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1.
Nat Commun ; 12(1): 1089, 2021 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-33597528

RESUMO

Cell-to-cell communication can be inferred from ligand-receptor expression in cell transcriptomic datasets. However, important challenges remain: global integration of cell-to-cell communication; biological interpretation; and application to individual cell population transcriptomic profiles. We develop ICELLNET, a transcriptomic-based framework integrating: 1) an original expert-curated database of ligand-receptor interactions accounting for multiple subunits expression; 2) quantification of communication scores; 3) the possibility to connect a cell population of interest with 31 reference human cell types; and 4) three visualization modes to facilitate biological interpretation. We apply ICELLNET to three datasets generated through RNA-seq, single-cell RNA-seq, and microarray. ICELLNET reveals autocrine IL-10 control of human dendritic cell communication with up to 12 cell types. Four of them (T cells, keratinocytes, neutrophils, pDC) are further tested and experimentally validated. In summary, ICELLNET is a global, versatile, biologically validated, and easy-to-use framework to dissect cell communication from individual or multiple cell-based transcriptomic profiles.


Assuntos
Comunicação Celular/genética , Biologia Computacional/métodos , Bases de Dados Factuais , Perfilação da Expressão Gênica/métodos , Transcriptoma/genética , Animais , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Neutrófilos/citologia , Neutrófilos/metabolismo , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Linfócitos T/citologia , Linfócitos T/metabolismo
2.
Nat Commun ; 11(1): 5067, 2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-33082341

RESUMO

Although acne is the most common human inflammatory skin disease, its pathogenic mechanisms remain incompletely understood. Here we show that GATA6, which is expressed in the upper pilosebaceous unit of normal human skin, is down-regulated in acne. GATA6 controls keratinocyte proliferation and differentiation to prevent hyperkeratinisation of the infundibulum, which is the primary pathological event in acne. When overexpressed in immortalised human sebocytes, GATA6 triggers a junctional zone and sebaceous differentiation program whilst limiting lipid production and cell proliferation. It modulates the immunological repertoire of sebocytes, notably by upregulating PD-L1 and IL10. GATA6 expression contributes to the therapeutic effect of retinoic acid, the main treatment for acne. In a human sebaceous organoid model GATA6-mediated down-regulation of the infundibular differentiation program is mediated by induction of TGFß signalling. We conclude that GATA6 is involved in regulation of the upper pilosebaceous unit and may be an actionable target in the treatment of acne.


Assuntos
Acne Vulgar/metabolismo , Fator de Transcrição GATA6/metabolismo , Glândulas Sebáceas/metabolismo , Acne Vulgar/genética , Acne Vulgar/patologia , Acne Vulgar/fisiopatologia , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Diferenciação Celular , Fator de Transcrição GATA6/genética , Homeostase , Humanos , Interleucina-10/genética , Interleucina-10/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Queratinócitos/patologia , Glândulas Sebáceas/citologia , Glândulas Sebáceas/patologia , Pele/citologia , Pele/metabolismo , Pele/patologia
3.
BMC Dermatol ; 20(1): 6, 2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32867747

RESUMO

BACKGROUND: Specific species of ceramides (Cer), major constituents of lipids in the stratum corneum (SC), are decreased and are correlated with SC barrier and water-holding functions in the skin of patients with atopic dermatitis (AD) or psoriasis (Pso). However, possible correlations between Cer subclass ratios and skin properties in barrier-disrupted skin and in healthy skin remain unclear. The objective of this study was to identify a new marker to evaluate skin properties and epidermal differentiation in SC not only in barrier-disrupted skin but also in healthy skin. METHODS: The Cer subclass ratios in the SC of healthy control subjects and in patients with AD or Pso were evaluated. Correlations with candidate markers and facial skin features of healthy Japanese females (20-74 years old, n = 210) were investigated. Variations of markers during epidermal differentiation were studied in human epidermis and in cultured keratinocytes. RESULTS: The ratios of Cer [NP]/[NS], Cer [NH]/[NS], Cer [NP]/[AS], Cer [NH]/[NS], Cer [NDS]/[AS], Cer [AH]/[AS] and Cer [EOP]/[AS] showed significant differences between non-lesional skin of AD patients and normal skin of healthy control subjects, as well as Pso patients and their healthy control subjects. The Cer [NP]/[NS] ratio was correlated with SC functional parameters (transepidermal water loss and capacitance) and with skin appearance (texture, scaling and color) even in the cheek skin of healthy female subjects. The Cer [NP]/[NS] ratio in the SC was approximately 18-times higher than in living keratinocytes, and it increased as they differentiated. CONCLUSIONS: The Cer [NP]/[NS] ratio in the SC is a potential marker for skin properties and epidermal differentiation in barrier-disrupted skin as well as in healthy skin.


