RESUMO
Recently, the increasing incidence of malignant melanoma has become a major public health concern owing to its poor prognosis and impact on quality of life. Consuming foods with potent antitumor compounds can help prevent melanoma and maintain skin health. Fucoxanthin (FX), a naturally occurring carotenoid found in brown algae, possesses antitumor properties. However, its bioavailability, safety risks, and in vivo effects and mechanisms against melanoma remain unclear. This research focused on evaluating the safety and prospective antimelanoma impact of simulated gastrointestinal digestion products (FX-ID) on HaCaT and A375 cells.The results indicate that FX-ID exerts negative effects on mitochondria in A375 cells, increases Bax expression, releases Cytochrome C, and activates cleaved caspase-3, ultimately promoting apoptosis. Additionally, FX-ID influences the mitogen-activated protein kinase (MAPK) pathway by enhancing cyclooxygenase-2 (COX-2) and nuclear factor kappa B (NF-κB) levels, consequently facilitating apoptosis and inflammation without significantly impacting HaCaT cells. These findings provide insight into inhibitory mechanism of FX-ID against melanoma, guiding the development of functional foods for prevention.
Assuntos
Apoptose , Queratinócitos , Melanoma , Xantofilas , Humanos , Melanoma/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Apoptose/efeitos dos fármacos , Xantofilas/farmacologia , Xantofilas/química , Linhagem Celular Tumoral , NF-kappa B/metabolismo , NF-kappa B/genética , Digestão , Modelos Biológicos , Ciclo-Oxigenase 2/metabolismo , Ciclo-Oxigenase 2/genética , Antineoplásicos/farmacologia , Antineoplásicos/química , Phaeophyceae/química , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 3/genéticaRESUMO
Disrupted N6-methyladenosine (m6A) modification modulates various inflammatory disorders. However, the role of m6A in regulating cutaneous inflammation remains elusive. Here, we reveal that the m6A and its methyltransferase METTL3 are down-regulated in keratinocytes in inflammatory skin diseases. Inducible deletion of Mettl3 in murine keratinocytes results in spontaneous skin inflammation and increases susceptibility to cutaneous inflammation with activation of neutrophil recruitment. Therapeutically, restoration of m6A alleviates the disease phenotypes in mice and suppresses inflammation in human biopsy specimens. We support a model in which m6A modification stabilizes the mRNA of the lipid-metabolizing enzyme ELOVL6 via the m6A reader IGF2BP3, leading to a rewiring of fatty acid metabolism with a reduction in palmitic acid accumulation and, consequently, suppressing neutrophil chemotaxis in cutaneous inflammation. Our findings highlight a previously unrecognized epithelial-intrinsic m6A modification-lipid metabolism pathway that is essential for maintaining epidermal and immune homeostasis and lay the basis for potential therapeutic targeting of m6A modulators to attenuate inflammatory skin diseases.
Assuntos
Adenosina , Homeostase , Queratinócitos , Metabolismo dos Lipídeos , Metiltransferases , Neutrófilos , Pele , Adenosina/análogos & derivados , Adenosina/metabolismo , Animais , Neutrófilos/metabolismo , Neutrófilos/imunologia , Camundongos , Queratinócitos/metabolismo , Humanos , Metiltransferases/metabolismo , Metiltransferases/genética , Pele/metabolismo , Pele/patologia , Pele/imunologia , Inflamação/metabolismo , Inflamação/patologia , Quimiotaxia , Elongases de Ácidos Graxos/metabolismo , Elongases de Ácidos Graxos/genéticaRESUMO
BACKGROUND AND RATIONALE: Ultraviolet-B (UVB) light induces dermal inflammation, although it is mostly absorbed in the epidermis. Recent reports suggest extracellular vesicles (EVs) act as a mediator of photodamage signaling. Melatonin is reported to be a protective factor against UV-induced damage. We hypothesized that EVs derived from UVB-irradiated keratinocytes might trigger proinflammatory responses in dermal cells and tested whether melatonin can ameliorate UVB-induced inflammation. METHODS: We used UVB-irradiated HaCaT cells, primary keratinocytes and STING knock-out mice to model production of EVs under photodamaging conditions and performed immunoblotting and ELISA to measure their effect on dermal macrophages. RESULTS: UVB-irradiated keratinocytes produce an increased number of EVs that contain higher concentrations of DNA and protein compared with controls. KC-derived EVs (KEVs) induced a STING- and inflammasome-mediated proinflammatory response in macrophages in vitro, and a pronounced inflammatory infiltrate in mouse dermis in vivo. Melatonin ameliorated KEVs inflammatory effect both in vitro and in vivo. CONCLUSIONS: This data suggests EVs are mediators in a crosstalk that takes place between keratinocytes and their neighboring cells as a result of photodamage. Further studies exploring EVs induced by damaging doses of UVB, and their impact on other cells will provide insight into photodamage and may help develop targeted therapeutic approaches.
