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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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Generation and characterization of SirT1co/co and SirT1ko/ko mice. (a) Mouse SirT1 wild-type allele (SirT1+), conditional targeted allele (SirT1co), and knockout allele (SirT1ko). B, BamHI. (b) Western blot analysis of SirT1 expression in wild-type (+/+), conditional targeted (co/co), heterozygote (+/ko), and knockout (ko/ko) murine embryonic fibroblasts (MEFs) and mammary tissues (MG). (c) Growth retardation in surviving SirT1ko/ko mice (open bar) using sibling mice or age-matched mice of control genotypes (filled bar). (d) The serum levels of insulin-like growth factor-1 (IGF-1) in wild-type (+/+), SirT1+/ko (+/ko), and SirT1ko/ko (ko/ko) mice. (e) The survival curves of wild-type (WT), SirT1co/co, SirT1ko/ko, and Ku70-/- embryonic stem cells after ionizing radiation. (f) The survival curve of wild-type (WT), SirT1ko/ko, Ku70-/-, and Atm-/- embryonic stem cells after treatment with hydrogen peroxide.
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Figure 1: Generation and characterization of SirT1co/co and SirT1ko/ko mice. (a) Mouse SirT1 wild-type allele (SirT1+), conditional targeted allele (SirT1co), and knockout allele (SirT1ko). B, BamHI. (b) Western blot analysis of SirT1 expression in wild-type (+/+), conditional targeted (co/co), heterozygote (+/ko), and knockout (ko/ko) murine embryonic fibroblasts (MEFs) and mammary tissues (MG). (c) Growth retardation in surviving SirT1ko/ko mice (open bar) using sibling mice or age-matched mice of control genotypes (filled bar). (d) The serum levels of insulin-like growth factor-1 (IGF-1) in wild-type (+/+), SirT1+/ko (+/ko), and SirT1ko/ko (ko/ko) mice. (e) The survival curves of wild-type (WT), SirT1co/co, SirT1ko/ko, and Ku70-/- embryonic stem cells after ionizing radiation. (f) The survival curve of wild-type (WT), SirT1ko/ko, Ku70-/-, and Atm-/- embryonic stem cells after treatment with hydrogen peroxide.

Mentions: The conditional targeted SirT1 mutant mice (SirT1co/co mice) carry an insertion mutation of the neomycin-resistant gene and lox sequences in the SirT1 gene flanking exon 4 that encodes a conserved Sir2 motif (Figure 1a). The mutation does not affect the expression of SirT1 in SirT1co/co mice (Figure 1b). As expected, SirT1co/co mice are phenotypically indistinguishable from wild-type mice. To convert the SirT1 co allele into the SirT1 ko allele, SirT1co/co mice were crossed with CMV-Cre transgenic mice to generate SirT1 heterozygotes carrying the SirT1+/ko, CMV-Cre+ genotype. The expression of the CMV-Cre transgene catalyzes the deletion of exon 4 in most lineages of cells, including germ cells (Figure 1a). This SirT1 ko allele should be identical to the previously described Δex4 mutation [23]. SirT1+/ko, Cre+ mice were backcrossed with wild-type mice to generate the mice harboring a germline-transmitted deletion mutation (that is, SirT1+/ko mice). The breeding of SirT1+/ko male mice and SirT1+/ko female mice resulted in SirT1ko/ko mice. The cells derived from either SirT1+/ko mice or SirT1ko/ko mice expressed a SirT1 mutant protein due to the inframe deletion of exon 4 (Figure 1b). This SirT1 mutant protein may not be functional, however, since SirT1+/ko mice are phenotypically indistinguishable from wild-type mice and SirT1Δex4/Δex4 mice were phenotypically identical to SirT1 mice [23].


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)

Generation and characterization of SirT1co/co and SirT1ko/ko mice. (a) Mouse SirT1 wild-type allele (SirT1+), conditional targeted allele (SirT1co), and knockout allele (SirT1ko). B, BamHI. (b) Western blot analysis of SirT1 expression in wild-type (+/+), conditional targeted (co/co), heterozygote (+/ko), and knockout (ko/ko) murine embryonic fibroblasts (MEFs) and mammary tissues (MG). (c) Growth retardation in surviving SirT1ko/ko mice (open bar) using sibling mice or age-matched mice of control genotypes (filled bar). (d) The serum levels of insulin-like growth factor-1 (IGF-1) in wild-type (+/+), SirT1+/ko (+/ko), and SirT1ko/ko (ko/ko) mice. (e) The survival curves of wild-type (WT), SirT1co/co, SirT1ko/ko, and Ku70-/- embryonic stem cells after ionizing radiation. (f) The survival curve of wild-type (WT), SirT1ko/ko, Ku70-/-, and Atm-/- embryonic stem cells after treatment with hydrogen peroxide.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1851382&req=5

Figure 1: Generation and characterization of SirT1co/co and SirT1ko/ko mice. (a) Mouse SirT1 wild-type allele (SirT1+), conditional targeted allele (SirT1co), and knockout allele (SirT1ko). B, BamHI. (b) Western blot analysis of SirT1 expression in wild-type (+/+), conditional targeted (co/co), heterozygote (+/ko), and knockout (ko/ko) murine embryonic fibroblasts (MEFs) and mammary tissues (MG). (c) Growth retardation in surviving SirT1ko/ko mice (open bar) using sibling mice or age-matched mice of control genotypes (filled bar). (d) The serum levels of insulin-like growth factor-1 (IGF-1) in wild-type (+/+), SirT1+/ko (+/ko), and SirT1ko/ko (ko/ko) mice. (e) The survival curves of wild-type (WT), SirT1co/co, SirT1ko/ko, and Ku70-/- embryonic stem cells after ionizing radiation. (f) The survival curve of wild-type (WT), SirT1ko/ko, Ku70-/-, and Atm-/- embryonic stem cells after treatment with hydrogen peroxide.
Mentions: The conditional targeted SirT1 mutant mice (SirT1co/co mice) carry an insertion mutation of the neomycin-resistant gene and lox sequences in the SirT1 gene flanking exon 4 that encodes a conserved Sir2 motif (Figure 1a). The mutation does not affect the expression of SirT1 in SirT1co/co mice (Figure 1b). As expected, SirT1co/co mice are phenotypically indistinguishable from wild-type mice. To convert the SirT1 co allele into the SirT1 ko allele, SirT1co/co mice were crossed with CMV-Cre transgenic mice to generate SirT1 heterozygotes carrying the SirT1+/ko, CMV-Cre+ genotype. The expression of the CMV-Cre transgene catalyzes the deletion of exon 4 in most lineages of cells, including germ cells (Figure 1a). This SirT1 ko allele should be identical to the previously described Δex4 mutation [23]. SirT1+/ko, Cre+ mice were backcrossed with wild-type mice to generate the mice harboring a germline-transmitted deletion mutation (that is, SirT1+/ko mice). The breeding of SirT1+/ko male mice and SirT1+/ko female mice resulted in SirT1ko/ko mice. The cells derived from either SirT1+/ko mice or SirT1ko/ko mice expressed a SirT1 mutant protein due to the inframe deletion of exon 4 (Figure 1b). This SirT1 mutant protein may not be functional, however, since SirT1+/ko mice are phenotypically indistinguishable from wild-type mice and SirT1Δex4/Δex4 mice were phenotypically identical to SirT1 mice [23].

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