RESUMO
BACKGROUND INFORMATION: Tumor stroma remodeling is a key feature of malignant tumors and can promote cancer progression. Laminins are major constituents of basement membranes that physically separate the epithelium from the underlying stroma. RESULTS: By employing mouse models expressing high and low levels of the laminin α1 chain (LMα1), we highlighted its implication in a tumor-stroma crosstalk, thus leading to increased colon tumor incidence, angiogenesis and tumor growth. The underlying mechanism involves attraction of carcinoma-associated fibroblasts by LMα1, VEGFA expression triggered by the complex integrin α2ß1-CXCR4 and binding of VEGFA to LM-111, which in turn promotes angiogenesis, tumor cell survival and proliferation. A gene signature comprising LAMA1, ITGB1, ITGA2, CXCR4 and VEGFA has negative predictive value in colon cancer. CONCLUSIONS: Together, we have identified VEGFA, CXCR4 and α2ß1 integrin downstream of LMα1 in colon cancer as of bad prognostic value for patient survival. SIGNIFICANCE: This information opens novel opportunities for diagnosis and treatment of colon cancer.
RESUMO
OBJECTIVE: Epidemiological and clinical data indicate that patients suffering from IBD with long-standing colitis display a higher risk to develop colorectal high-grade dysplasia. Whereas carcinoma invasion and metastasis rely on basement membrane (BM) disruption, experimental evidence is lacking regarding the potential contribution of epithelial cell/BM anchorage on inflammation onset and subsequent neoplastic transformation of inflammatory lesions. Herein, we analyse the role of the α6ß4 integrin receptor found in hemidesmosomes that attach intestinal epithelial cells (IECs) to the laminin-containing BM. DESIGN: We developed new mouse models inducing IEC-specific ablation of α6 integrin either during development (α6ΔIEC) or in adults (α6ΔIEC-TAM). RESULTS: Strikingly, all α6ΔIEC mutant mice spontaneously developed long-standing colitis, which degenerated overtime into infiltrating adenocarcinoma. The sequence of events leading to disease onset entails hemidesmosome disruption, BM detachment, IL-18 overproduction by IECs, hyperplasia and enhanced intestinal permeability. Likewise, IEC-specific ablation of α6 integrin induced in adult mice (α6ΔIEC-TAM) resulted in fully penetrant colitis and tumour progression. Whereas broad-spectrum antibiotic treatment lowered tissue pathology and IL-1ß secretion from infiltrating myeloid cells, it failed to reduce Th1 and Th17 response. Interestingly, while the initial intestinal inflammation occurred independently of the adaptive immune system, tumourigenesis required B and T lymphocyte activation. CONCLUSIONS: We provide for the first time evidence that loss of IECs/BM interactions triggered by hemidesmosome disruption initiates the development of inflammatory lesions that progress into high-grade dysplasia and carcinoma. Colorectal neoplasia in our mouse models resemble that seen in patients with IBD, making them highly attractive for discovering more efficient therapies.
Assuntos
Adenocarcinoma/fisiopatologia , Colite/fisiopatologia , Neoplasias Colorretais/fisiopatologia , Citocinas/metabolismo , Hemidesmossomos/fisiologia , Integrina alfa6/genética , Integrina alfa6beta4/metabolismo , Mucosa Intestinal/metabolismo , Imunidade Adaptativa , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animais , Linfócitos B , Membrana Basal/fisiopatologia , Caspase 1/metabolismo , Colite/genética , Colite/metabolismo , Colite/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Citocinas/genética , Células Epiteliais/metabolismo , Hemidesmossomos/genética , Homeostase/genética , Mucosa Intestinal/patologia , Mucosa Intestinal/fisiopatologia , Queratina-18/metabolismo , Queratina-8/metabolismo , Ativação Linfocitária , Camundongos , Muco/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Permeabilidade , Índice de Gravidade de Doença , Transdução de Sinais , Linfócitos TRESUMO
Besides digesting nutrients, the gut protects the host against invasion by pathogens. Enterocytes may be subjected to damage by both microbial and host defensive responses, causing their death. Here, we report a rapid epithelial response that alleviates infection stress and protects the enterocytes from the action of microbial virulence factors. Intestinal epithelia exposed to hemolysin, a pore-forming toxin secreted by Serratia marcescens, undergo an evolutionarily conserved process of thinning followed by the recovery of their initial thickness within a few hours. In response to hemolysin attack, Drosophila melanogaster enterocytes extrude most of their apical cytoplasm, including damaged organelles such as mitochondria, yet do not lyse. We identify two secreted peptides, the expression of which requires CyclinJ, that mediate the recovery phase in which enterocytes regain their original shape and volume. Epithelial thinning and recovery constitute a fast and efficient response to intestinal infections, with pore-forming toxins acting as alarm signals.
