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1.
Shanghai Kou Qiang Yi Xue ; 29(3): 257-261, 2020 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-33043341

RESUMO

PURPOSE: To preliminarily explore the cytotoxicological responses of keratinocytes derived from human embryonic stem cells(K-hESCs) to drugs, and to provide a basis for the establishment of a new biosafety evaluation model. METHODS: CCK-8 assay was used to detect cell viability and cytotoxicity. The detection of pharmacological response was observed and compared when K-hESCs directly derived from human embryonic stem cells, human gingival epithelial cells (HGECs), and human immortalized oral epithelial cells (HIOECs) were treated with retinoic acid (RA), 5-fluorouracil(5-FU), dexamethasone(DEX), and penicillin G(PG). Statistical analysis was performed using SPSS 16.0 software package. RESULTS: After drugs were applied to HGECs, HIOEC and K-hESCs, the half inhibitory concentrations (IC50) of RA was 6.1±0.03, 5.62±0.05 and 6.58±0.02, respectively. The IC50 of 5-FU was 1.65±0.02,3.00±0.02 and 1.72±0.04, respectively. The IC50 of DEX was 113.67±0.014,328±0.002 and 126.17±0.05, respectively. The IC50 of PG was 2200±1.34,3795±2.42 and 2880±1.5, respectively. The IC50 of the four drugs between HIOEC and HGECs had significant differences(P<0.05), but there was no significant difference in IC50 between K-hESCs and HGECs(P>0.05). The IC50 of K-hESCs and HIOEC also had significant differences(P<0.05). CONCLUSIONS: IC50 of K-hESCs was closer to HGECs than HIOEC. It was speculated that K-hESCs could simulate the response of normal human cells in cytotoxicity study.


Assuntos
Células-Tronco Embrionárias , Células-Tronco Embrionárias Humanas , Diferenciação Celular , Células Epiteliais , Humanos , Queratinócitos
2.
Adv Exp Med Biol ; 1268: 285-306, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32918224

RESUMO

Cutaneous malignancies including melanomas and keratinocyte carcinomas (KC) are the most common types of cancer, occurring at a rate of over one million per year in the United States. KC, which include both basal cell carcinomas and squamous cell carcinomas, are substantially more common than melanomas and form the subject of this chapter. Ultraviolet radiation (UVR), both UVB and UVA, as occurs with sunlight exposure is generally regarded as causal for these malignancies, but UVB is also required for vitamin D synthesis in the skin. Keratinocytes are the major cell in the epidermis. These cells not only produce vitamin D but contain the enzymatic machinery to metabolize vitamin D to its active metabolite, 1,25(OH)2D, and express the receptor for this metabolite, the vitamin D receptor (VDR). This allows the cell to respond to the 1,25(OH)2D that it produces. Based on our own data and that reported in the literature, we conclude that vitamin D signaling in the skin suppresses UVR-induced epidermal tumor formation. In this chapter we focus on four mechanisms by which vitamin D signaling suppresses tumor formation. They are inhibition of proliferation/stimulation of differentiation with discussion of the roles of hedgehog, Wnt/ß-catenin, and hyaluronan/CD44 pathways in mediating vitamin D regulation of proliferation/differentiation, regulation of the balance between oncogenic and tumor suppressor long noncoding RNAs, immune regulation, and promotion of DNA damage repair (DDR).


Assuntos
Receptores de Calcitriol/metabolismo , Pele/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Humanos , Queratinócitos/metabolismo , Pele/citologia , Neoplasias Cutâneas/metabolismo , Raios Ultravioleta/efeitos adversos , Vitamina D/metabolismo
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): 4788, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32963234

RESUMO

Regenerative proliferation capacity and poor differentiation are histological features usually linked to poor prognosis in head and neck squamous cell carcinoma (hnSCC). However, the pathways that regulate them remain ill-characterized. Here, we show that those traits can be triggered by the RHO GTPase activator VAV2 in keratinocytes present in the skin and oral mucosa. VAV2 is also required to maintain those traits in hnSCC patient-derived cells. This function, which is both catalysis- and RHO GTPase-dependent, is mediated by c-Myc- and YAP/TAZ-dependent transcriptomal programs associated with regenerative proliferation and cell undifferentiation, respectively. High levels of VAV2 transcripts and VAV2-regulated gene signatures are both associated with poor hnSCC patient prognosis. These results unveil a druggable pathway linked to the malignancy of specific SCC subtypes.


