Experiments from flight- and ground-based model systems suggest that unexpected alterations of the human lymphoblastoid cell line Jurkat, as well as effects on cell growth, metabolism, and apoptosis, can occur in altered gravity conditions. Using a desktop random positioning machine (RPM), we investigated the effects of simulated microgravity on Jurkat cells and their multidrug-resistant subline, Jurkat/A4 cells. The viability of Jurkat/A4 cells decreased after simulated microgravity in contrast with the Jurkat cells. At the same time, the viability between the experimental Jurkat cells and control Jurkat cells was not significantly different. Of note, Jurkat cells appeared as less susceptible to apoptosis than their multidrug-resistant clone Jurkat/A4 cells, whereas cell-cycle analysis showed that the percentage of Jurkat/A4 cells in the S-phase was increased after 72 and 96 h of RPM-simulated microgravity relative to their static counterparts. The differences in Jurkat cells at all phases between static and simulated microgravity were not significant. The surface expression of the intercellular adhesion molecule 3 (ICAM-3)—also known as cluster of differentiation (CD)50—protein was changed for Jurkat/A4 cells following exposure to the RPM. Changes in cell morphology were observed in the Jurkat/A4 cells after 96 h of RPM-simulated microgravity. Thus, we concluded that Jurkat/A4 cells are more sensitive to RPM-simulated microgravity as compared with the parental Jurkat cell line. We also suggest that intercellular adhesion molecule 3 may be an important adhesion molecule involved in the induction of leukocyte apoptosis. The Jurkat/A4 cells with an acquired multidrug resistance phenotype could be a useful model for studying the effects of simulated microgravity and testing anticancer drugs.
The accumulation of misfolded proteins in the endoplasmic reticulum (ER) lumen due to the disruption of the homeostatic system of the ER leads to the induction of the ER stress response. Cellular stress-induced pathways globally transform genes expression on both the transcriptional and post-transcriptional levels with small RNA involvement as regulators of the stress response. The modulation of small RNA processing might represent an additional layer of a complex stress response program. However, it is poorly understood. Here, we studied changes in expression and small RNAs processing upon ER stress in Jurkat T-cells. Induced by ER-stress, depletion of miRNAs among small RNA composition was accompanied by a global decrease of 3′ mono-adenylated, mono-cytodinylated and a global increase of 3′ mono-uridinylated miRNA isoforms. We observed the specific subset of differentially expressed microRNAs, and also the dramatic induction of 32-nt tRNA fragments precisely phased to 5′ and 3′ ends of tRNA from a subset of tRNA isotypes. The induction of these tRNA fragments was linked to Angiogenin RNase, which mediates translation inhibition. Overall, the global perturbations of the expression and processing of miRNAs and tiRNAs were the most prominent features of small RNA transcriptome changes upon ER stress.
Neuronal intracellular chloride concentration ([Cl À ] i) is a crucial determinant of transmission mediated by the c-aminobutyric acid type A receptor (GABA A R), which subserves synaptic and extrasynaptic inhibition as well as excitation. The Cl À ion is the main carrier of charge through the GABA A R; however, bicarbonate ions (HCO À 3) flowing in the opposite direction can also contribute to the net current. The direction of Cl À and HCO À 3 fluxes is determined by the underlying electrochemical gradient, which is controlled by Cl À transporters and channels. Accumulating evidence suggests that active mechanisms of chloride transport across the GABA A R pore can underlie the regulation of [Cl À ] i. Measurement of Cl À / HCO À 3-ATPase activity and Cl À transport in HEK 293FT cells expressing homomeric or heteromeric GABA A R ensembles (a2, b3, or c2) with fluorescent dye for chloride demonstrated that receptor subtypes containing the b3 subunit show enzymatic activity and participate in GABA-mediated or ATP-dependent Cl À transport. GABA-mediated flow of Cl À ions into and out of the cells occurred for a short time period but then rapidly declined. However, Cl À ion flux was stabilized for a long time period in the presence of HCO À 3 ions. The reconstituted b3 subunit isoform, purified as a fusion protein, confirmed that b3 is critical for ATPase; however, only the triplet variant showed the full receptor function. The high sensitivity of the enzyme to c-phosphate inhibitors led us to postulate that the b3 subunit is catalytic. Our discovery of a GABA A R type that requires ATP consumption for chloride movement provides new insight into the molecular mechanisms of inhibitory signaling.
