Chronic exposure to simulated space conditions predominantly affects cytoskeleton remodeling and oxidative stress response in mouse fetal fibroblasts
- Authors:
- Published online on: May 22, 2014 https://doi.org/10.3892/ijmm.2014.1785
- Pages: 606-615
Abstract
Introduction
In the present study, we established an in vitro model in which primary cultures of fetal fibroblasts from murine origin (PFC) were subjected for 65 h to simulated microgravity, chronic irradiation or a combination. Genome-wide gene expression changes were thereafter assessed by microarrays. For microgravity simulation, we used the random positioning machine (RPM), which is one of the most widely used instruments for this purpose and has proven valuable in many cell types (1–6). As far as cosmic radiation is concerned, simulating the wide variety of ions ranging from low to very high energies encountered in space is problematic, particularly if irradiation is combined with microgravity simulation models. At present, no facility offers the possibility of producing chronic exposures of very high-energy beams consisting of multiple charged particles. We therefore used a source of californium Cf-252 for low-dose rate long-term exposure consisting of a mixture of high-linear energy transfer (LET) neutrons and low-LET gamma-rays (7).
The large amount of data generated with a high-throughput technology such as microarrays constitutes a double-edged sword: whole expression pattern may be recorded, but extracting the relevant information becomes more challenging (8,9). To overcome this problem, analysis tools have been developed, such as single gene statistical analysis methods (SGA), which are widely used to determine the differentially expressed genes, and the gene set enrichment analysis (GSEA), which aims to identify gene expression differences in groups of genes, for instance in those acting synergistically in a cell process (9,10). The two analytical methods were used concomitantly in this study.
Materials and methods
Cell culture
All the animals were handled following the Belgian legislation after approval by the appropriate Ethics Committees (agreement number 08-002). BALB/cJ Rj (Janvier Laboratories, Saint-Berthevin, France) fetuses (three males and three females) originating from two different litters were dissected 17 days post-conception (day 0 being the fertilization day). Their skin was harvested and mechanically dissociated. The obtained tissue was enzymatically digested for 1 h at 37°C in phosphate-buffered saline (PBS; N.V. Invitrogen SA, Merelbeke, Belgium) solution containing 1 mg/ml of collagenase/dispase (Roche, Mannheim, Germany) and 5 mg/ml of trypsin 2,000 E/g (Merck KGaA, Darmstadt, Germany). The enzymatic reaction was subsequently stopped by adding fetal bovine serum (FBS; N.V. Invitrogen SA). The obtained cell suspension was subsequently centrifuged for 10 min at 350 × g and the cells were seeded in 6-well plates in F12 medium supplemented with 20% FBS and 1% penicillin/streptomycin (both from N.V. Invitrogen SA), one fetus skin in each well. The cells were allowed to grow for up to 3 or 4 passages at 37°C (5%, CO2) and were subsequently frozen in FBS with 10% dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO, USA). The primary cultures were then thawed and allowed to grow for two weeks. The cells were seeded at a density of ×105 cells in 12.5 cm2 flasks and allowed to adhere for 24 h prior to treatment.
Simulation of space conditions
Exposure to simulated space conditions included microgravity simulation using the desktop RPM (Dutch Space, Leiden, The Netherlands) and ionizing radiation (IR) (7). The exposure lasted for a period of 65 h. Four treatment conditions were used: controls (CTRL), microgravity simulation (RPM), irradiation and a combination of the treatment methods (RPM and IR). For microgravity simulation, the flasks were completely filled with medium, sealed and placed on the RPM at a rotational velocity between 55 and 65°/sec. Direction, speed and interval were set as random. The CTRL were placed in the same incubator under the same conditions as the treated samples. For chronic low-dose irradiation, the cells were exposed to a mixture of neutrons (98.2%) and gamma-rays (1.8%) directly or indirectly originating from a Cf-252 source were placed at 4.13 m from the incubator. The dosimetry was performed with bubble detectors as previously described (11) for neutron irradiation and with 600 cc ionization chamber (NE) coupled with a Farmer electrometer for gamma-rays. The total dose received was 55.94±19.70 mSv (862 μSv/h), which approximately corresponds to 35 times the dose rate measured on the International Space Station (ISS) (12), the total dose corresponding approximately to a stay of 100 days in the ISS.
