Characterization of two distinct immortalized endothelial cell lines, EA.hy926 and HMEC-1, for in vitro studies: exploring the impact of calcium electroporation, Ca2+ signaling and transcriptomic profiles.

BACKGROUND: Disruption of Ca2+ homeostasis after calcium electroporation (CaEP) in tumors has been shown to elicit an enhanced antitumor effect with varying impacts on healthy tissue, such as endothelium. Therefore, our study aimed to determine differences in Ca2+ kinetics and gene expression involved in the regulation of Ca2+ signaling and homeostasis, as well as effects of CaEP on cytoskeleton and adherens junctions of the established endothelial cell lines EA.hy926 and HMEC-1. METHODS: CaEP was performed on EA.hy926 and HMEC-1 cells with increasing Ca2+ concentrations. Viability after CaEP was assessed using Presto Blue, while the effect on cytoskeleton and adherens junctions was evaluated via immunofluorescence staining (F-actin, α-tubulin, VE-cadherin). Differences in intracellular Ca2+ regulation ([Ca2+]i) were determined with spectrofluorometric measurements using Fura-2-AM, exposing cells to DPBS, ionomycin, thapsigargin, ATP, bradykinin, angiotensin II, acetylcholine, LaCl3, and GdCl3. Molecular distinctions were identified by analyzing differentially expressed genes and pathways related to the cytoskeleton and Ca2+ signaling through RNA sequencing. RESULTS: EA.hy926 cells, at increasing Ca2+ concentrations, displayed higher CaEP susceptibility and lower survival than HMEC-1. Immunofluorescence confirmed CaEP-induced, time- and Ca2+-dependent morphological changes in EA.hy926's actin filaments, microtubules, and cell-cell junctions. Spectrofluorometric Ca2+ kinetics showed higher amplitudes in Ca2+ responses in EA.hy926 exposed to buffer, G protein coupled receptor agonists, bradykinin, and angiotensin II compared to HMEC-1. HMEC-1 exhibited significantly higher [Ca2+]i changes after ionomycin exposure, while responses to thapsigargin, ATP, and acetylcholine were similar in both cell lines. ATP without extracellular Ca2+ ions induced a significantly higher [Ca2+]i rise in EA.hy926, suggesting purinergic ionotropic P2X and metabotropic P2Y receptor activation. RNA-sequencing analysis showed significant differences in cytoskeleton- and Ca2+-related gene expression, highlighting upregulation of ORAI2, TRPC1, TRPM2, CNGA3, TRPM6, and downregulation of TRPV4 and TRPC4 in EA.hy926 versus HMEC-1. Moreover, KEGG analysis showed upregulated Ca2+ import and downregulated export genes in EA.hy926. CONCLUSIONS: Our finding show that significant differences in CaEP response and [Ca2+]i regulation exist between EA.hy926 and HMEC-1, which may be attributed to distinct transcriptomic profiles. EA.hy926, compared to HMEC-1, displayed higher susceptibility and sensitivity to [Ca2+]i changes, which may be linked to overexpression of Ca2+-related genes and an inability to mitigate changes in [Ca2+]i. The study offers a bioinformatic basis for selecting EC models based on research objectives.

Side-by-side comparison of published small molecule inhibitors against thapsigargin-induced store-operated Ca2+ entry in HEK293 cells.

Calcium (Ca2+) is a key second messenger in eukaryotes, with store-operated Ca2+ entry (SOCE) being the main source of Ca2+ influx into non-excitable cells. ORAI1 is a highly Ca2+-selective plasma membrane channel that encodes SOCE. It is ubiquitously expressed in mammals and has been implicated in numerous diseases, including cardiovascular disease and cancer. A number of small molecules have been identified as inhibitors of SOCE with a variety of potential therapeutic uses proposed and validated in vitro and in vivo. These encompass both nonselective Ca2+ channel inhibitors and targeted selective inhibitors of SOCE. Inhibition of SOCE can be quantified both directly and indirectly with a variety of assay setups, making an accurate comparison of the activity of different SOCE inhibitors challenging. We have used a fluorescence based Ca2+ addback assay in native HEK293 cells to generate dose-response data for many published SOCE inhibitors. We were able to directly compare potency. Most compounds were validated with only minor and expected variations in potency, but some were not. This could be due to differences in assay setup relating to the mechanism of action of the inhibitors and highlights the value of a singular approach to compare these compounds, as well as the general need for biorthogonal validation of novel bioactive compounds. The compounds observed to be the most potent against SOCE in our study were: 7-azaindole 14d (12), JPIII (17), Synta-66 (6), Pyr 3 (5), GSK5503A (8), CM4620 (14) and RO2959 (7). These represent the most promising candidates for future development of SOCE inhibitors for therapeutic use.

Benzomorphan and non-benzomorphan agonists differentially alter sigma-1 receptor quaternary structure, as does types of cellular stress.

