Maria Rosaria Mollo

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… Visualizza altro

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.
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We conclude that p63 gene mutations in AEC syndrome affect expression of specific target genes, including those encoding for epidermal proteins involved in cell adhesion and in mechanical resilience, causing extensive skin erosions. As a consequence of these lesions, epidermal cells produce inflammatory cytokines, leading to local and systemic inflammation. Because we found that extended skin erosions in AEC patients can also be associated with TSLP, measuring its serum levels in severely… Visualizza altro

We conclude that p63 gene mutations in AEC syndrome affect expression of specific target genes, including those encoding for epidermal proteins involved in cell adhesion and in mechanical resilience, causing extensive skin erosions. As a consequence of these lesions, epidermal cells produce inflammatory cytokines, leading to local and systemic inflammation. Because we found that extended skin erosions in AEC patients can also be associated with TSLP, measuring its serum levels in severely affected patients may help to design strategy to ameliorate their health conditions and to prevent a secondary autoimmune disease. Meno dettagli

Age-related macular degeneration (AMD) is a chronic, progressive degenerative disease associated reduction in visual acuity. Preventive and supportive strategies to slow progression of the disease are of primary importance. Oxidative stress contributes substantially to macular degeneration and is related to cellular damage caused by reactive oxygen species. Retinal structures, in fact, are susceptible to oxidative damage, which can be influenced by insufficient dietary intake of antioxidants… Visualizza altro

Age-related macular degeneration (AMD) is a chronic, progressive degenerative disease associated reduction in visual acuity. Preventive and supportive strategies to slow progression of the disease are of primary importance. Oxidative stress contributes substantially to macular degeneration and is related to cellular damage caused by reactive oxygen species. Retinal structures, in fact, are susceptible to oxidative damage, which can be influenced by insufficient dietary intake of antioxidants. Oral vitamin and mineral supplementation (especially vitamins C and E, lutein, zeaxanthin, zinc, and omega-3 fatty acids) has been widely investigated for its role in helping to slow disease progression. There is now general consensus that the AREDS1 and AREDS2 formulations exert their positive effects via antioxidant properties, and considering their potential benefits, commercially-available dietary supplements are now available based on the AREDS studies. Indeed, the AREDS2 formulation with 10 mg lutein/day and 2 mg zeaxanthin/day can be considered as standard of care to reduce the probability of advanced AMD in patients at high risk for progression to severe visual loss. Dietary supplements may help to delay the onset of AMD, and potentially progression of the disease. Clinicians caring for patients with AMD should be aware of the benefits of oral supplements for AMD.
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Cleft palate is a common congenital disorder that affects up to 1 in 2500 live births and results in considerable morbidity to affected individuals and their families. The aetiology of cleft palate is complex with both genetic and environmental factors implicated. Mutations in the transcription factor p63 are one of the major individual causes of cleft palate; however, the gene regulatory networks in which p63 functions remain only partially characterized. Our findings demonstrate that p63… Visualizza altro

Cleft palate is a common congenital disorder that affects up to 1 in 2500 live births and results in considerable morbidity to affected individuals and their families. The aetiology of cleft palate is complex with both genetic and environmental factors implicated. Mutations in the transcription factor p63 are one of the major individual causes of cleft palate; however, the gene regulatory networks in which p63 functions remain only partially characterized. Our findings demonstrate that p63 functions as an essential regulatory molecule in the spatio-temporal control of palatal epithelial cell fate to ensure appropriate fusion of the palatal shelves. Initially, p63 induces periderm formation and controls its subsequent maintenance to prevent premature adhesion between adhesion-competent, intra-oral epithelia. Subsequently, TGFβ3-induced down-regulation of p63 in the medial edge epithelia of the palatal shelves is a pre-requisite for palatal fusion by facilitating periderm migration from, and reducing the proliferative potential of, the midline epithelial seam thereby preventing cleft palate Meno dettagli

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… Visualizza altro

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. Meno dettagli

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… Visualizza altro

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. Meno dettagli

As the outermost tissue of the body, the epidermis is the first physical barrier for any pressure, stress or trauma. Several specialized cell-matrix and cell-cell adhesion structures, together with an intracellular network of dedicated intermediate filaments, are required to confer critical resilience to mechanical stress. The transcription factor p63 is a master regulator of gene expression in the epidermis and in other stratified epithelia. It has been extensively demonstrated that p63… Visualizza altro