RNA extraction
Immediately after treatment, adherent cells were washed in PBS, lysed in 350 ml of AllPrep DNA/RNA/Protein Mini kit lysis buffer (Qiagen, Hilden, Germany) and frozen at −80°C. RNA was extracted using the same kit and its concentration was measured using the Nanodrop spectrophotometer (Thermo Scientific, Waltham, MA, USA) while its quality (RNA integrity number, RIN) was determined with Agilent’s lab-on-chip Bioanalyzer 2100 (Agilent Technologies, Inc., Palo Alto, CA, USA). All the RNA samples had a RIN value of >9.0.
Affymetrix microarrays and data analysis
The RNA was treated using the GeneChip WT cDNA Synthesis and Amplification kit (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s instructions. The resulting RNA was hybridized onto Affymetrix Mouse Gene 1.0 ST arrays.
Raw data (.cel-files) were imported at exon level in Partek Genomics Suite v6.5 (Partek Incorporated, St. Louis, MO, USA). Briefly, robust Multi-array Average (RMA) background correction was applied, data were normalized by quantile normalization and probe set summarization was performed using the median polish method. Gene summarization was performed using One-Step Tukey’s Biweight method. These data were further analyzed with the Partek Genomics Suite software for SGA and by the GSEA software (v2.0, Broad Institute of Harvard and MIT, Cambridge, MA, USA).
For the single gene method, taking into consideration the scan date (also available for the litter), the fetus, the gender and the treatment as factors, a four-way ANOVA was performed to determine the genes that had a significantly altered expression for different conditions. For the pathway analysis, KEGG and PathArt databases were analyzed with ArrayTrack v3.3.0 (National Center for Toxicological Research, Jefferson, AR, USA).
For the GSEA, a selection of 144 gene sets from gene ontology (GO) databases was based on biological relevance (Table I). Gene sets were considered to be significantly differently regulated with a false discovery rate (FDR) when q<0.05.
Results
Single gene analysis revealed that 119 genes were downregulated and 55 genes were upregulated by >1.5-fold change (unadjusted p-value <0.01) across all the treatments (Fig. 1 and an exhaustive list of the differentially expressed genes can be found in Table II). KEGG and PathArt databases indicated that the 54 genes that were downregulated only by RPM treatment were mostly involved in cell cycle regulation (p53- and p21-mediated pathways), in cytoskeleton modeling, cell junctions and cell signaling via integrins, IL-1, and TGF-β. Within the list of individual genes that were downregulated after IR or RPM and IR treatments, no clear pathway was found. On the other hand, in the 52 genes that were upregulated following RPM and RPM and IR treatments, interleukin signaling (IL-11 and MMP) and glutathione metabolism were the most prominent pathways affected. Some genes were differentially expressed by RPM and RPM and IR, however, only a few genes were common between IR and RPM and IR. Six genes were upregulated (S1p3, Rab11b, Ptger3, Vldlr, Cnn1 and Serping1) and only one predicted gene of unknown function was downregulated (Gm13668) in both irradiated treatments (IR and RPM and IR). The upregulated genes were mostly membrane proteins, G-protein coupled (S1p3 and Ptger3) or involved in ligand endocytosis (Rab11b and Vldlr). Cnn1 and Serping1, involved in cytoskeleton organization and peptidase inhibition, respectively, were both upregulated in all the treatments, including RPM.
Table IIDown- and upregulated genes following IR, RPM or RPM and IR treatments (p<0.001, fold change >1.5). |
In contrast to the results obtained by SGA, GSEA revealed a high impact of IR on coordinately differentially expressed genes. A total of 63 gene sets were significantly downregulated following chronic low-dose irradiation. Of the 63 genes, 30 were exclusively enriched in irradiated samples (Fig. 2), although this number may be an overestimation due to redundancy between some of the gene sets. The gene sets that were specifically downregulated after irradiation conditions are mostly involved in DNA damage response, cell signaling, cell cycle, RNA processing and protein turnover (Table III). Moreover, we detected significantly downregulated gene sets involved in cell signaling, cell cycle, transcription, protein turnover, cell shape, adhesion, motility and communication for all the treatments. Of note, two gene sets involved in oxidative phosphorylation were significantly downregulated solely in the RPM and IR samples. No gene set was significantly upregulated in any of the treatments.
