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Presenilin 1 negatively regulates beta-catenin/T cell factor/lymphoid enhancer factor-1 signaling independently of beta-amyloid precursor protein and notch processing.

Soriano S, Kang DE, Fu M, Pestell R, Chevallier N, Zheng H, Koo EH - J. Cell Biol. (2001)

Bottom Line: Conversely, PS1 specifically represses LEF-dependent transcription in a dose-dependent manner.The hyperproliferative response can be reversed by reintroducing PS1 expression or overexpressing axin, but not a PS1 mutant that does not bind beta-catenin (PS1Deltacat) or by two different familial Alzheimer's disease mutants.Thus, PS1 adds to the molecules that are known to regulate the rapid turnover of beta-catenin.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosciences, University of California, San Diego, La Jolla, California 92093, USA.

ABSTRACT
In addition to its documented role in the proteolytic processing of Notch-1 and the beta-amyloid precursor protein, presenilin 1 (PS1) associates with beta-catenin. In this study, we show that this interaction plays a critical role in regulating beta-catenin/T Cell Factor/Lymphoid Enhancer Factor-1 (LEF) signaling. PS1 deficiency results in accumulation of cytosolic beta-catenin, leading to a beta-catenin/LEF-dependent increase in cyclin D1 transcription and accelerated entry into the S phase of the cell cycle. Conversely, PS1 specifically represses LEF-dependent transcription in a dose-dependent manner. The hyperproliferative response can be reversed by reintroducing PS1 expression or overexpressing axin, but not a PS1 mutant that does not bind beta-catenin (PS1Deltacat) or by two different familial Alzheimer's disease mutants. In contrast, PS1Deltacat restores Notch-1 proteolytic cleavage and Abeta generation in PS1-deficient cells, indicating that PS1 function in modulating beta-catenin levels can be separated from its roles in facilitating gamma-secretase cleavage of beta-amyloid precursor protein and in Notch-1 signaling. Finally, we show an altered response to Wnt signaling and impaired ubiquitination of beta-catenin in the absence of PS1, a phenotype that may account for the increased stability in PS1-deficient cells. Thus, PS1 adds to the molecules that are known to regulate the rapid turnover of beta-catenin.

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(a) Immunoblot analysis of β-catenin, cyclin D1, cyclin A, and Cdc2 from PS1+/− and −/− cells. β-catenin and cyclin D1 levels were elevated in PS1-deficient cells. (b) PS1-deficient cells showed higher levels of cyclin D1 mRNA. Semiquantitative RT-PCR analysis of β-catenin, cyclin D1, and actin from PS1−/− and +/− primary fibroblasts. 25 ng of total RNA-derived cDNA were used for PCR reactions. Controls without reverse transcriptase yielded no bands (not shown). Results from a representative experiment are shown. (c) Loss of PS1 did not affect the turnover rate of cyclin D1. The cells were labeled for 20 min and chased for 15, 30, and 60 min. (Middle) Because PS1−/− cells have higher constitutive levels of cyclin D1, a shorter film exposure of the same autoradiogram that is comparable with that seen in PS+/− cells is shown. Quantitation from three independent experiments is shown (Bottom). (d) BrdU incorporation in PS1+/− and −/− cells. Primary fibroblasts from embryonic day 15.5 embryos were incubated with BrdU for 45 min at 37°C and BrdU incorporation was determined as described in Materials and Methods. Results are shown as the proportion of total cells (calculated from propidium iodide-stained nuclei) that were positive for BrdU staining, and represent the average of two independent experiments, each performed in triplicate (1,000 cells from at least 10 independent fields were scored for each sample). (e) Enhanced β-catenin/LEF-induced activation of the cyclin D1 promoter in PS1-deficient cells. PS1+/− and PS1−/− fibroblasts were transfected with constructs containing luciferase reporter driven by the cyclin D1 promoters. 1 μg of −163CD1 (wild type) or −163ΔlefCD1 (lacking the main LEF binding sequence) were used together with 50 ng of pRL-TK renilla luciferase construct in each sample as an internal control for transcription efficiency. Results are shown as the ratio of reporter luciferase to control renilla luciferase and represent the mean ± SD of three independent experiments performed in triplicate.
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Figure 1: (a) Immunoblot analysis of β-catenin, cyclin D1, cyclin A, and Cdc2 from PS1+/− and −/− cells. β-catenin and cyclin D1 levels were elevated in PS1-deficient cells. (b) PS1-deficient cells showed higher levels of cyclin D1 mRNA. Semiquantitative RT-PCR analysis of β-catenin, cyclin D1, and actin from PS1−/− and +/− primary fibroblasts. 25 ng of total RNA-derived cDNA were used for PCR reactions. Controls without reverse transcriptase yielded no bands (not shown). Results from a representative experiment are shown. (c) Loss of PS1 did not affect the turnover rate of cyclin D1. The cells were labeled for 20 min and chased for 15, 30, and 60 min. (Middle) Because PS1−/− cells have higher constitutive levels of cyclin D1, a shorter film exposure of the same autoradiogram that is comparable with that seen in PS+/− cells is shown. Quantitation from three independent experiments is shown (Bottom). (d) BrdU incorporation in PS1+/− and −/− cells. Primary fibroblasts from embryonic day 15.5 embryos were incubated with BrdU for 45 min at 37°C and BrdU incorporation was determined as described in Materials and Methods. Results are shown as the proportion of total cells (calculated from propidium iodide-stained nuclei) that were positive for BrdU staining, and represent the average of two independent experiments, each performed in triplicate (1,000 cells from at least 10 independent fields were scored for each sample). (e) Enhanced β-catenin/LEF-induced activation of the cyclin D1 promoter in PS1-deficient cells. PS1+/− and PS1−/− fibroblasts were transfected with constructs containing luciferase reporter driven by the cyclin D1 promoters. 1 μg of −163CD1 (wild type) or −163ΔlefCD1 (lacking the main LEF binding sequence) were used together with 50 ng of pRL-TK renilla luciferase construct in each sample as an internal control for transcription efficiency. Results are shown as the ratio of reporter luciferase to control renilla luciferase and represent the mean ± SD of three independent experiments performed in triplicate.

