RESUMO
Barrett's esophagus (BE) and gastric intestinal metaplasia are related premalignant conditions in which areas of human stomach epithelium express mixed gastric and intestinal features. Intestinal transcription factors (TFs) are expressed in both conditions, with unclear causal roles and cis-regulatory mechanisms. Ectopic CDX2 reprogrammed isogenic mouse stomach organoid lines to a hybrid stomach-intestinal state transcriptionally similar to clinical metaplasia; squamous esophageal organoids resisted this CDX2-mediated effect. Reprogramming was associated with induced activity at thousands of previously inaccessible intestine-restricted enhancers, where CDX2 occupied DNA directly. HNF4A, a TF recently implicated in BE pathogenesis, induced weaker intestinalization by binding a novel shadow Cdx2 enhancer and hence activating Cdx2 expression. CRISPR/Cas9-mediated germline deletion of that cis-element demonstrated its requirement in Cdx2 induction and in the resulting activation of intestinal genes in stomach cells. dCas9-conjugated KRAB repression mapped this activity to the shadow enhancer's HNF4A binding site. Altogether, we show extensive but selective recruitment of intestinal enhancers by CDX2 in gastric cells and that HNF4A-mediated ectopic CDX2 expression in the stomach occurs through a conserved shadow cis-element. These findings identify mechanisms for TF-driven intestinal metaplasia and a likely pathogenic TF hierarchy.
Assuntos
Esôfago de Barrett , Fatores de Transcrição , Animais , Esôfago de Barrett/genética , Esôfago de Barrett/metabolismo , Esôfago de Barrett/patologia , Fator de Transcrição CDX2/genética , Proteínas de Homeodomínio/genética , Metaplasia/genética , Camundongos , Fatores de Transcrição/genéticaRESUMO
The generation of myotubes from fibroblasts upon forced MyoD expression is a classic example of transcription factor-induced reprogramming. We recently discovered that additional modulation of signaling pathways with small molecules facilitates reprogramming to more primitive induced myogenic progenitor cells (iMPCs). Here, we dissected the transcriptional and epigenetic dynamics of mouse fibroblasts undergoing reprogramming to either myotubes or iMPCs using a MyoD-inducible transgenic model. Induction of MyoD in fibroblasts combined with small molecules generated Pax7+ iMPCs with high similarity to primary muscle stem cells. Analysis of intermediate stages of iMPC induction revealed that extinction of the fibroblast program preceded induction of the stem cell program. Moreover, key stem cell genes gained chromatin accessibility prior to their transcriptional activation, and these regions exhibited a marked loss of DNA methylation dependent on the Tet enzymes. In contrast, myotube generation was associated with few methylation changes, incomplete and unstable reprogramming, and an insensitivity to Tet depletion. Finally, we showed that MyoD's ability to bind to unique bHLH targets was crucial for generating iMPCs but dispensable for generating myotubes. Collectively, our analyses elucidate the role of MyoD in myogenic reprogramming and derive general principles by which transcription factors and signaling pathways cooperate to rewire cell identity.
Assuntos
Desenvolvimento Muscular , Proteína MyoD , Animais , Diferenciação Celular/genética , Camundongos , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas , Músculo Esquelético , Proteína MyoD/genética , Proteína MyoD/metabolismo , Mioblastos/metabolismo , Células-Tronco/metabolismoRESUMO
Concomitant activation of the Wnt pathway and suppression of Mapk signalling by two small molecule inhibitors (2i) in the presence of leukaemia inhibitory factor (LIF) (hereafter termed 2i/L) induces a naive state in mouse embryonic stem (ES) cells that resembles the inner cell mass (ICM) of the pre-implantation embryo. Since the ICM exists only transiently in vivo, it remains unclear how sustained propagation of naive ES cells in vitro affects their stability and functionality. Here we show that prolonged culture of male mouse ES cells in 2i/L results in irreversible epigenetic and genomic changes that impair their developmental potential. Furthermore, we find that female ES cells cultured in conventional serum plus LIF medium phenocopy male ES cells cultured in 2i/L. Mechanistically, we demonstrate that the inhibition of Mek1/2 is predominantly responsible for these effects, in part through the downregulation of DNA methyltransferases and their cofactors. Finally, we show that replacement of the Mek1/2 inhibitor with a Src inhibitor preserves the epigenetic and genomic integrity as well as the developmental potential of ES cells. Taken together, our data suggest that, although short-term suppression of Mek1/2 in ES cells helps to maintain an ICM-like epigenetic state, prolonged suppression results in irreversible changes that compromise their developmental potential.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/enzimologia , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Animais , Blastocisto , Instabilidade Cromossômica , Metilação de DNA , Feminino , Impressão Genômica , Cariotipagem , Masculino , CamundongosRESUMO
Human embryonic stem cells (hESCs) can be captured in a primed state in which they resemble the postimplantation epiblast, or in a naive state where they resemble the preimplantation epiblast. Naive-cell-specific culture conditions allow the study of preimplantation development ex vivo but reportedly lead to chromosomal abnormalities, which compromises their utility in research and potential therapeutic applications. Although MEK inhibition is essential for the naive state, here we show that reduced MEK inhibition facilitated the establishment and maintenance of naive hESCs that retained naive-cell-specific features, including global DNA hypomethylation, HERVK expression, and two active X chromosomes. We further show that hESCs cultured under these modified conditions proliferated more rapidly; accrued fewer chromosomal abnormalities; and displayed changes in the phosphorylation levels of MAPK components, regulators of DNA damage/repair, and cell cycle. We thus provide a simple modification to current methods that can enable robust growth and reduced genomic instability in naive hESCs.
