Methods for Studying ER Stress and UPR Markers in Human Cells

Kennedy, Donna; Samali, Afshin; Jäger, Richard

doi:10.1007/978-1-4939-2522-3_1

Cited by 54 publications

(33 citation statements)

References 12 publications

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“…As shown in Figure 1A-E, TG treatment rapidly induced potent upregulation of GRP78, ATF4, CHOP, XBP1s and ATF6. Since the expression of ER stress-related genes is used as a marker of ER stress (Dey et al, 2010;Kennedy et al, 2015;Namba et al, 2007), these results indicate a successful induction of ER stress in our experimental condition. Associated with the increase of ER stress, mRNAs of CXCL10 and CCL2 were robustly increased in a time-dependent manner and reached peak expression at 3 to 6 hours after TG treatment ( Figure 1F and 1G).…”

Section: Endoplasmic Reticulum (Er) Stress Induces Cxcl10 and Ccl2 Prsupporting

confidence: 59%

PERK and XBP1 differentially regulate CXCL10 and CCL2 production

Zhu

Liu

Sha

et al. 2017

Experimental Eye Research

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Inflammation plays a key role in the pathogenesis of many retinal degenerative diseases related with photoreceptor dysfunction/degeneration. However the involvement of photoreceptor cells in inflammatory reactions is largely unknown as they are not considered as inflammatory cells. In this study, we assessed whether photoreceptor cells can produce CCL2 and CXCL10, two important players in inflammation during endoplasmic reticulum (ER) stress. After photoreceptor 661W cells were treated with ER stress inducer thapsigargin (TG), induction of ER stress increased CXCL10 and CCL2 expression at both mRNA and protein levels, which was significantly blocked by an ER stress blocker 4-phenylbutyrate. ER stress contains three pathways: PERK, ATF6 and IRE1α. Knockdown of PERK attenuated TG-induced CXCL10 and CCL2 mRNA expression, associated with significant decreases in phosphorylation of NF-κB RelA and STAT3. In contrast to PERK, knockdown of XBP1, which is activated by IRE1α-mediated splicing, robustly enhanced TG-induced CXCL10 and CCL2 expression and phosphorylation of NF-κB RelA and STAT3. Blockade of NF-κB or STAT3 markedly diminished TG-induced CXCL10 and CCL2 expression. The specific roles of PERK and XBP1 in CXCL10 and CCL2 expression were further investigated by treating photoreceptor cells with advanced glycation end products (AGE) and high glucose (HG), two of the major contributors to diabetic complications. Similarly, AGE and HG induced CXCL10 and CCL2 expression in which PERK was a positive regulator while XBP1 was a negative regulator. These studies suggest that photoreceptors may be involved in retinal inflammation by expressing chemokines CXCL10 and CCL2. PERK and IRE1α/XBP1 in the unfolded protein response differentially regulate the expression of CXCL10 and CCL2 likely through modulation of ER stress-induced NF-κB RelA and STAT3 activation.

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Section: Endoplasmic Reticulum (Er) Stress Induces Cxcl10 and Ccl2 Prsupporting

confidence: 59%

PERK and XBP1 differentially regulate CXCL10 and CCL2 production

Zhu

Liu

Sha

et al. 2017

Experimental Eye Research

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“…In several cell lines, ER stress has been found to be activated following UA exposure [37-39] and was reported to be involved in UA-induced apoptosis in one of these studies [39]. The results of the present study revealed that high levels of UA induced ER stress in cultured cardiomyocytes as well as rat myocardium, as evidenced by the increased GRP78, p-PERK, and CHOP expression, which are widely used as markers of ER stress [40-43]. …”

Section: Discussionmentioning

confidence: 55%

Uric Acid Induces Cardiomyocyte Apoptosis via Activation of Calpain-1 and Endoplasmic Reticulum Stress

