RESUMO
Cell proliferation is tightly controlled by inhibitors that block cell cycle progression until growth signals relieve this inhibition, allowing cells to divide. In several tissues, including the liver, cell proliferation is inhibited at mitosis by the transcriptional repressors E2F7 and E2F8, leading to formation of polyploid cells. Whether growth factors promote mitosis and cell cycle progression by relieving the E2F7/E2F8-mediated inhibition is unknown. We report here on a mechanism of cell division control in the postnatal liver, in which Wnt/ß-catenin signaling maintains active hepatocyte cell division through Tbx3, a Wnt target gene. The TBX3 protein directly represses transcription of E2f7 and E2f8, thereby promoting mitosis. This cascade of sequential transcriptional repressors, initiated by Wnt signals, provides a paradigm for exploring how commonly active developmental signals impact cell cycle completion.
Assuntos
Hepatócitos , Mitose , Proteínas Repressoras , Via de Sinalização Wnt , Animais , Hepatócitos/citologia , Hepatócitos/metabolismo , Camundongos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas com Domínio T/metabolismo , beta Catenina/metabolismoRESUMO
In the liver, Wnt/ß-catenin signaling is involved in regulating zonation and hepatocyte proliferation during homeostasis. We examined Wnt gene expression and signaling after injury, and we show by in situ hybridization that Wnts are activated by acute carbon tetrachloride (CCl4 ) toxicity. Following injury, peri-injury hepatocytes become Wnt-responsive, expressing the Wnt target gene axis inhibition protein 2 (Axin2). Lineage tracing of peri-injury Axin2+ hepatocytes shows that during recovery the injured parenchyma becomes repopulated and repaired by Axin2+ descendants. Using single-cell RNA sequencing, we show that endothelial cells are the major source of Wnts following acute CCl4 toxicity. Induced loss of ß-catenin in peri-injury hepatocytes results in delayed repair and ultimately injury-induced lethality, while loss of Wnt production from endothelial cells leads to a delay in the proliferative response after injury. Conclusion: Our findings highlight the importance of the Wnt/ß-catenin signaling pathway in restoring tissue integrity following acute liver toxicity and establish a role of endothelial cells as an important Wnt-producing regulator of liver tissue repair following localized liver injury.
Assuntos
Proteína Axina/genética , Regeneração Hepática/genética , Fígado/lesões , Proteínas Wnt/genética , Via de Sinalização Wnt/genética , beta Catenina/genética , Animais , Biópsia por Agulha , Tetracloreto de Carbono/farmacologia , Células Cultivadas , Modelos Animais de Doenças , Expressão Gênica/genética , Hepatócitos/citologia , Imuno-Histoquímica , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase/métodos , RNA/genética , Distribuição Aleatória , Valores de ReferênciaRESUMO
In the healthy adult liver, most hepatocytes proliferate minimally. However, upon physical or chemical injury to the liver, hepatocytes proliferate extensively in vivo under the direction of multiple extracellular cues, including Wnt and pro-inflammatory signals. Currently, liver organoids can be generated readily in vitro from bile-duct epithelial cells, but not hepatocytes. Here, we show that TNFα, an injury-induced inflammatory cytokine, promotes the expansion of hepatocytes in 3D culture and enables serial passaging and long-term culture for more than 6 months. Single-cell RNA sequencing reveals broad expression of hepatocyte markers. Strikingly, in vitro-expanded hepatocytes engrafted, and significantly repopulated, the injured livers of Fah-/- mice. We anticipate that tissue repair signals can be harnessed to promote the expansion of otherwise hard-to-culture cell-types, with broad implications.
Assuntos
Antígenos de Diferenciação/biossíntese , Técnicas de Cultura de Células , Proliferação de Células/efeitos dos fármacos , Hepatócitos/metabolismo , Esferoides Celulares/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Linhagem Celular Transformada , Células Hep G2 , Hepatócitos/transplante , Células Endoteliais da Veia Umbilical Humana , Humanos , Fígado/lesões , Fígado/metabolismo , Camundongos Knockout , Esferoides Celulares/transplante , Fatores de TempoRESUMO
The source of new hepatocytes in the uninjured liver has remained an open question. By lineage tracing using the Wnt-responsive gene Axin2 in mice, we identify a population of proliferating and self-renewing cells adjacent to the central vein in the liver lobule. These pericentral cells express the early liver progenitor marker Tbx3, are diploid, and thereby differ from mature hepatocytes, which are mostly polyploid. The descendants of pericentral cells differentiate into Tbx3-negative, polyploid hepatocytes, and can replace all hepatocytes along the liver lobule during homeostatic renewal. Adjacent central vein endothelial cells provide Wnt signals that maintain the pericentral cells, thereby constituting the niche. Thus, we identify a cell population in the liver that subserves homeostatic hepatocyte renewal, characterize its anatomical niche, and identify molecular signals that regulate its activity.
