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doi: 10.1242/10.1242/jcs.00334

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A ß-catenin survival signal is required for normal lobular development in the mammary gland

¹ Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030, USA
² Department of Biochemistry and Molecular Biology, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA
³ Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA

View larger version (27K): [in a new window]   Fig. 1. ß-catenin and ß-eng constructs and activity in HC11 cells. (A) Stabilized ß-catenin (*, SA, TA mutations in the N-terminal domain) and ß-eng constructs. The C-terminal domain of ß-catenin is replaced with the N-terminal repressor domain of Drosophila Engrailed to create ß-eng. These constructs were tested for ß-catenin signalling activity in HC11 mammary epithelial cells (B-C). Combinations of E-cadherin-luciferase reporter, either stabilized ß-catenin or ß-eng, and empty plasmid DNA were mixed to equivalent amounts of DNA and transfected into HC11 cells (B). These data show that stabilized ß-catenin upregulates transcription at the E-cadherin promoter, but ß-eng does not. Additionally, constant amounts of the E-cadherin reporter and stabilized ß-catenin were transfected into HC11 cells with increasing amounts of ß-eng (C). The activity of the reporter construct shows that the ß-catenin-mediated activation of E-cadherin transcription is effectively competed by ß-eng in stoichometric ratios with ß-catenin. The ß-eng chimera was cloned into two mammary-specific transgenic expression vectors, driven by the MMTV long terminal repeat or the WAP promoter (D). Both constructs contain six tandem myc tags, an intron 5' to the ß-eng construct and a growth hormone polyA sequence.

View larger version (115K): [in a new window]   Fig. 2. Morphology of the mammary gland and transgenic expression. Mammary glands from transgenic mice (A-I) and wild-type littermates (A'-I') isolated at day 10 (A,A',D,D'), day 13 (B,B',E,E') and day 16 of pregnancy (C,C',F,F'), and day 1 of lactation (G-I,G'-I') from lines WBK6414 and WBK6426. Haematoxylin and eosin staining of tissue sections (A-C,A'-C', G,H,G'H') and whole-mount haematoxylin staining (I,I') revealed reduced lobuloalveolar development of the mammary glands of transgenic mice compared with wild-type littermates. Immunohistochemistry using an antibody against the myc epitope tag in the transgene construct (antibody signal shown in black) (D-F, D'-F') reveals transgene expression at 10P (D, arrows) and 12P (E, arrows), but not at 16P (F). Note fragmented, apoptotic cells associated with transgene expression at 12P (E, arrows). Nontransgenic littermates (D'-F') show no specific antibody signal, and arrows in E' illustrate nonspecific antibody trapping in blood vessels. Bars, 25 µm (D-F,D'-F'); 100 µm (A-C,A'-C',H,H'); 500 µm (G,G',I,I').

View larger version (55K): [in a new window]   Fig. 3. Proliferation and apoptosis in MECs of ß-eng transgenic mice. Immunofluorescent detection of BrdU incorporation in mammary epithelium of wild-type (A) and transgenic (B) mice revealed decreased proliferation in ß-eng transgenics (C). Fluorescent TUNEL assay revealed increased apoptosis in MECs from transgenic (E) compared with wild type (D). BrdU and TUNEL signals are represented in green. Nuclei are visualized by DAPI stain (blue). Bars, 100 µm. *P<0.001.

View larger version (63K): [in a new window]   Fig. 4. ß-eng expression in HC11 cells induces apoptosis. Fluorescent microscopy revealed apoptotic (TUNEL-positive) cells (green), DAPI-labelled nuclei (blue) and antibody against myc tag (red). Mock-infected HC11 cells show very low levels of apoptosis (A,C), whereas infection of cells with ß-eng (B,D) induced apoptosis fivefold (C). Bars, 100 µm (A,B); 25 µm (D). *P<0.001.

View larger version (47K): [in a new window]   Fig. 5. Effect of ß-eng expression on HC11 cell proliferation. Western blot analysis of three replicate HC11 cell cultures infected with ß-catenin, ß-eng or mock-infected control (A). ß-catenin and ß-eng transgene expression in infected cells detected by an antibody against the mycepitope tag. Anti-ß-catenin antibody detected endogenous ß-catenin and served as a loading control. Anti-cyclin-D1 antibody detected no change in the level of cyclin D1 protein, whereas activated MAPK was upregulated compared with the total MAPK protein level in response to ß-eng expression. Quantitation of pMAPK activation by densitometry (C) revealed a 15-fold increase in activated MAPK compared with total MAPK. Proliferation in HC11 cells was measured by immunfluorescent detection of FITC-labelled BrdU incorporation. Quantitation of BrdU-labelled cells (B) revealed no change in proliferation in cells infected with ß-catenin or ß-eng compared with mock-infected cells.

View larger version (66K): [in a new window]   Fig. 6. Downstream signaling of ß-eng. Western blot analysis of three replicate HC11 cell cultures infected with ß-catenin, ß-eng or mock-infected control (A). Phospho-specific AKT antibody revealed no change in activated AKT (A, upper panel) compared with total AKT (A, lower panel). Quantification by densitometry (B) indicated that levels of pAKT relative to total AKT were not significantly changed as a consequence of the exogenous expression of ß-catenin or ß-eng. Semi-quantitative RT-PCR of target genes CD44 (C) and ITF-2 (E) at 18, 20 and 22 cycles of PCR (representative of three separate experiments). CD44 mRNA levels were increased slightly in HC11 cells infected with exogenous ß-catenin, whereas ITF-2 levels appeared unchanged. However, both CD44 and ITF-2 mRNA levels were decreased in cells expressing ß-eng (D,F). Quantitation of these results relative to the control L19 RNA showed that expression of ß-eng in HC11 cells downregulated the expression of both of these mRNAs by at least twofold.

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