With no adjust inside the levels of total protein as assayed by immunoblot (Fig. 6C). -catenin was also activated in these cells using lithium chloride (LiCl) following SLIT2 treatment and, once again, there was elevated -catenin membrane staining in SLIT2-treated samples and substantially decreased nuclear translocation (Fig. S4A). With each other, these studies recommend that SLIT/ROBO1 signaling influences -catenin’s subcellular localization. In cancer cells this happens via the Akt/PKB pathway (Prasad et al., 2008; Tseng et al., 2010), which negatively regulates glycogen synthase kinase 3-beta (GSK-3) downstream of development factor receptors (Cross et al., 1995). Similarly, we discovered that EGF and Insulin (GF) treatment of key MECs and LECs, as well as HME50 cells, improved the phosphorylation of Akt and GSK-3 (Figs. 6D, S4B)). Pre-treatment of cells with SLIT decreased this response in MECs and HME50 cells, but not in LECs. Decreased phosphorylation of GSK-3 activates it (Cross et al., 1995), favoring the accumulation of -catenin within the cytosol and membrane of these cells (Figs. 6A). Next, we probed entire MEC lysates with an antibody directed against active -catenin (ABC) (Staal et al., 2002), and observed a decrease in this form upon SLIT2 therapy (Fig. 6E). We made use of this antibody to examine the basal layer of +/+ organoids. In untreated organoids, there’s modest constructive staining in the nucleus. Treating cells with an activator of canonical WNT signaling, considerably elevated the nuclear staining of unphosphorylated -catenin, whereas treatment with SLIT2 decreased -catenin’s nuclear staining, while growing its membrane staining (Fig. 6F). These data indicate that SLIT2 inhibits nuclear translocation of -catenin, most likely decreasing its transcriptional functions. To Frizzled-8 Proteins Biological Activity evaluated LEF/TCF transcriptional targets by RT-qPCR and identified enhanced expression of Axin2, Cyclin D1 and Tcf1 mRNA in main MECs harvested from Robo1-/ glands, as well as a concordant reduce in mRNA from +/+ MECs treated with SLIT2 (Fig. 6G). A single of these transcripts also can be monitored in vivo employing Axin2lacZ/+ mice. These mice faithfully reflect -catenin signaling by reporting Axin2 expression in a number of tissues (Lustig et al., 2002). During branching morphogenesis, there is robust -gal staining in cap cells on the finish bud and basal MECs of subtending ducts (Fig. S4C) (Zeng and Nusse, 2010). We implanted SLIT2 and BSA pellets into Axin2lacZ/+ glands and observed substantially reduced -gal staining in MECs with SLIT2, but not BSA (Fig. 6H). These data indicate that SLIT2 inhibits the proliferation of ROBO1-expressing basal cells by opposing the activation of catenin. Taken together, sour data suggest a mechanism for restricting mammary branching morphogenesis by controlling cell quantity, specifically in the basal layer on the bi-layered mammary gland (Fig. 7).Dev Cell. Author manuscript; available in PMC 2012 June 14.Macias et al.PageDISCUSSIONOur studies define a mechanism governing mammary branching morphogenesis, whereby SLIT/ROBO1 signaling inhibits lateral branch formation by controlling the proliferation from the basal cell layer. Specificity of signaling is achieved by restricting the expression of ROBO1 to the basal layer and regulating it with TGF-1. This mechanism of SLIT regulating branching is different from the mechanisms identified in the nervous program, exactly where an extracellular source of SLIT signals to ROBO receptors expressed on growth cones or axo.
