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1.
Nature ; 545(7653): 238-242, 2017 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-28467820

RESUMO

The canonical Wnt/ß-catenin signalling pathway governs diverse developmental, homeostatic and pathological processes. Palmitoylated Wnt ligands engage cell-surface frizzled (FZD) receptors and LRP5 and LRP6 co-receptors, enabling ß-catenin nuclear translocation and TCF/LEF-dependent gene transactivation. Mutations in Wnt downstream signalling components have revealed diverse functions thought to be carried out by Wnt ligands themselves. However, redundancy between the 19 mammalian Wnt proteins and 10 FZD receptors and Wnt hydrophobicity have made it difficult to attribute these functions directly to Wnt ligands. For example, individual mutations in Wnt ligands have not revealed homeostatic phenotypes in the intestinal epithelium-an archetypal canonical, Wnt pathway-dependent, rapidly self-renewing tissue, the regeneration of which is fueled by proliferative crypt Lgr5+ intestinal stem cells (ISCs). R-spondin ligands (RSPO1-RSPO4) engage distinct LGR4-LGR6, RNF43 and ZNRF3 receptor classes, markedly potentiate canonical Wnt/ß-catenin signalling, and induce intestinal organoid growth in vitro and Lgr5+ ISCs in vivo. However, the interchangeability, functional cooperation and relative contributions of Wnt versus RSPO ligands to in vivo canonical Wnt signalling and ISC biology remain unknown. Here we identify the functional roles of Wnt and RSPO ligands in the intestinal crypt stem-cell niche. We show that the default fate of Lgr5+ ISCs is to differentiate, unless both RSPO and Wnt ligands are present. However, gain-of-function studies using RSPO ligands and a new non-lipidated Wnt analogue reveal that these ligands have qualitatively distinct, non-interchangeable roles in ISCs. Wnt proteins are unable to induce Lgr5+ ISC self-renewal, but instead confer a basal competency by maintaining RSPO receptor expression that enables RSPO ligands to actively drive and specify the extent of stem-cell expansion. This functionally non-equivalent yet cooperative interaction between Wnt and RSPO ligands establishes a molecular precedent for regulation of mammalian stem cells by distinct priming and self-renewal factors, with broad implications for precise control of tissue regeneration.


Assuntos
Autorrenovação Celular , Intestinos/citologia , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Trombospondinas/metabolismo , Proteínas Wnt/metabolismo , Animais , Linhagem da Célula , Proliferação de Células , Feminino , Humanos , Ligantes , Masculino , Camundongos , Organoides/citologia , Organoides/crescimento & desenvolvimento , Análise de Célula Única , Nicho de Células-Tronco , Transcriptoma , Ubiquitina-Proteína Ligases/metabolismo , beta Catenina/metabolismo
2.
Gastroenterology ; 145(2): 383-95.e1-21, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23644405

RESUMO

BACKGROUND & AIMS: Identification of intestinal stem cells (ISCs) has relied heavily on the use of transgenic reporters in mice, but this approach is limited by mosaic expression patterns and difficult to directly apply to human tissues. We sought to identify reliable surface markers of ISCs and establish a robust functional assay to characterize ISCs from mouse and human tissues. METHODS: We used immunohistochemistry, real-time reverse-transcription polymerase chain reaction, and fluorescence-activated cell sorting (FACS) to analyze intestinal epithelial cells isolated from mouse and human intestinal tissues. We compared different combinations of surface markers among ISCs isolated based on expression of Lgr5-green fluorescent protein. We developed a culture protocol to facilitate the identification of functional ISCs from mice and then tested the assay with human intestinal crypts and putative ISCs. RESULTS: CD44(+)CD24(lo)CD166(+) cells, isolated by FACS from mouse small intestine and colon, expressed high levels of stem cell-associated genes. Transit-amplifying cells and progenitor cells were then excluded based on expression of GRP78 or c-Kit. CD44(+)CD24(lo)CD166(+) GRP78(lo/-) putative stem cells from mouse small intestine included Lgr5-GFP(hi) and Lgr5-GFP(med/lo) cells. Incubation of these cells with the GSK inhibitor CHIR99021 and the E-cadherin stabilizer Thiazovivin resulted in colony formation by 25% to 30% of single-sorted ISCs. CONCLUSIONS: We developed a culture protocol to identify putative ISCs from mouse and human tissues based on cell surface markers. CD44(+)CD24(lo)CD166(+), GRP78(lo/-), and c-Kit(-) facilitated identification of putative stem cells from the mouse small intestine and colon, respectively. CD44(+)CD24(-/lo)CD166(+) also identified putative human ISCs. These findings will facilitate functional studies of mouse and human ISCs.


