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SirT1 modulates the estrogen-insulin-like growth factor-1 signaling for postnatal development of mammary gland in mice.

Li H, Rajendran GK, Liu N, Ware C, Rubin BP, Gu Y - Breast Cancer Res. (2007)

Bottom Line: The effect of exogenous estrogen was also examined by subcutaneous implantation of a slow-releasing pellet in the subscapular region.SirT1 deficiency deregulates the expression of IGF-1 binding protein-1 and attenuates the effect of IGF-1 signals, including estrogen-stimulated local IGF-1 signaling for the onset of ductal morphogenesis.These findings suggest that the enzymatic activity of SirT1 may influence both normal growth and malignant growth of mammary epithelial cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Radiation Oncology, University of Washington School of Medicine, Seattle, WA 98195, USA.

ABSTRACT

Introduction: Estrogen and insulin-like growth factor-1 (IGF-1) play important roles in mammary gland development and breast cancer. SirT1 is a highly conserved protein deacetylase that can regulate the insulin/IGF-1 signaling in lower organisms, as well as a growing number of transcription factors, including NF-kappaB, in mammalian cells. Whether SirT1 regulates the IGF-1 signaling for mammary gland development and function, however, is not clear. In the present study, this role of SirT1 was examined by studying SirT1-deficient mice.

Methods: SirT1-deficient (SirT1(ko/ko)) mice were generated by crossing a new strain of mice harboring a conditional targeted mutation in the SirT1 gene (SirT1(co/co)) with CMV-Cre transgenic mice. Whole mount and histology analyses, immunofluorescence staining, immunohistochemistry, and western blotting were used to characterize mammary gland development in virgin and pregnant mice. The effect of exogenous estrogen was also examined by subcutaneous implantation of a slow-releasing pellet in the subscapular region.

Results: Both male and female SirT1(ko/ko) mice can be fertile despite the growth retardation phenotype. Virgin SirT1(ko/ko) mice displayed impeded ductal morphogenesis, whereas pregnant SirT1(ko/ko) mice manifested lactation failure due to an underdeveloped lobuloalveolar network. Estrogen implantation was sufficient to rescue ductal morphogenesis. Exogenous estrogen reversed the increased basal level of IGF-1 binding protein-1 expression in SirT1(ko/ko) mammary tissues, but not that of IkappaB alpha expression, suggesting that increased levels of estrogen enhanced the production of local IGF-1 and rescued ductal morphogenesis. Additionally, TNFalpha treatment enhanced the level of the newly synthesized IkappaB alpha in SirT1(ko/ko) cells. SirT1 deficiency therefore affects the cellular response to multiple extrinsic signals.

Conclusion: SirT1 modulates the IGF-1 signaling critical for both growth regulation and mammary gland development in mice. SirT1 deficiency deregulates the expression of IGF-1 binding protein-1 and attenuates the effect of IGF-1 signals, including estrogen-stimulated local IGF-1 signaling for the onset of ductal morphogenesis. These findings suggest that the enzymatic activity of SirT1 may influence both normal growth and malignant growth of mammary epithelial cells.

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Mammary epithelial cell proliferation and apoptosis in SirT1ko/ko mice on lactation day 1. (a) Upper panel: bromodeoxyurdine (BrdU) staining of the mammary sections from SirT1ko/ko (ko/ko) and wild-type (+/+) mice on day 13 of pregnancy (P13) and lactation day 1 (L1). From left to right, three terminal end buds (TEBs) at a scale of 100 μm, a single TEB, ducts, and alveolus of SirT1ko/ko mice at a scale of 20 μm, and ducts and alveolus of wild-type mice on P13 and L1 at a scale of 20 μm. Lower panel: TUNEL (Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling) assay for apoptotic cells using sections neighbored to the sections for BrdU staining. (b) The quantitative analysis of BrdU-positive epithelial cells in TEBs, ducts, and alveoli of SirT1ko/ko mice on L1 (open bar) and wild-type mice on P13 (grey bar) and L1 (solid bar). (c) The quantitative analysis of TUNEL-positive cells in neighboring sections.
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Figure 5: Mammary epithelial cell proliferation and apoptosis in SirT1ko/ko mice on lactation day 1. (a) Upper panel: bromodeoxyurdine (BrdU) staining of the mammary sections from SirT1ko/ko (ko/ko) and wild-type (+/+) mice on day 13 of pregnancy (P13) and lactation day 1 (L1). From left to right, three terminal end buds (TEBs) at a scale of 100 μm, a single TEB, ducts, and alveolus of SirT1ko/ko mice at a scale of 20 μm, and ducts and alveolus of wild-type mice on P13 and L1 at a scale of 20 μm. Lower panel: TUNEL (Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling) assay for apoptotic cells using sections neighbored to the sections for BrdU staining. (b) The quantitative analysis of BrdU-positive epithelial cells in TEBs, ducts, and alveoli of SirT1ko/ko mice on L1 (open bar) and wild-type mice on P13 (grey bar) and L1 (solid bar). (c) The quantitative analysis of TUNEL-positive cells in neighboring sections.

