Highly Sensitive Terahertz Metasurface Based on Electromagnetically Induced Transparency-Like Resonance in Detection of Skin Cancer Cells.

Terahertz (THz) metasurfaces based on high Q-factor electromagnetically induced transparency-like (EIT-like) resonances are promising for biological sensing. Despite this potential, they have not often been investigated for practical differentiation between cancerous and healthy cells. The present methodology relies mainly on refractive index sensing, while factors of transmission magnitude and Q-factor offer significant information about the tumors. To address this limitation and improve sensitivity, we fabricated a THz EIT-like metasurface based on asymmetric resonators on an ultra-thin and flexible dielectric substrate. Bright-dark modes coupling at 1.96 THz was experimentally verified, and numerical results and theoretical analysis were presented. An enhanced theoretical sensitivity of 550 GHz/RIU was achieved for a sample with a thickness of 13 µm due to the ultra-thin substrate and novel design. A two-layer skin model was generated whereby keratinocyte cell lines were cultured on a base of collagen. When NEB1-shPTCH (basal cell carcinoma (BCC)) were switched out for NEB1-shCON cell lines (healthy) and when BCC's density was raised from 1 × 105 to 2.5 × 105, a frequency shift of 40 and 20 GHz were observed, respectively. A combined sensing analysis characterizes different cell lines. The findings may open new opportunities for early cancer detection with a fast, less-complicated, and inexpensive method.

Disseminating cells in human oral tumours possess an EMT cancer stem cell marker profile that is predictive of metastasis in image-based machine learning.

Cancer stem cells (CSCs) undergo epithelial-mesenchymal transition (EMT) to drive metastatic dissemination in experimental cancer models. However, tumour cells undergoing EMT have not been observed disseminating into the tissue surrounding human tumour specimens, leaving the relevance to human cancer uncertain. We have previously identified both EpCAM and CD24 as CSC markers that, alongside the mesenchymal marker Vimentin, identify EMT CSCs in human oral cancer cell lines. This afforded the opportunity to investigate whether the combination of these three markers can identify disseminating EMT CSCs in actual human tumours. Examining disseminating tumour cells in over 12,000 imaging fields from 74 human oral tumours, we see a significant enrichment of EpCAM, CD24 and Vimentin co-stained cells disseminating beyond the tumour body in metastatic specimens. Through training an artificial neural network, these predict metastasis with high accuracy (cross-validated accuracy of 87-89%). In this study, we have observed single disseminating EMT CSCs in human oral cancer specimens, and these are highly predictive of metastatic disease.

When oral cancers metastasise ­ that is, when tumour cells invade other parts of the body ­ they typically do so by first colonizing the lymph nodes present in the neck. As this event significantly reduces chances of survival, oral cancer patients often have their neck lymph nodes removed to prevent the spread of the disease. However, this surgery carries risks and leads to longer hospital stays, stressing the need for better ways to predict which oral tumours will metastasise. Evidence from lab-grown cells and mice studies suggest that, in oral cancer, metastasis occurs when some cells in the original tumour go through a process called the epithelial-mesenchymal transition (EMT for short). This transformation allows the cells to detach from the tumour and become invasive. However, it has so far been difficult to observe this process in actual human tumours; this is partly because cells undergoing EMT stop producing the proteins that scientists rely on to distinguish cancer and healthy cells. To address this knowledge gap, Youssef et al. focused on three proteins: two tumour markers, EpCAM and CD24; and Vimentin, which is produced in greater quantities in the invasive mesenchymal state. Previous work had shown that a specific population of oral tumour cells can continue to express all three proteins even when adopting a mesenchymal identity through EMT. Based on this knowledge, Youssef et al. hypothesised that tracking Vimentin, EpCAM and CD24 using fluorescence microscopy would allow them to identify metastasising cells in human samples. An analysis of over 12,000 images from 74 tumours obtained from surgeries revealed that, in the metastatic samples, the cells detaching from primary tumours were more likely to express these three proteins. Finally, Youssef et al. used these images to train a machine learning algorithm. When applied to data from new oral cancer patients, the programme was able to predict whether their tumours were likely to spread with 89% accuracy. If confirmed by further work, and in particular on larger samples, these findings could in the future help clinicians decide which patients with oral cancer would benefit the most from surgery to remove neck lymph nodes.