Assuntos
Proliferação de Células , Neoplasias de Cabeça e Pescoço/metabolismo , Proteínas Proto-Oncogênicas c-vav/genética , Proteínas Proto-Oncogênicas c-vav/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Animais , Diferenciação Celular , Modelos Animais de Doenças , Epiderme/metabolismo , Epiderme/patologia , GTP Fosfo-Hidrolases , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Hiperplasia/patologia , Queratinócitos/patologia , Camundongos , Camundongos Knockout , Membrana Mucosa/metabolismo , Prognóstico , RNA Mensageiro/metabolismo , Transdução de Sinais , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Transcriptoma
5.
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
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(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
8.
Gene ; 760: 145003, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32739587

RESUMO

Imiquimod (IMQ) is approved as a first-line treatment for genital warts caused by human papillomavirus (HPV) infection. However, the recurrence rate is very high. HPV E7 protein plays a critical role in HPV immune escape. However, the role of HPV11 E7 protein in genital warts recurrence during IMQ treatment is not clear. Here, we found that the expression profile of NHEK cells was obviously changed after IMQ treatment, and a large number of genes encoding cytokines and genes involved in cytokine-mediated signaling pathways and cellular metabolic signaling pathways were up- or downregulated. HPV11E7 overexpression inhibited the IMQ-induced production of of multiple chemokines and colony-stimulating factors in NHEK cells. Furthermore, we found that HPV11E7 could impair the activation of mitogen-activated protein kinase (MAPK) signaling pathway. Therefore, our results suggested that HPV11 E7 diminishes the production of chemokines, colony-stimulating factors and other cytokines via inhibition of the MAPK signaling pathway, which suppresses the therapeutic effect of IMQ and promotes the recurrence of diseases, such as condyloma acuminatum.


Assuntos
Imiquimode/farmacologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Quimiocinas/biossíntese , Quimiocinas/genética , Quimiocinas/metabolismo , Fatores Estimuladores de Colônias/biossíntese , Fatores Estimuladores de Colônias/metabolismo , Citocinas/metabolismo , Expressão Gênica/efeitos dos fármacos , Papillomavirus Humano 11/metabolismo , Humanos , Imiquimode/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Oncogênicas Virais/imunologia , Infecções por Papillomavirus/metabolismo , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
9.
PLoS Pathog ; 16(8): e1008792, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32813746

RESUMO

Tumor suppressors can exert pro-proliferation functions in specific contexts. In the beta human papillomavirus type 38 (HPV38) experimental model, the viral proteins E6 and E7 promote accumulation of a wild-type (WT) p53 form in human keratinocytes (HKs), promoting cellular proliferation. Inactivation of p53 by different means strongly decreases the proliferation of HPV38 E6/E7 HKs. This p53 form is phosphorylated at S392 by the double-stranded RNA-dependent protein kinase PKR, which is highly activated by HPV38. PKR-mediated S392 p53 phosphorylation promotes the formation of a p53/DNMT1 complex, which inhibits expression of integrin alpha 1 (ITGA1), a repressor of epidermal growth factor receptor (EGFR) signaling. Ectopic expression of ITGA1 in HPV38 E6/E7 HKs promotes EGFR degradation, inhibition of cellular proliferation, and cellular death. Itga1 expression was also inhibited in the skin of HPV38 transgenic mice that have an elevated susceptibility to UV-induced skin carcinogenesis. In summary, these findings reveal the existence of a specific WT p53 form that displays pro-proliferation properties.


Assuntos
Proteínas de Transporte/antagonistas & inibidores , Proliferação de Células , Queratinócitos/patologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/complicações , Proteínas Repressoras/metabolismo , Neoplasias Cutâneas/etiologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Células Cultivadas , Regulação para Baixo , Humanos , Queratinócitos/imunologia , Queratinócitos/virologia , Camundongos , Camundongos Transgênicos , Proteínas Oncogênicas Virais/genética , Papillomaviridae/isolamento & purificação , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/virologia , Proteínas Repressoras/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Proteína Supressora de Tumor p53/genética
10.
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
11.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao ; 42(3): 338-346, 2020 Jun 30.
Artigo em Chinês | MEDLINE | ID: mdl-32616129

