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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 23, 2002 as doi:10.1096/fj.01-1002fje. |
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2
Bloomfield Center for Research in Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, Montréal, Québec, Canada;
* Department of Anatomy and Cell Biology, McGill University, Montréal, Québec, Canada; and
Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, Louisville, Kentucky, USA
2Correspondence: Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, 570 South Preston St., Baxter Building, Room 304, Louisville, Kentucky 40202, USA. E-mail: Eugenia.Wang{at}Louisville.edu
SPECIFIC AIM
As a consequence of spaceflight, the expression of several muscle- and bone-specific genes is decreased, leading to musculoskeletal changes such as loss of muscle mass and decreased bone density. As these changes greatly impede our ability to conduct long-term manned space missions, we developed and implemented a new approach to microarrays to study the change in the gene expression of E-box binding transcription factor, interleukin (IL), and tumor necrosis factor (TNF) -related genes in normal WI38 human fibroblasts exposed to microgravity during the STS-93 Space Shuttle mission.
PRINCIPAL FINDINGS
1. Development of microarrays
To investigate changes in mRNA expression as a result of spaceflight, we developed and implemented a novel approach to microarray technology consisting of digoxigenin (DIG) labeling of cDNA using gene-specific primers and arrays printed on Hybond nylon membranes. After careful evaluation of several different publicly available databases, we chose 202 genes belonging to either the TNF, IL, or E-box binding gene families, as well as 9 housekeeping genes and a series of negative controls for arraying. Once genes of interest were identified, sense and antisense primers were designed for each gene with the help of Primer3 software by maintaining constant selection parameters for all genes. The average annealing temperature of all primers is 60.1 ± 0.09°C, the average size of PCR product for arraying is 441 ± 58 bp, and the average melting temperature is 80 ± 3°C. Selecting these parameters allowed us to conduct hybridization and posthybridization washing under stringent conditions, thereby decreasing the probability of cross-hybridization and reducing the background. Amplicons generated using these primers were arrayed onto Hybond membranes attached to glass slides.
2. Expression profiling of WI38 cells from ground and spaceflight samples using microarrays
Microarrays arrayed with either E-box-, TNF-, or IL-related gene amplicons were hybridized with DIG-labeled spaceflight or ground control cDNA samples. Three independent hybridizations were done for spaceflight and ground control samples for each type of microarray. Figure 1
A shows the average normalized expression levels for each type of array. Gene quantification and normalization was accomplished by averaging the intensities of the three replicated dots for each gene present on a single microarray, then normalizing each membrane according to the intensities of the housekeeping genes. Normalized intensities for each gene were then averaged for the three membranes.
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After microarray analysis, we identified 10 genes belonging to the TNF- or IL-related gene families whose expression was modified as a consequence of spaceflight. Genes for which changes were identified show reproducible results in all three hybridization experiments and include two genes from the IL array: interleukin-1 receptor antagonist (IL1RN) and interleukin-15 alpha chain (IL15RA); and eight genes from the TNF array, including two ligands from the TNF superfamily, TNF-related weak inducer of apoptosis (TWEAK), and TNF superfamily member 15 (TNFSF15); two TNF receptor associated proteins, protein-tyrosine phosphatase nonreceptor type 13 (PTPN13), and neutral sphingomyelinase activation-associated factor (NSMAF); three TNF-inducible genes, pentraxin 3 (PTX3), small inducible cytokine subfamily A member 13 (SCYA13), and ATP binding cassette member 50 (ABC50); and one modulator of TNF activity: TNF-
converting enzyme (TACE). No significant changes were detected in genes on the E-box chip. Figure 1B
shows a portion of a TNF array hybridized with WI38 fibroblast DIG-labeled cDNA from ground and spaceflight samples.
3. RT-PCR analysis of genes found to be differentially expressed by microarrays
After reverse transcription reaction, cDNA obtained from spaceflight and ground control samples were aliquoted in microtubes by fivefold serial dilutions and amplified by PCR in order to validate the results obtained by microarray techniques. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as control for the quantity of RNA as well as for gel loading. Figure 2
A shows representative gels for each PCR reaction and Fig. 2B
shows the quantification for each gene expression for ground and spaceflight samples. Using RT-PCR, we confirmed the up-regulation of TWEAK, TNFSF15, PTPN13, PTX3, SCYA13, ABC50, NSMAF, TACE, and IL1RN, and down-regulation of IL15RA gene expression in spaceflight samples compared with ground controls. These alterations in mRNA levels correlate with those registered by microarray techniques. The average correlation coefficient between RT-PCR and microarray data is 0.89.
