RESUMO
Many aspects of cellular behavior are defined by the content of information provided by association of the extracellular matrix (ECM) and with cell membrane receptors. When cultured in the presence of laminin-containing ECM and prolactin (Prl), normal mammary epithelial cells express the milk protein beta-casein. We have previously found that the minimal ECM- and Prl-responsive enhancer element BCE-1 was only active when stably integrated into chromatin, and that trichostatin A (TSA), a reagent that leads to alterations in chromatin structure, was able to activate the integrated enhancer element. We now show that endogenous beta-casein gene, which is controlled by a genetic assembly that is highly similar to that of BCE-1 and which is also activated by incubation in ECM and Prl, is instead inhibited by TSA. We provide evidence that the differing response of beta-casein and BCE-1 to TSA is neither due to an unusual effect of TSA on mammary epithelial cells, nor to secondary consequences from the expression of a separate gene, nor to a particular property of the BCE-1 construct. As a component of this investigation, we also showed that ECM mediated rapid histone deacetylation in mammary epithelial cells. These results are discussed in combination with previous work showing that TSA mediates the differentiation of many types of cancer cells but inhibits differentiation of some nonmalignant cell types.
Assuntos
Caseínas/antagonistas & inibidores , Caseínas/biossíntese , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Ácidos Hidroxâmicos/farmacologia , Glândulas Mamárias Animais/citologia , Inibidores da Síntese de Proteínas/farmacologia , Proteínas de Saccharomyces cerevisiae , Acetilação/efeitos dos fármacos , Acetiltransferases/biossíntese , Acetiltransferases/genética , Animais , Membrana Basal/química , Caseínas/genética , Caseínas/metabolismo , Bovinos , Linhagem Celular , Cromatina/efeitos dos fármacos , Cromatina/enzimologia , Cromatina/metabolismo , Elementos Facilitadores Genéticos/efeitos dos fármacos , Genes Reporter/efeitos dos fármacos , Histona Acetiltransferases , Histonas/metabolismo , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/farmacologia , Camundongos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/farmacologia , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacosRESUMO
The molecular pathways that participate in regulation of gene expression are being progressively unraveled. Extracellular signals, including the binding of extracellular matrix and soluble molecules to cell membrane receptors, activate specific signal transducers that process information inside the cell leading to alteration in gene expression. Some of these transducers when translocated to the cell nucleus may bind to transcription complexes and thereby modify the transcriptional activity of specific genes. However, the basic molecules involved in the regulation of gene expression are found in many different cell and tissue types; thus, the mechanisms underlying tissue-specific gene expression are still obscure. In this review we focus on the study of signals that are conveyed to the nucleus. We propose that the way in which extracellular signals are integrated may account for tissue-specific gene expression. We argue that the integration of signals depends on the nature of the structural organization of cells (i.e., extracellular matrix, membrane proteins, cytoskeleton, nucleus) that defines a particular cell type within a tissue. Thus, gene expression can be envisioned as being regulated by the mutual influence of extracellular and intracellular organizations, i.e., in context.
Assuntos
Núcleo Celular/metabolismo , Regulação da Expressão Gênica , Transdução de Sinais , Animais , Matriz Extracelular/metabolismo , Especificidade de ÓrgãosRESUMO
Nidogen-1 (entactin) acts as a bridge between the extracellular matrix molecules laminin-1 and type IV collagen, and thus participates in the assembly of basement membranes. To investigate the role of nidogen-1 in regulating cell-type-specific gene expression in mammary epithelium, we designed a culture microecosystem in which each component, including epithelial cells, mesenchymal cells, lactogenic hormones and extracellular matrix, could be controlled. We found that primary and established mesenchymal and myoepithelial cells synthesized and secreted nidogen-1, whereas expression was absent in primary and established epithelial cells. In an epithelial cell line containing mesenchymal cells, nidogen-1 was produced by the mesenchymal cells but deposited between the epithelial cells. In this mixed culture, mammary epithelial cells express (beta)-casein in the presence of lactogenic hormones. Addition of either laminin-1 plus nidogen-1, or laminin-1 alone, to mammary epithelial cells induced (beta)-casein production. We asked whether recombinant nidogen-1 alone could signal directly for (beta)-casein. Nidogen-1 did not induce (beta)-casein synthesis in epithelial cells, but it augmented the inductive capacity of laminin-1. These data suggest that nidogen-1 can cooperate with laminin-1 to regulate (beta)-casein expression. Addition of full-length nidogen-1 to the mixed cultures had no effect on (beta)-casein gene expression; however, a nidogen-1 fragment containing the laminin-1 binding domain, but lacking the type IV collagen-binding domain, had a dominant negative effect on (beta)-casein expression. These data point to a physiological role for nidogen-1 in the basement membrane-induced gene expression by epithelial cells.
