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1.
Int J Mol Sci ; 22(13)2021 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-34199056

RESUMO

Palmoplantar keratodermas (PPKs) are characterized by thickness of stratum corneum and epidermal hyperkeratosis localized in palms and soles. PPKs can be epidermolytic (EPPK) or non epidermolytic (NEPPK). Specific mutations of keratin 16 (K16) and keratin 1 (K1) have been associated to EPPK, and NEPPK. Cases of mosaicism in PPKs due to somatic keratin mutations have also been described in scientific literature. We evaluated a patient presenting hyperkeratosis localized monolaterally in the right palmar area, characterized by linear yellowish hyperkeratotic lesions following the Blaschko lines. No other relatives of the patient showed any dermatological disease. Light and confocal histological analysis confirmed the presence of epidermolityic hyperkeratosis. Genetic analysis performed demonstrates the heterozygous deletion NM_006121.4:r.274_472del for a total of 198 nucleotides, in KRT1 cDNA obtained by a palmar lesional skin biopsy, corresponding to the protein mutation NP_006112.3:p.Gly71_Gly137del. DNA extracted from peripheral blood lymphocytes did not display the presence of the mutation. These results suggest a somatic mutation causing an alteration in K1 N-terminal variable domain (V1). The deleted sequence involves the ISIS subdomain, containing a lysine residue already described as fundamental for epidermal transglutaminases in the crosslinking of IF cytoskeleton. Moreover, a computational analysis of the wild-type and V1-mutated K1/K10 keratin dimers, suggests an unusual interaction between these keratin filaments. The mutation taster in silico analysis also returned a high probability for a deleterious mutation. These data demonstrate once again the importance of the head domain (V1) of K1 in the formation of a functional keratinocyte cytoskeleton. Moreover, this is a further demonstration of the presence of somatic mutations arising in later stages of the embryogenesis, generating a mosaic phenotype.


Assuntos
Queratina-10/química , Queratina-1/química , Queratina-1/genética , Nevo/etiologia , Domínios e Motivos de Interação entre Proteínas , Deleção de Sequência , Neoplasias Cutâneas/etiologia , Sequência de Aminoácidos , Sequência de Bases , Biópsia , Análise Mutacional de DNA , Imunofluorescência , Humanos , Imuno-Histoquímica , Queratina-1/metabolismo , Queratina-10/metabolismo , Modelos Moleculares , Nevo/metabolismo , Nevo/patologia , Conformação Proteica , Multimerização Proteica , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Relação Estrutura-Atividade
2.
Front Immunol ; 12: 622216, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33936038

RESUMO

Background and aims: Patients with systemic lupus erythematosus (SLE) have a significantly higher incidence of atherosclerosis than the general population. Studies on atherosclerosis prediction models specific for SLE patients are very limited. This study aimed to build a risk prediction model for atherosclerosis in SLE. Methods: RNA sequencing was performed on 67 SLE patients. Subsequently, differential expression analysis was carried out on 19 pairs of age-matched SLE patients with (AT group) or without (Non-AT group) atherosclerosis using peripheral venous blood. We used logistic least absolute shrinkage and selection operator regression to select variables among differentially expressed (DE) genes and clinical features and utilized backward stepwise logistic regression to build an atherosclerosis risk prediction model with all 67 patients. The performance of the prediction model was evaluated by area under the curve (AUC), calibration curve, and decision curve analyses. Results: The 67 patients had a median age of 42.7 (Q1-Q3: 36.6-51.2) years, and 20 (29.9%) had atherosclerosis. A total of 106 DE genes were identified between the age-matched AT and Non-AT groups. Pathway analyses revealed that the AT group had upregulated atherosclerosis signaling, oxidative phosphorylation, and interleukin (IL)-17-related pathways but downregulated T cell and B cell receptor signaling. Keratin 10, age, and hyperlipidemia were selected as variables for the risk prediction model. The AUC and Hosmer-Lemeshow test p-value of the model were 0.922 and 0.666, respectively, suggesting a relatively high discrimination and calibration performance. The prediction model had a higher net benefit in the decision curve analysis than that when predicting with age or hyperlipidemia only. Conclusions: We built an atherosclerotic risk prediction model with one gene and two clinical factors. This model may greatly assist clinicians to identify SLE patients with atherosclerosis, especially asymptomatic atherosclerosis.