Assuntos
Ceramidas/análise , Dermatite Atópica/patologia , Epiderme/química , Psoríase/patologia , Adulto , Idoso , Biomarcadores/análise , Estudos de Casos e Controles , Diferenciação Celular , Células Cultivadas , Dermatite Atópica/diagnóstico , Epiderme/patologia , Feminino , Humanos , Queratinócitos/química , Queratinócitos/citologia , Lipídeos/análise , Pessoa de Meia-Idade , Psoríase/diagnóstico , Adulto Jovem
4.
Nat Commun ; 11(1): 4239, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32843640

RESUMO

How stem cells give rise to epidermis is unclear despite the crucial role the epidermis plays in barrier and appendage formation. Here we use single cell-RNA sequencing to interrogate basal stem cell heterogeneity of human interfollicular epidermis and find four spatially distinct stem cell populations at the top and bottom of rete ridges and transitional positions between the basal and suprabasal epidermal layers. Cell-cell communication modeling suggests that basal cell populations serve as crucial signaling hubs to maintain epidermal communication. Combining pseudotime, RNA velocity, and cellular entropy analyses point to a hierarchical differentiation lineage supporting multi-stem cell interfollicular epidermal homeostasis models and suggest that transitional basal stem cells are stable states essential for proper stratification. Finally, alterations in differentially expressed transitional basal stem cell genes result in severe thinning of human skin equivalents, validating their essential role in epidermal homeostasis and reinforcing the critical nature of basal stem cell heterogeneity.


Assuntos
Diferenciação Celular , Células Epidérmicas/citologia , Homeostase , Células-Tronco/citologia , Comunicação Celular/genética , Diferenciação Celular/genética , Linhagem da Célula/genética , Células Epidérmicas/metabolismo , Epiderme/metabolismo , Prepúcio do Pênis/citologia , Prepúcio do Pênis/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Recém-Nascido , Queratinócitos/citologia , Queratinócitos/metabolismo , Masculino , Modelos Biológicos , Transdução de Sinais , Células-Tronco/metabolismo
5.
PLoS One ; 15(8): e0238076, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32857768

RESUMO

Epidermal lineages and injury induced regeneration are controlled by transcriptional programs coordinating cellular signaling and epigenetic regulators, but the mechanism remains unclear. Previous studies showed that conditional deletion of the transcriptional coactivator Mediator 1 (Med1) changes epidermal lineages and accelerates wound re-epithelialization. Here, we studied a molecular mechanism by which Med1 facilitates these processes, in particular, by focusing on TGFß signaling through genome wide transcriptome analysis. The expression of the TGF ligands (Tgfß1/ß2) and their downstream target genes is decreased in both normal and wounded Med1 null skin. Med1 silencing in cultured keratinocytes likewise reduces the expression of the ligands (TGFß1/ß2) and diminishes activity of TGFß signaling as shown by decreased p-Smad2/3. Silencing Med1 increases keratinocyte proliferation and migration in vitro. Epigenetic studies using chromatin immuno-precipitation and next generation DNA sequencing reveals that Med1 regulates transcription of TGFß components by forming large clusters of enhancers called super-enhancers at the regulatory regions of the TGFß ligand and SMAD3 genes. These results demonstrate that Med1 is required for the maintenance of the TGFß signaling pathway. Finally, we show that pharmacological inhibition of TGFß signaling enhances epidermal lineages and accelerates wound re-epithelialization in skin similar to that seen in the Med1 null mice, providing new insights into epidermal regeneration.


