Gene expression profiling of human epidermal keratinocytes in simulated microgravity and recovery cultures

Abstract

Simulated microgravity (SMG) bioreactors and DNA microarray technology are powerful tools to identify "space genes" that play key roles in cellular response to microgravity. We applied these biotechnology tools to investigate SMG and post-SMG recovery effects on human epidermal keratinocytes by exposing cells to SMG for 3, 4, 9, and 10 d using the high aspect ratio vessel bioreactor followed by recovery culturing for 15, 50, and 60 d in normal gravity. As a result, we identified 162 differentially expressed genes, 32 of which were "center genes" that were most consistently affected in the time course experiments. Eleven of the center genes were from the integrated stress response pathways and were coordinately down-regulated. Another seven of the center genes, which are all metallothionein MT-I and MT-II isoforms, were coordinately up-regulated. In addition, HLA-G, a key gene in cellular immune response suppression, was found to be significantly up-regulated during the recovery phase. Overall, more than 80% of the differentially expressed genes from the shorter exposures (<or=4 d) recovered in 15 d; for longer (>or=9 d) exposures, more than 50 d were needed to recover to the impact level of shorter exposures. The data indicated that shorter SMG exposure duration would lead to quicker and more complete recovery from the microgravity effect.

Fig. 1

Morphology of HEK001 cells cultured in HARV bioreactors for SMG treatment and in conventional 2D cell culture flasks for normal gravity recovery. A and B. HEK001 cells cultured in normal gravity tissue culture flasks showing different photo fields and magnifications of the control cells. C. Cell aggregates from 3-day SMG culture. D. Cells recovering from 3-day SMG treatment in Panel C by 15-day growth in normal (1 g) gravity (culture flasks). E. Cell aggregates from 4-day SMG treatment. F. Cells recovering from 4-day SMG treatment through 15-day culture in normal gravity. G. Cells after 7-day recovery in normal gravity from 9-day SMG treatment. H. Cells after 16-day recovery in normal gravity from 10-day SMG treatment.

Fig. 2

MA scatter plots showing the average trend of the log ratio as a function of intensity of features in Arrays 1−5 using Lowess normalization. Each point represents one feature from the corresponding microarray. F633 represents the intensity of the red (R) laser wavelength (633 nm) that excites the cyanine 5 dye. F545 represents the intensity of the green (G) laser wavelength (545 nm) that excites the cyanine 3 dye. The MA plot shows the log intensity ratio M=log₂(R/G) versus the average intensity A= ½(log₂R+log₂G). A. MA plot for 3-day SMG (Array 1). B. MA plot for 4-day SMG (Array 2). C. MA plot for 4-day SMG followed by 15-day recovery at 1 g (Array 3). D and E. MA plots for 9-day and 10-day SMG followed by 50-day and 60-day recovery at 1 g, respectively (Arrays 4 and 5).

Fig. 3

Functional grouping and time course expression profiles of gravity sensitive genes. The gene expression profiles of the 162 putative gravity sensitive genes that were statistically significant (p≤0.05) and showed at least 1.5-fold up- or down-regulation from the five experiments were categorized into 18 functional groups and displayed as histograms. The red histograms represent the up-regulated genes and the blue histograms represent down-regulated genes. A. Category of 101 differentially regulated genes exposed to 3-day SMG. B. Category of 93 differentially regulated genes exposed to 4-day SMG. Note that the patterns of gene regulation exhibited in Panels A and B are very similar. C. Category of 18 differentially genes exposed to 4-day SMG followed by 15-day recovery at 1 g. D. Category of 105 differentially regulated genes exposed to 9-day SMG followed by 50-day recovery. E. Category of 95 differentially regulated genes exposed to 10-day SMG followed by 60-day recovery.

Fig. 4

Cluster analysis of 162 differentially regulated genes from five microarray experiments showing the trend in time course. A. The average-linkage hierarchal clustering of the expression data (log₂) of all the 162 significant genes. The rows represent individual genes categorized into five hierarchical clusters according to differential expression levels. The columns represent the five microarray experiments showing the gene expression pattern for each time point according to the five clusters. B. The time course trend of expression of all of the genes represented in each cluster.

Fig. 5

Venn diagrams of overlapping gene expression patterns of the 162 significant genes across the five time points. The red numbers represent up-regulated genes and the blue numbers represent down-regulated genes. A. Comparison of gene expression data of 3-day and 4-day SMG exposure with 4-day SMG exposure followed by 15-day recovery at 1 g. B. Comparison of gene expression data of 3-day and 4-day SMG exposure with 9-day SMG followed by 50-day recovery and 10-day SMG followed by 60-day recovery. The center of the Venn diagrams shows that there are 32 differentially regulated genes between all four array experiments. C. Heat map of the expression pattern for the 32 differentially regulated genes identified from the center of the Venn diagrams in Panel B.

Fig. 6

Validation of microarray results using Northern blotting analysis of genes differently expressed under modeledmicrogravity conditions. An amount of 10 μg total RNA from each sample was loaded onto a 1% formaldehyde agarosegel. Lanes 1−7 were loaded with samples as the following: Lane 1, conventional stationary control; Lane 2, three-day modeled microgravity; Lane 3, four-day modeled microgravity; Lane 4, three-day microgravity recovery; Lane 5, four-day microgravity recovery; Lane 6, nine-day microgravity recovery; Lane 7, ten-day microgravity recovery. The Northern blot was sequentially hybridized with ten different probes as indicated in Panels A−J: A. PDIA4; B. MT1X; C. TRIB3; D. AREG; E. MT2A: F. TRA1; G. MT1A: H. PCK2; I. ARMET; J. GAPDH. The expected mRNA bandfor each probe was indicated with an arrow to the right side of each panel.

Fig. 7

Quantitative comparison of gene expression data obtained from Northern blotting analysis and microarray analysis. The mRNA bands in Figure 6A−J were first normalized against the GAPDH level in each corresponding lane. The normalized levels of the mRNA in each panel were then plotted in histograms as shown in Figure 7A−I. In the histograms, the Northern blotting data (blue) were plotted side by side with the microarray data (red) to directly compare the expression levels of the same nine significant genes. The values of the correlation coefficient (R values) for each comparison were indicated in each panel.