Assuntos
Aterosclerose/diagnóstico , Lúpus Eritematoso Sistêmico/diagnóstico , Modelos Estatísticos , Adulto , Fatores Etários , Aterosclerose/epidemiologia , Feminino , Humanos , Interleucina-17/genética , Queratina-10/metabolismo , Modelos Logísticos , Lúpus Eritematoso Sistêmico/epidemiologia , Masculino , Pessoa de Meia-Idade , Fosforilação Oxidativa , Prognóstico , Risco , Análise de Sequência de RNA , Transdução de Sinais , Transcriptoma
3.
Eur J Histochem ; 65(1)2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33666385

RESUMO

This pilot study was aimed at comparing TLR7/TLR9 expression, cytoskeletal arrangement, and cell proliferation by indirect immunofluorescence in parallel lesional and non lesional skin samples of guttate psoriasis (PG) and psoriasis vulgaris (PV) in five male patients for each group (n=10). TLR7 expression was detected throughout all the epidermal compartment in PV samples, while in PG skin was restricted to the granular layer. TLR9 was present in the granular layer of non lesional skin and in the suprabasal layers of PV/PG lesional skin. Cell proliferation was localized in all the epidermal layers in lesional PG and PV, consistently with the immunopositivity for the "psoriatic keratin" K16. In the suprabasal layers of lesional PG and PV skin, a similar K17 expression was detected and K10 exhibited a patchy distribution. The present results suggest that TLR7 expression can be considered an intrinsic and differential histomorphological feature of PV.


Assuntos
Proliferação de Células/fisiologia , Citoesqueleto/metabolismo , Psoríase/metabolismo , Receptor 7 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Biomarcadores/metabolismo , Imunofluorescência , Humanos , Queratina-10/metabolismo , Queratina-16/metabolismo , Queratina-17/metabolismo , Queratinócitos/metabolismo , Masculino , Projetos Piloto , Psoríase/classificação , Psoríase/patologia , Pele/patologia
4.
J Invest Dermatol ; 141(4): 770-778, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33038352

RESUMO

Atopic Dermatitis is an inflammatory skin disease associated with broad defects in skin barrier function caused by increased levels of type-2 cytokines (IL-4 and IL-13) that repress keratinocyte (KC) differentiation. Although crucial in mediating allergic disease, the mechanisms for gene repression induced by type-2 cytokines remain unclear. In this study, we determined that gene repression requires the master regulator of the epidermal differentiation program, p63. We found that type-2 cytokine-mediated inhibition of the expression of genes involved in early KC differentiation, including keratin 1, keratin 10, and DSC-1, is reversed by p63 blockade. Type-2 cytokines, through p63, also regulate additional genes involved in KC differentiation, including CHAC-1, STC2, and CALML5. The regulation of the expression of these genes is ablated by p63 small interfering RNA as well. In addition, we found that IL-4 and IL-13 and Staphylococcus aureus lipoteichoic acid work in combination through p63 to further suppress the early KC differentiation program. Finally, we found that IL-4 and IL-13 also inhibit the activity of Notch, a transcription factor required to induce early KC differentiation. In conclusion, type-2 cytokine-mediated gene repression and blockade of KC differentiation are multifactorial, involving pathways that converge on transcription factors critical for epidermal development, p63 and Notch.


Assuntos
Diferenciação Celular/genética , Dermatite Atópica/imunologia , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Diferenciação Celular/imunologia , Células Cultivadas , Dermatite Atópica/genética , Dermatite Atópica/patologia , Desmocolinas/genética , Repressão Epigenética/efeitos dos fármacos , Repressão Epigenética/imunologia , Técnicas de Silenciamento de Genes , Humanos , Queratina-1/genética , Queratina-10/genética , Queratinócitos/imunologia , Queratinócitos/patologia , Lipopolissacarídeos/imunologia , Cultura Primária de Células , Receptores Notch/metabolismo , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Pele/imunologia , Pele/microbiologia , Pele/patologia , Staphylococcus aureus/imunologia , Ácidos Teicoicos/imunologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genética
5.
Macromol Biosci ; 21(3): e2000361, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33369081