Ovarian cancer (OC) is one of the most common types of cancer among malignancies of the female reproductive system. This pathology is asymptomatic until advanced stages and has a poor prognosis. Our study aimed to search for lncRNA–miRNA–mRNA competing triplets that promote ovarian tumorigenesis. For this purpose, we analyzed tumor samples from the TCGA database and verified the results experimentally in a set of 46 paired samples of tumor and matched histologically unchanged ovarian tissues from OC patients. The list of RNAs selected in silico for experimental studies included 13 mRNAs, 10 lncRNAs, and 5 miRNAs related to epithelial–mesenchymal transition and angiogenesis. We evaluated the expression of these RNAs by qRT-PCR and assessed the correlation between levels of miRNAs, mRNAs, and lncRNAs. Sixteen significant triplets were revealed, in some of which, e.g., OIP5-AS1–miR-203a–c-MET and OIP5-AS1–miR-203a–ZEB2, both lncRNA and mRNA had sites for miR-203a direct binding. Transfection of the OVCAR-3 and SKOV-3 cell lines with the miR-203a mimic was used to confirm the novel links of miR-203a with ZEB2 and c-MET in OC. These connections suggest that the interactomes have the potential for diagnostics of metastasis at early onset.
γ-Aminobutyric acid type A receptors (GABAARs) mediate primarily inhibitory synaptic transmission in the central nervous system. Following fast-paced activation, which provides the selective flow of mainly chloride (Cl−) and less bicarbonate (HCO3−) ions via the pore, these receptors undergo desensitization that is paradoxically prevented by the process of their recovery, referred to as resensitization. To clarify the mechanism of resensitization, we used the cortical synaptoneurosomes from the rat brain and HEK 293FT cells. Here, we describe the effect of γ-phosphate analogues (γPAs) that mimic various states of ATP hydrolysis on GABAAR-mediated Cl− and HCO3− fluxes in response to the first and repeated application of the agonist. We found that depending on the presence of bicarbonate, opened and desensitized states of the wild or chimeric GABAARs had different sensitivities to γPAs. This study presents the evidence that recovery of neuronal Cl− and HCO3− concentrations after desensitization is accompanied by a change in the intracellular ATP concentration via ATPase performance. The transition between the desensitization and resensitization states was linked to changes in both conformation and phosphorylation. In addition, the chimeric β3 isoform did not exhibit the desensitization of the GABAAR-mediated Cl− influx but only the resensitization. These observations lend a new physiological significance to the β3 subunit in the manifestation of GABAAR resensitization.
Zinc ions (Zn2+) are concentrated in various brain regions and can act as a neuromodulator, targeting a wide spectrum of postsynaptic receptors and enzymes. Zn2+ inhibits the GABAARs, and its potency is profoundly affected by the subunit composition and neuronal developmental stage. Although the extracellular amino acid residues of the receptor’s hetero-oligomeric structure are preferred for Zn2+ binding, there are intracellular sites that, in principle, could coordinate its potency. However, their role in modulating the receptor function during postembryonic development remains unclear. The GABAAR possesses an intracellular ATPase that enables the energy-dependent anion transport via a pore. Here, we propose a mechanistic and molecular basis for the inhibition of intracellular GABAAR/ATPase function by Zn2+ in neonatal and adult rats. The enzymes within the scope of GABAAR performance as Cl−ATPase and then as Cl−, HCO3−ATPase form during the first week of postnatal rat development. In addition, we have shown that the Cl−ATPase form belongs to the β1 subunit, whereas the β3 subunit preferably possesses the Cl−, HCO3−ATPase activity. We demonstrated that a Zn2+ with variable efficacy inhibits the GABAAR as well as the ATPase activities of immature or mature neurons. Using fluorescence recording in the cortical synaptoneurosomes (SNs), we showed a competitive association between Zn2+ and NEM in parallel changes both in the ATPase activity and the GABAAR-mediated Cl− and HCO3− fluxes. Finally, by site-directed mutagenesis, we identified in the M3 domain of β subunits the cysteine residue (C313) that is essential for the manifestation of Zn2+ potency.