Mentions: The finding of pregnancy-induced mammary gland development prompted us to investigate how mammary epithelial progenitor cells in pregnant SirT1ko/ko mice can differentiate so rapidly. Both BrdU labeling and the TUNEL (Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling) assay were used to measure the cell proliferation and apoptosis in the mammary tissues, respectively. We found in SirT1ko/ko mammary tissues that about one-third of the cells in TEBs were BrdU-positive as compared with the number of BrdU-positive cells in the newly differentiated ductal or alveolar epithelial cells (Figure 5a(upper panel), 5b). This BrdU-positive finding differs from that in wild-type mammary tissues, in which less than 3% of ductal epithelial cells and about 25% of alveolar epithelial cells were BrdU-positive. The newly generated ductal epithelial cells in SirT1ko/ko mice formed elongated ducts and side branches, while pushing TEBs towards the edge of mammary fat pad (Figure 3a, lower panel). The newly generated ductal epithelial cells can consequently further differentiate into a few observed alveolar cells in which milk proteins were detected (Figure 2c). These observations suggested that the proliferation and differentiation of epithelial progenitor cells, which is programmed to take place at different times of a female's reproductive life, could be concurrent at a late stage of pregnancy in SirT1ko/ko mice.


SirT1 modulates the estrogen-insulin-like growth factor-1 signaling for postnatal development of mammary gland in mice.

Li H, Rajendran GK, Liu N, Ware C, Rubin BP, Gu Y - Breast Cancer Res. (2007)

Mammary epithelial cell proliferation and apoptosis in SirT1ko/ko mice on lactation day 1. (a) Upper panel: bromodeoxyurdine (BrdU) staining of the mammary sections from SirT1ko/ko (ko/ko) and wild-type (+/+) mice on day 13 of pregnancy (P13) and lactation day 1 (L1). From left to right, three terminal end buds (TEBs) at a scale of 100 μm, a single TEB, ducts, and alveolus of SirT1ko/ko mice at a scale of 20 μm, and ducts and alveolus of wild-type mice on P13 and L1 at a scale of 20 μm. Lower panel: TUNEL (Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling) assay for apoptotic cells using sections neighbored to the sections for BrdU staining. (b) The quantitative analysis of BrdU-positive epithelial cells in TEBs, ducts, and alveoli of SirT1ko/ko mice on L1 (open bar) and wild-type mice on P13 (grey bar) and L1 (solid bar). (c) The quantitative analysis of TUNEL-positive cells in neighboring sections.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1851382&req=5