Assuntos
Células-Tronco Adultas/metabolismo , Antígenos de Superfície/metabolismo , Mucosa Intestinal/citologia , Molécula de Adesão de Leucócito Ativado/metabolismo , Animais , Antígeno CD24/metabolismo , Técnicas de Cultura de Células , Colo/citologia , Ensaio de Unidades Formadoras de Colônias , Chaperona BiP do Retículo Endoplasmático , Citometria de Fluxo , Proteínas de Choque Térmico/genética , Humanos , Receptores de Hialuronatos/metabolismo , Intestino Delgado/citologia , Camundongos , Proteínas Proto-Oncogênicas c-kit/metabolismo
3.
Gastroenterology ; 139(6): 2072-2082.e5, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20826154

RESUMO

BACKGROUND & AIMS: CD166 (also called activated leukocyte cell adhesion molecule [ALCAM]) is a marker of colorectal cancer (CRC) stem cells; it is expressed by aggressive tumors. Although the presence of CD166 at the tumor cell surface has been correlated with shortened survival, little is known about its function and expression in normal intestinal epithelia. METHODS: We characterized the expression pattern of CD166 in normal intestinal tissue samples from humans and mice using immunohistochemisty, flow cytometry, and quantitative reverse-transcriptase polymerase chain reaction. Human and mouse intestinal tumors were also analyzed. RESULTS: CD166 was expressed on the surface of epithelial cells within the stem cell niche and along the length of the intestine; expression was conserved across species. In the small intestine, CD166 was observed on crypt-based Paneth cells and intervening crypt-based columnar cells (putative stem cells). A subset of CD166-positive, crypt-based columnar cells coexpressed the stem cell markers Lgr5, Musashi-1, or Dcamkl-1. CD166 was located in the cytoplasm and at the surface of cells within human CRC tumors. CD166-positive cells were also detected in benign adenomas in mice; rare cells coexpressed CD166 and CD44 or epithelial-specific antigen. CONCLUSIONS: CD166 is highly expressed within the endogenous intestinal stem cell niche. CD166-positive cells appear at multiple stages of intestinal carcinoma progression, including benign and metastatic tumors. Further studies should investigate the function of CD166 in stem cells and the stem cell niche, which might have implications for normal intestinal homeostasis. CD166 has potential as a therapeutic target for CRC.


Assuntos
Adenocarcinoma/metabolismo , Antígenos CD/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Neoplasias Colorretais/metabolismo , Células Epiteliais/metabolismo , Proteínas Fetais/metabolismo , Mucosa Intestinal/metabolismo , Células-Tronco/metabolismo , Adenocarcinoma/secundário , Animais , Biomarcadores Tumorais/metabolismo , Biópsia , Colo/citologia , Colo/metabolismo , Neoplasias Colorretais/patologia , Células Epiteliais/citologia , Homeostase/fisiologia , Humanos , Mucosa Intestinal/citologia , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Células-Tronco/citologia
5.
BMC Gastroenterol ; 8: 57, 2008 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-19055726