Assuntos
Toxinas Bacterianas/toxicidade , Sistema Digestório/efeitos dos fármacos , Enterócitos/efeitos dos fármacos , Enterócitos/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Animais , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Sistema Digestório/imunologia , Sistema Digestório/microbiologia , Sistema Digestório/patologia , Modelos Animais de Doenças , Drosophila melanogaster , Enterócitos/patologia , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/toxicidade , Enteropatias/microbiologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Microvilosidades/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Infecções por Serratia , Serratia marcescens/metabolismo , Serratia marcescens/patogenicidade , Sobrevida , Varroidae , Fatores de VirulênciaRESUMO
Mechanical interaction between cells and their surrounding extracellular matrix (ECM) controls key processes such as proliferation, differentiation and motility. For many years, two-dimensional (2D) models were used to better understand the interactions between cells and their surrounding ECM. More recently, variation of the mechanical properties of tissues has been reported to play a major role in physiological and pathological scenarios such as cancer progression. The 3D architecture of the ECM finely tunes cellular behavior to perform physiologically relevant tasks. Technical limitations prevented scientists from obtaining accurate assessment of the mechanical properties of physiologically realistic matrices. There is therefore a need for combining the production of high-quality cell-derived 3D matrices (CDMs) and the characterization of their topographical and mechanical properties. Here, we describe methods that allow to accurately measure the young modulus of matrices produced by various cellular types. In the first part, we will describe and review several protocols for generating CDMs matrices from endothelial, epithelial, fibroblastic, muscle and mesenchymal stem cells. We will discuss tools allowing the characterization of the topographical details as well as of the protein content of such CDMs. In a second part, we will report the methodologies that can be used, based on atomic force microscopy, to accurately evaluate the stiffness properties of the CDMs through the quantification of their young modulus. Altogether, such methodologies allow characterizing the stiffness and topography of matrices deposited by the cells, which is key for the understanding of cellular behavior in physiological conditions.
Assuntos
Matriz Extracelular/fisiologia , Animais , Bovinos , Módulo de Elasticidade , Matriz Extracelular/química , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Células-Tronco Mesenquimais/fisiologia , Camundongos , Microscopia de Força Atômica , Miócitos de Músculo Liso/fisiologia , Células NIH 3T3RESUMO
The extracellular matrix (ECM) molecule tenascin-C (TNC) promotes tumor progression. This has recently been demonstrated in the stochastic murine RIP1-Tag2 insulinoma model, engineered to either express TNC abundantly or to be devoid of TNC. However, our knowledge about organization of the TNC microenvironment is scant. Here we determined the spatial distribution of TNC together with other ECM molecules in murine RIP1-Tag2 insulinoma and human cancer tissue (insulinoma and colorectal carcinoma). We found that TNC is organized in matrix tracks together with other ECM molecules of the AngioMatrix signature, a previously described gene expression profile that characterizes the angiogenic switch. Moreover, stromal cells including endothelial cells, fibroblasts and leukocytes were enriched in the TNC tracks. Thus, TNC tracks may provide niches for stromal cells and regulate their behavior. Given similarities of TNC rich niches for stromal cells in human insulinoma and colon cancer, we propose that the RIP1-Tag2 model may be useful for providing insights into the contribution of the tumor stroma specific ECM as promoter of cancer progression.
