RESUMEN
Skin identity is controlled by intrinsic features of the epidermis and dermis and their interactions. Modifying skin identity has clinical potential, such as the conversion of residual limb and stump (nonvolar) skin of amputees to pressure-responsive palmoplantar (volar) skin to enhance prosthesis use and minimize skin breakdown. Greater keratin 9 (KRT9) expression, higher epidermal thickness, keratinocyte cytoplasmic size, collagen length, and elastin are markers of volar skin and likely contribute to volar skin resiliency. Given fibroblasts' capacity to modify keratinocyte differentiation, we hypothesized that volar fibroblasts influence these features. Bioprinted skin constructs confirmed the capacity of volar fibroblasts to induce volar keratinocyte features. A clinical trial of healthy volunteers demonstrated that injecting volar fibroblasts into nonvolar skin increased volar features that lasted up to 5 months, highlighting a potential cellular therapy.
Asunto(s)
Refuerzo Biomédico , Bioimpresión , Dermis , Epidermis , Fibroblastos , Queratinocitos , Adulto , Femenino , Humanos , Masculino , Amputados , Diferenciación Celular , Colágeno/metabolismo , Dermis/citología , Dermis/metabolismo , Elastina/metabolismo , Epidermis/metabolismo , Fibroblastos/citología , Fibroblastos/trasplante , Mano , Queratina-9/metabolismo , Queratinocitos/citología , Queratinocitos/metabolismo , Refuerzo Biomédico/métodosRESUMEN
Natural and induced somatic mutations that accumulate in the genome during development record the phylogenetic relationships of cells; whether these lineage barcodes capture the complex dynamics of progenitor states remains unclear. We introduce quantitative fate mapping, an approach to reconstruct the hierarchy, commitment times, population sizes, and commitment biases of intermediate progenitor states during development based on a time-scaled phylogeny of their descendants. To reconstruct time-scaled phylogenies from lineage barcodes, we introduce Phylotime, a scalable maximum likelihood clustering approach based on a general barcoding mutagenesis model. We validate these approaches using realistic in silico and in vitro barcoding experiments. We further establish criteria for the number of cells that must be analyzed for robust quantitative fate mapping and a progenitor state coverage statistic to assess the robustness. This work demonstrates how lineage barcodes, natural or synthetic, enable analyzing progenitor fate and dynamics long after embryonic development in any organism.
Asunto(s)
Desarrollo Embrionario , Linaje de la Célula/genética , Estudios Retrospectivos , Filogenia , MutagénesisRESUMEN
Numerous small molecules (termed inducers), many of which are electrophiles, upregulate cytoprotective responses and inhibit pro-inflammatory pathways by activating nuclear factor-erythroid 2 p45-related factor 2 (NRF2). Key to NRF2 activation is the ability to chemically modifying critical sensor cysteines in the main negative regulator of NRF2, Kelch-like ECH-associated protein 1 (KEAP1), of which C151, C273 and C288 are best characterized. This study aimed to establish the requirement for these cysteine sensor(s) for the biological activities of the most potent NRF2 activators known to date, the cyclic cyanoenones, some of which are in clinical trials. It was found that C151 in KEAP1 is the main cysteine sensor for this class of inducers, irrespective of molecular size or shape. Furthermore, in primary macrophage cells expressing C151S mutant KEAP1, at low concentrations, the tricyclic cyanoenone TBE-31 is inactive as an activator of NRF2 as well as an inhibitor of lipopolysaccharide-stimulated gene expression of the pro-inflammatory cytokines IL6 and IL1ß. However, at high inducer concentrations, NRF2 activation proceeds in the absence of C151, albeit at a lower magnitude. Our findings highlight the intrinsic flexibility of KEAP1 and emphasize the critical importance of establishing the precise dose of NRF2 activators for maintaining on-target selectivity.
Asunto(s)
Cisteína/química , Proteína 1 Asociada A ECH Tipo Kelch/fisiología , Factor 2 Relacionado con NF-E2/metabolismo , Fenantrenos/farmacología , Activación Transcripcional/efectos de los fármacos , Animales , Células Cultivadas , Cisteína/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Macrófagos Peritoneales/citología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Factor 2 Relacionado con NF-E2/genética , Fenantrenos/química , Regulación hacia ArribaRESUMEN
The Keap1-Nrf2 system plays a central role in cytoprotection against electrophilic/oxidative stresses. Although Cys151, Cys273, and Cys288 of Keap1 are major sensor cysteine residues for detecting these stresses, it has not been technically feasible to evaluate the functionality of Cys273 or Cys288, since Keap1 mutants that harbor substitutions in these residues and maintain the ability to repress Nrf2 accumulation do not exist. To overcome this problem, we systematically introduced amino acid substitutions into Cys273/Cys288 and finally identified Cys273Trp and Cys288Glu mutations that do not affect Keap1's ability to repress Nrf2 accumulation. Utilizing these Keap1 mutants, we generated stable murine embryonic fibroblast (MEF) cell lines and knock-in mouse lines. Our analyses with the MEFs and peritoneal macrophages from the knock-in mice revealed that three major cysteine residues, Cys151, Cys273, and Cys288, individually and/or redundantly act as sensors. Based on the functional necessity of these three cysteine residues, we categorized chemical inducers of Nrf2 into four classes. Class I and II utilizes Cys151 and Cys288, respectively, while class III requires all three residues (Cys151/Cys273/Cys288), while class IV inducers function independently of all three of these cysteine residues. This study thus demonstrates that Keap1 utilizes multiple cysteine residues specifically and/or collaboratively as sensors for the detection of a wide range of environmental stresses.