Autophagy protects murine preputial glands against premature aging, and controls their sebum phospholipid and pheromone profile.

ABBREVIATIONS: ATG7: autophagy related 7; BODIPY: boron dipyrromethene; DAG: diacyl glycerides; DBI: diazepam binding inhibitor; GFP: green fluorescent protein; KRT14: keratin 14; HPLC-MS: high performance liquid chromatography-mass spectrometry; LD: lipid droplet; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MSI: mass spectrometric imaging; ORO: Oil Red O; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PG: preputial gland; PLIN2: perilipin 2; PtdIns: phosphatidylinositol; PL: phospholipids; POPC: 1-palmitoyl-2-oleoyl-PC; PS: phosphatidylserine; qRT-PCR: quantitative reverse transcribed PCR; SG: sebaceous gland; scRNAseq: single-cell RNA sequencing; TAG: triacylglycerides; TLC: thin layer chromatography.

The Whey Acidic Protein WFDC12 Is Specifically Expressed in Terminally Differentiated Keratinocytes and Regulates Epidermal Serine Protease Activity.

WFDC proteins such as peptidase inhibitor 3 and SLPI inhibit proteases in the epidermis and other tissues. In this study, we tested the hypothesis that further WFDC protein family members might contribute to epidermal homeostasis. We found that in addition to peptidase inhibitor 3 and SLPI, WFDC5 and WFDC12 were expressed in human epidermis. In contrast to WFDC5, the expression of WFDC12 was induced during the late differentiation of keratinocytes and was restricted to the outermost layer of live cells. Single-cell RNA sequencing demonstrated that WFDC12-positive keratinocytes were characterized by the upregulation of LCE mRNA expression and downregulated the expression of keratins and claudins. Immunogold-electron microscopy revealed the colocalization of WFDC12 with corneodesmosomes in the lower stratum corneum. WFDC12 was elevated in the affected skin of patients with psoriasis, atopic dermatitis, and Darier disease. By contrast, WFDC12 expression was strongly upregulated not only in the affected but even more so in clinically normal-appearing skin of patients with Netherton syndrome. Finally, functional analysis showed distinct inhibitory activity of WFDC12 on neutrophil elastase and epidermal kallikrein‒related peptidase. Altogether, our study identified WFDC12 as a marker of the last stage of epidermal keratinocyte differentiation and suggests that WFDC12 contributes to the control of protease activity in the stratum corneum.

Autophagy facilitates secretion and protects against degeneration of the Harderian gland.

The epithelial derived Harderian gland consists of 2 types of secretory cells. The more numerous type A cells are responsible for the secretion of lipid droplets, while type B cells produce dark granules of multilamellar bodies. The process of autophagy is constitutively active in the Harderian gland, as confirmed by our analysis of LC3 processing in GFP-LC3 transgenic mice. This process is compromised by epithelial deletion of Atg7. Morphologically, the Atg7 mutant glands are hypotrophic and degenerated, with highly vacuolated cells and pyknotic nuclei. The mutant glands accumulate lipid droplets coated with PLIN2 (perilipin 2) and contain deposits of cholesterol, ubiquitinated proteins, SQSTM1/p62 (sequestosome 1) positive aggregates and other metabolic products such as porphyrin. Immunofluorescence stainings show that distinct cells strongly aggregate both proteins and lipids. Electron microscopy of the Harderian glands reveals that its organized structure is compromised, and the presence of large intracellular lipid droplets and heterologous aggregates. We attribute the occurrence of large vacuoles to a malfunction in the formation of multilamellar bodies found in the less abundant type B Harderian gland cells. This defect causes the formation of large tertiary lysosomes of heterologous content and is accompanied by the generation of tight lamellar stacks of endoplasmic reticulum in a pseudo-crystalline form. To test the hypothesis that lipid and protein accumulation is the cause for the degeneration in autophagy-deficient Harderian glands, epithelial cells were treated with a combination of the proteasome inhibitor and free fatty acids, to induce aggregation of misfolded proteins and lipid accumulation, respectively. The results show that lipid accumulation indeed enhanced the toxicity of misfolded proteins and that this was even more pronounced in autophagy-deficient cells. Thus, we conclude autophagy controls protein and lipid catabolism and anabolism to facilitate bulk production of secretory vesicles of the Harderian gland.

NF-E2-related factor 2 regulates the stress response to UVA-1-oxidized phospholipids in skin cells.

