Dataset Information

Transcriptional Response in Human Jurkat T Lymphocytes to a near Physiological Hypergravity Environment and to One Common in Routine Cell Culture Protocols.

ABSTRACT: Cellular effects of hypergravity have been described in many studies. We investigated the transcriptional dynamics in Jurkat T cells between 20 s and 60 min of 9 g hypergravity and characterized a highly dynamic biphasic time course of gene expression response with a transition point between rapid adaptation and long-term response at approximately 7 min. Upregulated genes were shifted towards the center of the nuclei, whereby downregulated genes were shifted towards the periphery. Upregulated gene expression was mostly located on chromosomes 16-22. Protein-coding transcripts formed the majority with more than 90% of all differentially expressed genes and followed a continuous trend of downregulation, whereas retained introns demonstrated a biphasic time-course. The gene expression pattern of hypergravity response was not comparable with other stress factors such as oxidative stress, heat shock or inflammation. Furthermore, we tested a routine centrifugation protocol that is widely used to harvest cells for subsequent RNA analysis and detected a huge impact on the transcriptome compared to non-centrifuged samples, which did not return to baseline within 15 min. Thus, we recommend carefully studying the response of any cell types used for any experiments regarding the hypergravity time and levels applied during cell culture procedures and analysis.

SUBMITTER: Vahlensieck C

PROVIDER: S-EPMC9863927 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Transcriptional Response in Human Jurkat T Lymphocytes to a near Physiological Hypergravity Environment and to One Common in Routine Cell Culture Protocols.

Vahlensieck Christian C Thiel Cora Sandra CS Mosimann Meret M Bradley Timothy T Caldana Fabienne F Polzer Jennifer J Lauber Beatrice Astrid BA Ullrich Oliver O

International journal of molecular sciences 20230110 2

Cellular effects of hypergravity have been described in many studies. We investigated the transcriptional dynamics in Jurkat T cells between 20 s and 60 min of 9 g hypergravity and characterized a highly dynamic biphasic time course of gene expression response with a transition point between rapid adaptation and long-term response at approximately 7 min. Upregulated genes were shifted towards the center of the nuclei, whereby downregulated genes were shifted towards the periphery. Upregulated ge ...[more]

PMID: 36674869

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Project description:Cellular responses to micro- and hypergravity are rapid and complex and appear within the first few seconds of exposure. Transcriptomic analyses are a valuable tool to analyze these genome-wide cellular alterations. For a better understanding of the cellular dynamics upon altered gravity exposure, it is important to compare different time points. However, since most of the experiments are designed as endpoint measurements, the combination of cross-experiment meta-studies is inevitable. Microarray and RNA-Seq analyses are two of the main methods to study transcriptomics. In the field of altered gravity research, both methods are frequently used. However, the generation of these data sets is difficult and time-consuming and therefore the number of available data sets in this research field is limited. In this study, we investigated the comparability of microarray and RNA-Seq data and applied the results to a comparison of the transcriptomics dynamics between the hypergravity conditions during two real flight platforms and a centrifuge experiment to identify temporal adaptation processes. We performed a comparative study on an Affymetrix HTA2.0 microarray and a paired-end RNA-Seq data set originating from the same Jurkat T cell RNA samples from a short-term hypergravity experiment. The overall agreeability was high, with better sensitivity of the RNA-Seq analysis. The microarray data set showed weaknesses on the level of single upregulated genes, likely due to its normalization approach. On an aggregated level of biotypes, chromosomal distribution, and gene sets, both technologies performed equally well. The microarray showed better performance on the detection of altered gravity-related splicing events. We found that all initially altered transcripts fully adapted after 15 min to hypergravity and concluded that the altered gene expression response to hypergravity is transient and fully reversible. Based on the combined multiple-platform meta-analysis, we could demonstrate rapid transcriptional adaptation to hypergravity, the differential expression of the ATPase subunits ATP6V1A and ATP6V1D, and the cluster of differentiation (CD) molecules CD1E, CD2AP, CD46, CD47, CD53, CD69, CD96, CD164, and CD226 in hypergravity. We could experimentally demonstrate that it is possible to develop methodological evidence for the meta-analysis of individual data.

Project description:Jurkat T-lymphocytes comprise at least four intracellular Ca2+ pools. Pool I was agonist-sensitive and contained 23 +/- 8% (n = 18) of the total Ca(2+)-storage capacity, as shown in intact cells in the presence of EGTA. The time courses of the agonist-induced formation of Ins(1,4,5)P3 and of the Ca2+ release from pool I were nearly superimposable, indicating that the agonist-sensitive pool I is emptied by Ins(1,4,5)P3. Likewise, in permeabilized cells, the size of the Ins(1,4,5)P3-sensitive Ca2+ pool I was 27 +/- 11% (n = 14). Pool II contained 26 +/- 5% (n = 9) of intracellularly stored Ca2+ and was liberated by thapsigargin, an inhibitor of the endoplasmic-reticulum (ER) Ca(2+)-ATPase. Addition of thapsigargin before addition of agonist abolished the agonist-induced Ca2+ release in both intact and permeabilized cells, indicating that pool I is a subcompartment of the ER Ca2+ pool. The content of this ER Ca2+ pool (pools I and II) amounted to 51 +/- 15% (n = 9) in intact cells and 49 +/- 16% (n = 16) in permeabilized cells. Caffeine released Ca2+ even when the ER pool (pools I and II) was emptied by previous addition of thapsigargin, indicating the presence of a third pool independent of pools I and II. Pool III contained 23 +/- 6% (n = 8) in intact cells, but 41 +/- 8% (n = 5) in permeabilized cells. The remaining intracellularly stored Ca2+ was released by addition of the Ca2+ ionophore ionomycin. This fourth pool contained 27 +/- 8% (n = 9) in intact cells, but less than 10% in permeabilized cells. The size of pool III was increased when pools I and II were emptied before addition of caffeine, whereas the size of pool IV was decreased under such conditions. In conclusion, this first comprehensive description of intracellular Ca2+ pools in Jurkat T-lymphocytes demonstrates the presence of four different Ca2+ pools, provides estimates of their sizes and describes relationships between each other. Release of Ca2+ from pool I [Ins(1,4,5)P3-sensitive] has previously been shown to play a major role in T-cell activation, whereas the physiological role of pools II-IV remains to be established.

Dataset Information

Transcriptional Response in Human Jurkat T Lymphocytes to a near Physiological Hypergravity Environment and to One Common in Routine Cell Culture Protocols.

Publications

Transcriptional Response in Human Jurkat T Lymphocytes to a near Physiological Hypergravity Environment and to One Common in Routine Cell Culture Protocols.

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