Loss of Par3 leads to aberrant divisions and faster mitotic progression in differentiating primary murine keratinocytes

Dissertação de Mestrado, Oncobiologia: Mecanismos Moleculares do Cancro, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015

Melanosome transfer between melanocytes and keratinocytes

RESUMO: A pele é o maior órgão do corpo humano e a sua pigmentação é essencial para a sua coloração e proteção contra os efeitos nocivos da radiação ultravioleta (UV). A pigmentação da pele resulta essencialmente de três processos: a síntese e o armazenamento de melanina pelos melanócitos, em organelos especializados denominados melanossomas; o transporte dos melanossomas dentro dos melanócitos; e finalmente, a transferência dos melanossomas para os queratinócitos adjacentes. Nos queratinócitos, a melanina migra para a região perinuclear apical da célula para formar um escudo protetor,responsável pela proteção do DNA dos danos causados pela radiação UV. Os melanócitos estão localizados na camada basal da epiderme e contactam com 30-40 queratinócitos. Em conjunto, estas células formam a “unidade melano-epidérmica”. Apesar dos processos de síntese e transporte de melanina nos melanócitos estarem bastante bem caracterizados, os mecanismos moleculares subjacentes à transferência inter-celular de melanina são menos conhecidos e ainda controversos. Dados preliminares obtidos pelo nosso grupo, que se basearam na observação de amostras de pele humana por microscopia electrónica, indicam que a forma predominante de transferência de melanina na epiderme consiste na exocitose dos melanossomas pelos melanócitos e subsequente endocitose da melanina por queratinócitos. Para além disso sabe-se que as proteínas Rab, que controlam o tráfego membranar, estão envolvidas em várias etapas de pigmentação da pele, nomeadamente na biogénese e no transporte de melanina. Assim, dado o seu papel fundamental nestes processos, questionámo-nos sobre o seu envolvimento na transferência de melanina. Com este trabalho, propomo-nos a expandir o conhecimento atual sobre a transferência de melanina na pele, através do estudo detalhado dos seus mecanismos moleculares, identificando as proteínas Rab que regulam o processo. Pretendemos também confirmar o modelo de exo/endocitose como sendo o mecanismo principal de transferência de melanina. Primeiro, explorámos a regulação da secreção de melanina pelos melanócitos e analisámos o papel de proteínas Rab neste processo. Os resultados foram obtidos recorrendo a um método in vitro, desenvolvido previamente no laboratório, que avalia a quantidade de melanina segregada para o meio de cultura por espectrofotometria, e ainda por microscopia, contando o número de melanossomas transferidos para os queratinócitos. Através de co-culturas de melanócitos e queratinócitos, verificou-se que os queratinócitos estimulam a libertação de melanina dos melanócitos para o meio extra-celular, bem como a sua transferência para os queratinócitos. Além disso, a proteína Rab11b foi identificada como um regulador da exocitose de melanina e da sua transferência para os queratinócitos. De facto, a diminuição da expressão de Rab11b em melanócitos provocou a redução da secreção de melanina estimulada por queratinócitos, bem como da transferência desta. Em segundo lugar, para complementar o nosso estudo, centrámos a nossa investigação na internalização de melanina por queratinócitos. Especificamente, usando uma biblioteca de siRNA, explorámos o envolvimento de proteínas Rab na captação de melanina por queratinócitos. Como primeira abordagem, usámos esferas fluorescentes como substituto de melanina, avaliando os resultados por citometria de fluxo. No entanto, este método revelou-se ineficaz uma vez que a internalização destas esferas é independente do recetor PAR-2 (recetor 2 ativado por protease), que foi previamente descrito como essencial na captação de melanina por queratinócitos Posteriormente, foi desenvolvido um novo protocolo de endocitose baseado em microscopia, usando melanossomas sem a membrana envolvente (melanocores) purificados do meio de cultura de melanócitos, incluindo um programa informático especialmente desenhado para realizar uma análise semi-automatizada. Após internalização, os melanocores acumulam-se na região perinuclear dos queratinócitos, em estruturas que se assemelham ao escudo supranuclear observado na pele humana. Seguidamente, o envolvimento do recetor PAR-2 na captação de melanocores por queratinócitos foi confirmado, utilizando o novo protocolo de endocitose desenvolvido. Para além disso, a necessidade de quatro proteínas Rab foi identificada na internalização de melanocores por queratinócitos. A redução da expressão de Rab1a ou Rab5b em queratinócitos diminuiu significativamente o nível de internalização de melanocores, enquanto o silenciamento da expressão de Rab2a ou Rab14 aumentou a quantidade de melanocores internalizados por estas células. Em conclusão, os resultados apresentados corroboram as observações anteriores, obtidas em amostras de pele humana, e sugerem que o mecanismo de transferência predominante é a exocitose de melanina pelos melanócitos, induzida por queratinócitos, seguida por endocitose pelos queratinócitos. A pigmentação da pele tem implicações tanto ao nível da cosmética, como ao nível médico, relacionadas com foto-envelhecimento e com doenças pigmentares. Assim sendo, ao esclarecer quais os mecanismos moleculares que regulam a transferência de melanina na pele, este trabalho pode conduzir ao desenvolvimento de novas estratégias para modular a pigmentação da pele.