RESUMO

Despite the progress in chronic wound treatment, antibacterial cutaneous scaffold with high efficiency in wound healing is still the hot spot in the field. In present study, a functionalized silk fibroin (SF) cutaneous scaffold incorporated with natural medicine usnic acid (UA) is investigated, in which UA is used as an antibacterial and wound-healing reagent. Via electrospinning, UA-SF mixture is fabricated into UA-SF composite scaffold (USCS), which is composed of uniform nanofibers with average diameters of around 360 ± 10 nm. The interwoven nanofibers form mesh structure providing sufficient moisture permeability for scaffold. With methanol treatment, USCS presents improved mechanical properties and stability to protease XIV. In the presence of USCS, the growth rate of both Gram-positive and Gram-negative bacteria, including Staphylococcus aureus, Streptococci pyogenes, Escherichia coli, and Pseudomonas aeruginosa, is significantly inhibited in plate culture and suspension assays. In a cutaneous excisional mouse wound model, USCS presents a significant increase of wound closure rate, compared with pure SF scaffold and commercial dressing, Tegaderm Hydrocolloid 3M . The histological assessments further prove that USCS can enhance re-epithelialization, vascularization, and collagen deposition in wound site to promote the wound-healing process, which indicates the potential application of USCS in chronic wound treatment.


Assuntos
Benzofuranos/farmacologia , Fibroínas/farmacologia , Pele/patologia , Tecidos Suporte/química , Cicatrização/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Materiais Biocompatíveis/farmacologia , Fibroínas/ultraestrutura , Queratina-10/metabolismo , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Células NIH 3T3 , Espectroscopia de Infravermelho com Transformada de Fourier , Estresse Mecânico , Resistência à Tração , Fator A de Crescimento do Endotélio Vascular/metabolismo
6.
Mol Med Rep ; 22(4): 2715-2722, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32945375

RESUMO

Psoriasis is one of the most common chronic inflammatory skin diseases, it is characterized by hyperproliferation of keratinocytes and infiltration of inflammatory cells. Several in vitro studies have reported that interleukin (IL)­22 is involved in excessive proliferation and abnormal differentiation of human keratinocytes. However, the association between IL­22 and CCAAT enhancer binding protein α (C/EBPα) in the pathogenesis of psoriasis remains unclear. Therefore, the present study aimed to investigate the association between IL­22 and C/EBPα, and the effects of IL­22 on the proliferation and differentiation of keratinocytes. Keratinocytes were treated with different concentrations of IL­22 (30, 60 and 90 ng/ml) and subsequently cells were collected at different time intervals. The expression levels of the key molecules of the mitogen­activated protein kinase (MAPK) signaling pathway were detected using western blot analysis. In addition, the effect of IL­22 on the proliferation rate of keratinocytes and the mRNA expression levels of C/EBPα were determined using a Cell Counting Kit­8 assay and reverse transcription­quantitative PCR, respectively. Furthermore, keratinocytes were transfected with C/EBPα small interfering (si)RNA or control using Lipofectamine® 2000. The results revealed that IL­22 significantly induced the proliferation of keratinocytes and the expression of phosphorylated (p)­JNK, p­ERK and p­p38 (P<0.05). Additionally, IL­22 significantly inhibited the differentiation of keratinocytes, and the mRNA and protein expression of C/EBPα (P<0.05). Furthermore, downregulation of C/EBPα increased the proliferation rate of keratinocytes and reduced the expression levels of cytokeratin 10 and involucrin. Therefore, these results suggested that the effect of IL­22 on the proliferation and differentiation of keratinocytes may be mediated via the regulation of the MAPK signaling pathway and the expression of C/EBPα.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Interleucinas/farmacologia , Queratinócitos/citologia , Adolescente , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Criança , Relação Dose-Resposta a Droga , Prepúcio do Pênis/citologia , Prepúcio do Pênis/efeitos dos fármacos , Prepúcio do Pênis/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Queratina-10/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Cultura Primária de Células , Precursores de Proteínas/metabolismo
7.
Theranostics ; 10(19): 8807-8817, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32754279

