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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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Normal lactation in mammary glands from dab2 mosaic knockout mice.(A) Mammary glands from 4-month old, 5-day lactating mice of dab2 (f/df):Meox2-Cre genotype show mosaic Dab2 immunostaining. (B) Dab2 was absent in more than 99% of mammary epithelial cells from mice of dab2 (f/df):Sox2-Cre genotype. (C) DNA extracted from tail tissues was used to genotype flox (f) and delta flox (df) alleles of the dab2 gene in dab2 (+/df): Sox2-Cre, dab2 (f/df):Meox2-Cre, and dab2 (f/df):Sox2-Cre. (D) Milk was harvested from dab2 heterozygous (dab2 (+/df):Sox2-Cre) and knockout (dab2 (f/df):Sox2-Cre) mice. Total proteins were resolved by SDS-PAGE and stained with Coomassie blue. (E) Specific proteins, beta-casein and GC-globin (vitamin D binding protein), were analyzed by immunoblot.
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pone-0110737-g003: Normal lactation in mammary glands from dab2 mosaic knockout mice.(A) Mammary glands from 4-month old, 5-day lactating mice of dab2 (f/df):Meox2-Cre genotype show mosaic Dab2 immunostaining. (B) Dab2 was absent in more than 99% of mammary epithelial cells from mice of dab2 (f/df):Sox2-Cre genotype. (C) DNA extracted from tail tissues was used to genotype flox (f) and delta flox (df) alleles of the dab2 gene in dab2 (+/df): Sox2-Cre, dab2 (f/df):Meox2-Cre, and dab2 (f/df):Sox2-Cre. (D) Milk was harvested from dab2 heterozygous (dab2 (+/df):Sox2-Cre) and knockout (dab2 (f/df):Sox2-Cre) mice. Total proteins were resolved by SDS-PAGE and stained with Coomassie blue. (E) Specific proteins, beta-casein and GC-globin (vitamin D binding protein), were analyzed by immunoblot.

Mentions: To bypass the requirement of Dab2 in embryonic development [11], [32] and to study the roles of Dab2 in mammary glands and adult tissues, we developed a Cre-lox conditional Dab2 knockout mouse model [56]. Using Meox2-Cre [57] or Sox2-Cre [58] to spare extraembryonic tissues but to delete dab2 gene in embryonic proper in a mosaic fashion, we found that the resulting dab2 knockout mice were grossly normal [56]. By immunostaining, Dab2 deletion in adult mammary glands was estimated to be about 95% in dab2 (f/df): Meox2-Cre with Dab2-positive epithelial cells clustered focally (Figure 3A). However, the deletion was highly efficient in dab2 (f/df): Sox2-Cre mammary glands and essentially no Dab2 positive cells were detected (Figure 3B). The degree of deletion agreed with PCR genotyping of tail DNA (Figure 3C).


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

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

Normal lactation in mammary glands from dab2 mosaic knockout mice.(A) Mammary glands from 4-month old, 5-day lactating mice of dab2 (f/df):Meox2-Cre genotype show mosaic Dab2 immunostaining. (B) Dab2 was absent in more than 99% of mammary epithelial cells from mice of dab2 (f/df):Sox2-Cre genotype. (C) DNA extracted from tail tissues was used to genotype flox (f) and delta flox (df) alleles of the dab2 gene in dab2 (+/df): Sox2-Cre, dab2 (f/df):Meox2-Cre, and dab2 (f/df):Sox2-Cre. (D) Milk was harvested from dab2 heterozygous (dab2 (+/df):Sox2-Cre) and knockout (dab2 (f/df):Sox2-Cre) mice. Total proteins were resolved by SDS-PAGE and stained with Coomassie blue. (E) Specific proteins, beta-casein and GC-globin (vitamin D binding protein), were analyzed by immunoblot.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110737-g003: Normal lactation in mammary glands from dab2 mosaic knockout mice.(A) Mammary glands from 4-month old, 5-day lactating mice of dab2 (f/df):Meox2-Cre genotype show mosaic Dab2 immunostaining. (B) Dab2 was absent in more than 99% of mammary epithelial cells from mice of dab2 (f/df):Sox2-Cre genotype. (C) DNA extracted from tail tissues was used to genotype flox (f) and delta flox (df) alleles of the dab2 gene in dab2 (+/df): Sox2-Cre, dab2 (f/df):Meox2-Cre, and dab2 (f/df):Sox2-Cre. (D) Milk was harvested from dab2 heterozygous (dab2 (+/df):Sox2-Cre) and knockout (dab2 (f/df):Sox2-Cre) mice. Total proteins were resolved by SDS-PAGE and stained with Coomassie blue. (E) Specific proteins, beta-casein and GC-globin (vitamin D binding protein), were analyzed by immunoblot.
Mentions: To bypass the requirement of Dab2 in embryonic development [11], [32] and to study the roles of Dab2 in mammary glands and adult tissues, we developed a Cre-lox conditional Dab2 knockout mouse model [56]. Using Meox2-Cre [57] or Sox2-Cre [58] to spare extraembryonic tissues but to delete dab2 gene in embryonic proper in a mosaic fashion, we found that the resulting dab2 knockout mice were grossly normal [56]. By immunostaining, Dab2 deletion in adult mammary glands was estimated to be about 95% in dab2 (f/df): Meox2-Cre with Dab2-positive epithelial cells clustered focally (Figure 3A). However, the deletion was highly efficient in dab2 (f/df): Sox2-Cre mammary glands and essentially no Dab2 positive cells were detected (Figure 3B). The degree of deletion agreed with PCR genotyping of tail DNA (Figure 3C).

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