Нарушения фолдинга белков в эндоплазматическом ретикулуме (ЭПР) являются причиной развития особой формы протеотоксического клеточного стресса - стресса ЭПР. При этом незрелые и неправильно свернутые белки накапливаются в просвете ЭПР и образуют цитотоксичные агрегаты. При стрессе ЭПР в клетке запускается неспецифический защитный механизм - система сигнальных каскадов ответа на белки с нарушенными конформациями (Unfolded Protein Response, UPR). Важным элементом UPR является сигнальный путь, опосредуемый трансмембранным белком ЭПР IRE1, эндорибонуклеазный домен которого при активации вырезает интрон в мРНК XBP1, что приводит к синтезу транскрипционного фактора sXBP1, индуцирующего экспрессию целого ряда проадаптивных генов. Кроме цитопротекторной функции, IRE1 является ключевым регулятором ЭПР-стресс-индуцированной клеточной гибели. Предполагается, что при продолжительной активации IRE1 переключается с проадаптивной на проапототическую регуляцию. Цель . Исследование зависимости выживаемости клеток от длительности активности сигнального пути, опосредуемого рибонуклеазной активностью IRE1, при стрессе эндоплазматического ретикулума. Методика . С применением RT-qPCR, ингибирования эндорибонуклеазного домена IRE1a соединением STF-083010 проведен анализ зависимости выживаемости клеток от периода активности IRE1a при стрессе ЭПР различной интенсивности. Результаты . Установлено, IRE1a оказывает преимущественно цитопротекторное действие при интенсивном стрессе - ингибирование соединением STF-083010 снижает жизнеспособность клеток. Характер зависимости выживаемости клеток от периода активности IRE1a при стрессе ЭПР клеточноспецифичен: выживаемость несекретирующих Т-лимфобластов Jurkat была выше при ингибировании IRE1a на ранних сроках интенсивного стресса, нежели на поздних; для секретирующих эндотелиоцитоподобных клеток EA.hy926 наблюдалось обратное отношение . Protein unfolding in the endoplasmic reticulum (ER) induces a particular form of proteotoxic cellular stress - ER stress: immature and incorrectly folded proteins can accumulate in the ER lumen and form cytotoxic aggregates. Under ER stress, the non-specific protective mechanism, Unfolded Protein Response (UPR), is activated. The key element of UPR is the signaling pathway mediated by transmembrane ER protein IRE1. The activated endoribonuclease domain IRE1a causes non-canonic XBP1 mRNA splicing, which leads to the synthesis of an active transcription factor sXBP1. It induces the expression of proadaptive genes. In addition to its cytoprotective function, IRE1 is also a key regulator of ER stress-induced cell death. It is assumed that with prolonged activation, IRE1 switches from proadaptive to proapototic regulation. Aim. This paper is devoted to studying possible IRE1a switching from proadaptive to proapoptotic regulation. Using the inhibition of the IRE1a endoribonuclease domain by the compound STF-083010, we analyzed the dependence of cell survival on the period of IRE1a activity under ER stress of varying intensity. We observed the cell specificity of this dependence: in non-secreting Jurkat cells, inhibition of IRE1a in the early stages of intense stress was less toxic than in the later ones; in secreting EA.hy926 cells, an inverse relationship was observed. Purpose of the study. The study of the dependence of cell survival on the duration of the activity of the signaling pathway, mediated by the ribonuclease activity of IRE1, during endoplasmic reticulum stress. Methods. Using RT-qPCR, inhibition of the IRE1a endoribonuclease domain by compound STF-083010, the dependence of cell survival on the period of IRE1a activity during ER stress of various intensities was analyzed. Results. IRE1a exerts a predominantly cytoprotective effect under intense stress - inhibition by the compound STF-083010 reduces cell viability. The character of the dependence of cell survival on the period of IRE1a activity under ER stress is cell-specific: the survival of non-secretive T-lymphoblasts Jurkat was higher when IRE1a was inhibited in the early stages of intense stress than in the latter; for secreting endotheliocyte-like cells EA.hy926, an inverse relationship was observed.
