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American Journal of Physiology. Renal... Feb 2015ER stress has been implicated in the pathogenesis of both acute and chronic kidney diseases. However, the molecular regulation of ER stress in kidney cells and tissues...
ER stress has been implicated in the pathogenesis of both acute and chronic kidney diseases. However, the molecular regulation of ER stress in kidney cells and tissues remains poorly understood. In this study, we examined tunicamycin-induced ER stress in renal proximal tubular cells (RPTC). Tunicamycin induced the phosphorylation and activation of PERK and eIF2α within 2 h in RPTC, which was followed by the induction of GRP78 and CHOP. Consistently, tunicamycin also induced apoptosis in RPTC. Interestingly, mTOR was activated rapidly during tunicamycin treatment, as indicated by phosphorylation of both mTOR and p70S6K. Inhibition of mTOR with rapamycin partially suppressed the phosphorylation of PERK and eIF2a and the induction of CHOP and GRP78 induction during tunicamycin treatment. Rapamycin also inhibited apoptosis during tunicamycin treatment and increased cell survival. Collectively, the results suggest that mTOR plays a regulatory role in ER stress, and inhibition of mTOR may have potential therapeutic effects in ER stress-related renal diseases.
Topics: Animals; Apoptosis; Cell Survival; Cells, Cultured; Endoplasmic Reticulum Stress; Eukaryotic Initiation Factor-2; Kidney Tubules, Proximal; Rats; Signal Transduction; TOR Serine-Threonine Kinases; Tunicamycin
PubMed: 25428129
DOI: 10.1152/ajprenal.00629.2014 -
Genome Biology Oct 2021The endoplasmic reticulum (ER) is a membranous organelle that maintains proteostasis and cellular homeostasis, controlling the fine balance between health and disease....
BACKGROUND
The endoplasmic reticulum (ER) is a membranous organelle that maintains proteostasis and cellular homeostasis, controlling the fine balance between health and disease. Dysregulation of the ER stress response has been implicated in intestinal inflammation associated with inflammatory bowel disease (IBD), a chronic condition characterized by changes to the mucosa and alteration of the gut microbiota. While the microbiota and microbially derived metabolites have also been implicated in ER stress, examples of this connection remain limited to a few observations from pathogenic bacteria. Furthermore, the mechanisms underlying the effects of bacterial metabolites on ER stress signaling have not been well established.
RESULTS
Utilizing an XBP1s-GFP knock-in reporter colorectal epithelial cell line, we screened 399 microbiome-related metabolites for ER stress pathway modulation. We find both ER stress response inducers (acylated dipeptide aldehydes and bisindole methane derivatives) and suppressors (soraphen A) and characterize their activities on ER stress gene transcription and translation. We further demonstrate that these molecules modulate the ER stress pathway through protease inhibition or lipid metabolism interference.
CONCLUSIONS
Our study identified novel links between classes of gut microbe-derived metabolites and the ER stress response, suggesting the potential for these metabolites to contribute to gut ER homeostasis and providing insight into the molecular mechanisms by which gut microbes impact intestinal epithelial cell homeostasis.
Topics: Aldehydes; Apoptosis; Bacteria; Dipeptides; Endoplasmic Reticulum Stress; Gastrointestinal Microbiome; HT29 Cells; Humans; Indoles; Macrolides; Tunicamycin; Unfolded Protein Response
PubMed: 34654459
DOI: 10.1186/s13059-021-02496-8 -
Yakugaku Zasshi : Journal of the... 2022Although natural products are rich sources for drug discovery, only a small percentage of natural products themselves have been approved for clinical use, thus it is... (Review)
Review
Although natural products are rich sources for drug discovery, only a small percentage of natural products themselves have been approved for clinical use, thus it is necessary to modulate various properties, such as efficacy, toxicity, and metabolic stability. A question in natural product drug discovery is how to logically design natural product derivatives with desired biological properties. This review describes our recent studies regarding the medicinal chemistry of tunicamycin. Tunicamycin inhibits bacterial phospho-N-acetylmuramic acid (MurNAc)-pentapeptide translocase (MraY), which is an essential enzyme in bacteria and a good target for antibacterial drug discovery. The usefulness of tunicamycin as antibacterial agents is limited by off-target inhibition of human UDP-N-acetylglucosamine (GlcNAc): polyprenol phosphate translocase (GPT). We positioned the total synthesis of tunicamycin as a starting point for the research and have accomplished the synthesis of tunicamycin V by using the Achmatowicz reaction, [3,3] sigmatropic rearrangement of allyl cyanate, and stereoselective glycosylation as key reactions. Next, the minimum structural requirements for tunicamycin V for MraY inhibition were established by systematic structure-activity relationship studies with truncated analogs of tunicamycin V. Our collaborative study elucidated a crystal structure of human GPT in complex with tunicamycin. This structural information was then exploited to rationally design an MraY-specific inhibitor of tunicamycin V in which the GlcNAc moiety was modified to a MurNAc amide. The analog was identified as a highly selective MraY inhibitor.