----------------ABSTRACT: Skin pigmentation is achieved through the highly regulated production of the pigment melanin in specialized organelles, termed melanosomes within melanocytes. These are transported from their site of synthesis to the melanocyte periphery before being transferred to keratinocytes where melanin forms a supra-nuclear cap to protect the DNA from UVinduced damage. Together, melanocytes and keratinocytes form a functional complex, termed “epidermal-melanin unit”, that confers color and photoprotective properties to the skin. Skin pigmentation requires three processes: the biogenesis of melanin; its intracelular transport within the melanocyte to the cell periphery; and the melanin transfer to keratinocytes. The first two processes have been extensively characterized. However, despite significant advances that have been made over the past few years, the mechanisms underlying inter-cellular transfer of pigment from melanocytes to keratinocytes remain controversial.Preliminary studies from our group using electron microscopy and human skin samples found evidence for a mechanism of coupled exocytosis-endocytosis. Rab GTPases are master regulators of intracellular trafficking and have already been implicated in several steps of skin pigmentation. Thus, we proposed to explore and characterize the molecular mechanisms of melanin transfer and the role of Rab GTPases in this process. Moreover, we investigated whether the exo/endocytosis model is the main mechanism of melanin transfer. We first focused on melanin exocytosis by melanocytes. Then, we started to investigate the key regulatory Rab proteins involved in this step by establishing an in vitro tissue culture model of melanin secretion. Using co-cultures of melanocytes and keratinocytes, we found that keratinocytes stimulate melanin release and transfer. Moreover, depletion of Rab11b decreases keratinocyte-induced melanin exocytosis by melanocytes. In order to determine whether melanin exocytosis is a predominant mechanism of melanin transfer, the amount of melanin transferred to keratinocytes was then assayed in conditions where melanin exocytosis was inhibited. Indeed, Rab11b depletion resulted in a significant decrease in melanin uptake by keratinocytes. Taken together, these observations suggest that Rab11b mediates melanosome exocytosis from melanocytes and transfer to keratinocytes. To complement and extend our study, we of melanin by keratinocytes. Thus, we aimed to explore the effect of depleting Rab GTPases on melanin uptake and trafficking within keratinocytes. As a first approach, we used fluorescent microspheres as a melanin surrogate. However, the uptake of microspheres was observed to be independent of PAR-2, a receptor that is required for melanin uptakecentred our attention in the internalization of melanin by keratinocytes. Thus, we aimed to explore the effect of depleting Rab GTPases on melanin uptake and trafficking within keratinocytes. As a first approach, we used fluorescent microspheres as a melanin surrogate. However, the uptake of microspheres was observed to be independent of PAR-2, a receptor that is required for melanin uptake.Therefore, we concluded that microspheres were uptaken by keratinocytes through a different pathway than melanin. Subsequently, we developed a microscopy-based endocytosis assay using purified melanocores (melanosomes lacking the limiting membrane) from melanocytes, including a program to perform a semi-automated analysis. Melanocores are taken up by keratinocytes and accumulate in structures in the perinuclear area that resemble the physiological supranuclear cap observed in human skin. We then confirmed the involvement of PAR-2 receptor in the uptake of melanocores by keratinocytes, using the newly developed assay. Furthermore, we identified the role of four Rab GTPases on the uptake of melanocores by keratinocytes. Depletion of Rab1a and Rab5b from keratinocytes significantly reduced the uptake of melanocores, whereas Rab2a, and Rab14 silencing increased the amount the melanocores internalized by XB2 keratinocytes. In conclusion, we present evidence supporting keratinocyte-inducedmelanosome exocytosis from melanocytes, followed by endocytosis of the melanin core by keratinocytes as the predominant mechanism of melanin transfer in skin. Although advances have been made, there is a need for more effective and safer therapies directed at pigmentation disorders and also treatments for cosmetic applications. Hence, the understanding of the above mechanisms of skin pigmentation will lead to a greater appreciation of the molecular machinery underlying human skin pigmentation and could interest the pharmaceutical and cosmetic industries.