Эндотелиальные клетки, выстилающие стенки сосудов, являются одними из важнейших регуляторных элементов кровеносной системы. Непосредственно соприкасаясь с потоком крови, эти механочувствительные клетки способны детектировать свою деформацию через ее тангенциальный компонент (сдвиг) и составляющую, направленную по нормали к поверхности (растяжение). Деформация сдвига является ключевым индуктором комплекса сигнальных путей, опосредуемых тирозинкиназами, интегринами, ионными каналами, вовлекающих также мембранные липиды, гликокаликс и другие клеточные компоненты. На фоне достаточно большого количества данных о сигнальной трансдукции, в литературе меньше внимания уделено клеточной адаптации к сдвиговой деформации и сравнительно мало информации об участии генов стрессового ответа. Гидродинамические условия в определенных зонах сосудистой системы характеризуются значительной неоднородностью, что может приводить к ослаблению обратных связей, необходимых для поддержания гомеостаза в эндотелиальных клетках. Это может способствовать развитию заболеваний, например, таких, как атеросклероз. В обзоре обсуждаются новые аспекты и концепции, связанные с ответами эндотелиоцитов на сдвиговую деформацию и основные методы анализа эффектов сдвиговой деформации in vitro . Цель исследования. Обобщение современных данных о механизмах механочувствительности и механотрансдукции эндотелия. Результаты. В обзоре изложены основные механизмы механочувствительности клеток эндотелия, пути внутриклеточной передачи сигнала, рассмотрено вовлечение механизмов стрессового ответа клеток и адаптации. Обсуждаются эксперименты по изучению молекулярных основ механотрансдукции, в том числе белков и других молекул, вовлеченных в детектирование, передачу сигнала и клеточный ответ на сдвиговую деформацию. Endothelial cells lining the walls of blood vessels are one of the most important regulatory elements of the circulatory system. These mechanosensitive cells are in a direct contact with the flow of blood and able to detect deformation through its tangential component (shear) and the component directed along the normal to the surface (tension). Shear stress is the key inducer of the complex of signaling pathways mediated by tyrosine kinases, integrins, ion channels, involving also membrane lipids, glycocalyx and other cellular components. There are large amount of data on signal transduction in the literature, but less attention is paid to cellular adaptation to shear stress and there is relatively little information on the involvement of stress response genes in that process. Hydrodynamic conditions in certain zones of the vascular system are characterized by considerable heterogeneity, which can lead to weakening of feedbacks necessary for maintaining homeostasis in endothelial cells. This can contribute to the development of diseases such as atherosclerosis. This review presents new aspects and concepts related to the responses of endotheliocytes to shear stress and, in addition, highlights the basic methods of analyzing the effects of shear stress in vitro . Purpose of the study. Generalization of modern data on mechanisms of mechanosensitivity and mechanotransduction of the endothelium. Results. The review outlines the main mechanosensitivity mechanisms of endothelial cells, the pathways of intracellular signaling, the involvement of mechanisms of cellular stress response and adaptation. There are descriptions of experiments in which the molecular basis of mechanotransduction is identified, including the determination of proteins and other molecules involved in detection, signal transduction, and cellular response to shear stress.
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