Assuntos
Derme , Epiderme , Vesículas Extracelulares , Queratinócitos , Raios Ultravioleta , Vesículas Extracelulares/metabolismo , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Animais , Humanos , Epiderme/efeitos da radiação , Epiderme/metabolismo , Epiderme/patologia , Camundongos , Derme/patologia , Derme/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Melatonina/farmacologia , Melatonina/metabolismo , Macrófagos/metabolismo , Macrófagos/efeitos da radiação , Camundongos Knockout , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Células HaCaTRESUMO
OBJECTIVE: To explore the role of nuclear transcription factor E2-related factor 2(NRF2)-mediated reductive stress in arsenite induced malignant transformation in human keratinocytes. METHODS: HaCaT cells and fluorescent labeled mitochondrial glutathione HaCaT cells(Mito-Grx1-roGFP2 HaCaT) were cultured to 35 passages in medium containing 0.0 and 1.0 µmol/L NaAsO_2 to establish a model of malignant transformation of cells. Cellular and mitochondrial reduced glutathione/oxidized glutathione(GSH/GSSG) and reduced coenzyme II/oxidized coenzyme II(NADPH/NADP~+) ratios were measured in HaCaT cells. Cell doubling time, cell migration ability, soft agar clone formation ability and GSH/GSSG at different times in the 0 passage, the early stage(1st, 7th and 14th passages) and later stage(21st, 28th and 35th passages) were measured in Mito-Grx1-roGFP2 HaCaT cells. NaAsO_2 induced malignant transformation cells were transfected with NRF2 siRNA, and detected the expression level of NRF2 and the redox-related indexes and malignant transformation indexes. RESULTS: Compared with the control group, the GSH/GSSG ratio in 1.0 µmol/L NaAsO_2 treated HaCaT cells significantly decreased in the 1st and 7th generations, but significantly increased after the 21st generation, and the NADPH/NADP~+ ratio significantly increased in the 1st, 14th, 21st, 28th and 35th generations; The levels of GSH/GSSG in mitochondria significantly increased from 1st to 35th generation, and the levels of NADPH/NADP~+ in mitochondria significantly increased at 1st, 7th, 21st, 28th and 35th generation. After continuous treatment of Mito-Grx1-roGFP2 HaCaT cells with 0.0 or 1.0 µmol/L NaAsO_2 to 35 passages, the doubling time of cells treated with 1.0 µmol/L NaAsO_2 was significantly shortened, the cell migration rate was increased greatly, and more clones with larger volumes than the control cells formed. The GSH/GSSG ratio in mitochondria of Mito-Grx1-roGFP2 HaCaT cells showed a significant decrease in the 1st generation and increased from the 7th generation onwards(all P<0.05). After transfection of NaAsO_2 treated cells with NRF2 siRNA, the levels of hydrogen peroxide and superoxide increased compared with the siRNA controls. The levels of cell and mitochondrial NADPH/NADP~+ and GSH/GSSG decreased and the level of mitochondrial GSH/GSSG in Mito-Grx1-roGFP2 HaCaT cells decreased. Cell doubling time increased, cell migration rate and soft agar clone formation ability decreased(all P<0.05). The malignant phenotype was reversed. CONCLUSION: In the early stage(1st, 7th and 14th passages) of NaAsO_2 treated HaCaT cells, oxidative stress occurred with continuous high NRF2 expression. Later(21st, 28th and 35th passages), NRF2 induced reductive stress, leading to malignant transformation.
Assuntos
Transformação Celular Neoplásica , Queratinócitos , Fator 2 Relacionado a NF-E2 , Estresse Oxidativo , Humanos , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Queratinócitos/metabolismo , Queratinócitos/efeitos dos fármacos , Transformação Celular Neoplásica/induzido quimicamente , Estresse Oxidativo/efeitos dos fármacos , Oxirredução , Linhagem Celular , Arsênio/toxicidade , Arsênio/efeitos adversos , Glutationa/metabolismoRESUMO
Oral mucosal wounds exhibit accelerated healing with reduced scarring compared to cutaneous wounds, representing an optimal wound healing paradigm. However, the specific cellular subtypes orchestrating the efficient healing of mucosal tissues remain elusive. Through a comprehensive analysis integrating bulk-mRNA and single-cell sequencing data during the wound healing process in oral mucosa and skin, we have delineated a distinct set of genes markedly upregulated during tissue repair. This collection of wound healing-associated genesets was highly enriched in a specific keratinocyte subpopulation identified as STAT3-activated SPRR1B+ keratinocytes. Notably, despite the inherent rapidity of oral mucosal healing, the induction of SPRR1B+ keratinocytes is evident in both skin and mucosal wound healing processes in murine model. Intriguingly, these wound healing-promoting SPRR1B+ keratinocytes, which are induced via STAT3 activation, inherently abundant in unwounded normal mucosa but absent in normal skin. SPRR1B knockdown significantly inhibits mucosal keratinocyte migration, a critical attribute for effective wound healing. In summary, through analysis of human oral and skin wound healing processes at single-cell resolution, coupled with validation in murine model, suggests STAT3-activated SPRR1B+ keratinocytes are associated with the rapid mucosal repair process. This discovery underscores the potential application of SPRR1B+ keratinocytes in the therapeutic management of chronic or non-healing wounds.
