RESUMO
Wnt proteins modulate cell proliferation and differentiation and the self-renewal of stem cells by inducing ß-catenin-dependent signalling through the Wnt receptor frizzled (FZD) and the co-receptors LRP5 and LRP6 to regulate cell fate decisions and the growth and repair of several tissues. The 19 mammalian Wnt proteins are cross-reactive with the 10 FZD receptors, and this has complicated the attribution of distinct biological functions to specific FZD and Wnt subtype interactions. Furthermore, Wnt proteins are modified post-translationally by palmitoylation, which is essential for their secretion, function and interaction with FZD receptors. As a result of their acylation, Wnt proteins are very hydrophobic and require detergents for purification, which presents major obstacles to the preparation and application of recombinant Wnt proteins. This hydrophobicity has hindered the determination of the molecular mechanisms of Wnt signalling activation and the functional importance of FZD subtypes, and the use of Wnt proteins as therapeutic agents. Here we develop surrogate Wnt agonists, water-soluble FZD-LRP5/LRP6 heterodimerizers, with FZD5/FZD8-specific and broadly FZD-reactive binding domains. Similar to WNT3A, these Wnt agonists elicit a characteristic ß-catenin signalling response in a FZD-selective fashion, enhance the osteogenic lineage commitment of primary mouse and human mesenchymal stem cells, and support the growth of a broad range of primary human organoid cultures. In addition, the surrogates can be systemically expressed and exhibit Wnt activity in vivo in the mouse liver, regulating metabolic liver zonation and promoting hepatocyte proliferation, resulting in hepatomegaly. These surrogates demonstrate that canonical Wnt signalling can be activated by bi-specific ligands that induce receptor heterodimerization. Furthermore, these easily produced, non-lipidated Wnt surrogate agonists facilitate functional studies of Wnt signalling and the exploration of Wnt agonists for translational applications in regenerative medicine.
Assuntos
Transdução de Sinais , Proteínas Wnt/agonistas , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Animais , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Receptores Frizzled/metabolismo , Células HEK293 , Hepatócitos/citologia , Hepatomegalia/metabolismo , Hepatomegalia/patologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Intestinos/citologia , Ligantes , Fígado/metabolismo , Fígado/patologia , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Modelos Moleculares , Organoides/citologia , Organoides/metabolismo , Multimerização Proteica , Solubilidade , Técnicas de Cultura de TecidosRESUMO
Lgr5 was originally discovered as a common Wnt target gene in adult intestinal crypts and colon cancer. It was subsequently identified as an exquisite marker of multiple Wnt-driven adult stem cell types. Lgr5 and its homologs, Lgr4 and Lgr6, constitute the receptors for R-spondins, potent Wnt signal enhancers and stem cell growth factors. The Lgr5/R-spondin complex acts by neutralizing Rnf43 and Znrf3, two transmembrane E3 ligases that remove Wnt receptors from the stem cell surface. Rnf43/Znrf3 are themselves encoded by Wnt target genes and constitute a negative Wnt feedback loop. Thus, adult stem cells are controlled by an intricate interplay of potent Wnt agonists, antagonists, and anti-antagonists.
Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Trombospondinas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica , Humanos , Receptores Acoplados a Proteínas G/genética , Trombospondinas/genética , Ubiquitina-Proteína Ligases/genética , Via de Sinalização Wnt/genéticaRESUMO
Multiple myeloma (MM) is characterized by the expansion of malignant plasma cells in the bone marrow (BM). Most MMs display aberrant Wnt/ß-catenin signaling, which drives proliferation; however, they lack oncogenic Wnt pathway mutations, suggesting activation by autocrine Wnt ligands and/or paracrine Wnts from the BM microenvironment. Expression of the heparan sulfate (HS) proteoglycan syndecan-1 is a hallmark of MM. Syndecan-1 is a critical player in the complex reciprocal interaction between MM cells and their BM niche, mediating growth factor/cytokine binding and signaling by its HS chains. Here, by means of CRISPR/Cas9-mediated knockout and doxycycline-inducible short hairpin RNA-mediated knockdown of EXT1, a critical enzyme for HS polymerization, we demonstrate that the HS chains decorating syndecan-1 mediate aberrant Wnt pathway activation in MM. HS-deficient MM cells exhibited strongly decreased autocrine Wnt/ß-catenin pathway activity and reduced Wnt pathway-dependent proliferation. In addition, we demonstrate that Wnts bind to the HS side chains of syndecan-1 and that this binding contributes to paracrine Wnt pathway activation through the Wnt receptor Frizzled (Fzd). Furthermore, in an HS-dependent fashion, syndecan-1 also binds osteoblast-produced R-spondin, which represses Fzd degradation by activation of LGR4, an R-spondin receptor aberrantly expressed on MM cells. Costimulation with R-spondin and its binding to HS chains decorating syndecan-1 are indispensable for optimal stimulation of Wnt signaling in MM. Taken together, our results identify syndecan-1 as a crucial component of the Wnt signalosome in MM cells, binding Wnts and R-spondins to promote aberrant Wnt/ß-catenin signaling and cell growth, and suggest HS and its biosynthetic enzymes as potential targets in the treatment of MM.