Assuntos
Células-Tronco Embrionárias/metabolismo , Instabilidade Genômica , MAP Quinase Quinase Quinases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Metilação de DNA , Células-Tronco Embrionárias/enzimologia , Humanos , Proteoma , TranscriptomaRESUMO
The glandular stomach is composed of two regenerative compartments termed corpus and antrum, and our understanding of the transcriptional networks that maintain these tissues is incomplete. Here we show that cell types with equivalent functional roles in the corpus and antrum share similar transcriptional states including the poorly characterized stem cells of the isthmus region. To further study the isthmus, we developed a monolayer two-dimensional (2D) culture system that is continually maintained by Wnt-responsive isthmus-like cells capable of differentiating into several gastric cell types. Importantly, 2D cultures can be converted into conventional three-dimensional organoids, modelling the plasticity of gastric epithelial cells in vivo. Finally, we utilized the 2D culture system to show that Sox2 is both necessary and sufficient to generate enterochromaffin cells. Together, our data provide important insights into gastric homeostasis, establish a tractable culture system to capture isthmus cells and uncover a role for Sox2 in enterochromaffin cells.
Assuntos
Mucosa Gástrica , Estômago , Mucosa Gástrica/metabolismo , Diferenciação Celular , Células-Tronco/metabolismo , HomeostaseRESUMO
The epigenetic mechanisms that maintain differentiated cell states remain incompletely understood. Here we employed histone mutants to uncover a crucial role for H3K36 methylation in the maintenance of cell identities across diverse developmental contexts. Focusing on the experimental induction of pluripotency, we show that H3K36M-mediated depletion of H3K36 methylation endows fibroblasts with a plastic state poised to acquire pluripotency in nearly all cells. At a cellular level, H3K36M facilitates epithelial plasticity by rendering fibroblasts insensitive to TGFß signals. At a molecular level, H3K36M enables the decommissioning of mesenchymal enhancers and the parallel activation of epithelial/stem cell enhancers. This enhancer rewiring is Tet dependent and redirects Sox2 from promiscuous somatic to pluripotency targets. Our findings reveal a previously unappreciated dual role for H3K36 methylation in the maintenance of cell identity by integrating a crucial developmental pathway into sustained expression of cell-type-specific programmes, and by opposing the expression of alternative lineage programmes through enhancer methylation.
Assuntos
Epigênese Genética , Histonas , Metilação , Histonas/genética , Histonas/metabolismo , Diferenciação Celular/genética , Fibroblastos/metabolismo , Linhagem da Célula/genéticaRESUMO
Development and differentiation are associated with profound changes to histone modifications, yet their in vivo function remains incompletely understood. Here, we generated mouse models expressing inducible histone H3 lysine-to-methionine (K-to-M) mutants, which globally inhibit methylation at specific sites. Mice expressing H3K36M developed severe anaemia with arrested erythropoiesis, a marked haematopoietic stem cell defect, and rapid lethality. By contrast, mice expressing H3K9M survived up to a year and showed expansion of multipotent progenitors, aberrant lymphopoiesis and thrombocytosis. Additionally, some H3K9M mice succumbed to aggressive T cell leukaemia/lymphoma, while H3K36M mice exhibited differentiation defects in testis and intestine. Mechanistically, induction of either mutant reduced corresponding histone trimethylation patterns genome-wide and altered chromatin accessibility as well as gene expression landscapes. Strikingly, discontinuation of transgene expression largely restored differentiation programmes. Our work shows that individual chromatin modifications are required at several specific stages of differentiation and introduces powerful tools to interrogate their roles in vivo.
