ed contractility of p190B overexpressing MECs, increased ECM deposition, and elevated phosphorylated MLC2 and aSMA levels in the p190Bassociated fibroblasts are indicative of increased mechanical tension within the p190B overexpressing TEBs and adjacent stroma. These altered mechanical properties may also contribute to the activation of p190B-associated fibroblasts, although future experiments will be required to directly demonstrate the importance of altered mechanical forces in this process. In addition, although our conditioned medium and co-culture experiments show that p190B overexpressing MECs can induce TGF-b dependent fibroblast activation, 11259531 it is possible that this may lead to increased autocrine signaling in the fibroblasts to further promote their activation. Secretion of CTGF, a potent regulator of ECM production that PG-490 cooperates with TGF-b to regulate fibroblast activity, was upregulated in the p190Bassociated fibroblasts, suggesting that an autocrine loop may also be active. Proteolytic and integrin-mediated, cytoskeletal-dependent mechanisms 
have been shown to activate latent TGF-b. Our data demonstrating that p190B overexpressing MECs display elevated contractility suggests that an integrin/cytoskeletal-mediated mechanism may contribute to the increase in TGF-b activation by the p190B overexpressing MECs. However, future studies will be required to define the mechanisms by which p190B overexpression in MECs leads to increased activation of TGF-b. An important question that remains to be answered is how the altered stromal environment affects the developing mammary epithelium in the p190B overexpressing mammary glands. P190B overexpressing TEBs display increased proliferation, dysmorphic morphologies, and hyperbranching in association with fibroblast activation, increased ECM deposition, and potentially reorganization of the ECM by LOX. Increased ECM deposition and rigidity are known to disrupt MEC morphogenesis. However, it is currently unclear whether the aberrant TEB morphogenesis that occurs in p190B overexpressing mammary glands is MEC autonomous or whether mechanical cues resulting from the altered stroma contribute to these phenotypes. Activated fibroblasts also produce growth factors and cytokines that impact MEC morphogenesis. Thus, it is possible that the p190Bassociated fibroblasts secrete factors that impact proliferation and branching of the adjacent epithelium. Future studies will be required to define the relative contributions of mechanical and soluble signals generated by the p190B-associated fibroblasts as P190B Regulates the Mammary Gland Microenvironment well as epithelial autonomous effects to the p190B overexpressing TEB phenotypes. Rho GTPase expression and activity levels are elevated in human breast tumors, and aberrant Rho signaling affects transformation, tumor cell proliferation, and migration and invasion, all of which are important for tumorigenesis and metastasis. Our data as well as data from reported studies suggest that aberrant Rho signaling in the epithelial compartment may also play an important role in altering interactions between the epithelium and stroma, leading to fibroblast 26976569 activation and increased ECM deposition. Thus, stromal activation resulting from aberrant Rho signaling in the epithelial compartment may be another mechanism by which Rho signaling contributes to tumorigenesis. p190B-associated and control fibroblasts. The RT2 Profiler PCR Array Mouse Extracellular Matrix and
