Lumisterol (L3) is a stereoisomer of 7-dehydrocholesterol and is produced through the photochemical transformation of 7-dehydrocholesteol induced by high doses of UVB. L3 is enzymatically hydroxylated by CYP11A1, producing 20(OH)L3, 22(OH)L3, 20,22(OH)2L3, and 24(OH)L3. Hydroxylumisterols function as reverse agonists of the retinoic acid-related orphan receptors α and γ (RORα/γ) and can interact with the non-genomic binding site of the vitamin D receptor (VDR). These intracellular receptors are mediators of photoprotection and anti-inflammatory activity. In this study, we show that L3-hydroxyderivatives significantly increase the expression of VDR at the mRNA and protein levels in keratinocytes, both non-irradiated and after UVB irradiation. L3-hydroxyderivatives also altered mRNA and protein levels for RORα/γ in non-irradiated cells, while the expression was significantly decreased in UVB-irradiated cells. In UVB-irradiated keratinocytes, L3-hydroxyderivatives inhibited nuclear translocation of NFκB p65 by enhancing levels of IκBα in the cytosol. This anti-inflammatory activity mediated by L3-hydroxyderivatives through suppression of NFκB signaling resulted in the inhibition of the expression of UVB-induced inflammatory cytokines, including IL-17, IFN-γ, and TNF-α. The L3-hydroxyderivatives promoted differentiation of UVB-irradiated keratinocytes as determined from upregulation of the expression at the mRNA of involucrin (IVL), filaggrine (FLG), and keratin 14 (KRT14), downregulation of transglutaminase 1 (TGM1), keratins including KRT1, and KRT10, and stimulation of ILV expression at the protein level. We conclude that CYP11A1-derived hydroxylumisterols are promising photoprotective agents capable of suppressing UVB-induced inflammatory responses and restoring epidermal function through targeting the VDR and RORs.

Aims: Oxidative stress and mitochondrial dysfunction play a role in the process of skin photoaging via activation of matrix metalloproteases (MMPs) and the subsequent degradation of collagen. The activation of nuclear factor E2-related factor 2 (Nrf2), a transcription factor controlling antioxidant and cytoprotective defense systems, might offer a pharmacological approach to prevent skin photoaging. We therefore investigated a pharmacological approach to prevent skin photoaging, and also investigated a protective effect of the novel mitochondria-targeted hydrogen sulfide (H ₂ S) delivery molecules AP39 and AP123, and nontargeted control molecules, on ultraviolet A light (UVA)-induced photoaging in normal human dermal fibroblasts (NHDFs) in vitro and the skin of BALB/c mice in vivo . Results: In NHDFs, AP39 and AP123 (50–200 n M ) but not nontargeted controls suppressed UVA (8 J/cm ² )-mediated cytotoxicity and induction of MMP-1 activity, preserved cellular bioenergetics, and increased the expression of collagen and nuclear levels of Nrf2. In in vivo experiments, topical application of AP39 or AP123 (0.3–1 μ M /cm ² ; but not nontargeted control molecules) to mouse skin before UVA (60 J/cm ² ) irradiation prevented skin thickening, MMP induction, collagen loss of oxidative stress markers 8-hydroxy-2′-deoxyguanosine (8-OHdG), increased Nrf2-dependent signaling, as well as increased manganese superoxide dismutase levels and levels of the mitochondrial biogenesis marker peroxisome proliferator-activated receptor-gamma coactivator (PGC-1α). Innovation and Conclusion: Targeting H ₂ S delivery to mitochondria may represent a novel approach for the prevention and treatment of skin photoaging, as well as being useful tools for determining the role of mitochondrial H ₂ S in skin disorders and aging. Antioxid. Redox Signal . 36, 1268–1288.

Development of an automated process for quantifying proteins in multiple layers of skin is essential to research in the field of cell biology. Researchers aimed to quantify the cell density and levels of biomarkers on tissue samples. Immunofluorescence tissue staining is one of the standard methods used for the protein detection by tagging with the fluorescein isothiocyanate (FITC), the green fluorescent agent. Recently, the single-cell (monolayer tissue) analysis software automation has been obtained to quantify the protein levels. However, there are limitations for the analysis of overlapping multilayer tissue. Since the automatic analysis cannot be operated on the rough and muti-layered surface, the analysis is then manually performed using ImageJ software which process more than 5 minutes/image. Problems arise when many images of tissues need to be analyzed. Therefore, an automatic process has been developed for the image analysis to reduce time and error. This study developed the automatic process by adjusting the pixel intensity and splitting into the RGB channels and converting into gray with different intensities. The intensity can be measured on grayscale from 0 to 255 units based on the KODAK grayscale zone. The FITC staining is converted to be the grayscale intensity for the pixel intensity calculation. The gray level < 25 units was setup to be the exclusion criteria to solve the problem of over-exposure due to background noise. Therefore, this automated data processing enhances the technical capabilities and computational accuracy, which proposes an alternative approach to improve the tissue analysis for cell biology research.