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Vinculin is an essential component of cell adhesion complexes, where it regulates the strength and stability of adhesions. Whilst the role of vinculin in cell motility is well established, it remains unclear how vinculin contributes to other aspects of tissue function. Here we examine the role of vinculin in mammary epithelial cell phenotype. In these cells, correct adhesion to the extracellular matrix is essential for both the formation of polarised secretory acini and for the transcription of tissue-specific milk protein genes. We show that vinculin, through its interaction with talin, controls milk protein gene expression. However, vinculin is not required for the formation of polarised acini. This work reveals new roles for vinculin that are central to cellular differentiation, and for the ability of cells to interpret their extracellular microenvironment.

The role of the local microenvironment in influencing cell behavior is central to both normal development and cancer formation. Here, we show that sprouty 1 (SPRY1) modulates the microenvironment to enable proper mammary branching morphogenesis. This process occurs through negative regulation of epidermal growth factor receptor (EGFR) signaling in mammary stroma. Loss of SPRY1 resulted in up-regulation of EGFR-extracellular signal-regulated kinase (ERK) signaling in response to amphiregulin and transforming growth factor alpha stimulation. Consequently, stromal paracrine signaling and ECM remodeling is augmented, leading to increased epithelial branching in the mutant gland. By contrast, down-regulation of EGFR-ERK signaling due to gain of Sprouty function in the stroma led to stunted epithelial branching. Taken together, our results show that modulation of stromal paracrine signaling and ECM remodeling by SPRY1 regulates mammary epithelial morphogenesis during postnatal development.

The expression of tissue-specific genes during mammary gland differentiation relies on the coincidence of two distinct signaling events: the continued engagement of [beta]1 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 Pr1-induced synthesis of the milk protein [beta]-casein in primary mammary epithelial cells cultured as three-dimensional acini on basement membrane. Conversely, activated Racl rescues the defective [beta]-casein synthesis that occurs under conditions not normally permissive for mammary differentiation, either in [beta]1 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.

Integrin-mediated adhesion regulates the development and function of a range of tissues; however, little is known about its role in glandular epithelium. To assess the contribution of [beta]1 integrin, we conditionally deleted its gene in luminal epithelia during different stages of mouse mammary gland development and in cultured primary mammary epithelia. Loss of [beta]1 integrin in vivo resulted in impaired alveologenesis and lactation. Cultured [beta]1 integrin-null cells displayed abnormal focal adhesion function and signal transduction and could not form or maintain polarized acini. In vivo, epithelial cells became detached from the extracellular matrix but remained associated with each other and did not undergo overt apoptosis. [beta]1 integrin-null mammary epithelial cells did not differentiate in response to prolactin stimulation because of defective Stat5 activation. In mice where [beta]1 integrin was deleted after the initiation of differentiation, fewer defects in alveolar morphology occurred, yet major deficiencies were also observed in milk protein and milk fat production and Stat5 activation, indicating a permissive role for [beta]1 integrins in prolactin signaling. This study demonstrates that [beta]1 integrin is critical for the alveolar morphogenesis of a glandular epithelium and for maintenance of its differentiated function. Moreover, it provides genetic evidence for the cooperation between integrin and cytokine signaling pathways.

Myelination of the peripheral nervous system (PNS) requires the migration of Schwann cells during both development and regeneration. We have characterized the expression pattern of Schwann cell integrins and analyzed their role in migration on different ECM substrates known to be present within the PNS. We found that Schwann cells in cell culture express four [Beta]1 integrins, [Alpha]1[Beta]1, [Alpha]2[Beta]1, [Alpha]6[Beta]1, and another unidentified [Beta]1 integrin, as well as two [Alpha]v integrins, [Alpha]v[Beta]3 and [Alpha]v[Beta]8. Using the Varani migration assay, we found that laminin-1, laminin-2 (merosin), and fibronectin increased Schwann cell migration, while vitronectin and collagen did not increase migration compared to an uncoated plastic substrate. Schwann cell migration on laminin-1 and laminin-2 (merosin) was blocked by antibodies against [Beta]1 integrins, but not affected by RGD peptides or antibodies against [Beta]3 integrins. In contrast, migration on fibronectin was unaffected by antibodies against [Beta]1 and [Beta]3 integrins but was blocked by RGD peptides. This in vitro study shows that there is a division of labor of Schwann cell integrins in the regulation of migration on peripheral nerve ECM components; [Beta]1 integrins mediate migration on laminin-1 and laminin-2 (merosin), while [Alpha]v integrins mediate migration on fibronectin. Taken together, these results suggest that multiple interactions between Schwann cell integrins and ECM within the PNS will contribute to Schwann cell migration during myelination of the PNS.

The influence of the extracellular matrix in the expression of transforming growth factor-beta1 (TGF-beta1) by mammary epithelial cells was investigated. The absence of the basement membrane resulted in an upregulation of TGF-beta1 expression when the cells were maintained on a plastic substratum. Genetic analysis showed that the effect was due to enhanced transcription from the TGF-beta1 promoter gene. The basement membrane is thought to be part of a feedback loop that regulates TGF-beta1 expression and basement membrane formation.