RESUMO

Objective To observe the cell origin of N-methyl-D-aspartic acid(NMDA)receptor expression in skin after chronic ischemic pain modeling in rats and explore the role of NMDA receptor in type Ⅰ complex regional pain syndrome. Methods Forty-two adult male Sprague-Dawley rats were randomly divided into five groups:sham operation group(n=12),chronic post ischemia pain(CPIP)group(n=12),CPIP+normal saline(NS)group(n=6),CPIP+NMDA group(n=6),and CPIP+MK801 group(n=6).Six rats in the sham operation group and CPIP group were sacrificed under deep anesthesia one day after modeling.The plantar skin and L3-L5 spinal cord tissue were used for NR1(NMDA receptor)subunit immunofluorescence detection and for Western blotting of NR1,interleukin(IL)-1ß,and tumor necrosis factor(TNF)-α.For the remaining rats,the mechanical withdrawal threshold(MWT)values on the 2nd,6th,10th and 14th day after ischemia were recorded,and the corresponding drugs were injected subcutaneously from the 6th day after ischemia.The skin and L3-L5 spinal cords were collected on the 14th day,and the same detection methods were applied. Results Compared with the sham operation group,the CPIP group had significantly higher expressions of NR1(1.708±0.064;t=12.120, P<0.001),IL-1ß(2.575±0.305;t=5.158, P=0.003),and TNF-α(2.691±0.217;t=7.786, P<0.001)in the skin on the first day after modeling.After intervention with NMDA and MK801,the MWT value was [(20.37±0.95)g] in the CPIP+NS group,which was significantly higher than that in CPIP+NMDA group [(15.85±1.09)g;q=10.920, P<0.001] but significantly lower than that in CPIP+MK801 group[(22.95±0.96)g;q=6.421, P<0.001] 10 days after modeling.On the 14th day,compared with the MWT of the CPIP+NS group [(21.57±0.96)g],the CPIP+NMDA group had significantly decreased MWT value [(16.53±1.63)g;q=12.190, P<0.001],and the CPIP+MK801 group had significantly increased MWT value [(23.27±1.28)g;q=4.094, P=0.025].Compared with the sham operation group,the CPIP group had significantly increased NR1 expression(1.708±0.064;t=10.910, P<0.001)and the CPIP+NS group had significantly increased expressions of IL-1ß(2.518±0.147;q=11.010, P<0.001)and TNF-α(1.949±0.184;q=10.870, P<0.001).Compared with the CPIP+NS group,the CPIP+NMDA group had significantly increased expressions of IL-1ß(4.816±0.607;q=16.670, P=0.003)and TNF-α(2.629±0.349;q=7.790, P<0.001)and the CPIP+MK801 group had significantly decreased expressions of IL-1ß(1.048±0.257;q=10.660, P=0.003)and TNF-α(0.790±0.165;q=13.280, P<0.001). Conclusion NMDA receptor activation in skin keratinocytes after chronic ischemia in rats hinders the expression of inflammatory cytokines such as IL-1ß and TNF-α,which may be involved in central sensitization and pain conduction of type Ⅰ complex regional pain syndrome.


Assuntos
Queratinócitos , Animais , Interleucina-1beta , Masculino , Dor , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato , Medula Espinal , Fator de Necrose Tumoral alfa
12.
PLoS One ; 15(7): e0235295, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32687504

RESUMO

Spontaneous mutations in the SHANK-associated RH domain interacting protein (Sharpin) resulted in a severe autoinflammatory type of chronic proliferative dermatitis, inflammation in other organs, and lymphoid organ defects. To determine whether cell-type restricted loss of Sharpin causes similar lesions, a conditional null mutant was created. Ubiquitously expressing cre-recombinase recapitulated the phenotype seen in spontaneous mutant mice. Limiting expression to keratinocytes (using a Krt14-cre) induced a chronic eosinophilic dermatitis, but no inflammation in other organs or lymphoid organ defects. The dermatitis was associated with a markedly increased concentration of serum IgE and IL18. Crosses with S100a4-cre resulted in milder skin lesions and moderate to severe arthritis. This conditional null mutant will enable more detailed studies on the role of SHARPIN in regulating NFkB and inflammation, while the Krt14-Sharpin-/- provides a new model to study atopic dermatitis.