Observations that suggest a contribution of altered dermal papilla mitochondrial function to androgenetic alopecia.

Androgenetic alopecia (AGA) is a prevalent hair loss condition in males that develops due to the influence of androgens and genetic predisposition. With the aim of elucidating genes involved in AGA pathogenesis, we modelled AGA with three-dimensional culture of keratinocyte-surrounded dermal papilla (DP) cells. We co-cultured immortalised balding and non-balding human DP cells (DPCs) derived from male AGA patients with epidermal keratinocyte (NHEK) using multi-interfacial polyelectrolyte complexation technique. We observed up-regulated mitochondria-related gene expression in balding compared with non-balding DP aggregates which indicated altered mitochondria metabolism. Further observation of significantly reduced electron transport chain complex activity (complexes I, IV and V), ATP levels and ability to uptake metabolites for ATP generation demonstrated compromised mitochondria function in balding DPC. Balding DP was also found to be under significantly higher oxidative stress than non-balding DP. Our experiments suggest that application of antioxidants lowers oxidative stress levels and improves metabolite uptake in balding DPC. We postulate that the observed up-regulation of mitochondria-related genes in balding DP aggregates resulted from an over-compensatory effort to rescue decreased mitochondrial function in balding DP through the attempted production of new functional mitochondria. In all, our three-dimensional co-culturing revealed mitochondrial dysfunction in balding DPC, suggesting a metabolic component in the aetiology of AGA.

Human hair follicles operate an internal Cori cycle and modulate their growth via glycogen phosphorylase.

Hair follicles (HFs) are unique, multi-compartment, mini-organs that cycle through phases of active hair growth and pigmentation (anagen), apoptosis-driven regression (catagen) and relative quiescence (telogen). Anagen HFs have high demands for energy and biosynthesis precursors mainly fulfilled by aerobic glycolysis. Histochemistry reports the outer root sheath (ORS) contains high levels of glycogen. To investigate a functional role for glycogen in the HF we quantified glycogen by Periodic-Acid Schiff (PAS) histomorphometry and colorimetric quantitative assay showing ORS of anagen VI HFs contained high levels of glycogen that decreased in catagen. qPCR and immunofluorescence microscopy showed the ORS expressed all enzymes for glycogen synthesis and metabolism. Using human ORS keratinocytes (ORS-KC) and ex vivo human HF organ culture we showed active glycogen metabolism by nutrient starvation and use of a specific glycogen phosphorylase (PYGL) inhibitor. Glycogen in ORS-KC was significantly increased by incubation with lactate demonstrating a functional Cori cycle. Inhibition of PYGL significantly stimulated the ex vivo growth of HFs and delayed onset of catagen. This study defines translationally relevant and therapeutically targetable new features of HF metabolism showing that human scalp HFs operate an internal Cori cycle, synthesize glycogen in the presence of lactate and modulate their growth via PYGL activity.

Watching hair turn grey.

Analysing changes in hair pigmentation may lead to a better understanding of the impacts of 'life events' on human biology and aging.

Differential expression of phosphorylated MEK and ERK correlates with aggressive BCC subtypes.

