Temporal gene expression kinetics for human keratinocytes exposed to hyperthermic stress

Abstract

The gene expression kinetics for human cells exposed to hyperthermic stress are not well characterized. In this study, we identified and characterized the genes that are differentially expressed in human epidermal keratinocyte (HEK) cells exposed to hyperthermic stress. In order to obtain temporal gene expression kinetics, we exposed HEK cells to a heat stress protocol (44 °C for 40 min) and used messenger RNA (mRNA) microarrays at 0 h, 4 h and 24 h post-exposure. Bioinformatics software was employed to characterize the chief biological processes and canonical pathways associated with these heat stress genes. The data shows that the genes encoding for heat shock proteins (HSPs) that function to prevent further protein denaturation and aggregation, such as HSP40, HSP70 and HSP105, exhibit maximal expression immediately after exposure to hyperthermic stress. In contrast, the smaller HSPs, such as HSP10 and HSP27, which function in mitochondrial protein biogenesis and cellular adaptation, exhibit maximal expression during the "recovery phase", roughly 24 h post-exposure. These data suggest that the temporal expression kinetics for each particular HSP appears to correlate with the cellular function that is required at each time point. In summary, these data provide additional insight regarding the expression kinetics of genes that are triggered in HEK cells exposed to hyperthermic stress.

Figure 1

(A) A schematic of the heat shock exposure protocol and post analysis techniques. (B) Cellular MTT viability for keratinocytes exposed to hyperthermic stress. Viability percentage data (relative to untreated samples) was measured for wells with concentration densities ranging from 10,000 to 100,000 cells per well. Viability was measured at 0, 4 and 24 h post-heat treatment. (C) Microarray global gene expression profiles. The data shows that 762, 1,422 and 1,478 total genes were differentially expressed at 0, 4 and 24 h post-exposure, respectively. (D) Gene expression for minimal stress protein, heat shock protein 70 (HSPA6) using qRT-PCR. The mRNA expression fold values were measured for sham and heat treatment groups. For each time point, three samples (S1, S2 and S3) were assessed, each in triplicates. Values were calculated in relation to β-actin and normalized to a separate RNA calibrator.

Figure 2

Volcano plots of the gene expression profiles at each time point. The magnitude of differential expression (log₂ fold-change) is plotted versus the level of statistical significance (p-value) of all genes in the microarray for 0, 4 and 24 h post-heat exposure. Upregulated and downregulated genes with absolute log₂ ratios ≥1.5 and p-values ≤0.05 are depicted with green and red triangle symbols, respectively. Insignificant genes are denoted with light gray symbols.

Figure 3

Diagrams of cellular and molecular functions associated with genes expressed in human epidermal keratinocyte (HEK) cells exposed to heat shock. Following an Ingenuity Pathway Analysis (IPA) core analysis, the molecular and cellular functions linked to each recovery period were sorted and illustrated. (A) A list of the 23 biological functions common to all three time points and nine functions unique to one time point or shared between two time points; (B) Pie charts of gene functions at 0, 4 and 24 h post-exposure. The pie chart sectors represent the number of genes associated with each function.

Figure 4

Pie chart of the top five cellular and molecular functions at each time point. Each sector of the pie chart represents the total number of genes expressed for each cellular function. Genes were filtered for an absolute value log₂ ratio ≥1.5 and a significance value of p ≤ 0.05.

Figure 5

Identification of unique and shared genes. The Venn diagram indicates the number of unique and common differentially expressed genes at each time point. Biomarker comparison analysis was performed in IPA.

Figure 6

Pie chart of 50 shared biomarkers. Each sector of the pie represents the number of genes associated with each function. The symbols and number of genes are also provided for each function. Genes were filtered for an absolute value log₂ ratio ≥1.5 and a significance value of p ≤ 0.05.

Figure 7

The temporal expression kinetics for the genes with known gene ontology (GO). Asterisks indicate the genes with more than one biological process.

Figure 8

Gene family of the genes that are unique or are shared at two time points. The pie chart sectors represent the number of genes associated with each function indicated in the legend. The numbers of these genes are indicated for each function. The genes classified by IPA as “other” were not included in this representation. Genes were filtered for an absolute value log₂ ratio ≥1.5 and a significance value of p ≤ 0.05.

Figure 9

Gene expression profiles for a set of unique genes involved in several well-characterized pathways of the cellular stress response.

Figure 10

Classification of differentially expressed genes into various cellular stress response implicated canonical pathways. The [−log(p-value)] is the maximum p-value for all of the genes in each particular canonical pathway family. The ratio plot indicates the number of the genes expressed at each time point relative to the total number of genes in that particular canonical pathway group.