Heckman, B. M. et al. Crosstalk between the p190-B RhoGAP and IGF signaling pathways is required for embryonic mammary bud development. Dev. Biol. 309, 137-149

Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA.
Developmental Biology (Impact Factor: 3.55). 10/2007; 309(1):137-49. DOI: 10.1016/j.ydbio.2007.07.002
Source: PubMed


P190-B RhoGAP (p190-B, also known as ARHGAP5) has been shown to play an essential role in invasion of the terminal end buds (TEBs) into the surrounding fat pad during mammary gland ductal morphogenesis. Here we report that embryos with a homozygous p190-B gene deletion exhibit major defects in embryonic mammary bud development. Overall, p190-B-deficient buds were smaller in size, contained fewer cells, and displayed characteristics of impaired mesenchymal proliferation and differentiation. Consistent with the reported effects of p190-B deletion on IGF-1R signaling, IGF-1R-deficient embryos also displayed a similar small mammary bud phenotype. However, unlike the p190-B-deficient embryos, the IGF-1R-deficient embryos exhibited decreased epithelial proliferation and did not display mesenchymal defects. Because both IGF and p190-B signaling affect IRS-1/2, we examined IRS-1/2 double knockout embryonic mammary buds. These embryos displayed major defects similar to the p190-B-deficient embryos including smaller bud size. Importantly, like the p190-B-deficient buds, proliferation of the IRS-1/2-deficient mesenchyme was impaired. These results indicate that IGF signaling through p190-B and IRS proteins is critical for mammary bud formation and ensuing epithelial-mesenchymal interactions necessary to sustain mammary bud morphogenesis.


Available from: Tracy Vargo-Gogola
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    • "Both IRS1 and IRS2 were previously shown to play important roles during embryonic MG development (Chan and Lee, 2008; Heckman et al., 2007). Postnatally, both Irs1 and Irs2 null MGs are normal, possibly because they compensate for each other (Chan and Lee, 2008; Heckman et al., 2007). We previously generated Irs1/Irs2 double conditional knockout mice with the R26YFP reporter (Irs1 L/L ;Irs2 L/L ;R26YFP) and used K14-Cre and MMTV-Cre mouse lines to disrupt them in MECs (Figure S3A). "
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    ABSTRACT: Lineage tracing using Cre/lox transgenic mice provides a powerful tool for studying normal mammary epithelial cell (MEC) development and the cellular origins of mammary tumors under physiological settings. However, generation of new transgenic mice for lineage-tracing purposes is often time consuming. Here, we report a lineage-tracing tool for MECs based on intraductal injection of lineage-specific Cre-expressing adenovirus (Ad-Cre). Using well-characterized promoters for Keratin 8 and Keratin 14, we generated lineage-specific Ad-Cre lines for luminal and basal MECs, respectively. By pulse-chase lineage tracing using these Ad-Cre lines, we showed that luminal and basal lineages are largely self-sustained and that IRS1 and IRS2 are essential for maintaining the basal lineage; we also showed that heterogeneous mammary tumors can be induced from luminal MECs in mice carrying the Etv6-NTRK3 fusion gene. Overall, we validated the Ad-Cre system as a promising and efficient tool for fate mapping of normal and malignant cells in adult tissues.
    Stem Cell Reports 06/2014; 2(6). DOI:10.1016/j.stemcr.2014.04.004 · 5.37 Impact Factor
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    • "P190B Rho GTPase activating protein (GAP), an important regulator of Rac and RhoA, functions as a negative regulator by accelerating GTP hydrolysis [15], [16]. Our previous studies have shown that p190B plays a crucial role in the developing embryonic and postnatal mammary gland [17], [18], and intriguingly, that it has pro-tumorigenic functions during MMTV-Neu induced mammary tumor formation [19], [20]. Furthermore, we have demonstrated that p190B overexpression in the developing mammary gland promotes aberrant terminal end bud (TEB) morphogenesis and hyperbranching in association with alterations in the adjacent stroma [18]. "
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    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.
    PLoS ONE 05/2013; 8(5):e65105. DOI:10.1371/journal.pone.0065105 · 3.23 Impact Factor
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    • "Dlk1, an endogenous inhibitor of Notch, could act to repress Notch signals in the mammary primordial epithelium; Notch controls cell fate and tissue homeostasis in postnatal mammary epithelium and could be regulating stem cell function from the time that they arise [46]. Other genes of interest include Arhgap28 and Centd3 since another Rho-GTPase activating protein-encoding gene, Arhgap5, has been shown to regulate epithelial-mesenchymal interactions necessary to sustain mammary bud morphogenesis [47]. "
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    ABSTRACT: The mammary primordium forms during embryogenesis as a result of inductive interactions between its constitutive tissues, the mesenchyme and epithelium, and represents the earliest evidence of commitment to the mammary lineage. Previous studies of embryonic mouse mammary epithelium indicated that, by mid-gestation, these cells are determined to a mammary cell fate and that a stem cell population has been delimited. Mammary mesenchyme can induce mammary development from simple epithelium even across species and classes, and can partially restore features of differentiated tissue to mouse mammary tumours in co-culture experiments. Despite these exciting properties, the molecular identity of embryonic mammary cells remains to be fully characterised. Here, we define the transcriptome of the mammary primordium and the two distinct cellular compartments that comprise it, the mammary primordial bud epithelium and mammary mesenchyme. Pathway and network analysis was performed and comparisons of embryonic mammary gene expression profiles to those of both postnatal mouse and human mammary epithelial cell sub-populations and stroma were made. Several of the genes we have detected in our embryonic mammary cell signatures were previously shown to regulate mammary cell fate and development, but we also identified a large number of novel candidates. Additionally, we determined genes that were expressed by both embryonic and postnatal mammary cells, which represent candidate regulators of mammary cell fate, differentiation and progenitor cell function that could signal from mammary lineage inception during embryogenesis through postnatal development. Comparison of embryonic mammary cell signatures with those of human breast cells identified potential regulators of mammary progenitor cell functions conserved across species. These results provide new insights into genetic regulatory mechanisms of mammary development, particularly identification of novel potential regulators of mammary fate and mesenchymal-epithelial cross-talk. Since cancers may represent diseases of mesenchymal-epithelial communications, we anticipate these results will provide foundations for further studies into the fundamental links between developmental, stem cell and breast cancer biology.
    Breast cancer research: BCR 08/2011; 13(4):R79. DOI:10.1186/bcr2928 · 5.49 Impact Factor
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