Assuntos
Subunidade 1 do Complexo Mediador/genética , Regeneração/fisiologia , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta2/metabolismo , Animais , Linhagem da Célula , Movimento Celular , Proliferação de Células , Regulação para Baixo , Epiderme/fisiologia , Queratinócitos/citologia , Queratinócitos/metabolismo , Subunidade 1 do Complexo Mediador/antagonistas & inibidores , Subunidade 1 do Complexo Mediador/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Pele/metabolismo , Pele/patologia , Proteína Smad3/genética , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta2/genética , Regulação para Cima
6.
PLoS One ; 15(8): e0237985, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32822395

RESUMO

Allogeneic cultured epidermis (allo-CE) is a cultured keratinocyte sheet manufactured from donor cells and promotes wound healing when used in deep dermal burns, donor sites, and chronic ulcers and serves as a wound dressing. Allo-CE is usually cryopreserved to be ready to use. However, the cryopreservation procedure will damage the cell viability, and the influence of Allo-CE, according to its viability or wound healing process, has not been evaluated sufficiently. In this study, we aimed to prove the influence of keratinocyte viability contained in allo-CEs on wound healing. We prepared CEs with Green's method using keratinocytes obtained from a polydactyly patient and then prepared four kinds of CEs with different cell viabilities [fresh, cryopreserved, frozen, and FT (freeze and thaw)]. The cell viabilities of fresh, cryopreserved, frozen, and FT CEs were 95.7%, 59.9%, 16.7%, and 0.0%, respectively. The four CEs had homogeneous characteristics, except for small gaps found in the FT sheet by transmission electron microscopy observation. The four CEs were applied on the full-thickness skin defect of diabetic mice (BKS.Cg-Dock 7m +/+ Leprdb/Jcl), and the wound area and neoepithelium length were evaluated on days 4, 7, and 14. As a result, FT CEs without viable cells similarly promoted epithelialization on days 4 and 7 (p<0.05) and accelerated wound closure on day 7 (p<0.01) as fresh CEs compared with the control group. In conclusion, the promoting effect of allo-CE on wound healing does not depend on cell viability. Lyophilized CEs may be a suitable wound dressing with a long storage period at room temperature.


Assuntos
Diabetes Mellitus Experimental/patologia , Queratinócitos/transplante , Cicatrização , Animais , Sobrevivência Celular , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Humanos , Lactente , Queratinócitos/citologia , Queratinócitos/metabolismo , Masculino , Camundongos , Polidactilia/metabolismo , Polidactilia/patologia , Reepitelização
7.
PLoS One ; 15(7): e0235515, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32692781

RESUMO

BACKGROUND: The skin provides a predominant barrier against chemical, physical and microbial incursion. The intemperate exposure to ultraviolet A (UVA) radiation can cause excessive cellular oxidative stress, leading to skin damage, proteins damage and mitochondrial dysfunction. There is sufficient evidences supporting the proposal that mitochondria is highly implicated in skin photo-damage. METHODS: In the present study, a polysaccharide isolated from Astragalus membranaceus was further purified to be an α-glucan, which was further investigated its beneficial influence on UVA-induced photo-damage in HaCaT cells. RESULTS: Our results showed that the purified Astragalus membranaceus polysaccharide (AP) can protect HaCaT cells from UVA-induced photo-damage through reducing UVA-induced intracellular ROS production and mitochondrial membrane potential, thereby altering ATP content. It was found that the UVA induced damage in HaCaT cells could be effectively restored by co-treatment with AP. CONCLUSIONS: AP exhibited promising potential for advanced application as multifunctional skin care products and drugs.


Assuntos
Astragalus propinquus/química , Queratinócitos/efeitos dos fármacos , Queratinócitos/efeitos da radiação , Polissacarídeos/farmacologia , Protetores contra Radiação/farmacologia , Raios Ultravioleta/efeitos adversos , Trifosfato de Adenosina/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Polissacarídeos/química , Protetores contra Radiação/química , Espécies Reativas de Oxigênio/metabolismo
8.
Int J Nanomedicine ; 15: 4275-4288, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32606677