Sigma-1 receptor (S1R) is a calcium-sensitive, ligand-operated receptor chaperone present on the endoplasmic reticulum (ER) membrane. S1R plays an important role in ER-mitochondrial inter-organelle calcium signaling and cell survival. S1R and its agonists confer resilience against various neurodegenerative diseases; however, the molecular mechanism of S1R is not yet fully understood. At resting state, S1R is either in a monomeric or oligomeric state but the ratio of these concentrations seems to change upon activation of S1R. S1R is activated by either cellular stress, such as ER-calcium depletion, or ligands. While the effect of ligands on S1R quaternary structure remains unclear, the effect of cellular stress has not been studied. In this study we utilize cellular and an in-vivo model to study changes in quaternary structure of S1R upon activation. We incubated cells with cellular stressors (H2O2 and thapsigargin) or exogenous ligands, then quantified monomeric and oligomeric forms. We observed that benzomorphan-based S1R agonists induce monomerization of S1R and decrease oligomerization, which was confirmed in the liver tissue of mice injected with (+)-Pentazocine. Antagonists block this effect but do not induce any changes when used alone. Oxidative stress (H2O2) increases the monomeric/oligomeric S1R ratio whereas ER calcium depletion (thapsigargin) has no effect. We also analyzed the oligomerization ability of various truncated S1R fragments and identified the fragments favorizing oligomerization. In this publication we demonstrate that quaternary structural changes differ according to the mechanism of S1R activation. Therefore, we offer a novel perspective on S1R activation as a nuanced phenomenon dependent on the type of stimulus.

Endoplasmic reticulum stress is upregulated in inflammatory bowel disease and contributed TLR2 pathway-mediated inflammatory response.

OBJECTIVE: Endoplasmic reticulum stress (ERS) and Toll-like receptor 2 (TLR2) signaling play an important role in inflammatory bowel disease (IBD); however, the link between TLR2 and ERS in IBD is unclear. This study investigated whether Thapsigargin (TG) -induced ER protein expression levels contributed to TLR2-mediated inflammatory response. METHODS: The THP-1 cells were treated with TLR2 agonist (Pam3CSK4), ERS inducer Thapsigargin (TG) or inhibitor (TUDCA). The mRNA expressions of TLR1-TLR10 were detected by qPCR. The production and secretion of inflammatory factors were detected by PCR and ELISA. Immunohistochemistry was used to detect the expressions of GRP78 and TLR2 in the intestinal mucosa of patients with Crohn's disease (CD). The IBD mouse model was established by TNBS in the modeling group. ERS inhibitor (TUDCA) was used in the treatment group. RESULTS: The expression of TLRs was detected via polymerase chain reaction (PCR) in THP-1 cells treated by ERS agonist Thapsigargin (TG). According to the findings, TG could promote TLR2 and TLR5 expression. Subsequently, in TLR2 agonist Pam3CSK4 induced THP-1 cells, TG could lead to increased expression of the inflammatory factors such as TNF-α, IL-1ß and IL-8, and ERS inhibitor (TUDCA) could block this effect. However, Pam3CSK4 did not significantly impact the GRP78 and CHOP expression. Based upon the immunohistochemical results, TLR2 and GRP78 expression were significantly increased in the intestinal mucosa of patients with Crohn's disease (CD). For in vivo experiments, TUDCA displayed the ability to inhibit intestinal mucosal inflammation and reduce GRP78 and TLR2 proteins. CONCLUSIONS: ERS and TLR2 is upregulated in inflammatory bowel disease, ERS may promote TLR2 pathway-mediated inflammatory response. Moreover, ERS and TLR2 signaling could be novel therapeutic targets for IBD.

Cyclin-dependent kinase inhibitor 1 plays a more prominent role than activating transcription factor 4 or the p53 tumour suppressor in thapsigargin-induced G1 arrest.

Background: Thapsigargin (Tg) is a compound that inhibits the SERCA calcium transporter leading to decreased endoplasmic reticulum (ER) Ca2+ levels. Many ER chaperones are required for proper folding of membrane-associated and secreted proteins, and they are Ca2+ dependent. Therefore, Tg leads to the accumulation of misfolded proteins in the ER, activating the unfolded protein response (UPR) to help restore homeostasis. Tg reportedly induces cell cycle arrest and apoptosis in many cell types but how these changes are linked to the UPR remains unclear. The activating transcription factor 4 (ATF4) plays a key role in regulating ER stress-induced gene expression so we sought to determine if ATF4 is required for Tg-induced cell cycle arrest and apoptosis using ATF4-deficient cells. Methods: Two-parameter flow cytometric analysis of DNA replication and DNA content was used to assess the effects of Tg on cell cycle distribution in isogenic HCT116-derived cell lines either expressing or lacking ATF4. For comparison, we similarly assessed the Tg response in isogenic cell lines deleted of the p53 tumour suppressor and the p53-regulated p21WAF1 cyclin-dependent kinase inhibitor important in G1 and G2 arrests induced by DNA damage. Results: Tg led to a large depletion of the S phase population with a prominent increase in the proportion of HCT116 cells in the G1 phase of the cell cycle. Importantly, this effect was largely independent of ATF4. We found that loss of p21WAF1 but not p53 permitted Tg treated cells to enter S phase and synthesize DNA. Therefore, p21WAF1plays an important role in these Tg-induced cell cycle alterations while ATF4 and p53 do not. Remarkably, the ATF4-, p53-and p21WAF1-deficient cell lines were all more sensitive to Tg-induced apoptosis. Taken together, p21WAF1 plays a larger role in regulating Tg-induced G1 and G2 arrests than ATF4 or p53 but these proteins similarly contribute to protection from Tg-induced apoptosis. This work highlights the complex network of stress responses that are activated in response to ER stress.