Assuntos
Mieloma Múltiplo/metabolismo , Proteínas de Neoplasias/metabolismo , Sindecana-1/metabolismo , Trombospondinas/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Linhagem Celular Tumoral , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Heparitina Sulfato/genética , Heparitina Sulfato/metabolismo , Humanos , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Proteínas de Neoplasias/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Sindecana-1/genética , Trombospondinas/genética , Proteínas Wnt/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
The unrestrained growth of tumor cells is generally attributed to mutations in essential growth control genes, but tumor cells are also affected by, or even addicted to, signals from the microenvironment. As therapeutic targets, these extrinsic signals may be equally significant as mutated oncogenes. In multiple myeloma (MM), a plasma cell malignancy, most tumors display hallmarks of active Wnt signaling but lack activating Wnt-pathway mutations, suggesting activation by autocrine Wnt ligands and/or paracrine Wnts emanating from the bone marrow (BM) niche. Here, we report a pivotal role for the R-spondin/leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4) axis in driving aberrant Wnt/ß-catenin signaling in MM. We show that LGR4 is expressed by MM plasma cells, but not by normal plasma cells or B cells. This aberrant LGR4 expression is driven by IL-6/STAT3 signaling and allows MM cells to hijack R-spondins produced by (pre)osteoblasts in the BM niche, resulting in Wnt (co)receptor stabilization and a dramatically increased sensitivity to auto- and paracrine Wnts. Our study identifies aberrant R-spondin/LGR4 signaling with consequent deregulation of Wnt (co)receptor turnover as a driver of oncogenic Wnt/ß-catenin signaling in MM cells. These results advocate targeting of the LGR4/R-spondin interaction as a therapeutic strategy in MM.
Assuntos
Glicoproteínas de Membrana/metabolismo , Mieloma Múltiplo/metabolismo , Osteoblastos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Interleucina-6/metabolismo , Ligantes , Camundongos , Ligação Proteica/fisiologia , Fator de Transcrição STAT3/metabolismo , beta Catenina/metabolismoRESUMO
The adult stem cell marker Lgr5 and its relative Lgr4 are often co-expressed in Wnt-driven proliferative compartments. We find that conditional deletion of both genes in the mouse gut impairs Wnt target gene expression and results in the rapid demise of intestinal crypts, thus phenocopying Wnt pathway inhibition. Mass spectrometry demonstrates that Lgr4 and Lgr5 associate with the Frizzled/Lrp Wnt receptor complex. Each of the four R-spondins, secreted Wnt pathway agonists, can bind to Lgr4, -5 and -6. In HEK293 cells, RSPO1 enhances canonical WNT signals initiated by WNT3A. Removal of LGR4 does not affect WNT3A signalling, but abrogates the RSPO1-mediated signal enhancement, a phenomenon rescued by re-expression of LGR4, -5 or -6. Genetic deletion of Lgr4/5 in mouse intestinal crypt cultures phenocopies withdrawal of Rspo1 and can be rescued by Wnt pathway activation. Lgr5 homologues are facultative Wnt receptor components that mediate Wnt signal enhancement by soluble R-spondin proteins. These results will guide future studies towards the application of R-spondins for regenerative purposes of tissues expressing Lgr5 homologues.
Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Trombospondinas/metabolismo , Proteínas Wnt/metabolismo , Células-Tronco Adultas/metabolismo , Animais , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Receptores Frizzled/metabolismo , Deleção de Genes , Células HEK293 , Humanos , Camundongos , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/genética , Regeneração , Transdução de Sinais/genética , Proteínas Wnt/genética , Proteína Wnt3 , Proteína Wnt3ARESUMO
In colorectal cancer (CRC), a subpopulation of tumor cells, called cancer stem cell (CSC) fraction, is suggested to be responsible for tumor initiation, growth, and metastasis. The search for a reliable marker to identify these CSCs is ongoing as current markers, like CD44 and CD133, are more broadly expressed and therefore are not highly selective and currently also lack function in CSC biology. Here, we analyzed whether the Wnt target Lgr5, which has earlier been identified as a marker for murine intestinal stem cells, could potentially serve as a functional marker for CSCs. Fluorescence-activated cell sorting-based detection of Lgr5, using three newly developed antibodies, on primary colorectal tumor cells revealed a clear subpopulation of Epcam+ Lgr5+ cells. Similarly, primary CRC-derived spheroid cultures, known to be enriched for CSCs, contain high levels of Lgr5+ cells, which decrease upon in vitro differentiation of these CSCs. Selection of the Lgr5(high) CRC cells identified the clonogenic fraction in vitro as well as the tumorigenic population in vivo. Finally, we confirm that Lgr5 expression is dependent on the Wnt pathway and show that Lgr5 overexpression induces clonogenic growth. We thus provide evidence that Lgr5 is, next to a functional intestinal stem cell marker, a selective marker for human colorectal CSCs.
Assuntos
Anticorpos Monoclonais Murinos/química , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/patologia , Células-Tronco Neoplásicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Anticorpos Monoclonais Murinos/imunologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Diferenciação Celular , Separação Celular , Transformação Celular Neoplásica/metabolismo , Mapeamento de Epitopos , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Camundongos Nus , Mucina-2/metabolismo , Transplante de Neoplasias , Células-Tronco Neoplásicas/fisiologia , Ratos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/imunologia , Esferoides Celulares/metabolismo , Células Tumorais Cultivadas , Via de Sinalização WntRESUMO
Rapid identification of host genes essential for virus replication may expedite the generation of therapeutic interventions. Genetic screens are often performed in transformed cell lines that poorly represent viral target cells in vivo, leading to discoveries that may not be translated to the clinic. Intestinal organoids are increasingly used to model human disease and are amenable to genetic engineering. To discern which host factors are reliable anti-coronavirus therapeutic targets, we generate mutant clonal IOs for 19 host genes previously implicated in coronavirus biology. We verify ACE2 and DPP4 as entry receptors for SARS-CoV/SARS-CoV-2 and MERS-CoV respectively. SARS-CoV-2 replication in IOs does not require the endosomal Cathepsin B/L proteases, but specifically depends on the cell surface protease TMPRSS2. Other TMPRSS family members were not essential. The newly emerging coronavirus variant B.1.1.7, as well as SARS-CoV and MERS-CoV similarly depended on TMPRSS2. These findings underscore the relevance of non-transformed human models for coronavirus research, identify TMPRSS2 as an attractive pan-coronavirus therapeutic target, and demonstrate that an organoid knockout biobank is a valuable tool to investigate the biology of current and future emerging coronaviruses.
Assuntos
Enzima de Conversão de Angiotensina 2/genética , Bancos de Espécimes Biológicos , Sistemas CRISPR-Cas , Coronavirus , Dipeptidil Peptidase 4/genética , Organoides/metabolismo , Serina Endopeptidases/genética , COVID-19 , Linhagem Celular , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio , SARS-CoV-2 , Transcriptoma , Replicação ViralRESUMO
Mutational activation of the phosphatidylinositol 3-kinase (PI3K) pathway occurs in a wide variety of tumors, whereas activating Wnt pathway mutants are predominantly found in colon cancer. Because GSK3 is a key component of both pathways, it is widely assumed that active PI3K signaling feeds positively into the Wnt pathway by protein kinase B (PKB)-mediatefd inhibition of GSK3. In addition, PKB has been proposed to modulate the canonical Wnt signaling through direct stabilization and nuclear localization of beta-catenin. Here, we show that compartmentalization by Axin of GSK3 prohibits cross-talk between the PI3K and Wnt pathways and that Wnt-mediated transcriptional activity is not modulated by activation of the PI3K/PKB pathway.