Basal cell carcinoma (BCC) is associated with aberrant Hedgehog (HH) signalling through mutational inactivation of PTCH1; however, there is conflicting data regarding MEK/ERK signalling in BCC and the signalling pathway interactions in these carcinomas. To address this, expression of active phospho (p) MEK and ERK was examined in a panel of 15 non-aggressive and 14 aggressive BCCs. Although not uniformly expressed, both phospho-proteins were detected in the nuclei and/or cytoplasm of normal and tumour-associated epidermal cells however, whereas phospho-MEK (pMEK) was present in all non-aggressive BCCs (14/14), phospho-ERK (pERK) was rarely expressed (2/14). In contrast pERK expression was more prevalent in aggressive tumours (11/14). Interestingly, pMEK was only localized to the tumour mass whereas pERK was expressed in tumours and stroma of aggressive BCCs. Similarly, pERK (but not pMEK) was absent in mouse BCC-like tumours derived from X-ray irradiated Ptch1+/- mice with stromal pERK observed in myofibroblasts of the aggressive variant as well as in the tumour mass. RNA sequencing analysis of tumour epithelium and stroma of aggressive and non-aggressive BCC revealed the upregulation of epidermal growth factor receptor- and ERK-related pathways. Angiogenesis and immune response pathways were also upregulated in the stroma compared with the tumour. PTCH1 suppressed NEB1 immortalized keratinocytes (shPTCH1) display upregulated pERK that can be independent of MEK expression. Furthermore, epidermal growth factor pathway inhibitors affect the HH pathway by suppressing GLI1. These studies reveal differential expression of pERK between human BCC subtypes that maybe active by a pathway independent of MEK.

Early growth response 2 (EGR2) is a novel regulator of the senescence programme.

Senescence, a state of stable growth arrest, plays an important role in ageing and age-related diseases in vivo. Although the INK4/ARF locus is known to be essential for senescence programmes, the key regulators driving p16 and ARF transcription remain largely underexplored. Using siRNA screening for modulators of the p16/pRB and ARF/p53/p21 pathways in deeply senescent human mammary epithelial cells (DS HMECs) and fibroblasts (DS HMFs), we identified EGR2 as a novel regulator of senescence. EGR2 expression is up-regulated during senescence, and its ablation by siRNA in DS HMECs and HMFs transiently reverses the senescent phenotype. We demonstrate that EGR2 activates the ARF and p16 promoters and directly binds to both the ARF and p16 promoters. Loss of EGR2 down-regulates p16 levels and increases the pool of p16- p21- 'reversed' cells in the population. Moreover, EGR2 overexpression is sufficient to induce senescence. Our data suggest that EGR2 is a direct transcriptional activator of the p16/pRB and ARF/p53/p21 pathways in senescence and a novel marker of senescence.

Psoriatic skin inflammation induces a pre-diabetic phenotype via the endocrine actions of skin secretome.