Synergistic lethality in chronic myeloid leukemia - targeting oxidative phosphorylation and unfolded protein response effectively complements tyrosine kinase inhibitor treatment.

Chronic myeloid leukemia (CML) is effectively treated with tyrosine kinase inhibitors (TKIs), targeting the BCR::ABL1 oncoprotein. Still, resistance to therapy, relapse after treatment discontinuation, and side effects remain significant issues of long-term TKI treatment. Preliminary studies have shown that targeting oxidative phosphorylation (oxPhos) and the unfolded protein response (UPR) are promising therapeutic approaches to complement CML treatment. Here, we tested the efficacy of different TKIs, combined with the ATP synthase inhibitor oligomycin and the ER stress inducer thapsigargin in the CML cell lines K562, BV173, and KU812 and found a significant increase in cell death. Both, oligomycin and thapsigargin, triggered the upregulation of the UPR proteins ATF4 and CHOP, which was inhibited by imatinib. We observed comparable effects on cell death when combining TKIs with the ATP synthase inhibitor 8-chloroadenosine (8-Cl-Ado) as a potentially clinically applicable therapeutic agent. Stress-related apoptosis was triggered via a caspase cascade including the cleavage of caspase 3 and the inactivation of poly ADP ribose polymerase 1 (PARP1). The inhibition of PARP by olaparib also increased CML death in combination with TKIs. Our findings suggest a rationale for combining TKIs with 8-Cl-Ado or olaparib for future clinical studies in CML.

Intracellular remodeling associated with endoplasmic reticulum stress modifies biomechanical compliance of bladder cells.

Bladder cells face a challenging biophysical environment: mechanical cues originating from urine flow and regular contraction to enable the filling voiding of the organ. To ensure functional adaption, bladder cells rely on high biomechanical compliance, nevertheless aging or chronic pathological conditions can modify this plasticity. Obviously the cytoskeletal network plays an essential role, however the contribution of other, closely entangled, intracellular organelles is currently underappreciated. The endoplasmic reticulum (ER) lies at a crucial crossroads, connected to both nucleus and cytoskeleton. Yet, its role in the maintenance of cell mechanical stability is less investigated. To start exploring these aspects, T24 bladder cancer cells were treated with the ER stress inducers brefeldin A (10-40nM BFA, 24 h) and thapsigargin (0.1-100nM TG, 24 h). Without impairment of cell motility and viability, BFA and TG triggered a significant subcellular redistribution of the ER; this was associated with a rearrangement of actin cytoskeleton. Additional inhibition of actin polymerization with cytochalasin D (100nM CytD) contributed to the spread of the ER toward cell periphery, and was accompanied by an increase of cellular stiffness (Young´s modulus) in the cytoplasmic compartment. Shrinking of the ER toward the nucleus (100nM TG, 2 h) was related to an increased stiffness in the nuclear and perinuclear areas. A similar short-term response profile was observed also in normal human primary bladder fibroblasts. In sum, the ER and its subcellular rearrangement seem to contribute to the mechanical properties of bladder cells opening new perspectives in the study of the related stress signaling cascades. Video Abstract.

Lupenone attenuates thapsigargin-induced endoplasmic reticulum stress and apoptosis in pancreatic beta cells possibly through inhibition of protein tyrosine kinase 2 activity.

