The RhoA‐Rok‐myosin II pathway is involved in extracellular matrix‐mediated regulation of prolactin signaling in mammary epithelial cells

Abstract

In mammary epithelial cells (MECs), prolactin-induced signaling and gene expression requires integrin-mediated cell adhesion to basement membrane (BM). In the absence of proper cell–BM interactions, for example, culturing cells on collagen-coated plastic dishes, signal propagation is substantially impaired. Here we demonstrate that the RhoA-Rok-myosin II pathway accounts for the ineffectiveness of prolactin signaling in MECs cultured on collagen I. Under these culture conditions, the RhoA pathway is activated, leading to downregulation of prolactin receptor expression and reduced prolactin signaling. Enforced activation of RhoA in MECs cultured on BM suppresses prolactin receptor levels, and prevents prolactin-induced Stat5 tyrosine phosphorylation and β-casein expression. Overexpression of dominant negative RhoA in MECs cultured on collagen I, or inhibiting Rok activity, increases prolactin receptor expression, and enhances prolactin signaling. In addition, inhibition of myosin II ATPase activity by blebbistatin also exerts a beneficial effect on prolactin receptor expression and prolactin signaling, suggesting that tension exerted by the collagen substratum, in collaboration with the RhoA-Rok-myosin II pathway, contributes to the failure of prolactin signaling. Furthermore, MECs cultured on laminin-coated plastic have similar morphology and response to prolactin as those cultured on collagen I. They display high levels of RhoA activity and are inefficient in prolactin signaling, stressing the importance of matrix stiffness in signal transduction. Our results reveal that RhoA has a central role in determining the fate decisions of MECs in response to cell–matrix interactions. J. Cell. Physiol. 227: 1553–1560, 2012. © 2011 Wiley Periodicals, Inc.

Stiff Collagen Matrices Increase Tumorigenic Prolactin Signaling in Breast Cancer Cells

“…Under physiological conditions in vivo and in three-dimensional laminin culture in vitro, integrin input is independent of FAK but instead activates integrin-like kinase and RAC1 to enhance PRL-induced pSTAT5 and prompt mammary epithelia to undergo differentiation and produce milk proteins (45,46). When these normal cells are cultured in noncompliant matrices of either laminin or collagen I, PRLR, pSTAT5, and milk protein expression is reduced (78). In combination with our studies, these reports underscore the importance of stromal stiffness in determining the outcome of PRL exposure regardless of the target epithelium.…”

Milk Protein Synthesis in the Lactating Mammary Gland: Insights from Transcriptomics Analyses

“…In fact they showed an increase of histone acetylation (i.e., dissociation of histone from DNA allowing the latter to be accessible for transcription factors and polymerase) by the ECM. Very recently is was demonstrated that the RhoA-Rho kinase -myosin II pathway, involved in stress fiber formation, cellular contractility, cell migration, and polarity, has a negative effect on the activation of STAT5 by prolactin in mouse mammary epithelial cells [122]. The same study showed that mammary epithelial cells cultured on plastic or collagen I have a large activation of such pathways and, as a consequence, the stimulation of casein expression by prolactin is strongly impaired.…”

How integrins control breast biology

“…These signals guide tissue-specific gene expression in conjunction with temporal cues from the cytokine, prolactin [41–45]. In a separate mechanism, the elasticity of the local microenvironment and its effects on intracellular tension determine the levels of prolactin receptor — tension blocks transcription of the receptor [46 • ]. Tissue-specific gene expression is therefore controlled not only by biochemical signals, that is, soluble factors and ECM, but also by mechanical forces [47].…”