scholarly journals Dissection of thrombospondin-4 domains involved in intracellular adaptive ER Stress responsive signaling

2015 ◽  
pp. MCB.00607-15 ◽  
Author(s):  
Matthew J. Brody ◽  
Tobias G. Schips ◽  
Davy Vanhoutte ◽  
Onur Kanisicak ◽  
Jason Karch ◽  
...  
Keyword(s):  
Stress Response ◽  
Er Stress ◽  
Calcium Binding ◽  
Binding Motifs ◽  
Er Stress Response ◽  
Er Retention ◽  
Activating Transcription Factor ◽  
Thrombospondin 4 ◽  
Activating Transcription Factor 6Α ◽  
Stress Pathway

Thrombospondins are a family of stress-inducible secreted glycoproteins that underlie tissue remodeling. We recently reported that thrombospondin-4 (Thbs4) has a critical intracellular function where it regulates the adaptive endoplasmic reticulum (ER) stress pathway through activating transcription factor 6α (Atf6α). Here, we dissect the domains of Thbs4 that mediate interactions with ER proteins such as BiP (Grp78) and Atf6α, and the domains mediating activation of the ER stress response. Functionally, Thbs4 localized to the ER and post-ER vesicles and was actively secreted in cardiomyocytes, as were the T3R and TSP-C domains, while the LamG domain localized to the Golgi apparatus. We also mutated the major calcium-binding motifs within the T3R domain of full-length Thbs4, causing ER retention and secretion blockade. The T3R and TSP-C domains as well as wildtype Thbs4 and the calcium-binding mutant, interacted with Atf6α, induced an adaptive ER stress response, and caused expansion of intracellular vesicles. In contrast, overexpression of a related secreted oligomeric glycoprotein, Nell2, which lacks only the T3R and TSP-C domains, did not cause these effects. Finally, deletion of Atf6α abrogated Thbs4-induced vesicular expansion. Taken together, these data identify the critical intracellular functional domains of Thbs4, which was formerly thought to only have extracellular functions.

Abstract 412: Dissection of Thrombospondin-4 Domains Involved in Adaptive ER Stress Responsive Signaling and Secretory Pathway Functions

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Matthew J Brody ◽  
Tobias G Schips ◽  
Onur Kaniscak ◽  
Jason Karch ◽  
N. S Blair ◽  
...  
Keyword(s):  
Er Stress ◽  
Calcium Binding ◽  
Secretory Pathway ◽  
Intracellular Localization ◽  
Lectin Domain ◽  
Activating Transcription Factor ◽  
Thrombospondin 4 ◽  
Stress Pathway

Thrombospondins are a family of stress-inducible secreted glycoproteins with well-characterized roles in the extracellular matrix and tissue remodeling. We recently reported critical intracellular functions for thrombospondin-4 (Thbs4) by activation of an adaptive endoplasmic reticulum (ER) stress pathway in cardiomyocytes, in part by promoting activation of activating transcription factor 6α (Atf6α). Here, we dissect the domains of Thbs4 that mediate interactions with ER proteins and activation of this adaptive ER stress response, and determine the domains of Thbs4 involved in secretory pathway functions. We show that the N-terminal laminin G like domain (LamG) of Thbs4 exhibits intracellular localization almost exclusively at the Golgi apparatus and is robustly secreted in cultured cardiomyocytes, indicating rapid flux through the secretory pathway. We also generated a full length Thbs4 with mutations in calcium-binding motifs within the type III repeat (T3R) domain, including mutations homologous to human disease-causing mutations identified in THBS5/COMP. While wildtype Thbs4 localized to the ER and post-ER vesicles and was actively secreted in cardiomyocytes, the Thbs4 calcium-binding mutant localized predominately to the ER and was not secreted. We identified BiP (Grp78) as a novel binding partner of Thbs4 in cardiomyocytes in vitro and in vivo, and utilized adenoviruses overexpressing Thbs4 domain mutants in cardiomyocytes to map the TSP-C domain (L-type lectin domain) of Thbs4 as the BiP-interacting motif. Additionally, the TSP-C domain of Thbs4 bound Atf6α and overexpression of the TSP-C domain alone was sufficient to induce the adaptive ER stress pathway in cardiomyocytes, activate Atf6α, and protect against ER stress-induced cell death. These studies characterize the functional domains of Thbs4 and aid in parsing out the intracellular and extracellular roles of thrombospondins. Taken together, data indicate critical functions for the TSP-C and LamG domains of Thbs4 in ER stress-responsive and secretory pathway functions, respectively.