AIMS: Prolonged high levels of cytokines, glucose, or free fatty acids are associated with diabetes, elevation of cytosolic Ca2+ concentration ([Ca2+]C), and depletion of Ca2+ concentration in the endoplasmic reticulum (ER) of pancreatic beta cells. This Ca2+ imbalance induces ER stress and apoptosis. Lupenone, a lupan-type triterpenoid, is beneficial in diabetes; however, its mechanism of action is yet to be clarified. This study evaluated the protective mechanism of lupenone against thapsigargin-induced ER stress and apoptosis in pancreatic beta cells. MATERIALS AND METHODS: MIN6, INS-1, and native mouse islet cells were used. Western blot for protein expressions, measurement of [Ca2+]C, and in vivo glucose tolerance test were mainly performed. KEY FINDINGS: Thapsigargin increased the protein levels of cleaved caspase 3, cleaved PARP, and the phosphorylated form of JNK, ATF4, and CHOP. Thapsigargin increased the interaction between stromal interaction molecule1 (Stim1) and Orai1, enhancing store-operated calcium entry (SOCE). SOCE is further activated by protein tyrosine kinase 2 (Pyk2), which is Ca2+-dependent and phosphorylates the tyrosine residue at Y361 in Stim1. Lupenone inhibited thapsigargin-mediated Pyk2 activation, suppressed [Ca2+]C, ER stress, and apoptosis. Lupenone restored impaired glucose-stimulated insulin secretion effectuated by thapsigargin and glucose intolerance in a low-dose streptozotocin-induced diabetic mouse model. SIGNIFICANCE: These results suggested that lupenone attenuated thapsigargin-induced ER stress and apoptosis by inhibiting SOCE; this may be due to the hindrance of Pyk2-mediated Stim1 tyrosine phosphorylation. In beta cells that are inevitably exposed to frequent [Ca2+]C elevation, the attenuation of abnormally high SOCE would be beneficial for their survival.

miRNAs associated with endoplasmic reticulum stress and unfolded protein response during decidualization.

RESEARCH QUESTION: Do microRNAs (miRNAs) play a role in regulating endoplasmic reticulum stress (ERS) and unfolded protein response (UPR) in decidualized cells and endometrium associated with reproductive failures? DESIGN: Endometrial stromal cell line St-T1b was decidualized in vitro with 8-Br-cAMP over 5 days, or treated with the ERS inducer thapsigargin. Expression of ERS sensors, UPR markers and potential miRNA regulators was analysed by quantitative PCR. Endometrial biopsies from patients with recurrent pregnancy loss (RPL) and recurrent implantation failure (RIF) were investigated for the location of miRNA expression. RESULTS: Decidualization of St-T1b cells resulted in increased expression of ERS sensors including ATF6α, PERK and IRE1α, and the UPR marker, CHOP. TXNIP, which serves as a link between the ERS pathway and inflammation, as well as inflammasome NLRP3 and interleukin 1ß expression increased in decidualized cells. An in-silico analysis identified miR-17-5p, miR-21-5p and miR-193b-3p as miRNAs potentially involved in regulation of the ERS/UPR pathways and inflammation associated with embryo implantation. Their expression decreased significantly (P ≤ 0.0391) in non-decidualized cells in the presence of thapsigargin. Finally, expression of the selected miRNAs was localized by in-situ hybridization in stromal and glandular epithelial cells in endometrial samples from patients with RPL and RIF. Expression in stroma cells from patients with RPL was lower in comparison with stroma cells from patients with RIF. CONCLUSIONS: Decidualization in St-T1b cells is accompanied by ERS/UPR processes, associated with an inflammatory response that is potentially influenced by miR-17-5p, miR-21-5p and miR-193b-3p. These miRNAs are expressed differentially in stromal cells from patients with RPL and RIF, indicating an alteration in regulation of the ERS/UPR pathways.

Context-dependent effects of CCN2 on ß-cell mass expansion and indicators of cell stress in the setting of acute and chronic stress.

Stimulation of functional ß-cell mass expansion can be beneficial for the treatment of type 2 diabetes. Our group has previously demonstrated that the matricellular protein CCN2 can induce ß-cell mass expansion during embryogenesis, and postnatally during pregnancy and after 50% ß-cell injury. The mechanism by which CCN2 stimulates ß-cell mass expansion is unknown. However, CCN2 does not induce ß-cell proliferation in the setting of euglycemic and optimal functional ß-cell mass. We thus hypothesized that ß-cell stress is required for responsiveness to CCN2 treatment. In this study, a doxycycline-inducible ß-cell-specific CCN2 transgenic mouse model was utilized to evaluate the effects of CCN2 on ß-cell stress in the setting of acute (thapsigargin treatment ex vivo) or chronic [high-fat diet or leptin receptor haploinsufficiency (db/+) in vivo] cellular stress. CCN2 induction during 1 wk or 10 wk of high-fat diet or in db/+ mice had no effect on markers of ß-cell stress. However, CCN2 induction did result in a significant increase in ß-cell mass over high-fat diet alone when animals were fed high-fat diet for 10 wk, a duration known to induce insulin resistance. CCN2 induction in isolated islets treated with thapsigargin ex vivo resulted in upregulation of the gene encoding the Nrf2 transcription factor, a master regulator of antioxidant genes, suggesting that CCN2 further activates this pathway in the presence of cell stress. These studies indicate that the potential of CCN2 to induce ß-cell mass expansion is context-dependent and that the presence of ß-cell stress does not ensure ß-cell proliferation in response to CCN2.NEW & NOTEWORTHY CCN2 promotes ß-cell mass expansion in settings of suboptimal ß-cell mass. Here, we demonstrate that the ability of CCN2 to induce ß-cell mass expansion in the setting of ß-cell stress is context-dependent. Our results suggest that ß-cell stress is necessary but insufficient for CCN2 to increase ß-cell proliferation and mass. Furthermore, we found that CCN2 promotes upregulation of a key antioxidant transcription factor, suggesting that modulation of ß-cell oxidative stress contributes to the actions of CCN2.

