RESUMEN
The double-stranded DNA (dsDNA) sensor STING has been increasingly implicated in responses to "sterile" endogenous threats and pathogens without nominal DNA or cyclic di-nucleotide stimuli. Previous work showed an endoplasmic reticulum (ER) stress response, known as the unfolded protein response (UPR), activates STING. Herein, we sought to determine if ER stress generated a STING ligand, and to identify the UPR pathways involved. Induction of IFN-ß expression following stimulation with the UPR inducer thapsigargin (TPG) or oxygen glucose deprivation required both STING and the dsDNA-sensing cyclic GMP-AMP synthase (cGAS). Furthermore, TPG increased cytosolic mitochondrial DNA, and immunofluorescence visualized dsDNA punctae in murine and human cells, providing a cGAS stimulus. N-acetylcysteine decreased IFN-ß induction by TPG, implicating reactive oxygen species (ROS). However, mitoTEMPO, a mitochondrial oxidative stress inhibitor did not impact TPG-induced IFN. On the other hand, inhibiting the inositol requiring enzyme 1 (IRE1) ER stress sensor and its target transcription factor XBP1 decreased the generation of cytosolic dsDNA. iNOS upregulation was XBP1-dependent, and an iNOS inhibitor decreased cytosolic dsDNA and IFN-ß, implicating ROS downstream of the IRE1-XBP1 pathway. Inhibition of the PKR-like ER kinase (PERK) pathway also attenuated cytoplasmic dsDNA release. The PERK-regulated apoptotic factor Bim was required for both dsDNA release and IFN-ß mRNA induction. Finally, XBP1 and PERK pathways contributed to cytosolic dsDNA release and IFN-induction by the RNA virus, Vesicular Stomatitis Virus (VSV). Together, our findings suggest that ER stressors, including viral pathogens without nominal STING or cGAS ligands such as RNA viruses, trigger multiple canonical UPR pathways that cooperate to activate STING and downstream IFN-ß via mitochondrial dsDNA release.
Asunto(s)
Citosol , Estrés del Retículo Endoplásmico , Interferón beta , Proteínas de la Membrana , Nucleotidiltransferasas , Respuesta de Proteína Desplegada , Humanos , Animales , Ratones , Nucleotidiltransferasas/metabolismo , Citosol/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Interferón beta/metabolismo , ADN/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , eIF-2 Quinasa/metabolismo , Endorribonucleasas/metabolismo , Proteína 1 de Unión a la X-Box/metabolismo , Proteína 1 de Unión a la X-Box/genética , Tapsigargina/farmacología , Especies Reactivas de Oxígeno/metabolismo , Activación Transcripcional , ADN Mitocondrial/metabolismoRESUMEN
Swine enteric coronaviruses (SeCoVs) pose a significant threat to the global pig industry, but no effective drugs are available for treatment. Previous research has demonstrated that thapsigargin (TG), an ER stress inducer, has broad-spectrum antiviral effects on human coronaviruses. In this study, we investigated the impact of TG on transmissible gastroenteritis virus (TGEV) infection using cell lines, porcine intestinal organoid models, and piglets. The results showed that TG effectively inhibited TGEV replication both in vitro and ex vivo. Furthermore, animal experiments demonstrated that oral administration of TG inhibited TGEV infection in neonatal piglets and relieved TGEV-associated tissue injury. Transcriptome analyses revealed that TG improved the expression of the ER-associated protein degradation (ERAD) component and influenced the biological processes related to secretion, nutrient responses, and epithelial cell differentiation in the intestinal epithelium. Collectively, these results suggest that TG is a potential novel oral antiviral drug for the clinical treatment of TGEV infection, even for infections caused by other SeCoVs.