Assuntos
Queratinócitos , Mucosa Bucal , Fator de Transcrição STAT3 , Cicatrização , Animais , Humanos , Masculino , Camundongos , Movimento Celular , Queratinócitos/metabolismo , Camundongos Endogâmicos C57BL , Mucosa Bucal/metabolismo , Pele/metabolismo , Pele/lesões , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT3/genética , Cicatrização/genéticaRESUMO
Protease-activated receptor-2 (PAR2) is a class-A G protein-coupled receptor (GPCR) activated by serine proteases and is expressed by multiple tissues, including the skin. PAR2 is involved in the skin inflammatory response, promoting Th2 inflammation, delaying skin barrier repair, and affecting the differentiation of keratinocytes. It also participates in the transmission of itch and pain sensations in the skin. Increasing evidence indicates that PAR2 plays an important role in the pathogenesis of inflammatory skin diseases such as acne vulgaris, rosacea, psoriasis, and atopic dermatitis. Additional focus will be placed on potential targeted therapies based on PAR2. The Goal of this review is to outline the emerging effects of PAR2 activation in inflammatory skin disease and highlight the promise of PAR2 modulators.
Assuntos
Receptor PAR-2 , Humanos , Receptor PAR-2/metabolismo , Animais , Pele/metabolismo , Pele/imunologia , Pele/patologia , Dermatopatias/imunologia , Dermatopatias/metabolismo , Dermatite Atópica/imunologia , Dermatite Atópica/metabolismo , Transdução de Sinais , Queratinócitos/metabolismo , Queratinócitos/imunologia , Inflamação/imunologia , Inflamação/metabolismoRESUMO
Rosacea is a chronic inflammatory skin disease that typically affects the central facial area. Its main clinical symptoms include paroxysmal flushing, telangiectasia, and non-temporary erythema. Cell-free adipose tissue extracts (ATEs) are liquid components extracted from human adipose tissue that contain large amounts of growth factors. Despite the scar-reducing, anti-aging, and wound-healing effects of ATEs, the efficacy of ATEs in rosacea remains unknown. Therefore, the anti-rosacea effects of ATEs were investigated in human cathelicidin peptide (LL-37) induced rosacea mice and capsaicin (CAP)-stimulated HaCaT keratinocytes. In vitro, ATEs significantly reduced TRPV1 expression, intracellular calcium ions influx and the release of inflammatory factors (such as KLK5, IL-6, IL-8 and TNF-α) after intervening in CAP-stimulated cells. The in vivo results revealed that ATEs alleviated rosacea symptoms, such as erythema score, erythema area, transepidermal water loss, abnormal epidermal thickness, mast cell infiltration and telangiectasia upon downregulating TRPV1 and CD31 expression. Moreover, the up-regulated TRPV1 protein expression was also recovered by ATEs administration in vivo and in vitro. Meanwhile, ATEs demonstrated good biocompatibility. In summary, ATEs could be a potential therapeutic agent for rosacea by regulating inflammation and alleviating telangiectasia.
