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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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SirT1 deficiency derails NF-κB signaling in response to TNFα stimulation. (a) Western blot analysis of IκBα expression in SirT1ko/ko (ko/ko) and wild-type (+/+) murine embryonic fibroblasts (MEFs) within the first hour after TNFα stimulation. Actin is used as a loading control. (b) The relative levels of newly synthesized IκBα in three independent SirT1ko/ko MEF lines (red circles, blue and green triangles) and wild-type MEFs after TNFα stimulation. For each line of MEFs, the unit of IκBα protein at each time point is relative to the unit 0 minutes after normalization with actin.
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Figure 6: SirT1 deficiency derails NF-κB signaling in response to TNFα stimulation. (a) Western blot analysis of IκBα expression in SirT1ko/ko (ko/ko) and wild-type (+/+) murine embryonic fibroblasts (MEFs) within the first hour after TNFα stimulation. Actin is used as a loading control. (b) The relative levels of newly synthesized IκBα in three independent SirT1ko/ko MEF lines (red circles, blue and green triangles) and wild-type MEFs after TNFα stimulation. For each line of MEFs, the unit of IκBα protein at each time point is relative to the unit 0 minutes after normalization with actin.

Mentions: It was noted that increased levels of estrogen and local IGF-1 did not interfere with the expression of IκBα (Figure 4b). To determine whether deregulated IκBα expression in SirT1ko/ko cells can be reversed, we treated MEFs with TNFα and measured the kinetics of IκBα expression. Three independent lines of SirT1ko/ko MEFs were used, which displayed varying basal levels of increased IκBα expression. Following IκBα degradation induced by TNFα, the levels of newly synthesized IκBα in all three SirT1ko/ko MEFs were always higher than that of wild-type MEFs (Figure 6). This in vitro finding implies that SirT1 deficiency affects NF-κB signaling when the IκB kinase activation is normal.


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)

SirT1 deficiency derails NF-κB signaling in response to TNFα stimulation. (a) Western blot analysis of IκBα expression in SirT1ko/ko (ko/ko) and wild-type (+/+) murine embryonic fibroblasts (MEFs) within the first hour after TNFα stimulation. Actin is used as a loading control. (b) The relative levels of newly synthesized IκBα in three independent SirT1ko/ko MEF lines (red circles, blue and green triangles) and wild-type MEFs after TNFα stimulation. For each line of MEFs, the unit of IκBα protein at each time point is relative to the unit 0 minutes after normalization with actin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851382&req=5

Figure 6: SirT1 deficiency derails NF-κB signaling in response to TNFα stimulation. (a) Western blot analysis of IκBα expression in SirT1ko/ko (ko/ko) and wild-type (+/+) murine embryonic fibroblasts (MEFs) within the first hour after TNFα stimulation. Actin is used as a loading control. (b) The relative levels of newly synthesized IκBα in three independent SirT1ko/ko MEF lines (red circles, blue and green triangles) and wild-type MEFs after TNFα stimulation. For each line of MEFs, the unit of IκBα protein at each time point is relative to the unit 0 minutes after normalization with actin.
Mentions: It was noted that increased levels of estrogen and local IGF-1 did not interfere with the expression of IκBα (Figure 4b). To determine whether deregulated IκBα expression in SirT1ko/ko cells can be reversed, we treated MEFs with TNFα and measured the kinetics of IκBα expression. Three independent lines of SirT1ko/ko MEFs were used, which displayed varying basal levels of increased IκBα expression. Following IκBα degradation induced by TNFα, the levels of newly synthesized IκBα in all three SirT1ko/ko MEFs were always higher than that of wild-type MEFs (Figure 6). This in vitro finding implies that SirT1 deficiency affects NF-κB signaling when the IκB kinase activation is normal.

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