Asunto(s)
Antivirales , Gastroenteritis Porcina Transmisible , Tapsigargina , Virus de la Gastroenteritis Transmisible , Animales , Virus de la Gastroenteritis Transmisible/efectos de los fármacos , Virus de la Gastroenteritis Transmisible/fisiología , Porcinos , Gastroenteritis Porcina Transmisible/tratamiento farmacológico , Gastroenteritis Porcina Transmisible/virología , Antivirales/farmacología , Tapsigargina/farmacología , Línea Celular , Replicación Viral/efectos de los fármacosRESUMEN
The pathogenesis of non-alcoholic fatty liver disease (NAFLD) is influenced by a number of variables, including endoplasmic reticulum stress (ER). Thioredoxin domain-containing 5 (TXNDC5) is a member of the protein disulfide isomerase family and acts as an endoplasmic reticulum (ER) chaperone. Nevertheless, the function of TXNDC5 in hepatocytes under ER stress remains largely uncharacterized. In order to identify the role of TXNDC5 in hepatic wild-type (WT) and TXNDC5-deficient (KO) AML12 cell lines, tunicamycin, palmitic acid, and thapsigargin were employed as stressors. Cell viability, mRNA, protein levels, and mRNA splicing were then assayed. The protein expression results of prominent ER stress markers indicated that the ERN1 and EIF2AK3 proteins were downregulated, while the HSPA5 protein was upregulated. Furthermore, the ATF6 protein demonstrated no significant alterations in the absence of TXNDC5 at the protein level. The knockout of TXNDC5 has been demonstrated to increase cellular ROS production and its activity is required to maintain normal mitochondrial function during tunicamycin-induced ER stress. Tunicamycin has been observed to disrupt the protein levels of HSPA5, ERN1, and EIF2AK3 in TXNDC5-deficient cells. However, palmitic acid has been observed to disrupt the protein levels of ATF6, HSPA5, and EIF2AK3. In conclusion, TXNDC5 can selectively activate distinct ER stress pathways via HSPA5, contingent on the origin of ER stress. Conversely, the absence of TXNDC5 can disrupt the EIF2AK3 cascade.
Asunto(s)
Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico , Retículo Endoplásmico , Hepatocitos , Proteína Disulfuro Isomerasas , Transducción de Señal , Tunicamicina , Chaperón BiP del Retículo Endoplásmico/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Proteína Disulfuro Isomerasas/genética , Hepatocitos/metabolismo , Animales , Tunicamicina/farmacología , Retículo Endoplásmico/metabolismo , Ratones , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción Activador 6/metabolismo , Factor de Transcripción Activador 6/genética , Línea Celular , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Endorribonucleasas/metabolismo , Endorribonucleasas/genética , Ácido Palmítico/farmacología , Ácido Palmítico/metabolismo , Tapsigargina/farmacología , Humanos , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/patología , Tiorredoxinas/metabolismo , Tiorredoxinas/genética , Supervivencia Celular/efectos de los fármacosRESUMEN
Endoplasmic reticulum (ER) stress in oligodendrocyte (OL) linage cells contributes to several CNS pathologies including traumatic spinal cord injury (SCI) and multiple sclerosis. Therefore, primary rat OL precursor cell (OPC) transcriptomes were analyzed using RNASeq after treatments with two ER stress-inducing drugs, thapsigargin (TG) or tunicamycin (TM). Gene ontology term (GO) enrichment showed that both drugs upregulated mRNAs associated with the general stress response. The GOs related to ER stress were only enriched for TM-upregulated mRNAs, suggesting greater ER stress selectivity of TM. Both TG and TM downregulated cell cycle/cell proliferation-associated transcripts, indicating the anti-proliferative effects of ER stress. Interestingly, many OL lineage-enriched mRNAs were downregulated, including those for transcription factors that drive OL identity such as Olig2. Moreover, ER stress-associated decreases of OL-specific gene expression were found in mature OLs from mouse models of white matter pathologies including contusive SCI, toxin-induced demyelination, and Alzheimer's disease-like neurodegeneration. Taken together, the disrupted transcriptomic fingerprint of OL lineage cells may facilitate myelin degeneration and/or dysfunction when pathological ER stress persists in OL lineage cells.
The ER stress response compromises the transcriptomic identity of the OL lineage. Therefore, persistent, pathological ER stress may have a negative impact on structural and/or functional integrity of the white matter.