Assuntos
Tecido Adiposo , Rosácea , Canais de Cátion TRPV , Canais de Cátion TRPV/metabolismo , Rosácea/tratamento farmacológico , Rosácea/metabolismo , Rosácea/patologia , Animais , Humanos , Camundongos , Tecido Adiposo/metabolismo , Tecido Adiposo/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinócitos/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Capsaicina/farmacologia , Células HaCaT , Catelicidinas , Masculino , Modelos Animais de Doenças , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/metabolismoRESUMO
Skin psoriasis is defined as receiving external stimulation to activate skin dendritic cells (DCs) which can release interleukin 23 (IL-23) to interlink the innate and adaptive immunity as well as induce T helper 17 (Th17) cell differentiation leading to elevated production of interleukin 17 (IL-17) for keratinocytes over production. This autoimmune loop in psoriasis pathogenesis is influenced by G protein-coupled receptor (GPCR) signalling transduction, and in particular, function of adhesion molecule GPR97 in psoriasis endures to be utterly addressed. In this research, our team allocated GPR97 depletion (GPR97-/-), GPR97 conditional depletion on dendritic cell (DC-cKO), and keratin 14-conditional knockout (K14-cKO) mice models to explore the function of GPR97 which influences keratinocytes and skin immunity. It was found that significantly aggravated psoriasis-like lesion in GPR97-/- mice. In addition, hyperproliferative keratinocytes as well as accumulation of DCs and Th17 cells were detected in imiquimod (IMQ)-induced GPR97-/- mice, which was consistent with the results in DC-cKO and K14-cKO psoriasis model. Additional investigations indicated that beclomethasone dipropionate (BDP), an agonist of GPR97, attenuated the psoriasis-like skin disease and restricted HaCaT cells abnormal proliferation as well as Th17 cells differentiation. Particularly, we found that level of NF-κB p65 was increased in GPR97-/- DCs and BDP could inhibit p65 activation in DCs. Role of GPR97 is indispensable and this adhesion receptor may affect immune cell enrichment and function in skin and alter keratinocytes proliferation as well as differentiation in psoriasis.
Assuntos
Imiquimode , Interleucina-17 , Interleucina-23 , Queratinócitos , Camundongos Knockout , Psoríase , Receptores Acoplados a Proteínas G , Transdução de Sinais , Células Th17 , Animais , Psoríase/induzido quimicamente , Psoríase/patologia , Psoríase/genética , Psoríase/metabolismo , Interleucina-17/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/deficiência , Camundongos , Queratinócitos/metabolismo , Queratinócitos/patologia , Células Th17/imunologia , Células Th17/metabolismo , Interleucina-23/metabolismo , Células Dendríticas/metabolismo , Camundongos Endogâmicos C57BL , Humanos , Modelos Animais de Doenças , Pele/patologia , Pele/metabolismoRESUMO
BACKGROUND: Human epithelium-derived stem cells and induced pluripotent stem cells (hiPSCs) possess the capability to support tooth formation and differentiate into functional enamel-secreting ameloblasts, making them promising epithelial-component substitutes for future human tooth regeneration. However, current tissue recombination approaches are not only technically challenging, requiring precise induction procedures and sophisticated microsurgery, but also exhibit low success rates in achieving tooth formation and ameloblastic differentiation. METHODS: Suspended human keratinocyte stem cells (hKSCs) or cells from three hiPSC lines were directly mixed with dissociated embryonic mouse dental mesenchymal cells (mDMCs) that possess odontogenic potential in different proportions and reaggregated them to construct bioengineered tooth germs. The success rates of tooth formation and ameloblastic differentiation were confirmed after subrenal culture. The sorting capability, sequential development, and ameloblastic differentiation of stem cells were examined via GFP tracing, RT-PCR, and histological analysis, respectively. RESULTS: Our reaggregation approach achieved an impressive success rate of more than 90% in tooth formation and 100% in ameloblastic differentiation when the chimeric tooth germs contained 1%~10% hKSCs or 5% hiPSCs. In addition, we observed that hiPSCs, upon exposure to mDMCs, initially transformed into epidermal cells, as indicated by KRT14 and CD29 expression, before progressing into dental epithelial cells, as indicated by SP6 and SHH expression. We also found that epithelial-derived hiPSCs, when reaggregated with mDMCs, were more favorable for tooth formation than their mesenchymal-derived counterparts. CONCLUSIONS: This study establishes a simplified yet highly effective cell-cell reaggregation strategy for inducing stem cells to support tooth formation and differentiate into functional ameloblasts, paving the way for novel approaches for the development of stem cell-based tooth organoids and bioengineered tooth germs in vitro.
Assuntos
Ameloblastos , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas , Queratinócitos , Humanos , Ameloblastos/metabolismo , Ameloblastos/citologia , Animais , Camundongos , Queratinócitos/citologia , Queratinócitos/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Agregação Celular , Germe de Dente/citologia , Germe de Dente/metabolismo , Células CultivadasRESUMO
Many cell culture experiments are performed under light to evaluate the photodynamic or photosensitizing efficacy of various agents. In this study, the modulation of photosensitizing responses and phototoxicity under cell culture conditions by different medium components was investigated. The significant levels of reactive oxygen species (ROS) generated from DMEM, RPMI 1640, and MEM were observed under the irradiation of fluorescent light (FL) and white and blue LEDs, indicating that these media have their own photosensitizing properties; DMEM showed the most potent property. Phenol red-free DMEM (Pf-D) exhibited a stronger photosensitizing property than normal DMEM by 1.31 and 1.25 times under FL and blue LEDs, respectively; phenol red and riboflavin-free DMEM (PRbf-D) did not show any photosensitizing properties. The inhibitory effect on light transmission was more pronounced in DMEM than in RPMI, and the interference effect on green LED light was greatest at 57.8 and 27.4%, respectively; the effect disappeared in Pf-D. The media containing riboflavin induced strong phototoxicity in HaCaT keratinocytes by generating H2O2 under light irradiation, which was quenched by sodium pyruvate in the media. The presence of serum in the media was also reduced the phototoxicity; H2O2 levels in the media decreased serum content dependently. The phototoxicity of erythrosine B and protoporphyrin IX under FL was more sensitively pronounced in PRbf-D than in DMEM. The present results indicate that several medium components, including riboflavin, phenol red, sodium pyruvate, and serum, could modulate photosensitizing responses in a cell culture system by inducing photosensitizing activation and by interfering with irradiation efficacy and ROS generation.
