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?3 integrin-EGF receptor cross-talk activates p190RhoGAP in mouse mammary gland epithelial cells.

ABSTRACT: Active RhoA localizes to plasma membrane, where it stimulates formation of focal adhesions and stress fibers. RhoA activity is inhibited by p190RhoGAP following integrin-mediated cell attachment to allow sampling of new adhesive environments. p190RhoGAP is itself activated by Src-dependent tyrosine phosphorylation, which facilitates complex formation with p120RasGAP. This complex then translocates to the cell surface, where p190RhoGAP down-regulates RhoA. Here we demonstrate that the epidermal growth factor receptor (EGFR) cooperates with ?3 integrin to regulate p190RhoGAP activity in mouse mammary gland epithelial cells. Adhesion to fibronectin stimulates tyrosine phosphorylation of the EGFR in the absence of receptor ligands. Use of a dominant inhibitory EGFR mutant demonstrates that fibronectin-activated EGFR recruits p120RasGAP to the cell periphery. Expression of an inactive ?3 integrin subunit abolishes p190RhoGAP tyrosine phosphorylation, demonstrating a mechanistic link between ?3 integrin-activated Src and EGFR regulation of the RhoA inhibitor. The ?3 integrin/EGFR pathway also has a positive role in formation of filopodia. Together our data suggest that EGFR constitutes an important intrinsic migratory cue since fibronectin is a key component of the microenvironment in normal mammary gland development and breast cancer. Our data also suggest that EGFR expressed at high levels has a role in eliciting cell shape changes associated with epithelial-to-mesenchymal transition.

SUBMITTER: Balanis N

PROVIDER: S-EPMC3216655 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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β3 integrin-EGF receptor cross-talk activates p190RhoGAP in mouse mammary gland epithelial cells.

Balanis Nikolas N Yoshigi Masaaki M Wendt Michael K MK Schiemann William P WP Carlin Cathleen R CR

Molecular biology of the cell 20110921 22

Active RhoA localizes to plasma membrane, where it stimulates formation of focal adhesions and stress fibers. RhoA activity is inhibited by p190RhoGAP following integrin-mediated cell attachment to allow sampling of new adhesive environments. p190RhoGAP is itself activated by Src-dependent tyrosine phosphorylation, which facilitates complex formation with p120RasGAP. This complex then translocates to the cell surface, where p190RhoGAP down-regulates RhoA. Here we demonstrate that the epidermal g ...[more]

PMID: 21937717

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Project description:Cross-talk between the aryl hydrocarbon receptor (AHR) and the estrogen receptor (ER) plays a major role in signaling processes in female reproductive organs.We investigated the influence of the AHR ligand 3-methylcholanthrene (3-MC) on ER-mediated signaling in mammary gland tissue of ovariectomized (ovx) rats.After 14 days of hormonal decline, ovx rats were treated for 3 days with 4 ?g/kg 17?-estradiol (E2), 15 mg/kg 8-prenylnaringenin (8-PN), 15 mg/kg 3-MC, or a combination of these compounds (E2 + 3-MC, 8-PN + 3-MC). Whole-mount preparations of the mammary gland were used to count terminal end buds (TEBs). Protein expression studies (immunohistochemistry, immunofluorescence), a cDNA microarray, pathway analyses, and quantitative real-time polymerase chain reaction (qPCR) were performed to evaluate the interaction between AHR- and ER-mediated signaling pathways.E2 treatment increased the number of TEBs and the levels of Ki-67 protein and progesterone receptor (PR); this treatment also changed the expression of 325 genes by more than 1.5-fold. Although 3-MC treatment alone had marginal impact on gene or protein expression, when rats were co-treated with 3-MC and E2, 3-MC strongly inhibited E2-induced TEB development, protein synthesis, and the expression of nearly half of E2-induced genes. This inhibitory effect of 3-MC was partially mirrored when 8-PN was used as an ER ligand. The anti-estrogenicity of ligand-activated AHR was at least partly due to decreased protein levels of ER? in ductal epithelial cells.Our data show transcriptome-wide anti-estrogenic properties of ligand-activated AHR on ER-mediated processes in the mammary gland, thereby contributing an explanation for the chemopreventive and endocrine-disrupting potential of AHR ligands.Helle J, Bader MI, Keiler AM, Zierau O, Vollmer G, Chittur SV, Tenniswood M, Kretzschmar G. 2016. Cross-talk in the female rat mammary gland: influence of aryl hydrocarbon receptor on estrogen receptor signaling. Environ Health Perspect 124:601-610;?http://dx.doi.org/10.1289/ehp.1509680.

Project description:IntroductionBreast cancer is thought to arise in mammary epithelial stem cells. There is, therefore, a large amount of interest in identifying these cells. The breast is a complex tissue consisting of two epithelial layers (an outer myoepithelial/basal layer and an inner luminal epithelial layer) as well as a large non-epithelial component (fibroblasts, endothelial cells, lymphocytes, adipocytes, neurons and myocytes). The definitive identification of a mammary epithelial stem cell population is critically dependent on its purity. To date, this has been hampered by the lack of suitable markers to separate out the two epithelial layers, and to remove contaminating non-epithelial cells.MethodsMouse mammary glands were dissociated and stained with CD24. Cells were sorted into separate populations based on CD24 expression and assessed for luminal epithelial and myoepithelial/basal markers by direct fluorescent microscopy and real time PCR. The stem/progenitor potential of these cell populations was assessed in vivo by cleared mammary fat pad transplantation.ResultsThree populations of CD24 expressing cells were identified: CD24Negative, CD24Low and CD24High. Staining of these cells with cytokeratin markers revealed that these populations correspond to non-epithelial, myoepithelial/basal and luminal epithelial cells, respectively. Cell identities were confirmed by quantitative PCR. Cleared mammary fat pad transplantation of these cell populations revealed that extensive mammary fat pad repopulation capacity segregates with the CD24Low cells, whilst CD24High cells have limited repopulation capacity.ConclusionDifferential staining of mammary epithelial cells for CD24 can be used to simultaneously isolate pure populations of non-epithelial, myoepithelial/basal and luminal epithelial cells. Furthermore, mammary fat pad repopulation capacity is enriched in the CD24Low population. As separation is achieved using a single marker, it will be possible to incorporate additional markers to further subdivide these populations. This will considerably facilitate the further analysis of mammary epithelial subpopulations, whilst ensuring high purity, which is key for understanding mammary epithelial stem cells in normal tissue biology and carcinogenesis.

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?3 integrin-EGF receptor cross-talk activates p190RhoGAP in mouse mammary gland epithelial cells.

Publications

β3 integrin-EGF receptor cross-talk activates p190RhoGAP in mouse mammary gland epithelial cells.

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