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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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Induction of Dab2 by pregnancy and lactation in mammary glands.Mammary glands from virgin (A) and lactating (B) wildtype mice were harvested and used for histological analysis. Representative images of Dab2 immunostaining are shown. Arrows indicate the mammary epithelial cells. We also noted the positive staining of mammary adipocytes, which we have been confirmed to express Dab2 protein. (C) The induction of Dab2 proteins was confirmed by Western blot. Mammary glands were dissected and harvested from virgin, pregnant (Preg.) (12 days), and day 1 and day 5 of lactating (Lact.), and post-lactating/involuting (Invol.) wildtype (WT) mice. The post-lactation/involution samples were harvested from mothers that were weaned naturally. Specifically, the mice lactated for 3 weeks prior to separation from their pups for 1 (lane 9) or 3 (lane 10) days. Mammary tissues from conditional Dab2 knockout mice (dab2 (f/df):Meox2-Cre) (cKO) were used as controls for antibody specificity. The tissues were lysed in SDS buffer, and equal protein from each sample was used for electrophoresis and Western blot analysis of Dab2, beta-catenin, and E-cadherin. Arrows indicate Dab2 p96 and p67 isoforms and a band that is presumably IgG heavy chain.
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pone-0110737-g001: Induction of Dab2 by pregnancy and lactation in mammary glands.Mammary glands from virgin (A) and lactating (B) wildtype mice were harvested and used for histological analysis. Representative images of Dab2 immunostaining are shown. Arrows indicate the mammary epithelial cells. We also noted the positive staining of mammary adipocytes, which we have been confirmed to express Dab2 protein. (C) The induction of Dab2 proteins was confirmed by Western blot. Mammary glands were dissected and harvested from virgin, pregnant (Preg.) (12 days), and day 1 and day 5 of lactating (Lact.), and post-lactating/involuting (Invol.) wildtype (WT) mice. The post-lactation/involution samples were harvested from mothers that were weaned naturally. Specifically, the mice lactated for 3 weeks prior to separation from their pups for 1 (lane 9) or 3 (lane 10) days. Mammary tissues from conditional Dab2 knockout mice (dab2 (f/df):Meox2-Cre) (cKO) were used as controls for antibody specificity. The tissues were lysed in SDS buffer, and equal protein from each sample was used for electrophoresis and Western blot analysis of Dab2, beta-catenin, and E-cadherin. Arrows indicate Dab2 p96 and p67 isoforms and a band that is presumably IgG heavy chain.

Mentions: In a previous study of Dab2 using mouse models (Ref 56, and in more detail below), we observed that its expression in mammary glands varied greatly in different physiological stages. In virgin mice, the Dab2 protein was essentially undetectable in mammary epithelial cells by immunohistochemistry (Figure 1A, arrow), while Dab2 staining was robust and uniform in all mammary epithelial cells (arrow) of the mammary glands during lactation (Figure 1B, arrowhead). The induction of Dab2 protein isoforms in mammary glands was verified by Western blot analysis of tissue lysates (Figure 1C). While Dab2 was undetectable in virgin mammary glands, a low level appeared during pregnancy (15.5 days), and several isoforms, including p96 and p67, were massively induced upon lactation. In the involuting mammary glands, Dab2 proteins were lost (Figure 1C). Mammary tissue extracts from Dab2 conditional knockout mice were used to distinguish Dab2 isoforms from non-specific signals in the Western blots. Since mammary tissues contain multiple cell types, such as stromal, adipocytes, and immune cells, in addition to epithelial cells, we assayed E-cadherin as an indicator of epithelial content (Figure 1C). Beta-catenin was also probed, and it inversely correlated with E-cadherin levels. Based on equal protein loading and an equivalent beta-actin signal, the fraction of mammary epithelial cells was low in virgin, increased and remained similar in pregnant and day 1 lactating, and was highest in day 5 lactating mice (Figure 1C). In comparison, Dab2 proteins were not hormonally regulated in kidney epithelial or ovarian surface epithelial cells, suggesting a mammary-specific transcription co-factor(s) is required for regulation of Dab2 expression during pregnancy and lactation. The induction of Dab2 expression has been confirmed in multiple experiments using both Western blot, immunofluorescence microscopy, and immunohistochemistry, and these results indicate that in mammary glands, Dab2 expression commences in epithelia following pregnancy, reaches maximum during lactation, and wanes upon involution.