Topics: Bacterial Proteins; Biological Products; Humans; Transferases; Transferases (Other Substituted Phosphate Groups); Tunicamycin
PubMed: 35370192
DOI: 10.1248/yakushi.21-00185-2 -
Cellular Physiology and Biochemistry :... 2018Epithelial to mesenchymal transition (EMT) is a crucial process involved in pulmonary fibrosis. This study aimed to explore the role of histone deacetylases (HDACs) and...
BACKGROUND/AIMS
Epithelial to mesenchymal transition (EMT) is a crucial process involved in pulmonary fibrosis. This study aimed to explore the role of histone deacetylases (HDACs) and endoplasmic reticulum (ER) stress in EMT in human lung epithelial cells.
METHODS
Human lung adenocarcinoma A549 cells were treated with bleomycin and tunicamycin to induce EMT. The proliferation of A549 cells was detected by MTT assay. The expression of HDACs and EMT markers was detected by PCR and Western blot analysis. The secretion of TGF-β1 and collagen I was examined by ELISA.
RESULTS
A549 cells switched from a cobblestone-like appearance to an elongated fibroblast like appearance after exposure to tunicamycin or bleomycin, accompanied by increased expression of N-cadherin, α-SMA and Collagen I. Meanwhile, GRP78 was upregulated in A549 cells exposed to tunicamycin or bleomycin. These changes induced by tunicamycin or bleomycin could be abrogated by 4-PBA. Moreover, tunicamycin and bleomycin promoted the expression of HDAC2 and HDAC6, and HDACs inhibitor SAHA abrogated the morphological and biochemical changes in A549 cells. 4-PBA and SAHA inhibited the upregulation of pulmonary fibrosis factors TGF-β1 and IL-32 and the activation of Smad pathway induced by tunicamycin or bleomycin.
CONCLUSIONS
We provide the first evidence that tunicamycin and bleomycin induce ER stress and EMT in lung epithelial cells via the upregulation of HDACs. HDACs inhibitor could inhibit ER stress induced upregulation of pulmonary fibrosis factors and the activation of Smad pathway. HDACs inhibitors are promising agents for the therapy of pulmonary fibrosis.
Topics: A549 Cells; Bleomycin; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Epithelial-Mesenchymal Transition; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Lung; Respiratory Mucosa; Tunicamycin
PubMed: 29705800
DOI: 10.1159/000489367 -
American Journal of Physiology.... May 2023Hepatic lipotoxicity plays a central role in the pathogenesis of nonalcoholic fatty liver disease; however, the underlying mechanisms remain elusive. Here, using both...
Hepatic lipotoxicity plays a central role in the pathogenesis of nonalcoholic fatty liver disease; however, the underlying mechanisms remain elusive. Here, using both cultured hepatocytes (AML-12 cells and primary mouse hepatocytes) and the liver-specific gene knockout mice, we investigated the mechanisms underlying palmitate-elicited upregulation of CD36, a class B scavenger receptor mediating long-chain fatty acids uptake, and its role in palmitate-induced hepatolipotoxicity. We found that palmitate upregulates hepatic CD36 expression. Despite being a well-established target gene of PPARγ transactivation, our data demonstrated that the palmitate-induced CD36 upregulation in hepatocytes is in fact PPARγ-independent. We previously reported that the activation of ATF4, one of three canonical pathways activated upon endoplasmic reticulum (ER) stress induction, contributes to palmitate-triggered lipotoxicity in hepatocytes. In this study, our data revealed for the first time that ATF4 plays a critical role in mediating hepatic CD36 expression. Genetic inhibition of ATF4 attenuated CD36 upregulation induced by either palmitate or ER stress inducer tunicamycin in hepatocytes. In mice, tunicamycin upregulates liver CD36 expression, whereas hepatocyte-specific ATF4 knockout mice manifest lower hepatic CD36 expression when compared with control animals. Furthermore, we demonstrated that CD36 upregulation upon palmitate exposure represents a feedforward mechanism in that siRNA knockdown of CD36 in hepatocytes blunted ATF4 activation induced by both palmitate and tunicamycin. Finally, we confirmed that the ATF4-CD36 pathway activation contributes to palmitate-induced hepatolipotoxicity as genetic inhibition of either ATF4 or CD36 alleviated cell death and intracellular triacylglycerol accumulation. Collectively, our data demonstrate that CD36 upregulation by ATF4 activation contributes to palmitate-induced hepatic lipotoxicity. We provided the initial evidence that ATF4 is a principal transcription factor mediating hepatic CD36 expression in that both palmitate- and ER stress-elicited CD36 upregulation was blunted by ATF4 gene knockdown in hepatocytes, and hepatocyte-specific ATF4 knockout mice manifested lower hepatic CD36 expression. We further confirmed that the ATF4-CD36 pathway activation contributes to palmitate-induced hepatolipotoxicity as genetic inhibition of either ATF4 or CD36 alleviated cell death and intracellular triacylglycerol accumulation in response to exogenous palmitate exposure.