Asunto(s)
Estrés del Retículo Endoplásmico , Oligodendroglía , Tunicamicina , Animales , Estrés del Retículo Endoplásmico/fisiología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Oligodendroglía/metabolismo , Oligodendroglía/efectos de los fármacos , Ratas , Ratones , Tunicamicina/farmacología , Tapsigargina/farmacología , Ratas Sprague-Dawley , Ratones Endogámicos C57BL , Transcriptoma , Células Cultivadas , FemeninoRESUMEN
CD8+ T cells are essential for adaptive immunity against infection and tumors. Their ability to proliferate after stimulation is crucial to their functionality. Dendritic cells (DCs) are professional antigen-presenting cells that induce their proliferation. Here, we show that thapsigargin-induced LAD2 mast cell (MC) line-released products can impair the ability of monocyte-derived DCs to induce CD8+ T-cell proliferation and the generation of Th1 cytokine-producing T cells. We found that culture medium conditioned with LAD2 MCs previously stimulated with thapsigargin (thapsLAD2) induces maturation of DCs as determined by the maturation markers CD80, CD83, CD86, and HLA-DR. However, thapsLAD2-matured DCs produced no detectable TNFα or IL-12 during the maturation. In addition, although their surface expression of PD-L1 was comparable with the immature or TLR7/8-agonist (R848)-matured DCs, their TIM-3 expression was significantly higher than in immature DCs and even much higher than in R848-matured DCs. In addition, contrary to R848-matured DCs, the thapsLAD2-matured DCs only tended to induce enhanced proliferation of CD4+ T cells than immature DCs. For CD8+ T cells, this tendency was not even detected because thapsLAD2-matured and immature DCs comparably induced their proliferation, which contrasted with the significantly enhanced proliferation induced by R848-matured DCs. Furthermore, these differences were comparably recapitulated in the ability of the tested DCs to induce IFNγ- and IFNγ/TNFα-producing T cells. These findings show a novel mechanism of MC-mediated regulation of adaptive immune responses.
Asunto(s)
Linfocitos T CD8-positivos , Diferenciación Celular , Proliferación Celular , Células Dendríticas , Activación de Linfocitos , Mastocitos , Tapsigargina , Humanos , Células Dendríticas/inmunología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Tapsigargina/farmacología , Proliferación Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Mastocitos/inmunología , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Monocitos/inmunología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Receptor 2 Celular del Virus de la Hepatitis A/metabolismo , Citocinas/metabolismo , Imidazoles/farmacología , Línea CelularRESUMEN
Currently, more than 55 million people around the world suffer from dementia, and Alzheimer's Disease and Related Dementias (ADRD) accounts for nearly 60-70% of all those cases. The spread of Alzheimer's Disease (AD) pathology and progressive neurodegeneration in the hippocampus and cerebral cortex is strongly correlated with cognitive decline in AD patients; however, the molecular underpinning of ADRD's causality is still unclear. Studies of postmortem AD brains and animal models of AD suggest that elevated endoplasmic reticulum (ER) stress may have a role in ADRD pathology through altered neurocellular homeostasis in brain regions associated with learning and memory. To study the ER stress-associated neurocellular response and its effects on neurocellular homeostasis and neurogenesis, we modeled an ER stress challenge using thapsigargin (TG), a specific inhibitor of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), in the induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) of two individuals from our Mexican American Family Study (MAFS). High-content screening and transcriptomic analysis of the control and ER stress-challenged NSCs showed that the NSCs' ER stress response resulted in a significant decline in NSC self-renewal and an increase in apoptosis and cellular oxidative stress. A total of 2300 genes were significantly (moderated t statistics FDR-corrected p-value ≤ 0.05 and fold change absolute ≥ 2.0) differentially expressed (DE). The pathway enrichment and gene network analysis of DE genes suggests that all three unfolded protein response (UPR) pathways, protein kinase RNA-like ER kinase (PERK), activating transcription factor-6 (ATF-6), and inositol-requiring enzyme-1 (IRE1), were significantly activated and cooperatively regulated the NSCs' transcriptional response to ER stress. Our results show that IRE1/X-box binding protein 1 (XBP1) mediated transcriptional regulation of the E2F transcription factor 1 (E2F1) gene, and its downstream targets have a dominant role in inducing G1/S-phase cell cycle arrest in ER stress-challenged NSCs. The ER stress-challenged NSCs also showed the activation of C/EBP homologous protein (CHOP)-mediated apoptosis and the dysregulation of synaptic plasticity and neurotransmitter homeostasis-associated genes. Overall, our results suggest that the ER stress-associated attenuation of NSC self-renewal, increased apoptosis, and dysregulated synaptic plasticity and neurotransmitter homeostasis plausibly play a role in the causation of ADRD.