RESUMO

Purpose: Selenium nanoparticles (Se NPs) are promising antibacterial agents to tackle the growing problem of antimicrobial resistance. The aim of this study was to fabricate Se NPs with a net positive charge to enhance their antibacterial efficacy. Methods: Se NPs were coated with a positively charged protein - recombinant spider silk protein eADF4(κ16) - to give them a net positive surface charge. Their cytotoxicity and antibacterial activity were investigated, with negatively charged polyvinyl alcohol coated Se NPs as a control. Besides, these eADF4(κ16)-coated Se NPs were immobilized on the spider silk films, and the antibacterial activity of these films was investigated. Results: Compared to the negatively charged polyvinyl alcohol coated Se NPs, the positively charged eADF4(κ16)-coated Se NPs demonstrated a much higher bactericidal efficacy against the Gram-negative bacteria E. coli, with a minimum bactericidal concentration (MBC) approximately 50 times lower than that of negatively charged Se NPs. Cytotoxicity testing showed that the eADF4(κ16)-coated Se NPs are safe to both Balb/3T3 mouse embryo fibroblasts and HaCaT human skin keratinocytes up to 31 µg/mL, which is much higher than the MBC of these particles against E. coli (8 ± 1 µg/mL). In addition, antibacterial coatings were created by immobilising the eADF4(κ16)-coated Se NPs on positively charged spider silk films and these were shown to retain good bactericidal efficacy and overcome the issue of low particle stability in culture broth. It was found that these Se NPs needed to be released from the film surface in order to exert their antibacterial effects and this release can be regulated by the surface charge of the film, such as the change of the spider silk protein used. Conclusion: Overall, eADF4(κ16)-coated Se NPs are promising new antibacterial agents against life-threatening bacteria.


Assuntos
Antibacterianos/farmacologia , Nanopartículas/química , Proteínas Recombinantes/farmacologia , Selênio/farmacologia , Seda/farmacologia , Células 3T3 , Animais , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Contagem de Colônia Microbiana , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Nanopartículas/ultraestrutura , Tamanho da Partícula
10.
Food Chem ; 333: 127510, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32673958

RESUMO

Aqueous coenzyme Q10 (CoQ10) dispersions were prepared using sugary maize dendrimer-like glucan (SMDG) with solid-dispersion treatment. After measuring solubility, recovery rate and loading rate, the initial weight ratio of CoQ10:SMDG was optimized to be 1:27, with the solubility markedly increasing up 188.8-folds compared to pure CoQ10 solution. The structural characterizations of CoQ10-SMDG formulation showed crystal CoQ10 was entrapped in SMDG matrix for amorphous state, associated with the strong interactions with glucan chains. The antioxidant activity of CoQ10-SMDG was assessed via DPPH and FRAP assay. DPPH scavenging activity and FRAP value of it were as high as 95.1% and 0.87 mM, respectively. The cellular uptake of CoQ10 in CoQ10-SMDG group was significantly higher than that of natural CoQ10. CoQ10-SMDG also exhibited significant protective effects against cellular damage in H2O2-induced HaCaT cell model. The results indicated that dendrimer-like glucan is an excellent platform to encapsulate and improve biological activity of hydropholic compounds.


Assuntos
Antioxidantes/química , Glucanos/química , Nanopartículas/química , Ubiquinona/análogos & derivados , Antioxidantes/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Dendrímeros/química , Humanos , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/toxicidade , Queratinócitos/citologia , Queratinócitos/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Tamanho da Partícula , Solubilidade , Ubiquinona/química , Ubiquinona/metabolismo
11.
Sci Rep ; 10(1): 8863, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483193

RESUMO

The precise role of pre-mRNA processing factors (PRPs) in human tumorigenesis has not been yet explored. The object of the present study was to explore the effects of PRP3 in a common metastatic skin cancer, keratinocyte-derived cutaneous squamous cell carcinoma (cSCCs). RT-qPCR and western blotting were conducted to measure the expression levels of PRP3 in various cSCC cell lines and cSCC tissues. A benign epidermal keratinocyte cell line was transfected with a eukaryotic expression plasmid to overexpress PRP3. In addition, the endogenous expression level of PRP3 in cSCC cells was silenced using a short hairpin RNA method, and the role of PRP3 on cell proliferation and migration was examined by Cell Counting Kit-8, colony formation, wound healing assay and Transwell assays following knockdown in cSCC cells, and overexpression in keratinovcyte cells. Elevated levels of PRP3 mRNA and protein were noted in cSCC cell lines or cSCC tissues compared with actinic keratosis (AK) or benign epidermal keratinocyte cell line, respectively. Upregulation of PRP3 expression was found to be associated with poor clinical outcomes in patients with cSCCs. The upregulation of PRP3 promoted cell viability, metastasis and the activity of the JAK2/STAT3 pathway in epidermal keratinocyte cells. Interestingly, loss of PRP3 had no obvious impact on cell viability and migration in benign epidermal keratinocyte cells. Functionally, the inhibition of the JAK2/STAT3 pathway reversed the increased cell viability and migration of cSCC cells induced by PRP3. Taken together, the present observations indicated that PRP3 served as a tumor active factor in cSCCs by targeting the JAK2/STAT3 pathway. Moreover, it is implied that impeding the PRP3 activity may selectively constrain cancer cell growth and migration with limited effect on normal skin cells.


