RESUMO
The expanded hexanucleotide GGGGCC repeat mutation in the C9orf72 gene is the main genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Under one disease mechanism, sense and antisense transcripts of the repeat are predicted to bind various RNA-binding proteins, compromise their function and cause cytotoxicity. Here we identify phenylalanine-tRNA synthetase (FARS) subunit alpha (FARSA) as the main interactor of the CCCCGG antisense repeat RNA in cytosol. The aminoacylation of tRNAPhe by FARS is inhibited by antisense RNA, leading to decreased levels of charged tRNAPhe. Remarkably, this is associated with global reduction of phenylalanine incorporation in the proteome and decrease in expression of phenylalanine-rich proteins in cellular models and patient tissues. In conclusion, this study reveals functional inhibition of FARSA in the presence of antisense RNA repeats. Compromised aminoacylation of tRNA could lead to impairments in protein synthesis and further contribute to C9orf72 mutation-associated pathology.
Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Humanos , Aminoacilação de RNA de Transferência , Aminoacilação , Esclerose Lateral Amiotrófica/genética , Demência Frontotemporal/genética , Proteína C9orf72/genética , Fenilalanina/genética , RNA de Transferência de Fenilalanina , RNA AntissensoRESUMO
The alpha subunit of the cytoplasmic Phenylalanyl tRNA synthetase (α-PheRS, FARSA in humans) displays cell growth and proliferation activities and its elevated levels can induce cell fate changes and tumor-like phenotypes that are neither dependent on the canonical function of charging tRNAPhe with phenylalanine nor on stimulating general translation. In intestinal stem cells of Drosophila midguts, α-PheRS levels are naturally slightly elevated and human FARSA mRNA levels are elevated in multiple cancers. In the Drosophila midgut model, elevated α-PheRS levels caused the accumulation of many additional proliferating cells resembling intestinal stem cells (ISCs) and enteroblasts (EBs). This phenotype partially resembles the tumor-like phenotype described as Notch RNAi phenotype for the same cells. Genetic interactions between α-PheRS and Notch suggest that their activities neutralize each other and that elevated α-PheRS levels attenuate Notch signaling when Notch induces differentiation into enterocytes, type II neuroblast stem cell proliferation, or transcription of a Notch reporter. These non-canonical functions all map to the N-terminal part of α-PheRS which accumulates naturally in the intestine. This truncated version of α-PheRS (α-S) also localizes to nuclei and displays weak sequence similarity to the Notch intracellular domain (NICD), suggesting that α-S might compete with the NICD for binding to a common target. Supporting this hypothesis, the tryptophan (W) residue reported to be key for the interaction between the NICD and the Su(H) BTD domain is not only conserved in α-PheRS and α-S, but also essential for attenuating Notch signaling.
Assuntos
Fenilalanina-tRNA Ligase , Animais , Drosophila/genética , Fenilalanina , Fenilalanina-tRNA Ligase/química , Fenilalanina-tRNA Ligase/genética , Fenilalanina-tRNA Ligase/metabolismo , RNA de Transferência de Fenilalanina/química , RNA de Transferência de Fenilalanina/metabolismoRESUMO
Aminoacyl transfer RNA (tRNA) synthetases (aaRSs) not only load the appropriate amino acid onto their cognate tRNAs, but many of them also perform additional functions that are not necessarily related to their canonical activities. Phenylalanyl tRNA synthetase (PheRS/FARS) levels are elevated in multiple cancers compared to their normal cell counterparts. Our results show that downregulation of PheRS, or only its α-PheRS subunit, reduces organ size, whereas elevated expression of the α-PheRS subunit stimulates cell growth and proliferation. In the wing disc system, this can lead to a 67% increase in cells that stain for a mitotic marker. Clonal analysis of twin spots in the follicle cells of the ovary revealed that elevated expression of the α-PheRS subunit causes cells to grow and proliferate â¼25% faster than their normal twin cells. This faster growth and proliferation did not affect the size distribution of the proliferating cells. Importantly, this stimulation proliferation turned out to be independent of the ß-PheRS subunit and the aminoacylation activity, and it did not visibly stimulate translation.This article has an associated First Person interview with the joint first authors of the paper.
