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Hormonal induction and roles of Disabled-2 in lactation and involution.

Tao W, Moore R, Smith ER, Xu XX - PLoS ONE (2014)

Bottom Line: Loss of Dab2 had subtle effects on lactation, but Dab2-deficient mammary glands showed a strikingly delayed cell clearance during involution.However, Dab2 deletion did not affect Smad2 phosphorylation; rather TGF-beta-stimulated MAPK activation was enhanced in Dab2-deficient cells.We conclude that Dab2 expression is induced by hormones and Dab2 plays a role in modulating TGF-beta signaling to enhance apoptotic clearance of mammary epithelial cells during involution.

View Article: PubMed Central - PubMed

Affiliation: Sylvester Comprehensive Cancer Center, and Department of Cell Biology, Graduate Program in Molecular Cell and Developmental Biology, University of Miami Miller School of Medicine, Miami, Florida, United States of America.

ABSTRACT
Disabled-2 (Dab2) is a widely expressed endocytic adaptor that was first isolated as a 96 KDa phospho-protein, p96, involved in MAPK signal transduction. Dab2 expression is lost in several cancer types including breast cancer, and Dab2 is thought to have a tumor suppressor function. In mammary epithelia, Dab2 was induced upon pregnancy and further elevated during lactation. We constructed mutant mice with a mosaic Dab2 gene deletion to bypass early embryonic lethality and to investigate the roles of Dab2 in mammary physiology. Loss of Dab2 had subtle effects on lactation, but Dab2-deficient mammary glands showed a strikingly delayed cell clearance during involution. In primary cultures of mouse mammary epithelial cells, Dab2 proteins were also induced by estrogen, progesterone, and/or prolactin. Dab2 mammary epithelial cells were refractory to growth suppression induced by TGF-beta. However, Dab2 deletion did not affect Smad2 phosphorylation; rather TGF-beta-stimulated MAPK activation was enhanced in Dab2-deficient cells. We conclude that Dab2 expression is induced by hormones and Dab2 plays a role in modulating TGF-beta signaling to enhance apoptotic clearance of mammary epithelial cells during involution.

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Hormonal induction of Dab2 expression in primary mammary epithelial cells.Mammary epithelial cells were prepared from virgin wildtype control (fl/+) and Dab2 conditional knockout (df/df) mice inheriting Meox2-Cre. The cells were treated with 17 beta estradiol (1 nM), progesterone (1 µM), and prolactin (1 nM, or 50 ng/ml), individually or in combination for 4 days in culture. (A) Dab2 and beta-actin from primary mammary epithelial cells of virgin mice were analyzed by Western blot. (B) The signals of Dab2 proteins from the Western blot were quantified using NIH Image J software using beta-actin as normalization control. Relative values were plotted with the value of untreated cells defined as 1.0. (C) Dab2 and beta-actin from primary mammary epithelial cells isolated from pregnant mice were analyzed by Western blot. Dab2 protein was absent in cells from the conditional knockout mice. (D) The relative intensity of Dab2 proteins on Western blots was quantified using NIH Image J software, using beta-actin for normalization.
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pone-0110737-g002: Hormonal induction of Dab2 expression in primary mammary epithelial cells.Mammary epithelial cells were prepared from virgin wildtype control (fl/+) and Dab2 conditional knockout (df/df) mice inheriting Meox2-Cre. The cells were treated with 17 beta estradiol (1 nM), progesterone (1 µM), and prolactin (1 nM, or 50 ng/ml), individually or in combination for 4 days in culture. (A) Dab2 and beta-actin from primary mammary epithelial cells of virgin mice were analyzed by Western blot. (B) The signals of Dab2 proteins from the Western blot were quantified using NIH Image J software using beta-actin as normalization control. Relative values were plotted with the value of untreated cells defined as 1.0. (C) Dab2 and beta-actin from primary mammary epithelial cells isolated from pregnant mice were analyzed by Western blot. Dab2 protein was absent in cells from the conditional knockout mice. (D) The relative intensity of Dab2 proteins on Western blots was quantified using NIH Image J software, using beta-actin for normalization.

Mentions: Because Dab2 expression coincides with lactation, we speculated that Dab2 levels in mammary epithelial cells are regulated by reproductive hormones during lactogenic differentiation of mammary epithelial cells. We tested this possibility using primary mouse mammary epithelial cell cultures (Figure 2). In mammary epithelial cells isolated from mammary glands of virgin mice, progesterone but not estrogen or prolactin induced an about 4 folds increase in Dab2 proteins (Figure 2A, B). Progesterone and prolactin were synergistic in a higher induction to about 10 folds (Figure 2A, B). The mammary epithelial cell were isolated from expanded mammary glands of pregnant mice in order to produce sufficient number of cells for additional experiments, and the preparations were found to be more than 90% cytokeratin-positive. In these cultured cells, we found that Dab2 was regulated by physiological levels of estrogen, progesterone, and prolactin, while the combination of progesterone and prolactin was most potent in inducing Dab2 expression (Figure 2C). Several likely Dab2 isoforms, including the p96 and p67, were induced following exposure to hormones for 4 days. Mammary epithelial cells isolated from Dab2 knockout mice (more detail below) were used as controls for the specificity of Dab2 proteins in Western blot. The maximal induction of Dab2 proteins by prolactin plus progesterone was estimated to be 22-fold (Figure 2D), and the increase was similar in three repeat experiments.