OBJECTIVE: Psoriasis is a chronic inflammatory skin disease that is thought to affect ∼2% of the global population. Psoriasis has been associated with ∼30% increased risk of developing type 2 diabetes (T2D), with numerous studies reporting that psoriasis is an independent risk-factor for T2D, separate from underlying obesity. Separately, studies of skin-specific transgenic mice have reported altered whole-body glucose homeostasis in these models. These studies imply a direct role for skin inflammation and dysfunction in mediating the onset of T2D in psoriasis patients, potentially via the endocrine effects of the skin secretome on key metabolic tissues. We used a combination of in vivo and ex vivo mouse models and ex vivo human imiquimod (IMQ) models to investigate the effects of psoriasis-mediated changes in the skin secretome on whole-body metabolic function. METHODS: To induce psoriatic skin inflammation, mice were topically administered 75 mg of 5% IMQ cream (or Vaseline control) to a shaved dorsal region for 4 consecutive days. On day 5, mice were fasted for glucose and insulin tolerance testing, or sacrificed in the fed state with blood and tissues collected for analysis. To determine effects of the skin secretome, mouse skin was collected at day 5 from IMQ mice and cultured for 24 h. Conditioned media (CM) was collected and used 1:1 with fresh media to treat mouse explant subcutaneous adipose tissue (sAT) and isolated pancreatic islets. For human CM experiments, human skin was exposed to 5% IMQ cream for 20 min, ex vivo, to induce a psoriatic phenotype, then cultured for 24 h. CM was collected, combined 1:1 with fresh media and used to treat human sAT ex vivo. Markers of tissue inflammation and metabolic function were determined by qPCR. Beta cell function in isolated islets was measured by dynamic insulin secretion. Beta-cell proliferation was determined by measurement of Ki67 immunofluorescence histochemistry and BrDU uptake, whilst islet apoptosis was assessed by caspase 3/7 activity. All data is expressed as mean ± SEM. RESULTS: Topical treatment with IMQ induced a psoriatic-like phenotype in mouse skin, evidenced by thickening, erythema and inflammation of the skin. Topical IMQ treatment induced inflammation and signs of metabolic dysfunction in sub-cutaneous and epidydimal adipose tissue, liver, skeletal muscle and gut tissue. However, consistent with islet compensation and a pre-diabetic phenotype, IMQ mice displayed improved glucose tolerance, increased insulin and c-peptide response to glucose, and increased beta cell proliferation. Treatment of sAT with psoriatic mouse or human skin-CM replicated the in vivo phenotype, leading to increased inflammation and metabolic dysfunction in mouse and human sAT. Treatment of pancreatic islets with psoriatic mouse skin-CM induced increases in beta-proliferation and apoptosis, thus partially replicating the in vivo phenotype. CONCLUSIONS: Psoriasis-like skin inflammation induces a pre-diabetic phenotype, characterised by tissue inflammation and markers of metabolic dysfunction, together with islet compensation in mice. The in vivo phenotype is partially replicated by exposure of sAT and pancreatic islets to psoriatic-skin conditioned media. These results support the hypothesis that psoriatic skin inflammation, potentially via the endocrine actions of the skin secretome, may constitute a novel pathophysiological pathway mediating the development of T2D.

An alternative modular 'click-SNAr-click' approach to develop subcellular localised fluorescent probes to image mobile Zn2+ .

Zn2+ is involved in a number of biological processes and its wide-ranging roles at the subcellular level, especially in specific organelles, have not yet been fully established due to a lack of tools to image it effectively. We report a new and efficient modular double 'click' approach towards a range of sub-cellular localised probes for mobile zinc. Through this methodology, endoplasmic reticulum, mitochondria and lysosome localised probes were successfully prepared which show good fluorescence responses to mobile Zn2+in vitro and in cellulo whilst a non-targeting probe was synthesized as a control. The methodology appears to have wide-utility for the generation of sub-cellular localised probes by incorporating specific organelle targeting vectors for mobile Zn2+ imaging.

Endoplasmic reticulum targeting fluorescent probes to image mobile Zn2.

Zn2+ plays an important role in the normal function of the endoplasmic reticulum (ER) and its deficiency can cause ER stress, which is related to a wide range of diseases. In order to provide tools to better understand the role of mobile Zn2+ in ER processes, the first custom designed ER-localised fluorescent Zn2+ probes have been developed through the introduction of a cyclohexyl sulfonylurea as an ER-targeting unit with different Zn2+ receptors. Experiments in vitro and in cellulo show that both probes have a good fluorescence switch on response to Zn2+, high selectivity over other cations, low toxicity, ER-specific targeting ability and are efficacious imaging agents for mobile Zn2+ in four different cell lines. Probe 9 has been used to detect mobile Zn2+ changes under ER stress induced by both tunicamycin or thapsigargin, which indicates that the new probes should allow a better understanding of the mechanisms cells use to respond to dysfunction of zinc homeostasis in the ER and its role in the initiation and progression of diseases to be developed.

Biotin-tagged fluorescent sensor to visualize 'mobile' Zn2+ in cancer cells.

A cancer cell-targeting fluorescent sensor has been developed to image mobile Zn2+ by introducing a biotin group. It shows a highly selective response to Zn2+in vitro, no toxicity in cellulo and images 'mobile' Zn2+ specifically in cancer cells. We believe this probe has the potential to help improve our understanding of the role of Zn2+ in the processes of cancer initiation and development.