Assuntos
Carcinoma de Células Escamosas/patologia , Janus Quinase 2/metabolismo , Proteínas Nucleares/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Fator de Transcrição STAT3/metabolismo , Neoplasias Cutâneas/patologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/mortalidade , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células , Humanos , Janus Quinase 2/antagonistas & inibidores , Queratinócitos/citologia , Queratinócitos/metabolismo , Ceratose Actínica/metabolismo , Ceratose Actínica/patologia , Pessoa de Meia-Idade , Metástase Neoplásica , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Prognóstico , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/antagonistas & inibidores , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Transdução de Sinais , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/mortalidade , Taxa de Sobrevida , Tirfostinas/farmacologia , Regulação para Cima
12.
J Vis Exp ; (159)2020 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-32478738

RESUMO

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported for in vitro differentiation of keratinocytes cultured in two-dimensional (2D) submerged manners using various induction conditions. Described here is a procedure for 2D in vitro keratinocyte differentiation method by contact inhibition and subsequent molecular characterization by RNA-seq. In brief, keratinocytes are grown in defined keratinocyte medium supplemented with growth factors until they are fully confluent. Differentiation is induced by close contacts between the keratinocytes and further stimulated by excluding growth factors in the medium. Using RNA-seq analyses, it is shown that both 1) differentiated keratinocytes exhibit distinct molecular signatures during differentiation and 2) the dynamic gene expression pattern largely resembles cells during epidermal stratification. As for comparison to normal keratinocyte differentiation, keratinocytes carrying mutations of the transcription factor p63 exhibit altered morphology and molecular signatures, consistent with their differentiation defects. In conclusion, this protocol details the steps for 2D in vitro keratinocyte differentiation and its molecular characterization, with an emphasis on bioinformatic analysis of RNA-seq data. Because RNA extraction and RNA-seq procedures have been well-documented, it is not the focus of this protocol. The experimental procedure of in vitro keratinocyte differentiation and bioinformatic analysis pipeline can be used to study molecular events during epidermal differentiation in healthy and diseased keratinocytes.


Assuntos
Queratinócitos/citologia , Diferenciação Celular , Células Cultivadas , Humanos , RNA-Seq
13.
Nat Commun ; 11(1): 2988, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32532976

RESUMO

Tissue homeostasis requires regulation of cell-cell communication, which relies on signaling molecules and cell contacts. In skin epidermis, keratinocytes secrete factors transduced by melanocytes into signaling cues promoting their pigmentation and dendrite outgrowth, while melanocytes transfer melanin pigments to keratinocytes to convey skin photoprotection. How epidermal cells integrate these functions remains poorly characterized. Here, we show that caveolae are asymmetrically distributed in melanocytes and particularly abundant at the melanocyte-keratinocyte interface in epidermis. Caveolae in melanocytes are modulated by ultraviolet radiations and keratinocytes-released factors, like miRNAs. Preventing caveolae formation in melanocytes increases melanin pigment synthesis through upregulation of cAMP signaling and decreases cell protrusions, cell-cell contacts, pigment transfer and epidermis pigmentation. Altogether, we identify that caveolae serve as molecular hubs that couple signaling outputs from keratinocytes to mechanical plasticity of pigment cells. The coordination of intercellular communication and contacts by caveolae is thus crucial to skin pigmentation and tissue homeostasis.


Assuntos
Cavéolas/metabolismo , Queratinócitos/metabolismo , Melanócitos/metabolismo , Pigmentação da Pele/fisiologia , Pele/metabolismo , Caveolina 1/metabolismo , Comunicação Celular/fisiologia , Comunicação Celular/efeitos da radiação , Células Cultivadas , Técnicas de Cocultura , Células Epidérmicas/metabolismo , Epiderme/metabolismo , Epiderme/ultraestrutura , Células HeLa , Humanos , Queratinócitos/citologia , Melanócitos/citologia , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Transdução de Sinais/fisiologia , Transdução de Sinais/efeitos da radiação , Pele/citologia , Pele/ultraestrutura , Raios Ultravioleta
14.
Braz Oral Res ; 34: e033, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32267290