RESUMO

Rationale: Inflammatory heart disorders are among the causes of human death. The causative factors of heart inflammation are to be further elucidated. House dust mite (HDM)-derived protein antigens are involved in the pathogenesis of many human diseases. This study aims to investigate the role of HDM-specific autoantibodies in the pathogenesis of heart inflammation. Methods: Human heart tissue samples were obtained from surgically removed hearts in heart transplantation. The interaction of the heart tissues with HDM-specific antibodies was assessed by pertinent immune analysis. The role of HDM-specific autoantibodies in the induction of heart inflammation was assessed with a murine model. Results: HDM-specific IgG (mIgG) was detected in the serum of patients with myocarditis (Mcd); the mIgG titers were positively correlated with the neutrophil counts in the heart tissues. The mIgG specifically bound to keratin-10 (KRT10) in heart vascular endothelial cells and the heart tissue protein extracts. The amounts of C3a, C5a and C5b-9 were increased in the mouse heart tissues after exposing to mIgG. In the presence of the complement-containing serum, mIgG bound cardiovascular epithelial monolayers to impair the barrier functions. Administration of mIgG or HDM induced the Mcd-like inflammation in the heart, in which neutrophils were the dominant cellular components in the infiltration of inflammatory cells. Conclusions: Mcd patients with neutrophilic inflammation in the heart had higher serum levels of mIgG. The mIgG bound heart endothelial cells to impair the endothelial barrier functions and induce neutrophilic inflammation in the heart.


Assuntos
Alérgenos/efeitos adversos , Autoanticorpos/sangue , Miocardite/imunologia , Neutrófilos/metabolismo , Pyroglyphidae/imunologia , Adulto , Alérgenos/imunologia , Animais , Autoanticorpos/efeitos adversos , Proteínas do Sistema Complemento/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Imunoglobulina G/sangue , Queratina-10/metabolismo , Masculino , Camundongos , Miocardite/sangue , Miocardite/etiologia , Adulto Jovem
8.
J Histochem Cytochem ; 68(8): 561-570, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32618487

RESUMO

Cells of the human breast gland express an array of keratins, of which some are used for characterizing both normal and neoplastic breast tissue. However, the expression pattern of certain keratins has yet to be detailed. Here, the expression of a differentiation marker of epidermal epithelium, keratin 10 (K10), was investigated in the human breast gland. While in normal breast tissue generally less than 1% of luminal epithelial cells expressed K10, in women >30 years of age glandular structures with K10-positive (K10pos) cells were found at higher frequency than in younger women. K10pos cells belong to a mature luminal compartment as they were negative for cKIT, positive for Ks20.8, and mostly non-cycling. In breast cancer, around 16% of primary breast carcinomas tested were positive for K10 by immunohistochemistry. Interestingly, K10pos tumor cells generally exhibit features of differentiation similar to their normal counterparts. Although this suggests that K10 is a marker of tumor differentiation, data based on gene expression analysis imply that high levels of K10 dictate a worse outcome for breast cancer patients. These findings can form the basis of future studies that should unravel which role K10 may play as a marker of breast cancer.


Assuntos
Neoplasias da Mama/patologia , Mama/citologia , Mama/patologia , Diferenciação Celular , Epiderme/patologia , Regulação Neoplásica da Expressão Gênica , Queratina-10/genética , Biomarcadores/metabolismo , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Humanos , Células MCF-7 , Prognóstico
11.
Invest Ophthalmol Vis Sci ; 61(3): 54, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32232349

RESUMO

Purpose: To investigate the effects and mechanisms of the peroxisome proliferator-activated receptor alpha (PPAR-α) agonist fenofibrate on the formation of ocular surface squamous metaplasia induced by topical benzalkonium chloride (BAC) in a mouse model. Methods: Ocular surface squamous metaplasia was induced in 16 days by topical BAC application in mice. During the period of induction, mice were divided into four groups: no additional treatment (BAC+UT), topical vehicle (BAC+Vehicle), topical fenofibrate (BAC+Feno), or topical fenofibrate plus intraperitoneal injection of MK886 (BAC+Feno+MK886). The parameters of tear film were evaluated on day 16, and eye specimens were collected. Histologic investigation; PAS assays; immunostaining for cytokeratin 10 (K10), Ki67, and F4/80; and PCR assays for TNF-α and IL-6 were performed. Cell Counting Kit 8 (CCK-8) assays were performed to evaluate the inhibitory effects of fenofibrate on RAW264.7 cells. Results: Fenofibrate suppressed the formation of BAC-induced instable tear film. In the BAC+Feno group, the expression of K10 and Ki67 was lower than in the other three groups. The number of goblet cells was reduced in eyes of the BAC+UT and BAC+Vehicle groups but was maintained in eyes of the BAC+Feno group. The number of F4/80-positive cells and the levels of TNF-α and IL-6 mRNA were significantly reduced in the cornea of the BAC+Feno group. These effects of fenofibrate could be attenuated by MK886. The cell viability of RAW264.7 cells could be significantly inhibited by fenofibrate in a dose-dependent pattern. Conclusions: Topical application of fenofibrate suppressed the formation of ocular surface squamous metaplasia, which might be mediated through the PPAR-α signaling pathway.