MicroRNA and transcriptome analysis in periocular Sebaceous Gland Carcinoma.

Sebaceous gland carcinoma (SGC) is a rare, but life-threatening condition with a predilection for the periocular region. Eyelid SGC can be broadly categorised into two subtypes, namely either nodular or pagetoid with the latter being more aggressive and requiring radical excision to save life. We have identified key altered microRNAs (miRNA) involved in SGC shared by both subtypes, hsa-miR-34a-5p and hsa-miR-16-5p. However, their gene targets BCL2 and MYC were differentially expressed with both overexpressed in pagetoid but unchanged in nodular suggesting different modes of action of these two miRNAs on BCL/MYC expression. Hsa-miR-150p is nodular-specifically overexpressed, and its target ZEB1 was significantly downregulated in nodular SGC suggesting a tumour suppressor role. Invasive pagetoid subtype demonstrated specific overexpression of hsa-miR-205 and downregulation of hsa-miR-199a. Correspondingly, miRNA gene targets, EZH2 (by hsa-miR-205) and CD44 (by hsa-miR-199a), were both overexpressed in pagetoid SGC. CD44 has been identified as a potential cancer stem cell marker in head and neck squamous cell carcinoma and its overexpression in pagetoid cells represents a novel treatment target. Aberrant miRNAs and their gene targets have been identified in both SGC subtypes, paving the way for better molecular understanding of these tumours and identifying new treatment targets.

Culture of the human pilosebaceous unit, hair follicle and sebaceous gland.

Terence Kealey first pioneered the isolation and organ maintenance of human eccrine and sebaceous glands in the early to mid-1980. This led to subsequent methods describing the isolation and culture of human hair follicles, the human pilosebaceous unit as well as the sebaceous duct. The importance of these models in the study of the biology of human skin glands and appendages has been demonstrated in numerous publications and their importance as models for animal replacement, refinement and reduction (3Rs) is increasingly important. In particular, in vitro (ex vivo) hair follicle culture has played a significant part in helping elucidate the role of signalling molecules in regulating hair growth and hair fibre formation and has been especially useful in understanding metabolic aspects of hair growth. However, obtaining sufficient numbers of hair follicles is becoming increasingly difficult as plastic surgery becomes less invasive and smaller skin samples provided. There is therefore an urgent requirement for the next generation of in vitro models using cell lines and tissue engineering, and this has led to the development of immortalised cell lines as well as attempts to model hair follicle embryogenesis in vitro and development of skin on a chip.

Tissue stiffening promotes keratinocyte proliferation through activation of epidermal growth factor signaling.

Tissue biomechanics regulate a wide range of cellular functions, but the influences on epidermal homeostasis and repair remain unclear. Here, we examined the role of extracellular matrix stiffness on human keratinocyte behavior using elastomeric substrates with defined mechanical properties. Increased matrix stiffness beyond normal physiologic levels promoted keratinocyte proliferation but did not alter the ability to self-renew or terminally differentiate. Activation of epidermal growth factor (EGF) signaling mediated the proliferative response to matrix stiffness and depended on focal adhesion assembly and cytoskeletal tension. Comparison of normal skin with keloid scar tissue further revealed an upregulation of EGF signaling within the epidermis of stiffened scar tissue. We conclude that matrix stiffness regulates keratinocyte proliferation independently of changes in cell fate and is mediated by EGF signaling. These findings provide mechanistic insights into how keratinocytes sense and respond to their mechanical environment, and suggest that matrix biomechanics may play a role in the pathogenesis keloid scar formation.

Analysis of hedgehog signaling in periocular sebaceous carcinoma.