Topics: Animals; Mice; Palmitates; Up-Regulation; Transcriptional Activation; PPAR gamma; Tunicamycin; Hepatocytes; Endoplasmic Reticulum Stress; Mice, Knockout; Triglycerides
PubMed: 36852918
DOI: 10.1152/ajpgi.00163.2022 -
American Journal of Physiology. Cell... Aug 2021Prolonged endoplasmic reticulum (ER) stress can mediate inflammatory myopathies and insulin signaling pathways. The double-stranded RNA (dsRNA)-activated protein kinase...
Prolonged endoplasmic reticulum (ER) stress can mediate inflammatory myopathies and insulin signaling pathways. The double-stranded RNA (dsRNA)-activated protein kinase R (PKR) has been implicated in skeletal muscle dysfunction. However, pathological roles of PKR in ER stress in muscle are not fully understood. The current study aimed to investigate the effect of imoxin (IMX), a selective PKR inhibitor, on tunicamycin (TN)-induced promotion of ER stress and suppression of insulin signaling in C2C12 myotubes. Cells were pretreated with 5 µM IMX for 1 h and exposed to 0.5 µg/mL TN for 23 h. A subset of cells was stimulated with 100 nM insulin for the last 15 min. mRNA expression and protein levels involved in ER stress were measured by RT-PCR and Western blotting, respectively. TN significantly augmented PKR phosphorylation by 231%, which was prevented by IMX. In addition, IMX reduced mRNA and protein levels of ER stress-related markers, including CCAAT-enhancer-binding protein homologous protein (CHOP, mRNA: 95% decrease; protein: 98% decrease), activating transcription factor 4 (ATF4, mRNA: 69% decrease; protein: 99% decrease), cleavage of ATF6, and spliced X-box-binding protein 1 (XBP-1s, mRNA: 88% decrease; protein: 79% decrease), which were induced by TN. Furthermore, IMX ameliorated TN-induced suppression of phospho-insulin receptor β (317% increase) and Akt phosphorylation (by 36% at Ser473 and 30% at Thr308) in myotubes, while augmenting insulin-stimulated AS160 phosphorylation and glucose uptake (by ∼30%). These findings suggest that IMX may protect against TN-induced skeletal muscle ER stress and insulin resistance, which are potentially mediated by PKR.
Topics: Animals; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Imidazoles; Indoles; Insulin; Insulin Resistance; Mice; Muscle Fibers, Skeletal; Phosphorylation; Signal Transduction; Tunicamycin
PubMed: 34077277
DOI: 10.1152/ajpcell.00544.2020 -
Scientific Reports Sep 2022We have previously shown that Kyoto University Substances (KUSs), valosin-containing protein (VCP) modulators, suppress cell death in retinal ganglion cells of glaucoma...
We have previously shown that Kyoto University Substances (KUSs), valosin-containing protein (VCP) modulators, suppress cell death in retinal ganglion cells of glaucoma mouse models through alterations of various genes expressions. In this study, among the genes whose expression in retinal ganglion cells was altered by KUS treatment in the N-methyl-D-aspartic acid (NMDA) injury model, we focused on two genes, endothelin-1 (Edn1) and endothelin receptor type B (Ednrb), whose expression was up-regulated by NMDA and down-regulated by KUS treatment. First, we confirmed that the expression of Edn1 and Ednrb was upregulated by NMDA and suppressed by KUS administration in mice retinae. Next, to clarify the influence of KUSs on cell viability in relation to the endothelin signaling, cell viability was examined with or without antagonists or agonists of endothelin and with or without KUS in 661W retinal cells under stress conditions. KUS showed a significant protective effect under glucose-free conditions and tunicamycin-induced stress. This protective effect was partially attenuated in the presence of an endothelin antagonist or agonist under glucose-free conditions. These results suggest that KUSs protect cells partially by suppressing the upregulated endothelin signaling under stress conditions.
Topics: Animals; Endothelin Receptor Antagonists; Endothelin-1; Mice; N-Methylaspartate; Neuroprotection; Retinal Ganglion Cells; Tunicamycin; Valosin Containing Protein
PubMed: 36171250
DOI: 10.1038/s41598-022-20497-w -
Cell Stress & Chaperones Jul 2019Endoplasmic reticulum stress (ER stress) is involved in lipid metabolism and lipotoxicity and can lead to apoptosis. Resveratrol, a sirtuin 1 (SIRT1) agonist, prevents...