Asunto(s)
Enfermedad de Alzheimer , Estrés del Retículo Endoplásmico , Humanos , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Tapsigargina/farmacología , Demencia/genética , Demencia/metabolismo , Demencia/patología , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismo , Masculino , Factor de Transcripción Activador 6/metabolismo , Factor de Transcripción Activador 6/genética , Neurogénesis , Proteína 1 de Unión a la X-Box/metabolismo , Proteína 1 de Unión a la X-Box/genética , Femenino , Respuesta de Proteína Desplegada , Factor de Transcripción CHOPRESUMEN
Although the unfolded protein response (UPR) contributes to survival by removing misfolded proteins, endoplasmic reticulum (ER) stress also activates proapoptotic pathways. Changed sensitivity to normal developmental stimuli may underlie observed cardiomyocyte apoptosis in the healthy perinatal heart. We determined in vitro sensitivity to thapsigargin in sheep cardiomyocytes from four perinatal ages. In utero cardiac activation of ER stress and apoptotic pathways was determined at these same ages. Thapsigargin-induced phosphorylation of eukaryotic initiation factor 2 (EIF2A) was decreased by 72% between 135 and 143 dGA (P = 0.0096) and remained low at 1 dPN (P = 0.0080). Conversely, thapsigargin-induced caspase cleavage was highest around the time of birth: cleaved caspase 3 was highest at 1 dPN (3.8-fold vs. 135 dGA, P = 0.0380; 7.8-fold vs. 5 dPN, P = 0.0118), cleaved caspase 7 and cleaved caspase 12 both increased between 135 and 143 dGA (25-fold and 6.9-fold respectively, both P < 0.0001) and remained elevated at 1 dPN. Induced apoptosis, measured by TdT-mediated dUTP nick-end labeling (TUNEL) assay, was highest around the time of birth (P < 0.0001). There were changes in myocardial ER stress pathway components in utero. Glucose (78 kDa)-regulated protein (GRP78) protein levels were high in the fetus and declined after birth (P < 0.0001). EIF2A phosphorylation was profoundly depressed at 1 dPN (vs. 143 dGA, P = 0.0113). In conclusion, there is dynamic regulation of ER proteostasis, ER stress, and apoptosis cascade in the perinatal heart. Apoptotic signaling is more readily activated in fetal cardiomyocytes near birth, leading to widespread caspase cleavage in the newborn heart. These pathways are important for the regulation of normal maturation in the healthy perinatal heart.NEW & NOTEWORTHY Cardiomyocyte apoptosis occurs even in the healthy, normally developing perinatal myocardium. As cardiomyocyte number is a critical contributor to heart health, the sensitivity of cardiomyocytes to endoplasmic reticulum stress leading to apoptosis is an important consideration. This study suggests that the heart has less robust protective mechanisms in response to endoplasmic reticulum stress immediately before and after birth, and that more cardiomyocyte death can be induced by stress in this period.