MicroRNAs Regulate Ca2+ Homeostasis in Murine Embryonic Stem Cells.

MicroRNAs (miRNAs) are important regulators of embryonic stem cell (ESC) biology, and their study has identified key regulatory mechanisms. To find novel pathways regulated by miRNAs in ESCs, we undertook a bioinformatics analysis of gene pathways differently expressed in the absence of miRNAs due to the deletion of Dicer, which encodes an RNase that is essential for the synthesis of miRNAs. One pathway that stood out was Ca2+ signaling. Interestingly, we found that Dicer-/- ESCs had no difference in basal cytoplasmic Ca2+ levels but were hyperresponsive when Ca2+ import into the endoplasmic reticulum (ER) was blocked by thapsigargin. Remarkably, the increased Ca2+ response to thapsigargin in ESCs resulted in almost no increase in apoptosis and no differences in stress response pathways, despite the importance of miRNAs in the stress response of other cell types. The increased Ca2+ response in Dicer-/- ESCs was also observed during purinergic receptor activation, demonstrating a physiological role for the miRNA regulation of Ca2+ signaling pathways. In examining the mechanism of increased Ca2+ responsiveness to thapsigargin, neither store-operated Ca2+ entry nor Ca2+ clearance mechanisms from the cytoplasm appeared to be involved. Rather, it appeared to involve an increase in the expression of one isoform of the IP3 receptors (Itpr2). miRNA regulation of Itpr2 expression primarily appeared to be indirect, with transcriptional regulation playing a major role. Therefore, the miRNA regulation of Itpr2 expression offers a unique mechanism to regulate Ca2+ signaling pathways in the physiology of pluripotent stem cells.

Obtaining Functional Proteomics Insights From Thermal Proteome Profiling Through Optimized Melt Shift Calculation and Statistical Analysis With InflectSSP.

Thermal proteome profiling (TPP) is an invaluable tool for functional proteomics studies that has been shown to discover changes associated with protein-ligand, protein-protein, and protein-RNA interaction dynamics along with changes in protein stability resulting from cellular signaling. The increasing number of reports employing this assay has not been met concomitantly with new approaches leading to advancements in the quality and sensitivity of the corresponding data analysis. The gap between data acquisition and data analysis tools is important to fill as TPP findings have reported subtle melt shift changes related to signaling events such as protein posttranslational modifications. In this study, we have improved the Inflect data analysis pipeline (now referred to as InflectSSP, available at https://CRAN.R-project.org/package=InflectSSP) to increase the sensitivity of detection for both large and subtle changes in the proteome as measured by TPP. Specifically, InflectSSP now has integrated statistical and bioinformatic functions to improve objective functional proteomics findings from the quantitative results obtained from TPP studies through increasing both the sensitivity and specificity of the data analysis pipeline. InflectSSP incorporates calculation of a "melt coefficient" into the pipeline with production of average melt curves for biological replicate studies to aid in identification of proteins with significant melts. To benchmark InflectSSP, we have reanalyzed two previously reported datasets to demonstrate the performance of our publicly available R-based program for TPP data analysis. We report new findings following temporal treatment of human cells with the small molecule thapsigargin that induces the unfolded protein response as a consequence of inhibition of sarcoplasmic/endoplasmic reticulum calcium ATPase 2A. InflectSSP analysis of our unfolded protein response study revealed highly reproducible and statistically significant target engagement over a time course of treatment while simultaneously providing new insights into the possible mechanisms of action of the small molecule thapsigargin.

Expression of HMGCS2 in intestinal epithelial cells is downregulated in inflammatory bowel disease associated with endoplasmic reticulum stress.

Introduction: The Unfolded Protein Response, a mechanism triggered by the cell in response to Endoplasmic reticulum stress, is linked to inflammatory responses. Our aim was to identify novel Unfolded Protein Response-mechanisms that might be involved in triggering or perpetuating the inflammatory response carried out by the Intestinal Epithelial Cells in the context of Inflammatory Bowel Disease. Methods: We analyzed the transcriptional profile of human Intestinal Epithelial Cell lines treated with an Endoplasmic Reticulum stress inducer (thapsigargin) and/or proinflammatory stimuli. Several genes were further analyzed in colonic biopsies from Ulcerative Colitis patients and healthy controls. Lastly, we generated Caco-2 cells lacking HMGCS2 by CRISPR Cas-9 and analyzed the functional implications of its absence in Intestinal Epithelial Cells. Results: Exposure to a TLR ligand after thapsigargin treatment resulted in a powerful synergistic modulation of gene expression, which led us to identify new genes and pathways that could be involved in inflammatory responses linked to the Unfolded Protein Response. Key differentially expressed genes in the array also exhibited transcriptional alterations in colonic biopsies from active Ulcerative Colitis patients, including NKG2D ligands and the enzyme HMGCS2. Moreover, functional studies showed altered metabolic responses and epithelial barrier integrity in HMGCS2 deficient cell lines. Conclusion: We have identified new genes and pathways that are regulated by the Unfolded Protein Response in the context of Inflammatory Bowel Disease including HMGCS2, a gene involved in the metabolism of Short Chain Fatty Acids that may have an important role in intestinal inflammation linked to Endoplasmic Reticulum stress and the resolution of the epithelial damage.