Assuntos
Proteína Axina/metabolismo , Diploide , Hepatócitos/citologia , Hepatócitos/metabolismo , Homeostase , Fígado/citologia , Animais , Biomarcadores/metabolismo , Linhagem da Célula , Proliferação de Células , Células Clonais/citologia , Células Clonais/metabolismo , Células Endoteliais/metabolismo , Feminino , Fígado/irrigação sanguínea , Masculino , Camundongos , Poliploidia , Regeneração , Coloração e Rotulagem , Nicho de Células-Tronco/fisiologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Proteínas com Domínio T/deficiência , Proteínas com Domínio T/metabolismo , Fatores de Tempo , Veias/citologia , Veias/metabolismo , Via de Sinalização WntRESUMO
Lipid-modified Wnt/Wingless (Wg) proteins can signal to their target cells in a short- or long-range manner. How these hydrophobic proteins travel through the extracellular environment remains an outstanding question. Here, we report on a Wg binding protein, Secreted Wg-interacting molecule (Swim), that facilitates Wg diffusion through the extracellular matrix. Swim, a putative member of the Lipocalin family of extracellular transport proteins, binds to Wg with nanomolar affinity in a lipid-dependent manner. In quantitative signaling assays, Swim is sufficient to maintain the solubility and activity of purified Wg. In Drosophila, swim RNAi phenotypes resemble wg loss-of-function phenotypes in long-range signaling. We propose that Swim is a cofactor that promotes long-range Wg signaling in vivo by maintaining the solubility of Wg.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Lipocalinas/metabolismo , Transdução de Sinais/fisiologia , Proteína Wnt1/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico/fisiologia , Primers do DNA/genética , Drosophila/genética , Proteínas de Drosophila/genética , Ensaio de Imunoadsorção Enzimática , Matriz Extracelular/metabolismo , Imuno-Histoquímica , Lipocalinas/genética , Dados de Sequência Molecular , Plasmídeos/genética , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de Sequência , Transdução de Sinais/genética , SolubilidadeRESUMO
Wnt/beta-catenin signals orchestrate cell fate and behavior throughout the animal kingdom. Aberrant Wnt signaling impacts nearly the entire spectrum of human disease, including birth defects, cancer, and osteoporosis. If Wnt signaling is to be effectively manipulated for therapeutic advantage, we first must understand how Wnt signals are normally controlled. Naked cuticle (Nkd) is a novel and evolutionarily conserved inducible antagonist of Wnt/beta-catenin signaling that is crucial for segmentation in the model genetic organism, the fruit fly Drosophila melanogaster. Nkd can bind and inhibit the Wnt signal transducer Dishevelled (Dsh), but the mechanism by which Nkd limits Wnt signaling in the fly embryo is not understood. Here we show that nkd mutants exhibit elevated levels of the beta-catenin homolog Armadillo but no alteration in Dsh abundance or distribution. In the fly embryo, Nkd and Dsh are predominantly cytoplasmic, although a recent report suggests that vertebrate Dsh requires nuclear localization for activity in gain-of-function assays. While Dsh-binding regions of Nkd contribute to its activity, we identify a conserved 30-amino-acid motif, separable from Dsh-binding regions, that is essential for Nkd function and nuclear localization. Replacement of the 30-aa motif with a conventional nuclear localization sequence rescued a small fraction of nkd mutant animals to adulthood. Our studies suggest that Nkd targets Dsh-dependent signal transduction steps in both cytoplasmic and nuclear compartments of cells receiving the Wnt signal.
Assuntos
Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/química , Drosophila/metabolismo , Sinais de Localização Nuclear/fisiologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Wnt/antagonistas & inibidores , Transporte Ativo do Núcleo Celular/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Motivos de Aminoácidos/fisiologia , Sequência de Aminoácidos , Animais , Anopheles/genética , Proteínas do Domínio Armadillo/metabolismo , Citoplasma/metabolismo , Proteínas Desgrenhadas , Drosophila/embriologia , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Embrião não Mamífero , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Mutantes/fisiologia , Mutação , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Distribuição Tecidual , Fatores de Transcrição/metabolismo , Proteínas Wnt/metabolismo , Proteína Wnt1 , beta Catenina/metabolismoRESUMO
The frizzled (fz) gene in Drosophila controls two distinct signaling pathways: it directs the planar cell polarization (PCP) of epithelia and it regulates cell fate decisions through Armadillo (Arm) by acting as a receptor for the Wnt protein Wingless (Wg). With the exception of dishevelled (dsh), the genes functioning in these two pathways are distinct. We have taken a genetic approach, based on a series of new and existing fz alleles, for identifying individual amino acids required for PCP or Arm signaling. For each allele, we have attempted to quantify the strength of signaling by phenotypic measurements. For PCP signaling, the defect was measured by counting the number of cells secreting multiple hairs in the wing. We then examined each allele for its ability to participate in Arm signaling by the rescue of fz mutant embryos with maternally provided fz function. For both PCP and Arm signaling we observed a broad range of phenotypes, but for every allele there is a strong correlation between its phenotypic strength in each pathway. Therefore, even though the PCP and Arm signaling pathways are genetically distinct, the set of signaling-defective fz alleles affected both pathways to a similar extent. This suggests that fz controls these two different signaling activities by a common mechanism. In addition, this screen yielded a set of missense mutations that identify amino acids specifically required for fz signaling function.