Assuntos
Regulação da Expressão Gênica , Laminina/fisiologia , Glândulas Mamárias Animais/fisiologia , Glicoproteínas de Membrana/fisiologia , Animais , Caseínas/antagonistas & inibidores , Caseínas/biossíntese , Células Cultivadas , Técnicas de Cocultura , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Feminino , Laminina/metabolismo , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Mesoderma/metabolismo , Camundongos , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/fisiologia , Ligação Proteica , Proteínas Recombinantes/farmacologia , Transdução de Sinais/fisiologiaRESUMO
We have previously described a 160-bp enhancer (BCE-1) in the bovine beta-casein gene that is activated in the presence of prolactin and extracellular matrix (ECM). Here we report the characterization of the enhancer by deletion and site-directed mutagenesis, electrophoretic mobility shift analysis, and in vivo footprinting. Two essential regions were identified by analysis of mutant constructions: one binds C/EBP-beta and the other binds MGF/STAT5 and an as-yet-unidentified binding protein. However, no qualitative or quantitative differences in the binding of these proteins were observed in electrophoretic mobility shift analysis using nuclear extracts derived from cells cultured in the presence or absence of ECM with or without prolactin, indicating that prolactin- and ECM-induced transcription was not dependent on the availability of these factors in the functional cell lines employed. An in vivo footprinting analysis of the factors bound to nuclear chromatin in the presence or absence of ECM and/or prolactin found no differences in the binding of C/EBP-beta but did not provide definitive results for the other factors. Neither ECM nor prolactin activated BCE-1 in transient transfections, suggesting that the chromosomal structure of the integrated template may be required for ECM-induced transcription. Further evidence is that treatment of cells with inhibitors of histone deacetylase was sufficient to induce transcription of integrated BCE-1 in the absence of ECM. Together, these results suggest that the ECM induces a complex interaction between the enhancer-bound transcription factors, the basal transcriptional machinery, and a chromosomally integrated template responsive to the acetylation state of the histones.
Assuntos
Caseínas/genética , Elementos Facilitadores Genéticos , Matriz Extracelular/fisiologia , Regulação da Expressão Gênica , Histonas/metabolismo , Proteínas do Leite , Prolactina/fisiologia , Acetilação , Animais , Sequência de Bases , Proteínas Estimuladoras de Ligação a CCAAT , Bovinos , Linhagem Celular , Cromatina/metabolismo , DNA , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Inibidores de Histona Desacetilases , Histona Desacetilases/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Nucleares/metabolismo , Ligação Proteica , Fator de Transcrição STAT5 , Deleção de Sequência , Moldes Genéticos , Transativadores/metabolismo , Ativação Transcricional , TransfecçãoRESUMO
In the normal colon, myofibroblasts are closely apposed to colonocytes where they deposit type IV collagen, the main basement membrane component. In colon carcinomas, this epithelial-mesenchymal association is physically disrupted, leading to the production of an abnormal, type IV collagen defective, basement membrane. Tumor-infiltrating myofibroblasts are migratory cells that accumulate at the invasive front of the colorectal carcinomas. They produce lytic enzymes able to degrade the basement membrane surrounding tumor glands. They also participate in the synthesis of the extracellular matrix components of the tumor stroma, which could subsequently alter the adhesive and migratory properties of the epithelial colon cancer cells. These results suggest that tumor-infiltrating myofibroblasts play a role in the invasion and metastasis of colorectal tumor cells.
Assuntos
Carcinoma/patologia , Neoplasias do Colo/patologia , Fibroblastos/patologia , Músculo Liso/citologia , Músculo Liso/patologia , Invasividade Neoplásica/patologia , Células Estromais/patologia , Animais , Humanos , Metástase Neoplásica/patologiaRESUMO
Alternative splicing of primary fibronectin (FN) mRNA results in the synthesis of different isoforms. ED-A+ and ED-B+ FN isoforms are absent from plasma FN and are representative of cellular FN. Their expression was studied in human and rat normal colon, in human colorectal carcinomas, and in transplanted tumors derived from a chemically-induced rat colon cancer. In normal colon, only the ED-A+ FN isoform was expressed as a thin deposit between crypt colonocytes and pericryptal myofibroblasts. Conversely, heavy ED-A+ FN deposits and lighter ED-B+ FN expression were found in the stroma of colorectal tumors in association with myofibroblasts surrounding tumor glands. Some colonic cancer cells also contained intracellular FN isoform granules and expressed FN mRNA. Tumor-associated myofibroblasts and some cancer cell lines were able to synthesize and deposit extracellular ED-A+ and ED-B+ FN in vitro. FN isoform deposition by tumor-associated myofibroblasts was not modulated by colon cancer cell-conditioned medium, but was strongly enhanced when myofibroblasts were cultured on colon cancer cell extracellular matrix or on laminin. These results show that the ED-A+ and ED-B+ FN isoforms were overexpressed in colorectal cancer. Cancer cells can deposit these FN isoforms directly and also stimulate their deposition by tumor-associated myofibroblasts.
