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Human skin as a model for signalling during cellular differentiation

A central research theme within the Systems Biology Laboratory is investigating how cells use molecular mechanisms to control tissue function.

Cursons J, Gao J, Hurley DG, Dunbar PR, Jacobs MD, Crampin EJ. Regulation of ERK-MAPK signaling in human epidermis. BMC Syst Biol. 2015;9(1):41.

Through collaboration with Prof. Rod Dunbar’s laboratory at the Maurice Wilkins Centre we have been investigating the changes that occur to cells within the outermost layer of the skin, known as the epidermis.

Figure 1

The outermost layer of the skin is known as the epidermis

Specifically, we are interested in the terminal differentiation of cells (changes that occur as the cells age and adopt a specialised function) known as keratinocytes, which largely comprise the interfollicular epidermis. The molecular and morphological changes that occur to keratinocytes during differentiation are essential for the epidermal barrier function, which provides protection against environmental insult.

The interfollicular epidermis consists of several discrete tissue layers where cells show very different characteristics. Fully differentiated keratinocytes, known as corneocytes, are lost at the superficial surface due to mechanical abrasion, and these cells are replaced by the slow, but continuous proliferation of cells within the deepest basal layers. Cellular proliferation within the deepest layers creates a continuous movement of cells towards the outer surface, and over approximately two-three weeks a keratinocyte will traverse the depth of the epidermis and undergo the required changes, establishing a keratinocyte differentiation gradient.

Figure 2

Keratinocytes within the interfollicular epidermis maintain a differentiation gradient which is necessary for tissue function

We have been using imaging methods to investigate the relative abundance and distribution of selected proteins within human skin. Much of our work has focused on changes in activity for the ERK-MAPK signalling cascade which has been extensively studied in cell culture experiments, and previously implicated in regulating keratinocyte proliferation and differentiation. The large number of in vitro studies have been essential for understanding how these signalling pathway components interact at a molecular level. However, we are yet to fully elucidate how signalling pathways are modulated within human tissues, and how they influence the development and maintenance of tissue structures.

For this work, we developed computational methods which allow us to convert the fluorescence image data obtained from confocal microscopy into a quantitative format which is amenable to mathematical analyses. We incorporated histological features which allowed us to identify the distinct epidermal layers, and measured the signal intensity (as an indicator of relative protein abundance) within each of these tissue layers, at specific sub-cellular localisations.

Figure 3

Computational methods were used to sample the fluorescence image data and convert it into a quantitative format for mathematical analysis

By incorporating these histological features, we were able to combine data collected across different experiments using single-target labelling. Furthermore, this transformation allowed us to more effectively compare data collected from different patients which showed histological differences in skin structure.

Intriguingly, these experimental data showed evidence of co-ordinated and sustained activation of ERK-MAPK signalling across the depth of human epidermis, or along the gradient of keratinocyte terminal differentiation. It has been known for some time that increasing extracellular calcium can induce terminal differentiation of keratinocytes grown in vitro. Furthermore, imaging studies by other groups have shown an increase in the relative abundance of Ca2+ across the depth of the epidermis, peaking within the late granular layers.

Given these results, we proposed the hypothesis that changes in calcium signalling may drive the graded activation of ERK-MAPK which we observed across the depth of the epidermis. A model was developed describing canonical ERK-MAPK interactions with regulation by Ca2+ and calmodulin.

Figure 4

Computational methods were used to sample the fluorescence image data and convert it into a quantitative format for mathematical analysis

This model was implemented using a normalized-Hill differential equation formalism with default values for all but four parameters, which were estimated using a non-linear least squares approach within MATLAB.

Intriguingly, this relatively simple model shows that the epidermal gradient of calcium and changes in the abundance of plasma-membrane localised calmodulin appear sufficient to drive the extended gradient of ERK-MAPK signalling which we observed within our imaging data.

Figure 5

Computational methods were used to sample the fluorescence image data and convert it into a quantitative format for mathematical analysis

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