Asunto(s)
Animales Recién Nacidos , Apoptosis , Miocitos Cardíacos , Tapsigargina , Animales , Apoptosis/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Ovinos , Tapsigargina/farmacología , Femenino , Factor 2 Eucariótico de Iniciación/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Fosforilación , Chaperón BiP del Retículo Endoplásmico , Embarazo , Respuesta de Proteína Desplegada , Células Cultivadas , Proteínas de Choque Térmico/metabolismo , Transducción de Señal , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/efectos de los fármacosRESUMEN
BACKGROUND: SARS-Co-V2 infection can induce ER stress-associated activation of unfolded protein response (UPR) in host cells, which may contribute to the pathogenesis of COVID-19. To understand the complex interplay between SARS-Co-V2 infection and UPR signaling, we examined the effects of acute pre-existing ER stress on SARS-Co-V2 infectivity. METHODS: Huh-7 cells were treated with Tunicamycin (TUN) and Thapsigargin (THA) prior to SARS-CoV-2pp transduction (48 h p.i.) to induce ER stress. Pseudo-typed particles (SARS-CoV-2pp) entry into host cells was measured by Bright GloTM luciferase assay. Cell viability was assessed by cell titer Glo® luminescent assay. The mRNA and protein expression was evaluated by RT-qPCR and Western Blot. RESULTS: TUN (5 µg/mL) and THA (1 µM) efficiently inhibited the entry of SARS-CoV-2pp into host cells without any cytotoxic effect. TUN and THA's attenuation of virus entry was associated with differential modulation of ACE2 expression. Both TUN and THA significantly reduced the expression of stress-inducible ER chaperone GRP78/BiP in transduced cells. In contrast, the IRE1-XBP1s and PERK-eIF2α-ATF4-CHOP signaling pathways were downregulated with THA treatment, but not TUN in transduced cells. Insulin-mediated glucose uptake and phosphorylation of Ser307 IRS-1 and downstream p-AKT were enhanced with THA in transduced cells. Furthermore, TUN and THA differentially affected lipid metabolism and apoptotic signaling pathways. CONCLUSIONS: These findings suggest that short-term pre-existing ER stress prior to virus infection induces a specific UPR response in host cells capable of counteracting stress-inducible elements signaling, thereby depriving SARS-Co-V2 of essential components for entry and replication. Pharmacological manipulation of ER stress in host cells might provide new therapeutic strategies to alleviate SARS-CoV-2 infection.
Asunto(s)
Apoptosis , SARS-CoV-2 , Tapsigargina , Tunicamicina , Respuesta de Proteína Desplegada , Humanos , Apoptosis/efectos de los fármacos , COVID-19/virología , COVID-19/metabolismo , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/fisiología , Transducción de Señal/efectos de los fármacos , Tapsigargina/farmacología , Tunicamicina/farmacología , Respuesta de Proteína Desplegada/efectos de los fármacos , Internalización del Virus/efectos de los fármacosRESUMEN
Cells sense, respond, and adapt to environmental conditions that cause stress. In a previous study using HeLa cells, we isolated reporter cells responding to the endoplasmic reticulum (ER) stress inducers, thapsigargin and tunicamycin, using a highly sensitive promoter trap vector system. Splinkerette PCR and 5' rapid amplification of cDNA ends (5' RACE) identified a novel transcript that is upregulated by ER stress. Its endogenous expression increased approximately 10-fold in response to thapsigargin and tunicamycin within 1 h, but was down-regulated after 4 h. Because the transcript starts from an intron of a long noncoding RNA known as LINC-PINT, we designated the newly identified transcript TISPL (transcript induced by stressors from LINC-PINTlocus). TISPL was also expressed under several other stress conditions. It was particularly increased > 10-fold upon glucose starvation and 7-fold by arsenite exposure. Furthermore, in silico analyses, including a ChIP-atlas search, revealed that there is an ATF4-binding region with a c/ebp-Atf response element (CARE) downstream of the transcription start site of TISPL. Based on these results, we hypothesized that TISPL may be induced by the phospho-eIF2α and ATF4- axis of the integrated stress response pathway, which is known to be activated by the stress conditions listed above. As expected, knockout of ATF4 abolished the stress-induced upregulation of TISPL. Our results indicate that TISPL may be a useful biomarker for detecting stress conditions that activate ATF4. Our highly sensitive trap vector system proved beneficial in discovering new biomarkers.