Assuntos
Movimento Celular/fisiologia , Neoplasias Colorretais/metabolismo , Matriz Extracelular/metabolismo , Células Estromais/patologia , Tenascina/metabolismo , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Fibroblastos/metabolismo , Humanos , Camundongos TransgênicosRESUMO
Laminins (LM), basement membrane molecules and mediators of epithelial-stromal communication, are crucial in tissue homeostasis. Inflammatory Bowel Diseases (IBD) are multifactorial pathologies where the microenvironment and in particular LM play an important yet poorly understood role in tissue maintenance, and in cancer progression which represents an inherent risk of IBD. Here we showed first that in human IBD colonic samples and in murine colitis the LMα1 and LMα5 chains are specifically and ectopically overexpressed with a concomitant nuclear p53 accumulation. Linked to this observation, we provided a mechanism showing that p53 induces LMα1 expression at the promoter level by ChIP analysis and this was confirmed by knockdown in cell transfection experiments. To mimic the human disease, we induced colitis and colitis-associated cancer by chemical treatment (DSS) combined or not with a carcinogen (AOM) in transgenic mice overexpressing LMα1 or LMα5 specifically in the intestine. We demonstrated that high LMα1 or LMα5 expression decreased susceptibility towards experimentally DSS-induced colon inflammation as assessed by histological scoring and decrease of pro-inflammatory cytokines. Yet in a pro-oncogenic context, we showed that LM would favor tumorigenesis as revealed by enhanced tumor lesion formation in both LM transgenic mice. Altogether, our results showed that nuclear p53 and associated overexpression of LMα1 and LMα5 protect tissue from inflammation. But in a mutation setting, the same LM molecules favor progression of IBD into colitis-associated cancer. Our transgenic mice represent attractive new models to acquire knowledge about the paradoxical effect of LM that mediate either tissue reparation or cancer according to the microenvironment. In the early phases of IBD, reinforcing basement membrane stability/organization could be a promising therapeutic approach.
Assuntos
Carcinoma/metabolismo , Neoplasias do Colo/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Laminina/metabolismo , Animais , Células CACO-2 , Citocinas/metabolismo , Células HCT116 , Células HT29 , Humanos , Laminina/genética , Camundongos , Proteína Supressora de Tumor p53/metabolismoRESUMO
The extracellular matrix molecule tenascin-C (TNC) is a major component of the cancer-specific matrix, and high TNC expression is linked to poor prognosis in several cancers. To provide a comprehensive understanding of TNC's functions in cancer, we established an immune-competent transgenic mouse model of pancreatic ß-cell carcinogenesis with varying levels of TNC expression and compared stochastic neuroendocrine tumor formation in abundance or absence of TNC. We show that TNC promotes tumor cell survival, the angiogenic switch, more and leaky vessels, carcinoma progression, and lung micrometastasis. TNC downregulates Dickkopf-1 (DKK1) promoter activity through the blocking of actin stress fiber formation, activates Wnt signaling, and induces Wnt target genes in tumor and endothelial cells. Our results implicate DKK1 downregulation as an important mechanism underlying TNC-enhanced tumor progression through the provision of a proangiogenic tumor microenvironment.