Assuntos
Epitélio Corneano/efeitos dos fármacos , Fenofibrato/farmacologia , Hipolipemiantes/farmacologia , PPAR alfa/agonistas , Animais , Anti-Infecciosos Locais/toxicidade , Compostos de Benzalcônio/toxicidade , Proteínas de Ligação ao Cálcio/metabolismo , Contagem de Células , Linhagem Celular , Sobrevivência Celular , Modelos Animais de Doenças , Epitélio Corneano/metabolismo , Epitélio Corneano/patologia , Humanos , Imuno-Histoquímica , Interleucina-6/genética , Queratina-10/metabolismo , Antígeno Ki-67/metabolismo , Masculino , Metaplasia/induzido quimicamente , Metaplasia/tratamento farmacológico , Metaplasia/metabolismo , Metaplasia/patologia , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase em Tempo Real , Receptores Acoplados a Proteínas G/metabolismo , Fator de Necrose Tumoral alfa/genética
12.
J Dermatol Sci ; 98(1): 35-40, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32113649

RESUMO

BACKGROUND: Ichthyosis with confetti (IWC) is an extremely rare autosomal-dominant genodermatosis characterized by erythroderma with numerous confetti-like pale spots. IWC is caused by mutations in KRT10 (IWC-I) or KRT1 (IWC-II) which affect their tail domains. In IWC-I, the mutations lead to replacement of glycine/serine-rich keratin 10 (K10) tail with arginine- or alanine-rich frameshift motifs, causing K10 mis-localization which might trigger loss of the mutant KRT10 allele via mitotic recombination, leading to genetic reversion. OBJECTIVE: To investigate mutations in five IWC-I patients and their functional consequences. METHODS: We performed Sanger sequencing of KRT1 and KRT10 in peripheral blood samples of five patients, with highly polymorphic KRT10 SNPs genotyped to confirm loss-of-heterozygosity in the epidermis of pale spots. K10 expression pattern was examined in both patient skin biopsies and HaCaT cells overexpressing mutant KRT10-enhanced green fluorescence protein fusion. RESULTS: Four novel and one recurrent KRT10 mutations were identified in patient peripheral blood samples but not in the corresponding pale spot epidermis. Two of the mutations, c.1696_1699dupCACA and c.1676dupG, affected residues close to K10 carboxyl terminus and encoded only 3 and 6 arginine residues, which were far fewer than reported previously. Interestingly, imaging analyses for K10 in HaCaT cells overexpressing either of these two mutations and in the corresponding patients' affected skin, showed a remarkably lower level of K10 mis-localization compared to that of other mutations reported in this study. CONCLUSIONS: Our findings suggest that the number of arginine residues in the mutant tail may correlate with the level of K10 mis-localization in IWC-I keratinocytes. These results expand the genotypic and phenotypic spectrum of IWC-I.


Assuntos
Epiderme/patologia , Ictiose/genética , Queratina-10/genética , Queratinócitos/patologia , Adolescente , Sequência de Aminoácidos/genética , Arginina/genética , Biópsia , Núcleo Celular/patologia , Pré-Escolar , Análise Mutacional de DNA , Feminino , Mutação da Fase de Leitura , Glicina/genética , Células HaCaT , Humanos , Ictiose/sangue , Ictiose/patologia , Queratina-10/sangue , Queratina-10/metabolismo , Queratinócitos/citologia , Perda de Heterozigosidade , Masculino , Serina/genética
13.
J Cell Mol Med ; 24(8): 4819-4829, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32168425