Assuntos
Meios de Cultura , Fármacos Fotossensibilizantes , Espécies Reativas de Oxigênio , Humanos , Meios de Cultura/química , Espécies Reativas de Oxigênio/metabolismo , Fármacos Fotossensibilizantes/farmacologia , Riboflavina/farmacologia , Luz , Peróxido de Hidrogênio/farmacologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/efeitos da radiação , Queratinócitos/metabolismo , Técnicas de Cultura de Células/métodos , Linhagem CelularRESUMO
TLR3 is expressed in human skin and keratinocytes, and given its varied role in skin inflammation, development, and regeneration, we sought to determine the cellular response in normal human keratinocytes to TLR3 activation. We investigated this mechanism by treating primary human keratinocytes with both UVB, an endogenous and physiologic TLR3 activator, and poly(I:C), a synthetic and selective TLR3 ligand. TLR3 activation with either UVB or poly(I:C) altered keratinocyte morphology, coinciding with the key features of epithelial-to-mesenchymal transition: increased epithelial-to-mesenchymal transition gene expression, enhanced migration, and increased invasion properties. These results confirm and extend previous studies demonstrating that in addition to its classical role in the innate immune response, TLR3 signaling also regulates stem cell-like properties and developmental programs.
Assuntos
Movimento Celular , Transição Epitelial-Mesenquimal , Queratinócitos , Poli I-C , Transdução de Sinais , Receptor 3 Toll-Like , Humanos , Receptor 3 Toll-Like/metabolismo , Receptor 3 Toll-Like/genética , Queratinócitos/metabolismo , Transição Epitelial-Mesenquimal/genética , Poli I-C/farmacologia , Movimento Celular/genética , Raios Ultravioleta/efeitos adversos , Células Cultivadas , Pele/metabolismo , Pele/citologiaRESUMO
Malassezia, the most abundant fungal commensal on the mammalian skin, has been linked to several inflammatory skin diseases such as atopic dermatitis, seborrheic dermatitis and psoriasis. This study reveals that epicutaneous application with Malassezia globosa (M. globosa) triggers skin inflammation in mice. RNA-sequencing of the resulting mouse lesions indicates activation of Interleukin-17 (IL-17) signaling and T helper 17 (Th17) cells differentiation pathways by M. globosa. Furthermore, our findings demonstrate a significant upregulation of IL-23, IL-23R, IL-17A, and IL-22 expressions, along with an increase in the proportion of Th17 and pathogenic Th17 cells in mouse skin exposed to M. globosa. In vitro experiments illustrate that M. globosa prompts human primary keratinocytes to secrete IL-23 via TLR2/MyD88/NF-κB signaling. This IL-23 secretion by keratinocytes is shown to be adequate for inducing the differentiation of pathogenic Th17 cells in the skin. Overall, these results underscore the significant role of Malassezia in exacerbating skin inflammation by stimulating IL-23 secretion by keratinocytes and promoting the differentiation of pathogenic Th17 cells.
Assuntos
Diferenciação Celular , Interleucina-23 , Queratinócitos , Malassezia , Células Th17 , Malassezia/imunologia , Queratinócitos/microbiologia , Queratinócitos/imunologia , Queratinócitos/metabolismo , Células Th17/imunologia , Animais , Interleucina-23/metabolismo , Humanos , Camundongos , Transdução de Sinais , NF-kappa B/metabolismo , Receptor 2 Toll-Like/metabolismo , Interleucina-17/metabolismo , Pele/microbiologia , Pele/patologia , Pele/imunologia , Modelos Animais de Doenças , Fator 88 de Diferenciação Mieloide/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Células Cultivadas , Camundongos Endogâmicos C57BL , Interleucina 22RESUMO
Somatosensory neurons can sense external temperature by converting sensation of temperature information to neural activity via afferent input to the central nervous system. Various populations of somatosensory neurons have specialized gene expression, including expression of thermosensitive transient receptor potential (TRP) ion channels. Thermosensitive TRP channels are responsible for thermal transduction at the peripheral ends of somatosensory neurons and can sense a wide range of temperatures. Here we focus on several thermosensitive TRP channels including TRPV1, TRPV4, TRPM2, TRPM3, TRPM8, TRPC5, and TRPA1 in sensory neurons. TRPV3, TRPV4, and TRPC5 are also involved in somatosensation in nonneuronal cells and tissues. In particular, we discuss whether skin senses ambient temperatures through TRPV3 and TRPV4 activation in skin keratinocytes and the involvement of TRPM2 expressed by hypothalamic neurons in thermosensation in the brain.
