19 research outputs found
Molecular mechanisms at the basis of epidermal defects and skin fragility in AEC syndrome
AEC syndrome is a rare autosomal dominant disorder, characterized by skin erosions, ectodermal dysplasia and cleft lip and/or palate. Missense mutations in the p63 gene, a key regulator of stratified epithelia, are causative of the disorder. The pathogenesis and the biological mechanisms underlying the skin erosions of AEC syndrome have been elusive. We generated a conditional knock-in mouse model carrying an inducible L514F mutation, found in AEC patients. p63+/floxL514F mice were crossed with the K14-Cre knock-in mouse line to obtain p63L514F expression soon before birth. Newborn mutant mice were indistinguishable from their wild-type littermates, however few days after birth focal skin blistering and scaling was observed accompanied by weight loss and often by death. Histological analysis and dye penetration assays revealed focal disruption of the epidermal barrier, followed by severe skin inflammation and epidermal hyperplasia. Consistently with the focal gaps in the epidermis of mutant mice, we found a strong reduction of desmosomal component Dsg1, Dsc3 and Dsp and a downregulation of two components of adherens junctions, Pvrl1 and Pvrl4 in AEC mutant keratinocytes. Similar results were obtained in human keratinocytes derived from AEC patients, indicating impaired cell-cell adhesion is AEC syndrome. In addition, we found an unbalance in the basal keratins. The intermediate filaments keratin5 (Krt5) and keratin14 (Krt14) are essential to withstand mechanical stress in the epidermis and are known p63 target genes. Both Krt5 and Krt14 expression was strongly downregulated in AEC mutant keratinocytes and in p63 knockdown, whereas in AEC mutant epidermis only Krt5 was significantly affected indicating that in vivo p63 plays a crucial function in Krt5 regulation. Importantly, we found a strong reduction in KRT5 expression also in human AEC patients, thus indicating impaired intermediate filaments network in AEC syndrome. Consistent with a reduction in Krt5, a reduced number of keratin bundles were observed by tissue-electron microscopy (TEM) in the basal compartment of the epidermis.
Skin fragility lead to focal failure of epidermal barrier, causing a progressive inflammation characterized by hyperplasia and hyperkeratosis in the skin of mutant mice. Clear signs of severe inflammation were preceded by strongly elevated levels of Thymic stromal lymphopoietin (Tslp), an IL-7 like cytokine, known to cause systemic defects which were observed in AEC mice and in at least one AEC patient. Strong induction of Tslp was associated with reduced Notch expression. Interestingly, reactivation of the Notch pathway could reduce Tslp expression in mutant keratinocytes.
Together these data support the hypothesis that the basal cell fragility and blistering observed in p63 mutant mice and AEC patients are due to alteration in adhesion molecules belonging to different categories. These results together with reduced Notch signaling lead to excessive production of the proinflammatory molecule Tslp
Research Techniques Made Simple: Identification and Characterization of Long Noncoding RNA in Dermatological Research
Long noncoding RNAs (lncRNAs) are a functionally heterogeneous and abundant class of RNAs acting in all cellular compartments that can form complexes with DNA, RNA, and proteins. Recent advances in high-throughput sequencing and techniques leading to the identification of DNA-RNA, RNA-RNA, and RNA-protein complexes have allowed the functional characterization of a small set of lncRNAs. However, characterization of the full repertoire of lncRNAs playing essential roles in a number of normal and dysfunctional cellular processes remains an important goal for future studies. Here we describe the most commonly used techniques to identify lncRNAs, and to characterize their biological functions. In addition, we provide examples of these techniques applied to cutaneous research in healthy skin, that is, epidermal differentiation, and in diseases such as cutaneous squamous cell carcinomas and psoriasis. As with protein-coding RNA transcripts, lncRNAs are differentially regulated in disease, and can serve as novel biomarkers for the diagnosis and prognosis of skin diseases
Research Techniques Made Simple: Skin Carcinogenesis Models: Xenotransplantation Techniques