RESUMO

Psoriasis is a chronic immune-mediated inflammatory dermatosis. Recently, ozone therapy has been applicated to psoriasis treatment; however, the mechanism by which ozone therapy improves psoriasis remains unclear. The excessive proliferation and the differentiation of basal keratinocytes have been considered critical issues during pathological psoriasis process, in which keratin 6 (KRT6) and KRT10 might be involved. In the present study, KRT6, IL-17 and IL-22 protein within psoriasis lesions was decreased, while KRT10 and Tp63 protein in psoriasis lesions was increased by ozone treatment in both patient and IMQ mice psoriatic tissues. In the meantime, ozone treatment down-regulated KRT6 mRNA and protein expression while up-regulated KRT10 mRNA and protein expression within IL-22 treated primary KCs; the cell viability of KCs was suppressed by ozone treatment. Moreover, Tp63 bound to KRT10 promoter region to activate its transcription in basal keratinocytes; the promotive effects of ozone on Tp63 and KRT10 were significantly reversed by Tp63 silence. Both TP63 and KRT10 mRNA expression were significantly increased by ozone treatment in psoriasis lesions; there was a positive correlation between Tp63 and KRT10 expression within tissue samples, suggesting that ozone induces the expression of Tp63 to enhance the expression of KRT10 and the differentiation of keratinocytes, therefore improving the psoriasis. In conclusion, the application of ozonated oil could be an efficient and safe treatment for psoriasis; ozone promotes the differentiation of keratinocytes via increasing Tp63-mediated transcription of KRT10, therefore improving psoriasis.


Assuntos
Queratina-10/genética , Queratina-6/genética , Ozônio/farmacologia , Psoríase/terapia , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Adulto , Animais , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dermatite/genética , Dermatite/patologia , Dermatite/terapia , Modelos Animais de Doenças , Feminino , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/patologia , Masculino , Camundongos , Ozônio/uso terapêutico , Cultura Primária de Células , Psoríase/genética , Psoríase/patologia , Pele/efeitos dos fármacos , Pele/patologia
14.
Sci Rep ; 10(1): 4829, 2020 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-32179842

RESUMO

Abnormal keratinocyte differentiation is fundamental to pathologies such as skin cancer and mucosal inflammatory diseases. The ability to grow keratinocytes in vitro allows the study of differentiation however any translational value is limited if keratinocytes get altered by the culture method. Although serum lipids (SLPs) and phenol red (PR) are ubiquitous components of culture media their effect on differentiation is largely unknown. We show for the first time that PR and SLP themselves suppress expression of differentiation-specific keratins K1, K10 and K2 in normal human epidermal keratinocytes (NHEK) and two important cell lines, HaCaT and N/TERT-1. Removal of SLP increased expression of K1, K10 and K2 in 2D and 3D cultures, which was further enhanced in the absence of PR. The effect was reversed for K1 and K10 by adding all-trans retinoic acid (ATRA) but increased for K2 in the absence of PR. Furthermore, retinoid regulation of differentiation-specific keratins involves post-transcriptional mechanisms as we show KRT2 mRNA is stabilised whilst KRT1 and KRT10 mRNAs are destabilised in the presence of ATRA. Taken together, our results indicate that the presence of PR and SLP in cell culture media may significantly impact in vitro studies of keratinocyte differentiation.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Queratina-10/genética , Queratina-10/metabolismo , Queratina-1/genética , Queratina-1/metabolismo , Queratina-2/genética , Queratina-2/metabolismo , Queratinócitos/metabolismo , Queratinócitos/fisiologia , Lipídeos/fisiologia , Fenolsulfonaftaleína/farmacologia , Tretinoína/farmacologia , Células Cultivadas , Células HaCaT , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
15.
J Cutan Pathol ; 47(6): 524-529, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32045015

