded by a phospholipid monolayer that is coated with associated proteins. Compared with other tissues, they tend to be smaller in size and more numerous in numbers. These LDs are enriched with PLIN1c, PLIN2 and PLIN3. Both CIDE A and B are found in mouse ovary. Free cholesterol (FC) released upon hormone stimulation from LDs is the preferred source of cholesterol substrate for steroidogenesis, and HSL is the major neutral cholesterol esterase mediating the conversion of CEs to FC. Through the interaction of HSL with vimentin and StAR, FC is translocated to mitochondria for steroid hormone production. Proteomic analyses of LDs isolated from loaded primary ovarian granulosa cells, mouse MLTC-1 Leydig tumor cells and mouse testes revealed LD associated proteins that are actively involved in modulating lipid homeostasis along with a number of steroidogenic enzymes. Microscopy analysis confirmed the localization of many of these proteins to LDs. These studies broaden the role of LDs to include being a platform for functional steroidogenic enzyme activity or as a port for transferring steroidogenic enzymes and/or steroid intermediates, in addition to being a storage depot for CEs. [ABSTRACT FROM AUTHOR]

Cytosolic lipid droplets (LDs) are observed in enterocytes of jejunum during lipid absorption. One important function of the intestine is to secrete chylomicrons, which provide dietary lipids throughout the body, from digested lipids in meals. The current hypothesis is that cytosolic LDs in enterocytes constitute a transient pool of stored lipids that provides lipids during interprandial period while lowering chylomicron production during the post-prandial phase. This smoothens the magnitude of peaks of hypertriglyceridemia. Here, we review the composition and functions of lipids and associated proteins of enterocyte LDs, the known physiological functions of LDs as well as the role of LDs in pathological processes in the context of the intestine. [ABSTRACT FROM AUTHOR]

Exocrine pancreas acinar cells have a highly developed endoplasmic reticulum (ER), accommodating their high protein production rate. Overload of dietary fat (typical to obesity) is a recognized risk factor in pancreatitis and pancreatic cancer. Dietary fat, especially saturated fat, has been suggested by others and us to induce an acinar lipotoxic effect. The effect of different dietary fatty acids on the ER stress response is unknown. We studied the effect of acute (24 h) challenge with different fatty acids (saturated, mono and poly-unsaturated) at different concentrations (between 200 and 500 µM, typical to normal and obese states, respectively), testing fat accumulation, ER stress indicators, X-box binding protein 1 ( Xbp1 ) splicing and nuclear translocation, as well as unfolded protein response (UPR) transcripts and protein levels using exocrine pancreas acinar AR42J and primary cells. Acute exposure of AR42J cells to different fatty acids caused increased accumulation of triglycerides, dependent on the type of fat. Different FAs had different effects on ER stress: most notably, saturated palmitic acid significantly affected the UPR response, as demonstrated by altered Xbp1 splicing, elevation in transcript levels of UPR ( Xbp, CHOP, Bip ) and immune factors ( Tnfα, Tgfβ ) , and enhanced Xbp1 protein levels and Xbp1 time-dependent nuclear translocation. Poly-unsaturated FAs caused milder elevation of ER stress markers, while mono-unsaturated oleic acid attenuated the ER stress response. Thus, various fatty acids differentially affect acinar cell fat accumulation and, apart from oleic acid, induce ER stress. The differential effect of the various fatty acids could have potential nutritional and therapeutic implications. [ABSTRACT FROM AUTHOR]