Assuntos
Epigênese Genética , Histonas/metabolismo , Leucemia de Células T/genética , Lisina/metabolismo , Metionina/metabolismo , Teratoma/genética , Animais , Transplante de Medula Óssea , Linhagem da Célula/genética , Modelos Animais de Doenças , Doxiciclina/farmacologia , Células Eritroides/metabolismo , Células Eritroides/patologia , Feminino , Granulócitos/metabolismo , Granulócitos/patologia , Histonas/genética , Leucemia de Células T/induzido quimicamente , Leucemia de Células T/metabolismo , Leucemia de Células T/patologia , Masculino , Metilação , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/patologia , Mutação , Transdução de Sinais , Análise de Sobrevida , Linfócitos T/metabolismo , Linfócitos T/patologia , Teratoma/induzido quimicamente , Teratoma/metabolismo , Teratoma/patologiaRESUMO
Post-transcriptional mechanisms have the potential to influence complex changes in gene expression, yet their role in cell fate transitions remains largely unexplored. Here, we show that suppression of the RNA helicase DDX6 endows human and mouse primed embryonic stem cells (ESCs) with a differentiation-resistant, "hyper-pluripotent" state, which readily reprograms to a naive state resembling the preimplantation embryo. We further demonstrate that DDX6 plays a key role in adult progenitors where it controls the balance between self-renewal and differentiation in a context-dependent manner. Mechanistically, DDX6 mediates the translational suppression of target mRNAs in P-bodies. Upon loss of DDX6 activity, P-bodies dissolve and release mRNAs encoding fate-instructive transcription and chromatin factors that re-enter the ribosome pool. Increased translation of these targets impacts cell fate by rewiring the enhancer, heterochromatin, and DNA methylation landscapes of undifferentiated cell types. Collectively, our data establish a link between P-body homeostasis, chromatin organization, and stem cell potency.
Assuntos
Diferenciação Celular/genética , Plasticidade Celular/genética , RNA Helicases DEAD-box/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Linhagem Celular , Montagem e Desmontagem da Cromatina/genética , RNA Helicases DEAD-box/genética , Metilação de DNA , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica/genética , Ontologia Genética , Homeostase/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/enzimologia , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína Homeobox Nanog/metabolismo , Organoides/citologia , Organoides/diagnóstico por imagem , Organoides/metabolismo , Biossíntese de Proteínas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/metabolismo , RNA-Seq , Ribonucleoproteínas/genética , Ribossomos/metabolismoRESUMO
The embryonic stem cell (ESC) transition from naive to primed pluripotency is marked by major changes in cellular properties and developmental potential. ISY1 regulates microRNA (miRNA) biogenesis, yet its role and relevance to ESC biology remain unknown. Here, we find that highly dynamic ISY1 expression during the naive-to-primed ESC transition defines a specific phase of "poised" pluripotency characterized by distinct miRNA and mRNA transcriptomes and widespread poised cell contribution to mouse chimeras. Loss- and gain-of-function experiments reveal that ISY1 promotes exit from the naive state and is necessary and sufficient to induce and maintain poised pluripotency, and that persistent ISY1 overexpression inhibits the transition from the naive to the primed state. We identify a large subset of ISY1-dependent miRNAs that can rescue the inability of miRNA-deficient ESCs to establish the poised state and transition to the primed state. Thus, dynamic ISY1 regulates poised pluripotency through miRNAs to control ESC fate.
Assuntos
Células-Tronco Embrionárias/metabolismo , MicroRNAs/metabolismo , Células A549 , Animais , Diferenciação Celular , Células Cultivadas , Células-Tronco Embrionárias/citologia , Feminino , Humanos , Masculino , Camundongos , Proteínas de Ligação a RNA/metabolismo , TransfecçãoRESUMO
Skeletal muscle harbors quiescent stem cells termed satellite cells and proliferative progenitors termed myoblasts, which play pivotal roles during muscle regeneration. However, current technology does not allow permanent capture of these cell populations in vitro. Here, we show that ectopic expression of the myogenic transcription factor MyoD, combined with exposure to small molecules, reprograms mouse fibroblasts into expandable induced myogenic progenitor cells (iMPCs). iMPCs express key skeletal muscle stem and progenitor cell markers including Pax7 and Myf5 and give rise to dystrophin-expressing myofibers upon transplantation in vivo. Notably, a subset of transplanted iMPCs maintain Pax7 expression and sustain serial regenerative responses. Similar to satellite cells, iMPCs originate from Pax7+ cells and require Pax7 itself for maintenance. Finally, we show that myogenic progenitor cell lines can be established from muscle tissue following small-molecule exposure alone. This study thus reports on a robust approach to derive expandable myogenic stem/progenitor-like cells from multiple cell types.