As the outermost tissue of the body, the epidermis is the first physical barrier for any pressure, stress or trauma. Several specialized cell-matrix and cell-cell adhesion structures, together with an intracellular network of dedicated intermediate filaments, are required to confer critical resilience to mechanical stress. The transcription factor p63 is a master regulator of gene expression in the epidermis and in other stratified epithelia. It has been extensively demonstrated that p63 positively controls a large number of tissue-specific genes, including those encoding a large fraction of tissue-restricted cell adhesion molecules. Consistent with p63 functions in cell adhesion and in epidermal differentiation, heterozygous mutations clustered mainly in the p63 C-terminus are causative of AEC syndrome, an autosomal dominant disorder characterized by cleft palate, ankyloblepharon and ectodermal dysplasia associated with severe skin erosions, bleeding and infections. The molecular basis of skin erosions in AEC patients is not fully understood, although defects in desmosomes and in other cell junctions are likely to be involved. Here, we provide an extensive review of the different epidermal cell junctions that cooperate to withstand mechanical stress and on the mechanisms by which p63 regulates gene expression of their components in healthy skin and in AEC syndrome. Collectively, advancement in understanding the molecular mechanisms by which epidermal cell junctions precisely exert their functions and how p63 orchestrates their coordinated expression, will ultimately lead to insight into developing future strategies for the treatment of AEC syndrome and more in generally for diseases that share an overlapping phenotype.
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Nectins are immunoglobulin-like cell adhesion molecules mainly localized in adherens junctions. The transcription factor p63 is a master regulator of gene expression in stratified epithelia and controls several molecular processes. Since mutations in the Pvrl1 and Pvrl4 genes encoding for nectins cause genetic disorders with phenotypes similar to p63-related syndromes, we investigated whether these proteins might be under p63 transcriptional control. Here we show that in p63-null skin Pvrl1… Visualizza altro

Nectins are immunoglobulin-like cell adhesion molecules mainly localized in adherens junctions. The transcription factor p63 is a master regulator of gene expression in stratified epithelia and controls several molecular processes. Since mutations in the Pvrl1 and Pvrl4 genes encoding for nectins cause genetic disorders with phenotypes similar to p63-related syndromes, we investigated whether these proteins might be under p63 transcriptional control. Here we show that in p63-null skin Pvrl1 gene expression is strongly reduced, whereas Pvrl4 expression is unaffected. In human and mouse primary keratinocytes p63 depletion leads to a specific downregulation of the Pvrl1 gene. Consistent with a direct regulation, Chromatin Immunoprecipitation experiments (ChIP) indicate that p63 binds to two conserved intronic Pvrl1 enhancer regions. AEC syndrome is a rare autosomal dominant disorder, caused by mutations in p63 gene, mainly characterized by skin fragility. To test whether nectins may be affected in AEC syndrome, their expression was measured in keratinocytes obtained from AEC patients or from a conditional mouse model for AEC syndrome. Pvrl1 expression was reduced in AEC keratinocytes, consistent with impaired p63 function. Surprisingly Pvrl4 expression was similarly affected, in parallel with decreased expression of the transcription factor Irf6. Consistent with the well-characterized role of Irf6 in keratinocyte differentiation and its strong downregulation in AEC syndrome, Irf6 depletion caused reduced expression of Pvrl4 in wild-type keratinocytes. Taken together our results indicate that Pvrl1 is a bona fide target gene of the transcription factor p63, whereas Pvrl4 regulation is linked to epidermal differentiation and is under Irf6 control. Meno dettagli

Ankyloblepharon, ectodermal defects, cleft lip/palate (AEC) syndrome is a rare autosomal dominant disorder caused by mutations in the p63 gene, essential for embryonic development of stratified epithelia. The most severe cutaneous manifestation of this disorder is the long-lasting skin fragility associated with severe skin erosions after birth. Using a knock-in mouse model for AEC syndrome, we found that skin fragility was associated with microscopic blistering between the basal and suprabasal… Visualizza altro

Ankyloblepharon, ectodermal defects, cleft lip/palate (AEC) syndrome is a rare autosomal dominant disorder caused by mutations in the p63 gene, essential for embryonic development of stratified epithelia. The most severe cutaneous manifestation of this disorder is the long-lasting skin fragility associated with severe skin erosions after birth. Using a knock-in mouse model for AEC syndrome, we found that skin fragility was associated with microscopic blistering between the basal and suprabasal compartments of the epidermis and reduced desmosomal contacts. Expression of desmosomal cadherins and desmoplakin was strongly reduced in AEC mutant keratinocytes and in newborn epidermis. A similar impairment in desmosome gene expression was observed in human keratinocytes isolated from AEC patients, in p63-depleted keratinocytes and in p63 null embryonic skin, indicating that p63 mutations causative of AEC syndrome have a dominant-negative effect on the wild-type p63 protein. Among the desmosomal components, desmocollin 3, desmoplakin and desmoglein 1 were the most significantly reduced by mutant p63 both at the RNA and protein levels. Chromatin immunoprecipitation experiments and transactivation assays revealed that p63 controls these genes at the transcriptional level. Consistent with reduced desmosome function, AEC mutant and p63-deficient keratinocytes had an impaired ability to withstand mechanical stress, which was alleviated by epidermal growth factor receptor inhibitors known to stabilize desmosomes. Our study reveals that p63 is a crucial regulator of a subset of desmosomal genes and that this function is impaired in AEC syndrome. Reduced mechanical strength resulting from p63 mutations can be alleviated pharmacologically by increasing desmosome adhesion with possible therapeutic implications.
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