Assuntos
Caenorhabditis elegans/metabolismo , Núcleo Celular/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans , Linhagem Celular , Núcleo Celular/genética , Ativação Enzimática/fisiologia , Humanos , Fosfatidilinositol 3-Quinases/genética , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transcrição Gênica/fisiologia , Proteínas Wnt/genética , beta Catenina/genéticaRESUMO
Modulation of Wnt signaling has untapped potential in regenerative medicine due to its essential functions in stem cell homeostasis. However, Wnt lipidation and Wnt-Frizzled (Fzd) cross-reactivity have hindered translational Wnt applications. Here, we designed and engineered water-soluble, Fzd subtype-specific "next-generation surrogate" (NGS) Wnts that hetero-dimerize Fzd and Lrp6. NGS Wnt supports long-term expansion of multiple different types of organoids, including kidney, colon, hepatocyte, ovarian, and breast. NGS Wnts are superior to Wnt3a conditioned media in organoid expansion and single-cell organoid outgrowth. Administration of Fzd subtype-specific NGS Wnt in vivo reveals that adult intestinal crypt proliferation can be promoted by agonism of Fzd5 and/or Fzd8 receptors, while a broad spectrum of Fzd receptors can induce liver zonation. Thus, NGS Wnts offer a unified organoid expansion protocol and a laboratory "tool kit" for dissecting the functions of Fzd subtypes in stem cell biology.
Assuntos
Receptores Frizzled , Organoides , Hepatócitos , Células-Tronco , Via de Sinalização WntRESUMO
The intestinal epithelium is a self-renewing tissue that represents a unique model for studying interconnected cellular processes such as proliferation, differentiation, cell migration and carcinogenesis. This review covers work from the past decade and highlights the importance of the canonical Wnt pathway in regulating multiple aspects of intestinal homeostasis. Numerous in vivo studies combined with gene profiling experiments have shown that Wnt signaling promotes maintenance of epithelial stem cells and early progenitors by driving transcription of genes associated with proliferation. These studies also revealed strong similarities between the genetic program initiated by Wnt signals in normal crypt progenitors and in colorectal cancer cells. More recently it has become apparent that Wnts do not act alone but rather cooperate with Notch signals in maintaining progenitor cell populations. Processes associated with differentiated epithelial cells also appear to be regulated by Wnt signals. For instance, Paneth cells employ active Wnt signals for terminal differentiation. Moreover, through transcriptional regulation of members of the Eph and Ephrin families, Wnt signaling promotes compartmentalization of epithelial cells along the crypt-villus axis. The Eph/Ephrin system also operates to limit progression of colorectal cancer beyond the early stages.
Assuntos
Neoplasias Colorretais/genética , Mucosa Intestinal/metabolismo , Transdução de Sinais , Proteínas Wnt/fisiologia , Animais , Colo/citologia , Neoplasias Colorretais/metabolismo , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/embriologia , Intestinos/embriologia , Camundongos , Mutação , Receptores Notch/metabolismo , Células-Tronco/metabolismoRESUMO
Zinc RING finger 3 (ZNRF3) and its homolog RING finger 43 (RNF43) antagonize Wnt signaling in adult stem cells by ubiquitinating Frizzled receptors (FZD), which leads to endocytosis of the Wnt receptor. Conversely, binding of ZNRF3/RNF43 to LGR4-6 - R-spondin blocks Frizzled ubiquitination and enhances Wnt signaling. Here, we present crystal structures of the ZNRF3 ectodomain and its complex with R-spondin 1 (RSPO1). ZNRF3 binds RSPO1 and LGR5-RSPO1 with micromolar affinity via RSPO1 furin-like 1 (Fu1) domain. Anonychia-related mutations in RSPO4 support the importance of the observed interface. The ZNRF3-RSPO1 structure resembles that of LGR5-RSPO1-RNF43, though Fu2 of RSPO1 is variably oriented. The ZNRF3-binding site overlaps with trans-interactions observed in 2:2 LGR5-RSPO1 complexes, thus binding of ZNRF3/RNF43 would disrupt such an arrangement. Sequence conservation suggests a single ligand-binding site on ZNRF3, consistent with the proposed competing binding role of ZNRF3/RNF43 in Wnt signaling.