Assuntos
Reprogramação Celular , Fibroblastos/citologia , Músculo Esquelético/citologia , Células-Tronco/citologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Autorrenovação Celular/efeitos dos fármacos , Reprogramação Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Camundongos , Desenvolvimento Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/patologia , Distrofia Muscular Animal/patologia , Proteína MyoD/metabolismo , Fator de Transcrição PAX7/metabolismo , Regeneração/efeitos dos fármacos , Células Satélites de Músculo Esquelético/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Nicho de Células-Tronco/efeitos dos fármacos , Transplante de Células-Tronco , Células-Tronco/efeitos dos fármacos , TransgenesRESUMO
Blastocyst-derived embryonic stem cells (ESCs) and gonad-derived embryonic germ cells (EGCs) represent two classic types of pluripotent cell lines, yet their molecular equivalence remains incompletely understood. Here, we compare genome-wide methylation patterns between isogenic ESC and EGC lines to define epigenetic similarities and differences. Surprisingly, we find that sex rather than cell type drives methylation patterns in ESCs and EGCs. Cell fusion experiments further reveal that the ratio of X chromosomes to autosomes dictates methylation levels, with female hybrids being hypomethylated and male hybrids being hypermethylated. We show that the X-linked MAPK phosphatase DUSP9 is upregulated in female compared to male ESCs, and its heterozygous loss in female ESCs leads to male-like methylation levels. However, male and female blastocysts are similarly hypomethylated, indicating that sex-specific methylation differences arise in culture. Collectively, our data demonstrate the epigenetic similarity of sex-matched ESCs and EGCs and identify DUSP9 as a regulator of female-specific hypomethylation.
Assuntos
Fosfatases de Especificidade Dupla/metabolismo , Células-Tronco Pluripotentes/metabolismo , Cromossomo X/metabolismo , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Metilação de DNA/genética , Metilação de DNA/fisiologia , Fosfatases de Especificidade Dupla/genética , Células Germinativas Embrionárias/efeitos dos fármacos , Células Germinativas Embrionárias/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Impressão Genômica/genética , Masculino , Camundongos , Modelos Biológicos , Células-Tronco Pluripotentes/citologiaRESUMO
Loricrin is a major component of the cornified cell envelope, a highly insoluble structure composed of covalently cross-linked proteins. Although loricrin knockout mice only exhibit a mild transient phenotype at birth, they show a marked delay in the formation of an epidermal barrier in utero. We recently discovered that induction of a compensatory response to repair the defective barrier is initiated by amniotic fluid via activation of NF-E2-related factor 2 and identified Sprr2d and Sprr2h as direct transcriptional targets. Proteomic analysis suggested that other proteins were also incorporated into the loricrin knockout cell envelope, in addition to the small proline rich proteins. Here we present evidence suggesting that the late cornified envelope 1 proteins are also compensatory components as determined by their localization within the loricrin knockout cell envelope via immunoelectron microscopy. We also demonstrate that late cornified envelope 1 genes are upregulated at the transcriptional level in loricrin knockout mouse skin and confirm that late cornified envelope 1 genes are transcriptional targets of NRF2. Our present study further highlights the complexity and importance of a compensatory mechanism that evolved in terrestrial animals to ensure the formation of a functional epidermal barrier.
