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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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Mild effects of Dab2 deletion on endocytic components and polarity markers in mammary epithelia.Mammary glands from lactating (5 days) mice of dab2 heterozygous controls and mosaic knockouts (dab2 (f/df):Meox2-Cre) were analyzed by confocal immunofluorescence microscopy for several endocytosis and polarity markers. Where possible, markers were compared between Dab2-positive and Dab2-negative cells in the same section of the mosaic dab2-deleted tissues. (A) Adaptin alpha and Dab2 double staining of mammary glands from a mosaic dab2-deleted mouse, comparing Dab2-positive (arrow) and negative (arrowhead) cells; (B) Clathrin and Dab2 co-staining of mammary glands from a mosaic dab2-deleted mouse, comparing Dab2-positive (arrow) and negative (arrowhead) cells; (C) Dab2 and E-cadherin staining: E-cadherin is localized more in the cytoplasm in Dab2-deleted cells (arrowhead); (D) NPT2b, a sodium-phosphate cotransporter; and (E) PMCA2 ATPase, a calcium pump, in Dab2-positive (arrow) and negative (arrowhead) cells. The results are the representation of multiple slides from 3 control (wildtype and heterozygous) and 3 dab2 conditional knockout mice.
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pone-0110737-g004: Mild effects of Dab2 deletion on endocytic components and polarity markers in mammary epithelia.Mammary glands from lactating (5 days) mice of dab2 heterozygous controls and mosaic knockouts (dab2 (f/df):Meox2-Cre) were analyzed by confocal immunofluorescence microscopy for several endocytosis and polarity markers. Where possible, markers were compared between Dab2-positive and Dab2-negative cells in the same section of the mosaic dab2-deleted tissues. (A) Adaptin alpha and Dab2 double staining of mammary glands from a mosaic dab2-deleted mouse, comparing Dab2-positive (arrow) and negative (arrowhead) cells; (B) Clathrin and Dab2 co-staining of mammary glands from a mosaic dab2-deleted mouse, comparing Dab2-positive (arrow) and negative (arrowhead) cells; (C) Dab2 and E-cadherin staining: E-cadherin is localized more in the cytoplasm in Dab2-deleted cells (arrowhead); (D) NPT2b, a sodium-phosphate cotransporter; and (E) PMCA2 ATPase, a calcium pump, in Dab2-positive (arrow) and negative (arrowhead) cells. The results are the representation of multiple slides from 3 control (wildtype and heterozygous) and 3 dab2 conditional knockout mice.

Mentions: We further examined the impact of Dab2 deletion on the distribution of other endocytic components. In lactating mammary glands with a mosaic dab2 deletion, we compared adaptin-alpha (Figure 4A) and clathrin (Figure 4B) in adjacent Dab2-positive and negative epithelial cells in the same section, and noticed slight but consistent differences in the distribution of these endocytic proteins. Dab2-positive cells had a more intense apical localization of adaptin-alpha and clathrin (Figure 4A and B, arrow) than Dab2-negative cells (Figure 4A and B, arrowhead). Since Dab2 has a role in maintaining polarity and epithelial organization of extraembryonic endoderm [12], we further examined E-cadherin and other polarity markers in mammary epithelial cells. E-cadherin showed an overwhelmingly basolateral distribution in the Dab2-positive mammary epithelial cells; however the staining was more cytoplasmic and punctated in Dab2-negative cells (Figure 4C, arrowhead). No obvious changes were detected in the distribution of the apical marker, sodium/phosphate co-transporter NPT2b [61] (Figure 4D). Nevertheless, a loss or reduced apical distribution of the calcium pump PMCA2 [62] was evident in the Dab2-negative (Figure 4E, arrowhead) compared to adjacent Dab2-positive cells (Figure 4E, arrow). We have consistently detected the genotype-dependent changes in multiple slides from 3 controls (both wildtype and heterozygous) and 3 conditional deletions. Additionally, when available, we used slides containing regions showing Dab2-positive and negative adjacent cells for analysis to demonstrate a change depending on Dab2 expression.