Assuntos
Dermatite Atópica/genética , Inflamação/genética , Queratina-14/genética , Proteínas do Tecido Nervoso/genética , Proteína A4 de Ligação a Cálcio da Família S100/genética , Animais , Apoptose/genética , Artrite/genética , Artrite/patologia , Dermatite Atópica/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Humanos , Imunoglobulina E/genética , Inflamação/patologia , Integrases/genética , Interleucina-18/genética , Queratinócitos/metabolismo , Queratinócitos/patologia , Camundongos , NF-kappa B/genética , Fenótipo , Transdução de Sinais
13.
Proc Natl Acad Sci U S A ; 117(30): 17796-17807, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32651268

RESUMO

Fluctuation in signal transduction pathways is frequently observed during mammalian development. However, its role in regulating stem cells has not been explored. Here we tracked spatiotemporal ERK MAPK dynamics in human epidermal stem cells. While stem cells and differentiated cells were distinguished by high and low stable basal ERK activity, respectively, we also found cells with pulsatile ERK activity. Transitions from Basalhi-Pulselo (stem) to Basalhi-Pulsehi, Basalmid-Pulsehi, and Basallo-Pulselo (differentiated) cells occurred in expanding keratinocyte colonies and in response to differentiation stimuli. Pharmacological inhibition of ERK induced differentiation only when cells were in the Basalmid-Pulsehi state. Basal ERK activity and pulses were differentially regulated by DUSP10 and DUSP6, leading us to speculate that DUSP6-mediated ERK pulse down-regulation promotes initiation of differentiation, whereas DUSP10-mediated down-regulation of mean ERK activity promotes and stabilizes postcommitment differentiation. Levels of MAPK1/MAPK3 transcripts correlated with DUSP6 and DUSP10 transcripts in individual cells, suggesting that ERK activity is negatively regulated by transcriptional and posttranslational mechanisms. When cells were cultured on a topography that mimics the epidermal-dermal interface, spatial segregation of mean ERK activity and pulses was observed. In vivo imaging of mouse epidermis revealed a patterned distribution of basal cells with pulsatile ERK activity, and down-regulation was linked to the onset of differentiation. Our findings demonstrate that ERK MAPK signal fluctuations link kinase activity to stem cell dynamics.


Assuntos
Diferenciação Celular , Células Epidérmicas/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células-Tronco/metabolismo , Animais , Técnicas de Cultura de Células , Proliferação de Células , Ativação Enzimática , Células Epidérmicas/citologia , Queratinócitos/metabolismo , Mamíferos , Camundongos , Fosfoproteínas Fosfatases/metabolismo , Transdução de Sinais , Células-Tronco/citologia
14.
Sci Total Environ ; 744: 140830, 2020 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-32721671

RESUMO

Because of the deficiency of water caused by the regional disparities of rainfall due to global warming, attention has been given to the use of well water as drinking water in developing countries. Our fieldwork study in Afghanistan showed that there was a maximum value of 3371 µg/L and an average value of 233 µg/L of lithium in well drinking water. Since the level of lithium in well water is higher than the levels in other countries, we investigated the health risk of lithium. After confirming no influence of ≤1000 µM lithium on cell viability, we found that lithium at concentrations of 100 and 500 µM promoted anchorage-independent growth of human immortalized keratinocytes (HaCaT) and lung epithelial cells (BEAS-2B) but not that of human keratinocytic carcinoma cells (HSC-5) or lung epithelial carcinoma cells (A549). The same concentrations of lithium also promoted phosphorylation of c-SRC and MEK/ERK but not that of AKT in the keratinocytes. Inhibitors of c-SRC (PP2) and MEK (PD98059) suppressed the lithium-induced increase in anchorage-independent growth of the keratinocytes. Our results suggested that lithium promoted transformation of nontumorigenic cells rather than progression of tumorigenic cells with preferential activation of the c-SRC/MEK/ERK pathway. Since previous pharmacokinetics studies indicated that it is possible for the serum level of lithium to reach 100 µM by drinking 2.5 L of water containing 3371 µg/L of lithium per day, the high level of lithium contamination in well drinking water in Kabul might be a potential oncogenic risk in humans.


Assuntos
Transformação Celular Neoplásica , Lítio , Afeganistão , Linhagem Celular , Humanos , Queratinócitos
15.
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
16.
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
17.
PLoS One ; 15(7): e0232565, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32722676