EGFR signalling as a negative regulator of Notch1 gene transcription and function in proliferating keratinocytes and cancer.

The Notch1 gene has an important role in mammalian cell-fate decision and tumorigenesis. Upstream control mechanisms for transcription of this gene are still poorly understood. In a chemical genetics screen for small molecule activators of Notch signalling, we identified epidermal growth factor receptor (EGFR) as a key negative regulator of Notch1 gene expression in primary human keratinocytes, intact epidermis and skin squamous cell carcinomas (SCCs). The underlying mechanism for negative control of the Notch1 gene in human cells, as well as in a mouse model of EGFR-dependent skin carcinogenesis, involves transcriptional suppression of p53 by the EGFR effector c-Jun. Suppression of Notch signalling in cancer cells counteracts the differentiation-inducing effects of EGFR inhibitors while, at the same time, synergizing with these compounds in induction of apoptosis. Thus, our data reveal a key role of EGFR signalling in the negative regulation of Notch1 gene transcription, of potential relevance for combinatory approaches for cancer therapy.

The epithelial sodium channel mediates the directionality of galvanotaxis in human keratinocytes.

Cellular directional migration in an electric field (galvanotaxis) is one of the mechanisms guiding cell movement in embryogenesis and in skin epidermal repair. The epithelial sodium channel (ENaC), in addition to its function of regulating sodium transport in kidney, has recently been found to modulate cell locomotory speed. Here we tested whether ENaC has an additional function of mediating the directional migration of galvanotaxis in keratinocytes. Genetic depletion of ENaC completely blocks only galvanotaxis and does not decrease migration speed. Overexpression of ENaC is sufficient to drive galvanotaxis in otherwise unresponsive cells. Pharmacologic blockade or maintenance of the open state of ENaC also decreases or increases, respectively, galvanotaxis, suggesting that the channel open state is responsible for the response. Stable lamellipodial extensions formed at the cathodal sides of wild-type cells at the start of galvanotaxis; these were absent in the ENaC knockout keratinocytes, suggesting that ENaC mediates galvanotaxis by generating stable lamellipodia that steer cell migration. We provide evidence that ENaC is required for directional migration of keratinocytes in an electric field, supporting a role for ENaC in skin wound healing.

