Comparative Medicine Faculty Papers

Permanent URI for this collectionhttps://digital.lib.washington.edu/handle/1773/11646

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SirT1 modulates the estrogen-insulin-like growth factor-1 signaling for postnatal development of mammary gland in mice
(2007) Li, Hongzhe; Rajendran, Grace K.; Liu, Ninning; Ware, Carol B.; Rubin, Brian P.; Gu, Yansong
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-[kappa]B, 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[super]ko/ko) mice were generated by crossing a new strain of mice harboring a conditional targeted mutation in the SirT1 gene (SirT1[super]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[super]ko/ko mice can be fertile despite the growth retardation phenotype. Virgin SirT1[super]ko/ko mice displayed impeded ductal morphogenesis, whereas pregnant SirT1[super]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[super]ko/ko mammary tissues, but not that of I[kappa]B[alpha] expression, suggesting that increased levels of estrogen enhanced the production of local IGF-1 and rescued ductal morphogenesis. Additionally, TNF[alpha] treatment enhanced the level of the newly synthesized I[kappa]B[alpha] in SirT1[super]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.
Late gestation modulation of fetal glucocorticoid effects requires the receptor for leukemia inhibitory factor: an observational study
(2003) Ware, Carol B.; Nelson, Angelique M.; Liggitt, Denny
Background: Ablation of the low-affinity receptor subunit for leukemia inhibitory factor (LIFR) causes multi-systemic defects in the late gestation fetus. Because corticosterone is known to have a broad range of effects and LIF function has been associated with the hypothalamo-pituitaryadrenal axis, this study was designed to determine the role for LIFR in the fetus when exposed to the elevated maternal glucocorticoid levels of late gestation. Uncovering a requirement for LIFR in appropriate glucocorticoid response will further understanding of control of glucocorticoid function. Methods: Maternal adrenalectomy or RU486 administration were used to determine the impact of the maternal glucocorticoid surge on fetal development in the absence of LIFR. The mice were analyzed by a variety of histological techniques including immunolabeling and staining techniques (hematoxylin and eosin, Alizarin red S and alcian blue). Plasma corticosterone was assayed using radioimmunoassay. Results: Maternal adrenalectomy does not improve the prognosis for LIFR null pups and exacerbates the effects of LIFR loss. RU486 noticeably improves many of the tissues affected by LIFR loss: bone density, skeletal muscle integrity and glial cell formation. LIFR null pups exposed during late gestation to RU486 in utero survive natural delivery, unlike LIFR null pups from untreated litters. But RU486 treated LIFR null pups succumb within the first day after birth, presumably due to neural deficit resulting in an inability to suckle. Conclusion: LIFR plays an integral role in modulating the fetal response to elevated maternal glucocorticoids during late gestation. This role is likely to be mediated through the glucocorticoid receptor and has implications for adult homeostasis as a direct tie between immune, neural and hormone function.
Hepatocyte-specific inhibition of NF-κB leads to apoptosis after TNF treatment, but not after partial hepatectomy
(2002-07-15) Chaisson, Michelle L.; Brooling, John T.; Ladiges, Warren; Tsai, Sophia; Fausto, Nelson
One of the earliest TNF-dependent events to occur during liver regeneration is the activation of the transcription factor NF-κB through TNF receptor type 1. NF-κB activation in the liver can have both antiapoptotic and proliferative effects, but it is unclear which liver cell types, hepatocytes or nonparenchymal cells (NPCs), contribute to these effects. To specifically evaluate the role of hepatocyte NF-κB, we created GLVP/ΔN-IκBα transgenic mice, in which expression of a deletion mutant of IκBα (ΔN-IκBα) was induced in hepatocytes after injection of mifepristone. In control mice, injection of 25 μg/kg TNF caused NF-κB nuclear translocation in virtually all hepatocytes by 30 minutes and no detectable apoptosis, while in mice expressing ΔN-IκBα, NF-κB nuclear translocation was blocked in 45% of hepatocytes, leading to apoptosis 4 hours after TNF injection. In contrast, expression of ΔN-IκBα in hepatocytes during the first several hours after partial hepatectomy did not lead to apoptosis or decreased proliferation. As NF-κB activation was not inhibited in liver NPCs, it is likely that these cells are responsible for mediating the proliferative and antiapoptotic effects of NF-κB during liver regeneration.
Discovery of induced point mutations in maize genes by TILLING
(2004) Till, Bradley J.; Reynolds, Steven H.; Weil, Clifford; Springer, Nathan; Burtner, Chris; Young, Kim; Bowers, Elisabeth; Codomo, Christine A.; Enns, Linda C.; Odden, Anthony R.; Greene, Elizabeth A.; Comail, Luca; Henikoff, Steven
Background: Going from a gene sequence to its function in the context of a whole organism requires a strategy for targeting mutations, referred to as reverse genetics. Reverse genetics is highly desirable in the modern genomics era; however, the most powerful methods are generally restricted to a few model organisms. Previously, we introduced a reverse-genetic strategy with the potential for general applicability to organisms that lack well-developed genetic tools. Our TILLING (Targeting Induced Local Lesions IN Genomes) method uses chemical mutagenesis followed by screening for single-base changes to discover induced mutations that alter protein function. TILLING was shown to be an effective reverse genetic strategy by the establishment of a highthroughput TILLING facility and the delivery of thousands of point mutations in hundreds of Arabidopsis genes to members of the plant biology community. Results: We demonstrate that high-throughput TILLING is applicable to maize, an important crop plant with a large genome but with limited reverse-genetic resources currently available. We screened pools of DNA samples for mutations in 1-kb segments from 11 different genes, obtaining 17 independent induced mutations from a population of 750 pollen-mutagenized maize plants. One of the genes targeted was the DMT102 chromomethylase gene, for which we obtained an allelic series of three missense mutations that are predicted to be strongly deleterious. Conclusions: Our findings indicate that TILLING is a broadly applicable and efficient reversegenetic strategy. We are establishing a public TILLING service for maize modeled on the existing Arabidopsis TILLING Project.
Hepatocyte-specific inhibition of NF-κB leads to apoptosis after TNF treatment, but not after partial hepatectomy
(2002) Chaisson, Michelle L.; Brooling, John T.; Ladiges, Warren; Tsai, Sophia; Fausto, Nelson
One of the earliest TNF-dependent events to occur during liver regeneration is the activation of the transcription factor NF-κB through TNF receptor type 1. NF-κB activation in the liver can have both antiapoptotic and proliferative effects, but it is unclear which liver cell types, hepatocytes or nonparenchymal cells (NPCs), contribute to these effects. To specifically evaluate the role of hepatocyte NF-κB, we created GLVP/ΔN-IκBα transgenic mice, in which expression of a deletion mutant of IκBα (ΔN-IκBα) was induced in hepatocytes after injection of mifepristone. In control mice, injection of 25 μg/kg TNF caused NF-κB nuclear translocation in virtually all hepatocytes by 30 minutes and no detectable apoptosis, while in mice expressing ΔN-IκBα, NF-κB nuclear translocation was blocked in 45% of hepatocytes, leading to apoptosis 4 hours after TNF injection. In contrast, expression of ΔN-IκBα in hepatocytes during the first several hours after partial hepatectomy did not lead to apoptosis or decreased proliferation. As NF-κB activation was not inhibited in liver NPCs, it is likely that these cells are responsible for mediating the proliferative and antiapoptotic effects of NF-κB during liver regeneration.

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