RESUMO

In vitro scratch wound healing assay, a simple and low-cost technique that works along with other image analysis tools, is one of the most widely used 2D methods to determine the cellular migration and proliferation in processes such as regeneration and disease. There are open-source programs such as imageJ to analyze images of in vitro scratch wound healing assays, but these tools require manual tuning of various parameters, which is time-consuming and limits image throughput. For that reason, we developed an optimized plugin for imageJ to automatically recognize the wound healing size, correct the average wound width by considering its inclination, and quantify other important parameters such as: area, wound area fraction, average wound width, and width deviation of the wound images obtained from a scratch/ wound healing assay. Our plugin is easy to install and can be used with different operating systems. It can be adapted to analyze both individual images and stacks. Additionally, it allows the analysis of images obtained from bright field, phase contrast, and fluorescence microscopes. In conclusion, this new imageJ plugin is a robust tool to automatically standardize and facilitate quantification of different in vitro wound parameters with high accuracy compared with other tools and manual identification.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Software , Cicatrização , Linhagem Celular , Movimento Celular , Meios de Cultivo Condicionados/farmacologia , Humanos , Queratinócitos/efeitos dos fármacos , Células-Tronco Mesenquimais/química , Reprodutibilidade dos Testes , Cicatrização/efeitos dos fármacos
18.
Chin J Dent Res ; 23(2): 119-130, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32548603

RESUMO

OBJECTIVE: To investigate and characterise the differences between the open chromatin regions of oral and epidermal keratinocytes. METHODS: Human immortalised oral epithelial cell lines (HIOECs) were used as the standard model for oral keratinocytes, and primary normal human epidermal keratinocytes (NHEKs) were chosen as the model for epidermal keratinocytes. Assay for transposase accessible chromatin using sequencing (ATAC-seq) and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) were used to evaluate the dynamic changes in open chromatin regions and active enhancers during oral keratinocyte differentiation. In silico prediction and dual-luciferase assays were used to evaluate the enriched motifs and maintain enhancer activity in specific enriched HIOECs. Integration and comparison of HIOEC ATAC-seq with NHEK ATAC-seq were used to identify oral keratinocyte-enriched open chromatin regions along with key motifs governing differential enhancer activity. The genomic regulatory elements and GWAS overlap algorithm was used to compare the annotation rate of HIOEC-overlapped craniofacial enhancers with other craniofacial enhancers for orofacial cleft-associated variants. RESULTS: During the differentiation of HIOECs, 14933 open chromatin regions became more accessible. Grainyhead-like (GRHL) and Krüppel-like factor (KLF) motifs were overrepresented in maintaining HIOEC-specific activity. Compared with NHEKs, 16161 open chromatin regions were uniquely accessible in HIOECs. Within these regions, the C/EBP motif governed HIOEC-specific enhancer regulating SOX2 and PITX2, which enhanced oral keratinocyte wound healing. When intersected with human craniofacial super-enhancers, open chromatin regions in HIOECS can better annotate the common variants associated with orofacial cleft. CONCLUSION: The intrinsic differences between the open chromatin regions of human oral and epidermal keratinocytes are directly maintained by a set of transcription factors.


Assuntos
Cromatina , Fenda Labial , Diferenciação Celular , Humanos , Queratinócitos , Fatores de Transcrição/genética
19.
Nat Commun ; 11(1): 2711, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483135

RESUMO

p16INK4a (CDKN2A) is a central tumor suppressor, which induces cell-cycle arrest and senescence. Cells expressing p16INK4a accumulate in aging tissues and appear in premalignant lesions, yet their physiologic effects are poorly understood. We found that prolonged expression of transgenic p16INK4a in the mouse epidermis induces hyperplasia and dysplasia, involving high proliferation rates of keratinocytes not expressing the transgene. Continuous p16INK4a expression increases the number of epidermal papillomas formed after carcinogen treatment. Wnt-pathway ligands and targets are activated upon prolonged p16INK4a expression, and Wnt inhibition suppresses p16INK4a-induced hyperplasia. Senolytic treatment reduces p16INK4a-expressing cell numbers, and inhibits Wnt activation and hyperplasia. In human actinic keratosis, a precursor of squamous cell carcinoma, p16INK4a-expressing cells are found adjacent to dividing cells, consistent with paracrine interaction. These findings reveal that chronic p16INK4a expression is sufficient to induce hyperplasia through Wnt-mediated paracrine stimulation, and suggest that this tumor suppressor can promote early premalignant epidermal lesion formation.


Assuntos
Transformação Celular Neoplásica/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Epiderme/metabolismo , Via de Sinalização Wnt/genética , Animais , Proliferação de Células/genética , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Humanos , Hiperplasia/genética , Hiperplasia/metabolismo , Queratinócitos/metabolismo , Ceratose/genética , Ceratose/metabolismo , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Papiloma/genética , Papiloma/metabolismo , Papiloma/patologia
20.
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
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