Inhibition of 125I-epidermal growth factor binding to murine fibroblasts and keratinocytes by irradiation with ultraviolet-B light

Treatment of murine Swiss 3T3 fibroblasts and XB/2 keratinocytes with UV-B light (302 nm) resulted in a dose-dependent inhibition of [125I] epidermal growth factor (EGF) binding. The light dose required to achieve 50% inhibition of binding in both cell types was 80–85 J/m2 Decreased [125I] platelet-derived growth factor binding was not evoked even by light doses of up to 280 J/m2 UV-B irradiation did not stimultate phosphorylation of the 80 kd protein substrate for protein kinase C. Furthermore, its effect on [125I]EGF binding was not altered as a consequence of protein kinase C down-regulation following prolonged exposure of cells to phorbol esters. These results indicate that UV-B-induced transmodulation of the epidermal growth factor receptor is a specific event mediated through a protein kinase C-indepen dent pathway. Transfer of culture medium from irradiated cells to untreated control cells showed this effect was not induced as a result of transforming growth factor α release and subsequent binding to the EGF receptor in these cells.

The Autoimmune Regulator (AIRE), Which Is Defective in Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy Patients, Is Expressed in Human Epidermal and Follicular Keratinocytes and Associates With the Intermediate Filament Protein Cytokeratin 17

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome, which is caused by mutation of the autoimmune regulator (AIRE) gene, is a highly variable disease characterized by multiple endocrine failure, chronic mucocutaneous candidiasis, and various ectodermal defects. AIRE is a transcriptional regulator classically expressed in medullary thymic epithelial cells, monocytes, macrophages, and dendritic cells. Previous studies have suggested that AIRE can shuttle between the nucleus and cytoplasm of cells, although its cytoplasmic functions are poorly characterized. Through mass spectrometry analysis of proteins co-immunoprecipitating with cytoplasmic AIRE, we identified a novel association of AIRE with the intermediate filament protein cytokeratin 17 (K17) in the THP-1 monocyte cell line. We confirmed AIRE expression in HaCaT epidermal keratinocytes, as well as its interaction with K17. Confocal microscopy of human fetal and adult scalp hair follicles demonstrated a cytoplasmic pattern of AIRE staining that moderately colocalized with K17. The cytoplasmic association of AIRE with the intermediate filament network in human epidermal and follicular keratinocytes may provide a new path to understanding the ectodermal abnormalities associated with the APECED syndrome. (Am J Pathol 2011, 178:983-988; DOI: 10.1016/j.ajpath.2010.12.007)

Expression of human papillomavirus type 16 E7 oncoprotein alters keratinocytes expression profile in response to tumor necrosis factor-alpha

Acute expression of E7 oncogene from human papillomavirus (HPV) 16 or HPV18 is sufficient to overcome tumor necrosis factor (TNF)-alpha cytostatic effect on primary human keratinocytes. In the present study, we investigated the molecular basis of E7-induced TNF resistance through a comparative analysis of the effect of this cytokine on the proliferation and global gene expression of normal and E7-expressing keratinocytes. Using E7 functional mutants, we show that E7-induced TNF resistance correlates with its ability to mediate pRb degradation and cell transformation. On the other hand, this effect does not depend on E7 sequences required to override DNA damage-induced cell cycle arrest or extend keratinocyte life span. Furthermore, we identified a group of 66 genes whose expression pattern differs between normal and E7-expressing cells upon cytokine treatment. These genes are mainly involved in cell cycle regulation suggesting that their altered expression may contribute to sustained cell proliferation even in the presence of a cytostatic stimulus. Differential expression of TCN1 (transcobalamin I), IFI44 (Interferon-induced protein 44), HMGB2 (high-mobility group box 2) and FUS [Fusion (involved in t(12; 16) in malignant liposarcoma)] among other genes were further confirmed by western-blot and/or real-time polymerase chain reaction. Moreover, FUS upregulation was detected in HPV-positive cervical high-grade squamous intraepithelial lesions when compared with normal cervical tissue. Further evaluation of the role of such genes in TNF resistance and HPVassociated disease development is warranted.