Figure 5: Mammary epithelial cell proliferation and apoptosis in SirT1ko/ko mice on lactation day 1. (a) Upper panel: bromodeoxyurdine (BrdU) staining of the mammary sections from SirT1ko/ko (ko/ko) and wild-type (+/+) mice on day 13 of pregnancy (P13) and lactation day 1 (L1). From left to right, three terminal end buds (TEBs) at a scale of 100 μm, a single TEB, ducts, and alveolus of SirT1ko/ko mice at a scale of 20 μm, and ducts and alveolus of wild-type mice on P13 and L1 at a scale of 20 μm. Lower panel: TUNEL (Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling) assay for apoptotic cells using sections neighbored to the sections for BrdU staining. (b) The quantitative analysis of BrdU-positive epithelial cells in TEBs, ducts, and alveoli of SirT1ko/ko mice on L1 (open bar) and wild-type mice on P13 (grey bar) and L1 (solid bar). (c) The quantitative analysis of TUNEL-positive cells in neighboring sections.
Mentions: The finding of pregnancy-induced mammary gland development prompted us to investigate how mammary epithelial progenitor cells in pregnant SirT1ko/ko mice can differentiate so rapidly. Both BrdU labeling and the TUNEL (Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling) assay were used to measure the cell proliferation and apoptosis in the mammary tissues, respectively. We found in SirT1ko/ko mammary tissues that about one-third of the cells in TEBs were BrdU-positive as compared with the number of BrdU-positive cells in the newly differentiated ductal or alveolar epithelial cells (Figure 5a(upper panel), 5b). This BrdU-positive finding differs from that in wild-type mammary tissues, in which less than 3% of ductal epithelial cells and about 25% of alveolar epithelial cells were BrdU-positive. The newly generated ductal epithelial cells in SirT1ko/ko mice formed elongated ducts and side branches, while pushing TEBs towards the edge of mammary fat pad (Figure 3a, lower panel). The newly generated ductal epithelial cells can consequently further differentiate into a few observed alveolar cells in which milk proteins were detected (Figure 2c). These observations suggested that the proliferation and differentiation of epithelial progenitor cells, which is programmed to take place at different times of a female's reproductive life, could be concurrent at a late stage of pregnancy in SirT1ko/ko mice.

Bottom Line: The effect of exogenous estrogen was also examined by subcutaneous implantation of a slow-releasing pellet in the subscapular region.SirT1 deficiency deregulates the expression of IGF-1 binding protein-1 and attenuates the effect of IGF-1 signals, including estrogen-stimulated local IGF-1 signaling for the onset of ductal morphogenesis.These findings suggest that the enzymatic activity of SirT1 may influence both normal growth and malignant growth of mammary epithelial cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Radiation Oncology, University of Washington School of Medicine, Seattle, WA 98195, USA.

ABSTRACT

Introduction: Estrogen and insulin-like growth factor-1 (IGF-1) play important roles in mammary gland development and breast cancer. SirT1 is a highly conserved protein deacetylase that can regulate the insulin/IGF-1 signaling in lower organisms, as well as a growing number of transcription factors, including NF-kappaB, in mammalian cells. Whether SirT1 regulates the IGF-1 signaling for mammary gland development and function, however, is not clear. In the present study, this role of SirT1 was examined by studying SirT1-deficient mice.

Methods: SirT1-deficient (SirT1(ko/ko)) mice were generated by crossing a new strain of mice harboring a conditional targeted mutation in the SirT1 gene (SirT1(co/co)) with CMV-Cre transgenic mice. Whole mount and histology analyses, immunofluorescence staining, immunohistochemistry, and western blotting were used to characterize mammary gland development in virgin and pregnant mice. The effect of exogenous estrogen was also examined by subcutaneous implantation of a slow-releasing pellet in the subscapular region.

Results: Both male and female SirT1(ko/ko) mice can be fertile despite the growth retardation phenotype. Virgin SirT1(ko/ko) mice displayed impeded ductal morphogenesis, whereas pregnant SirT1(ko/ko) mice manifested lactation failure due to an underdeveloped lobuloalveolar network. Estrogen implantation was sufficient to rescue ductal morphogenesis. Exogenous estrogen reversed the increased basal level of IGF-1 binding protein-1 expression in SirT1(ko/ko) mammary tissues, but not that of IkappaB alpha expression, suggesting that increased levels of estrogen enhanced the production of local IGF-1 and rescued ductal morphogenesis. Additionally, TNFalpha treatment enhanced the level of the newly synthesized IkappaB alpha in SirT1(ko/ko) cells. SirT1 deficiency therefore affects the cellular response to multiple extrinsic signals.

Conclusion: SirT1 modulates the IGF-1 signaling critical for both growth regulation and mammary gland development in mice. SirT1 deficiency deregulates the expression of IGF-1 binding protein-1 and attenuates the effect of IGF-1 signals, including estrogen-stimulated local IGF-1 signaling for the onset of ductal morphogenesis. These findings suggest that the enzymatic activity of SirT1 may influence both normal growth and malignant growth of mammary epithelial cells.

Show MeSH
Related in: MedlinePlus