Assuntos
Complexos Multiproteicos/química , Trombospondinas/química , Ubiquitina-Proteína Ligases/química , Via de Sinalização Wnt , Células-Tronco Adultas/metabolismo , Animais , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Humanos , Camundongos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Estrutura Quaternária de Proteína , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Trombospondinas/genética , Trombospondinas/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Leucine-rich repeat-containing G protein-coupled receptors 4-6 (LGR4-LGR6) are receptors for R-spondins, potent Wnt agonists that exert profound trophic effects on Wnt-driven stem cells compartments. We present crystal structures of a signaling-competent fragment of R-spondin 1 (Rspo1) at a resolution of 2.0 Å and its complex with the LGR5 ectodomain at a resolution of 3.2 Å. Ecto-LGR5 binds Rspo1 at its concave leucine-rich-repeat (LRR) surface, forming a dimeric 2:2 complex. Fully conserved residues on LGR4-LGR6 explain promiscuous binding of R-spondins. A phenylalanine clamp formed by Rspo1 Phe106 and Phe110 pinches Ala190 of LGR5 and is critical for binding. Mutations related to congenital anonychia reduce signaling, but not binding of Rspo1 to LGR5. Furthermore, antibody binding to the extended loop of the C-terminal LRR cap of LGR5 activates signaling in a ligand-independent manner. Thus, our data reveal binding of R-spondins to conserved sites on LGR4-LGR6 and, in analogy to FSHR and related receptors, suggest a direct signaling role for LGR4-LGR6 in addition to its formation of Wnt receptor and coreceptor complexes.
Assuntos
Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Trombospondinas/química , Trombospondinas/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Modelos Moleculares , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , Relação Estrutura-Atividade , Trombospondinas/genética , TransfecçãoRESUMO
The four vertebrate R-spondin proteins are secreted agonists of the canonical Wnt/ß-catenin signaling pathway. These proteins are approximately 35 kDa, and are characterized by two amino-terminal furin-like repeats, which are necessary and sufficient for Wnt signal potentiation, and a thrombospondin domain situated more towards the carboxyl terminus that can bind matrix glycosaminoglycans and/or proteoglycans. Although R-spondins are unable to initiate Wnt signaling, they can potently enhance responses to low-dose Wnt proteins. In humans, rare disruptions of the gene encoding R-spondin1 cause a syndrome of XX sex reversal (phenotypic male), palmoplantar keratosis (a thickening of the palms and soles caused by excess keratin formation) and predisposition to squamous cell carcinoma of the skin. Mutations in the gene encoding R-spondin4 cause anonychia (absence or hypoplasia of nails on fingers and toes). Recently, leucine-rich repeat-containing G-protein-coupled receptor (Lgr)4, Lgr5 and Lgr6, three closely related orphans of the leucine-rich repeat family of G-protein-coupled receptors, have been identified as receptors for R-spondins. Lgr5 and Lgr6 are markers for adult stem cells. Because R-spondins are potent stimulators of adult stem cell proliferation in vivo and in vitro, these findings might guide the therapeutic use of R-spondins in regenerative medicine.
Assuntos
Família Multigênica , Mutação , Trombospondinas/genética , Transtornos Testiculares 46, XX do Desenvolvimento Sexual/genética , Animais , Carcinoma de Células Escamosas/genética , Humanos , Unhas Malformadas/congênito , Unhas Malformadas/genética , Receptores Acoplados a Proteínas G/genética , Neoplasias Cutâneas/genética , Proteínas Wnt/genética , Via de Sinalização Wnt/genética , beta Catenina/genéticaRESUMO
Peyer's patches consist of domains of specialized intestinal epithelium overlying gut-associated lymphoid tissue (GALT). Luminal antigens reach the GALT by translocation through epithelial gatekeeper cells, the so-called M cells. We recently demonstrated that all epithelial cells required for the digestive functions of the intestine are generated from Lgr5-expressing stem cells. Here, we show that M cells also derive from these crypt-based Lgr5 stem cells. The Ets family transcription factor SpiB, known to control effector functions of bone marrow-derived immune cells, is specifically expressed in M cells. In SpiB(-/-) mice, M cells are entirely absent, which occurs in a cell-autonomous fashion. It has been shown that Tnfsf11 (RankL) can induce M cell development in vivo. We show that in intestinal organoid ("minigut") cultures, stimulation with RankL induces SpiB expression within 24 h and expression of other M cell markers subsequently. We conclude that RankL-induced expression of SpiB is essential for Lgr5 stem cell-derived epithelial precursors to develop into M cells.