Assuntos
Regulação para Baixo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Tenascina/metabolismo , Proteínas Wnt/metabolismo , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Modelos Animais de Doenças , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica , Tumores Neuroendócrinos/metabolismo , Tumores Neuroendócrinos/patologia , Transdução de Sinais , Tenascina/deficiência , Tenascina/genética , Proteínas Wnt/antagonistas & inibidoresAssuntos
Membrana Basal/metabolismo , Laminina/metabolismo , Transdução de Sinais , Animais , Membrana Basal/fisiologia , Adesão Celular , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Matriz Extracelular/metabolismo , Humanos , Laminina/genética , Camundongos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Polimerização , Mapeamento de Interação de ProteínasRESUMO
Laminins (LM) are extracellular matrix molecules that contribute to and are required for the formation of basement membranes. They participate in the modulation of epithelial/mesenchymal interactions and are implicated in organogenesis and maintenance of organ homeostasis. Among the LM molecules, the LM α5 chain (LMα5) is one of the most widely distributed LM in the developing and mature organism. Its presence in some basement membranes during embryogenesis is absolutely required for maintenance of basement membrane integrity and thus for proper organogenesis. LMα5 also regulates the expression of genes important for major biological processes, in part by repressing or activating signaling pathways, depending upon the physiological context.
Assuntos
Padronização Corporal , Regulação da Expressão Gênica no Desenvolvimento , Laminina/metabolismo , Organogênese , Animais , Membrana Basal/citologia , Membrana Basal/metabolismo , Sítios de Ligação , Diferenciação Celular , Movimento Celular , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Laminina/genética , Camundongos , Ligação Proteica , Células-Tronco/citologia , Células-Tronco/metabolismo , Sindecana-4/genética , Sindecana-4/metabolismo , Via de Sinalização WntRESUMO
Laminins are major constituents of basement membranes and are essential for tissue homeostasis. Laminin-511 is highly expressed in the intestine and its absence causes severe malformation of the intestine and embryonic lethality. To understand the mechanistic role of laminin-511 in tissue homeostasis, we used RNA profiling of embryonic intestinal tissue of lama5 knockout mice and identified a lama5 specific gene expression signature. By combining cell culture experiments with mediated knockdown approaches, we provide a mechanistic link between laminin α5 gene deficiency and the physiological phenotype. We show that laminin α5 plays a crucial role in both epithelial and mesenchymal cell behavior by inhibiting Wnt and activating PI3K signaling. We conclude that conflicting signals are elicited in the absence of lama5, which alter cell adhesion, migration as well as epithelial and muscle differentiation. Conversely, adhesion to laminin-511 may serve as a potent regulator of known interconnected PI3K/Akt and Wnt signaling pathways. Thus deregulated adhesion to laminin-511 may be instrumental in diseases such as human pathologies of the gut where laminin-511 is abnormally expressed as it is shown here.
Assuntos
Mucosa Intestinal/metabolismo , Intestinos/citologia , Laminina/deficiência , Laminina/genética , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/genética , Proteínas Wnt/metabolismo , Animais , Biomarcadores/metabolismo , Adesão Celular/genética , Diferenciação Celular/genética , Técnicas de Inativação de Genes , Humanos , Mucosa Intestinal/citologia , Intestinos/embriologia , Camundongos , Células Musculares/citologia , Organogênese/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , Transcriptoma/genéticaRESUMO
The interaction of endothelial cells and pericytes with their microenvironment, in particular with the basement membrane, plays a crucial role during vasculogenesis and angiogenesis. In this review, we focus on laminins, a major family of extracellular matrix molecules present in basement membranes. Laminins interact with cell surface receptors to trigger intracellular signalling that shapes cell behaviour. Each laminin exerts a distinct effect on endothelial cells and pericytes which largely depends on the adhesion receptor profile expressed on the cell surface. Moreover, proteolytic cleavage of laminins may affect their role in angiogenesis. We report in vitro and in vivo data on laminin-111, -411, -511 and -332 and their associated signalling that regulates cell behaviour and angiogenesis under normal and pathological conditions. We also discuss how tissue-specific deletion of laminin genes affects the behaviour of endothelial cells and pericytes and thus angiogenesis. Finally, we examine how coculture systems with defined laminin expression contribute to our understanding of the roles of laminins in normal and pathological vasculogenesis and angiogenesis.
