Biology subjects

O'Shaughnessy, R. F.

Publications and source records attributed to O'Shaughnessy, R. F..

3 recordsLinked to original sources

bioRxiv2023

Loss of cholesterol in Junctional Epidermolysis Bullosa skin identifies a key role for Laminin-332 in actomyosin mediated cholesterol transport

Individuals with Junctional Epidermolysis Bullosa (JEB), a rare genetic skin disease characterised by loss of function mutations in the Laminin332 (Lam332), do not survive beyond their first birthday. Here we report that loss of Lam332 leads to absence of cholesterol lipid from the epidermis in vitro and in vivo. Using 3D skin equivalents, a JEB mouse model and JEB patient samples we confirmed changes in epidermal lipid synthesis, which was further explored using lipidomics. Cholesterol biosynthesis genes were increased with loss of Laminin-332 in vitro, however a decrease in immunofluorescence lipid staining was observed. Cholesterol transport in Laminin-332 knockdown keratinocytes was revealed to be disrupted, which in keratinocytes is dependent on the actomyosin network. In conclusion these findings suggest a role for the basement membrane protein Laminin-332 in lipid metabolism in the skin, and a broader role for epidermal homeostasis and barrier formation. Restoration of cholesterol transport in epidermal keratinocytes of JEB patients offers the potential to improve their skin barrier.

cell biology↗

bioRxiv2022

Phosphoproteomic analysis of the AKT signalling axis in cutaneous squamous carcinoma progression reveals novel therapeutic targets

Cutaneous Squamous Cell Carcinoma (cSCC) represents about 20% of all non-melanoma skin cancers. Whilst generally low risk to patients, metastases are associated with a poor prognosis. cSCC incidence is increasing, owing to an ageing population, greater exposure to UV radiation, and more patients receiving immunosuppressive treatments associated with organ transplants. Therefore, there is interest in identifying new biomarkers that may be to track progression of the disease and to exploit as therapeutic vulnerabilities. We show dynamic changes in AKT expression in precursor lesions and in SCC tumour tissue, with initial loss of AKT activity followed by progressive and widespread increase in AKT activity in SCC. Phosphoproteomic analysis and kinase substrate enrichment analysis on a panel of isogenic cSCC cell lines representing different stages of the disease from premalignancy to metastasis revealed several up-regulated kinases and AKT-targets. From this analysis we chose DNA dependent protein kinase (DNA-PK), a key kinase upstream of AKT phoshorlyation, and N-Myc downstream-regulated gene 2 (NDRG2) a downstream AKT phosphorylation target, to investigate in further detail. Both proteins were up-regulated and mis-expressed in a panel of SCC tissue from different patients. We therefore explored the potential of inhibiting DNA-PK and NDRG2 as cSCC treatments. Treatment with the iron chelator Dp44mT decreased levels of phosphorylated NDRG2 and led to significant losses to viability and reduced migration in our cSCC cell lines, while DNA-PK inhibition promoted the differentiation of premalignant and early-stage SCC cell lines. Our results suggest that NDRG2 and DNA-PK may be viable targets in cSCC treatment, with effectiveness at different stages of SCC progression.

cancer biology↗

bioRxiv2022

Chronic activation of Toll-like receptor 2 induces an ichthyotic skin phenotype

Ichthyosis defines a group of chronic conditions that manifest phenotypically as a thick layer of fish-like scales in response to disorders of cornification and often affects the entire skin. While the gene mutations that lead to ichthyosis are well documented, the actual signalling mechanisms that lead to scaling are poorly characterised, however recent publications suggest that there are common mechanisms active in ichthyotic tissue, and in analogous models of ichthyosis. Combining gene expression analysis of gene-specific shRNA knockdowns of more severe autosomal recessive congenital ichthyoses (ARCI) and proteomic analysis of skin scale from ARCI patients, we identified a common activation of the Toll-like receptor (TLR) 2 pathway. Exogenous activation of TLR2 led to increased expression of important cornified envelope genes and in organotypic culture caused hyperkeratosis. Conversely blockade of TLR2 signalling in ichthyosis patient keratinocytes and our shRNA models reduced the expression of keratin 1, a structural protein overexpressed in ichthyosis scale. A time-course of Tlr2 activation in rat epidermal keratinocytes revealed that although there was rapid initial activation of innate immune pathways, this was rapidly superseded by widespread up-regulation of epidermal differentiation related proteins. Both NF{kappa}{beta} phosphorylation and the Gata3 up-regulation was associated with this switch and Gata3 overexpression was sufficient to increase Keratin 1 expression. Taken together, these data define a dual role for Tlr2 during epidermal barrier repair, that may be a useful therapeutic modality in treating diseases of epidermal barrier dysfunction.

cell biology↗