Mitochondria-Endoplasmic Reticulum Contact Sites Dynamics and Calcium Homeostasis Are Differentially Disrupted in PINK1-PD or PRKN-PD Neurons.

BACKGROUND: It is generally believed that the pathogenesis of PINK1/parkin-related Parkinson's disease (PD) is due to a disturbance in mitochondrial quality control. However, recent studies have found that PINK1 and Parkin play a significant role in mitochondrial calcium homeostasis and are involved in the regulation of mitochondria-endoplasmic reticulum contact sites (MERCSs). OBJECTIVE: The aim of our study was to perform an in-depth analysis of the role of MERCSs and impaired calcium homeostasis in PINK1/Parkin-linked PD. METHODS: In our study, we used induced pluripotent stem cell-derived dopaminergic neurons from patients with PD with loss-of-function mutations in PINK1 or PRKN. We employed a split-GFP-based contact site sensor in combination with the calcium-sensitive dye Rhod-2 AM and applied Airyscan live-cell super-resolution microscopy to determine how MERCSs are involved in the regulation of mitochondrial calcium homeostasis. RESULTS: Our results showed that thapsigargin-induced calcium stress leads to an increase of the abundance of narrow MERCSs in wild-type neurons. Intriguingly, calcium levels at the MERCSs remained stable, whereas the increased net calcium influx resulted in elevated mitochondrial calcium levels. However, PINK1-PD or PRKN-PD neurons showed an increased abundance of MERCSs at baseline, accompanied by an inability to further increase MERCSs upon thapsigargin-induced calcium stress. Consequently, calcium distribution at MERCSs and within mitochondria was disrupted. CONCLUSIONS: Our results demonstrated how the endoplasmic reticulum and mitochondria work together to cope with calcium stress in wild-type neurons. In addition, our results suggests that PRKN deficiency affects the dynamics and composition of MERCSs differently from PINK1 deficiency, resulting in differentially affected calcium homeostasis. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

PAQR9 regulates glucose homeostasis in diabetic mice and modulates insulin secretion in ß cells in vitro under stress conditions.

Progesterone and adipoQ receptor 9 (PAQR9) is an endoplasmic reticulum (ER)-localized membrane protein that is involved in protein quality control of ER by interacting with BAG6. One of the physiological functions of PAQR9 is regulation of fasting-induced ketogenesis and fatty acid oxidation in the liver via modulating protein degradation of PPARα. However, it is currently unknown whether or not PAQR9 impacts glucose homeostasis. We addressed this question using a Paqr9-deleted mouse model in which type 1 diabetes was induced by streptozotocin injection and type 2 diabetes was induced by high-fat diet (HFD) with streptozotocin injection. Paqr9 deletion improved hyperglycemia and glucose tolerance in both of the diabetic mouse models. In the pancreatic islets, Paqr9 deletion reduced apoptosis of ß cells in type 2 diabetic mice. Paqr9 deletion also reduced HFD-induced hepatic steatosis and adiposity of white adipose tissue. In Min6 cells, overexpression of DUF3538 domain of BAG6 to block the interaction of PAQR9 with BAG6 was able to enhance glucose-stimulated insulin secretion upon treatment with inflammatory factors or thapsigargin, an ER stress inducer. Thapsigargin-induced ER stress markers were also reduced by overexpression of DUF3538 domain. Collectively, these results indicate that PAQR9 has a modulatory role in glucose homeostasis, associated with regulation on insulin secretion of ß cells in vitro under stress conditions.

Functional differences in agonist-induced plasma membrane expression of Orai1α and Orai1ß.