Assuntos
Nódulos Linfáticos Agregados/citologia , Nódulos Linfáticos Agregados/metabolismo , Proteínas Proto-Oncogênicas c-ets/metabolismo , Ligante RANK/metabolismo , Receptores Acoplados a Proteínas G/biossíntese , Animais , Diferenciação Celular , Proteínas de Ligação a DNA/biossíntese , Desenvolvimento Embrionário , Proteínas de Fluorescência Verde/genética , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Intestinos/embriologia , Camundongos , Camundongos Knockout , Nódulos Linfáticos Agregados/embriologia , Células-Tronco/metabolismo , Fatores de Transcrição/biossínteseRESUMO
Barrett's esophagus (BE) affects approximately 2% of the Western population and progresses to esophageal adenocarcinoma (EAC) in 0.5% of these patients each year. In BE, the stratified epithelium is replaced by an intestinal-type epithelium owing to chronic gastroduodenal reflux. Since self-renewal of intestinal crypts is driven by Notch signaling, we investigated whether this pathway was active in the proliferative crypts of BE. Immunohistochemistry confirmed the presence of an intact and activated Notch signaling pathway in metaplastic BE epithelium, but not in the normal human esophagus. Similar observations were made in two well-known human Barrett's-derived EAC cell lines, OE33 and SKGT-5. We then sought to investigate the effects of Notch inhibition by systemic treatment with a gamma-secretase inhibitor in a well-validated rodent model for BE. As we have shown previously in normal intestinal epithelium, Notch inhibition converted the proliferative Barrett's epithelial cells into terminally differentiated goblet cells, whereas the squamous epithelium remained intact. These data imply that local application of gamma-secretase inhibitors may present a simple therapeutic strategy for this increasingly common pre-malignant condition.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Esôfago de Barrett/enzimologia , Esôfago de Barrett/patologia , Epitélio/patologia , Células Caliciformes/patologia , Mitose , Animais , Biópsia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Dibenzazepinas/farmacologia , Modelos Animais de Doenças , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/metabolismo , Humanos , Metaplasia , Mitose/efeitos dos fármacos , Ratos , Receptores Notch/antagonistas & inibidores , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Developmental studies in model organisms have revealed that cell fate decisions are governed by only a handful of highly conserved signal transduction cascades. Recent data indicate that at least two of these, the Wnt and the Notch cascades, have been recruited by the vertebrate immune system to control early lymphopoiesis.
Assuntos
Diferenciação Celular , Linfócitos/citologia , Linfócitos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Proteínas de Peixe-Zebra , Animais , Linhagem da Célula , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Fator 1 de Ligação ao Facilitador Linfoide , Camundongos , Camundongos Knockout , Timo/citologia , Timo/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas WntRESUMO
The transactivation of TCF target genes induced by Wnt pathway mutations constitutes the primary transforming event in colorectal cancer (CRC). We show that disruption of beta-catenin/TCF-4 activity in CRC cells induces a rapid G1 arrest and blocks a genetic program that is physiologically active in the proliferative compartment of colon crypts. Coincidently, an intestinal differentiation program is induced. The TCF-4 target gene c-MYC plays a central role in this switch by direct repression of the p21(CIP1/WAF1) promoter. Following disruption of beta-catenin/TCF-4 activity, the decreased expression of c-MYC releases p21(CIP1/WAF1) transcription, which in turn mediates G1 arrest and differentiation. Thus, the beta-catenin/TCF-4 complex constitutes the master switch that controls proliferation versus differentiation in healthy and malignant intestinal epithelial cells.