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Deaf-1 regulates epithelial cell proliferation and side-branching in the mammary gland.

Barker HE, Smyth GK, Wettenhall J, Ward TA, Bath ML, Lindeman GJ, Visvader JE - BMC Dev. Biol. (2008)

Bottom Line: In addition, the ratio of the progesterone receptor isoforms PRA and PRB, previously implicated in regulating ductal side-branching, was altered.Furthermore, MMTV-Deaf-1 transgenic mammary glands were found to have elevated levels of Rac3 mRNA, suggesting that it is a bona fide target.Transgenic mammary glands overexpressing Deaf-1 exhibited a modest side-branching phenotype, accompanied by an increase in the number of BrdU-positive cells and a decrease in the proportion of PRA-expressing cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3050, Australia. barker@wehi.edu.au

ABSTRACT

Background: The transcription factor DEAF-1 has been identified as a high affinity binding partner of the LIM-only protein LMO4 that plays important roles in mammary gland development and breast cancer. Here we investigated the influence of DEAF-1 on human and mouse mammary epithelial cells both in vitro and in vivo and identified a potential target gene.

Results: Overexpression of DEAF-1 in human breast epithelial MCF10A cells enhanced cell proliferation in the mammary acini that develop in 3D cultures. To investigate the effects of Deaf-1 on mammary gland development and oncogenesis, we generated MMTV-Deaf-1 transgenic mice. Increased ductal side-branching was observed in young virgin mammary glands, accompanied by augmented cell proliferation. In addition, the ratio of the progesterone receptor isoforms PRA and PRB, previously implicated in regulating ductal side-branching, was altered. Affymetrix gene profiling studies revealed Rac3 as a potential target gene and quantitative RT-PCR analysis confirmed that Rac3 was upregulated by Deaf-1 in immortalized mouse mammary epithelial cells. Furthermore, MMTV-Deaf-1 transgenic mammary glands were found to have elevated levels of Rac3 mRNA, suggesting that it is a bona fide target.

Conclusion: We have demonstrated that overexpression of Deaf-1 enhances the proliferation of human breast epithelial cells in vitro and mouse epithelial cells in vivo. Transgenic mammary glands overexpressing Deaf-1 exhibited a modest side-branching phenotype, accompanied by an increase in the number of BrdU-positive cells and a decrease in the proportion of PRA-expressing cells. Although proliferation was enhanced in Deaf-1 transgenic mice, overexpression of this gene was not sufficient to induce the formation of mammary tumors. In addition, our studies identified Rac3, encoding a small Rho-like GTPase, as a potential target of Deaf-1 in mouse mammary epithelial cells.

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No phenotype is evident in Deaf-1 transgenic mice during pregnancy, lactation and involution. A) Wholemounts of inguinal mammary glands from Deaf-1 transgenic (a, c and e) or FVB/N wild-type mice (b, d and f). Whole-mounted glands at different time-points: 12 days of pregnancy (12 dP; a and b), 1 day of lactation (1 dL; c and d) and 4 days of involution (4 dI; e and f). Original magnification ×7.5. B) Sections of glands from Deaf-1 transgenic (a, c and e) and FVB/N wild-type mice (b, d and f) at 12 days of pregnancy (12 dP; a and b), 1 day of lactation (1 dL; c and d) and 4 days of involution (4 dI; e and f). Original magnification ×100. Tg, transgenic; wt, wild-type.
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Figure 4: No phenotype is evident in Deaf-1 transgenic mice during pregnancy, lactation and involution. A) Wholemounts of inguinal mammary glands from Deaf-1 transgenic (a, c and e) or FVB/N wild-type mice (b, d and f). Whole-mounted glands at different time-points: 12 days of pregnancy (12 dP; a and b), 1 day of lactation (1 dL; c and d) and 4 days of involution (4 dI; e and f). Original magnification ×7.5. B) Sections of glands from Deaf-1 transgenic (a, c and e) and FVB/N wild-type mice (b, d and f) at 12 days of pregnancy (12 dP; a and b), 1 day of lactation (1 dL; c and d) and 4 days of involution (4 dI; e and f). Original magnification ×100. Tg, transgenic; wt, wild-type.

Mentions: Wholemounts and histological sections were prepared from transgenic (Deaf-29 and Deaf-42) and wild-type mice at the following stages of mammary gland development: 12 and 18 days of pregnancy, 1 and 8 days of lactation, and 1 and 4 days of involution. All transgenic mammary glands appeared morphologically normal. Figure 4 shows representative wholemounts and sections harvested from transgenic and wild-type mice at pregnancy, lactation and involution. At least three mice of each genotype were analyzed for each stage.