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CONCLUSION
Although many methods exist to study multiple changes in gene expression, microarrays seem to be the best method available, as they can promptly characterize changes in many genes simultaneously. Despite the fast progress in the field of microarray technology, a major obstacle to their broader implementation is the requirement for large amounts of mRNA. Since we had only limited quantities of spaceflight WI38 mRNA, we developed and implemented microarrays enabling us to study gene changes using minimal amounts of biological material. To decrease the demand for RNA, we used gene-specific antisense primers to produce DIG-labeled cDNA and printed our arrays on nylon membranes.
Using our microarrays, we studied the expression of genes from three functional classes of genes (TNF, IL, and E-box binding), which we believed to be important in adaptation to spaceflight and aging. For instance, variations in the expression of crucial transcription factors could be important for driving global gene expression whereas modulation of TNF and IL genes may represent inflammatory or general stress responses, resulting in the musculoskeletal changes seen in spaceflight and aging. Consistent with the published literature, our array failed to revealed any differential expression of E-box genes. However, we identified 10 genes belonging to the TNF- and IL-related gene families that are differentially expressed as a consequence of spaceflight (Fig. 3
). Although TNF- mRNA expression remains constant, up-regulation of TACE mRNA is detected in spaceflight samples. As TACE is necessary to produce mature TNF-, its up-regulation may result in an augmentation of TNF- secretion without changes in the level of TNF- mRNA; this may explain why TNF secretion is increased in space flown macrophages. Two newly identified TNF superfamily members (TWEAK and TNFSF15) are also up-regulated as a consequence of spaceflight. Like TNF, TWEAK has apoptotic ability; however, its main function is believed to be to induce the NF
ß pathway rather than apoptosis. TNFSF15 is also capable of activating NFß, but in normal human fibroblasts it induces cell division.
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Although TNF ligands mediate their cellular responses by first interacting with TNF surface receptor, none of the 20 TNF receptors represented on our TNF array was up- or down-regulated as a consequence of spaceflight. However, probably as a result of TACE up-regulation, increased expression of three TNF-inducible genes (PTX 3, SCYA13, and ABC50) is observed in spaceflight samples. Changes in the expression of these three genes may reflect stress or proinflammatory responses of fibroblasts to microgravity conditions, as PTX3 and SCYA13 participate in inflammatory reactions. On the other hand, down-regulation of IL15RA may indicate an attempt by cells to counteract this developing proinflammatory situation, as IL15, its ligand, is a regulator of macrophage proinflammatory cytokine.
Using our microarray, we have identified an up-regulation in IL1RN mRNA under spaceflight conditions. IL1RN is a cytokine receptor antagonist functioning to neutralize the biological activity of IL1 and IL1ß by competing for binding to the IL1 receptor. It is possible that the up-regulation of IL1RN observed in spaceflight samples is part of a protective mechanism attempting to counteract the effect of IL1ß, a potent stimulator of bone resorption involved in the pathogenesis of osteoporosis, whose biological effect depends on the IL1ß/IL1RN ratio, to diminish the magnitude of bone loss during spaceflight. Increased mRNA expression of two TNF receptor-associated proteins (NSMAF and PTPN13) also occurs as a consequence of spaceflight. Whereas NSMAF regulates ceramide production, PTPN13 negatively regulates Fas-induced apoptosis. As Fas was recently shown to block calcium influx in lymphocytes through the activation of acidic sphingomyelinase and ceramide release, it is possible this up-regulation of PTPN13 and NSMAF may also function to counteract factors contributing to bone loss in space.
By comparing the level of expression of E-box binding, TNF-, and IL-related genes between spaceflight and ground control WI38 human fibroblasts, we identified a complex series of changes in response to microgravity. These changes reveal that various signaling pathways involved in the regulation of apoptosis and proinflammation are modified in fibroblasts as a consequence of spaceflight. Changes in TNF- and IL-related genes may underlie a mechanism attempting to counteract the bone loss occurring as a result of spaceflight. However, it remains to be determined whether these changes indeed contribute to the regulation of apoptosis, and thereby bone mass during spaceflight, and, most important, whether some of the changes in gene expression detected by microarray occur during normal aging.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-1002fje; to cite this article, use FASEB J. (April 23, 2002) 10.1096/fj.01-1002fje. 
This article has been cited by other articles:
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  C. Dogra, H. Changotra, N. Wedhas, X. Qin, J. E. Wergedal, and A. Kumar TNF-related weak inducer of apoptosis (TWEAK) is a potent skeletal muscle-wasting cytokine FASEB J, June 1, 2007; 21(8): 1857 - 1869. [Abstract] [Full Text] [PDF]
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phage DNA negative control; 3, the 2xSSC negative controls, 4, TWEAK; 5, NSMAF; 6, PTPN13; 7, PTX3.