Assuntos
Laminina/fisiologia , Neovascularização Patológica/fisiopatologia , Neovascularização Fisiológica/fisiologia , Animais , Membrana Basal/fisiologia , Adesão Celular/fisiologia , Técnicas de Cocultura , Células Endoteliais/fisiologia , Humanos , Laminina/química , Modelos Biológicos , Pericitos/fisiologia , Processamento de Proteína Pós-Traducional , Transdução de Sinais/fisiologiaRESUMO
We had developed a conditional Laminin α 1 knockout-mouse model (Lama1(cko)) bypassing embryonic lethality of Lama1 deficient mice to study the role of this crucial laminin chain during late developmental phases and organogenesis. Here, we report a strong defect in the organization of the adult cerebellum of Lama1(cko) mice. Our study of the postnatal cerebellum of Lama1(cko) animals revealed a disrupted basement membrane correlated to an unexpected excessive proliferation of granule cell precursors in the external granular layer (EGL). This was counteracted by a massive cell death occurring between the postnatal day 7 (P7) and day 20 (P20) resulting in a net balance of less cells and a smaller cerebellum. Our data show that the absence of Lama1 has an impact on the Bergmann glia scaffold that aberrantly develops. This phenotype is presumably responsible for the observed misplacing of granule cells that may explain the overall perturbation of the layering of the cerebellum and an aberrant folia formation.
Assuntos
Cerebelo/crescimento & desenvolvimento , Laminina/fisiologia , Animais , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Cerebelo/citologia , Cerebelo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Antígeno Ki-67/metabolismo , Laminina/deficiência , Laminina/genética , Laminina/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Neuroglia/metabolismo , Neurônios/metabolismo , Neurônios/fisiologiaRESUMO
BACKGROUND: Functional loss of the tumor suppressor Smad4 is involved in pancreatic and colorectal carcinogenesis and has been associated with the acquisition of invasiveness. We have previously demonstrated that the heterotrimeric basement membrane protein laminin-332 is a Smad4 target. Namely, Smad4 functions as a positive transcriptional regulator of all three genes encoding laminin-332; its loss is thus implicated in the reduced or discontinuous deposition of the heterotrimeric basement membrane molecule as evident in carcinomas. Uncoupled expression of laminin genes, on the other hand, namely overexpression of the laminin-gamma2 chain is an impressive marker at invasive edges of carcinomas where tumor cells are maximally exposed to signals from stromal cell types like macrophages. As Smad4 is characterized as an integrator of multiple extracellular stimuli in a strongly contextual manner, we asked if loss of Smad4 may also be involved in uncoupled expression of laminin genes in response to altered environmental stimuli. Here, we address Smad4 dependent effects of the prominent inflammatory cytokine TNFalpha on tumor cells. RESULTS: Smad4-reconstituted colon carcinoma cells like adenoma cells respond to TNFalpha with an increased expression of all three chains encoding laminin-332; coincubation with TGFbeta and TNFalpha leads to synergistic induction and to the secretion of large amounts of the heterotrimer. In contrast, in Smad4-deficient cells TNFalpha can induce expression of the gamma2 and beta3 but not the alpha3 chain. Surprisingly, this uncoupled induction of laminin-332 chains in Smad4-negative cells rather than causing intracellular accumulation is followed by the release of gamma2 into the medium, either in a monomeric form or in complexes with as yet unknown proteins. Soluble gamma2 is associated with increased cell migration. CONCLUSIONS: Loss of Smad4 may lead to uncoupled induction of laminin-gamma2 in response to TNFalpha and may therefore represent one of the mechanisms which underlie accumulation of laminin-gamma2 at the invasive margin of a tumor. The finding, that gamma2 is secreted from tumor cells in significant amounts and is associated with increased cell migration may pave the way for further investigation to better understand its functional relevance for tumor progression.
