Abstract
Rho GTPases mediate stromal-epithelial interactions that are important for mammary epithelial cell (MEC) morphogenesis. Increased extracellular matrix (ECM) deposition and reorganization affect MEC morphogenesis in a Rho GTPase-dependent manner. Although the effects of altered ECM on MEC morphogenesis have been described, how MECs regulate stromal deposition is not well understood. Previously, we showed that p190B RhoGAP overexpression disrupts mammary gland morphogenesis by inducing hyperbranching in association with stromal alterations. We therefore hypothesized that MEC overexpression of p190B regulates paracrine interactions to impact fibroblast activation. Using a combination of in vivo morphometric and immunohistochemical analyses and primary cell culture assays, we found that p190B overexpression in MECs activates fibroblasts leading to increased collagen, fibronectin, and laminin production and elevated expression of the collagen crosslinking enzyme lysyl oxidase. Phosphorylation of the TGF-β effector SMAD2 and expression of the TGF-β target gene αSma were increased in p190B-associated fibroblasts, suggesting that elevated TGF-β signaling promoted fibroblast activation. Mechanical tension and TGF-β cooperate to activate fibroblasts. Interestingly, active TGF-β was elevated in conditioned medium from p190B overexpressing MECs compared to control MECs, and p190B overexpressing MECs exhibited increased contractility in a collagen gel contraction assay. These data suggest that paracrine signaling from the p190B overexpressing MECs may activate TGF-β signaling in adjacent fibroblasts. In support of this, transfer of conditioned medium from p190B overexpressing MECs onto wildtype fibroblasts or co-culture of p190B overexpressing MECs with wildtype fibroblasts increased SMAD2 phosphorylation and mRNA expression of ECM genes in the fibroblasts when compared to fibroblasts treated with control CM or co-cultured with control MECs. The increased ECM gene expression and SMAD2 phosphorylation were blocked by treatment with a TGF-β receptor inhibitor. Taken together, these data suggest that p190B overexpression in the mammary epithelium induces fibroblast activation via elevated TGF-β paracrine signaling.
Highlights
Stromal-epithelial interactions are critical for mammary gland development, and soluble and mechanical cues originating in the stroma impact mammary epithelial cell (MEC) morphogenesis [1]
We previously reported that extracellular matrix (ECM) deposition appeared to be increased in association with dysmorphic, hyperbranched terminal end buds (TEBs) in the p190B overexpressing mammary glands [18]
Condensed stroma is typically found adjacent to the neck region of the TEB, whereas the tip of the TEB is usually surrounded by only a thin basement membrane [22]
Summary
Stromal-epithelial interactions are critical for mammary gland development, and soluble and mechanical cues originating in the stroma impact MEC morphogenesis [1]. The effects of matrix rigidity and cancer-associated fibroblasts on epithelial morphogenesis and tumor formation have been described, the mechanisms by which the epithelium regulates fibroblast activity and ECM production during normal and neoplastic mammary gland development are not well understood. Rho GTPases have been shown to be critical mediators of interactions between the stromal and epithelial compartments during normal tissue morphogenesis and tumor formation [6]. Targeted overexpression of ROCK in the epidermis increases actomyosin contractility leading to stromal activation, increased collagen density and ECM stiffness, and tumor formation [14] These studies suggest that Rho signaling affects tissue morphogenesis and homeostasis by regulating a bidirectional mechanical signaling loop between the stromal and epithelial compartments. RhoA and ROCK have been shown to be important regulators of mechanical signaling, the contribution of other Rho family GTPases and their regulators is unclear
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