RESUMO

BACKGROUND: The canonical Wnt signaling pathway is a known regulator of cell proliferation during development and maintenance of the intestinal epithelium. Perturbations in this pathway lead to aberrant epithelial proliferation and intestinal cancer. In the mature intestine, proliferation is confined to the relatively quiescent stem cells and the rapidly cycling transient-amplifying cells in the intestinal crypts. Although the Wnt signal is believed to regulate all proliferating intestinal cells, surprisingly, this has not been thoroughly demonstrated. This important determination has implications on intestinal function, especially during epithelial expansion and regeneration, and warrants an extensive characterization of Wnt-activated cells. METHODS: To identify intestinal epithelial cells that actively receive a Wnt signal, we analyzed intestinal Wnt-reporter expression patterns in two different mouse lines using immunohistochemistry, enzymatic activity, in situ hybridization and qRT-PCR, then corroborated results with reporter-independent analyses. Wnt-receiving cells were further characterized for co-expression of proliferation markers, putative stem cell markers and cellular differentiation markers using an immunohistochemical approach. Finally, to demonstrate that Wnt-reporter mice have utility in detecting perturbations in intestinal Wnt signaling, the reporter response to gamma-irradiation was examined. RESULTS: Wnt-activated cells were primarily restricted to the base of the small intestinal and colonic crypts, and were highest in numbers in the proximal small intestine, decreasing in frequency in a gradient toward the large intestine. Interestingly, the majority of the Wnt-reporter-expressing cells did not overlap with the transient-amplifying cell population. Further, while Wnt-activated cells expressed the putative stem cell marker Musashi-1, they did not co-express DCAMKL-1 or cell differentiation markers. Finally, gamma-irradiation stimulated an increase in Wnt-activated intestinal crypt cells. CONCLUSION: We show, for the first time, detailed characterization of the intestine from Wnt-reporter mice. Further, our data show that the majority of Wnt-receiving cells reside in the stem cell niche of the crypt base and do not extend into the proliferative transient-amplifying cell population. We also show that the Wnt-reporter mice can be used to detect changes in intestinal epithelial Wnt signaling upon physiologic injury. Our findings have an important impact on understanding the regulation of the intestinal stem cell hierarchy during homeostasis and in disease states.


Assuntos
Perfilação da Expressão Gênica , Genes Reporter/genética , Homeostase/genética , Mucosa Intestinal/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Animais , Proliferação de Células , Colo/citologia , Colo/metabolismo , Colo/efeitos da radiação , Raios gama , Regulação da Expressão Gênica/efeitos da radiação , Homeostase/fisiologia , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Intestino Delgado/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Células-Tronco/efeitos da radiação
6.
Artigo em Inglês | MEDLINE | ID: mdl-29928673

RESUMO

BACKGROUND & AIMS: Continual renewal of the intestinal epithelium is dependent on active- and slow-cycling stem cells that are confined to the crypt base. Tight regulation of these stem cell populations maintains homeostasis by balancing proliferation and differentiation to support critical intestinal functions. The hierarchical relation of discrete stem cell populations in homeostasis or during regenerative epithelial repair remains controversial. Although recent studies have supported a model for the active-cycling leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5)+ intestinal stem cell (ISC) functioning upstream of the slow-cycling B lymphoma Mo-MLV insertion region 1 homolog (Bmi1)-expressing cell, other studies have reported the opposite relation. Tools that facilitate simultaneous analyses of these populations are required to evaluate their coordinated function. METHODS: We used novel monoclonal antibodies (mAbs) raised against murine intestinal epithelial cells in conjunction with ISC-green fluorescent protein (GFP) reporter mice to analyze relations between ISC populations by microscopy. Ex vivo 3-dimensional cultures, flow cytometry, and quantitative reverse-transcription polymerase chain reaction analyses were performed. RESULTS: Two novel mAbs recognized distinct subpopulations of the intestinal epithelium and when used in combination permitted isolation of discrete Lgr5GFP and Bmi1GFP-enriched populations with stem activity. Growth from singly isolated Lgr5GFP ISCs gave rise to small spheroids. Spheroids did not express Lgr5GFP and instead up-regulated Bmi1GFP expression. Conversely, Bmi1-derived spheroids initiated Lgr5GFP expression as crypt domains were established. CONCLUSIONS: These data showed the functional utility of murine mAbs in the isolation and investigation of Lgr5GFP and Bmi1GFP ISC-enriched populations. Ex vivo analyses showed hierarchical plasticity between different ISC-expressing states; specifically Lgr5GFP ISCs gave rise to Bmi1GFP cells, and vice versa. These data highlight the impact of temporal and physiological context on unappreciated interactions between Lgr5GFP and Bmi1GFP cells during crypt formation.