Assuntos
Neoplasias Colorretais/metabolismo , Laminina/metabolismo , Proteína Smad4/deficiência , Fator de Necrose Tumoral alfa/farmacologia , Adenoma/metabolismo , Sequência de Aminoácidos , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Sinergismo Farmacológico , Técnicas de Silenciamento de Genes , Humanos , Laminina/química , Espectrometria de Massas , Dados de Sequência Molecular , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Conformação Proteica , Proteína Smad4/genética , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , CalininaRESUMO
The Neuromutagenesis Facility at the Jackson Laboratory generated a mouse model of retinal vasculopathy, nmf223, which is characterized clinically by vitreal fibroplasia and vessel tortuosity. nmf223 homozygotes also have reduced electroretinogram responses, which are coupled histologically with a thinning of the inner nuclear layer. The nmf223 locus was mapped to chromosome 17, and a missense mutation was identified in Lama1 that leads to the substitution of cysteine for a tyrosine at amino acid 265 of laminin alpha1, a basement membrane protein. Despite normal localization of laminin alpha1 and other components of the inner limiting membrane, a reduced integrity of this structure was suggested by ectopic cells and blood vessels within the vitreous. Immunohistochemical characterization of nmf223 homozygous retinas demonstrated the abnormal migration of retinal astrocytes into the vitreous along with the persistence of hyaloid vasculature. The Y265C mutation significantly reduced laminin N-terminal domain (LN) interactions in a bacterial two-hybrid system. Therefore, this mutation could affect interactions between laminin alpha1 and other laminin chains. To expand upon these findings, a Lama1 null mutant, Lama1(tm1.1Olf), was generated that exhibits a similar but more severe retinal phenotype than that seen in nmf223 homozygotes. The increased severity of the Lama1 null mutant phenotype is probably due to the complete loss of the inner limiting membrane in these mice. This first report of viable Lama1 mouse mutants emphasizes the importance of this gene in retinal development. The data presented herein suggest that hypomorphic mutations in human LAMA1 could lead to retinal disease.
Assuntos
Laminina , Mutação de Sentido Incorreto , Isoformas de Proteínas , Retina , Doenças Retinianas , Vasos Retinianos , Adulto , Sequência de Aminoácidos , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Membrana Basal/citologia , Membrana Basal/metabolismo , Eletrorretinografia , Feminino , Teste de Complementação Genética , Humanos , Laminina/genética , Laminina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Dados de Sequência Molecular , Fenótipo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Retina/anormalidades , Retina/anatomia & histologia , Retina/fisiologia , Doenças Retinianas/genética , Doenças Retinianas/patologia , Vasos Retinianos/anormalidades , Vasos Retinianos/anatomia & histologia , Vasos Retinianos/fisiologia , Alinhamento de Sequência , TransgenesRESUMO
Several techniques have been used to study the expression of basement membranes molecules but none of them allow distinguishing the cellular origin of the deposition of a single molecule at the subepithelial basement membrane. For this purpose, we designed an experimental model using recombinants between chick and mouse embryonic intestines. Following constructions of interspecies endodermal/mesenchymal associations in culture, developmental growth was achieved by in vivo transplantation in the chick embryo. Immunocytochemistry, using species-specific antibodies recognizing either chick or mouse basement membrane molecules, was then performed on cryosections made through the developed hybrid intestines.The use of this experimental design permits determination of the precise expression/secretion in the intestinal basement membrane region of the individual constituents: interestingly some of them are strictly of epithelial or of mesenchymal origin, while others are of dual origin. Furthermore, we could show that each of these molecules is expressed in a peculiar development-dependent pattern. Such interspecies as well as heterotopic recombinants (from different levels of the gastrointestinal tract) can also be used successfully to approach the regulation of the expression of functional markers, i.e., digestive enzymes.
