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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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Increased side-branching and proliferation in the mammary glands of young Deaf-1 transgenic mice. A) Wholemounts of thoracic mammary glands harvested from transgenic (a) and wild-type (b) 8 week-old virgin mice. Original magnification, ×7.5. Sections of inguinal mammary glands from transgenic (c and e) and wild-type (d and f) mice stained with haematoxylin and eosin. Original magnification, ×40 (c and d) and ×400 (e and f). B) Immunostaining of mammary sections from transgenic (a) and wild-type (b) 8 week-old mice with anti-BrdU. Original magnification ×400. C) Percent BrdU-positive cells was calculated by counting greater than 1,000 nuclei in 10 random fields for each mouse. At least 5 mice were analyzed for both transgenic and wild-type genotypes. The percentage of BrdU-positive cells in transgenic glands (Tg) was 24.2% ± 7.9% versus 2.5% ± 1.2% in control (wt) glands. Error bars represent standard deviation from the mean. D) Immunostaining of sections from transgenic (a and b) and wild-type (c and d) mammary glands of 8 week-old mice using anti-PRA antibody. Original magnification ×400. E) Percent PRA-positive cells was calculated by counting greater than 1,000 nuclei in 10 random fields for each mouse. At least 4 mice were analyzed for each genotype. The percentage of PRA-positive cells in transgenic glands (Tg) was 32.1% ± 3.0% versus 61.8% ± 2.9% in control (wt) glands. Error bars represent the standard deviation from the mean.
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Figure 3: Increased side-branching and proliferation in the mammary glands of young Deaf-1 transgenic mice. A) Wholemounts of thoracic mammary glands harvested from transgenic (a) and wild-type (b) 8 week-old virgin mice. Original magnification, ×7.5. Sections of inguinal mammary glands from transgenic (c and e) and wild-type (d and f) mice stained with haematoxylin and eosin. Original magnification, ×40 (c and d) and ×400 (e and f). B) Immunostaining of mammary sections from transgenic (a) and wild-type (b) 8 week-old mice with anti-BrdU. Original magnification ×400. C) Percent BrdU-positive cells was calculated by counting greater than 1,000 nuclei in 10 random fields for each mouse. At least 5 mice were analyzed for both transgenic and wild-type genotypes. The percentage of BrdU-positive cells in transgenic glands (Tg) was 24.2% ± 7.9% versus 2.5% ± 1.2% in control (wt) glands. Error bars represent standard deviation from the mean. D) Immunostaining of sections from transgenic (a and b) and wild-type (c and d) mammary glands of 8 week-old mice using anti-PRA antibody. Original magnification ×400. E) Percent PRA-positive cells was calculated by counting greater than 1,000 nuclei in 10 random fields for each mouse. At least 4 mice were analyzed for each genotype. The percentage of PRA-positive cells in transgenic glands (Tg) was 32.1% ± 3.0% versus 61.8% ± 2.9% in control (wt) glands. Error bars represent the standard deviation from the mean.

Mentions: Analysis of young virgin transgenic mice (8 weeks of age) revealed an increase in the number of side-branches by wholemount and histological analyses (Fig. 3A). Since fluctuation in ductal branching occurs during the estrous cycle, vaginal smears were stained with haematoxylin and eosin to ensure that mammary glands were harvested from mice at the same stage of the estrous cycle. For strain Deaf-29, 5 out of 12 mice exhibited mammary glands with substantially increased side-branching compared with 2 out of 5 Deaf-42 transgenic mice (Table 1). Although some variation in the degree of side-branching was noted amongst the sixteen control (wild-type) mice analyzed, transgenic mice exhibited profoundly abnormal mammary glands compared to wild-type glands.


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)

Increased side-branching and proliferation in the mammary glands of young Deaf-1 transgenic mice. A) Wholemounts of thoracic mammary glands harvested from transgenic (a) and wild-type (b) 8 week-old virgin mice. Original magnification, ×7.5. Sections of inguinal mammary glands from transgenic (c and e) and wild-type (d and f) mice stained with haematoxylin and eosin. Original magnification, ×40 (c and d) and ×400 (e and f). B) Immunostaining of mammary sections from transgenic (a) and wild-type (b) 8 week-old mice with anti-BrdU. Original magnification ×400. C) Percent BrdU-positive cells was calculated by counting greater than 1,000 nuclei in 10 random fields for each mouse. At least 5 mice were analyzed for both transgenic and wild-type genotypes. The percentage of BrdU-positive cells in transgenic glands (Tg) was 24.2% ± 7.9% versus 2.5% ± 1.2% in control (wt) glands. Error bars represent standard deviation from the mean. D) Immunostaining of sections from transgenic (a and b) and wild-type (c and d) mammary glands of 8 week-old mice using anti-PRA antibody. Original magnification ×400. E) Percent PRA-positive cells was calculated by counting greater than 1,000 nuclei in 10 random fields for each mouse. At least 4 mice were analyzed for each genotype. The percentage of PRA-positive cells in transgenic glands (Tg) was 32.1% ± 3.0% versus 61.8% ± 2.9% in control (wt) glands. Error bars represent the standard deviation from the mean.
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Figure 3: Increased side-branching and proliferation in the mammary glands of young Deaf-1 transgenic mice. A) Wholemounts of thoracic mammary glands harvested from transgenic (a) and wild-type (b) 8 week-old virgin mice. Original magnification, ×7.5. Sections of inguinal mammary glands from transgenic (c and e) and wild-type (d and f) mice stained with haematoxylin and eosin. Original magnification, ×40 (c and d) and ×400 (e and f). B) Immunostaining of mammary sections from transgenic (a) and wild-type (b) 8 week-old mice with anti-BrdU. Original magnification ×400. C) Percent BrdU-positive cells was calculated by counting greater than 1,000 nuclei in 10 random fields for each mouse. At least 5 mice were analyzed for both transgenic and wild-type genotypes. The percentage of BrdU-positive cells in transgenic glands (Tg) was 24.2% ± 7.9% versus 2.5% ± 1.2% in control (wt) glands. Error bars represent standard deviation from the mean. D) Immunostaining of sections from transgenic (a and b) and wild-type (c and d) mammary glands of 8 week-old mice using anti-PRA antibody. Original magnification ×400. E) Percent PRA-positive cells was calculated by counting greater than 1,000 nuclei in 10 random fields for each mouse. At least 4 mice were analyzed for each genotype. The percentage of PRA-positive cells in transgenic glands (Tg) was 32.1% ± 3.0% versus 61.8% ± 2.9% in control (wt) glands. Error bars represent the standard deviation from the mean.
Mentions: Analysis of young virgin transgenic mice (8 weeks of age) revealed an increase in the number of side-branches by wholemount and histological analyses (Fig. 3A). Since fluctuation in ductal branching occurs during the estrous cycle, vaginal smears were stained with haematoxylin and eosin to ensure that mammary glands were harvested from mice at the same stage of the estrous cycle. For strain Deaf-29, 5 out of 12 mice exhibited mammary glands with substantially increased side-branching compared with 2 out of 5 Deaf-42 transgenic mice (Table 1). Although some variation in the degree of side-branching was noted amongst the sixteen control (wild-type) mice analyzed, transgenic mice exhibited profoundly abnormal mammary glands compared to wild-type glands.

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