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

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

Induction of Dab2 by pregnancy and lactation in mammary glands.Mammary glands from virgin (A) and lactating (B) wildtype mice were harvested and used for histological analysis. Representative images of Dab2 immunostaining are shown. Arrows indicate the mammary epithelial cells. We also noted the positive staining of mammary adipocytes, which we have been confirmed to express Dab2 protein. (C) The induction of Dab2 proteins was confirmed by Western blot. Mammary glands were dissected and harvested from virgin, pregnant (Preg.) (12 days), and day 1 and day 5 of lactating (Lact.), and post-lactating/involuting (Invol.) wildtype (WT) mice. The post-lactation/involution samples were harvested from mothers that were weaned naturally. Specifically, the mice lactated for 3 weeks prior to separation from their pups for 1 (lane 9) or 3 (lane 10) days. Mammary tissues from conditional Dab2 knockout mice (dab2 (f/df):Meox2-Cre) (cKO) were used as controls for antibody specificity. The tissues were lysed in SDS buffer, and equal protein from each sample was used for electrophoresis and Western blot analysis of Dab2, beta-catenin, and E-cadherin. Arrows indicate Dab2 p96 and p67 isoforms and a band that is presumably IgG heavy chain.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110737-g001: Induction of Dab2 by pregnancy and lactation in mammary glands.Mammary glands from virgin (A) and lactating (B) wildtype mice were harvested and used for histological analysis. Representative images of Dab2 immunostaining are shown. Arrows indicate the mammary epithelial cells. We also noted the positive staining of mammary adipocytes, which we have been confirmed to express Dab2 protein. (C) The induction of Dab2 proteins was confirmed by Western blot. Mammary glands were dissected and harvested from virgin, pregnant (Preg.) (12 days), and day 1 and day 5 of lactating (Lact.), and post-lactating/involuting (Invol.) wildtype (WT) mice. The post-lactation/involution samples were harvested from mothers that were weaned naturally. Specifically, the mice lactated for 3 weeks prior to separation from their pups for 1 (lane 9) or 3 (lane 10) days. Mammary tissues from conditional Dab2 knockout mice (dab2 (f/df):Meox2-Cre) (cKO) were used as controls for antibody specificity. The tissues were lysed in SDS buffer, and equal protein from each sample was used for electrophoresis and Western blot analysis of Dab2, beta-catenin, and E-cadherin. Arrows indicate Dab2 p96 and p67 isoforms and a band that is presumably IgG heavy chain.
Mentions: In a previous study of Dab2 using mouse models (Ref 56, and in more detail below), we observed that its expression in mammary glands varied greatly in different physiological stages. In virgin mice, the Dab2 protein was essentially undetectable in mammary epithelial cells by immunohistochemistry (Figure 1A, arrow), while Dab2 staining was robust and uniform in all mammary epithelial cells (arrow) of the mammary glands during lactation (Figure 1B, arrowhead). The induction of Dab2 protein isoforms in mammary glands was verified by Western blot analysis of tissue lysates (Figure 1C). While Dab2 was undetectable in virgin mammary glands, a low level appeared during pregnancy (15.5 days), and several isoforms, including p96 and p67, were massively induced upon lactation. In the involuting mammary glands, Dab2 proteins were lost (Figure 1C). Mammary tissue extracts from Dab2 conditional knockout mice were used to distinguish Dab2 isoforms from non-specific signals in the Western blots. Since mammary tissues contain multiple cell types, such as stromal, adipocytes, and immune cells, in addition to epithelial cells, we assayed E-cadherin as an indicator of epithelial content (Figure 1C). Beta-catenin was also probed, and it inversely correlated with E-cadherin levels. Based on equal protein loading and an equivalent beta-actin signal, the fraction of mammary epithelial cells was low in virgin, increased and remained similar in pregnant and day 1 lactating, and was highest in day 5 lactating mice (Figure 1C). In comparison, Dab2 proteins were not hormonally regulated in kidney epithelial or ovarian surface epithelial cells, suggesting a mammary-specific transcription co-factor(s) is required for regulation of Dab2 expression during pregnancy and lactation. The induction of Dab2 expression has been confirmed in multiple experiments using both Western blot, immunofluorescence microscopy, and immunohistochemistry, and these results indicate that in mammary glands, Dab2 expression commences in epithelia following pregnancy, reaches maximum during lactation, and wanes upon involution.

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