Assuntos
Membrana Basal/metabolismo , Matriz Extracelular/metabolismo , Animais , Embrião de Galinha , CamundongosRESUMO
Lutheran blood group and basal cell adhesion molecule (Lu/BCAM) has been recognized as a unique receptor for laminin alpha5 chain in human red blood cells and as a coreceptor in epithelial, endothelial, and smooth muscle cells. Because limited information is available regarding the function of this adhesion glycoprotein in vivo, we generated Lu/BCAM-null mice and looked for abnormalities in red blood cells as well as in kidney and intestine, two tissues showing alteration in laminin alpha5 chain-deficient mice. We first showed that, in contrast to humans, wild-type murine red blood cells failed to express Lu/BCAM. Lu/BCAM-null mice were healthy and developed normally. However, although no alteration of the renal function was evidenced, up to 90% of the glomeruli from mutant kidneys exhibited abnormalities characterized by a reduced number of visible capillary lumens and irregular thickening of the glomerular basement membrane. Similarly, intestine analysis of mutant mice revealed smooth muscle coat thickening and disorganization. Because glomerular basement membrane and smooth muscle coat express laminin alpha5 chain and are in contact with cell types expressing Lu/BCAM in wild-type mice, these results provide evidence that Lu/BCAM, as a laminin receptor, is involved in vivo in the maintenance of normal basement membrane organization in the kidney and intestine.
Assuntos
Membrana Basal/anormalidades , Intestinos/anormalidades , Rim/anormalidades , Laminina/metabolismo , Glicoproteínas de Membrana/genética , Animais , Membrana Basal/metabolismo , Membrana Basal/ultraestrutura , Moléculas de Adesão Celular , Células Endoteliais/metabolismo , Eritrócitos/metabolismo , Feminino , Expressão Gênica , Membrana Basal Glomerular/anormalidades , Membrana Basal Glomerular/metabolismo , Membrana Basal Glomerular/ultraestrutura , Mucosa Intestinal/metabolismo , Rim/metabolismo , Testes de Função Renal , Glomérulos Renais/anormalidades , Glomérulos Renais/ultraestrutura , Laminina/análise , Sistema do Grupo Sanguíneo Lutheran , Masculino , Glicoproteínas de Membrana/metabolismo , Células Mesangiais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Músculo Liso/anormalidades , Músculo Liso/ultraestrutura , Miócitos de Músculo Liso/metabolismo , Podócitos/metabolismo , Receptores de Laminina/metabolismoRESUMO
DNA-binding transcription factors bind to promoters that carry their binding sites. Transcription factors therefore function as nodes in gene regulatory networks. In the present work we used a bioinformatic approach to search for transcription factors that might function as nodes in gene regulatory networks during the differentiation of the small intestinal epithelial cell. In addition we have searched for connections between transcription factors and the villus metabolome. Transcriptome data were generated from mouse small intestinal villus, crypt, and fetal intestinal epithelial cells. Metabolome data were generated from crypt and villus cells. Our results show that genes that are upregulated during fetal to adult and crypt to villus differentiation have an overrepresentation of potential hepatocyte nuclear factor (HNF)-4 binding sites in their promoters. Moreover, metabolome analyses by magic angle spinning (1)H nuclear magnetic resonance spectroscopy showed that the villus epithelial cells contain higher concentrations of lipid carbon chains than the crypt cells. These findings suggest a model where the HNF-4 transcription factor influences the villus metabolome by regulating genes that are involved in lipid metabolism. Our approach also identifies transcription factors of importance for crypt functions such as DNA replication (E2F) and stem cell maintenance (c-Myc).
