Naeem, AS; (2014) The role of Akt1 in skin barrier formation. Doctoral thesis , UCL (University College London).

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Abstract

Atopic Dermatitis (AD) is a chronic inflammatory disease characterised by pruritus, hyperkeratosis, parakeratosis and dry skin. Association of filaggrin mutations with AD has been reported, however not all AD patients have filaggrin mutations suggesting other mechanisms give rise to the barrier defect present in AD. Akt1 activity is essential for cornified envelope formation and correct profilaggrin processing, although the functional role of Akt1 in these processes remains unclear. The aim of this study was to investigate the role of downstream targets of Akt1 signaling in profilaggrin processing and cornified envelope formation. Using shRNA to knock down Akt1 activity in rat epidermal keratinocyte cell lines, an in vitro organotypic model was created that phenocopies AD displaying hyperkeratosis, parakeratosis and impaired filaggrin processing. Results show that reduced Akt activity led to decreased lamin A/C degradation disrupting nuclear disintegration process giving rise to parakeratosis. HspB1 is reported to interact with filaggrin and also involved in filaggrin processing. Inhibition of Akt activity demonstrated a switch between HspB1-filaggrin to HspB1- actin interaction in the upper epidermis, which may interfere with filaggrin processing. Cathepsin H (Ctsh) was down regulated 4-fold in our Akt1 knockdown cultures and its expression co-localized with filaggrin in the granular layer of human epidermis. Filaggrin processing was impaired in Ctsh shRNA knockdown cell lines, and both Ctsh+/− and Ctsh−/− mice displayed hyperkeratosis and reduced filaggrin expression. Inhibition of RAPTOR (a component of mTORC1) has previously been reported to increase Akt1 activity. Raptor overexpression in REKs showed a decrease in Akt phosphorylation, Ctsh and filaggrin processing, and treatment with Rapamycin, an mTORC1 inhibitor, reverses these effects. In uninvolved AD skin, increase in RAPTOR correlated with decrease in both Akt phosphorylation and filaggrin expression. This thesis presents a novel mechanism where increase in RAPTOR can lead to a reduction in epidermal granular Akt1 phosphorylation leading to impaired filaggrin processing and barrier defects in AD.

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