Endoplasmic reticulum stress (ER stress) is involved in lipid metabolism and lipotoxicity and can lead to apoptosis. Resveratrol, a sirtuin 1 (SIRT1) agonist, prevents ER stress and improves ER stress-induced hepatic steatosis and cell death. Clusterin is a secreted chaperone and has roles in various physiological processes. However, changes in the expression of clusterin upon ER stress and the connection between SIRT1 and clusterin in protection against ER stress are not well known. In cells treated with tunicamycin, resveratrol increased the expression of clusterin mRNA and protein and the secreted clusterin protein level in conditioned medium. Resveratrol decreased protein expression of the ER stress markers, p-PERK, p-IRE1α, and CHOP, and increased the expression of the ER-associated degradation (ERAD) factors, SEL1L and HRD1, in tunicamycin-treated cells. However, no changes in the expression of these genes were observed in clusterin siRNA-transfected cells. Moreover, increased LAMP2 and LC3 expression and decreased Rubicon expression were observed in cells treated with resveratrol or secreted clusterin. These data suggest that SIRT1 activation by resveratrol attenuates ER stress by promoting protective processes such as ERAD and autophagy pathways and that these protective effects are mediated by clusterin.
Topics: Autophagy; Biomarkers; Clusterin; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Hep G2 Cells; Humans; Proteins; Resveratrol; Sirtuin 1; Tunicamycin; Ubiquitin-Protein Ligases
PubMed: 31183612
DOI: 10.1007/s12192-019-01012-z -
Molecules (Basel, Switzerland) May 2020Four new variants of L1210 cells resistant to endoplasmic reticulum (ER) stressors, tunicamycin (S), thapsigargin (S), bortezomib (S), and MG-132 (S), were developed via...
Four new variants of L1210 cells resistant to endoplasmic reticulum (ER) stressors, tunicamycin (S), thapsigargin (S), bortezomib (S), and MG-132 (S), were developed via an 18-month periodic cultivation in culture medium with a gradual increase in substance concentration. Multidrug resistance was generated for S (to tunicamycin, bortezomib and MG-132), S (to tunicamycin, thapsigargin and MG-132), S (to bortezomib and MG-132), and S (to bortezomib and MG-132). These cells were compared to the original L1210 cells and another two variants, which expressed P-gp due to induction with vincristine or transfection with the gene encoding P-gp, in terms of the following properties: sensitivity to either vincristine or the ER stressors listed above, proliferative activity, expression of resistance markers and proteins involved in the ER stress response, and proteasome activity. The resistance of the new cell variants to ER stressors was accompanied by a decreased proliferation rate and increased proteasome activity. The most consistent change in protein expression was the elevation of GRP78/BiP at the mRNA and protein levels in all resistant variants of L1210 cells. In conclusion, the mechanisms of resistance to these stressors have certain common features, but there are also specific differences.
Topics: Animals; Bortezomib; Cell Line, Tumor; Drug Resistance, Multiple; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Leupeptins; Mice; Thapsigargin; Tunicamycin; Vincristine
PubMed: 32481618
DOI: 10.3390/molecules25112517 -
Acta Biochimica Polonica Mar 2022To clarify the regulatory effect of GRP-78 induced by tunicamycin on endoplasmic reticulum (ER) stress.
OBJECTIVE
To clarify the regulatory effect of GRP-78 induced by tunicamycin on endoplasmic reticulum (ER) stress.
METHODS
Tunicamycin was used to induce ER stress in trabecular meshwork cells (HTMC and GTM3). Cell apoptosis and ROS content were detected by flow cytometry to reveal the effect of tunicamycin on trabecular meshwork cells.
RESULTS
Tunicamycin could significantly increase the ROS content and the apoptosis rate in HTMC and GTM3 (p<0.01). The results showed that tunicamycin could increase the Ca2+ flow in cells. Tunicamycin can also increase expression levels of GRP78,VDAC1, ATF4, PERK, eIF2a, and CHOP (p<0.01). Overexpression of GRP78 protected cells from ER stress. Co-IP test showed that GRP78 directly bound to eIF2. These results suggest that GRP78 may play a regulatory role by regulating eIF2.
CONCLUSION
Tunicamycin induces oxidative stress in trabecular meshwork cells, and the increase in GRP78 expression can protect the cells during ER stress by regulating eIF2.
Topics: Anti-Bacterial Agents; Apoptosis; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Eukaryotic Initiation Factor-2; Humans; Oxidative Stress; Reactive Oxygen Species; Trabecular Meshwork; Transcription Factor CHOP; Tunicamycin
PubMed: 35233582
DOI: 10.18388/abp.2020_5624