PURPOSE: Sebaceous carcinoma (SC) is a clinical masquerader of benign conditions resulting in significant eye morbidity, sometimes leading to extensive surgical treatment including exenteration, and even mortality. Little is known about the genetic or molecular basis of SC. This study identifies the involvement of Hedgehog (Hh) signaling in periocular SC. METHODS: Fifteen patients with periocular SC patients were compared to 15 patients with eyelid nodular basal cell carcinoma (nBCC; a known Hh tumor), alongside four normal individuals as a control for physiological Hh expression. Expression of Patched 1 (PTCH1), Smoothened (SMO), and glioma-associated zinc transcription factors (Gli1 and Gli2) were assessed in histological sections using immunohistochemistry and immunofluorescence (IF) techniques. Antibody specificity was verified using Western-blot analysis of a Gli1 over-expressed cancer cell line, LNCaP-Gli1. Semi-quantification compared tumors and control tissue using IF analysis by ImageJ software. RESULTS: Expression of the Hh pathway was observed in SC for all four major components of the pathway. PTCH1, SMO, and Gli2 were more significantly upregulated in SC (P < 0.01) compared to nBCC. Stromal expression of PTCH1 and Gli2 was observed in SC (P < 0.01). In contrast, stromal expression of these proteins in nBCC was similar or down-regulated compared to physiological Hh controls. CONCLUSIONS: The Hh signaling pathway is significantly more upregulated in periocular SC compared to nBCC, a known aberrant Hh pathway tumor. Furthermore, the stroma of the SC demonstrated Hh upregulation, in particular Gli2, compared to nBCC. Targeting of this pathway may be a potential treatment strategy for SC.

Can the skin make you fat? A role for the skin in regulating adipose tissue function and whole-body glucose and lipid homeostasis.

Prevalence of obesity and related complications such as type 2 diabetes (T2D) has increased dramatically in recent decades. Metabolic complications of obesity arise in part due to subcutaneous adipose tissue (SAT) dysfunction. However, it is currently unclear why some obese individuals develop insulin resistance and T2D and others do not. In this review, we discuss the role of the skin in regulating SAT function, and whether presence of inflammatory skin diseases such as psoriasis represent a novel risk mechanism mediating development of obesity-related complications.

Meta-analysis identifies novel risk loci and yields systematic insights into the biology of male-pattern baldness.

Male-pattern baldness (MPB) is a common and highly heritable trait characterized by androgen-dependent, progressive hair loss from the scalp. Here, we carry out the largest GWAS meta-analysis of MPB to date, comprising 10,846 early-onset cases and 11,672 controls from eight independent cohorts. We identify 63 MPB-associated loci (P<5 × 10-8, METAL) of which 23 have not been reported previously. The 63 loci explain ∼39% of the phenotypic variance in MPB and highlight several plausible candidate genes (FGF5, IRF4, DKK2) and pathways (melatonin signalling, adipogenesis) that are likely to be implicated in the key-pathophysiological features of MPB and may represent promising targets for the development of novel therapeutic options. The data provide molecular evidence that rather than being an isolated trait, MPB shares a substantial biological basis with numerous other human phenotypes and may deserve evaluation as an early prognostic marker, for example, for prostate cancer, sudden cardiac arrest and neurodegenerative disorders.

GLI2 Is a Regulator of ß-Catenin and Is Associated with Loss of E-Cadherin, Cell Invasiveness, and Long-Term Epidermal Regeneration.