RESUMO

BACKGROUND: Epidermolytic acanthoma (EA) is a rare acquired lesion demonstrating a characteristic histopathological pattern of epidermal degeneration referred to as epidermolytic hyperkeratosis (EHK). On histopathological analysis, EA appears nearly identical to inherited EHK-associated dermatoses such as epidermolytic ichthyosis and ichthyosis bullosa of Siemens. While it has been speculated that EA is caused by mutations in KRT10, KRT1, or KRT2 found in these inherited dermatoses, none have yet been identified. Herein, we aim to identify the contributions of keratin mutations to EA. METHODS: Using genomic DNA extracted from paraffin-embedded samples from departmental archives, we evaluated a discovery cohort using whole-exome sequencing (WES) and assessed remaining samples using Sanger sequencing screening and restriction fragment length polymorphism (RFLP) analysis. RESULTS: DNA from 16/20 cases in our sample was of sufficient quality for polymerase chain reaction amplification. WES of genomic DNA from lesional tissue revealed KRT10 c.466C > T, p.Arg156Cys mutations in 2/3 samples submitted for examination. RFLP analysis of these samples as well as eight additional samples confirmed the mutations identified via WES and identified four additional cases with Arg156 mutations. In sum, 6/11 screened cases showed hotspot mutation in KRT10. CONCLUSIONS: Hotspot mutations in the Arg156 position of KRT10, known to cause epidermolytic ichthyosis, also underlie EA.


Assuntos
Acantoma/congênito , Hiperceratose Epidermolítica/genética , Queratina-10/genética , Neoplasias Cutâneas/patologia , Acantoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Genômica/métodos , Humanos , Hiperceratose Epidermolítica/patologia , Ictiose Bolhosa de Siemens/patologia , Queratinas/genética , Masculino , Pessoa de Meia-Idade , Mutação , Sequenciamento Completo do Exoma/métodos
16.
J Invest Dermatol ; 140(7): 1346-1354.e5, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-31945349

RESUMO

Epidermal keratinocytes are primarily involved in the expression of semaphorin (Sema) 3A, which is involved in the regulation of cutaneous innervation. However, the mechanisms underlying the intracellular signaling of Sema3A expression in keratinocytes remain unknown. We herein investigated the signaling mechanisms for the induction of Sema3A expression in normal human epidermal keratinocytes (NHEKs). Sema3A expression is transiently increased in calcium-stimulated NHEKs, whereas it is markedly decreased in terminally differentiated NHEKs. Sema3A mRNA is mainly localized in the stratum basale and stratum suprabasale of the epidermis. We cloned the 5'-flanking region of the Sema3A gene and identified a critical region for Sema3A promoter activity within -134 base pairs of the start codon. We found transcription factor binding sites, including that for activator protein (AP)-1, in this region. Sema3A expression was increased by the co-overexpression of JunB and Fra-2 in the presence of 0.1 or 1.4 mM calcium. The calcium-mediated transient upregulation of Sema3A expression was significantly suppressed by mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) 1/2 or AP-1 inhibitors. These results demonstrate that the calcium-mediated transient upregulation of Sema3A in NHEKs is involved in the MEK/ERK and AP-1 signaling axis. Therefore, Sema3A mRNA may be expressed in the lower epidermis under controlled conditions by calcium via the MAPK-AP-1 axis.


Assuntos
Queratinócitos/metabolismo , Sistema de Sinalização das MAP Quinases , Semaforina-3A/metabolismo , Fator de Transcrição AP-1/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Diferenciação Celular , Linhagem Celular , Células Epidérmicas/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Humanos , Queratina-10/metabolismo , Queratina-14/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais
17.
Biochim Biophys Acta Mol Cell Res ; 1867(2): 118574, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31682865

RESUMO

During keratinocyte stratification and wound healing, keratinocytes undergo a switch between differentiation and motility. However, limited knowledge exists on the mechanisms of the switch. We have previously demonstrated that the expression of CD9 was changed in different wound stages and involved in the regulation of keratinocyte migration. In this study, we showed that CD9 expression was increased in both human and mouse keratinocytes undergoing differentiation. CD9 overexpression in keratinocytes stimulated terminal differentiation and reduced cell motility. CD9 silencing inhibited calcium-induced keratinocyte differentiation and increased cell motility. Furthermore, CD9 overexpression recruited E-cadherin to the plasma membrane and subsequently activated PI3K/Akt signaling, while CD9 knockdown inhibited the recruitment of E-cadherin to the plasma membrane and PI3K/Akt activation. Importantly, silencing E-cadherin expression or inhibiting PI3K/Akt signaling reversed CD9 overexpression-induced differentiation and -reduced motility. These results demonstrate that CD9 acts as an important node that regulates keratinocyte differentiation and motility. The recruitment of E-cadherin to the plasma membrane and activation of the PI3K/Akt signaling pathway mediated by CD9 play an important role in these processes.