RESUMO

The aim of our study was to isolate populations of keratinocyte stem cells based on the expression of cell surface markers and to investigate whether the culture could affect their phenotype. keratinocytes from human oral mucosa were sorted based on the expression of the epithelial stem cell markers p75NTR and CD71. We also examined the co-expression of other epithelial stem markers such as integrins ß1 and α6 and their stem cell-like proprieties in in vitro assays. Three passages after being sorted by MACS, more than 93% of the p75NTR+ve cells lost the expression of p75NTR, while 5.46% of the p75NTR-ve gained it. Within the small population of the p75NTR+ve cells, 88% co-expressed other epithelial stem cell markers such as integrins ß1 and α6, while only 28% of p75NTR-ve cells co-expressed these markers. These results were confirmed by sorting cells by FACS. Additionally, when double staining was used for sorting cells, 99% of the p75NTR+veCD71-ve and 33% of the p75NTR-veCD71+ve cells expressed both integrins, but just one week after culture, only 1.74% of the p75NTR+veCD71-ve cells still expressed p75NTR and only 0.32% still expressed CD71. Similar results were obtained when co-culturing p75NTR+ve and p75NTR-ve populations before analysis. Our results suggest that phenotype changes may be part of an intrinsic cellular mechanism to conserve levels of protein expression as they may found in the human body. In addition, in vitro culture may not offer ideal conditions for epithelial stem cell maintenance due to phenotype changes under standard culture conditions.


Assuntos
Técnicas de Cultura de Células/métodos , Células Epiteliais/citologia , Queratinócitos/citologia , Mucosa Bucal/citologia , Fenótipo , Células-Tronco/citologia , Antígenos CD/análise , Biomarcadores/análise , Separação Celular/métodos , Citometria de Fluxo/métodos , Humanos , Proteínas do Tecido Nervoso/análise , Receptores de Fator de Crescimento Neural/análise , Receptores da Transferrina/análise , Reprodutibilidade dos Testes
15.
Adv Wound Care (New Rochelle) ; 9(5): 233-244, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32226648

RESUMO

Objectives: To investigate the effect of suppressor of fused (Sufu) on epidermal and dermal cellular properties and in wound healing. Approach: Transgenic (TG) mice overexpressing human Sufu (hSufu) in the epidermis were applied to investigate the effects of Sufu on epidermal and dermal cellular properties and in wound healing. Results: Histological staining revealed a reduction of epidermal and dermal thickness and an increase of hypodermal adipose tissue in homozygous K14-hSufu TG mice when compared with wild-type (WT) controls. TG mice exhibited significantly delayed skin wound healing. Moreover, the migratory and proliferative capabilities of cultured keratinocytes were decreased in K14-hSufuTG mice. Transforming growth factor-ß treatment increased the expression of α-smooth muscle actin more in WT than in TG fibroblasts. Sufu overexpression significantly decreased the expression of ß-catenin, glioma transcription factor 1 (Gli1), and matrix metalloproteinase-3 in wounds of K14-hSufu TG mice when compared with controls, probably indicating a delaying effect of Sufu on wound healing via blocking the hedgehog (Hh)/Gli and Wnt/ß-catenin pathway. Innovation: Our results indicate a new property of Sufu in the process of skin wound healing. It provides an important basis for Sufu as a potential target for skin wound healing. Conclusion: Our findings suggest that Sufu overexpression in the epidermis impairs wound healing via dampening the Hh/Gli and Wnt/ß-catenin signaling pathway. These data provide an important basis for further analyses of Sufu in skin wound healing.


Assuntos
Fibroblastos/metabolismo , Queratinócitos/metabolismo , Proteínas Repressoras/metabolismo , Via de Sinalização Wnt , Cicatrização , Animais , Diferenciação Celular , Modelos Animais de Doenças , Células Epidérmicas , Epiderme/crescimento & desenvolvimento , Epiderme/metabolismo , Fibroblastos/citologia , Proteínas Hedgehog/metabolismo , Queratinócitos/citologia , Masculino , Camundongos , Camundongos Transgênicos , Envelhecimento da Pele/patologia , Envelhecimento da Pele/fisiologia , Fator de Crescimento Transformador beta/metabolismo
16.
PLoS One ; 15(4): e0231418, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32267900

