Galactomyces ferment filtrate upregulates anchoring junctions and stabilizes actin to maintain the Young's modulus of skin cells in vitro

OBJECTIVE: Skin is exposed to multiple external factors such as solar radiation and environmental oxidative stressors. Ageing and the accumulation of stress damage lead to reduced skin health and loss of skin firmness, which contribute to wrinkling and sagging. We investigated if Galactomyces ferment filtrate (GFF) could modulate skin cell mechanical properties through changes to the actin cytoskeleton and anchoring junctions. METHODS: Proteomic analysis was performed to identify age-associated changes in cellular components in skin biopsies and GFF-mediated changes in full thickness 3D skin equivalent models. Nanoindentation was used to measure the Young's modulus (resistance to elastic deformation) of keratinocyte cells under Latrunculin B (LatB)-induced destabilization of the actin cytoskeleton. Immunofluorescence and confocal microscopy were performed on skin equivalents to validate the GFF-induced changes observed in proteomic analysis and identify expression profiles of key structural proteins within the skin. RESULTS: Aged skin had decreased expression of internal cellular components 'actin cytoskeleton' and 'focal adhesion'. Destabilization of actin cytoskeleton by LatB significantly reduced the Young's modulus of keratinocyte cells. Pre-treatment of keratinocytes with GFF was able to prevent LatB-induced decline of skin cell mechanical properties. Proteomic analysis of full thickness 3D skin equivalent models revealed that GFF increased the expression of 'actin cytoskeleton' and 'anchoring junctions', especially the actin-binding focal adhesion proteins. Immunofluorescence staining confirmed that GFF upregulated expression of the actin stabilizer, calponin 2 and integrin α2 in basal keratinocytes. GFF increased integrin localisation to the dermal-epidermal junction (DEJ) and colocalised with collagen IV in vitro, suggesting increased linkage between the epidermis and dermis. CONCLUSION: Our data establish the actin cytoskeleton and anchoring junctions which bind it as important cellular structures which decline with age. We also identified that the cellular structures are required for the maintenance of keratinocyte Young's modulus, which could be an important property impacting skin firmness. Furthermore, we have identified that GFF promotes actin stability and builds a rigid holding structure of the anchoring junctions to increase cell-cell and epidermis-dermis anchoring in skin equivalent models. Therefore, GFF has the potential to help maintain and restore skin firmness in young and old skin, respectively.