Utilising silk fibroin membranes as scaffolds for the growth of tympanic membrane keratinocytes, and application to myringoplasty surgery

Background: Chronic tympanic membrane perforations can cause significant morbidity. The term myringoplasty describes the operation used to close such perforations. A variety of graft materials are available for use in myringoplasty, but all have limitations and few studies report post-operative hearing outcomes. Recently, the biomedical applications of silk fibroin protein have been studied. This material’s biocompatibility, biodegradability and ability to act as a scaffold to support cell growth prompted an investigation of its interaction with human tympanic membrane keratinocytes. Methods and materials: Silk fibroin membranes were prepared and human tympanic membrane keratinocytes cultured. Keratinocytes were seeded onto the membranes and immunostained for a number of relevant protein markers relating to cell proliferation, adhesion and specific epithelial differentiation. Results: The silk fibroin scaffolds successfully supported the growth and adhesion of keratinocytes, whilst also maintaining their cell lineage. Conclusion: The properties of silk fibroin make it an attractive option for further research, as a potential alternative graft in myringoplasty.

In vitro biocompatibility of poly(vinylidene fluoride-trifluoroethylene)/barium titanate composite using cultures of human periodontal ligament fibroblasts and keratinocytes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Porphyromonas gingivalis LPS stimulation downregulates DNMT1, DNMT3a, and JMJD3 gene expression levels in human HaCaT keratinocytes

Objective: The role of epigenetic regulation in inflammatory diseases such as periodontitis is poorly known. The aim of this study was to assess whether Porphyromonas gingivalis lipopolysaccharide (LPS) can modulate gene expression levels of the some enzymes that promote epigenetic events in cultures of the human keratinocytes and gingival fibroblasts. In addition, the same enzymes were evaluated in gingival samples from healthy and periodontitis-affected individuals. Materials and methods: Primary gingival fibroblast and keratinocyte (HaCaT) cultures were treated with medium containing P. gingivalis LPS or P. gingivalis LPS vehicle for 24 h. After this period, cell viability was assessed by MTT test and total RNA extracted to evaluate gene expression levels of the following enzymes by qRT-PCR: DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), histone demethylases Jumonji domain containing 3 (JMJD3) and ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX). To evaluate gene expression in healthy and periodontitis-affected individuals, total RNA was extracted from biopsies of gingival tissue from healthy and periodontitis sites, and gene expression of DNMT1, DNAMT3a, JMJD3, and UTX was evaluated by qRT-PCR. Results: No significant differences were found in the gene expression analysis between healthy and periodontitis-affected gingival samples. The results showed that LPS downregulated DNMT1 (p < 0. 05), DNMT3a (p < 0. 05), and JMJD3 (p < 0. 01) gene expression in HaCaT cells, but no modulation was observed in gingival fibroblasts. Conclusion: P. gingivalis LPS exposure to human HaCaT keratinocytes downregulates gene expression of the enzymes that promote epigenetic events. Clinical relevance: The advance knowledge about epigenetic modifications caused by periodontopathogens may to possibly led to the development of new periodontal therapies. © 2012 Springer-Verlag.

Biostimulatory effect of low-level laser therapy on keratinocytes in vitro

Epithelial cells play an important role in reparative events. Therefore, therapies that can stimulate the proliferation and metabolism of these cells could accelerate the healing process. To evaluate the effects of low-level laser therapy (LLLT), human keratinocytes were irradiated with an InGaAsP diode laser prototype (LASERTable; 780 ± 3 nm; 40 mW) using 0.5, 1.5, 3, 5, and 7 J/cm2 energy doses. Irradiations were done every 24 h totaling three applications. Evaluation of cell metabolism (MTT assay) showed that LLLT with all energy doses promoted an increase of cell metabolism, being more effective for 0.5, 1.5, and 3 J/cm2. The highest cell counts (Trypan blue assay) were observed with 0.5, 3, and 5 J/cm2. No statistically significant difference for total protein (TP) production was observed and cell morphology analysis by scanning electron microscopy revealed that LLLT did not promote morphological alterations on the keratinocytes. Real-time polymerase chain reaction (qPCR) revealed that LLLT also promoted an increase of type I collagen (Col-I) and vascular endothelial growth factor (VEGF) gene expression, especially for 1.5 J/cm2, but no change on fibroblast growth factor-2 (FGF-2) expression was observed. LLLT at energy doses ranging from 0.5 to 3 J/cm2 promoted the most significant biostimulatory effects on cultured keratinocytes. © 2012 Springer-Verlag London Ltd.