7.
Sci Adv ; 4(9): eaat7828, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30214939

RESUMO

High lethality rates associated with metastatic cancer highlight an urgent medical need for improved understanding of biologic mechanisms driving metastatic spread and identification of biomarkers predicting late-stage progression. Numerous neoplastic cell intrinsic and extrinsic mechanisms fuel tumor progression; however, mechanisms driving heterogeneity of neoplastic cells in solid tumors remain obscure. Increased mutational rates of neoplastic cells in stressed environments are implicated but cannot explain all aspects of tumor heterogeneity. We present evidence that fusion of neoplastic cells with leukocytes (for example, macrophages) contributes to tumor heterogeneity, resulting in cells exhibiting increased metastatic behavior. Fusion hybrids (cells harboring hematopoietic and epithelial properties) are readily detectible in cell culture and tumor-bearing mice. Further, hybrids enumerated in peripheral blood of human cancer patients correlate with disease stage and predict overall survival. This unique population of neoplastic cells provides a novel biomarker for tumor staging, as well as a potential therapeutic target for intervention.


Assuntos
Carcinoma Ductal Pancreático/patologia , Células Neoplásicas Circulantes/patologia , Neoplasias Pancreáticas/patologia , Animais , Biomarcadores Tumorais/sangue , Carcinoma Ductal Pancreático/mortalidade , Fusão Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Células Epiteliais/patologia , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Células Híbridas , Cariotipagem , Macrófagos/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias Pancreáticas/mortalidade , Microambiente Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Cell Mol Gastroenterol Hepatol ; 3(3): 389-409, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28462380

RESUMO

BACKGROUND & AIMS: Intestinal epithelial homeostasis is maintained by active-cycling and slow-cycling stem cells confined within an instructive crypt-based niche. Exquisite regulating of these stem cell populations along the proliferation-to-differentiation axis maintains a homeostatic balance to prevent hyperproliferation and cancer. Although recent studies focus on how secreted ligands from mesenchymal and epithelial populations regulate intestinal stem cells (ISCs), it remains unclear what role cell adhesion plays in shaping the regulatory niche. Previously we have shown that the cell adhesion molecule and cancer stem cell marker, CD166/ALCAM (activated leukocyte cell adhesion molecule), is highly expressed by both active-cycling Lgr5+ ISCs and adjacent Paneth cells within the crypt base, supporting the hypothesis that CD166 functions to mediate ISC maintenance and signal coordination. METHODS: Here we tested this hypothesis by analyzing a CD166-/- mouse combined with immunohistochemical, flow cytometry, gene expression, and enteroid culture. RESULTS: We found that animals lacking CD166 expression harbored fewer active-cycling Lgr5+ ISCs. Homeostasis was maintained by expansion of the transit-amplifying compartment and not by slow-cycling Bmi1+ ISC stimulation. Loss of active-cycling ISCs was coupled with deregulated Paneth cell homeostasis, manifested as increased numbers of immature Paneth progenitors due to decreased terminal differentiation, linked to defective Wnt signaling. CD166-/- Paneth cells expressed reduced Wnt3 ligand expression and depleted nuclear ß-catenin. CONCLUSIONS: These data support a function for CD166 as an important cell adhesion molecule that shapes the signaling microenvironment by mediating ISC-niche cell interactions. Furthermore, loss of CD166 expression results in decreased ISC and Paneth cell homeostasis and an altered Wnt microenvironment.

9.
Cell Stem Cell ; 21(1): 78-90.e6, 2017 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-28686870

RESUMO

Several cell populations have been reported to possess intestinal stem cell (ISC) activity during homeostasis and injury-induced regeneration. Here, we explored inter-relationships between putative mouse ISC populations by comparative RNA-sequencing (RNA-seq). The transcriptomes of multiple cycling ISC populations closely resembled Lgr5+ ISCs, the most well-defined ISC pool, but Bmi1-GFP+ cells were distinct and enriched for enteroendocrine (EE) markers, including Prox1. Prox1-GFP+ cells exhibited sustained clonogenic growth in vitro, and lineage-tracing of Prox1+ cells revealed long-lived clones during homeostasis and after radiation-induced injury in vivo. Single-cell mRNA-seq revealed two subsets of Prox1-GFP+ cells, one of which resembled mature EE cells while the other displayed low-level EE gene expression but co-expressed tuft cell markers, Lgr5 and Ascl2, reminiscent of label-retaining secretory progenitors. Our data suggest that the EE lineage, including mature EE cells, comprises a reservoir of homeostatic and injury-inducible ISCs, extending our understanding of cellular plasticity and stemness.


