Abstract Introduction and aims Skin ageing is a universal process that can increase susceptibility to inflammation and disease. Basement membrane collagens (BMCs) are reduced during 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 linker of nucleoskeleton and cytoskeleton (LINC) complex (composed of nesprin and sun proteins), nuclear lamina and lamina-associated chromatin. Our hypothesis is that BMC loss during ageing leads to a deformation of the nucleocytoskeleton and disrupts chromatin organization in keratinocytes, leading to impairment of epidermal differentiation and inability to withstand mechanical stress. Methods To explore the role of BMC in mechanical stress response, stable lentiviral short hairpin (sh)RNA knockdowns of Col7 (shCol7) and Col17 (shCol17) were generated in immortalized N/TERT keratinocytes. Western blot (WB) validated knockdowns were compared with shC nontargeting control. IncuCyte assays showed shCol17 had a hyperproliferative phenotype compared with shC. Sun2 was reduced in shCol17 (P < 0.001) compared with shC (n = 5 experimental replicates). The mechanical stretch effect on cells on a flexible membrane was studied using immunofluorescence and WB analysis. 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. Results We observed collagen-specific responses to stretch-induced stress with changes to actin, components of the nuclear membrane, and LINC complex proteins. Additionally, work on investigating chromatin changes with BMC knockdown using Cleavage Under Targets & Tagmentation and Cleavage Under Targeted Accessible Chromatin assays is ongoing. Conclusions Our data show an altered nucleocytoskeleton in the context of BMC loss and mechanotransduction in skin. These data could provide novel insights into epidermal regulation by BMC in ageing.

Background: The composition of lipids within the stratum corneum (SC) is intrinsically linked to the quality of the barrier and to skin hydration. Differences in ceramides, fatty acids and cholesterol have been linked with dry skin and susceptibility to conditions such as atopic dermatitis. Most of the existing literature on skin lipid profiling is from studies in lighter skin tones. There is conflicting evidence for differences in lipid profiles between skin tones, and the relationship with skin dryness has not been addressed.

Recent studies have linked endogenous double-stranded RNA (dsRNA) to the development of inflammatory and autoimmune diseases and cancer. However, the scale of dsRNA formation and its biological consequences is still unclear. Malignant melanoma is the most lethal skin cancer, with an increasing prevalence worldwide. Despite the development of immunotherapy and targeted therapies for melanoma, treatment options are still limited for most patients. dsRNA derived from repetitive DNA elements has the potential to stimulate pattern recognition receptors (PRRs). These PRRs can trigger antiviral signalling cascades and induce an interferon response that renders resistant melanoma sensitive to immunotherapy. Consequently, dsRNA can add a new approach to treating melanoma. Tissue culture and molecular biology techniques were used to induce dsRNA formation in melanoma cell lines (with different mitogen-activated protein kinas-activating mutations) using PRRs agonist poly (I:C), azacitidine (Aza) and hydrogen peroxide (H2O2) as stressors. The impact was measured by quantifying cytosolic dsRNA sensors, also known as PRRs (pPKR, MDA5, RIG1 and ADAR1), using reverse transcriptase quantitative polymerase chain reaction and Western blotting. Immunofluorescence and high-resolution microscopy were then applied to investigate dsRNA and its sensors in skin biopsy samples from patients with melanoma. Moreover, the nature of dsRNA in melanoma was studied by isolating native dsRNA from multiple melanoma cell lines by immune enrichment followed by RNA sequencing. Analysis revealed that inducing endogenous dsRNA significantly activates PRR transcripts and protein levels. However, this activation showed different trends. A375 (BRAFV600E) showed robust MDA5 and RIG1 stimulation to poly (I:C) and a mild ADAR1 and PKR response to Aza and H2O2. Meanwhile, C8161 [BRAF wild type (WT)] was resistant to poly (I:C) with a favourable ADAR1 and RIG1 response to Aza. Immunofluorescence analysis of early stages I and II melanoma skin biopsies showed a significant reduction in ADAR1, which melts dsRNA, with increased dsRNA within BRAF-mutant biopsies. RNAseq analysis showed that A375 (BRAFV600E) produced significantly more dsRNA than C8161 (BRAF WT) and primary dermal fibroblasts. The increase was predominantly due to the enhanced production of dsRNA from nuclear DNA. However, about 11% of the reads mapped to the mitochondrial genome vs. 14% and 19% in C8161 and fibroblasts, respectively. In addition, compared to primary dermal fibroblasts, the dsRNA transcriptome of melanoma cell lines shows unique characteristics and altered dsRNA signalling pathways. To summarize, our data support the hypothesis that endogenous dsRNA activates dsRNA sensors and triggers innate immune signalling. Hence, dsRNA signalling may be explored as a potential therapeutic strategy to treat resistant melanoma with an attack from within.