Assuntos
Proteínas Ricas em Prolina do Estrato Córneo/metabolismo , Epiderme/metabolismo , Proteínas de Membrana/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Motivos de Aminoácidos , Animais , Proteínas Ricas em Prolina do Estrato Córneo/genética , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Microscopia Imunoeletrônica , Fator 2 Relacionado a NF-E2/genética , Regiões Promotoras Genéticas , Proteômica , Transgenes , Regulação para CimaRESUMO
Sox2 expression marks gastric stem and progenitor cells, raising important questions regarding the genes regulated by Sox2 and the role of Sox2 itself during stomach homeostasis and disease. By using ChIP-seq analysis, we have found that the majority of Sox2 targets in gastric epithelial cells are tissue specific and related to functions such as endoderm development, Wnt signaling, and gastric cancer. Unexpectedly, we found that Sox2 itself is dispensable for gastric stem cell and epithelial self-renewal, yet Sox2(+) cells are highly susceptible to tumorigenesis in an Apc/Wnt-driven mouse model. Moreover, Sox2 loss enhances, rather than impairs, tumor formation in Apc-deficient gastric cells in vivo and in vitro by inducing Tcf/Lef-dependent transcription and upregulating intestinal metaplasia-associated genes, providing a mechanistic basis for the observed phenotype. Together, these data identify Sox2 as a context-dependent tumor suppressor protein that is dispensable for normal tissue regeneration but restrains stomach adenoma formation through modulation of Wnt-responsive and intestinal genes.
Assuntos
Adenoma/genética , Proteína da Polipose Adenomatosa do Colo/genética , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição SOXB1/genética , Neoplasias Gástricas/genética , Adenoma/metabolismo , Adenoma/patologia , Proteína da Polipose Adenomatosa do Colo/deficiência , Animais , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/genética , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Camundongos , Camundongos Transgênicos , Fatores de Transcrição SOXB1/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Via de Sinalização WntRESUMO
Brief expression of pluripotency-associated factors such as Oct4, Klf4, Sox2 and c-Myc (OKSM), in combination with differentiation-inducing signals, has been reported to trigger transdifferentiation of fibroblasts into other cell types. Here we show that OKSM expression in mouse fibroblasts gives rise to both induced pluripotent stem cells (iPSCs) and induced neural stem cells (iNSCs) under conditions previously shown to induce only iNSCs. Fibroblast-derived iNSC colonies silenced retroviral transgenes and reactivated silenced X chromosomes, both hallmarks of pluripotent stem cells. Moreover, lineage tracing with an Oct4-CreER labeling system demonstrated that virtually all iNSC colonies originated from cells transiently expressing Oct4, whereas ablation of Oct4(+) cells prevented iNSC formation. Lastly, an alternative transdifferentiation cocktail that lacks Oct4 and was reportedly unable to support induced pluripotency yielded iPSCs and iNSCs carrying the Oct4-CreER-derived lineage label. Together, these data suggest that iNSC generation from fibroblasts using OKSM and other pluripotency-related reprogramming factors requires passage through a transient iPSC state.
Assuntos
Transdiferenciação Celular/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Feminino , Fibroblastos/fisiologia , Inativação Gênica , Fator 4 Semelhante a Kruppel , Masculino , Camundongos , Camundongos Transgênicos , TransgenesRESUMO
The transcription factor Nrf2 is a key regulator of the cellular stress response, and pharmacological Nrf2 activation is a promising strategy for skin protection and cancer prevention. We show here that prolonged Nrf2 activation in keratinocytes causes sebaceous gland enlargement and seborrhea in mice due to upregulation of the growth factor epigen, which we identified as a novel Nrf2 target. This was accompanied by thickening and hyperkeratosis of hair follicle infundibula. These abnormalities caused dilatation of infundibula, hair loss, and cyst development upon aging. Upregulation of epigen, secretory leukocyte peptidase inhibitor (Slpi), and small proline-rich protein 2d (Sprr2d) in hair follicles was identified as the likely cause of infundibular acanthosis, hyperkeratosis, and cyst formation. These alterations were highly reminiscent to the phenotype of chloracne/"metabolizing acquired dioxin-induced skin hamartomas" (MADISH) patients. Indeed, SLPI, SPRR2, and epigen were strongly expressed in cysts of MADISH patients and upregulated by dioxin in human keratinocytes in an NRF2-dependent manner. These results identify novel Nrf2 activities in the pilosebaceous unit and point to a role of NRF2 in MADISH pathogenesis.
