Confirmation of gene expression responses to cold stress and heat shock
Samples from six independently performed experiments were submitted for microarray analysis (a total of 36 gene chips; 3 conditions per experiment
2 time points per experiment
36 chips). The temperatures measured during these six experiments were (mean
SD): cold stress, 31.0
12, or two measurements per experiment); heat shock, 43.1
12); control, 37.0
18, or three measurements per experiment); and recovery, 37.0
6). Cell survival was measured by the Trypan blue assay both at the end of the thermal stress period and again after recovery and ranged from 94.5% to 97.4%.
Figure provides a representative example of the heat- and cold-induced changes in the expression of control genes (as ascertained by RT-PCR) that occurred in the sample sets submitted for microarray analysis. There was a noticeable increase in expression of CIRBP after cold stress but not after heat shock. By contrast, HSP70B’ showed a substantial increase in expression after heat shock but not hypothermia and furthermore, demonstrated maximal expression after a period of recovery at 37°C. Expression of cyclophilin A appeared to be uniform under all conditions tested.
Fig. 1 Reverse transcription PCR showing the expression responses to heat shock and cold stress from one of the independent experiments submitted for microarray analysis. The numbers (0, 3) denote the recovery time at 37°C, in hours, after heat shock (more ...)
Thermal stress produced extensive gene expression changes that included a large component of downregulation
Of 22,283 sequences on the U133A array, 6,609 (29.7%) showed changes in expression that were both statistically significant and relevant to the experimental hypothesis, as determined by two-way repeated-measures ANOVA. Of these, 3,572 (54%) showed an effect of thermal stress condition only (a “main effect” of heat, cold, or control temperature), 1,407 (21%) showed both a main effect and an interactive effect between time and thermal stress condition, and 1,630 (25%) showed only an interactive effect. Another 1,275 (5.7%) of the 22,283 sequences on the array showed only a significant effect of time (i.e., no main effect and no interactive effect). This last number corresponds closely to what would be expected by random chance alone and represents changes that occurred independently of thermal stress (and therefore not of direct relevance to the experimental hypothesis). Accordingly, these 1,275 sequences were excluded from further consideration.
One-way ANOVA was performed at each of the time points to help identify the source(s) of the differences found by two-way ANOVA. This analysis revealed that, of the 6,609 sequences identified by two-way ANOVA, 5,379 also showed a statistically significant effect of temperature (heat, cold, control) at one or both of the time points examined. Post-hoc analysis found a significant difference between controls and at least one of the thermal stress conditions (heat, cold) for 4,253 sequences, of which 3,256 were affected by cold stress at one or both time points and 1,749 by heat shock.
Tables and and Fig. present complimentary displays of how the 4,253 sequences in this experiment were affected by time and thermal stress; Table lists affected sequences by time and thermal condition, Table parses these sequences into those with increased and decreased expression, and Fig. presents a graphical display that allows a visual comparison of the number of sequences affected by the alternative forms of thermal stress. Because many sequences fell into more than one category (for example, some were affected both by heat and by cold at a given time point or were affected by a given stress at both time points), the totals in the tables and in the figure add up to more than 4,253. Unless otherwise specified, the magnitude of change was not specifically accounted for in these presentations of the data; so, for example, a gene whose expression was significantly increased both by heat and by cold at T
0 would be listed as “similarly affected by heat and cold,” regardless of the actual magnitude of the changes.
Pattern of expression of the sequences affected by cold stress and heat shock
Effect of time on gene expression responses to moderate hypothermia and heat shock
Venn diagrams illustrating the time dependence of gene expression after moderate hypothermia and heat shock. Only a minority of sequences at each time point showed sustained increases or decreases in expression
Cold stress and heat shock produced different expression patterns
Table compares the number of sequences affected by hypothermia to those affected by heat shock. The gene expression response to cold stress was somewhat more extensive than the response to heat shock, as evidenced by the observation that hypothermia affected more sequences than did heat shock at both time points examined. The difference in the number of sequences affected was greater at T
0, the end of the thermal stress period, than after 3 h of recovery at 37°C (Table ).
Additionally, the total number of sequences affected by heat shock increased during recovery at 37°C (as expected), whereas the number of sequences affected by hypothermia decreased over the same time period. These differences held true even when we excluded sequences that showed changes in expression of less than twofold relative to controls (Table ).
The gene expression responses to both thermal stress conditions also included substantial components of decreased expression. About half of the sequences affected by cold stress and about two thirds of the sequences affected by heat shock showed decreased expression relative to controls (Table ).