RESUMO

The telomerase reverse transcriptase (TERT) gene is responsible for telomere maintenance in germline and stem cells, and is re-expressed in 90% of human cancers. CpG methylation in the TERT promoter (TERTp) was correlated with TERT mRNA expression. Furthermore, two hotspot mutations in TERTp, dubbed C228T and C250T, have been revealed to facilitate binding of transcription factor ETS/TCF and subsequent TERT expression. This study aimed to elucidate the combined contribution of epigenetic (promoter methylation and chromatin accessibility) and genetic (promoter mutations) mechanisms in regulating TERT gene expression in healthy skin samples and in melanoma cell lines (n = 61). We unexpectedly observed that the methylation of TERTp was as high in a subset of healthy skin cells, mainly keratinocytes, as in cutaneous melanoma cell lines. In spite of the high promoter methylation fraction in wild-type (WT) samples, TERT mRNA was only expressed in the melanoma cell lines with either high methylation or intermediate methylation in combination with TERT mutations. TERTp methylation was positively correlated with chromatin accessibility and TERT mRNA expression in 8 melanoma cell lines. Cooperation between epigenetic and genetic mechanisms were best observed in heterozygous mutant cell lines as chromosome accessibility preferentially concerned the mutant allele. Combined, these results suggest a complex model in which TERT expression requires either a widely open chromatin state in TERTp-WT samples due to high methylation throughout the promoter or a combination of moderate methylation fraction/chromatin accessibility in the presence of the C228T or C250T mutations.


Assuntos
Cromatina/metabolismo , Metilação de DNA , Telomerase/metabolismo , Alelos , Linhagem Celular , Cromatina/química , Montagem e Desmontagem da Cromatina , Ilhas de CpG , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Melanoma/genética , Melanoma/patologia , Mutação , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Telomerase/genética , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
17.
Nat Commun ; 11(1): 1472, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32193376

RESUMO

The Hippo TEAD-transcriptional regulators YAP1 and TAZ are central for cell renewal and cancer growth; however, the specific downstream gene networks involved in their activity are not completely understood. Here we introduce TEADi, a genetically encoded inhibitor of the interaction of YAP1 and TAZ with TEAD, as a tool to characterize the transcriptional networks and biological effects regulated by TEAD transcription factors. Blockage of TEAD activity by TEADi in human keratinocytes and mouse skin leads to reduced proliferation and rapid activation of differentiation programs. Analysis of gene networks affected by TEADi and YAP1/TAZ knockdown identifies KLF4 as a central transcriptional node regulated by YAP1/TAZ-TEAD in keratinocyte differentiation. Moreover, we show that TEAD and KLF4 can regulate the activity of each other, indicating that these factors are part of a transcriptional regulatory loop. Our study establishes TEADi as a resource for studying YAP1/TAZ-TEAD dependent effects.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Redes Reguladoras de Genes , Homeostase , Fatores de Transcrição Kruppel-Like/metabolismo , Pele/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Pontos de Checagem do Ciclo Celular/genética , Diferenciação Celular/genética , Proliferação de Células/genética , Células HEK293 , Homeostase/genética , Humanos , Inflamação/patologia , Queratinócitos/citologia , Queratinócitos/metabolismo , Camundongos , Modelos Animais , Modelos Biológicos , Ligação Proteica , Células-Tronco/citologia , Células-Tronco/metabolismo , Transcrição Genética
18.
Radiat Res ; 193(5): 481-496, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32196412

RESUMO

During fractionated radiotherapy, epithelial cell populations are thought to decrease initially, followed by accelerated repopulation to compensate cell loss. However, previous findings in skin with daily 1.1 Gy dose fractions indicate continued and increasing cell depletion. Here we investigated epidermal keratinocyte response with daily 2 Gy fractions as well as accelerated and hypofractionation. Epidermal interfollicular melanocytes were also assessed. Skin-punch biopsies were collected from breast cancer patients before, during and after mastectomy radiotherapy to the thoracic wall with daily 2 Gy fractions for 5 weeks. In addition, 2.4 Gy radiotherapy four times per week and 4 Gy fractions twice per week for 5 weeks, and two times 2 Gy daily for 2.5 weeks, were used. Basal keratinocyte density of the interfollicular epidermis was determined and immunostainings of keratinocytes for DNA double-strand break (DSB) foci, growth arrest, apoptosis and mitosis were quantified. In addition, interfollicular melanocytes were counted. Initially minimal keratinocyte loss was observed followed by pronounced depletion during the second half of treatment and full recovery at 2 weeks post treatment. DSB foci per cell peaked towards the end of treatment. p21-stained cell counts increased during radiotherapy, especially the second half. Apoptotic frequency was low throughout radiotherapy but increased at treatment end. Mitotic cell count was significantly suppressed throughout radiotherapy and did not recover during weekend treatment gaps, but increased more than threefold compared to unexposed skin 2 weeks post-radiotherapy. The number of melanocytes remained constant over the study period. Germinal keratinocyte loss rate increased gradually during daily 2 Gy fractions for 5 weeks, and similarly for hypofractionation. DSB foci number after 2 Gy irradiation revealed an initial radioresistance followed by increasing radiosensitivity. Growth arrest mediated by p21 strongly suggests that cells within or recruited into the cell cycle during treatment are at high risk of loss and do not contribute significantly to repopulation. It is possible that quiescent (G0) cells at treatment completion accounted for the accelerated post-treatment repopulation. Recent knowledge of epidermal tissue regeneration and cell cycle progression during genotoxic and mitogen stress allows for a credible explanation of the current finding. Melanocytes were radioresistant regarding cell depletion.