Table IIIDownregulated gene sets revealed by GSEA, based on the list of gene sets provided by Fig. 2. |
Discussion
In this study, primary cultures of murine fetal fibroblasts were chronically exposed (65 h) to simulated space conditions including simulated microgravity via RPM and a low-dose mixture of neutrons and gamma-rays (IR). The duration of the experiment was chosen to allow cellular adaptation to the simulated microgravity environment for instance for cytoskeleton remodeling (13,14), in order to decrease the primary stress response mechanisms and to better characterize the effects of chronic exposure to these conditions. Microarrays were performed on RNA harvested from CTRL, IR, RPM and RPM and IR conditions. Microarrays generate a substantial amount of information on the gene expression pattern of cells subjected to a defined treatment. However, a <2-fold difference in the gene expression is often not sufficient to meet the requirements for statistical significance (8). Identification of moderate gene expression differences in groups of genes acting together in a cell process can nevertheless be achieved by means of GSEA. For this reason, we analyzed our microarray output data using the single gene analysis method as well as GSEA.
The RPM has a dominant impact on single gene expression
The SGA method revealed a significant impact of 65 h of simulated microgravity on gene expression in murine fetal fibroblasts. The combination of RPM and IR triggered a differential expression of fewer genes than RPM alone. Only a few genes had an altered expression in IR samples, suggesting that such a low dose of radiation exerted a moderate impact on the expression of individual genes. It was also noted that only a few genes were commonly differentially expressed in all irradiated treatments (IR and RPM and IR), of which there were only six known genes, all upregulated (S1p3, Rab11b, Ptger3, Vldlr, Cnn1 and Serping1), with most of them being involved in cell signaling. No explanation can be provided for the fact that few genes were commonly up- or downregulated in the irradiated treatments (with or without RPM). However the strong effect of RPM may have concealed a more subtle effect of IR, making it statistically less significant.
Among the upregulated genes following RPM treatment, glutathione-S-transferases α 1 and 2 (Gstα1 and Gstα2) were prominent enzymes for the detoxification of breakdown products of oxidative stress (15). However, since the Affymetrix arrays cannot distinguish between the two isoforms due to their very high sequence homology (97%), we cannot dismiss the possibility that only one of the two isoforms was actually affected by the treatment. The modifier subunit of glutathione-cysteine ligase (Gclm) was significantly upregulated as well. The protein encoded by this gene was shown to play an important role in controlling the rate of glutathione synthesis in murine fetal fibroblasts (16). We also report upregulation of the heme oxygenase 1 (Hmox1), a cytoprotective enzyme against oxidative stress (17). In murine fibroblasts, its upregulation by curcumin was found to block radiation-induced reactive oxygen species (ROS) generation (18). Notably, these three genes are targets of the nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) which induces transcription of cytoprotective genes containing antioxidant response elements (19). The transcription factor Nrf2 may therefore play a cytoprotective role against a possible oxidative stress induced by the RPM, which is in line with previous observations of increased oxidative stress in simulated microgravity (20–22).
After RPM treatment, two members of the actin filament family, Actg2 and Acta1 were downregulated. These genes were described in smooth (23) or skeletal muscles (24), respectively. Calponin 1 (Cnn1), a gene coding for a protein involved in the cytoskeleton organization (25), and four and a half LIM domains 1 (Fhl1), which functions in adherens junctions signaling to the cytoskeleton (26), were also downregulated. Notably, the four genes were shown to be regulated by the serum response factor (SRF). SRF was shown to be mediated by the Rho signaling pathway (25–27), which may have been triggered by the RPM. Rho signaling is believed to be an important pathway for focal adhesion assembly and cytoskeleton remodeling in response to cellular tension stress (28) and has been suggested to play a role in the microgravity response (21,29–31). Furthermore, Rho GTPase activities were shown to be increased in dermal fibroblasts subjected to simulated microgravity for 30 and 120 min, thereafter decreasing to reach similar values to those of the CTRL at 48 h of treatment (32). Our hypothesis is that a 65-h exposure to RPM induced downregulation of the Rho signaling pathway, which decreased the activity of the transcription factor SRF, decreasing in turn the expression of genes involved in cytoskeleton organization (Cnn1) and adherens junctions (Fhl1).