scholarly journals Endoplasmic Reticulum Stress-Related Inflammation and Cardiovascular Diseases

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Tomomi Gotoh ◽  
Motoyoshi Endo ◽  
Yuichi Oike
Keyword(s):  
Endoplasmic Reticulum ◽  
Stress Response ◽  
Cardiovascular Diseases ◽  
Inflammatory Response ◽  
Er Stress ◽  
Er Stress Response ◽  
Stress Response Pathway ◽  
Apoptosis Pathways ◽  
Novel Target ◽  
Stress Pathway

The endoplasmic reticulum (ER) is the site of synthesis and maturation of proteins designed for secretion or for localization on the cell membrane. Various types of stress from both inside and outside cells disturb ER function, thus causing unfolded or misfolded proteins to accumulate in the ER. To improve and maintain the ER functions against such stresses, the ER stress response pathway is activated. However, when the stress is prolonged or severe, apoptosis pathways are activated to remove damaged cells. It was recently reported that the ER stress pathway is also involved in the inflammatory response, whereby inflammation induces ER stress, and ER stress induces an inflammatory response. Therefore, the ER stress response pathway is involved in various diseases, including cardiovascular diseases such as atherosclerosis and ischemic diseases, in various ways. The ER stress pathway may represent a novel target for the treatment of these diseases.

scholarly journals Sigmar1 regulates endoplasmic reticulum stress-induced C/EBP-homologous protein expression in cardiomyocytes

2017 ◽  
Vol 37 (4) ◽  
Author(s):  
Shafiul Alam ◽  
Chowdhury S. Abdullah ◽  
Richa Aishwarya ◽  
A. Wayne Orr ◽  
James Traylor ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Stress Response ◽  
Er Stress ◽  
Neonatal Rat ◽  
Cellular Toxicity ◽  
Sigma 1 Receptor ◽  
Homologous Protein ◽  
Er Stress Response ◽  
Chop Expression ◽  
Stress Pathway

C/EBP-homologous protein (CHOP) is a ubiquitously expressed stress-inducible transcription factor robustly induced by maladaptive endoplasmic reticulum (ER) stresses in a wide variety of cells. Here, we examined a novel function of Sigma 1 receptor (Sigmar1) in regulating CHOP expression under ER stress in cardiomyocytes. We also defined Sigmar1-dependent activation of the adaptive ER-stress pathway in regulating CHOP expression. We used adenovirus-mediated Sigmar1 overexpression as well as Sigmar1 knockdown by siRNA in neonatal rat ventricular cardiomyocytes (NRCs); to induce ER stress, cardiomyocytes were treated with tunicamycin. Sigmar1-siRNA knockdown significantly increased the expression of CHOP and significantly induced cellular toxicity by sustained activation of ER stress in cardiomyocytes. Sigmar1 overexpression decreased the expression of CHOP and significantly decreased cellular toxicity in cells. Using biochemical and immunocytochemical experiments, we also defined the specific ER-stress pathway associated with Sigmar1-dependent regulation of CHOP expression and cellular toxicity. We found that Sigmar1 overexpression significantly increased inositol requiring kinase 1α (IRE1α) phosphorylation and increased spliced X-box-binding proteins (XBP1s) expression as well as nuclear localization. In contrast, Sigmar1 knockdown significantly decreased IRE1α phosphorylation and decreased XBP1s expression as well as nuclear transport. Taken together, these results indicate that Sigmar1-dependent activation of IRE1α-XBP1s ER-stress response pathways are associated with inhibition of CHOP expression and suppression of cellular toxicity. Hence, Sigmar1 is an essential component of the adaptive ER-stress response pathways eliciting cellular protection in cardiomyocytes.