The sequences affected by each thermal stress varied over time
Cold stress and heat shock produced time-dependent changes in expression. As noted previously, slightly less than half of the sequences identified by the two-way ANOVA analysis showed evidence of an effect of time on gene expression. Similar conclusions were reached in the one-way ANOVA analysis, as illustrated in detail in Table and Fig. . Of the 4,253 sequences identified as showing expression that was significantly different from controls at one or both time points, 3,256 were affected by cold stress and 1,749 by heat shock. However, less than one third (978 of 3,256) of the sequences affected by hypothermia and less than one eighth (198 of 1,749) of the sequences affected by heat shock showed sustained changes in expression (i.e., significant changes in expression in the same direction at both of the time points studied, Fig. ). Very few sequences (<1%) exhibited a bidirectional response to thermal stress (i.e., significantly decreased at one time point, increased at the other, Table ).
These expression patterns showed greater overlap during the recovery period
Some sequences showed changes in the same direction (increased or decreased) in response to both cold stress and heat shock (Table and Fig. ). The proportion of sequences displaying congruent changes in expression roughly doubled during recovery at 37°C. Specifically, 5% (160 of 2,987) of the significantly affected sequences showed changes in the same direction immediately after cold stress and heat shock, as compared to 11% (296 of 2,632) after 3 h of recovery at 37°C.
Fig. 3 Effect of heat shock and hypothermia on gene expression. The ordinate represents the number of sequences affected in each category. Sequences were said to be similarly affected by the two thermal stress conditions if both hypothermia and heat shock produced (more ...)
Effect of thermal stress on negative and positive control sequences
Several important negative control sequences corresponding to genes that are generally unaffected by a wide variety of different stimuli (“housekeeping genes”) were likewise unaffected by thermal stress in this experiment. Supplemental Table 2
details the effect of moderate hypothermia and heat shock on a list of such control sequences, including beta-actin, GAPDH, cyclophilin A, a number of other sequences found in previous gene chip array experiments to demonstrate stable expression after heat shock (Sonna et al. 2002b
) and hypoxia (Sonna et al. 2003
), and several of the ribosomal proteins most highly expressed in this cell line as determined by signal intensity on the microarrays. Of the 19 genes represented by the 25 sequences in this table, only three (beta-actin, GAPDH, and ribosomal protein L30) showed statistically significant changes in expression by two-way ANOVA. However, the magnitudes of the expression changes were very small; none of the geometric mean expression ratios in these three genes exceeded 1.2-fold.
Additionally, sequences corresponding to genes known to respond to thermal stress (positive controls) showed significant changes in expression. Specifically, sequences corresponding to the cold-responsive genes CIRBP and RBM3 were increased in response to hypothermia. Two sequences corresponding to CIRBP were increased 4.8-fold and 3.5-fold, respectively, during cold stress; these sequences were increased 3.5-fold and 2.7-fold after recovery. A sequence corresponding to RBM3 was increased 2.5-fold during cold stress and 2.4-fold after recovery. None of these sequences were significantly affected by heat. Likewise, as discussed below, many sequences corresponding to heat shock proteins (including HSP70B’) showed increases in expression in response to heat shock.
Effect of thermal stress and recovery on expression of heat shock proteins
The two-way ANOVA analysis identified 76 sequences corresponding to heat shock proteins (HSPs), chaperonins, and co-chaperonins that showed statistically significant changes in expression. Subsequent one-way ANOVA with post-hoc analysis revealed a source of the difference(s) for 67 of these sequences.
Heat shock and hypothermia produced different effects on the expression patterns of sequences corresponding to HSPs and related proteins. Whereas heat shock typically produced increases in HSP mRNA expression that became larger and more extensive after 3 h of recovery at 37°C, hypothermia often had an inhibitory response on HSP expression, followed by a mild increase in HSP expression during the recovery period. Specifically, heat shock produced increases in the expression of 15 sequences during thermal stress and of 37 sequences after recovery. One HSP sequence (corresponding to sacsin) was slightly (though significantly) decreased by heat shock during thermal stress (expression ratio 0.70) and two (corresponding to sacsin and DNAJA3) decreased during recovery (expression ratios, 0.44 and 0.75, respectively). By contrast, moderate hypothermia led predominantly to decreased HSP expression at the end of thermal stress (16 decreased sequences, as compared to five increased sequences), followed by increases in expression relative to controls after recovery (a total of 27 increased sequences, as compared to three decreased sequences). A total of 16 sequences were significantly increased both by moderate hypothermia and heat shock after 3 h of recovery at 37°C.