IL-2, IL-5, TNF-α and IFN-γ mRNA expression in epidermal keratinocytes of systemic lupus erythematosus skin lesions

OBJECTIVE: To analyze cytokine gene expression in keratinocytes from patients with systemic lupus erythematosus (SLE). INTRODUCTION: Keratinocytes represent 95% of epidermal cells and can secrete several cytokines. METHODS: Keratinocytes were obtained by laser microdissection from 21 patients with SLE (10 discoid and 11 acute lesions) at involved and uninvolved sites. All patients were receiving a low/moderate prednisone dose and 18 were receiving chloroquine diphosphate. IL-2, IL-5, TNF-α and IFN-γ gene expression was evaluated by real-time PCR and expressed as the ratio (R) to a pool of skin samples from 12 healthy volunteers. RESULTS: Heterogeneity in cytokine gene expression was found among patients with SLE. Eighteen of 38 valid SLE samples (47%) presented overexpression (R>1) of at least one cytokine. Lesional skin samples tended to show higher cytokine expression than samples from uninvolved skin (p = 0.06). IL-5 and IFN-γ were the most commonly overexpressed cytokines. Samples with cytokine overexpression corresponded to more extensive and severe lesions. Prednisone dose did not differ between samples without cytokine overexpression (15.71±3.45 mg/day) and those with overexpressed cytokines (12.68±5.41 mg/day) (p = 0.216). Samples from all patients not receiving diphosphate chloroquine had at least one overexpressed cytokine. CONCLUSIONS: The heterogeneous keratinocyte cytokine gene expression reflects the complex immunological and inflammatory background in SLE. Patients with severe/extensive skin lesions showed a higher frequency of cytokine gene overexpression. Increased IFN-γ and IL-5 expression suggests that Th1 and Th2 cells are involved in SLE skin inflammation. The possibility that prednisone and antimalarial drugs may have contributed to low cytokine gene expression in some samples cannot be ruled out.

Effects of Soft Denture Liners on L929 Fibroblasts, HaCaT Keratinocytes, and RAW 264.7 Macrophages

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Changes in nuclear morphology and chromatin texture of basal keratinocytes in melasma

BackgroundThe pathogenesis of melasma and the role of keratinocytes in disease development and maintenance are not completely understood. Dermal abnormalities, the expression of inflammatory mediators, growth factors, epithelial expression of melanocortin and sexual hormones receptors suggest that not only melanocytes, but entire epidermal melanin unit is involved in melasma physiopathology.ObjectivesTo compare nuclear morphological features and chromatin texture between basal keratinocytes in facial melasma and adjacent normal skin.MethodsWe took facial skin biopsies (2mm melasma and adjacent normal skin) from women processed for haematoxylin and eosin. Thirty non-overlapping basal keratinocyte nuclei were segmented and descriptors of area, highest diameter, perimeter, circularity, pixel intensity, profilometric index (Ra) and fractal dimension were extracted using ImageJ software.ResultsBasal keratinocyte nuclei from facial melasma epidermis displayed larger size, irregular shape, hyperpigmentation and chromatin heterogeneity by fractal dimension than perilesional skin.ConclusionBasal keratinocytes from facial melasma display changes in nuclear form and chromatin texture, suggesting that the phenotype differences between melasma and adjacent facial skin can result from complete epidermal melanin unit alterations, not just hypertrophic melanocytes.

Activation of the low molecular weight protein tyrosine phosphatase in keratinocytes exposed to hyperosmotic stress

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)