An acidic tumor microenvironment exists widely in solid tumors. However, the detailed mechanism of cell survival under acidic stress remains unclear. The aim of this study is to clarify whether acid-induced autophagy exists and to determine the function and mechanism of autophagy in lung cancer cells. We have found that acute low pH stimulated autophagy by increasing LC3-positive punctate vesicles, increasing LC3 II expression levels and reducing p62 protein levels. Additionally, autophagy was inhibited by the addition of Baf or knockdown of Beclin 1, and cell apoptosis was increased markedly. In mouse tumors, the expression of cleaved caspase3 and p62 was enhanced by oral treatment with sodium bicarbonate, which can raise the intratumoral pH. Furthermore, the protein levels of ER stress markers, including p-PERK, p-eIF2α, CHOP, XBP-1s and GRP78, were also increased in response to acidic pH. The antioxidant NAC, which reduces ROS accumulation, alleviated acid-mediated ER stress and autophagy, and knocking down GRP78 reduced autophagy activation under acidic conditions, which suggests that autophagy was induced by acidic pH through ER stress. Taken together, these results indicate that the acidic microenvironment in non-small cell lung cancer cells promotes autophagy by increasing ROS-ER stress, which serves as a survival adaption in this setting. [ABSTRACT FROM AUTHOR]

Background Environmental conditions or chemical agents can interfere with the function of the endoplasmic reticulum, and the resulting endoplasmic reticulum (ER) stress can be toxic to the cell if it is not relieved. The classical compensatory response to ER stress is the unfolded protein response (UPR) that reduces protein load in the ER. However, autophagy may also compensate by removing large insoluble protein aggregates. Agents that stress the ER can have anti-cancer activity, and novel applications of ER stress inducing agents are being investigated. Plant stilbenes are a class of stress responsive molecules that includes resveratrol, which are being investigated as potential therapeutics in humans for conditions such as aging or cancer. Results We performed a screen of 1726 small, drug like molecules to identify those that could activate an ER-stress responsive luciferase gene. After secondary screening, we determined that the plant stilbenes pterostilbene and piceatannol were the most potent inducers of ER stress from this group. ER stress can be particularly toxic to cells with high ER load, so we examined their effect on cells expressing the Wnt family of secreted glycoprotein growth factors. Molecular analysis determined that these ER stress-inducing stilbenes could block Wnt processing and also induce autophagy in acute lymphoblastic leukemia cells expressing Wnt16. Combining pterostilbene (to induce ER stress) with chloroquine (to inhibit autophagy) lead to significant cellular toxicity in cells from aggressive acute lymphoblastic leukemia. Conclusions Plant stilbenes are potent inducers of ER stress. However, their toxicity is more pronounced in cancer cells expressing Wnt growth factors. The toxicity of stilbenes in these ALL cells can be potentiated by the addition of autophagy inhibitors, suggesting a possible therapeutic application. [ABSTRACT FROM AUTHOR]

After CNS injury, astrocytes and mesenchymal cells attempt to restore the disrupted glia limitans by secreting proteoglycans and extracellular matrix proteins (ECMs), forming the so-called glial scar. Although the glial scar is important in sealing the lesion, it is also a physical and functional barrier that prevents axonal regeneration. The synthesis of secretory proteins in the RER is under the control of the initiation factor of translation eIF2α. Inhibiting the synthesis of secretory proteins by increasing the phosphorylation of eIF2α, might be a pharmacologically efficient way of reducing proteoglycans and other profibrotic proteins present in the glial scar. Salubrinal, a neuroprotective drug, decreased the expression and secretion of proteoglycans and other profibrotic proteins induced by EGF or TGFβ, maintaining eIF2α phosphorylated. Besides, Salubrinal also reduced the transcription of proteoglycans and other profibrotic proteins, suggesting that it induced the degradation of non-translated mRNA. In a model in vitro of the glial scar, cortical neurons grown on cocultures of astrocytes and fibroblasts with TGFβ treated with Salubrinal, showed increased neurite outgrowth compared to untreated cells. Our results suggest that Salubrinal may be considered of therapeutic value facilitating axonal regeneration, by reducing overproduction and secretion of proteoglycans and profibrotic protein inhibitors of axonal growth. [ABSTRACT FROM AUTHOR]