Figure illustrates these effects in the subset of HSPs most strongly affected by heat shock or hypothermia (i.e., sequences, which, among other criteria, showed expression ratios ≥2 or ≤0.5).1
Fig. 4 Effect of hypothermia and heat shock on the expression of selected HSPs, chaperonins, and co-chaperonins. For illustrative purposes, the sequences shown were significantly affected at one or more time points in the ANOVA analysis had a geometric mean (more ...)
Effect of thermal stress on expression of heat shock factors
Hypothermia also had an effect on the expression of a sequence corresponding to heat shock factor-1 (HSF-1), the major transcription factor responsible for increased expression of HSPs during thermal stress (Cotto and Morimoto 1999
). Hypothermia produced a small but statistically significant decrease in expression of a sequence corresponding to heat shock factor-1 (expression ratio 0.74) during thermal stress that was no longer significantly different from control after recovery. By contrast, heat shock did not produce any changes in HSF-1 RNA expression.
A sequence corresponding to heat shock factor-2 was significantly decreased by a small amount (expression ratio 0.63) after recovery from heat shock but not affected at either time point by moderate hypothermia.
We did not identify any sequences corresponding to heat shock factor-4 that were significantly affected by heat shock.
Biological processes affected by thermal stress
We used the Onto-Express software package (Draghici et al. 2003
) to gain additional insight into the biological processes affected by hypothermia and rewarming. Figure presents the biological processes most strongly affected by genes whose expression were increased during or after thermal stress, and Fig. presents those which were decreased. These figures only depict pathways that are statistically overrepresented at one or both of the time points examined. Because the total number of increased sequences was different for each combination of thermal stress and time (Tables and ), it is possible for two time points to have the same number of affected sequences in a given biological process and yet for one not to be statistically significant. Furthermore, because the categories were generated by a statistical algorithm, some of them are overlapping or are subsets of others (e.g., “lipid metabolism” and “metabolism”). Because of this, it is possible for a gene to be represented in more than one category and as might be expected, it is also possible for multifunctional genes to be included in more than one of the categories presented. The Gene Ontology Annotation numbers corresponding to the categories shown, and their consensus definitions, are listed in Supplemental Table 3
Fig. 5 Functional categories most highly affected by increases in gene expression after heat shock and hypothermia. N.S. the number of affected sequences in that category at the time point in question does not differ from what would be expected from a random (more ...)
Fig. 6 Functional categories most highly affected by decreases in gene expression after heat shock and hypothermia. N.S. the number of affected sequences in that category at the time point in question does not differ from what would be expected from a random (more ...)
As might be expected, the sequences increased by heat shock affected biological processes known to be involved in protein biosynthesis, folding, and in the response to unfolded protein; furthermore, the largest effect was seen after a period of recovery at 37°C (Fig. ). By contrast, and consistent with our manual analysis of HSPs (above), hypothermia did not significantly affect the response to unfolded protein until after recovery at 37°C. Hypothermia-induced genes heavily affected pathways involved in cell replication (including cell cycle, DNA replication, mitosis, etc.), signal transduction, and the ubiquitin cycle. We also detected substantial effects of hypothermia on pathways involved in RNA splicing and processing.
A similar analysis was performed on genes whose expression was decreased by heat shock and hypothermia (Fig. ). Pathways involved in transcription and the regulation of transcription were the ones most extensively affected both by heat shock and hypothermia, but this association was only significant after 3 h of recovery at 37°C. A gene expression signature of the metabolic response to hypothermia was clearly detected. Hypothermia led to decreased expression of sequences of key metabolic pathways, including those of lipid metabolism and carbohydrate metabolism (Fig. ).
An analysis of KEGG pathways affected by hypothermia was performed using DAVID version 6 (Table ). Consistent with the results of our Onto-Express analysis, a strong effect of hypothermia was noted on pathways involved in cell cycle, signal transduction (including the p53 signaling pathway, the insulin signaling pathway, and the phosphatidylinositol signaling system), ubiquitin-mediated proteolysis, and several metabolic pathways.