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

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

Mild effects of Dab2 deletion on endocytic components and polarity markers in mammary epithelia.Mammary glands from lactating (5 days) mice of dab2 heterozygous controls and mosaic knockouts (dab2 (f/df):Meox2-Cre) were analyzed by confocal immunofluorescence microscopy for several endocytosis and polarity markers. Where possible, markers were compared between Dab2-positive and Dab2-negative cells in the same section of the mosaic dab2-deleted tissues. (A) Adaptin alpha and Dab2 double staining of mammary glands from a mosaic dab2-deleted mouse, comparing Dab2-positive (arrow) and negative (arrowhead) cells; (B) Clathrin and Dab2 co-staining of mammary glands from a mosaic dab2-deleted mouse, comparing Dab2-positive (arrow) and negative (arrowhead) cells; (C) Dab2 and E-cadherin staining: E-cadherin is localized more in the cytoplasm in Dab2-deleted cells (arrowhead); (D) NPT2b, a sodium-phosphate cotransporter; and (E) PMCA2 ATPase, a calcium pump, in Dab2-positive (arrow) and negative (arrowhead) cells. The results are the representation of multiple slides from 3 control (wildtype and heterozygous) and 3 dab2 conditional knockout mice.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110737-g004: Mild effects of Dab2 deletion on endocytic components and polarity markers in mammary epithelia.Mammary glands from lactating (5 days) mice of dab2 heterozygous controls and mosaic knockouts (dab2 (f/df):Meox2-Cre) were analyzed by confocal immunofluorescence microscopy for several endocytosis and polarity markers. Where possible, markers were compared between Dab2-positive and Dab2-negative cells in the same section of the mosaic dab2-deleted tissues. (A) Adaptin alpha and Dab2 double staining of mammary glands from a mosaic dab2-deleted mouse, comparing Dab2-positive (arrow) and negative (arrowhead) cells; (B) Clathrin and Dab2 co-staining of mammary glands from a mosaic dab2-deleted mouse, comparing Dab2-positive (arrow) and negative (arrowhead) cells; (C) Dab2 and E-cadherin staining: E-cadherin is localized more in the cytoplasm in Dab2-deleted cells (arrowhead); (D) NPT2b, a sodium-phosphate cotransporter; and (E) PMCA2 ATPase, a calcium pump, in Dab2-positive (arrow) and negative (arrowhead) cells. The results are the representation of multiple slides from 3 control (wildtype and heterozygous) and 3 dab2 conditional knockout mice.
Mentions: We further examined the impact of Dab2 deletion on the distribution of other endocytic components. In lactating mammary glands with a mosaic dab2 deletion, we compared adaptin-alpha (Figure 4A) and clathrin (Figure 4B) in adjacent Dab2-positive and negative epithelial cells in the same section, and noticed slight but consistent differences in the distribution of these endocytic proteins. Dab2-positive cells had a more intense apical localization of adaptin-alpha and clathrin (Figure 4A and B, arrow) than Dab2-negative cells (Figure 4A and B, arrowhead). Since Dab2 has a role in maintaining polarity and epithelial organization of extraembryonic endoderm [12], we further examined E-cadherin and other polarity markers in mammary epithelial cells. E-cadherin showed an overwhelmingly basolateral distribution in the Dab2-positive mammary epithelial cells; however the staining was more cytoplasmic and punctated in Dab2-negative cells (Figure 4C, arrowhead). No obvious changes were detected in the distribution of the apical marker, sodium/phosphate co-transporter NPT2b [61] (Figure 4D). Nevertheless, a loss or reduced apical distribution of the calcium pump PMCA2 [62] was evident in the Dab2-negative (Figure 4E, arrowhead) compared to adjacent Dab2-positive cells (Figure 4E, arrow). We have consistently detected the genotype-dependent changes in multiple slides from 3 controls (both wildtype and heterozygous) and 3 conditional deletions. Additionally, when available, we used slides containing regions showing Dab2-positive and negative adjacent cells for analysis to demonstrate a change depending on Dab2 expression.

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