As a widely used anti-bacterial agent and a metabolic inhibitor as well as AMP-activated protein kinase (AMPK) activator, berberine (BBR) has been shown to cross the blood–brain barrier. Its efficacy has been investigated in various disease models of the central nervous system. Neurite outgrowth is critical for nervous system development and is a highly energy-dependent process regulated by AMPK-related pathways. In the present study, we aimed to investigate the effects of BBR on AMPK activation and neurite outgrowth in neurons. The neurite outgrowth of primary rat cortical neurons at different stages of polarization was monitored after exposure of BBR. Intracellular energy level, AMPK activation and polarity-related pathways were also inspected. The results showed that BBR suppressed neurite outgrowth and affected cytoskeleton stability in the early stages of neuronal polarization, which was mediated by lowered energy status and AMPK activation. Liver kinase B1 and PI3K–Akt–GSK3β signaling pathways were also involved. In addition, mitochondrial dysfunction and endoplasmic reticulum stress contributed to the lowered energy status induced by BBR. This study highlighted the knowledge of the complex activities of BBR in neurons and corroborated the significance of energy status during the neuronal polarization. [ABSTRACT FROM AUTHOR]

Background Human choriocarcinoma, a highly curable solid tumour, is exceptionally sensitive to methotrexate-based chemotherapy at the metastatic stage. The present study aimed to investigate the molecular basis for this resistance to methotrexate therapy occurs in some cases, and these patients subsequently die from progressive and advanced disease. Methods The autophagy and apoptotic activity regulated by PERK/ATF4 axis in methotrexate-resistant JEG-3 and parental cells were evaluated with western blotting and chromatin immunoprecipitation (ChIP). The regulatory relationships among the reactive oxygen species (ROS), JNK/p62 axis, PERK/ATF4-mediated apoptosis and autophagy were assessed with western blotting, RT-PCR, fluorescence-activated cell sorting as well as ChIP. Results The decreased apoptosis in methotrexate-resistant JEG-3 cells was observed with an up-regulation of protective autophagy, suggesting a switch from apoptosis to autophagy, which was regulated via the PERK/ATF4 pathway under condition of endoplasmic reticulum (ER) stress. Further experiments demonstrated that this cell death switch was regulated by ROS-mediated JNK/p62 pathway and subsequently lead to the resistance of choriocarcinoma cells to methotrexate treatment. Conclusions This study provides evidence to explain a survival mechanism of the switch from ER stress-induced apoptosis to autophagy via ROS-mediated JNK/p62 signals in methotrexate-resistant choriocarcinoma cells and may implicate the chemotherapy of methotrexate resistance in choriocarcinoma. [ABSTRACT FROM AUTHOR]

This review provides insight into the intraneuronal transport of the Amyloid-β Precursor Protein (APP), the prototype of an extensively posttranslationally modified and proteolytically cleaved transmembrane protein. Uncovering the intricacies of APP transport proves to be a challenging endeavor of cell biology research, deserving increased priority, since APP is at the core of the pathogenic process in Alzheimer׳s disease. After being synthesized in the endoplasmic reticulum in the neuronal soma, APP enters the intracellular transport along the secretory, endocytic, and recycling routes. Along these routes, APP undergoes cleavage into defined sets of fragments, which themselves are transported - mostly independently - to distinct sites in neurons, where they exert their functions. We review the currently known routes and mechanisms of transport of full-length APP, and of APP fragments, commenting largely on the experimental challenges posed by studying transport of extensively cleaved proteins. The review emphasizes the interrelationships between the proteolytic and posttranslational modifications, the intracellular transport, and the functions of the APP species. A goal remaining to be addressed in the future is the incorporation of the various views on APP transport into a coherent picture. In this review, the disease context is only marginally addressed; the focus is on the basic biology of APP transport under normal conditions. As shown, the studies of APP transport uncovered numerous mechanisms of transport, some of them conventional, and others, novel, awaiting exploration. [ABSTRACT FROM AUTHOR]