KEGG Pathways affected by hypothermia
Effect of thermal stress on other sequences
Supplemental Tables 4
present lists of genes not traditionally considered heat shock proteins (“non-HSPs”) whose expression was maximally affected by moderate hypothermia and by heat shock. The lists of genes in these tables again suggest that heat shock and moderate hypothermia produced contrasting effects on gene expression. Importantly, some of the expression changes in non-HSPs observed have been previously reported in studies of thermal stress. As noted previously, hypothermia produced changes in expression of genes widely acknowledged to be cold responsive, including CIRBP and RBM3. Likewise, the non-HSPs affected by heat stress included several genes previously found to be highly heat responsive in a previous study in peripheral blood mononuclear cells (Sonna et al. 2002b
), including Rad (Supplemental Table 7
), the phosphatidylserine receptor (Supplemental Table 7
), and NF-kappa B repressing factor (increased after recovery from heat stress by 2.3-fold but unaffected by moderate hypothermia).
Comparison to the responses of THP-1 cells
It is generally acknowledged that cellular responses to heat involve a number of changes that are likely shared by many different cell types (Kregel 2002
; Lindquist 1986
; Parsell and Lindquist 1993
; Sonna et al. 2002a
). However, the extent to which cold-induced changes in expression are similarly independent of cell type is not well described. We compared the findings in HepG2 cells to sequences that were identified in previous work as being affected by moderate hypothermia (32°C × 24 h) in the acute monocytic leukemia cell line THP-1 (Sonna et al. 2006
). Because the experiments with THP-1 cells did not involve rewarming, only the changes in expression at the end of cold exposure in HepG2 cells were used for comparison.
Of the 1,297 sequences that showed statistically significant increases in expression by moderate hypothermia in HepG2 cells at the end of thermal stress (Table ), 456 (35%) were also significantly increased in THP-1 cells (irrespective of post-hoc filter criteria). Of the 143 sequences that showed significant expression ratios of twofold or greater in HepG2 cells as a result of moderate hypothermia, 51 were also significantly increased in THP-1 cells (irrespective of magnitude), of which 12 also met all post-hoc expression call filter criteria in the THP-1 cells (a change of twofold or greater, expression calls of “present” or “marginal” in at least half of the cells exposed to hypothermia). By contrast, of these 143 sequences, only three showed statistically significant decreases in expression in THP-1 cells (P
0.001 by z
test of proportions with Yates’ correction).
Of the 1,301 sequences that showed statistically significant decreases in expression at the end of moderate hypothermia in HepG2 cells, 484 (37%) were also significantly decreased in THP-1 cells (irrespective of post-hoc filter criteria). Of the 266 sequences in HepG2 cells that showed significant expression ratios of 0.5 or less, 96 were also significantly decreased in THP-1 cells (irrespective of magnitude), of which 13 also met the post-hoc filter criteria in THP-1 cells outline above. By contrast, of these 266 sequences, only 11 were increased by moderate hypothermia in THP-1 cells (P
0.001 by z
test of proportions with Yates’ correction).
Thus, 25 sequences (12 increased, 13 decreased) representing 23 genes showed changes in expression in both cell lines that were statistically significant and that also met the post-hoc filter criteria of the respective studies, including geometric mean changes in expression of at least twofold, Table . Importantly, the sequences in this consensus list include both CIRBP and RBM3, which are known to be increased by moderate hypothermia (Danno et al. 1997
; Nishiyama et al. 1997a
; Sonna et al. 2002a
). Additionally, HSP70-1 was significantly decreased during the period of cold exposure in both experiments (though as noted previously, HSP70-1 showed increases in expression upon rewarming).
Nonspecific cold response genes
Confirmatory reverse transcription PCR
Figure presents qualitative confirmatory experimental data that illustrate the time course of the change in expression of several of the genes identified that were affected by moderate hypothermia and heat shock in our microarray studies. Our objective here was to confirm only change in direction and to confirm qualitative differences in the responses to heat shock and hypothermia, not to perform a formal correlation between magnitudes of change observed on the microarray and by PCR (which would require qrPCR).
Confirmation of selected microarray findings by reverse transcription PCR. Cells were subjected to heat shock or cold stress and allowed to recover at 37°C for 0, 1, 3, or 6 h. Arrows point to the bright 500 bp ladder band
Cells in culture were exposed to thermal stress conditions as described previously and allowed to recover at 37°C for varying amounts of time, at which point RNA was isolated and subjected to expression analysis by conventional reverse transcription PCR. The directions of the changes observed were congruent with the findings made by microarray. Additionally, as predicted by the microarray, the effects of heat shock on the expression of these genes differed noticeably from the effects of moderate hypothermia in the direction of change, the time course, or both.