Intrinsic differences between oral and skin keratinocytes

Abstract

Keratinocytes cover both the skin and some oral mucosa, but the morphology of each tissue and the behavior of the keratinocytes from these two sites are different. One significant dissimilarity between the two sites is the response to injury. Oral mucosal wounds heal faster and with less inflammation than equivalent cutaneous wounds. We hypothesized that oral and skin keratinocytes might have intrinsic differences at baseline as well as in the response to injury, and that such differences would be reflected in gene expression profiles.

Figure 1. Hierarchical clustering of genes expressed more than A) 10-fold and B) 50-fold in oral and skin epithelium.

The heatmap indicates gene expression in the skin and oral epithelium is significantly different. The color key shows the assignment of color to the expression intensity value. FDR<0.01, N = 4. The clustering is based on hierarchical clustering with squared Euclidean distance between the samples and complete linkage. The heatmaps were generated using package "gplots” in R (http://www.r-project.org).

Figure 2. Top Biological Functions at each location.

Fischer's exact test was used to calculate a p-value (<0.01) determining the probability each biological function assigned to data set is due to chance alone. Ingenuity Pathway Analysis software.

Figure 3. Oral and Skin keratinocyte wound closure after In Vitro wound scratching.

Human primary oral and skin keratinocyte monolayers were wounded by scratches. A) The defined areas were photographed at 0, 6 and 24 hours after wounding. B) The number of cells in the denuded initial area were counted. Number of cells 6 hours post scratch: oral 83.4±10.9 vs skin 23.8±6.4. Number of cells 24 hours post scratch: oral 317.3±24.8 vs skin 118.0±37.6 *P<0.01. Student's t-test was used to compare the difference between oral and skin wounds at each time point and two-way ANOVA was used to evaluate grouped data over time. N = 3.

Figure 4. Comparison of skin and oral keratinocyte proliferation after In Vitro wounding.

Isolated primary paired human keratinocytes (skin and hard palate) were cultured for In Vitro scratch assays. Keratinocyte proliferation was measured at time points of 0, 24, and 48 hours post scratch with CellTiter96 Aqueous One Solution Cell Proliferation Assay. Proliferation ratio was determined by the formula: OD_{X hrs post-scratch}/OD_{0 hrs post-scratch}. OD was read at an absorbance of 490 nm. 24 hours post scratch skin 1.190±0.03 vs oral 1.413± 0.02; 48 hours post scratch skin 1.370±0.02 vs oral 1.408±0.01 * p<0.05 by two-way ANOVA and Bonferroni post-test in skin vs oral wounds. N = 3. *Compared to 24 hr skin.

Figure 5. Comparison of skin and oral keratinocyte migration by Gold-Colloidal Migration Assay.

Photographs are representative examples of the tracks made by single cells. The sum of the track areas in the field was divided by the total area of the field and multiplied by 100 to yield the percentage of each field taken up by tracks. This percentage was called the migration index (MI). A) Representative migration assay slides of paired human skin and oral keratinocytes. B) Skin keratinocyte MI 5.1±1.5 and oral keratinocyte MI 13.0±2.7 respectively. Average migration fold ratio of (2.6 fold) oral/skin of N = 3 independent paired oral and skin keratinocyte migration assays.

Figure 6. VEGF, IL-15, and AKT3 expression in skin and oral keratinocytes treated with IL-1β, IL-6, and DMOG (HIF 1α stimulant).

Results were based on 3 individual donors' paired skin and oral mucosal keratinocytes. A) Baseline VEGF expression in oral and skin keratinocytes by RT PCR analyses. B) Relative VEGF expression in keratinocytes following stimulation. C) Baseline IL-15 expression in oral and skin keratinocytes by RT PCR analyses. D) Relative IL-15 expression in keratinocytes following stimulation. E) Baseline AKT3 expression in oral and skin keratinocytes. F) Relative AKT3 expression in keratinocytes following stimulation.