Xenotransplantation is a widely used technique to test the tumorigenic potential of human cells in vivo using immunodeficient mice. Here we describe basic technologies and recent advances in xenotransplantation applied to study squamous cell carcinomas (SCCs) of the skin. SCC cells isolated from tumors can either be cultured to generate a cell line or injected directly into mice. Several immunodeficient mouse models are available for selection based on the experimental design and the type of tumorigenicity assay. Subcutaneous injection is the most widely used technique for xenotransplantation because it involves a simple procedure allowing the use of a large number of cells, although it may not mimic the original tumor environment. SCC cell injections at the epidermal-to-dermal junction or grafting of organotypic cultures containing human stroma have also been used to more closely resemble the tumor environment. Mixing of SCC cells with cancer-associated fibroblasts can allow the study of their interaction and reciprocal influence, which can be followed in real time by intradermal ear injection using conventional fluorescent microscopy. In this article, we will review recent advances in xenotransplantation technologies applied to study behavior of SCC cells and their interaction with the tumor environment in vivo
A B-Lymphoproliferative disorder is associated with severe skin erosions in AEC syndrome
Functional and Mechanistic Insights into the Pathogenesis of P63-Associated Disorders
The p53 family member p63 is a master regulator of gene expression in stratified epithelia, such as the epidermis. One of the main functions of p63 is to sustain mechanical resistance, positively regulating several epidermal genes involved in cell-matrix adhesion and cell-cell adhesion (Ferone et al., 2015)
Research Techniques Made Simple: Skin Carcinogenesis Models: Xenotransplantation Techniques
Interaction of the NRF2 and p63 transcription factors promotes keratinocyte proliferation in the epidermis
Epigenetic regulation of cell and tissue function requires the coordinated action of transcription factors. However, their combinatorial activities during regeneration remain largely unexplored. Here, we discover an unexpected interaction between the cytoprotective transcription factor NRF2 and p63- a key player in epithelial morphogenesis. Chromatin immunoprecipitation combined with sequencing and reporter assays identifies enhancers and promoters that are simultaneously activated by NRF2 and p63 in human keratinocytes. Modeling of p63 and NRF2 binding to nucleosomal DNA suggests their chromatin-assisted interaction. Pharmacological and genetic activation of NRF2 increases NRF2-p63 binding to enhancers and promotes keratinocyte proliferation, which involves the common NRF2-p63 target cyclin-dependent kinase 12. These results unravel a collaborative function of NRF2 and p63 in the control of epidermal renewal and suggest their combined activation as a strategy to promote repair of human skin and other stratified epithelia.ISSN:1362-4962ISSN:0301-561
In silico identification and experimental validation of novel anti-Alzheimer's multitargeted ligands from marine source featuring a "2-amino-imidazole plus aromatic group" scaffold
Multitargeting/polypharmacological approaches, looking for single chemical entities retaining the ability to bind two or more molecular targets, are a potentially powerful strategy to fight complex, multifactorial pathologies. Unfortunately, the search for multiligand agents is challenging, because only a small subset of molecules contained in molecular databases are bioactive, and even fewer are active on a preselected set of multiple targets. However, collections of natural compounds feature a significantly higher fraction of bioactive molecules than synthetic ones. In this view, we searched our library of 1,175 natural compounds from marine sources for molecules including a 2-aminoimidazole+aromatic group motif, found in known compounds active on single relevant targets for Alzheimer's disease (AD). This identified two molecules, a pseudozoanthoxanthin (1) and a bromo-pyrrole alkaloid (2), which were predicted by a computational approach to possess interesting multitarget profiles on AD target proteins. Biochemical assays experimentally confirmed their biological activities. The two compounds inhibit acetylcholinesterase, butyrylcholinesterase and \u3b2-secretase enzymes in high- to sub-micromolar range. They are also able to prevent and revert \u3b2-Amyloid (A\u3b2) aggregation of both A\u3b21-40 and A\u3b21-42 peptides, with 1 being more active than 2. Preliminary in vivo studies suggest that compound 1 is able to restore cholinergic cortico-hippocampal functional connectivity