Deaf-1 regulates epithelial cell proliferation and side-branching in the mammary gland.

Barker HE, Smyth GK, Wettenhall J, Ward TA, Bath ML, Lindeman GJ, Visvader JE - BMC Dev. Biol. (2008)

No phenotype is evident in Deaf-1 transgenic mice during pregnancy, lactation and involution. A) Wholemounts of inguinal mammary glands from Deaf-1 transgenic (a, c and e) or FVB/N wild-type mice (b, d and f). Whole-mounted glands at different time-points: 12 days of pregnancy (12 dP; a and b), 1 day of lactation (1 dL; c and d) and 4 days of involution (4 dI; e and f). Original magnification ×7.5. B) Sections of glands from Deaf-1 transgenic (a, c and e) and FVB/N wild-type mice (b, d and f) at 12 days of pregnancy (12 dP; a and b), 1 day of lactation (1 dL; c and d) and 4 days of involution (4 dI; e and f). Original magnification ×100. Tg, transgenic; wt, wild-type.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2570686&req=5

Figure 4: No phenotype is evident in Deaf-1 transgenic mice during pregnancy, lactation and involution. A) Wholemounts of inguinal mammary glands from Deaf-1 transgenic (a, c and e) or FVB/N wild-type mice (b, d and f). Whole-mounted glands at different time-points: 12 days of pregnancy (12 dP; a and b), 1 day of lactation (1 dL; c and d) and 4 days of involution (4 dI; e and f). Original magnification ×7.5. B) Sections of glands from Deaf-1 transgenic (a, c and e) and FVB/N wild-type mice (b, d and f) at 12 days of pregnancy (12 dP; a and b), 1 day of lactation (1 dL; c and d) and 4 days of involution (4 dI; e and f). Original magnification ×100. Tg, transgenic; wt, wild-type.
Mentions: Wholemounts and histological sections were prepared from transgenic (Deaf-29 and Deaf-42) and wild-type mice at the following stages of mammary gland development: 12 and 18 days of pregnancy, 1 and 8 days of lactation, and 1 and 4 days of involution. All transgenic mammary glands appeared morphologically normal. Figure 4 shows representative wholemounts and sections harvested from transgenic and wild-type mice at pregnancy, lactation and involution. At least three mice of each genotype were analyzed for each stage.

Bottom Line: In addition, the ratio of the progesterone receptor isoforms PRA and PRB, previously implicated in regulating ductal side-branching, was altered.Furthermore, MMTV-Deaf-1 transgenic mammary glands were found to have elevated levels of Rac3 mRNA, suggesting that it is a bona fide target.Transgenic mammary glands overexpressing Deaf-1 exhibited a modest side-branching phenotype, accompanied by an increase in the number of BrdU-positive cells and a decrease in the proportion of PRA-expressing cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3050, Australia. barker@wehi.edu.au

ABSTRACT

Background: The transcription factor DEAF-1 has been identified as a high affinity binding partner of the LIM-only protein LMO4 that plays important roles in mammary gland development and breast cancer. Here we investigated the influence of DEAF-1 on human and mouse mammary epithelial cells both in vitro and in vivo and identified a potential target gene.

Results: Overexpression of DEAF-1 in human breast epithelial MCF10A cells enhanced cell proliferation in the mammary acini that develop in 3D cultures. To investigate the effects of Deaf-1 on mammary gland development and oncogenesis, we generated MMTV-Deaf-1 transgenic mice. Increased ductal side-branching was observed in young virgin mammary glands, accompanied by augmented cell proliferation. In addition, the ratio of the progesterone receptor isoforms PRA and PRB, previously implicated in regulating ductal side-branching, was altered. Affymetrix gene profiling studies revealed Rac3 as a potential target gene and quantitative RT-PCR analysis confirmed that Rac3 was upregulated by Deaf-1 in immortalized mouse mammary epithelial cells. Furthermore, MMTV-Deaf-1 transgenic mammary glands were found to have elevated levels of Rac3 mRNA, suggesting that it is a bona fide target.

Conclusion: We have demonstrated that overexpression of Deaf-1 enhances the proliferation of human breast epithelial cells in vitro and mouse epithelial cells in vivo. Transgenic mammary glands overexpressing Deaf-1 exhibited a modest side-branching phenotype, accompanied by an increase in the number of BrdU-positive cells and a decrease in the proportion of PRA-expressing cells. Although proliferation was enhanced in Deaf-1 transgenic mice, overexpression of this gene was not sufficient to induce the formation of mammary tumors. In addition, our studies identified Rac3, encoding a small Rho-like GTPase, as a potential target of Deaf-1 in mouse mammary epithelial cells.

Show MeSH
Related in: MedlinePlus