Rebecca Justiniano

The Nrf2-inducers tanshinone I and dihydrotanshinone protect human skin cells and reconstructed human skin against solar simulated UV.

Exposure to solar ultraviolet (UV) radiation is a causative factor in skin photocarcinogenesis and photoaging, and an urgent need exists for improved strategies for skin photoprotection. The redox-sensitive transcription factor Nrf2 (nuclear factor-E2-related factor 2), a master regulator of the cellular antioxidant defense against environmental electrophilic insult, has recently emerged as an important determinant of cutaneous damage from solar UV, and the concept of pharmacological activation of Nrf2 has attracted considerable attention as a novel approach to skin photoprotection. In this study, we examined feasibility of using tanshinones, a novel class of phenanthrenequinone-based cytoprotective Nrf2 inducers derived from the medicinal plant Salvia miltiorrhiza, for protection of cultured human skin cells and reconstructed human skin against solar simulated UV. Using a dual luciferase reporter assay in human Hs27 dermal fibroblasts pronounced transcriptional activation of Nrf2 by four major tanshinones [tanshinone I (T-I), dihydrotanshinone (DHT), tanshinone IIA (T-II-A) and cryptotanshinone (CT)] was detected. In fibroblasts, the more potent tanshinones T-I and DHT caused a significant increase in Nrf2 protein half-life via blockage of ubiquitination, ultimately resulting in upregulated expression of cytoprotective Nrf2 target genes (GCLC, NQO1) with the elevation of cellular glutathione levels. Similar tanshinone-induced changes were also observed in HaCaT keratinocytes. T-I and DHT pretreatment caused significant suppression of skin cell death induced by solar simulated UV and riboflavin-sensitized UVA. Moreover, feasibility of tanshinone-based cutaneous photoprotection was tested employing a human skin reconstruct exposed to solar simulated UV (80 mJ/cm2 UVB; 1.53 J/cm2 UVA). The occurrence of markers of epidermal solar insult (cleaved procaspase 3, pycnotic nuclei, eosinophilic cytoplasm, acellular cavities) was significantly attenuated in DHT-treated reconstructs that displayed increased immunohistochemical staining for Nrf2 and γ-GCS together with the elevation of total glutathione levels. Taken together, our data suggest the feasibility of achieving tanshinone-based cutaneous Nrf2-activation and photoprotection.

The Tryptophan-Derived Endogenous Aryl Hydrocarbon Receptor Ligand 6-Formylindolo[3,2-b]Carbazole Is a Nanomolar UVA Photosensitizer in Epidermal Keratinocytes

Endogenous UVA-chromophores may act as sensitizers of oxidative stress underlying cutaneous photoaging and photocarcinogenesis, but the molecular identity of non-DNA key chromophores displaying UVA-driven photodyamic activity in human skin remains largely undefined. Here we report that 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan photoproduct and endogenous high affinity aryl hydrocarbon receptor (AhR) agonist, acts as a nanomolar photosensitizer potentiating UVA-induced oxidative stress irrespective of AhR ligand activity. In human HaCaT and primary epidermal keratinocytes, photodynamic induction of apoptosis was elicited by the combined action of solar simulated UVA and FICZ, whereas exposure to the isolated action of UVA or FICZ did not impair viability. In a human epidermal tissue reconstruct, FICZ/UVA-cotreatment caused pronounced phototoxicity inducing keratinocyte cell death, and FICZ photodynamic activity was also substantiated in a murine skin exposure model. Array analysis revealed pronounced potentiation of cellular heat shock, ER stress, and oxidative stress response gene expression observed only upon FICZ/UVA-cotreatment. FICZ photosensitization caused intracellular oxidative stress, and comet analysis revealed introduction of formamidopyrimidine-DNA glycosylase (FPG)-sensitive oxidative DNA lesions suppressible by antioxidant cotreatment. Taken together, our data demonstrate that the endogenous AhR ligand FICZ displays nanomolar photodynamic activity representing a molecular mechanism of UVA-induced photooxidative stress potentially operative in human skin.

The B6-vitamer Pyridoxal is a Sensitizer of UVA-induced Genotoxic Stress in Human Primary Keratinocytes and Reconstructed Epidermis

UVA‐driven photooxidative stress in human skin may originate from excitation of specific endogenous chromophores acting as photosensitizers. Previously, we have demonstrated that 3‐hydroxypyridine‐derived chromophores including B6‐vitamers (pyridoxine, pyridoxamine and pyridoxal) are endogenous photosensitizers that enhance UVA‐induced photooxidative stress in human skin cells. Here, we report that the B6‐vitamer pyridoxal is a sensitizer of genotoxic stress in human adult primary keratinocytes (HEKa) and reconstructed epidermis. Comparative array analysis indicated that exposure to the combined action of pyridoxal and UVA caused upregulation of heat shock (HSPA6, HSPA1A, HSPA1L, HSPA2), redox (GSTM3, EGR1, MT2A, HMOX1, SOD1) and genotoxic (GADD45A, DDIT3, CDKN1A) stress response gene expression. Together with potentiation of UVA‐induced photooxidative stress and glutathione depletion, induction of HEKa cell death occurred only in response to the combined action of pyridoxal and UVA. In addition to activational phosphorylation indicative of genotoxic stress [p53 (Ser15) and γ‐H2AX (Ser139)], comet analysis indicated the formation of Fpg‐sensitive oxidative DNA lesions, observable only after combined exposure to pyridoxal and UVA. In human reconstructed epidermis, pyridoxal preincubation followed by UVA exposure caused genomic oxidative base damage, procaspase 3 cleavage and TUNEL positivity, consistent with UVA‐driven photooxidative damage that may be relevant to human skin exposed to high concentrations of B6‐vitamers.