Asunto(s)
Factor de Transcripción Activador 4 , Estrés del Retículo Endoplásmico , ARN Largo no Codificante , Humanos , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Arsenitos/toxicidad , Arsenitos/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/genética , Células HeLa , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Tapsigargina/farmacología , Tunicamicina/farmacología , Regulación hacia ArribaRESUMEN
The inhibition of the unfolded protein response (UPR), which usually protects cancer cells from stress, may be exploited to potentiate the cytotoxic effect of drugs inducing ER stress. However, in this study, we found that ER stress and UPR activation by thapsigargin or tunicamycin promoted the lysosomal degradation of mutant (MUT) TP53 and that the inhibition of the UPR sensor ATF6, but not of ERN1/IRE1 or EIF2AK3/PERK, counteracted such an effect. ATF6 activation was indeed required to sustain the function of lysosomes, enabling the execution of chaperone-mediated autophagy (CMA) as well as of macroautophagy, processes involved in the degradation of MUT TP53 in stressed cancer cells. At the molecular level, by pharmacological and genetic approaches, we demonstrated that the inhibition of ATF6 correlated with the activation of MTOR and with TFEB and LAMP1 downregulation in thapsigargin-treated MUT TP53 carrying cells. We hypothesize that the rescue of MUT TP53 expression by ATF6 inhibition, could further activate MTOR and maintain lysosomal dysfunction, further inhibiting MUT TP53 degradation, in a vicious circle. The findings of this study suggest that the presence of MUT TP53, which often exerts oncogenic properties, should be considered before approaching treatments combining ER stressors with ATF6 inhibitors against cancer cells, while it could represent a promising strategy against cancer cells that harbor WT TP53.
Asunto(s)
Factor de Transcripción Activador 6 , Estrés del Retículo Endoplásmico , Lisosomas , Serina-Treonina Quinasas TOR , Tapsigargina , Proteína p53 Supresora de Tumor , Respuesta de Proteína Desplegada , Factor de Transcripción Activador 6/metabolismo , Factor de Transcripción Activador 6/genética , Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Humanos , Proteína p53 Supresora de Tumor/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/genética , Tapsigargina/farmacología , Respuesta de Proteína Desplegada/efectos de los fármacos , Respuesta de Proteína Desplegada/genética , Serina-Treonina Quinasas TOR/metabolismo , Autofagia Mediada por Chaperones/efectos de los fármacos , Autofagia Mediada por Chaperones/genética , Mutación/genética , Línea Celular Tumoral , Autofagia/efectos de los fármacos , Autofagia/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Neoplasias/genética , Neoplasias/patología , Neoplasias/metabolismo , Tunicamicina/farmacología , Proteína 1 de la Membrana Asociada a los LisosomasRESUMEN
Autophagy is a double-edged sword for cells; it can lead to both cell survival and death. Calcium (Ca2+) signalling plays a crucial role in regulating various cellular behaviours, including cell migration, proliferation and death. In this study, we investigated the effects of modulating cytosolic Ca2+ levels on autophagy using chemical and optogenetic methods. Our findings revealed that ionomycin and thapsigargin induce Ca2+ influx to promote autophagy, whereas the Ca2+ chelator BAPTA-AM induces Ca2+ depletion and inhibits autophagy. Furthermore, the optogenetic platform allows the manipulation of illumination parameters, including density, frequency, duty cycle and duration, to create different patterns of Ca2+ oscillations. We used the optogenetic tool Ca2+-translocating channelrhodopsin, which is activated and opened by 470 nm blue light to induce Ca2+ influx. These results demonstrated that high-frequency Ca2+ oscillations induce autophagy. In addition, autophagy induction may involve Ca2+-activated adenosine monophosphate (AMP)-activated protein kinases. In conclusion, high-frequency optogenetic Ca2+ oscillations led to cell death mediated by AMP-activated protein kinase-induced autophagy.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Autofagia , Calcio , Optogenética , Proteínas Quinasas Activadas por AMP/metabolismo , Calcio/metabolismo , Señalización del Calcio , Activación Enzimática , Ionomicina/farmacología , Optogenética/métodos , Tapsigargina/farmacologíaRESUMEN