IR has a dominant effect on gene sets
At the gene set level, GSEA did not detect any upregulation, except for the structural constituents of the ribosome in IR-treated samples. This result is noteworthy as it did not occur with SGA. Since SGA and GSEA are purely statistical methods, it is unlikely that this result originates from an experimental issue, which may have affected both methods. We also examined the gene set selection, however, a screening of all the gene sets of GO provided the same result. Since the experimental design involved long-term irradiation, it is possible that a feedback loop occurred and decreased the expression pattern of the gene sets.
We identified a significant downregulation of 63 gene sets in response to low-dose IR, although single gene analysis did not reveal any important effects. Of the 63 gene sets, 30 were specifically enriched in IR-treated samples (Fig. 2). These latter gene sets are involved in DNA damage response, cell signaling, cell cycle, RNA processing, protein turnover or cell motility. Of note, the DNA damage response gene sets were downregulated, which may be explained by the long duration of continuous irradiation at an extremely slow-dose rate. It is possible that an adaptation mechanism of the cells to irradiation triggered a feedback loop to decrease the expression of these pathways, as was observed at the gene level (SGA) for SRF responsive genes in response to the RPM. Various other gene sets involved in the same cell processes were also enriched in the RPM, and RPM and IR treatments.
Many of the downregulated gene sets are involved in cell signaling, including Rho and Ras GTPases, inositol and phosphatidylinositol, JNK and insulin receptor-mediated pathways. The downregulation of these signaling pathways may lead to an alteration of the cell cycle (33). In addition to its major role in the cell response to radiation (34,35), the regulation of the cell cycle has been shown to be affected by simulated microgravity (36). GSEA revealed that gene sets involved in the positive regulation of the cell cycle were downregulated in all treatments. However, cells that were only irradiated exhibited a significant downregulation of gene sets involved in cell cycle arrest, indicating no trend towards a pro- or anti-proliferative expression profile, while both RPM and RPM and IR showed an anti-proliferative expression profile. We suggest that all the treatments may have induced a general stress response that decreased the expression of cell cycle progression pathways, while irradiation alone also reduced the expression of genes involved in cell cycle arrest. This hypothesis is in agreement with the decreased expression of DNA damage response pathways that we also detected. In RPM and IR, the effect of the RPM may have concealed the cell cycle arrest gene set downregulation.
In addition, many gene sets involved in the composition of the cytoskeleton (actin and microtubule) and inter- (cell junctions) and extracellular connections (extracellular matrix) were affected by all the treatments. While it has been shown in various cell types that cytoskeleton remodeling starts immediately after exposure to simulated or real microgravity (21,29–31), few studies investigated the effects of IR on the cytoskeleton. However, therapeutic doses of irradiation were shown to affect cell permeability of microvascular endothelial cells through Rho-mediated cytoskeleton remodeling (37). More recently, Rho-mediated focal adhesion and fibronectin adhesion were shown to be increased in endothelial cells in response to radiation (38). As Rho GTPases intervene in a number of additional cell pathways (e.g., cell cycle arrest, and regulation of apoptosis) (39), Rho GTPases potentially play a pivotal role in the cell response to simulated space conditions. In agreement with this hypothesis, GSEA revealed that Rho GTPases activity was downregulated in IR-treated samples. Notably, gene sets involved in integrin and receptor binding were specifically downregulated following treatment using the RPM. The results of this study confirm therefore that integrins play a significant role in the cellular response to simulated microgravity.
In conclusion, this study has shown that continuous exposure to simulated microgravity affects fetal murine fibroblasts, especially at the single gene level, by increasing the expression of oxidative stress responsive genes and decreasing the expression of genes involved in cytoskeleton remodeling. As far as irradiation is concerned, we detected a decreased expression of gene sets involved in cytoskeleton mechanisms, in cell signaling and DNA damage response after a chronic low-dose rate of irradiation, particularly at the gene set level. The results indicate that the effects of the combination of the two treatments did not result in a synergism between the two separate effects, since many genes or gene sets that were altered by RPM or IR treatment, were not changed by the combined treatment (RPM and IR).
Acknowledgements
This study was supported by the ESA Topical Team on ‘Developmental Biology in Vertebrates’ and 4 PRODEX/ESA contracts [C90-303, C90-380, C90-391 and 42-000-90-380].
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