Parkinson's Disease Skin Fibroblasts Display Signature Alterations in Growth, Redox Homeostasis, Mitochondrial Function, and Autophagy

The discovery of biomarkers for Parkinsons disease (PD) is challenging due to the heterogeneous nature of this disorder, and a poor correlation between the underlying pathology and the clinically expressed phenotype. An ideal biomarker would inform on PD-relevant pathological changes via an easily assayed biological characteristic, which reliably tracks clinical symptoms. Human dermal (skin) fibroblasts are accessible peripheral cells that constitute a patient-specific system, which potentially recapitulates the PD chronological and epigenetic aging history. Here, we compared primary skin fibroblasts obtained from individuals diagnosed with late-onset sporadic PD, and healthy age-matched controls. These fibroblasts were studied from fundamental viewpoints of growth and morphology, as well as redox, mitochondrial, and autophagic function. It was observed that fibroblasts from PD subjects had higher growth rates, and appeared distinctly different in terms of morphology and spatial organization in culture, compared to control cells. It was also found that the PD fibroblasts exhibited significantly compromised mitochondrial structure and function when assessed via morphological and oxidative phosphorylation assays. Additionally, a striking increase in baseline macroautophagy levels was seen in cells from PD subjects. Exposure of the skin fibroblasts to physiologically relevant stress, specifically ultraviolet irradiation (UVA), further exaggerated the autophagic dysfunction in the PD cells. Moreover, the PD fibroblasts accumulated higher levels of reactive oxygen species (ROS) coupled with lower cell viability upon UVA treatment. In essence, these studies highlight primary skin fibroblasts as a patient-relevant model that captures fundamental PD molecular mechanisms, and supports their potential utility to develop diagnostic and prognostic biomarkers for the disease.

A Topical Zinc Ionophore Blocks Tumorigenic Progression in UV-exposed SKH-1 High Risk Mouse Skin

Nonmelanoma skin cancer (NMSC) is the most common malignancy in the United States representing a considerable public health burden. Pharmacological suppression of skin photocarcinogenesis has shown promise in preclinical and clinical studies, but more efficacious photochemopreventive agents are needed. Here, we tested feasibility of harnessing pharmacological disruption of intracellular zinc homeostasis for photochemoprevention in vitro and in vivo. Employing the zinc ionophore and FDA‐approved microbicidal agent zinc pyrithione (ZnPT), used worldwide in over‐the‐counter (OTC) topical consumer products, we first demonstrated feasibility of achieving ZnPT‐based intracellular Zn2+ overload in cultured malignant keratinocytes (HaCaT‐ras II‐4; SCC‐25) employing membrane‐permeable fluorescent probes. Zinc overload was accompanied by induction of intracellular oxidative stress, associated with mitochondrial superoxide release as substantiated by MitoSOX Red™ fluorescence microscopy. ZnPT‐induced cell death observable in malignant keratinocytes was preceded by induction of metal (MT2A), proteotoxic (HSPA6, HSPA1A, DDIT3, HMOX1) and genotoxic stress response (GADD45A, XRCC2) gene expression at the mRNA and protein levels. Comet analysis revealed introduction of formamidopyrimidine‐DNA glycosylase (Fpg)‐sensitive oxidative DNA lesions. In a photocarcinogenesis model (UV‐exposed SKH‐1 high‐risk mouse skin), topical ZnPT administration post‐UV caused epidermal zinc overload and stress response gene expression with pronounced blockade of tumorigenesis. Taken together, these data suggest feasibility of repurposing a topical OTC drug for zinc‐directed photochemoprevention of solar UV‐induced NMSC.

The Aryl Hydrocarbon Receptor (AhR) as an Environmental Stress Sensor and Regulator of Skin Barrier Function: Molecular Mechanisms and Therapeutic Opportunities

The aryl hydrocarbon receptor (AhR) is a transcription factor responsive to both xenobiotic and endogenous ligands involved in skin barrier adaptations in response to environmental and endogenous stressors. Due to the fundamental role that AhR-controlled signaling plays in skin barrier formation, homeostasis, resilience to environmental stressors, and damage repair, AhR-directed pharmacological strategies that aim at AhR-orchestrated signaling for anti-inflammatory, immune-modulatory, cancer chemopreventive, and barrier enhancing intervention show great therapeutic promise, delivering unique patient-directed benefit by targeting specific skin pathologies (including psoriasis, atopic dermatitis, seborrheic dermatitis, and solar radiation-induced skin photodamage) that have remained elusive and difficult to treat. The ever expanding and accessible range of chemically diverse physiological and synthetic AhR-modulators that differ with regard to pharmacokinetic and pharmacodynamic profile, AhR-directed potency, metabolic stability, and off-target effects through engagement of other signaling pathways provides a versatile and accessible compound platform of prototype agents and therapeutic leads for experimental intervention through AhR engagement, potentially representing breakthrough therapeutics that can quickly be optimized, developed, and formulated into novel AhR-directed cutaneous therapeutic entities.