Uncontrolled hedgehog (HH)/glioma-associated oncogene (GLI) and WNT/ß-catenin signaling are important events in the genesis of many cancers including skin cancer and are often implicated in tumor progression, invasion, and metastasis. However, because of the complexity and context dependency of both pathways, little is known about HH and WNT interactions in human carcinogenesis. In the current study, we provide evidence of HH/glioma-associated oncogene family zinc finger 2 (GLI2)-WNT/ß-catenin signaling crosstalk in human keratinocytes. Overexpression of GLI2ΔN in human keratinocytes resulted in cytoplasmic accumulation and nuclear relocalization of ß-catenin in vitro and in 3D organotypic cultures, accompanied by upregulation of WNT genes. Induction of GLI2ΔN enhanced the ß-catenin-dependent transcriptional activation and the subsequent activation of ß-catenin target genes including cyclin-D1. Additionally, GLI2 overexpression was associated with decreased E-cadherin protein levels; increased expression of SNAIL, matrix metalloproteinase 2, and integrin ß1; and increased cell invasion in 3D organotypic cultures. Invasion was reduced by WNT inhibition, thus unveiling the direct role of GLI2/WNT crosstalk in cell invasion. We show that GLI2 overexpression supported long-term epidermal regeneration in 3D organotypic cultures, and resulted in the manifestation of an undifferentiated basal/stem cell-associated phenotype in human keratinocytes. Both these observations are consistent with the role of ß-catenin and SNAIL in epidermal stem cell maintenance. This work suggests that GLI2 is a regulator of ß-catenin and provides insights into its role in tumorigenesis.

Differential Expression between Human Dermal Papilla Cells from Balding and Non-Balding Scalps Reveals New Candidate Genes for Androgenetic Alopecia.

Androgenetic alopecia (AGA) is a common heritable and androgen-dependent hair loss condition in men. Twelve genetic risk loci are known to date, but it is unclear which genes at these loci are relevant for AGA. Dermal papilla cells (DPCs) located in the hair bulb are the main site of androgen activity in the hair follicle. Widely used monolayer-cultured primary DPCs in hair-related studies often lack dermal papilla characteristics. In contrast, immortalized DPCs have high resemblance to intact dermal papilla. We derived immortalized human DPC lines from balding (BAB) and non-balding (BAN) scalp. Both BAB and BAN retained high proportions of dermal papilla signature gene and versican protein expression. We performed expression analysis of BAB and BAN and annotated AGA risk loci with differentially expressed genes. We found evidence for AR but not EDA2R as the candidate gene at the AGA risk locus on chromosome X. Further, our data suggest TWIST1 (twist family basic helix-loop-helix transcription factor 1) and SSPN (sarcospan) to be the functionally relevant AGA genes at the 7p21.1 and 12p12.1 risk loci, respectively. Down-regulated genes in BAB compared to BAN were highly enriched for vasculature-related genes, suggesting that deficiency of DPC from balding scalps in fostering vascularization around the hair follicle may contribute to the development of AGA.

Constitutive Autophagy and Nucleophagy during Epidermal Differentiation.

Epidermal keratinocytes migrate through the epidermis up to the granular layer where, on terminal differentiation, they progressively lose organelles and convert into anucleate cells or corneocytes. Our report explores the role of autophagy in ensuring epidermal function providing the first comprehensive profile of autophagy marker expression in developing epidermis. We show that autophagy is constitutively active in the epidermal granular layer where by electron microscopy we identified double-membrane autophagosomes. We demonstrate that differentiating keratinocytes undergo a selective form of nucleophagy characterized by accumulation of microtubule-associated protein light chain 3/lysosomal-associated membrane protein 2/p62 positive autolysosomes. These perinuclear vesicles displayed positivity for histone interacting protein, heterochromatin protein 1α, and localize in proximity with Lamin A and B1 accumulation, whereas in newborn mice and adult human skin, we report LC3 puncta coincident with misshaped nuclei within the granular layer. This process relies on autophagy integrity as confirmed by lack of nucleophagy in differentiating keratinocytes depleted from WD repeat domain phosphoinositide interacting 1 or Unc-51 like autophagy activating kinase 1. Final validation into a skin disease model showed that impaired autophagy contributes to the pathogenesis of psoriasis. Lack of LC3 expression in psoriatic skin lesions correlates with parakeratosis and deregulated expression or location of most of the autophagic markers. Our findings may have implications and improve treatment options for patients with epidermal barrier defects.