Rac1 links integrin-mediated adhesion to the control of lactational differentiation in mammary epithelia

The expression of tissue-specific genes during mammary gland differentiation relies on the coincidence of two distinct signaling events: the continued engagement of β1 integrins with the extracellular matrix (ECM) and a hormonal stimulus from prolactin (Prl). How the integrin and Prl receptor (PrlR) systems integrate to regulate milk protein gene synthesis is unknown. In this study, we identify Rac1 as a key link. Dominant-negative Rac1 prevents Prl-induced synthesis of the milk protein β-casein in primary mammary epithelial cells cultured as three-dimensional acini on basement membrane. Conversely, activated Rac1 rescues the defective β-casein synthesis that occurs under conditions not normally permissive for mammary differentiation, either in β1 integrin–null cells or in wild-type cells cultured on collagen. Rac1 is required downstream of integrins for activation of the PrlR/Stat5 signaling cascade. Cdc42 is also necessary for milk protein synthesis but functions via a distinct mechanism to Rac1. This study identifies the integration of signals provided by ECM and hormones as a novel role for Rho family guanosine triphosphatases

How integrins control mammary epithelial differentiation: A possible role for the ILK–PINCH–Parvin complex

AbstractDifferentiation into tissue-specific cell types occurs in response to numerous external signals. Integrins impart signals from the extracellular matrix microenvironment that are required for cell differentiation. However, the precise cytoplasmic transducers of these signals are yet to be understood properly. In lactating mammary epithelial cells, integrin-linked kinase has been identified as an indispensable integrin-signalling adaptor that enables the activation of Rac1, which is necessary for prolactin-induced milk protein expression. Here we use examples from various tissues to summarise possible mechanisms by which ILK and its binding partners PINCH and Parvin (ILK–PINCH–Parvin complex) could be required for Rac activation and mammary epithelial differentiation

Insulin-like growth factors and insulin-like growth factor binding proteins in mammary gland function

Insulin-like growth factor (IGF)-mediated proliferation and survival are essential for normal development in the mammary gland during puberty and pregnancy. IGFs interact with IGF-binding proteins and regulate their function. The present review focuses on the role of IGFs and IGF-binding proteins in the mammary gland and describes how modulation of their actions occurs by association with hormones, other growth factors and the extracellular matrix. The review will also highlight the involvement of the IGF axis in breast cancer

Integrin-mediated survival signals regulate the apoptotic function of Bax through its conformation and subcellular localization

Most normal cells require adhesion to extracellular matrix for survival, but the molecular mechanisms that link cell surface adhesion events to the intracellular apoptotic machinery are not understood. Bcl-2 family proteins regulate apoptosis induced by a variety of cellular insults through acting on internal membranes. A pro-apoptotic Bcl-2 family protein, Bax, is largely present in the cytosol of many cells, but redistributes to mitochondria after treatment with apoptosis-inducing drugs. Using mammary epithelial cells as a model for adhesion-regulated survival, we show that detachment from extracellular matrix induced a rapid translocation of Bax to mitochondria concurrent with a conformational change resulting in the exposure of its BH3 domain. Bax translocation and BH3 epitope exposure were reversible and occurred before caspase activation and apoptosis. Pp125FAK regulated the conformation of the Bax BH3 epitope, and PI 3-kinase and pp60src prevented apoptosis induced by defective pp125FAK signaling. Our results provide a mechanistic connection between integrin-mediated adhesion and apoptosis, through the kinase-regulated subcellular distribution of Bax

Vinculin controls focal adhesion formation by direct interactions with talin and actin