Long-wavelength ultraviolet (UVA-1) radiation causes oxidative stress that modifies cellular molecules. To defend themselves against noxious oxidation products, skin cells produce detoxifying enzymes and antioxidants. We have recently shown that UVA-1 oxidized the abundant membrane phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC), which then induced the stress-response protein heme oxygenase 1 (HO-1) in dermal fibroblasts. Here we examined the effects of UVA-1- and UV-oxidized phospholipids on global gene expression in human dermal fibroblasts and keratinocytes. We identified a cluster of genes that were coinduced by UVA-1-oxidized PAPC and UVA-1 radiation. The cluster included HO-1, glutamate-cysteine ligase modifier subunit, aldo-keto reductases-1-C1 and -C2, and IL-8. These genes are members of the cellular stress response system termed "antioxidant response." Accordingly, the regulatory regions of all of these genes contain binding sites for NF-E2-related factor 2 (NRF2), a major regulator of the antioxidant response. Both UVA-1 irradiation and treatment with oxidized lipids led to increased nuclear accumulation and DNA binding of NRF2. Silencing and deficiency of NRF2 suppressed the antioxidant response. Taken together, our data show that UVA-1-mediated lipid oxidation induces expression of antioxidant response genes, which is dependent on the redox-regulated transcription factor NRF2. Our findings suggest a different view on UV-generated lipid mediators that were commonly regarded as detrimental

Photooxidation generates biologically active phospholipids that induce heme oxygenase-1 in skin cells.

Heme oxygenase-1 (HO-1) is a key enzyme in the cellular response to tissue injury and oxidative stress. HO-1 enzymatic activity results in the formation of the cytoprotective metabolites CO and biliverdin. In the skin, HO-1 is strongly induced after long wave ultraviolet radiation (UVA-1). Here we show that UVA-1 irradiation generates oxidized phospholipids derived from 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC) that mediate the expression of HO-1 in skin cells. Using EO6 antibodies that recognize oxidized phospholipids, we show that UVA-1 irradiation of dermal fibroblasts generates oxidation-specific epitopes. Irradiation of arachidonate-containing phospholipids with UVA-1 led to formation of defined lipid oxidation products including epoxyisoprostane-phosphatidylcholine that induced HO-1 expression in dermal fibroblasts, in keratinocytes, and in a three-dimensional epidermal equivalent model. In addition, we demonstrate that the oxidation of PAPC by UVA-1 is a singlet oxygen-dependent mechanism. Together, we present a novel mechanism of UVA-1-induced HO-1 expression that is mediated by the generation of biologically active phospholipid oxidation products. Because UVA-1 irradiation is a mainstay treatment of several inflammatory skin diseases, structural identification of UVA-1-generated biomolecules with HO-1-inducing capacity should lead to the development of drugs that could substitute for irradiation.

Screening for amblyopia in preverbal children with photoscreening photographs: IV. Interobserver variability in photograph grading: origin and method of reduction.

BACKGROUND: Earlier detection of childhood vision disorders is a prominent goal of vision screening. The Medical Technology and Innovation (MTI) PhotoScreener addresses this objective. Use of this camera does not require verbal feedback and may be administered early in a child's development. Decreasing the variability in photograph grading results will boost the utility of any photoscreening system. This report aims to understand and to decrease intra- and inter-observer variability in grading photoscreening photographs. METHODS: We dissected the photograph grading process and quantified the intra- and inter-observer agreement using intraclass plot correlation coefficients. We evaluated the outcome of a two grader verification system vs. adjudicated measurements with Receiver Operator Characteristic (ROC) curves. PARTICIPANTS: Data on 955 children under 5 years of age, normal except for refractive error, each with complete photoscreening and eye examination data, culled from two previous studies. MAIN OUTCOME MEASURES: Intra- and inter-observer agreement in measuring bright crescent dimensions and pupillary diameters. Sensitivity and specificity of detection of hyperopia. RESULTS: Measurements of bright crescents are associated with greater variability than are measurements of pupillary diameters. Recognition and omission of light "rim" measurements from photograph grading will result in superior inter-observer agreement. Photograph independent errors increase variability and may be corrected by remeasurement. A verification system in which the most discrepant 5% of measurements are redone results in ROC curves similar to adjudicated dimension. CONCLUSIONS: We conclude: 1) two novices grading photographs can do as well as one expert in most cases; 2) the proposed grading methodology has undergone statistical validation and can be used in other areas of ophthalmology and medicine; and 3) inter-observer variability, one of the limitations of photoscreening photograph grading, can be reduced. For 95% of the photographs, two novices achieve similar true positive and true negative values with or without adjudication.

Retinoic acid increases the expression of p53 and proapoptotic caspases and sensitizes keratinocytes to apoptosis: a possible explanation for tumor preventive action of retinoids.