Assuntos
Drosophila melanogaster/enzimologia , Drosophila melanogaster/crescimento & desenvolvimento , Fenilalanina-tRNA Ligase/metabolismo , Biossíntese de Proteínas , Aminoácidos/metabolismo , Aminoacilação , Animais , Proliferação de Células , Técnicas de Silenciamento de Genes , Mitose , Tamanho do Órgão , OrganogêneseRESUMO
PURPOSE: One of the features in cancer development is the migration of cancer cells to form metastatic lesions. CYR61 protein promotes migration and the epithelial-mesenchymal transition in several cancer cell types. Evidence suggests that CYR61 and dexamethasone are relevant to colorectal cancer. However, relationships between them and colorectal cancer are still unclear. Understanding the molecular mechanism of colorectal cancer progression related with CYR61 and dexamethasone, which is widely used for combination chemotherapy, is necessary for improved therapy. MATERIALS AND METHODS: We used colorectal cancer cells, HCT116, co-treated with transforming growth factor ß1 (TGF-ß1) and dexamethasone to examine the inhibitory migration effect of dexamethasone by migratory assay. Alternatively, both migratory pathways, expression of AKT and ERK, and the target factor CYR61 was also tested by co-treatment with TGF-ß1 and dexamethasone. RESULTS: We report that dexamethasone significantly inhibited TGF-ß1-induced cell migration, without affecting cell proliferation. Importantly, we observed that TGF-ß1 promoted the epithelial-mesenchymal transition process and that dexamethasone co-treatment abolished this effect. ERK and AKT signaling pathways were found to mediate TGF-ß1-induced migration, which was inhibited by dexamethasone. In addition, TGF-ß1 treatment induced CYR61 expression whereas dexamethasone reduced it. These observations were compatible with the modulation of migration observed following treatment of HCT116 cells with human recombinant CYR61 and anti-CYR61 antibody. Our results also indicated that TGF-ß1 enhanced collagen I and reduced matrix metalloproteinase 1 expression, which was reversed by dexamethasone treatment. CONCLUSION: These findings suggested that dexamethasone inhibits AKT and ERK phosphorylation, leading to decreased CYR61 expression, which in turn blocks TGF-ß1-induced migration.
Assuntos
Antineoplásicos Hormonais/farmacologia , Movimento Celular/efeitos dos fármacos , Neoplasias Colorretais/patologia , Proteína Rica em Cisteína 61/metabolismo , Dexametasona/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismo , Anticorpos/farmacologia , Caderinas , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Proteína Rica em Cisteína 61/antagonistas & inibidores , Progressão da Doença , Regulação para Baixo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HCT116 , Humanos , Metaloproteinase 1 da Matriz/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Recombinantes/metabolismo , Regulação para CimaRESUMO
Camphor ((1R)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-one), a bicyclic monoterpene, is one of the major constituents of essential oils from various herbs such as rosemary, lavender, and sage. In this study, we investigated the beneficial effects of camphor as a botanical ingredient in cosmetics. Camphor induced the proliferation of human primary dermal fibroblasts in a dose-dependent manner via the PI3K/AKT and ERK signaling pathways. Camphor attenuated the elevation of senescence associated with ß-galactosidase (SA-ß-gal) activity. Elastase activity decreased, while the total amount of collagen increased, in a dose- and time-dependent manner in human primary dermal fibroblasts treated with camphor. Camphor induced the expression of collagen IA, collagen IIIA, collagen IVA, and elastin in human primary dermal fibroblasts. In addition, posttreatment with 26 and 52 mM camphor for 2 weeks led to a significant reduction in the expression of MMP1 but increases in the expression of collagen IA, IIIA, and elastin in mouse skin exposed to UV for 4 weeks. These posttreatments also reduced the depths of the epidermis and subcutaneous fat layer in UV-exposed mouse skin. Taken together, these findings suggest camphor to be a potent wound healing and antiwrinkle agent with considerable potential for use in cosmeceuticals.
Assuntos
Cânfora/farmacologia , Senescência Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Envelhecimento da Pele/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Criança , Colágeno/metabolismo , Elastina/metabolismo , Humanos , Masculino , Matricaria/química , Metaloproteinase 13 da Matriz/metabolismo , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Pele/efeitos dos fármacos , Pele/efeitos da radiação , Envelhecimento da Pele/efeitos da radiação , Raios Ultravioleta/efeitos adversos , beta-Galactosidase/metabolismoRESUMO
BACKGROUND: Wound healing is an intricate process whereby the skin repairs itself after injury. The epithelial-mesenchymal transition (EMT) is associated with wound healing and tissue regeneration. Naphthochalcone derivatives have various pharmaceutical properties. We investigated the effect of a novel naphthochalcone derivative, 2-(5-(2,4,6-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)naphthalen-1-ol (TDPN), on dermal wound healing in vivo and the migration of keratinocytes in vitro. RESULT: We investigated the effect of TDPN on signaling pathway and epithelial-mesenchymal transition through protein and transcriptional expression. The TDPN treatment accelerated dermal closure about 3 days and remodeling of dermis. We found that treatment with TDPN induced the migration of keratinocytes but not cytotoxicity. TDPN induced the phosphorylation of ERK and AKT. TDPN-treated cells showed loss of adherence protein and showed induction of the transcriptional factor Slug, mesenchymal marker, and fibronectin. Moreover, TDPN treatment induced the expression of matrix metalloproteinase-1 (MMP-1), which degrades specific components of the extracellular matrix, thereby providing new substrates that facilitate migration and invasion. MMP expression is considered to be one of the major attributes acquired by cells after EMT. CONCLUSION: We propose that a novel naphthochalcone derivative TDPN is capable of promoting keratinocyte migration via the induction of EMT resulting acceleration of wound closure and matrix remodeling.