Assuntos
Cloracne/metabolismo , Queratinócitos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Células Cultivadas , Cloracne/genética , Modelos Animais de Doenças , Epigen/genética , Epigen/metabolismo , Folículo Piloso/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Fator 2 Relacionado a NF-E2/genética , Inibidor Secretado de Peptidases Leucocitárias/genética , Inibidor Secretado de Peptidases Leucocitárias/metabolismoRESUMO
Aurora kinase-A (Aurora-A) promotes timely entry into mitosis, centrosome maturation, and formation of bipolar spindles. To address the role of Aurora-A in skin development and homeostasis, we interbred a floxed Aurora-A (Aurora-A(fl)) mouse with the Cre-deleter strain, K14.Cre. Aurora-A(fl/fl);Krt14.Cre (Aurora-A(-/-)) mice died shortly after birth. These mice had translucent skin, and histological evaluation showed that the dorsal skin was very thin and fragile with frank erosions. Although the expression of the basal layer marker keratin 14 and the differentiation marker keratin 1 was evident in Aurora-A(-/-) epidermis, there was a marked reduction in the number of suprabasal layers and basal keratinocytes. Dye exclusion assays also showed defects in barrier function. Unlike wild-type cells, Aurora-A(-/-) basal progenitors were delayed in forming two layers at embryonic day (E)13.5 when embryonic skin begins to stratify. Increased numbers of mitotic cells, apoptotic bodies, and polyploid keratinocytes were evident in Aurora-A(-/-) epidermis, indicating that a deficiency in Aurora-A promotes aberrant mitosis, mitotic slippage, and cell death. Finally, Aurora-A(-/-) keratinocytes displayed centrosomal abnormalities that included centrosomes located at nonapical sites in basal cells. Thus, the deletion of Aurora-A in the developing epidermis alters centrosome function of basal keratinocytes and markedly impairs their ability to divide and stratify.
Assuntos
Divisão Celular/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Pele/enzimologia , Pele/crescimento & desenvolvimento , Animais , Apoptose/fisiologia , Aurora Quinase A , Aurora Quinases , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Divisão Celular/genética , Centrossomo/enzimologia , Deleção de Genes , Queratina-1 , Queratina-14/biossíntese , Queratinócitos/fisiologia , Queratinas Específicas do Cabelo/biossíntese , Camundongos , Poliploidia , Proteínas Serina-Treonina Quinases/genética , Pele/patologia , Células-Tronco/fisiologiaRESUMO
The loss of loricrin, a major component of the cornified envelope, results in a delay of epidermal barrier formation. Therefore, the living layers of the epidermis are aberrantly exposed to late-stage amniotic fluid, which may serve as the signal to upregulate genes that functionally compensate for the loss of loricrin. Consistent with this hypothesis, metabolomic studies revealed marked changes in amniotic fluid between E14.5 and E16.5 days postcoitum. In addition, we discovered that the Nrf2/Keap1 pathway detects these compositional changes and directly upregulates the expression of genes involved in the compensatory response, thus ensuring postnatal survival. In support of this finding, we demonstrate that genetically blocking the Nrf2 pathway abolishes the compensatory response and that preemptively activating Nrf2 pharmacologically rescues the delay in barrier formation in utero. Our findings reveal that the functions of Nrf2 and the composition of amniotic fluid have coevolved to ensure the formation of a functional barrier.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Líquido Amniótico/fisiologia , Proteínas do Citoesqueleto/metabolismo , Epiderme/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Queratinócitos/metabolismo , Proteínas de Membrana/fisiologia , Fator 2 Relacionado a NF-E2/metabolismo , Líquido Amniótico/química , Animais , Células Cultivadas , Proteínas Ricas em Prolina do Estrato Córneo/genética , Epiderme/metabolismo , Proteínas Filagrinas , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/fisiologia , Proteína 1 Associada a ECH Semelhante a Kelch , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Fator 2 Relacionado a NF-E2/genética , Regiões Promotoras Genéticas , Transdução de Sinais , Regulação para CimaRESUMO
The skin provides an efficient permeability barrier and protects from microbial invasion and oxidative stress. Here, we show that these essential functions are linked through the Nrf2 transcription factor. To test the hypothesis that activation of Nrf2 provides skin protection under stress conditions, we determined the consequences of pharmacological or genetic activation of Nrf2 in keratinocytes. Surprisingly, mice with enhanced Nrf2 activity in keratinocytes developed epidermal thickening, hyperkeratosis and inflammation resembling lamellar ichthyosis. This resulted from upregulation of the cornified envelope proteins small proline-rich proteins (Sprr) 2d and 2h and of secretory leukocyte peptidase inhibitor (Slpi), which we identified as novel Nrf2 targets in keratinocytes. Since Sprrs are potent scavengers of reactive oxygen species and since Slpi has antimicrobial activities, their upregulation contributes to Nrf2's protective function. However, it also caused corneocyte fragility and impaired desquamation, followed by alterations in the epidermal lipid barrier, inflammation and overexpression of mitogens that induced keratinocyte hyperproliferation. These results identify an unexpected role of Nrf2 in epidermal barrier function, which needs to be considered for pharmacological use of Nrf2 activators.