Differential Temperature-Induced Responses in Immortalized Oral and Skin Keratinocytes

Abstract

The epidermis of the skin and oral mucosa is constantly exposed to various environmental stimuli, including temperature changes. In particularly extreme conditions, such as excess heat or cold, significant injury may occur. Oral and skin keratinocytes exhibit tissue-specific differences in wound healing outcomes and the transcriptomic response to injury. This study investigated if skin and oral keratinocytes also have differential responses to heat- and cold-induced injury. Oral keratinocytes (TIGKs) were found to exhibit an enhanced viability following heat-induced injury compared to skin keratinocytes (HaCaTs). However, there were no discernible differences between skin and oral keratinocyte viability following cold-induced injury. To examine the transcriptomic differences between skin and oral keratinocytes in response to temperature-induced injury, we generated an mRNA-sequencing gene expression dataset. Differentially expressed genes (DEGs) including heat shock proteins (HSPs) were identified between HaCaTs and TIGKs at baseline (37 °C) and after heat- (60 °C) or cold-induced (-25 °C) injury. Our comparative analyses suggest that skin and oral keratinocytes exhibit transcriptomic differences at baseline and in their responses to heat or cold exposure. The enhanced heat tolerance of TIGKs relative to HaCaTs may be due to an advantageous expression of a subset of HSPs at baseline in TIGKs. Our work also provides a source of skin and oral keratinocyte gene expression data following heat- and cold-induced injury that can be used for future analyses.

Keywords: keratinocyte; oral mucosa; skin; temperature-induced injury; transcriptomics.

Figure 1

Oral keratinocytes are more tolerant to heat-induced injury than skin keratinocytes. (A) Viability of oral keratinocytes (TIGKs) and skin keratinocytes (HaCaTs) under hot and cold conditions. N = 6–12. Data shown are pooled from 2–4 repeated experiments. (B) Representative photos of HaCaTs and TIGKs exposed to hot and cold conditions. (C,D) HaCaTs and TIGKs were harvested after being exposed to 60 °C or −25 °C for 10 min. Cell death assessment was performed using an Annexin V Apoptosis Detection kit analyzed by flow cytometry. Representative images of flow cytometry analysis. Scale bar = 50 μm. (C). Q1: necrotic cells, Q2: late apoptotic cells, Q3: early apoptotic cells, Q4: live cells. Summary of cell death assessment (D). N = 3. Baseline values for HaCaTs and TIGKs are shown in the dashed red and blue lines, respectively. The data shown is representative of 2 experiments. * = p < 0.05, ** = p < 0.01. Two-way ANOVA was used for A and Multiple t-test was used for D.

Figure 2

Comparative analysis of the transcriptomic response of oral and skin keratinocytes to injury reveals they cluster by cell type and temperature-induced injury. (A) Principal component analysis plot of mRNA-sequencing expression data. Each sample is represented by a colored point on the graph. The x-axis and y-axis are the first and second principal components, respectively. (B) Heatmap representing similarities of the mean gene expression profiles grouped by keratinocyte cell type (oral vs skin) and temperature condition: −25 °C, 37 °C, and 60 °C. Each square and its color represents aggregate Pearson’s correlation coefficient values between each experimental group (cell type and temperature condition).

Figure 3

Oral and skin keratinocytes exhibit significant transcriptomic differences at baseline and following heat- or cold-induced injury. (A) Volcano plot showing the number of differentially expressed genes (DEGs) between HaCaTs and TIGKs at each temperature comparison: 37 °C, 60 °C, and −25 °C. (B) Volcano plot showing the number of DEGs in HaCaTs or TIGKs after exposure to 60 °C for 10 min relative to 37 °C or (C) to −25 °C for 10 min relative to 37 °C. (D) Venn diagram comparing the DEGs upregulated in HaCaTs and TIGKs after exposure to 60 °C for 10 min relative to 37 °C or to −25 °C for 10 min relative to 37 °C. (E) Venn diagram comparing the DEGs upregulated after exposure to 60 °C for 10 min relative to 37 °C and the DEGs upregulated after exposure to −25 °C for 10 min relative to 37 °C for HaCaTs and TIGKs.

Figure 4

Oral and skin keratinocytes exhibit significant differences in their expression of heat shock proteins (HSPs) at baseline and following heat- or cold-induced injury. (A) Heatmap for all differentially expressed HSPs in HaCaTs and TIGKs at each temperature comparison: 37 °C, 60 °C, and −25 °C. (B) Heatmap of the HSPs differentially expressed in HaCaTs or TIGKs after exposure to 60 °C for 10 min relative to 37 °C or (C) to −25 °C for 10 min relative to 37 °C. Groups are denoted on both axes of the heatmap and each column or row represents an independent sample. (D) Venn diagram comparing the HSPs that are upregulated in HaCaTs and TIGKs after exposure to 60 °C for 10 min relative to 37 °C or to −25 °C for 10 min relative to 37 °C. (E) Venn diagram comparing the HSPs upregulated after exposure to 60 °C for 10 min relative to 37 °C and the HSPs upregulated after exposure to −25 °C for 10 min relative to 37 °C for HaCaTs and TIGKs.

Figure 5

Oral keratinocytes have an advantageous expression profile of heat shock proteins that relate to survival at baseline. (A) Venn diagram comparing the shared HSPs that are upregulated in TIGKs relative to HaCaTs at 37 °C and in HaCaTs after exposure to 60 °C for 10 min relative to 37 °C. (B) Heatmap showing expression of HSPs upregulated in TIGKs relative to HaCaTs at 37 °C and in HaCaTs after exposure to 60 °C for 10 min relative to 37 °C. Groups are denoted on both axes of the heatmap and each column or row represents an independent sample.