scholarly journals Gut bacterial metabolites modulate endoplasmic reticulum stress

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Xiaobo Ke ◽  
Kwontae You ◽  
Matthieu Pichaud ◽  
Henry J. Haiser ◽  
Daniel B. Graham ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Stress Response ◽  
Epithelial Cell ◽  
Er Stress ◽  
Pathogenic Bacteria ◽  
Molecular Mechanisms ◽  
Protease Inhibition ◽  
Bacterial Metabolites ◽  
Er Stress Response ◽  
Stress Pathway

Abstract 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.

Abstract 257: Endogenous Activating Transcription Factor 6 Preserves Heart Structure and Function in a Mouse Model of Myocardial Infarction-induced Heart Failure

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Winston T Stauffer ◽  
Khalid Azizi ◽  
Erik A Blackwood ◽  
Randal J Kaufman ◽  
Christopher C Glembotski
Keyword(s):  
Myocardial Infarction ◽  
Heart Failure ◽  
Transcription Factor ◽  
Stress Response ◽  
Er Stress ◽  
Heart Function ◽  
Structure And Function ◽  
Er Stress Response ◽  
Activating Transcription Factor ◽  
And Function

Rationale: The ER stress response is activated by the accumulation of misfolded, toxic proteins in the endoplasmic reticulum (ER), and upregulates proteins that restore ER protein-folding capacity. The ER-transmembrane protein, activating transcription factor 6 (ATF6) senses ER stress and responds by transcriptionally inducing many of these genes and is thus a key component of the adaptive ER stress response. We previously showed that in the heart, ischemia activates ATF6. Furthermore, transgenic mouse hearts expressing a conditionally activated form of ATF6, and subjected to ex vivo ischemia/reperfusion, exhibited preserved heart function and smaller infarcts. Our lab also showed that by serving as a novel inducer of a global anti-oxidant gene program, endogenous ATF6 limits cardiac damage caused by reactive oxygen species during reperfusion. However, the effect of endogenous ATF6 in the failing heart is not known. Given that acute ischemia caused by occlusion of the coronary arteries is the cause of myocardial infarction (MI), we hypothesized that endogenous ATF6 limits infarct size and preserves heart function during MI. Additionally, since deleterious cardiac remodeling and heart failure can be long-term consequences of MI, we hypothesized that ATF6 can mitigate these effects. Objective/Methods: To examine the role of endogenous ATF6 in heart failure, in vivo, we used a mouse model of MI-induced heart failure in mice with a global deletion of the ATF6 gene (ATF6 KO). Infarct size was measured by TTC staining and heart function was observed via longitudinal echocardiogram. Results: We found that following infarction, ATF6 KO mouse hearts had larger infarcts compared to control. Thus, ischemic cardiac tissue in the peri-infarct region requires ATF6 to limit cardiac myocyte death. Interestingly, ejection fraction following MI decreased more over 13 weeks in ATF6 KO mice relative to control. While control and ATF6 KO mouse hearts hypertrophied to a similar degree, KO mice showed greater cardiac dilation. Conclusions: Together these findings show for the first time that endogenous ATF6 acts to preserve heart structure and function in an MI model of heart failure, suggesting that ATF6 may be a viable therapeutic target for treatment of this disease.

scholarly journals Differential regulation of the endoplasmic reticulum stress response in pancreatic β-cells exposed to long-chain saturated and monounsaturated fatty acids

2008 ◽  
Vol 197 (3) ◽  
pp. 553-563 ◽  
Author(s):  
Eleftheria Diakogiannaki ◽  
Hannah J Welters ◽  
Noel G Morgan
Keyword(s):  
Fatty Acids ◽  
Endoplasmic Reticulum ◽  
Stress Response ◽  
Er Stress ◽  
Initiation Factor ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Er Stress Response ◽  
Stress Pathway ◽  
Long Chain