Assuntos
Caderinas/metabolismo , Diferenciação Celular , Membrana Celular/metabolismo , Transdução de Sinais , Tetraspanina 29/metabolismo , Animais , Caderinas/antagonistas & inibidores , Caderinas/genética , Cálcio/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Queratina-10/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Pele/patologia , Tetraspanina 29/antagonistas & inibidores , Tetraspanina 29/genética
18.
J Invest Dermatol ; 140(4): 774-784.e11, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31626786

RESUMO

Varicella zoster virus (VZV) is a skin-tropic virus that infects epidermal keratinocytes and causes chickenpox. Although common, VZV infection can be life-threatening, particularly in the immunocompromized. Therefore, understanding VZV-keratinocyte interactions is important to find new treatments beyond vaccination and antiviral drugs. In VZV-infected skin, kallikrein 6 and the ubiquitin ligase MDM2 are upregulated concomitant with keratin 10 (KRT10) downregulation. MDM2 binds to KRT10, targeting it for degradation via the ubiquitin-proteasome pathway. Preventing KRT10 degradation reduced VZV propagation in culture and prevented epidermal disruption in skin explants. KRT10 knockdown induced expression of NR4A1 and enhanced viral propagation in culture. NR4A1 knockdown prevented viral propagation in culture, reduced LC3 levels, and increased LAMP2 expression. We therefore describe a drug-able pathway whereby MDM2 ubiquitinates and degrades KRT10, increasing NR4A1 expression and allowing VZV replication and propagation.


Assuntos
Regulação da Expressão Gênica , Herpes Zoster/genética , Herpes Zoster/metabolismo , Herpesvirus Humano 3/fisiologia , Queratina-10/genética , Queratinócitos/patologia , RNA/genética , Replicação Viral , Herpes Zoster/virologia , Humanos , Queratina-10/biossíntese , Queratinócitos/metabolismo , Queratinócitos/virologia
20.
J Cell Mol Med ; 23(12): 8442-8452, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31638346

RESUMO

Ichthyosis with confetti (IWC) is a genodermatosis associated with dominant-negative variants in keratin 10 (KRT10) or keratin 1 (KRT1). These frameshift variants result in extended aberrant proteins, localized to the nucleus rather than the cytoplasm. This mislocalization is thought to occur as a result of the altered carboxy (C)-terminus, from poly-glycine to either a poly-arginine or -alanine tail. Previous studies on the type of C-terminus and subcellular localization of the respective mutant protein are divergent. In order to fully elucidate the pathomechanism of IWC, a greater understanding is critical. This study aimed to establish the consequences for localization and intermediate filament formation of altered keratin 10 (K10) C-termini. To achieve this, plasmids expressing distinct KRT10 variants were generated. Sequences encoded all possible reading frames of the K10 C-terminus as well as a nonsense variant. A keratinocyte line was transfected with these plasmids. Additionally, gene editing was utilized to introduce frameshift variants in exon 6 and exon 7 at the endogenous KRT10 locus. Cellular localization of aberrant K10 was observed via immunofluorescence using various antibodies. In each setting, immunofluorescence analysis demonstrated aberrant nuclear localization of K10 featuring an arginine-rich C-terminus. However, this was not observed with K10 featuring an alanine-rich C-terminus. Instead, the protein displayed cytoplasmic localization, consistent with wild-type and truncated forms of K10. This study demonstrates that, of the various 3' frameshift variants of KRT10, exclusively arginine-rich C-termini lead to nuclear localization of K10.


Assuntos
Arginina/genética , Núcleo Celular/genética , Eritrodermia Ictiosiforme Congênita/genética , Queratina-10/genética , Mutação , Transporte Ativo do Núcleo Celular/genética , Alanina/genética , Alanina/metabolismo , Arginina/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Éxons/genética , Mutação da Fase de Leitura , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Eritrodermia Ictiosiforme Congênita/metabolismo , Eritrodermia Ictiosiforme Congênita/patologia , Queratina-10/química , Queratina-10/metabolismo , Queratinócitos/metabolismo , Microscopia Confocal
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