Assuntos
Sensação Térmica , Canais de Potencial de Receptor Transitório , Humanos , Sensação Térmica/fisiologia , Sensação Térmica/genética , Animais , Canais de Potencial de Receptor Transitório/metabolismo , Canais de Potencial de Receptor Transitório/genética , Canais de Potencial de Receptor Transitório/fisiologia , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/fisiologia , Canais de Cátion TRPV/metabolismo , Canais de Cátion TRPV/genética , Pele/metabolismo , Pele/inervação , Canais de Cátion TRPM/metabolismo , Canais de Cátion TRPM/genética , Queratinócitos/metabolismoRESUMO
Cell-cell junctions, and specifically desmosomes, are crucial for robust intercellular adhesion. Desmosomal function is compromised in the autoimmune blistering skin disease pemphigus vulgaris. We combine whole-genome knockout screening and a promotor screen of the desmosomal gene desmoglein 3 in human keratinocytes to identify novel regulators of intercellular adhesion. Kruppel-like-factor 5 (KLF5) directly binds to the desmoglein 3 regulatory region and promotes adhesion. Reduced levels of KLF5 in patient tissue indicate a role in pemphigus vulgaris. Autoantibody fractions from patients impair intercellular adhesion and reduce KLF5 levels in in vitro and in vivo disease models. These effects were dependent on increased activity of histone deacetylase 3, leading to transcriptional repression of KLF5. Inhibiting histone deacetylase 3 increases KLF5 levels and protects against the deleterious effects of autoantibodies in murine and human pemphigus vulgaris models. Together, KLF5 and histone deacetylase 3 are regulators of desmoglein 3 gene expression and intercellular adhesion and represent potential therapeutic targets in pemphigus vulgaris.
Assuntos
Adesão Celular , Desmogleína 3 , Queratinócitos , Fatores de Transcrição Kruppel-Like , Pênfigo , Humanos , Pênfigo/metabolismo , Pênfigo/patologia , Pênfigo/imunologia , Desmogleína 3/metabolismo , Desmogleína 3/genética , Animais , Queratinócitos/metabolismo , Camundongos , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Autoanticorpos/imunologia , Desmossomos/metabolismo , Modelos Animais de Doenças , Histona Desacetilases/metabolismo , Histona Desacetilases/genética , Regulação da Expressão Gênica , Regiões Promotoras Genéticas/genética , MasculinoRESUMO
Introduction: Ionizing radiation has been widely used in industry, medicine, military and agriculture. Radiation-induced skin injury is a significant concern in the context of radiotherapy and accidental exposure to radiation. The molecular changes at the single-cell level and intercellular communications during radiation-induced skin injury are not well understood. Methods: This study aims to illustrate this information in a murine model and human skin samples from a radiation accident using single-cell RNA sequencing (scRNA-Seq). We further characterize the functional significance of key molecule, which may provide a potential therapeutic target. ScRNA-Seq was performed on skin samples from a nuclear accident patient and rats exposed to ionizing radiation. Bioinformatic tools were used to analyze the cellular heterogeneity and preferential mRNAs. Comparative analysis was performed to identify dysregulated pathways, regulators, and ligand-receptor interactions in fibroblasts. The function of key molecule was validated in skin cells and in three mouse models of radiation-induced skin injury. Results: 11 clusters in human skin and 13 clusters of cells in rat skin were depicted respectively. Exposure to ionizing radiation caused changes in the cellular population (upregulation of fibroblasts and endothelial cells, downregulation of keratinocytes). Fibroblasts and keratinocytes possessed the most interaction pairs with other cell lineages. Among the five DEGs common to human and rat skins, Nur77 was highly expressed in fibroblasts, which mediated radiosensitivity by cell apoptosis and modulated crosstalk between macrophages, keratinocytes and endothelial cells in radiation-induced skin injury. In animal models, Nur77 knock-out mice (Nur77 -/-) showed more severe injury after radiation exposure than wild-type counterparts in three models of radiation-induced skin injury with complex mechanisms. Conclusion: The study reveals a single-cell transcriptional framework during radiation-induced skin injury, which provides a useful resource to uncover key events in its progression. Nur77 is a novel target in radiation-induced skin injury, which provides a potential therapeutic strategy against this disease.
