P16 How does skin ageing-related basement membrane collagen loss affect the nucleus?

Abstract Skin ageing effects, such as inflammation, oxidative stress, disrupted wound healing and disease, are universal issues. Basement membrane collagens (BMCs) are reduced by ageing and the consequences are poorly understood. BMC sensing of mechanical stress, vital for skin homeostasis, elicits signalling sensed via cytoskeletal actin filaments to the LINC complex (composed of nesprin and sun proteins), nuclear lamina and lamina-associated chromatin. RNAseq data from collagen 7 (Col7) or collagen 17 (Col17) small interfering (si)RNA knockdown primary keratinocytes revealed an opposing enrichment of nuclear function genes. Nesprin-2 was reduced with siCol7 (P < 0.01) vs. siC nontargeting control. We hypothesized that BMC loss in keratinocytes disrupts the nucleocytoskeleton and chromatin organization, dysregulating gene expression and signalling, leading to altered epidermal differentiation and skin barrier impairment. To explore the role of BMC in mechanical stress response, stable short hairpin (sh)RNA knockdowns of Col7 (shCol7) and Col17 (shCol17), and nontargeting control (shC) were generated in immortalized N/TERT cells. Western blot validated knockdowns compared to shC. MTT assays showed a hyperproliferative phenotype with shCol17 compared to shC. Mechanical stretch effect on cells on a flexible membrane was studied by immunofluorescence and Western blot (WB). Images taken from the IN Cell Analyzer confocal microscope were analysed to detect changes in protein expression and localization. Nuclear and cytoplasmic fractions made before and after stretch were analysed by WB. We observed collagen-specific responses to stretch-induced stress with changes to actin and nuclear membrane proteins: emerin and lamin B1, and sun2. Our data are the first to show an altered nucleocytoskeleton in the context of BMC loss and mechanotransduction in skin. This study could provide novel insights into ageing epidermal regulation by BMCs.