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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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Dab2 modulates TGF-beta-stimulated growth regulation and signaling in primary mammary epithelial cells.Mammary epithelial cells were prepared from pregnant control (+/df) and dab2 conditional knockout (df/df) mice inheriting Sox2-Cre. The cells were treated with or without TGF-beta (10 ng/ml). (A) Growth of the cells was determined by WST assay over a 6-day period. Student-t test indicated that the difference in cell growth was statistically significant for at day 3 to 6 for dab2 (+/df) (p<0.005) but not dab2 (df/df) cells. (B) Protein lysates prepared from the primary cultures were analyzed by Western blots for Dab2, phospho-Smad2, E-cadherin, N-cadherin, phospho-Erk1/2, total Erk1/2, PCNA, Bcl-2, Bcl-xl, Bax, activated caspase-3, and beta-actin. (C) The relative protein level was quantified from the Western blots using NIH Image J software, and the values of the optical density (O.D.) critical markers (p-Smad2, p = Erk1/2, and Bcl-2) were compared. (D) Co-immunoprecipitation was performed to determine the association between Grb2 and Sos or Dab2. The primary cells were stimulated by TGF-beta for a time course of 0, 5, 15, 30, and 90 min. At each time point, the monolayer was washed with ice-cold PBS, lysed, and the post-nuclear supernatants were used for immunoprecipitation with antibodies against Grb2. The eluted proteins from the immunoprecipitation were separated by SDS-PAGE, and immunoprobed for Sos, Dab2, and Grb2. The immunoprecipitation experiments were performed twice and similar results were obtained. (E) Relative Dab2 and Sos protein amounts in the Western blot were estimated using NIH Image J program and the O.D. values were plotted.
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pone-0110737-g007: Dab2 modulates TGF-beta-stimulated growth regulation and signaling in primary mammary epithelial cells.Mammary epithelial cells were prepared from pregnant control (+/df) and dab2 conditional knockout (df/df) mice inheriting Sox2-Cre. The cells were treated with or without TGF-beta (10 ng/ml). (A) Growth of the cells was determined by WST assay over a 6-day period. Student-t test indicated that the difference in cell growth was statistically significant for at day 3 to 6 for dab2 (+/df) (p<0.005) but not dab2 (df/df) cells. (B) Protein lysates prepared from the primary cultures were analyzed by Western blots for Dab2, phospho-Smad2, E-cadherin, N-cadherin, phospho-Erk1/2, total Erk1/2, PCNA, Bcl-2, Bcl-xl, Bax, activated caspase-3, and beta-actin. (C) The relative protein level was quantified from the Western blots using NIH Image J software, and the values of the optical density (O.D.) critical markers (p-Smad2, p = Erk1/2, and Bcl-2) were compared. (D) Co-immunoprecipitation was performed to determine the association between Grb2 and Sos or Dab2. The primary cells were stimulated by TGF-beta for a time course of 0, 5, 15, 30, and 90 min. At each time point, the monolayer was washed with ice-cold PBS, lysed, and the post-nuclear supernatants were used for immunoprecipitation with antibodies against Grb2. The eluted proteins from the immunoprecipitation were separated by SDS-PAGE, and immunoprobed for Sos, Dab2, and Grb2. The immunoprecipitation experiments were performed twice and similar results were obtained. (E) Relative Dab2 and Sos protein amounts in the Western blot were estimated using NIH Image J program and the O.D. values were plotted.

Mentions: Since TGF-beta signaling is known to be critical in mammary involution [48], [63], [64] and several reports suggest a role of Dab2 in the regulation of this pathway. We investigated TGF-beta signaling and growth control in primary mammary epithelial cells isolated from dab2 knockout and control mice. Unlike involution in vivo, TGF-beta failed to induce significant cell death in cultures of primary mammary epithelial cells. Nevertheless, upon TGF-beta exposure, the wildtype mammary epithelial cells showed a reduced cell proliferation (Figure 7A). However, Dab2-deficient cells exhibited an unsuppressed proliferation and were refractory to TGF-beta induced growth inhibition (Figure 7A).