Assuntos
Queratinócitos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , RNA-Seq , Análise de Célula Única , Pele , Animais , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Humanos , Camundongos , Ratos , Pele/efeitos da radiação , Pele/patologia , Pele/metabolismo , Pele/lesões , Queratinócitos/efeitos da radiação , Queratinócitos/metabolismo , Fibroblastos/efeitos da radiação , Fibroblastos/metabolismo , Masculino , Camundongos Knockout , Radiação Ionizante , Lesões por Radiação/genética , Lesões por Radiação/patologia , Análise da Expressão Gênica de Célula ÚnicaRESUMO
Skin aging is influenced by intrinsic and extrinsic factors that progressively impair skin functionality over time. Investigating the skin aging process requires thorough research using innovative technologies. This review explores the use of in vitro human 3D culture models, serving as valuable alternatives to animal ones, in skin aging research. The aim is to highlight the benefits and necessity of improving the methodology in analyzing the molecular mechanisms underlying human skin aging. Traditional 2D models, including monolayers of keratinocytes, fibroblasts, or melanocytes, even if providing cost-effective and straightforward methods to study critical processes such as extracellular matrix degradation, pigmentation, and the effects of secretome on skin cells, fail to replicate the complex tissue architecture with its intricated interactions. Advanced 3D models (organoid cultures, "skin-on-chip" technologies, reconstructed human skin, and 3D bioprinting) considerably enhance the physiological relevance, enabling a more accurate representation of skin aging and its peculiar features. By reporting the advantages and limitations of 3D models, this review highlights the importance of using advanced in vitro systems to develop practical anti-aging preventive and reparative approaches and improve human translational research in this field. Further exploration of these technologies will provide new opportunities for previously unexplored knowledge on skin aging.
Assuntos
Envelhecimento da Pele , Humanos , Envelhecimento da Pele/fisiologia , Pele/metabolismo , Melanócitos/metabolismo , Melanócitos/citologia , Queratinócitos/citologia , Queratinócitos/metabolismo , Fibroblastos/metabolismo , Fibroblastos/citologia , Modelos Biológicos , Impressão Tridimensional , Bioimpressão/métodos , Técnicas de Cultura de Células em Três Dimensões/métodosRESUMO
Background: Esophageal organoids from a variety of pathologies including cancer are grown in Advanced Dulbecco's Modified Eagle Medium-Nutrient Mixture F12 (hereafter ADF). However, the currently available ADF-based formulations are suboptimal for normal human esophageal organoids, limiting the ability to compare normal esophageal organoids with those representing a given disease state. Methods: We have utilized immortalized normal human esophageal epithelial cell (keratinocyte) lines EPC1 and EPC2 and endoscopic normal esophageal biopsies to generate three-dimensional (3D) organoids. To optimize the ADF-based medium, we evaluated the requirement of exogenous epidermal growth factor (EGF) and inhibition of transforming growth factor-(TGF)-ß receptor-mediated signaling, both key regulators of the proliferation of human esophageal keratinocytes. We have modeled human esophageal epithelial pathology by stimulating esophageal 3D organoids with interleukin (IL)-13, an inflammatory cytokine, or UAB30, a novel pharmacological activator of retinoic acid signaling. Results: The formation of normal human esophageal 3D organoids was limited by excessive EGF and intrinsic TGFß-receptor-mediated signaling. Optimized HOME0 improved normal human esophageal organoid formation. In the HOME0-grown organoids, IL-13 and UAB30 induced epithelial changes reminiscent of basal cell hyperplasia, a common histopathologic feature in broad esophageal disease conditions including eosinophilic esophagitis. Conclusions: HOME0 allows modeling of the homeostatic differentiation gradient and perturbation of the human esophageal epithelium while permitting a comparison of organoids from mice and other organs grown in ADF-based media.
Assuntos
Esôfago , Homeostase , Organoides , Humanos , Organoides/efeitos dos fármacos , Organoides/metabolismo , Esôfago/metabolismo , Esôfago/patologia , Esôfago/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Fator de Crescimento Epidérmico/metabolismo , Queratinócitos/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/citologia , Transdução de Sinais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Modelos Biológicos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Receptores de Fatores de Crescimento Transformadores beta/metabolismoRESUMO
The therapeutic usage of physical stimuli is framed in a highly heterogeneous research area, with variable levels of maturity and of translatability into clinical application. In particular, electrostimulation is deeply studied for its application on the autonomous nervous system, but less is known about the anti- inflammatory effects of such stimuli beyond the inflammatory reflex. Further, reproducibility and meta-analyses are extremely challenging, owing to the limited rationale on dosage and experimental standardization. It is specifically to address the fundamental question on the anti-inflammatory effects of electricity on biological systems, that we propose a series of controlled experiments on the effects of direct and alternate current delivered on a standardized 3D bioconstruct constituted by fibroblasts and keratinocytes in a collagen matrix, in the presence or absence of TNF-α as conventional inflammation inducer. This selected but systematic exploration, with transcriptomics backed by metabolomics at specific time points allows to obtain the first systemic overview of the biological functions at stake, highlighting the differential anti-inflammatory potential of such approaches, with promising results for 5 V direct current stimuli, correlating with the wound healing process. With our results, we wish to set the base for a rigorous systematic approach to the problem, fundamental towards future elucidations of the detailed mechanisms at stake, highlighting both the healing and damaging potential of such approaches.