Retinoids influence growth and differentiation of keratinocytes (KCs) and are widely used for the management of skin diseases and for prevention of nonmelanoma skin cancer (NMSC) in predisposed patients. Here we investigated the effect of all-trans-retinoic acid (ATRA) on KC apoptosis. When KCs were cultured in confluent monolayers for several days, they acquired resistance against UVB-induced apoptosis. In contrast, when the cells were treated with 1 micromol/L ATRA for 6 days and subsequently irradiated with different doses of UVB, they underwent massive apoptosis as assessed by morphology, expression of activated caspase-3, and DNA fragmentation. The same effect was observed when doxorubicin was used instead of UVB. Analysis by real-time PCR and Western blot revealed that ATRA treatment strongly increased the mRNA and protein expression of p53 and caspase-3, -6, -7, and -9, which are key regulators of apoptosis. UVB irradiation of ATRA-treated cells but not of control cells led to the accumulation of p53 protein and of its target gene Noxa. Inhibition of p53 and caspases with alpha-pifithrin and z-Val-Ala-Asp-fluoromethyl ketone, respectively, blocked UVB- and doxorubicin-induced apoptosis in ATRA-treated KCs. Analogous to the observed ATRA effects in monolayer cultures, in vitro-generated organotypic skin cultures reacted with up-regulation of p53 and proapoptotic caspases and displayed increased sensitivity to UVB-induced apoptosis. The ability of retinoic acid to regulate the expression of proapoptotic genes and to sensitize KCs to apoptosis may play a role in their prevention of NMSC in transplant patients and patients with DNA-repair deficiencies.

Caspase-14 expression by epidermal keratinocytes is regulated by retinoids in a differentiation-associated manner.

Caspase-14 is the only member of the caspase family that shows a restricted tissue expression. It is mainly confined to epidermal keratinocytes and in contrast to other caspases, is not activated during apoptosis induced by ultraviolet irradiation or cytotoxic substances. As it is cleaved under conditions leading to terminal differentiation of keratinocytes we suggested that caspase-14 plays a part in the physiologic cell death of keratinocytes leading to skin barrier formation. Here we show that retinoic acid, at concentrations inhibiting terminal differentiation of keratinocytes, strongly suppressed caspase-14 mRNA and protein expression by keratinocytes in monolayer culture and in a three-dimensional in vitro model of differentiating human epidermis (skin equivalent). By contrast, the expression of the caspases 3 and 8, which are both activated during conventional apoptosis, was increased and unchanged, respectively, after retinoic acid treatment. In addition to inhibition of differentiation in skin equivalents, retinoic acid treatment led to keratinocyte apoptosis and activation of caspase-3, both of which were undetectable in differentiated control skin equivalents. As this occurred in the absence of detectable caspase-14, our data demonstrate that caspase-14 is dispensable for keratinocyte apoptosis. The fact that in contrast to caspase-3 and caspase-8, caspase-14, similarly to other keratinocyte differentiation-associated proteins, is downregulated by retinoids, strongly suggests that this caspase, but not caspase-3 and -8, plays a part in terminal keratinocyte differentiation and skin barrier formation.

Hepatocyte growth factor/scatter factor inhibits UVB-induced apoptosis of human keratinocytes but not of keratinocyte-derived cell lines via the phosphatidylinositol 3-kinase/AKT pathway.

Acute irreparable UV-induced DNA damage leads to apoptosis of epidermal keratinocytes (KC) and the formation of sunburn cells, whereas less severely damaged cells survive but harbor the potential of tumor formation. Here we report that hepatocyte growth factor/scatter factor (HGF/SF) prevents UVB-induced apoptosis in primary KC cultured in vitro. When we analyzed the signaling pathways initiated by the HGF/SF receptor c-met, we found that the phosphatidylinositol (PI) 3-kinase and its downstream-element AKT and the mitogen-activated protein (MAP) kinase were activated. Inhibition of PI 3-kinase led to a complete abrogation of the anti-apoptotic effect of HGF/SF, whereas blockade of the MAP kinase pathway had no effect. In contrast to the observation with primary KC, HGF/SF could not enhance survival after UVB irradiation of HaCaT and A431 cell lines, despite the fact that in these cells the PI 3-kinase and MAP kinase pathways were also activated by HGF/SF. Cell cycle analysis of KC revealed a G(2)/M arrest after UVB irradiation and a complete loss of proliferating cells. Because HGF/SF in the skin is produced by dermal fibroblasts, our findings suggest that the HGF/SF-mediated rescue of KC from apoptosis represents an important paracrine loop by which UVB-damaged KC can be kept alive to maintain the epidermal barrier function but cannot further proliferate, thereby preventing the induction of epithelial skin tumors.