Mentions: We previously reported that the turnover rate of β-catenin in PS1-deficient immortalized fibroblasts was slower when compared with hemizygous PS1+/− cells, resulting in higher levels of cytosolic β-catenin (Kang et al. 1999). Accordingly, based on the recent finding that cyclin D1 transcription is activated by β-catenin/LEF, we predicted comparable alterations in cyclin D1 transcription in primary fibroblasts from early passages. Western blotting showed that PS1−/− cells contained higher amounts of β-catenin and cyclin D1 protein (Fig. 1 a). In contrast, levels of cdc2 and cyclin A, two other related genes not subject to β-catenin–mediated transcription (Tetsu and McCormick 1999), were not elevated in PS1−/− cells. This was confirmed by quantitative RT-PCR, which showed that PS1−/− cells expressed ∼ 2.5-fold higher levels of cyclin D1 mRNA than that of PS1+/− controls (2.53 ± 0.1, mean ± SD, n = 3) while β-catenin mRNA levels were unchanged (Fig. 1 b). Consistent with our previous report using immortalized PS1-deficient cell lines (Kang et al. 1999), pulse-chase experiments in primary fibroblasts showed that β-catenin turnover was reduced in PS1−/− cells (data not shown, see Fig. 3). No difference in cyclin D1 turnover was detected (Fig. 1 c), demonstrating that differences in cyclin D1 levels occurred at the transcriptional level.


Presenilin 1 negatively regulates beta-catenin/T cell factor/lymphoid enhancer factor-1 signaling independently of beta-amyloid precursor protein and notch processing.

Soriano S, Kang DE, Fu M, Pestell R, Chevallier N, Zheng H, Koo EH - J. Cell Biol. (2001)

