Function following form: functional differentiation of mammary epithelial cells requires laminin-induced polarization of PI3-kinase.

The biochemical and structural composition of the extracellular matrix (ECM) provides contextual information that guides tissue morphogenesis and controls cellular function. The specific processes by which specific signals from the ECM control cellular phenotype have been studied extensively in mammary epithelial cells (MEC). Isolated from the lactating mammary glands and plated on conventional tissue culture plastic, MEC rapidly lose mammary-specific characteristics and stop synthesizing milk proteins.1 However, when provided with lactogenic hormones and cultured in laminin-111 (LN1)-rich 3-dimensional (3D) gels, isolated MEC can be induced to form polarized acinar structures and to generate milk proteins, markers of functional differentiation,2 showing that specific signals from the ECM are responsible for the maintenance of the differentiated state. While subsequent experiments have provided much information about the many different mechanisms induced by LN1, many aspects of the signaling pathway connecting extracellular LN1 to the activation of milk protein gene transcription remain unknown. LN1 is a major component of the basement membrane, a specialized ECM that surrounds the epithelial cells in the mammary gland. Studies using transgenic models have elucidated the critical role of the LN1 receptors, dystroglycan and β1-integrin, and their downstream signaling pathways in the control of glandular development, tissue organization, and cellular differentiation.3,4 Parallel studies using cell culture models have helped to define the specific signals activated by cell association with LN1, showing that dystroglycan and β1-integrin serve both to physically connect LN1 molecules in the basement membrane to the cytoskeleton and also to function as activators of key signaling pathways.3,4 Prolactin is a lactogenic hormone mainly produced by the pituitary gland. Canonical prolactin signal transduction is initiated by binding of the hormone to the prolactin receptor, which activates JAK2-mediated phosphorylation and nuclear translocation of the transcription factor STAT5.5 However, LN is required for this pathway to be functional in MEC, as cells cultured on plastic do not show sustained phosphorylation of STAT5 in response to prolactin.6,7 In a previous study, Xu et al set out to determine why prolactin fails to activate STAT5 in 2D culture, finding that the prolactin receptor is mainly basolaterally localized, and in 2D culture, apically localized prolactin could not bind to and activate the basolateral receptor, preventing robust activation of STAT5.8 The authors also found that exposure to LN1 led to MEC reorganization into polarized acini, in which the prolactin receptor was oriented toward the basolateral surface where it could be more effectively activated by soluble hormone.8 In the study, Xu et al. identified phosphoinositide-3 kinase (PI3K) as the key signaling molecule linking LN1 and prolactin signaling pathways. They found that when cultured in plates coated with polyHEMA, which prevents adhesion to the plastic substratum, MEC form unpolarized cell aggregates that show only transient activation of STAT5 and no stimulation of β-casein gene expression in response to prolactin. By contrast, they found that LN1-induced structural polarization of the MEC was associated with functional activation of the prolactin signaling pathway: sustained activation of STAT5 and stimulation of β-casein gene expression. They evaluated PI3K as a potential mediator of this effect due to its role as a key mediator of integrin-induced signaling pathways, and found that inhibition of PI3K blocked LN1-mediated activation of prolactin signaling and also blocked LN1-mediated activation of Rac1, a key downstream effector of PI3K which was previously found to be crucial for STAT5 activation and functional differentiation of MECs.9 Now that PI3K has been identified as the key signaling integrator of LN1 and prolactin signaling pathways, it will be important to determine exactly how LN1 stimulates PI3K during MEC functional differentiation. Xu et al. speculate that β1-integrin or dystroglycan could be leading to activation of PI3K through stimulation of focal adhesion kinase (FAK) or integrin-linked kinase (ILK). Another possibility could be an indirect activation of cell surface signaling receptors that can activate PI3K such as EGFR. Another important question would be to determine whether LN1-induced PI3K activation is also involved in the structural polarization that appears to be a prerequisite for functional differentiation. As such pathways play critical roles in both mammary gland development and in tumor progression, these studies may provide key insight into the differential response of normal and cancer cells to ECM and soluble signaling factors, and better understanding of why the former respond by arresting growth and functionally differentiating, while the latter continue to proliferate and fail to differentiate. The elegant cell culture system described by Xu et al. their recent study should make these questions much more tractable.