| dc.description.abstract | Atopic eczema is the most common inflammatory dermatosis, affecting up to 20% of children. Loss of function mutations in the Filaggrin (FLG) gene are the most strongly implicated genetic risk factor for eczema, but so far attempts to replace or enhance the FLG protein have been unsuccessful. In this PhD I assessed the impact of loss of FLG on the cellular signalling environment and other barrier proteins. The FLG gene sits with 62 related genes on a locus of Chromosome 1 called the Epidermal Differentiation Complex (EDC). Using shRNA to knock down (KD) the FLG gene in vitro, I showed that loss of cellular FLG, causes a reduction of other Keratin and EDC proteins. I then went on to examine these finding in patients with atopic eczema. To do this I optimised a technique to extract and quantify barrier proteins from tape strip samples. Although there was no clear change in Keratin or EDC expression from the patient samples, there was a negative correlation between FLG and phospho-SMAD1/5/9 expression. Further analysis of patient samples suggests that this is mediated by BMP2 and BMP6. I then went on to explore mechanistically the effect of BMP2 and BMP6 treatment on the cellular environment, and found that this increased FLG and Keratin expression, suggesting it has a protective role. Following this, I performed phosphoproteomic analysis on FLG shRNA KD cell lines. Enrichment analysis showed that loss of FLG changes the AKT1, ERK1/2 and p38 signalling pathways. This suggests that FLG has a direct effect on cellular signalling. The protein cFOS was downregulated following FLG KD and correlated with FLG expression in patient samples and repository datasets. cFOS was also downregulated following BMP6 treatment, suggesting that the Activator Protein 1 (AP-1) pathway may be an important link between FLG and phospho-SMAD1/5/9 expression. | en_US |