Assuntos
Transição Epitelial-Mesenquimal/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Metaloproteinase 1 da Matriz/genética , Naftóis/administração & dosagem , Pirazóis/administração & dosagem , Cicatrização/efeitos dos fármacos , Animais , Movimento Celular/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Metaloproteinase 1 da Matriz/biossíntese , Naftalenos/administração & dosagem , Fosforilação , Ratos , Transdução de Sinais/efeitos dos fármacos , Pele/efeitos dos fármacos , Pele/patologia , Cicatrização/genéticaRESUMO
Wound healing plays an important role in protecting the human body from external infection. Cell migration and proliferation of keratinocytes and dermal fibroblasts are essential for proper wound healing. Recently, several studies have demonstrated that secondary compounds produced in plants could affect skin cells migration and proliferation. In this study, we identified a novel compound DK223 ([1E,2E-1,2-bis(6-methoxy-2H-chromen-3-yl)methylene]hydrazine) that concomitantly induced human keratinocyte migration and dermal fibroblast proliferation. We evaluated the regulation of epithelial and mesenchymal protein markers, such as E-cadherin and Vimentin, in human keratinocytes, as well as extracellular matrix (ECM) secretion and metalloproteinase families in dermal fibroblasts. DK223 upregulated keratinocyte migration and significantly increased the epithelial marker E-cadherin in a time-dependent manner. We also found that reactive oxygen species (ROS) increased significantly in keratinocytes after 2 h of DK223 exposure, returning to normal levels after 24 h, which indicated that DK223 had an early shock effect on ROS production. DK223 also stimulated fibroblast proliferation, and induced significant secretion of ECM proteins, such as collagen I, III, and fibronectin. In dermal fibroblasts, DK223 treatment induced TGF-ß1, which is involved in a signaling pathway that mediates proliferation. In conclusion, DK223 simultaneously induced both keratinocyte migration via ROS production and fibroblast proliferation via TGF-ß1 induction.
Assuntos
Benzopiranos/farmacologia , Fibroblastos/efeitos dos fármacos , Hidrazinas/farmacologia , Queratinócitos/efeitos dos fármacos , Benzopiranos/síntese química , Benzopiranos/química , Caderinas/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Humanos , Hidrazinas/síntese química , Hidrazinas/química , Queratinócitos/citologia , Queratinócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Fatores de Tempo , Fator de Crescimento Transformador beta/metabolismo , Vimentina/metabolismoRESUMO
Flavonoids are major active ingredients in plants and are considered components of food that provide medical or health benefits. They have diversified structures and have effects on human health, including wound healing induction. More than a hundred flavonoids were screened for HaCaT keratinocytes cellular migration measurements and the relationships between their structural properties and the effects promoting cellular migration were examined. Here, among flavonoids used in the previous structure-activity relationship calculations, 4',6,7-trimethoxyisoflavone (TMF) was one of the compounds showing the best activity, so that its molecular mechanism of the wound healing effect on HaCaT keratinocytes was investigated in more detail. Our data revealed that TMF increased the wound healing rate, but not the proliferation rate, in a dose-dependent manner. Treatment of keratinocytes with TMF influenced signaling pathways, affecting the phosphorylation of AKT and ERK in a time-dependent manner. TMF also induced the cell-cell adhesion protein E-cadherin, which is essential for promoting collective cell migration. Furthermore, the TMF treatment group also showed higher ROS and NOX2 transcriptional and protein levels. Correlating with matrix metalloproteinase induction by TMF, levels of extracellular matrix proteins such as collagens I and III were significantly lower in the treatment group. To confirm that the effects of TMF occur through the NOX2 pathway, we co-treated cells with TMF plus an NADPH inhibitor (DPI) or a ROS scavenger (NAC). Western blotting revealed that DPI and NAC attenuated the effect of TMF, suggesting that TMF induces ROS through the NOX2 pathway and regulates keratinocyte migration. In summary, TMF promotes wound healing through NOX2 induction, which leads to collective migration and MMP activation.