Abstract
In adherent cells, cell-substratum interactions are essential for the propagation of some growth factor signaling events. However, it has not been resolved to what extent different types of extracellular matrix regulate the signals elicited by different soluble ligands. Our previous work has shown that prolactin signaling in mammary epithelium requires a specific cell interaction with the basement membrane and does not occur in cells plated on collagen I. We have now investigated whether the proximal signaling pathways triggered by insulin, epidermal growth factor (EGF), and interferon-gamma are differentially regulated in primary mammary epithelial cell cultures established on basement membrane and collagen I. Two distinct signaling pathways triggered by insulin exhibited a differential requirement for cell-matrix interactions. Activation of insulin receptor substrate (IRS) and phosphatidylinositol 3-kinase was restricted to cells contacting basement membrane, whereas the phosphorylation of Erk occurred equally in cells on both substrata. The amplitude and duration of insulin-triggered IRS-1 phosphorylation and its association with phosphatidylinositol 3-kinase were strongly enhanced by cell-basement membrane interactions. The mechanism for inhibition of IRS-1 phosphorylation in cells cultured on collagen I may in part be mediated by protein-tyrosine phosphatase activity since vanadate treatment somewhat alleviated this effect. In contrast to the results with insulin, cell adhesion to collagen I conferred greater response to EGF, leading to higher levels of tyrosine phosphorylation of the EGF receptor and Erk. The mechanism for increased EGF signaling in cells adhering to collagen I was partly through an increase in EGF receptor expression. The interferon-gamma-activated tyrosine phosphorylation of Jak2 and Stat3 was independent of the extracellular matrix. It is well recognized that the cellular environment determines cell phenotype. We now suggest that this may occur through a selective modulation of growth factor signal transduction resulting from different cell-matrix interactions.
Highlights
In mammary gland, the secretory epithelium resides on basement membrane (BM), which is required for milk protein gene expression and cell survival [7]
Growth Factor-activated Tyrosine Phosphorylation Is Differentially Regulated by extracellular matrix (ECM)—We have previously shown that prolactin-activated signal transduction in mammary epithelial cells is dependent on cell-BM interactions, but does not occur when cells are plated on tissue culture plastic or dishes coated with collagen I [13]
To examine whether this reflected a general response of mammary epithelium to growth factors, cells cultured on collagen I and BM were stimulated by various growth factors, and protein tyrosine phosphorylation was monitored by Western blot analysis
Summary
To discriminate between these two possibilities, we examined the proximal signaling events triggered by insulin, EGF, and interferon-␥ (IFN-␥) in mammary epithelial cells plated on either collagen I or BM. In contrast to other studies that assessed growth factor signaling in cells plated on ECM for very short periods of time [3, 4, 6, 14], cell signaling in long-term cultures was investigated. We examined growth factor signaling in cells that had established stable cell-matrix interactions over several days, mimicking the type of interactions they would experience in vivo. We investigated the signaling responses of wild-type primary cell cultures, rather than those of reconstituted cell lines transfected with vectors overexpressing signaling components, as has largely been performed previously. Our results show that the cellular microenvironment selectively modulates the response of mammary cells to different soluble signaling ligands and that this occurs through different mechanisms
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