Orai1 is the pore-forming subunit of the store-operated Ca2+ release-activated Ca2+ (CRAC) channels involved in a variety of cellular functions. Two Orai1 variants have been identified, the long form, Orai1α, containing 301 amino acids, and the short form, Orai1ß, which arises from alternative translation initiation from methionines 64 or 71, in Orai1α. Orai1 is mostly expressed in the plasma membrane, but a subset of Orai1 is located in intracellular compartments. Here we show that Ca2+ store depletion leads to trafficking and insertion of compartmentalized Orai1α in the plasma membrane via a mechanism that is independent on changes in cytosolic free-Ca2+ concentration, as demonstrated by cell loading with the fast intracellular Ca2+ chelator dimethyl BAPTA in the absence of extracellular Ca2+ . Interestingly, thapsigargin (TG) was found to be unable to induce translocation of Orai1ß to the plasma membrane when expressed individually; by contrast, when Orai1ß is co-expressed with Orai1α, cell treatment with TG induced rapid trafficking and insertion of compartmentalized Orai1ß in the plasma membrane. Translocation of Orai1 forms to the plasma membrane was found to require the integrity of the actin cytoskeleton. Finally, expression of a dominant negative mutant of the small GTPase ARF6, and ARF6-T27N, abolished the translocation of compartmentalized Orai1 variants to the plasma membrane upon store depletion. These findings provide new insights into the mechanism that regulate the plasma membrane abundance of Orai1 variants after Ca2+ store depletion.

Store-operated Ca2+ entry-sensitive glycolysis regulates neutrophil adhesion and phagocytosis in dairy cows with subclinical hypocalcemia.

Hypocalcemia in dairy cows is associated with a decrease of neutrophil adhesion and phagocytosis, an effect driven partly by changes in the expression of store-operated Ca2+ entry (SOCE)-related molecules. It is well established in nonruminants that neutrophils obtain the energy required for immune function through glycolysis. Whether glycolysis plays a role in the acquisition of energy by neutrophils during hypocalcemia in dairy cows is unknown. To address this relationship, we performed a cohort study and then a clinical trial. Neutrophils were isolated at 2 d postcalving from lactating Holstein dairy cows (average 2.83 ± 0.42 lactations, n = 6) diagnosed as clinically healthy (CON) or with plasma concentrations of Ca2+ <2.0 mmol/L as a criterion for diagnosing subclinical hypocalcemia (HYP, average 2.83 ± 0.42 lactations, n = 6). In the first experiment, neutrophils were isolated from blood of CON and HYP cows and used to analyze aspects of adhesion and phagocytosis function through quantitative reverse-transcription PCR along with confocal laser scanning microscopy, mRNA expression of the glycolysis-related gene hexokinase 2 (HKII), and components of the SOCE moiety ORAI calcium release-activated calcium modulator 1 (ORAI1, ORAI2, ORAI3, stromal interaction molecule 1 [STIM1], and STIM2). Results showed that adhesion and phagocytosis function were reduced in HYP cows. The mRNA expression of adhesion-related syndecan-4 (SDC4), integrin ß9 (ITGA9), and integrin ß3 (ITGB3) and phagocytosis-related molecules complement component 1 R subcomponent (C1R), CD36, tubulinß1 (TUBB1) were significantly decreased in the HYP group. In the second experiment, to address how glycolysis affects neutrophil adhesion and phagocytosis, neutrophils isolated from CON and HYP cows were treated with 2 µM HKII inhibitor benserazide-d3 or 1 µM fructose-bisphosphatase 1 (FBP1) inhibitor MB05032 for 1 h. Results revealed that the HKII inhibitor benserazide-d3 reduced phagocytosis and the mRNA abundance of ITGA9, and CD36 in the HYP group. The FBP1 inhibitor MB05032 increased adhesion and phagocytosis and increased mRNA abundance of HKII, ITGA9, and CD36 in the HYP group. Finally, to investigate the mechanism whereby SOCE-sensitive glycolysis affects neutrophil adhesion and phagocytosis, isolated neutrophils were treated with 1 µM SOCE activator thapsigargin or 50 µM inhibitor 2-APB for 1 h. Results showed that thapsigargin increased mRNA abundance of HKII, ITGA9, and CD36, and increased adhesion and phagocytosis in the HYP group. In contrast, 2-APB decreased mRNA abundance of HKII and both adhesion and phagocytosis of neutrophils in the CON group. Overall, the data indicated that SOCE-sensitive intracellular Ca2+ levels affect glycolysis and help regulate adhesion and phagocytosis of neutrophils during hypocalcemia in dairy cows.

Overexpression of salusin­α upregulates AdipoR2 and activates the PPARα/ApoA5/SREBP­1c pathway to inhibit lipid synthesis in HepG2 cells.