Assuntos
Antígenos de Diferenciação/metabolismo , Células Enteroendócrinas/metabolismo , Mucosa Intestinal/lesões , Mucosa Intestinal/metabolismo , Jejuno/lesões , Jejuno/metabolismo , Células-Tronco/metabolismo , Animais , Antígenos de Diferenciação/genética , Células Enteroendócrinas/patologia , Regulação da Expressão Gênica , Mucosa Intestinal/patologia , Jejuno/patologia , Camundongos , Camundongos Transgênicos , Células-Tronco/patologia
10.
PLoS One ; 9(3): e93310, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24667776

RESUMO

Olanzapine is a first line medication for the treatment of schizophrenia, but it is also one of the atypical antipsychotics carrying the highest risk of weight gain. Metformin was reported to produce significant attenuation of antipsychotic-induced weight gain in patients, while the study of preventing olanzapine-induced weight gain in an animal model is absent. Berberine, an herbal alkaloid, was shown in our previous studies to prevent fat accumulation in vitro and in vivo. Utilizing a well-replicated rat model of olanzapine-induced weight gain, here we demonstrated that two weeks of metformin or berberine treatment significantly prevented the olanzapine-induced weight gain and white fat accumulation. Neither metformin nor berberine treatment demonstrated a significant inhibition of olanzapine-increased food intake. But interestingly, a significant loss of brown adipose tissue caused by olanzapine treatment was prevented by the addition of metformin or berberine. Our gene expression analysis also demonstrated that the weight gain prevention efficacy of metformin or berberine treatment was associated with changes in the expression of multiple key genes controlling energy expenditure. This study not only demonstrates a significant preventive efficacy of metformin and berberine treatment on olanzapine-induced weight gain in rats, but also suggests a potential mechanism of action for preventing olanzapine-reduced energy expenditure.


Assuntos
Antipiréticos/efeitos adversos , Benzodiazepinas/efeitos adversos , Berberina/farmacologia , Metformina/farmacologia , Aumento de Peso/efeitos dos fármacos , Tecido Adiposo Branco/citologia , Animais , Ingestão de Alimentos/efeitos dos fármacos , Feminino , Canais Iônicos/genética , Proteínas Mitocondriais/genética , Olanzapina , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Transcriptoma/efeitos dos fármacos , Proteína Desacopladora 1
11.
PLoS One ; 8(1): e55572, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23383228

RESUMO

Following transplantation of hematopoietic lineage cells, genetic markers unique to the transplanted cells have been detected in non-hematopoietic recipient cells of human liver, vascular endothelium, intestinal epithelium and brain. The underlying mechanisms by which this occurs are unclear. Evidence from mice suggests it is due in part to fusion between cells of hematopoietic and non-hematopoietic origins; however, direct evidence for this in humans is scant. Here, by quantitative and statistical analysis of X- and Y-chromosome numbers in epithelial and non-epithelial intestinal cells from gender-mismatched hematopoietic cell transplant patients, we provide evidence that transplanted cells of the hematopoietic lineage incorporate into human intestinal epithelium through cell fusion. This is the first definitive identification of cell fusion between hematopoietic cells and any epithelial cell type in humans, and provides the basis for further understanding the physiological and potential pathological consequences of cell fusion in humans.


Assuntos
Células da Medula Óssea/metabolismo , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/citologia , Adulto , Transplante de Medula Óssea , Fusão Celular , Cromossomos Humanos X , Feminino , Humanos , Cariótipo , Masculino , Reprodutibilidade dos Testes , Doadores de Tecidos
12.
Cancer Res ; 71(4): 1497-505, 2011 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-21303980