(a) Immunoblot analysis of β-catenin, cyclin D1, cyclin A, and Cdc2 from PS1+/− and −/− cells. β-catenin and cyclin D1 levels were elevated in PS1-deficient cells. (b) PS1-deficient cells showed higher levels of cyclin D1 mRNA. Semiquantitative RT-PCR analysis of β-catenin, cyclin D1, and actin from PS1−/− and +/− primary fibroblasts. 25 ng of total RNA-derived cDNA were used for PCR reactions. Controls without reverse transcriptase yielded no bands (not shown). Results from a representative experiment are shown. (c) Loss of PS1 did not affect the turnover rate of cyclin D1. The cells were labeled for 20 min and chased for 15, 30, and 60 min. (Middle) Because PS1−/− cells have higher constitutive levels of cyclin D1, a shorter film exposure of the same autoradiogram that is comparable with that seen in PS+/− cells is shown. Quantitation from three independent experiments is shown (Bottom). (d) BrdU incorporation in PS1+/− and −/− cells. Primary fibroblasts from embryonic day 15.5 embryos were incubated with BrdU for 45 min at 37°C and BrdU incorporation was determined as described in Materials and Methods. Results are shown as the proportion of total cells (calculated from propidium iodide-stained nuclei) that were positive for BrdU staining, and represent the average of two independent experiments, each performed in triplicate (1,000 cells from at least 10 independent fields were scored for each sample). (e) Enhanced β-catenin/LEF-induced activation of the cyclin D1 promoter in PS1-deficient cells. PS1+/− and PS1−/− fibroblasts were transfected with constructs containing luciferase reporter driven by the cyclin D1 promoters. 1 μg of −163CD1 (wild type) or −163ΔlefCD1 (lacking the main LEF binding sequence) were used together with 50 ng of pRL-TK renilla luciferase construct in each sample as an internal control for transcription efficiency. Results are shown as the ratio of reporter luciferase to control renilla luciferase and represent the mean ± SD of three independent experiments performed in triplicate.
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Figure 1: (a) Immunoblot analysis of β-catenin, cyclin D1, cyclin A, and Cdc2 from PS1+/− and −/− cells. β-catenin and cyclin D1 levels were elevated in PS1-deficient cells. (b) PS1-deficient cells showed higher levels of cyclin D1 mRNA. Semiquantitative RT-PCR analysis of β-catenin, cyclin D1, and actin from PS1−/− and +/− primary fibroblasts. 25 ng of total RNA-derived cDNA were used for PCR reactions. Controls without reverse transcriptase yielded no bands (not shown). Results from a representative experiment are shown. (c) Loss of PS1 did not affect the turnover rate of cyclin D1. The cells were labeled for 20 min and chased for 15, 30, and 60 min. (Middle) Because PS1−/− cells have higher constitutive levels of cyclin D1, a shorter film exposure of the same autoradiogram that is comparable with that seen in PS+/− cells is shown. Quantitation from three independent experiments is shown (Bottom). (d) BrdU incorporation in PS1+/− and −/− cells. Primary fibroblasts from embryonic day 15.5 embryos were incubated with BrdU for 45 min at 37°C and BrdU incorporation was determined as described in Materials and Methods. Results are shown as the proportion of total cells (calculated from propidium iodide-stained nuclei) that were positive for BrdU staining, and represent the average of two independent experiments, each performed in triplicate (1,000 cells from at least 10 independent fields were scored for each sample). (e) Enhanced β-catenin/LEF-induced activation of the cyclin D1 promoter in PS1-deficient cells. PS1+/− and PS1−/− fibroblasts were transfected with constructs containing luciferase reporter driven by the cyclin D1 promoters. 1 μg of −163CD1 (wild type) or −163ΔlefCD1 (lacking the main LEF binding sequence) were used together with 50 ng of pRL-TK renilla luciferase construct in each sample as an internal control for transcription efficiency. Results are shown as the ratio of reporter luciferase to control renilla luciferase and represent the mean ± SD of three independent experiments performed in triplicate.
Mentions: We previously reported that the turnover rate of β-catenin in PS1-deficient immortalized fibroblasts was slower when compared with hemizygous PS1+/− cells, resulting in higher levels of cytosolic β-catenin (Kang et al. 1999). Accordingly, based on the recent finding that cyclin D1 transcription is activated by β-catenin/LEF, we predicted comparable alterations in cyclin D1 transcription in primary fibroblasts from early passages. Western blotting showed that PS1−/− cells contained higher amounts of β-catenin and cyclin D1 protein (Fig. 1 a). In contrast, levels of cdc2 and cyclin A, two other related genes not subject to β-catenin–mediated transcription (Tetsu and McCormick 1999), were not elevated in PS1−/− cells. This was confirmed by quantitative RT-PCR, which showed that PS1−/− cells expressed ∼ 2.5-fold higher levels of cyclin D1 mRNA than that of PS1+/− controls (2.53 ± 0.1, mean ± SD, n = 3) while β-catenin mRNA levels were unchanged (Fig. 1 b). Consistent with our previous report using immortalized PS1-deficient cell lines (Kang et al. 1999), pulse-chase experiments in primary fibroblasts showed that β-catenin turnover was reduced in PS1−/− cells (data not shown, see Fig. 3). No difference in cyclin D1 turnover was detected (Fig. 1 c), demonstrating that differences in cyclin D1 levels occurred at the transcriptional level.

Bottom Line: Conversely, PS1 specifically represses LEF-dependent transcription in a dose-dependent manner.The hyperproliferative response can be reversed by reintroducing PS1 expression or overexpressing axin, but not a PS1 mutant that does not bind beta-catenin (PS1Deltacat) or by two different familial Alzheimer's disease mutants.Thus, PS1 adds to the molecules that are known to regulate the rapid turnover of beta-catenin.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosciences, University of California, San Diego, La Jolla, California 92093, USA.

ABSTRACT
In addition to its documented role in the proteolytic processing of Notch-1 and the beta-amyloid precursor protein, presenilin 1 (PS1) associates with beta-catenin. In this study, we show that this interaction plays a critical role in regulating beta-catenin/T Cell Factor/Lymphoid Enhancer Factor-1 (LEF) signaling. PS1 deficiency results in accumulation of cytosolic beta-catenin, leading to a beta-catenin/LEF-dependent increase in cyclin D1 transcription and accelerated entry into the S phase of the cell cycle. Conversely, PS1 specifically represses LEF-dependent transcription in a dose-dependent manner. The hyperproliferative response can be reversed by reintroducing PS1 expression or overexpressing axin, but not a PS1 mutant that does not bind beta-catenin (PS1Deltacat) or by two different familial Alzheimer's disease mutants. In contrast, PS1Deltacat restores Notch-1 proteolytic cleavage and Abeta generation in PS1-deficient cells, indicating that PS1 function in modulating beta-catenin levels can be separated from its roles in facilitating gamma-secretase cleavage of beta-amyloid precursor protein and in Notch-1 signaling. Finally, we show an altered response to Wnt signaling and impaired ubiquitination of beta-catenin in the absence of PS1, a phenotype that may account for the increased stability in PS1-deficient cells. Thus, PS1 adds to the molecules that are known to regulate the rapid turnover of beta-catenin.

Show MeSH
Related in: MedlinePlus