Assuntos
Apoptose/efeitos da radiação , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Epiderme/efeitos da radiação , Queratinócitos/efeitos da radiação , Melanócitos/efeitos da radiação , Tolerância a Radiação , Proliferação de Células/efeitos da radiação , Relação Dose-Resposta à Radiação , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Melanócitos/citologia , Melanócitos/metabolismo , Fatores de Tempo
19.
PLoS One ; 15(3): e0230380, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32163511

RESUMO

Epidermal morphogenesis and hair follicle (HF) development depend on the ability of keratinocytes to adhere to the basement membrane (BM) and migrate along the extracellular matrix. Integrins are cell-matrix receptors that control keratinocyte adhesion and migration, and are recognized as major regulators of epidermal homeostasis. How integrins regulate the behavior of keratinocytes during epidermal morphogenesis remains insufficiently understood. Here, we show that α-parvin (α-pv), a focal adhesion protein that couples integrins to actin cytoskeleton, is indispensable for epidermal morphogenesis and HF development. Inactivation of the murine α-pv gene in basal keratinocytes results in keratinocyte-BM detachment, epidermal thickening, ectopic keratinocyte proliferation and altered actin cytoskeleton polarization. In vitro, α-pv-null keratinocytes display reduced adhesion to BM matrix components, aberrant spreading and stress fibers formation, and impaired directed migration. Together, our data demonstrate that α-pv controls epidermal homeostasis by facilitating integrin-mediated adhesion and actin cytoskeleton organization in keratinocytes.


Assuntos
Membrana Basal/metabolismo , Epiderme/crescimento & desenvolvimento , Folículo Piloso/metabolismo , Queratinócitos/metabolismo , Proteínas dos Microfilamentos/fisiologia , Morfogênese/fisiologia , Actinas/metabolismo , Animais , Membrana Basal/citologia , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Células Cultivadas , Adesões Focais/metabolismo , Integrinas/metabolismo , Queratinócitos/citologia , Camundongos , Camundongos Transgênicos
20.
Sci Rep ; 10(1): 4123, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32139717

RESUMO

Drug screening studies for inflammatory skin diseases are currently performed using model systems that only partially recapitulate human diseased skin. Here, we developed a new strategy to incorporate T cells into human 3D skin constructs (HSCs), which enabled us to closely monitor and quantitate T cell responses. We found that the epidermis promotes the activation and infiltration of T cells into the skin, and provides a directional cue for their selective migration towards the epidermis. We established a psoriatic HSC (pHSC) by incorporating polarized Th1/Th17 cells or CCR6+CLA+ T cells derived from psoriasis patients into the constructs. These pHSCs showed a psoriatic epidermal phenotype and characteristic cytokine profiles, and responded to various classes of psoriasis drugs, highlighting the potential utility of our model as a drug screening platform. Taken together, we developed an advanced immunocompetent 3D skin model to investigate epidermal-T cell interactions and to understand the pathophysiology of inflammatory skin diseases in a human-relevant and patient-specific context.


Assuntos
Psoríase/imunologia , Pele/citologia , Pele/metabolismo , Células Cultivadas , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Fibroblastos/citologia , Fibroblastos/imunologia , Fibroblastos/metabolismo , Citometria de Fluxo , Humanos , Queratinócitos/citologia , Queratinócitos/imunologia , Queratinócitos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores CCR6/genética , Receptores CCR6/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/imunologia , Células Th1/metabolismo , Células Th17/metabolismo
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