RESUMO

The most deadly phase in cancer progression is attributed to the inappropriate acquisition of molecular machinery leading to metastatic transformation and spread of disease to distant organs. Although it is appreciated that metastasis involves epithelial-mesenchymal interplay, the underlying mechanism defining this process is poorly understood. Specifically, how cancer cells evade immune surveillance and gain the ability to navigate the circulatory system remains a focus. One possible mechanism underlying metastatic conversion is fusion between blood-derived immune cells and cancer cells. While this notion is a century old, in vivo evidence that cell fusion occurs within tumors and imparts genetic or physiologic changes remains controversial. We have previously demonstrated in vivo cell fusion between blood cells and intestinal epithelial cells in an injury setting. Here, we hypothesize that immune cells, such as macrophages, fuse with tumor cells imparting metastatic capabilities by transferring their cellular identity. We used parabiosis to introduce fluorescent-labeled bone marrow-derived cells to mice with intestinal tumors, finding that fusion between circulating blood-derived cells and tumor epithelium occurs during the natural course of tumorigenesis. Moreover, we identify the macrophage as a key cellular partner for this process. Interestingly, cell fusion hybrids retain a transcriptome identity characteristic of both parental derivatives, while also expressing a unique subset of transcripts. Our data supports the novel possibility that tumorigenic cell fusion may impart physical behavior attributed to migratory macrophages, including navigation of circulation and immune evasion. As such, cell fusion may represent a promising novel mechanism underlying the metastatic conversion of cancer cells.


Assuntos
Reprogramação Celular/fisiologia , Células Epiteliais/patologia , Mucosa Intestinal/patologia , Macrófagos/patologia , Neoplasias/patologia , Animais , Fusão Celular/métodos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Células Cultivadas , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Perfilação da Expressão Gênica , Mucosa Intestinal/metabolismo , Macrófagos/metabolismo , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise em Microsséries , Modelos Biológicos , Metástase Neoplásica , Neoplasias/genética
13.
PLoS One ; 4(8): e6530, 2009 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-19657387

RESUMO

Cell fusion between circulating bone marrow-derived cells (BMDCs) and non-hematopoietic cells is well documented in various tissues and has recently been suggested to occur in response to injury. Here we illustrate that inflammation within the intestine enhanced the level of BMDC fusion with intestinal progenitors. To identify important microenvironmental factors mediating intestinal epithelial cell fusion, we performed bone marrow transplantation into mouse models of inflammation and stimulated epithelial proliferation. Interestingly, in a non-injury model or in instances where inflammation was suppressed, an appreciable baseline level of fusion persisted. This suggests that additional mediators of cell fusion exist. A rigorous temporal analysis of early post-transplantation cellular dynamics revealed that GFP-expressing donor cells first trafficked to the intestine coincident with a striking increase in epithelial proliferation, advocating for a required fusogenic state of the host partner. Directly supporting this hypothesis, induction of augmented epithelial proliferation resulted in a significant increase in intestinal cell fusion. Here we report that intestinal inflammation and epithelial proliferation act together to promote cell fusion. While the physiologic impact of cell fusion is not yet known, the increased incidence in an inflammatory and proliferative microenvironment suggests a potential role for cell fusion in mediating the progression of intestinal inflammatory diseases and cancer.


Assuntos
Fusão Celular , Proliferação de Células , Inflamação/patologia , Mucosa Intestinal/patologia , Animais , Sequência de Bases , Células da Medula Óssea/patologia , Transplante de Medula Óssea , Primers do DNA , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
14.
Proc Natl Acad Sci U S A ; 103(16): 6321-5, 2006 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-16606845

RESUMO

Transplanted adult bone marrow-derived cells (BMDCs) have been shown to adopt the phenotype and function of several nonhematopoietic cell lineages and promote tumorigenesis. Beyond its cancer enhancing potential, cell fusion has recently emerged as an explanation of how BMDCs regenerate diseased heptocytes, contribute to Purkinje neurons and skeletal and cardiac muscle cells, and participate in skin and heart regeneration. Although bone marrow-derived epithelial cells also have been observed in the intestine, fusion as a mechanism has not been investigated. Here, we show that transplanted BMDCs fuse with both normal and neoplastic intestinal epithelium. Long-term repopulation by donor-derived cells was detected in all principal intestinal epithelial lineages including enterocytes, goblet cells, Paneth cells, and enteroendocrine cells, suggesting that the fusion partners of the BMDCs are long-lived intestinal progenitors or stem cells. Fusion of BMDCs with neoplastic epithelium did not result in tumor initiation. Our findings suggest an unexpected role for BMDCs in both regeneration and tumorigenesis of the intestine.