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

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

Dab2 modulates TGF-beta-stimulated growth regulation and signaling in primary mammary epithelial cells.Mammary epithelial cells were prepared from pregnant control (+/df) and dab2 conditional knockout (df/df) mice inheriting Sox2-Cre. The cells were treated with or without TGF-beta (10 ng/ml). (A) Growth of the cells was determined by WST assay over a 6-day period. Student-t test indicated that the difference in cell growth was statistically significant for at day 3 to 6 for dab2 (+/df) (p<0.005) but not dab2 (df/df) cells. (B) Protein lysates prepared from the primary cultures were analyzed by Western blots for Dab2, phospho-Smad2, E-cadherin, N-cadherin, phospho-Erk1/2, total Erk1/2, PCNA, Bcl-2, Bcl-xl, Bax, activated caspase-3, and beta-actin. (C) The relative protein level was quantified from the Western blots using NIH Image J software, and the values of the optical density (O.D.) critical markers (p-Smad2, p = Erk1/2, and Bcl-2) were compared. (D) Co-immunoprecipitation was performed to determine the association between Grb2 and Sos or Dab2. The primary cells were stimulated by TGF-beta for a time course of 0, 5, 15, 30, and 90 min. At each time point, the monolayer was washed with ice-cold PBS, lysed, and the post-nuclear supernatants were used for immunoprecipitation with antibodies against Grb2. The eluted proteins from the immunoprecipitation were separated by SDS-PAGE, and immunoprobed for Sos, Dab2, and Grb2. The immunoprecipitation experiments were performed twice and similar results were obtained. (E) Relative Dab2 and Sos protein amounts in the Western blot were estimated using NIH Image J program and the O.D. values were plotted.
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Related In: Results  -  Collection

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pone-0110737-g007: Dab2 modulates TGF-beta-stimulated growth regulation and signaling in primary mammary epithelial cells.Mammary epithelial cells were prepared from pregnant control (+/df) and dab2 conditional knockout (df/df) mice inheriting Sox2-Cre. The cells were treated with or without TGF-beta (10 ng/ml). (A) Growth of the cells was determined by WST assay over a 6-day period. Student-t test indicated that the difference in cell growth was statistically significant for at day 3 to 6 for dab2 (+/df) (p<0.005) but not dab2 (df/df) cells. (B) Protein lysates prepared from the primary cultures were analyzed by Western blots for Dab2, phospho-Smad2, E-cadherin, N-cadherin, phospho-Erk1/2, total Erk1/2, PCNA, Bcl-2, Bcl-xl, Bax, activated caspase-3, and beta-actin. (C) The relative protein level was quantified from the Western blots using NIH Image J software, and the values of the optical density (O.D.) critical markers (p-Smad2, p = Erk1/2, and Bcl-2) were compared. (D) Co-immunoprecipitation was performed to determine the association between Grb2 and Sos or Dab2. The primary cells were stimulated by TGF-beta for a time course of 0, 5, 15, 30, and 90 min. At each time point, the monolayer was washed with ice-cold PBS, lysed, and the post-nuclear supernatants were used for immunoprecipitation with antibodies against Grb2. The eluted proteins from the immunoprecipitation were separated by SDS-PAGE, and immunoprobed for Sos, Dab2, and Grb2. The immunoprecipitation experiments were performed twice and similar results were obtained. (E) Relative Dab2 and Sos protein amounts in the Western blot were estimated using NIH Image J program and the O.D. values were plotted.
Mentions: Since TGF-beta signaling is known to be critical in mammary involution [48], [63], [64] and several reports suggest a role of Dab2 in the regulation of this pathway. We investigated TGF-beta signaling and growth control in primary mammary epithelial cells isolated from dab2 knockout and control mice. Unlike involution in vivo, TGF-beta failed to induce significant cell death in cultures of primary mammary epithelial cells. Nevertheless, upon TGF-beta exposure, the wildtype mammary epithelial cells showed a reduced cell proliferation (Figure 7A). However, Dab2-deficient cells exhibited an unsuppressed proliferation and were refractory to TGF-beta induced growth inhibition (Figure 7A).

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