Focal adhesions (FAs) regulate cell migration. Vinculin, with its many potential binding partners, can interconnect signals in FAs. Despite the well-characterized structure of vinculin, the molecular mechanisms underlying its action have remained unclear. Here, using vinculin mutants, we separate the vinculin head and tail regions into distinct functional domains. We show that the vinculin head regulates integrin dynamics and clustering and the tail regulates the link to the mechanotransduction force machinery. The expression of vinculin constructs with unmasked binding sites in the head and tail regions induces dramatic FA growth, which is mediated by their direct interaction with talin. This interaction leads to clustering of activated integrin and an increase in integrin residency time in FAs. Surprisingly, paxillin recruitment, induced by active vinculin constructs, occurs independently of its potential binding site in the vinculin tail. The vinculin tail, however, is responsible for the functional link of FAs to the actin cytoskeleton. We propose a new model that explains how vinculin orchestrates FAs

Spatial and temporal changes in Bax subcellular localization during anoikis

Bax, a member of the Bcl-2 family, translocates to mitochondria during apoptosis, where it forms oligomers which are thought to release apoptogenic factors such as cytochrome c. Using anoikis as a model system, we have examined spatial and temporal changes in Bax distribution. Bax translocates to mitochondria within 15 min of detaching cells from extracellular matrix, but mitochondrial permeabilization does not occur for a number of hours. The formation of Bax oligomers and perimitochondrial clusters occurs concomitant with caspase activation and loss of mitochondrial membrane potential, before nuclear condensation. Cells can be rescued from apoptosis if they are replated onto extracellular matrix within an hour, whereas cells detached for longer could not. The loss of ability to rescue cells from anoikis occurs after Bax translocation, but before the formation of clusters and cytochrome c release. Our data suggest that Bax regulation occurs at several levels, with formation of clusters a late event, and with critical changes determining cell fate occurring earlier

Analysis of inhibitor of apoptosis protein family expression during mammary gland development

<p>Abstract</p> <p>Background</p> <p>Inhibitors-of-Apoptosis-Proteins (IAPs) are an evolutionarily conserved family of proteins capable of regulating several facets of apoptosis. IAPs are frequently dysregulated in cancer, but their role in the regulation of apoptosis during developmental processes is not fully understood. Here we examined the expression of IAPs during the post-natal development of the mouse mammary gland, which is a tissue that exhibits a profound induction of apoptosis during involution.</p> <p>Results</p> <p>Six out of eight mammalian IAP family members are expressed in the mammary gland. Notably, quantitative PCR and immunoblotting revealed that XIAP, c-IAP1 and c-IAP2 are down-regulated in pregnancy and lactation, and prior to the onset of involution. In cultured mammary epithelial cells (MECs), XIAP levels decreased in response to inhibition of growth factor signalling. Maintaining XIAP levels in MECs by expressing exogenous XIAP protected them from all apoptotic stimuli tested.</p> <p>Conclusions</p> <p>These data suggest that the developmental regulation of IAP expression <it>in vivo </it>contributes to naturally occurring programmes of cell death.</p

Elevated EDAR signalling promotes mammary gland tumourigenesis with squamous metaplasia

Ectodysplasin A receptor (EDAR) is a death receptor in the Tumour Necrosis Factor Receptor (TNFR) superfamily with roles in the development of hair follicles, teeth and cutaneous glands. Here we report that human Oestrogen Receptor (ER) negative breast carcinomas which display squamous differentiation express EDAR strongly. Using a mouse model with a high Edar copy number, we show that elevated EDAR signalling results in a high incidence of mammary tumours in breeding female mice. These tumours resemble the EDAR-high human tumours in that they are characterised by a lack of oestrogen receptor expression, contain extensive squamous metaplasia, and display strong β-catenin transcriptional activity. In the mouse model, all of the tumours carry somatic deletions of the third exon of the CTNNB1 gene that encodes β-catenin. Deletion of this exon yields unconstrained β-catenin signalling activity. We also demonstrate that β-catenin activity is required for transformed cell growth, showing that increased EDAR signalling creates an environment in which β-catenin activity can readily promote tumourigenesis. Together, this work identifies a novel death receptor oncogene in breast cancer, whose mechanism of transformation is based on the interaction between the WNT and Ectodysplasin A (EDA) pathways