Salusin­α and adiponectin, are vasoactive peptides with numerous similar biological effects related to lipid metabolism. Adiponectin has been shown to reduce fatty acid oxidation and to inhibit lipid synthesis of liver cells through its receptor, adiponectin receptor 2 (AdipoR2), but whether salusin­α is able to interact with AdipoR2, was not previously reported. To investigate this, in vitro experiments were carried out. The overexpression and interference recombinant plasmids were constructed with salusin­α. The lentiviral expression systems of salusin­α overexpression and interference were respectively synthesized in 293T cells, and 293T cells were infected with the lentivirus. Finally, the association between salusin­α and AdipoR2 was analyzed by semi­quantitative PCR. Subsequently, HepG2 cells were also infected with these viruses. The expression levels of AdipoR2, peroxisome proliferator­activated receptor­α (PPARα), apolipoprotein A5 (ApoA5) and sterol regulatory element­binding transcription factor 1 (SREBP­1c) were detected by western blotting, and AdipoR2 inhibitor (thapsigargin) and agonist [4­phenyl butyric acid (PBA)] were used to observe the resultant changes in the aforementioned molecules. The results obtained revealed that the overexpression of salusin­α increased the level of AdipoR2 in 293T and HepG2 cells, led to an upregulation of the levels of PPARα and ApoA5, and inhibited the expression of SREBP­1c, whereas the salusin­α interference lentivirus exerted the opposite effects. Notably, thapsigargin inhibited the expression of AdipoR2, PPARα and ApoA5 in HepG2 cells of pHAGE­Salusin­α group, and caused an increase in the level of SREBP­1c, whereas the opposite effects were observed in pLKO.1­shSalusin­α#1 group upon treatment with PBA. Taken together, these data demonstrated that overexpression of salusin­α upregulated AdipoR2, which in turn activated the PPARα/ApoA5/SREBP­1c signaling pathway to inhibit lipid synthesis in HepG2 cells, thereby providing theoretical data on which to base the clinical application of salusin­α as a novel peptide for molecular intervention in fatty liver disease.

Apigenin Alleviates Endoplasmic Reticulum Stress-Mediated Apoptosis in INS-1 ß-Cells.

The improvement of type 2 diabetes mellitus induced by naturally occurring polyphenols, known as flavonoids, has received considerable attention. However, there is a dearth of information regarding the effect of the trihydroxyflavone apigenin on pancreatic ß-cell function. In the present study, the anti-diabetic effect of apigenin on pancreatic ß-cell insulin secretion, apoptosis, and the mechanism underlying its anti-diabetic effects, were investigated in the INS-ID ß-cell line. The results showed that apigenin concentration-dependently facilitated 11.1-mM glucose-induced insulin secretion, which peaked at 30 µM. Apigenin also concentration-dependently inhibited the expression of endoplasmic reticulum (ER) stress signaling proteins, CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) and cleaved caspase-3, which was elevated by thapsigargin in INS-1D cells, with peak suppression at 30 µM. This was strongly correlated with the results of flow cytometric analysis of annexin V/propidium iodide (PI) staining and DNA fragmentation analysis. Moreover, the increased expression of thioredoxin-interacting protein (TXNIP) induced by thapsigargin was remarkably reduced by apigenin in a concentration-dependent manner. These results suggest that apigenin is an attractive candidate with remarkable and potent anti-diabetic effects on ß-cells, which are mediated by facilitating glucose-stimulated insulin secretion and preventing ER stress-mediated ß-cell apoptosis, the latter of which may be possibly mediated by reduced expression of CHOP and TXNIP, thereby promoting ß-cell survival and function.

Sex differences in islet stress responses support female ß cell resilience.

OBJECTIVE: Pancreatic ß cells play a key role in maintaining glucose homeostasis; dysfunction of this critical cell type causes type 2 diabetes (T2D). Emerging evidence points to sex differences in ß cells, but few studies have examined male-female differences in ß cell stress responses and resilience across multiple contexts, including diabetes. Here, we address the need for high-quality information on sex differences in ß cell and islet gene expression and function using both human and rodent samples. METHODS: In humans, we compared ß cell gene expression and insulin secretion in donors with T2D to non-diabetic donors in both males and females. In mice, we generated a well-powered islet RNAseq dataset from 20-week-old male and female siblings with similar insulin sensitivity. Our unbiased gene expression analysis pointed to a sex difference in the endoplasmic reticulum (ER) stress response. Based on this analysis, we hypothesized female islets would be more resilient to ER stress than male islets. To test this, we subjected islets isolated from age-matched male and female mice to thapsigargin treatment and monitored protein synthesis, cell death, and ß cell insulin production and secretion. Transcriptomic and proteomic analyses were used to characterize sex differences in islet responses to ER stress. RESULTS: Our single-cell analysis of human ß cells revealed sex-specific changes to gene expression and function in T2D, correlating with more robust insulin secretion in human islets isolated from female donors with T2D compared to male donors with T2D. In mice, RNA sequencing revealed differential enrichment of unfolded protein response pathway-associated genes, where female islets showed higher expression of genes linked with protein synthesis, folding, and processing. This differential expression was physiologically significant, as islets isolated from female mice were more resilient to ER stress induction with thapsigargin. Specifically, female islets showed a greater ability to maintain glucose-stimulated insulin production and secretion during ER stress compared with males. CONCLUSIONS: Our data demonstrate sex differences in ß cell gene expression in both humans and mice, and that female ß cells show a greater ability to maintain glucose-stimulated insulin secretion across multiple physiological and pathological contexts.