Assuntos
Células da Medula Óssea/fisiologia , Transformação Celular Neoplásica/patologia , Neoplasias Intestinais/patologia , Intestinos/citologia , Células-Tronco/citologia , Animais , Células da Medula Óssea/citologia , Transplante de Medula Óssea , Fusão Celular , Feminino , Proteínas de Fluorescência Verde/análise , Intestinos/patologia , Masculino , Células-Tronco/patologia , Cromossomo Y/química , Cromossomo Y/genética
15.
J Biol Chem ; 279(24): 25085-92, 2004 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-15044462

RESUMO

Iron is essential for life in almost all organisms and, in mammals, is absorbed through the villus cells of the duodenum. Using a human colonic carcinoma cell line that has many duodenal characteristics, HT29, we show that genes involved in intestinal iron transport are endogenously expressed. When stably transfected to express the hereditary hemochromatosis protein HFE these cells have increased ferritin levels. We demonstrate that this is not due to an effect on the transferrin (TF)-mediated iron uptake pathway but rather due to inhibition of iron efflux from the cell. The effect of HFE was independent of its interaction with TF receptor 1 as indicated by similar results using both the wild type HFE and the W81A mutant that binds TF receptor 1 with greatly reduced affinity. HFE expression did not affect the mRNA levels of most of the genes involved in iron absorption that were tested; however, it did correspond to a decrease in hephaestin message levels. These results point to a role for HFE in inhibition of iron efflux in HT29 cells. This is a distinct role from that in HeLa and human embryonic kidney 293 cells where HFE has been shown to inhibit TF-mediated iron uptake resulting in decreased ferritin levels. Such a distinction suggests a multifunctional role for HFE that is dependent upon expression levels of proteins involved in iron transport.


Assuntos
Ferritinas/sangue , Antígenos de Histocompatibilidade Classe I/fisiologia , Ferro/metabolismo , Proteínas de Membrana/fisiologia , Antígenos CD , Transporte Biológico , Polaridade Celular , Células HT29 , Proteína da Hemocromatose , Homeostase , Humanos , RNA Mensageiro/análise , Receptores da Transferrina/fisiologia
16.
Proc Natl Acad Sci U S A ; 100(16): 9500-5, 2003 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-12874382

RESUMO

The mechanisms by which the hereditary hemochromatosis protein, HFE, decreases transferrin-mediated iron uptake were examined. Coimmunoprecipitation studies using solubilized cell extracts demonstrated that transferrin (Tf) competed with HFE for binding to the transferrin receptor (TfR) similar to previous in vitro studies using soluble truncated forms of HFE and the TfR. At concentrations of Tf approaching those found in the blood, no differences in Tf binding to cells were detected, which is consistent with the lower binding constant of HFE for TfR versus Tf. However, cells expressing HFE still showed a decrease in Tf-mediated iron uptake at concentrations of Tf sufficient to dissociate HFE from the TfR. These results indicate that the association of HFE with TfR is not essential for its ability to lower intracellular iron stores. To test the effect of HFE on lowering intracellular iron levels independently of its association with TfR, a mutated HFE (fW81AHFE) that shows greatly reduced affinity for the TfR was transfected into tetracycline-controlled transactivator HeLa cells. HeLa cells expressing fW81AHFE behaved in a similar manner to cells expressing wild-type HFE with respect to decreased intracellular iron levels measured by iron regulatory protein gel-shift assays and ferritin levels. The results indicate that HFE can lower intracellular iron levels independently of its interaction with the TfR.


Assuntos
Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/fisiologia , Ferro/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Mutação , Ligação Competitiva , Western Blotting , Linhagem Celular , Regulação para Baixo , Células HeLa , Proteína da Hemocromatose , Humanos , Microscopia de Fluorescência , Testes de Precipitina , Ligação Proteica , Receptores da Transferrina/metabolismo , Fatores de Tempo , Ativação Transcricional , Transferrina/metabolismo
17.
Curr Microbiol ; 45(2): 115-7, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12070689

RESUMO

Acidithiobacillus ferrooxidans ATCC 23270 possesses two copies of form I ribulose bisphosphate carboxylase/oxygenase (RuBisCO). The nucleotide sequence identity between the two large and two small subunit peptides was 75% and 58%, respectively. It is proposed that the two copies resulted from lateral gene transfer.


