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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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Delayed apoptosis of Dab2-deficient mammary epithelial cells during involution.(A) The day 3 involuting mammary glands from control (dab2 heterozygous) and dab2 knockout (dab2 (f/df):Sox2-Cre) mice were analyzed. Apoptotic cell death in situ indicated by immunostaining for activated caspase-3, comparing wildtype (arrow) and Dab2 deficient cells (arrowhead). (B) A representative immunofluorescence microscopy staining for phospho-Erk1/2 in Dab2 heterozygous and knockout mammary glands on day 3 of involution. Phospho-Erk1/2 (red) overlaying on DAPI (blue) stainings are shown. (C) Immunofluorescence microscopy for Bcl-2 (red) and DAPI (blue) staining of day 3 involuting mammary glands. (D) Dab2 expression was determined by Western blot of tissue extracts of involuting mammary glands from dab2 heterozygous mice at 0, 1, 3, 5, and 7 days following forced involution. (E) Western blot analysis of E-cadherin, Bax, Bcl-xl, and activated caspase-3. Mammary protein extracts from heterozygous lactating (day 12) and forced involuting (day 3) mice were analyzed. (F) Western blot analysis of Bcl-2, Bcl-xl, Bax, phospho-Smad2, total Smad2, phospho-Erk1/2, and total Erk1/2 in protein lysates extracted from mammary glands following 3 and 5 days of forced involution. Two independent samples (duplicate) of each genotype from different mice are shown in the blot.
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pone-0110737-g006: Delayed apoptosis of Dab2-deficient mammary epithelial cells during involution.(A) The day 3 involuting mammary glands from control (dab2 heterozygous) and dab2 knockout (dab2 (f/df):Sox2-Cre) mice were analyzed. Apoptotic cell death in situ indicated by immunostaining for activated caspase-3, comparing wildtype (arrow) and Dab2 deficient cells (arrowhead). (B) A representative immunofluorescence microscopy staining for phospho-Erk1/2 in Dab2 heterozygous and knockout mammary glands on day 3 of involution. Phospho-Erk1/2 (red) overlaying on DAPI (blue) stainings are shown. (C) Immunofluorescence microscopy for Bcl-2 (red) and DAPI (blue) staining of day 3 involuting mammary glands. (D) Dab2 expression was determined by Western blot of tissue extracts of involuting mammary glands from dab2 heterozygous mice at 0, 1, 3, 5, and 7 days following forced involution. (E) Western blot analysis of E-cadherin, Bax, Bcl-xl, and activated caspase-3. Mammary protein extracts from heterozygous lactating (day 12) and forced involuting (day 3) mice were analyzed. (F) Western blot analysis of Bcl-2, Bcl-xl, Bax, phospho-Smad2, total Smad2, phospho-Erk1/2, and total Erk1/2 in protein lysates extracted from mammary glands following 3 and 5 days of forced involution. Two independent samples (duplicate) of each genotype from different mice are shown in the blot.

Mentions: In control day-3 involuting mammary glands, intensive focal staining of cleaved caspase-3 indicated active apoptosis (Figure 6A, arrow); however in comparison, the staining of many Dab2-deficient mammary epithelial cells appeared lighter and diffuse, and few clear caspase-3-positive cells were seen (Figure 6A, arrowhead). The Dab2 mammary glands showed an increased activation of Erk1/2 since that 16% of the cells were phospho-Erk1/2 positive in nuclei; in contrast, few (<1%) cells were positive for nuclear phospho-Erk1/2 in control mammary glands (Figure 6B). Consistently with an increased Erk1/2 activation, 82% of the day-3 involuting Dab2 mammary cells were positive for Bcl-2, compared to 26% in control (dab2 heterozygous) cells (Figure 6C).


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

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

Delayed apoptosis of Dab2-deficient mammary epithelial cells during involution.(A) The day 3 involuting mammary glands from control (dab2 heterozygous) and dab2 knockout (dab2 (f/df):Sox2-Cre) mice were analyzed. Apoptotic cell death in situ indicated by immunostaining for activated caspase-3, comparing wildtype (arrow) and Dab2 deficient cells (arrowhead). (B) A representative immunofluorescence microscopy staining for phospho-Erk1/2 in Dab2 heterozygous and knockout mammary glands on day 3 of involution. Phospho-Erk1/2 (red) overlaying on DAPI (blue) stainings are shown. (C) Immunofluorescence microscopy for Bcl-2 (red) and DAPI (blue) staining of day 3 involuting mammary glands. (D) Dab2 expression was determined by Western blot of tissue extracts of involuting mammary glands from dab2 heterozygous mice at 0, 1, 3, 5, and 7 days following forced involution. (E) Western blot analysis of E-cadherin, Bax, Bcl-xl, and activated caspase-3. Mammary protein extracts from heterozygous lactating (day 12) and forced involuting (day 3) mice were analyzed. (F) Western blot analysis of Bcl-2, Bcl-xl, Bax, phospho-Smad2, total Smad2, phospho-Erk1/2, and total Erk1/2 in protein lysates extracted from mammary glands following 3 and 5 days of forced involution. Two independent samples (duplicate) of each genotype from different mice are shown in the blot.
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Related In: Results  -  Collection

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pone-0110737-g006: Delayed apoptosis of Dab2-deficient mammary epithelial cells during involution.(A) The day 3 involuting mammary glands from control (dab2 heterozygous) and dab2 knockout (dab2 (f/df):Sox2-Cre) mice were analyzed. Apoptotic cell death in situ indicated by immunostaining for activated caspase-3, comparing wildtype (arrow) and Dab2 deficient cells (arrowhead). (B) A representative immunofluorescence microscopy staining for phospho-Erk1/2 in Dab2 heterozygous and knockout mammary glands on day 3 of involution. Phospho-Erk1/2 (red) overlaying on DAPI (blue) stainings are shown. (C) Immunofluorescence microscopy for Bcl-2 (red) and DAPI (blue) staining of day 3 involuting mammary glands. (D) Dab2 expression was determined by Western blot of tissue extracts of involuting mammary glands from dab2 heterozygous mice at 0, 1, 3, 5, and 7 days following forced involution. (E) Western blot analysis of E-cadherin, Bax, Bcl-xl, and activated caspase-3. Mammary protein extracts from heterozygous lactating (day 12) and forced involuting (day 3) mice were analyzed. (F) Western blot analysis of Bcl-2, Bcl-xl, Bax, phospho-Smad2, total Smad2, phospho-Erk1/2, and total Erk1/2 in protein lysates extracted from mammary glands following 3 and 5 days of forced involution. Two independent samples (duplicate) of each genotype from different mice are shown in the blot.
Mentions: In control day-3 involuting mammary glands, intensive focal staining of cleaved caspase-3 indicated active apoptosis (Figure 6A, arrow); however in comparison, the staining of many Dab2-deficient mammary epithelial cells appeared lighter and diffuse, and few clear caspase-3-positive cells were seen (Figure 6A, arrowhead). The Dab2 mammary glands showed an increased activation of Erk1/2 since that 16% of the cells were phospho-Erk1/2 positive in nuclei; in contrast, few (<1%) cells were positive for nuclear phospho-Erk1/2 in control mammary glands (Figure 6B). Consistently with an increased Erk1/2 activation, 82% of the day-3 involuting Dab2 mammary cells were positive for Bcl-2, compared to 26% in control (dab2 heterozygous) cells (Figure 6C).

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