Yan

Chen

et al. 2018

Cell Physiol Biochem

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Background/Aims: Hyperuricemia is associated with an increased risk for multiple cardiovascular diseases, but the underlying mechanisms remain largely elusive. Calpain-1 is a protease that is implicated in several pathological conditions that affect the heart. The aim of this current study was to test the effects of uric acid (UA) on cardiomyocyte survival and cardiac function and to investigate the role of calpain-1 in the UA-induced effects in the heart and their underlying mechanisms. Methods: In vivo, hyperuricemia was induced by oxonic acid (OA) administration in Sprague-Dawley rats for 16 weeks; TUNEL staining was used to identify apoptotic cells. Left ventricular (LV) sections were stained with Sirius Red to evaluate interstitial fibrosis. Cardiac catheterization was performed to evaluate cardiac function. In vitro, cultured H9c2 cells were incubated with different UA concentrations. MTT assays and flow cytometry were used to evaluate cell viability and apoptosis. All related gene expression levels were analyzed by quantitative real-time PCR (qRT-PCR), and all protein expression levels were analyzed by western blotting. Results: Hyperuricemia induction in vivo resulted in cellular apoptosis, interstitial fibrosis and diastolic dysfunction in the rat hearts, as well as increased activation of calpain-1 and endoplasmic reticulum (ER) stress, while allopurinol treatment mitigated the above changes. UA administration in vitro increased apoptosis and decreased H9c2 cell viability in a dose-dependent manner. Increased activation of calpain-1 and ER stress was also observed in the groups with high UA levels. Calpain-1 siRNA and the calpain inhibitor CI-III alleviated UA-induced ER stress and apoptosis, while inhibiting ER stress by tauroursodeoxycholic acid (TUDCA) mitigated UA-induced apoptosis without affecting calpain-1 expression or activity. Conclusions: These findings suggest that UA induces cardiomyocyte apoptosis through activation of calpain-1 and ER stress. These results may provide new insights into the mechanisms of hyperuricemia-associated cardiovascular risks and hopefully identify new treatment targets.

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“…A previous study has suggested that the IRE1 pathway is involved in the ER stress response (15). The RNase activity of IRE1α is activated following ER stress and its spliced form may reflect the activation of IRE1α (16).…”

Section: Involvement Of the Ire1 Pathway With Tsa Treatmentmentioning

confidence: 93%

Trichostatin A induces p53‑dependent endoplasmic reticulum stress in human colon cancer cells

Dai

Zhang

et al. 2018

Oncol Lett

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Trichostatin A (TSA) has been demonstrated to exhibit various anticancer effects that influence cell cycle arrest, cell proliferation and apoptosis of cancer cells. A potential association between TSA and endoplasmic reticulum (ER) function has been suggested but its anticancer mechanism involving the induction of ER stress is unknown. p53 has previously been demonstrated to regulate ER function in response to stress but its role involving TSA and ER stress in cancer cells is poorly understood. The current study identified that TSA induced ER stress in wild type (WT) HCT116 human colon cancer cells. Following TSA treatment, the ER stress markers GRP78 and GRP94 significantly increased without hyperacetylation of their promoter regions. The inositol-requiring enzyme 1 α (IRE1α)/X-box binding protein 1 (XBP1) pathway was implicated due to an association of phosphorylated IRE1α and spliced XBP1 with ER stress. However, luciferase reporter assay indicated that splicing events were attenuated in HCT116 TP53(-/-) cells. Furthermore, cell viability and apoptosis were revealed to depend on p53 during TSA treatment. Cell viability increased and the apoptosis rate decreased in HCT116 TP53(-/-) cells compared with WT HCT116 cells undergoing TSA treatment. In conclusion, the current study revealed that TSA may induce ER stress via a p53-dependent mechanism in colon cancer cells. This provides information that may assist the development of treatments that exploit the anticancer function of TSA.

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Methods for Studying ER Stress and UPR Markers in Human Cells

Cited by 54 publications

References 12 publications

PERK and XBP1 differentially regulate CXCL10 and CCL2 production

PERK and XBP1 differentially regulate CXCL10 and CCL2 production

Uric Acid Induces Cardiomyocyte Apoptosis via Activation of Calpain-1 and Endoplasmic Reticulum Stress

Trichostatin A induces p53‑dependent endoplasmic reticulum stress in human colon cancer cells

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