Assuntos
Enterócitos/metabolismo , Regulação da Expressão Gênica/fisiologia , Fator 4 Nuclear de Hepatócito/fisiologia , Transcrição Gênica/fisiologia , Algoritmos , Animais , Sítios de Ligação , Diferenciação Celular/genética , Clonagem Molecular , Replicação do DNA/genética , DNA Recombinante/genética , DNA Recombinante/metabolismo , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/fisiologia , Endoderma/citologia , Enterócitos/citologia , Enterócitos/ultraestrutura , Genes myc , Genômica/métodos , Íleo/citologia , Mucosa Intestinal/citologia , Mucosa Intestinal/embriologia , Mucosa Intestinal/crescimento & desenvolvimento , Metabolismo dos Lipídeos/genética , Espectroscopia de Ressonância Magnética , Metaloendopeptidases/genética , Camundongos , Camundongos Endogâmicos C57BL , Microvilosidades/metabolismo , Modelos Genéticos , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-myc/fisiologia , Células-Tronco/citologia , Biologia de Sistemas/métodosRESUMO
The deleted in colorectal cancer (DCC) gene encodes a 170- to 190-kDa protein of the Immunoglobulin superfamily. Firstly identified as a tumor suppressor gene in human colorectal carcinomas, the main function for DCC has been described in the nervous system as part of a receptor complex for netrin-1. Moreover, roles in mucosecretory cell differentiation and as inducer of apoptosis have also been reported. DCC knockout mice supported a crucial role for this gene in axonal migration, yet questioned its implication in tumor suppression and mucosecretory differentiation. The work presented here demonstrates that a DCC-transfected HT-29 colonic human cell line (HT-29/DCC) displays an increase in cell-cell adhesion to the detriment of cell-matrix interactions: HT-29/DCC cells exhibit more and better-structured desmosomes while focal adhesions and hemidesmosomes are disrupted. HT-29/DCC cells show no changes in adherent junctions but upon treatment with TPA, HT-29/DCC cells show resistance to scattering, and maintain E-cadherin in the membrane. In addition, the actin cytoskeleton is affected in HT-29/DCC cells: stress fibers are disrupted while cortical actin remains intact. We identified a putative ERM-M (ezrin/radixin/moesin and merlin) binding domain in the juxtamembrane region of the DCC protein. In vitro pull-down assays demonstrate the interaction of the DCC cytoplasmic domain with the N-terminal region of ezrin and merlin, and co-immunoprecipitation assays in transiently DCC-transfected COS-1 cells showed that the interaction between DCC and ezrin also takes place in vivo. Altogether, our results suggest that DCC could regulate cell adhesion and migration through its association with ERM-M proteins.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Western Blotting , Adesão Celular/fisiologia , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Receptor DCC , Desmossomos/metabolismo , Desmossomos/ultraestrutura , Matriz Extracelular/metabolismo , Células HT29 , Humanos , Imuno-Histoquímica , Imunoprecipitação , Proteínas dos Microfilamentos/metabolismo , Microscopia Eletrônica , Modelos Genéticos , Dados de Sequência Molecular , Neurofibromina 2/metabolismo , Ligação Proteica , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/fisiologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/fisiologia , Transfecção/métodos , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/fisiologiaRESUMO
Intestinal epithelial cells are characterized by continuous renewal and differentiation events, which may be influenced by the basement membrane, and in particular laminins, which are major components of this specialized extracellular matrix. The function and signaling pathways of laminins in these processes are still poorly documented. In this study, we investigated the possible role and the subcellular localization of nucleolin, a nuclear shuttling protein, in relation to differentiation of human intestinal epithelial Caco2/TC7 cells triggered by exogenous laminin-1. Immunofluorescence and Western blot analysis indicated that laminin-1 induced early differentiation of the cells concomitantly to a decrease in nuclear nucleolin and its a cell surface location. We also showed that both effects of laminin-1 on Caco2/TC7 cells--induction of the differentiation marker sucrase-isomaltase and redistribution of nucleolin--could be mediated by a beta1-integrin dependent cascade that implicated activation of the p38 MAPK pathway. In addition, knock-down of nucleolin expression by the small interfering RNA strategy mimicked the effect of laminin-1 as it resulted in the induction of cell polarization and differentiation. Thus, our study suggests that changes in the subcellular distribution and expression level of nucleolin play an important role in intestinal cell differentiation and relay the signaling pathway induced by laminin-1.