Assuntos
Estimulação Elétrica , Fibroblastos , Inflamação , Queratinócitos , Cicatrização , Humanos , Estimulação Elétrica/métodos , Inflamação/metabolismo , Inflamação/terapia , Fibroblastos/metabolismo , Queratinócitos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Anti-Inflamatórios/farmacologia , Terapia por Estimulação Elétrica/métodos , Metabolômica/métodos , Colágeno/metabolismoRESUMO
Although oxidative stress is closely associated with tumor invasion and metastasis, its' exact role and mechanism in the initial stage of oral cancer remain ambiguous. Glutamine uptake mediated by alanine-serine-cysteine transporter 2 (ASCT2) participates in glutathione synthesis to resolve oxidative stress. Currently, we firstly found that ASCT2 deletion caused oxidative stress in oral mucosa and promoted oral carcinogenesis induced by 4-Nitroquinoline-1-oxide (4-NQO) using transgenic mice of ASCT2 knockout in oral epithelium. Subsequently, we identified an upregulated gene Thbs1 linked to macrophage infiltration by mRNA sequencing and immunohistochemistry. Importantly, multiplex immunohistochemistry showed M1-like tumor-associated macrophages (TAMs) were enriched in cancerous area. Mechanically, targeted ASCT2 effectively curbed glutamine uptake and caused intracellular reactive oxygen species (ROS) accumulation, which upregulated Thbs1 in oral keratinocytes and then activated p38, Akt and SAPK/JNK signaling to polarize M1-like TAMs via exosome-transferred pathway. Moreover, we demonstrated M1-like TAMs promoted malignant progression of oral squamous cell carcinoma (OSCC) both in vitro and in vivo by a DOK transformed cell line induced by 4-NQO. All these results establish that oxidative stress triggered by ASCT2 deletion promotes oral carcinogenesis through Thbs1-mediated M1 polarization, and indicate that restore redox homeostasis is a new approach to prevent malignant progression of oral potentially malignant disorders.
Assuntos
Neoplasias Bucais , Estresse Oxidativo , Trombospondina 1 , Macrófagos Associados a Tumor , Animais , Camundongos , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Neoplasias Bucais/genética , Humanos , Trombospondina 1/metabolismo , Trombospondina 1/genética , Macrófagos Associados a Tumor/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Carcinogênese/metabolismo , Carcinogênese/genética , 4-Nitroquinolina-1-Óxido/toxicidade , Queratinócitos/metabolismo , Queratinócitos/patologia , Linhagem Celular TumoralRESUMO
Excessive melanogenesis leads to hyperpigmentation-related cosmetic problems. UV exposure increases oxidative stress, which promotes melanogenesis-related signal pathways such as the PKA, microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), tyrosinase-related protein-1 (TRP1), and tyrosinase-related protein-2 (TRP2) pathways. Glycine is a source of endogenous antioxidants, including glutathione. Fermented fish collagen (FC) contains glycine; thus, we evaluated the effect of FC on decreasing melanogenesis via decreasing oxidative stress. The glycine receptor (GlyR) and glycine transporter-1 (GlyT1) levels were decreased in UV-irradiated keratinocytes; however, the expression levels of these proteins increased upon treatment with FC. The FC decreased oxidative stress, as indicated by the decreasing expression of NOX1/2/4, increased expression of GSH/GSSG, increased SOD activity, and decreased 8-OHdG expression in UV-irradiated keratinocytes. Administration of conditioned media from FC-treated keratinocytes to melanocytes led to decreased p38, PKC, MITF, TRP1, and TRP2 expression. These changes induced by the FC were also observed in UV-irradiated animal skin. FC treatment increased the expression of GlyR and GlyT, which was accompanied by decreased oxidative stress in the UV-irradiated skin. Moreover, the FC negatively regulated the melanogenesis signaling pathways, leading to decreased melanin content in the UV-irradiated skin. In conclusion, FC decreased UV-induced oxidative stress and melanogenesis in melanocytes and animal skin. FC could be used in the treatment of UV-induced hyperpigmentation problems.