Assuntos
Gammaproteobacteria/genética , Ribulose-Bifosfato Carboxilase/genética , Gammaproteobacteria/enzimologia , Transferência Genética Horizontal , Mapeamento por Restrição
18.
Biochem J ; 373(Pt 1): 145-53, 2003 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-12667138

RESUMO

HFE, the protein mutated in hereditary haemochromatosis type 1, is known to interact with the transferrin receptor (TfR) on the cell surface and during endocytosis [Gross, Irrinki, Feder and Enns (1998) J. Biol. Chem. 273, 22068-22074; Roy, Penny, Feder and Enns (1999) J. Biol. Chem. 274, 9022-9028]. However, whether they are capable of interacting with each other once inside the cell is not known. In the present study we present several lines of evidence that they do interact in endosome compartments. Cells expressing a chimaera of HFE protein with the cytoplasmic domain of lysosomal-associated membrane protein 1 (LAMP1) in place of its own (HFE-LAMP) show a decrease in the half-life of the TfR. This implies that the interaction between HFE and TfR in endosomes targets the TfR to lysosomal compartments. The interaction between TfR and HFE-LAMP was confirmed by immunoprecipitation, in addition to immunofluorescence studies. Addition of transferrin (Tf) to HFE-LAMP-expressing cells competes with HFE for binding to the TfR, thereby increasing the half-life of TfR and confirming that the HFE-LAMP-TfR complex reaches the cell surface prior to entering the endosomal vesicles and trafficking to the lysosome. These results raise the possibility that interaction of HFE and TfR in intracellular vesicles may play an important role in determining the function of HFE in iron homoeostasis, which is still unknown. Analysis of endosomal pH and the iron content of internalized Tf indicated that HFE does not appear to alter the unloading of iron from Tf in the endosome.


Assuntos
Endocitose/fisiologia , Endossomos/fisiologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Proteínas de Membrana/metabolismo , Receptores da Transferrina/metabolismo , Sequência de Aminoácidos , Membrana Celular/fisiologia , Primers do DNA , Meia-Vida , Células HeLa , Proteína da Hemocromatose , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Ferro/metabolismo , Cinética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transferrina/metabolismo
19.
J Cell Physiol ; 190(2): 218-26, 2002 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11807826

RESUMO

Iron regulatory proteins (IRPs), the cytosolic proteins involved in the maintenance of cellular iron homeostasis, bind to stem loop structures found in the mRNA of key proteins involved iron uptake, storage, and metabolism and regulate the expression of these proteins in response to changes in cellular iron needs. We have shown previously that HFE-expressing fWTHFE/tTA HeLa cells have slightly increased transferrin receptor levels and dramatically reduced ferritin levels when compared to the same clonal cell line without HFE (Gross et al., 1998, J Biol Chem 273:22068-22074). While HFE does not alter transferrin receptor trafficking or non-transferrin mediated iron uptake, it does specifically reduce (55)Fe uptake from transferrin (Roy et al., 1999, J Biol Chem 274:9022-9028). In this report, we show that IRP RNA binding activity is increased by up to 5-fold in HFE-expressing cells through the activation of both IRP isoforms. Calcein measurements show a 45% decrease in the intracellular labile iron pool in HFE-expressing cells, which is in keeping with the IRP activation. These results all point to the direct effect of the interaction of HFE with transferrin receptor in lowering the intracellular labile iron pool and establishing a new set point for iron regulation within the cell.


Assuntos
Antígenos HLA/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Ferro/metabolismo , Proteínas de Membrana , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Ferritinas/antagonistas & inibidores , Ferritinas/biossíntese , Fluoresceínas , Corantes Fluorescentes , Células HeLa , Proteína da Hemocromatose , Humanos , Membranas Intracelulares/metabolismo , Proteína 1 Reguladora do Ferro , Proteína 2 Reguladora do Ferro , Proteínas Reguladoras de Ferro
20.
Curr Microbiol ; 46(2): 115-9, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12520366

RESUMO

The order of genes in the carboxysome gene clusters of four thiobacilli was examined and the possibility of the cluster forming an operon evaluated. Furthermore, carboxysome peptide homologs were compared with respect to similarities in primary sequence, and the unique structural features of the shell protein CsoS2 were described.


Assuntos
Proteínas de Bactérias/genética , Genes Bacterianos , Organelas/metabolismo , Proteobactérias/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Thiobacillus/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Dados de Sequência Molecular , Família Multigênica , Óperon , Proteobactérias/genética , Análise de Sequência de DNA , Thiobacillus/genética
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