Electrical Impedance Spectroscopy Quantifies Skin Barrier Function in Organotypic In Vitro Epidermis Models.

Three-dimensional human epidermal equivalents (HEEs) are a state-of-the-art organotypic culture model in preclinical investigative dermatology and regulatory toxicology. In this study, we investigated the utility of electrical impedance spectroscopy (EIS) for noninvasive measurement of HEE epidermal barrier function. Our setup comprised a custom-made lid fit with 12 electrode pairs aligned on the standard 24-transwell cell culture system. Serial EIS measurements for 7 consecutive days did not impact epidermal morphology, and readouts showed comparable trends with HEEs measured only once. We determined 2 frequency ranges in the resulting impedance spectra: a lower frequency range termed EISdiff correlated with keratinocyte terminal differentiation independent of epidermal thickness and a higher frequency range termed EISSC correlated with stratum corneum thickness. HEEs generated from CRISPR/Cas9-engineered keratinocytes that lack key differentiation genes FLG, TFAP2A, AHR, or CLDN1 confirmed that keratinocyte terminal differentiation is the major parameter defining EISdiff. Exposure to proinflammatory psoriasis- or atopic dermatitis-associated cytokine cocktails lowered the expression of keratinocyte differentiation markers and reduced EISdiff. This cytokine-associated decrease in EISdiff was normalized after stimulation with therapeutic molecules. In conclusion, EIS provides a noninvasive system to consecutively and quantitatively assess HEE barrier function and to sensitively and objectively measure barrier development, defects, and repair.

The Aryl Hydrocarbon Receptor Regulates Epidermal Differentiation through Transient Activation of TFAP2A.

The aryl hydrocarbon receptor (AHR) is an evolutionary conserved environmental sensor identified as an indispensable regulator of epithelial homeostasis and barrier organ function. Molecular signaling cascade and target genes upon AHR activation and their contribution to cell and tissue function are however not fully understood. Multiomics analyses using human skin keratinocytes revealed that upon ligand activation, AHR binds open chromatin to induce expression of transcription factors, for example, TFAP2A, as a swift response to environmental stimuli. The terminal differentiation program, including upregulation of barrier genes, FLG and keratins, was mediated by TFAP2A as a secondary response to AHR activation. The role of AHR-TFAP2A axis in controlling keratinocyte terminal differentiation for proper barrier formation was further confirmed using CRISPR/Cas9 in human epidermal equivalents. Overall, the study provides additional insights into the molecular mechanism behind AHR-mediated barrier function and identifies potential targets for the treatment of skin barrier diseases.

The aryl hydrocarbon receptor regulates epidermal differentiation through transient activation of TFAP2A.

The aryl hydrocarbon receptor (AHR) is an evolutionary conserved environmental sensor identified as indispensable regulator of epithelial homeostasis and barrier organ function. Molecular signaling cascade and target genes upon AHR activation and their contribution to cell and tissue function are however not fully understood. Multi-omics analyses using human skin keratinocytes revealed that, upon ligand activation, AHR binds open chromatin to induce expression of transcription factors (TFs), e.g., Transcription Factor AP-2α (TFAP2A), as a swift response to environmental stimuli. The terminal differentiation program including upregulation of barrier genes, filaggrin and keratins, was mediated by TFAP2A as a secondary response to AHR activation. The role of AHR-TFAP2A axis in controlling keratinocyte terminal differentiation for proper barrier formation was further confirmed using CRISPR/Cas9 in human epidermal equivalents. Overall, the study provides novel insights into the molecular mechanism behind AHR-mediated barrier function and potential novel targets for the treatment of skin barrier diseases.

Expression of Oncogenic Drivers in 3D Cell Culture Depends on Nuclear ATP Synthesis by NUDT5.

The growth of cancer cells as oncospheres in three-dimensional (3D) culture provides a robust cell model for understanding cancer progression, as well as for early drug discovery and validation. We have previously described a novel pathway in breast cancer cells, whereby ADP (Adenosine diphosphate)-ribose derived from hydrolysis of poly (ADP-Ribose) and pyrophosphate (PPi) are converted to ATP, catalysed by the enzyme NUDT5 (nucleotide diphosphate hydrolase type 5). Overexpression of the NUDT5 gene in breast and other cancer types is associated with poor prognosis, increased risk of recurrence and metastasis. In order to understand the role of NUDT5 in cancer cell growth, we performed phenotypic and global expression analysis in breast cancer cells grown as oncospheres. Comparison of two-dimensional (2D) versus 3D cancer cell cultures from different tissues of origin suggest that NUDT5 increases the aggressiveness of the disease via the modulation of several key driver genes, including ubiquitin specific peptidase 22 (USP22), RAB35B, focadhesin (FOCAD) and prostagladin E synthase (PTGES). NUDT5 functions as a master regulator of key oncogenic pathways and of genes involved in cell adhesion, cancer stem cell (CSC) maintenance and epithelial to mesenchyme transition (EMT). Inhibiting the enzymatic activities of NUDT5 prevents oncosphere formation and precludes the activation of cancer driver genes. These findings highlight NUDT5 as an upstream regulator of tumour drivers and may provide a biomarker for cancer stratification, as well as a novel target for drug discovery for combinatorial drug regimens for the treatment of aggressive cancer types and metastasis.