Exposure of pancreatic β-cells to long-chain fatty acids leads to the activation of some components of the endoplasmic reticulum (ER) stress pathway and this mechanism may underlie the ability of certain fatty acids to promote β-cell death. We have studied ER stress in BRIN-BD11 β-cells exposed to either the saturated fatty acid palmitate (C16:0) or the monounsaturated palmitoleate (C16:1). Palmitate (0.025–0.25 mM) induced the expression of various markers of the RNA-dependent protein kinase-like ER eukaryotic initiation factor 2α (eIF2α) kinase (PERK)-dependent pathway of ER stress (phospho-eIF2α; ATF4, activating transcription factor 4 and C/EBP homologous protein (CHOP-10)) although it failed to promote the expression of the ER chaperone GRP78. By contrast, palmitoleate did not induce any markers of the ER stress pathway even at concentrations as high as 1 mM. When palmitate and palmitoleate were added in combination, a marked attenuation of the ER stress response occurred. Under these conditions, the levels of phospho-eIF2α, ATF4 and CHOP-10 were reduced to less than those found in control cells. Palmitoleate also attenuated the ER stress response to the protein glycosylation inhibitor, tunicamycin, and improved the viability of the cells exposed to this agent. Exposure of the BRIN-BD11 cells to the protein phosphatase inhibitor, salubrinal, in the absence of fatty acids resulted in increased eIF2α phosphorylation but this was abolished by co-incubation with palmitoleate. We conclude that saturated fatty acids activate components of the PERK-dependent ER stress pathway in β-cells, ultimately leading to increased apoptosis. This effect is antagonised by monounsaturates that may exert their anti-apoptotic actions by regulating the activity of one or more kinase enzymes involved in mediating the phosphorylation of eIF2α.

scholarly journals Defective Flux of Thrombospondin-4 through the Secretory Pathway Impairs Cardiomyocyte Membrane Stability and Causes Cardiomyopathy

2018 ◽  
Vol 38 (14) ◽  
Author(s):  
Matthew J. Brody ◽  
Davy Vanhoutte ◽  
Tobias G. Schips ◽  
Justin G. Boyer ◽  
Chinmay V. Bakshi ◽  
...  
Keyword(s):  
Stress Response ◽  
Er Stress ◽  
Secretory Pathway ◽  
Tissue Injury ◽  
Wild Type ◽  
Content Type ◽  
Er Stress Response ◽  
Defective Mutant ◽  
Intracellular Vesicles ◽  
Thrombospondin 4

ABSTRACT Thrombospondins are stress-inducible secreted glycoproteins with critical functions in tissue injury and healing. Thrombospondin-4 (Thbs4) is protective in cardiac and skeletal muscle, where it activates an adaptive endoplasmic reticulum (ER) stress response, induces expansion of the ER, and enhances sarcolemmal stability. However, it is unclear if Thbs4 has these protective functions from within the cell, from the extracellular matrix, or from the secretion process itself. In this study, we generated transgenic mice with cardiac cell-specific overexpression of a secretion-defective mutant of Thbs4 to evaluate its exclusive intracellular and secretion-dependent functions. Like wild-type Thbs4, the secretion-defective mutant upregulates the adaptive ER stress response and expands the ER and intracellular vesicles in cardiomyocytes. However, only the secretion-defective Thbs4 mutant produces cardiomyopathy with sarcolemmal weakness and rupture that is associated with reduced adhesion-forming glycoproteins in the membrane. Similarly, deletion of Thbs4 in the mdx mouse model of Duchenne muscular dystrophy enhances cardiomyocyte membrane instability and cardiomyopathy. Finally, overexpression of the secretion-defective Thbs4 mutant in Drosophila , but not wild-type Thbs4, impaired muscle function and sarcomere alignment. These results suggest that transit through the secretory pathway is required for Thbs4 to augment sarcolemmal stability, while ER stress induction and vesicular expansion mediated by Thbs4 are exclusively intracellular processes.

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