Hormonal induction and roles of Disabled-2 in lactation and involution.

Tao W, Moore R, Smith ER, Xu XX - PLoS ONE (2014)

Hormonal induction of Dab2 expression in primary mammary epithelial cells.Mammary epithelial cells were prepared from virgin wildtype control (fl/+) and Dab2 conditional knockout (df/df) mice inheriting Meox2-Cre. The cells were treated with 17 beta estradiol (1 nM), progesterone (1 µM), and prolactin (1 nM, or 50 ng/ml), individually or in combination for 4 days in culture. (A) Dab2 and beta-actin from primary mammary epithelial cells of virgin mice were analyzed by Western blot. (B) The signals of Dab2 proteins from the Western blot were quantified using NIH Image J software using beta-actin as normalization control. Relative values were plotted with the value of untreated cells defined as 1.0. (C) Dab2 and beta-actin from primary mammary epithelial cells isolated from pregnant mice were analyzed by Western blot. Dab2 protein was absent in cells from the conditional knockout mice. (D) The relative intensity of Dab2 proteins on Western blots was quantified using NIH Image J software, using beta-actin for normalization.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4216001&req=5

pone-0110737-g002: Hormonal induction of Dab2 expression in primary mammary epithelial cells.Mammary epithelial cells were prepared from virgin wildtype control (fl/+) and Dab2 conditional knockout (df/df) mice inheriting Meox2-Cre. The cells were treated with 17 beta estradiol (1 nM), progesterone (1 µM), and prolactin (1 nM, or 50 ng/ml), individually or in combination for 4 days in culture. (A) Dab2 and beta-actin from primary mammary epithelial cells of virgin mice were analyzed by Western blot. (B) The signals of Dab2 proteins from the Western blot were quantified using NIH Image J software using beta-actin as normalization control. Relative values were plotted with the value of untreated cells defined as 1.0. (C) Dab2 and beta-actin from primary mammary epithelial cells isolated from pregnant mice were analyzed by Western blot. Dab2 protein was absent in cells from the conditional knockout mice. (D) The relative intensity of Dab2 proteins on Western blots was quantified using NIH Image J software, using beta-actin for normalization.
Mentions: Because Dab2 expression coincides with lactation, we speculated that Dab2 levels in mammary epithelial cells are regulated by reproductive hormones during lactogenic differentiation of mammary epithelial cells. We tested this possibility using primary mouse mammary epithelial cell cultures (Figure 2). In mammary epithelial cells isolated from mammary glands of virgin mice, progesterone but not estrogen or prolactin induced an about 4 folds increase in Dab2 proteins (Figure 2A, B). Progesterone and prolactin were synergistic in a higher induction to about 10 folds (Figure 2A, B). The mammary epithelial cell were isolated from expanded mammary glands of pregnant mice in order to produce sufficient number of cells for additional experiments, and the preparations were found to be more than 90% cytokeratin-positive. In these cultured cells, we found that Dab2 was regulated by physiological levels of estrogen, progesterone, and prolactin, while the combination of progesterone and prolactin was most potent in inducing Dab2 expression (Figure 2C). Several likely Dab2 isoforms, including the p96 and p67, were induced following exposure to hormones for 4 days. Mammary epithelial cells isolated from Dab2 knockout mice (more detail below) were used as controls for the specificity of Dab2 proteins in Western blot. The maximal induction of Dab2 proteins by prolactin plus progesterone was estimated to be 22-fold (Figure 2D), and the increase was similar in three repeat experiments.

Bottom Line: Loss of Dab2 had subtle effects on lactation, but Dab2-deficient mammary glands showed a strikingly delayed cell clearance during involution.However, Dab2 deletion did not affect Smad2 phosphorylation; rather TGF-beta-stimulated MAPK activation was enhanced in Dab2-deficient cells.We conclude that Dab2 expression is induced by hormones and Dab2 plays a role in modulating TGF-beta signaling to enhance apoptotic clearance of mammary epithelial cells during involution.

View Article: PubMed Central - PubMed

Affiliation: Sylvester Comprehensive Cancer Center, and Department of Cell Biology, Graduate Program in Molecular Cell and Developmental Biology, University of Miami Miller School of Medicine, Miami, Florida, United States of America.

ABSTRACT
Disabled-2 (Dab2) is a widely expressed endocytic adaptor that was first isolated as a 96 KDa phospho-protein, p96, involved in MAPK signal transduction. Dab2 expression is lost in several cancer types including breast cancer, and Dab2 is thought to have a tumor suppressor function. In mammary epithelia, Dab2 was induced upon pregnancy and further elevated during lactation. We constructed mutant mice with a mosaic Dab2 gene deletion to bypass early embryonic lethality and to investigate the roles of Dab2 in mammary physiology. Loss of Dab2 had subtle effects on lactation, but Dab2-deficient mammary glands showed a strikingly delayed cell clearance during involution. In primary cultures of mouse mammary epithelial cells, Dab2 proteins were also induced by estrogen, progesterone, and/or prolactin. Dab2 mammary epithelial cells were refractory to growth suppression induced by TGF-beta. However, Dab2 deletion did not affect Smad2 phosphorylation; rather TGF-beta-stimulated MAPK activation was enhanced in Dab2-deficient cells. We conclude that Dab2 expression is induced by hormones and Dab2 plays a role in modulating TGF-beta signaling to enhance apoptotic clearance of mammary epithelial cells during involution.

Show MeSH
Related in: MedlinePlus