Laminin-111 and the Level of Nuclear Actin Regulate Epithelial Quiescence via Exportin-6.

Nuclear actin (N-actin) is known to participate in the regulation of gene expression. We showed previously that N-actin levels mediate the growth and quiescence of mouse epithelial cells in response to laminin-111 (LN1), a component of the mammary basement membrane (BM). We know that BM is defective in malignant cells, and we show here that it is the LN1/N-actin pathway that is aberrant in human breast cancer cells, leading to continuous growth.

Identification of fluorescent compounds with non-specific binding property via high throughput live cell microscopy.

Introduction: Compounds exhibiting low non-specific intracellular binding or non-stickiness are concomitant with rapid clearing and in high demand for live-cell imaging assays because they allow for intracellular receptor localization with a high signal/noise ratio. The non-stickiness property is particularly important for imaging intracellular receptors due to the equilibria involved.

Method: Three mammalian cell lines with diverse genetic backgrounds were used to screen a combinatorial fluorescence library via high throughput live cell microscopy for potential ligands with high in- and out-flux properties.

Nuclear actin: A key player in extracellular matrix-nucleus communication.

Decades of research have shown that there is an intimate relationship between the extracellular matrix (ECM) and cellular phenotype. While the existence of this relationship remains inarguably clear, the exact details through which the extracellular matrix controls phenotypic behavior at the gene expression level are, for the most part, elusive. In a recent study on mammary epithelial cells, nuclear actin was identified as a key effector protein through which laminin Type III (LN1) attenuates RNA polymerase activity to promote growth arrest.

Actin-towards a deeper understanding of the relationship between tissue context, cellular function and tumorigenesis.

It is well-established that the actin cytoskeleton plays an important role in tumor development yet the contribution made by nuclear actin is ill-defined. In a recent study, nuclear actin was identified as a key mediator through which laminin type III (LN1) acts to control epithelial cell growth. In the breast, epithelial tumors are surrounded by an environment which lacks LN1.

Depletion of nuclear actin is a key mediator of quiescence in epithelial cells.

Functional differentiation is orchestrated by precise growth-regulatory controls conveyed by the tissue microenvironment. Cues from laminin 111 (LN1) lower transcription and suppress mammary epithelial cell growth in culture, but how LN1 induces quiescence is unknown. Recent literature points to involvement of nuclear β-actin in transcriptional regulation.

Laminin regulates PI3K basal localization and activation to sustain STAT5 activation.

Extracellular matrix (ECM) is a key regulator of tissue morphogenesis and functional differentiation in the mammary gland. We showed recently that laminin-111 (LN1) together with prolactin induces β-casein expression in mammary epithelial cells (MECs) by sustaining STAT5 activation. Others have shown that Rac1 is required for integrin-mediated STAT5 activation, but molecules upstream of Rac1 remain to be elucidated.

Gene expression in the third dimension: the ECM-nucleus connection.

Decades ago, we and others proposed that the dynamic interplay between a cell and its surrounding environment dictates cell phenotype and tissue structure. Whereas much has been discovered about the effects of extracellular matrix molecules on cell growth and tissue-specific gene expression, the nuclear mechanisms through which these molecules promote these physiological events remain unknown. Using mammary epithelial cells as a model, the purpose of this review is to discuss how the extracellular matrix influences nuclear structure and function in a three-dimensional context to promote epithelial morphogenesis and function in the mammary gland.

Laminin and biomimetic extracellular elasticity enhance functional differentiation in mammary epithelia.

In the mammary gland, epithelial cells are embedded in a 'soft' environment and become functionally differentiated in culture when exposed to a laminin-rich extracellular matrix gel. Here, we define the processes by which mammary epithelial cells integrate biochemical and mechanical extracellular cues to maintain their differentiated phenotype. We used single cells cultured on top of gels in conditions permissive for beta-casein expression using atomic force microscopy to measure the elasticity of the cells and their underlying substrata.

Epigenetic reversion of breast carcinoma phenotype is accompanied by changes in DNA sequestration as measured by AluI restriction enzyme.

The importance of microenvironment and context in regulation of tissue-specific genes is well established. DNA exposure to or the sequestration from nucleases detects differences in higher order chromatin structure in intact cells without disturbing cellular or tissue architecture. To investigate the relationship between chromatin organization and tumor phenotype, we used an established three-dimensional assay in which normal and malignant human breast cells can be easily distinguished by the morphology of the structures they make (acinus-like versus tumor-like, respectively).

Extracellular matrix, nuclear and chromatin structure, and gene expression in normal tissues and malignant tumors: a work in progress.

Almost three decades ago, we presented a model where the extracellular matrix (ECM) was postulated to influence gene expression and tissue-specificity through the action of ECM receptors and the cytoskeleton. This hypothesis implied that ECM molecules could signal to the nucleus and that the unit of function in higher organisms was not the cell alone, but the cell plus its microenvironment. We now know that ECM invokes changes in tissue and organ architecture and that tissue, cell, nuclear, and chromatin structure are changed profoundly as a result of and during malignant progression.

Extracellular matrix-regulated gene expression requires cooperation of SWI/SNF and transcription factors.

Extracellular cues play crucial roles in the transcriptional regulation of tissue-specific genes, but whether and how these signals lead to chromatin remodeling is not understood and subject to debate. Using chromatin immunoprecipitation assays and mammary-specific genes as models, we show here that extracellular matrix molecules and prolactin cooperate to induce histone acetylation and binding of transcription factors and the SWI/SNF complex to the beta- and gamma-casein promoters. Introduction of a dominant negative Brg1, an ATPase subunit of SWI/SNF complex, significantly reduced both beta- and gamma-casein expression, suggesting that SWI/SNF-dependent chromatin remodeling is required for transcription of mammary-specific genes.

Estrogen regulation of trefoil factor 1 expression by estrogen receptor alpha and Sp proteins.

Estrogen-responsive genes in human breast cancer cells often have an estrogen response element (ERE) positioned next to an Sp1 binding site. In chromatin immunoprecipitation (ChIP) assays, we investigated the binding of estrogen receptor alpha (ER), Sp1, and Sp3 to the episomal and native estrogen-responsive trefoil factor 1 (TFF1; formerly pS2) promoter in MCF-7 breast cancer cells. Mutation of the Sp site upstream of the ERE reduced estrogen responsiveness and prevented binding of Sp1 and Sp3, but not ER to the episomal promoter.

Chromatin immunoprecipitation: a tool for studying histone acetylation and transcription factor binding.

The function of a protein in gene expression can often be explained, in part, by the location of that protein along a specific gene sequence. In recent years, the chromatin immunoprecipitation (ChIP) assay has been developed to study the association of proteins located within 2 A of DNA such as transcription factors and modified histones. Numerous important findings have been published using the ChIP assay and many questions about transcription have been answered.

Measurement of histone acetyltransferase and histone deacetylase activities and kinetics of histone acetylation.

Dynamic histone acetylation has a role in chromatin remodeling and in the regulation of transcription. Histone deacetylases (HDACs) and histone acetyltransferases (HATs) catalyze reversible histone acetylation. HATs and HDACs exist as multiprotein complexes that have coactivator and corepressor activities, respectively.