Assuntos
Neoplasias do Colo/metabolismo , Neoplasias Colorretais/metabolismo , Fibroblastos/metabolismo , Fibronectinas/biossíntese , Actinas/análise , Animais , Western Blotting , Colo/química , Colo/metabolismo , Neoplasias do Colo/patologia , Neoplasias Colorretais/patologia , Matriz Extracelular/química , Fibroblastos/química , Fibroblastos/patologia , Fibronectinas/análise , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Hibridização In Situ , Mucosa Intestinal/química , Mucosa Intestinal/metabolismo , Laminina , Transplante de Neoplasias , Ratos , Células Tumorais CultivadasRESUMO
BACKGROUND/AIMS: Colonic mucosa basement membrane results from a cooperation between epithelial cells and pericryptal fibroblasts characterized as myofibroblasts. This cooperation may be abnormal in colorectal carcinoma resulting in basement membrane alteration. METHODS: Basement membrane composition and myofibroblast distribution were studied in normal rat colon and two colon carcinoma models by immunohistochemistry. Colon cancer cells and tumor-associated myofibroblasts were also studied for their capacity to deposit three basement membrane components (laminin, heparan sulfate proteoglycan, and type IV collagen) in vitro. RESULTS: A continuous, type IV collagen-containing basement membrane, such as that observed in normal colon, was found only in the most differentiated tumor model and was restricted to the areas in which myofibroblasts were closely apposed to carcinoma cells. In other areas of this tumor and in the poorly differentiated tumor model, myofibroblasts dissociated from the epithelial cells and the basement membrane was devoid of type IV collagen. In vitro, carcinoma cells deposited laminin and heparan sulfate proteoglycan but not type IV collagen. Tumor-associated myofibroblasts deposited type IV collagen only in the presence of tumor cell extracellular matrix or laminin coating. CONCLUSIONS: The colon cancer basement membrane defect in type IV collagen may result from a physical disruption in the association between epithelial cancer cells and myofibroblasts.
Assuntos
Adenocarcinoma/patologia , Membrana Basal/ultraestrutura , Carcinoma/patologia , Neoplasias do Colo/patologia , Animais , Carcinoma/metabolismo , Colágeno/metabolismo , Colo/metabolismo , Colo/patologia , Neoplasias do Colo/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Imuno-Histoquímica/métodos , Laminina , Músculo Liso/metabolismo , Músculo Liso/patologia , Transplante de Neoplasias , Ratos , Valores de Referência , Coloração e RotulagemRESUMO
In various cell systems, an inverse relationship was found between expression of E-cadherin, a molecule involved in the Ca(2+)-dependent homophylic cell-to-cell attachment of epithelial cells, and the capacity to invade extracellular matrix gels or normal tissues in vitro. DHD/K12/TRb (PROb) cells, maintained as a cell line derived from a rat colon carcinoma, homogeneously expressed in vitro immunoreactive E-cadherin, which was functional as shown in cell dissociation-reassociation assays. PROb cells were found to be non-invasive in 3 different assays in vitro. However, tumors resulting from a s.c. injection of PROb cells into syngeneic BD-IX rats were invasive, although PROb cells maintained E-cadherin expression in the tumors. Cells from a freshly dissociated PROb tumor showed, not only PROb cells but also tumor-associated myofibroblasts and were able to cross a Matrigel-coated filter. PROb tumors were indeed infiltrated by numerous myofibroblasts, mainly located at the invasive edge of the tumor. Cells from an established culture of tumor-infiltrating myofibroblasts were able to confer upon PROb cells invasiveness through Matrigel-coated filter or into chick-heart fragments. PROb cells maintained their capacity to express E-cadherin after myofibroblast-enhanced Matrigel invasion. Tumor-associated myofibroblasts, but not PROb cells, secreted a 72-kDa collagenase that could play a role in tumor-cell invasion. These results strongly suggest that cells from the tumor stroma, and more specifically myofibroblasts, may be involved in the invasiveness of epithelial tumor cells in vivo, even when E-cadherin expression prevents tumor-cell invasiveness in different in vitro assays.