The Endoplasmic Reticulum (ER) is associated with many cellular functions, from post-transcriptional modifications to the proper folding of proteins, and disruption of these functions causes ER stress. Although the relationship between epileptic seizures and ER stress has been reported, the contribution of ER stress pathways to epileptogenesis is still unclear. This study aimed to investigate the possible effects of ER stress-related molecular pathways modulated by mild- and high-dose Thapsigargin (Tg) on absence epileptic activity, CACNA1H and immune responses in WAG/Rij rats. For this purpose, rats were divided into four groups; mild-dose (20 ng) Tg, high-dose (200 ng) Tg, saline, and DMSO and drugs administered intracerebroventriculary. EEG activity was recorded for 1 h and 24 h after drug administration following the baseline recording. In cortex and thalamus tissues, GRP78, ERp57, GAD153 protein changes (Western Blot), Eif2ak3, XBP-1, ATF6, CACNA1H mRNA expressions (RT-PCR), NF-κB and TNF-α levels (ELISA) were measured. Mild-dose-Tg administration resulted in increased spike-wave discharge (SWD) activity at the 24th hour compared to administration of saline, and high-dose-Tg and it also significantly increased the amount of GRP78 protein, the expression of Eif2ak3, XBP-1, and CACNA1H mRNA in the thalamus tissue. In contrast, high-dose-Tg administration suppressed SWD activity and significantly increased XBP-1 and ATF6 mRNA expression in the thalamus, and increased NF-κB and TNF-α levels. In conclusion, our findings indicate that Tg affects SWD occurrence by modulating the unfolded protein response pathway and activating inflammatory processes in a dose-dependent manner.
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Relación Dosis-Respuesta a Droga , Estrés del Retículo Endoplásmico , Tapsigargina , Respuesta de Proteína Desplegada , Animales , Tapsigargina/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Ratas , Masculino , Respuesta de Proteína Desplegada/efectos de los fármacos , FN-kappa B/metabolismo , Inmunidad/efectos de los fármacos , Electroencefalografía , eIF-2 Quinasa/metabolismo , eIF-2 Quinasa/genéticaRESUMEN
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.
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Acetilcolina , Calcio , Calcio/metabolismo , Acetilcolina/metabolismo , Acetilcolina/farmacología , Angiotensina II/farmacología , Bradiquinina/farmacología , Ionomicina/metabolismo , Ionomicina/farmacología , Tapsigargina/metabolismo , Línea Celular , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Perfilación de la Expresión Génica , Electroporación , Adenosina Trifosfato/metabolismoRESUMEN
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.
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Calcio , Inhibidores de Fusión de VIH , Animales , Humanos , Células HEK293 , Tapsigargina , Bioensayo , Calcio de la Dieta , MamíferosRESUMEN
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.
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Benzomorfanos , Calcio , Animales , Ratones , Peróxido de Hidrógeno , Receptor Sigma-1 , Tapsigargina , Señalización del CalcioRESUMEN
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.
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Enfermedad de Crohn , Enfermedades Inflamatorias del Intestino , Ácido Tauroquenodesoxicólico , Ratones , Animales , Humanos , Receptor Toll-Like 2/metabolismo , Chaperón BiP del Retículo Endoplásmico , Tapsigargina/farmacología , Estrés del Retículo EndoplásmicoRESUMEN
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.
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Factor de Transcripción Activador 4 , Proteína p53 Supresora de Tumor , Humanos , Proteína p53 Supresora de Tumor/genética , Tapsigargina/farmacología , Factor de Transcripción Activador 4/genética , Línea Celular Tumoral , ADN , Quinasas Ciclina-Dependientes/metabolismoRESUMEN
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.
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Leucemia Mielógena Crónica BCR-ABL Positiva , Humanos , Proteínas de Fusión bcr-abl , Fosforilación Oxidativa , Tapsigargina/farmacología , Tapsigargina/uso terapéutico , Resistencia a Antineoplásicos , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores Enzimáticos/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Oligomicinas/farmacología , Adenosina Trifosfato/metabolismo , ApoptosisRESUMEN
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.
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Retículo Endoplásmico , Vejiga Urinaria , Humanos , Estrés del Retículo Endoplásmico , Citoesqueleto , Tapsigargina/farmacologíaRESUMEN
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.