RESUMO
The senescence-associated protein p16INK4A acts as a limiter element in cell-cycle progression. The loss of p16INK4A function is causally related to cellular immortalization. The increase in p16INK4A levels with advancing age was demonstrated in melanocytes. However, the characteristic difference between young and senescent melanocytes affecting immortalization of melanocytes remains unclear. In this study, we generated 10 different cell lines in total from newborn (NB) and adult (AD) primary normal human epidermal melanocytes (NHEM) using four different methods, transduction of CDK4R24C and cyclin D1 (K4D), K4D with TERT (K4DT), SV40 T-antigen (SV40T), and HPV16 E6 and E7 (E6/E7) and performed whole transcriptome sequencing analysis (RNA-Seq) to elucidate the differences of genome-wide expression profiles among cell lines. The analysis data revealed distinct differences in expression pattern between cell lines from NB and AD although no distinct biological differences were detected in analyses such as comparison of cell morphology, evaluation of cell proliferation, and cell cycle profiles. This study may provide useful in vitro models to benefit the understanding of skin-related diseases.
RESUMO
BACKGROUND: Although vitamins or their derivatives (Vits), such as panthenyl ethyl ether, tocopherol acetate, and pyridoxine, have been widely used in topical hair care products, their efficacy and mode of action have been insufficiently studied. OBJECTIVE: To elucidate the biological influence of Vits on hair follicles and determine the underlying mechanisms. METHODS: A mouse vibrissa hair follicle organ culture model was utilized to evaluate the effects of Vits on hair shaft elongation. Gene and protein expression analyses and histological investigations were conducted to elucidate the responsible mechanisms. A human hair follicle cell culture was used to assess the clinical relevance. RESULTS: In organ culture models, the combination of panthenyl ethyl ether, tocopherol acetate, and pyridoxine (namely, PPT) supplementation significantly promoted hair shaft elongation. PPT treatment enhanced hair matrix cell proliferation by 1.9-fold compared to controls, as demonstrated by Ki67-positive immunoreactivity. PPT-treated mouse dermal papillae exhibited upregulated Placental growth factor (Plgf) by 1.6-fold compared to controls. Importantly, the addition of PlGF neutralizing antibodies to the ex vivo culture diminished the promotive effect on hair growth and increase in VEGFR-1 phosphorylation achieved by PPT. A VEGFR-1 inhibitor also inhibited the promotion of hair growth. Microarray analysis suggested synergistic summation of individual Vits' bioactivity, putatively explaining the effect of PPT. Moreover, PPT increased PlGF secretion in cultured human dermal papilla cells. CONCLUSION: Our findings suggested that PPT promoted hair shaft elongation by activating PlGF/VEGFR-1 signalling. The current study can shed light on the previously underrepresented advantage of utilizing Vits in hair care products.
Assuntos
Preparações para Cabelo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Humanos , Feminino , Camundongos , Animais , Fator de Crescimento Placentário/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/farmacologia , Vitaminas/farmacologia , Vitaminas/metabolismo , alfa-Tocoferol/farmacologia , Piridoxina/metabolismo , Piridoxina/farmacologia , Cabelo , Folículo Piloso/metabolismo , Células Cultivadas , Vitamina A/farmacologia , Preparações para Cabelo/metabolismo , Preparações para Cabelo/farmacologiaRESUMO
BACKGROUND: Testosterone signaling mediates various diseases, such as androgenetic alopecia and prostate cancer. Testosterone signaling is mediated by the androgen receptor (AR). In this study, we fortuitously found that primary and immortalized dermal papilla cells suppressed AR expression, although dermal papilla cells express AR in vivo. To analyze the AR signaling pathway, we exogenously introduced the AR gene via a retrovirus into immortalized dermal papilla cells and comprehensively compared their expression profiles with and without AR expression. RESULTS: Whole-transcriptome profiling revealed that the focal adhesion pathway was mainly affected by the activation of AR signaling. In particular, we found that caveolin-1 gene expression was downregulated in AR-expressing cells, suggesting that caveolin-1 is controlled by AR. CONCLUSION: Our whole transcriptome data is critical resources for discovery of new therapeutic targets for testosterone-related diseases.
Assuntos
Caveolina 1 , Receptores Androgênicos , Caveolina 1/genética , Perfilação da Expressão Gênica , Humanos , Masculino , Receptores Androgênicos/genética , Testosterona , Congêneres da Testosterona , Transcriptoma/genéticaRESUMO
The immortalized cell is an essential research tool that uses robust growth properties for the functional investigation of gene products. Immortalized mammalian cells have mainly been established using three methods: expression of simian vacuolating virus 40 T antigen (the SV40 method); human papilloma virus-derived oncoprotein E6/E7 (the E6/E7 method); or combinatorial expression of R24C mutant cyclin-dependent kinase 4, cyclin D1, and telomerase reverse transcriptase (the K4DT method). However, it is unclear as to which method is optimal for an in vitro model. Here, we compared the biological characteristics and genome-wide expression profiles of immortalized human dermal papilla cells generated by the SV40, E6/E7, or K4DT method. To our knowledge, this is the first study to comprehensively compare expression profiles to determine the optimal immortalization method for maintaining the original nature of the wild-type cells. These data would be valuable to scientists aiming to establish new immortalized cell lines.
RESUMO
Human DNA polymerase η (HsPolη) plays an important role in translesion synthesis (TLS), which allows for replication past DNA damage such as UV-induced cis-syn cyclobutane pyrimidine dimers (CPDs). Here, we characterized ApPolη from the thermophilic worm Alvinella pompejana, which inhabits deep-sea hydrothermal vent chimneys. ApPolη shares sequence homology with HsPolη and contains domains for binding ubiquitin and proliferating cell nuclear antigen. Sun-induced UV does not penetrate Alvinella's environment; however, this novel DNA polymerase catalyzed efficient and accurate TLS past CPD, as well as 7,8-dihydro-8-oxoguanine and isomers of thymine glycol induced by reactive oxygen species. In addition, we found that ApPolη is more thermostable than HsPolη, as expected from its habitat temperature. Moreover, the activity of this enzyme was retained in the presence of a higher concentration of organic solvents. Therefore, ApPolη provides a robust, human-like Polη that is more active after exposure to high temperatures and organic solvents.