The skin serves an important barrier function performed by epidermal lipids, cornified epithelial cells and cell–cell adhesions. This function is compromised in old age and exhibits certain abnormalities such as flattened dermal–epidermal junction or improper lipid metabolism, and is also associated with mitochondrial dysfunction. The levels of a basement membrane protein laminin 332 have been shown to decrease as the skin ages. We have previously demonstrated that loss of laminin 332 leads to an upregulation of genes involved in cholesterol biosynthesis and is associated with an altered skin lipid profile. We showed that this change is caused by an abnormal intracellular cholesterol transport that is actin-dependent. To further elucidate the mechanism around cholesterol transport, we have identified five proteins of interest for further investigation. We have shown that two of those proteins, NPC2 and Myo5b, are decreased with the loss of the α3 laminin 332 subunit in laminin α3 knockdown nTERT keratinocytes vs. shC controls. Interestingly, the NPC2 binding partner, NPC1, appears to be increased in the laminin α3 knockdown nTERT keratinocytes. Our analysis has also demonstrated that e-cadherin, a crucial cell–cell adhesion protein, as well as several differentiation markers, are altered with the loss of laminin α3. We have carried out laminin 332 recovery experiments in two (2D) and three dimensions, which showed that the addition of recombinant laminin 332 restores the proper levels of myo5b and e-cadherin, suggesting a potential therapeutic effect. We have also performed proteomic, lipidomic, and transcriptomic studies in 2D, which demonstrated statistically significant changes (Q-value ≤ 0.01) in proteins involved in actin and microtubule regulation, cell–cell adhesion and showed unexpected mitochondrial metabolic changes associated with the loss of laminin α3. These findings show that laminin α3 plays numerous, previously unreported roles in epidermal lipid transport, cell–cell adhesion, differentiation and metabolism, and suggests a potential therapeutic role of the protein in skin ageing.

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.

Junctional epidermolysis bullosa (JEB) caused by loss of function mutations in basement membrane genes, including that encoding laminin-332, is among the most severe forms of epidermolysis bullosa. Affected individuals suffer from blistering from birth, leading to scarring, granulation tissue and susceptibility to infection. In generalized severe JEB, there is a 50% mortality rate in the first 2 years of life, often due to failure to thrive or overwhelming infection. Using cells with knockdown of laminin-α3 we generated a RNAseq data set and have identified a new pathway altered in JEB, the regulation of cholesterol biosynthesis. It has been shown that loss of laminin-332 results in reduced epidermal lipid barrier specifically due to loss of cholesterol transport within keratinocytes. Cholesterol biosynthesis and cholesterol trafficking are fundamental processes in a wide range of cells and tissues, especially in the formation of the normal epidermal lipid barrier. A cholesterol transport screening assay has been developed and used to analyse the ability of 10 specifically selected compounds to restore cholesterol transport in our in vitro model of JEB. Results of this small-scale screen showed that nine of 10 of the selected drugs were able to restore cholesterol transport up to 50% of control. Six drugs were selected for further analysis within our three-dimensional (3D) skin-equivalent model of JEB. Organotypic skin equivalents of normal skin (siControl) and JEB skin (siLaminin-α3) were treated daily for 7 days with either dimethyl sulfoxide or 1 µmol L–1 of selected drugs. In 3D, drugs 1, 3, 5 and 6 displayed an increase in stratum corneum lipids shown by Nile red staining. Drugs 4 and 7 did not increase stratum corneum lipids above levels seen in the untreated JEB organotypics. Further analysis of skin differentiation by keratin, involucrin and tranglutaminase staining were assessed to narrow drug selection for further testing. These data also suggest that JEB is not just a disorder of epidermal–dermal adhesion, but a disorder of aberrant cholesterol trafficking causing a markedly impaired skin barrier. These findings suggests that drugs to target cholesterol transport within keratinocytes could be a beneficial treatment for JEB, helping to improve the crucial epidermal lipid barrier and prevent water loss and infection at the early stages of life.

When properly formulated into lotions and cleansers to enable deposition and delivery into skin it has been previously shown that skin-natural fatty acids (palmitic acid and stearic acid) elongate into longer chain fatty acids and ceramides which are critical for skin barrier health. In separate studies, 12-hydroxystearic acid (12-HSA) has been shown to act as a PPAR agonist to help with epidermal homeostasis and barrier function. Exploring potential synergy between topically applied skin-natural fatty acids and 12-HSA is of interest to understand mechanisms for improving barrier health and develop more efficacious products. To determine if 12-HSA, applied topically with deuterated palmitic acid (C16:0), promotes its elongation and integration into skin ceramides and sphingosine. Monolayer keratinocytes were dosed with 1 uM of deuterated palmitic acid and varying levels of 12-HSA for 7 days prior to cell harvesting and lipid extraction. Ex vivo human skin explants were dosed topically with 3% deuterated palmitic acid, with or without 3% 12-HSA, for 5 days prior to lipid extraction. Analysis was conducted by LC/MS/MS. Deuterated fatty acids with chain lengths higher than palmitic were detected in each of the combinations of palmitic acid and 12-HSA tested in keratinocytes. Correspondingly, 12-HSA boosted levels of deuterated longer chain fatty acids in skin explants compared with the controls without 12-HSA and increased integration of deuterated palmitic acid into ceramides. Topical application of 12-HSA and skin-natural fatty acids show synergistic metabolic effect by enhancing lipid elongation and ceramide production, consistent with building and supporting a healthy barrier.