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The mTOR pathway controls cell proliferation by regulating the FoxO3a transcription factor via SGK1 kinase.

Mori S, Nada S, Kimura H, Tajima S, Takahashi Y, Kitamura A, Oneyama C, Okada M - PLoS ONE (2014)

Bottom Line: Although Akt was activated by mTORC1 inactivation, FoxO3a was upregulated via an epigenetic mechanism and hypophosphorylated at Ser314, which resulted in its nuclear accumulation.Consistently, mTORC1 inactivation induced downregulation of serum- and glucocorticoid-inducible kinase 1 (SGK1), the kinase responsible for Ser314 phosphorylation.SGK1 overexpression suppressed CDKI expression in p18-deficient cells, whereas SGK1 knockdown induced CDKI expression in wild-type cells, resulting in the suppression of cell proliferation.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka, Japan.

ABSTRACT
The mechanistic target of rapamycin (mTOR) functions as a component of two large complexes, mTORC1 and mTORC2, which play crucial roles in regulating cell growth and homeostasis. However, the molecular mechanisms by which mTOR controls cell proliferation remain elusive. Here we show that the FoxO3a transcription factor is coordinately regulated by mTORC1 and mTORC2, and plays a crucial role in controlling cell proliferation. To dissect mTOR signaling, mTORC1 was specifically inactivated by depleting p18, an essential anchor of mTORC1 on lysosomes. mTORC1 inactivation caused a marked retardation of cell proliferation, which was associated with upregulation of cyclin-dependent kinase inhibitors (CDKIs). Although Akt was activated by mTORC1 inactivation, FoxO3a was upregulated via an epigenetic mechanism and hypophosphorylated at Ser314, which resulted in its nuclear accumulation. Consistently, mTORC1 inactivation induced downregulation of serum- and glucocorticoid-inducible kinase 1 (SGK1), the kinase responsible for Ser314 phosphorylation. Expression of FoxO3a mutated at Ser314 suppressed cell proliferation by inducing CDKI expression. SGK1 overexpression suppressed CDKI expression in p18-deficient cells, whereas SGK1 knockdown induced CDKI expression in wild-type cells, resulting in the suppression of cell proliferation. These results suggest that mTORC1, in coordination with mTORC2, controls cell proliferation by regulating FoxO3a gene expression and SGK1-mediated phosphorylation of FoxO3a at Ser314.

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Expression of FoxO3a is regulated by DNA methylation.(A) WT cells were treated with trichostatin A (TSA), 5-aza-deoxycytidine (5-Aza), or a combination of TSA and 5-Aza for 48 h, and FoxO3a mRNA levels were determined by quantitative real-time PCR. Fold change in mRNA levels was calculated after normalization against β-tubulin mRNA (internal control). **P<0.01 and *P<0.05 (Student's t-test). (B) DNA methylation status of FoxO3 CpG islands in p18KO, p18Rev, and WT cells was analyzed by bisulfite sequencing. The methylation status in the gray boxed region was determined for ten clones obtained from each cell line. Individual clones are indicated by lines with circles. Methylated and non-methylated cytosines are indicated by closed and open circles, respectively. (C) A series of fragments from the intronic region of the mouse FoxO3a gene were subcloned upstream of a luciferase reporter gene. Each construct was transfected into MEFs, and luciferase activity was measured and normalized against pRLTK activity. Normalized luciferase activity is expressed as means±SD (n = 3). ***P<0.001 and **P<0.01 (Student's t-test).
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pone-0088891-g004: Expression of FoxO3a is regulated by DNA methylation.(A) WT cells were treated with trichostatin A (TSA), 5-aza-deoxycytidine (5-Aza), or a combination of TSA and 5-Aza for 48 h, and FoxO3a mRNA levels were determined by quantitative real-time PCR. Fold change in mRNA levels was calculated after normalization against β-tubulin mRNA (internal control). **P<0.01 and *P<0.05 (Student's t-test). (B) DNA methylation status of FoxO3 CpG islands in p18KO, p18Rev, and WT cells was analyzed by bisulfite sequencing. The methylation status in the gray boxed region was determined for ten clones obtained from each cell line. Individual clones are indicated by lines with circles. Methylated and non-methylated cytosines are indicated by closed and open circles, respectively. (C) A series of fragments from the intronic region of the mouse FoxO3a gene were subcloned upstream of a luciferase reporter gene. Each construct was transfected into MEFs, and luciferase activity was measured and normalized against pRLTK activity. Normalized luciferase activity is expressed as means±SD (n = 3). ***P<0.001 and **P<0.01 (Student's t-test).

Mentions: Because the upregulation of FoxO3a was a chronic event, we investigated the role of the epigenetic mechanisms for FoxO3a upregulation in p18KO cells. Treatment of WT cells with 5-aza-deoxycytidine (5-aza-dCA), an inhibitor of DNA methyltransferase, significantly upregulated the transcription of FoxO3a; trichostatin A (TSA), an inhibitor of histone deacetylase, also upregulated Foxo3 transcription, albeit to a lesser extent (Figure 4A). This observation suggests that the expression of the Foxo3 gene is affected by its DNA methylation status. Bisulfite sequencing analyses of Foxo3 CpG islands revealed that DNA demethylation of a specific region at the 5′ end of the second intron occurred specifically in p18KO cells. Luciferase reporter assays revealed that the enhancer activity was located close to the 3′ end of the methylated region (Figure 4C). Thus, it is possible that DNA methylation in the adjacent region would interfere with enhancer activity, and hence inactivate the expression of Foxo3 gene. These observations suggest that mTORC1 is involved in the regulation of gene expression by controlling the DNA methylation status of the Foxo3 gene.


The mTOR pathway controls cell proliferation by regulating the FoxO3a transcription factor via SGK1 kinase.

Mori S, Nada S, Kimura H, Tajima S, Takahashi Y, Kitamura A, Oneyama C, Okada M - PLoS ONE (2014)

Expression of FoxO3a is regulated by DNA methylation.(A) WT cells were treated with trichostatin A (TSA), 5-aza-deoxycytidine (5-Aza), or a combination of TSA and 5-Aza for 48 h, and FoxO3a mRNA levels were determined by quantitative real-time PCR. Fold change in mRNA levels was calculated after normalization against β-tubulin mRNA (internal control). **P<0.01 and *P<0.05 (Student's t-test). (B) DNA methylation status of FoxO3 CpG islands in p18KO, p18Rev, and WT cells was analyzed by bisulfite sequencing. The methylation status in the gray boxed region was determined for ten clones obtained from each cell line. Individual clones are indicated by lines with circles. Methylated and non-methylated cytosines are indicated by closed and open circles, respectively. (C) A series of fragments from the intronic region of the mouse FoxO3a gene were subcloned upstream of a luciferase reporter gene. Each construct was transfected into MEFs, and luciferase activity was measured and normalized against pRLTK activity. Normalized luciferase activity is expressed as means±SD (n = 3). ***P<0.001 and **P<0.01 (Student's t-test).
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pone-0088891-g004: Expression of FoxO3a is regulated by DNA methylation.(A) WT cells were treated with trichostatin A (TSA), 5-aza-deoxycytidine (5-Aza), or a combination of TSA and 5-Aza for 48 h, and FoxO3a mRNA levels were determined by quantitative real-time PCR. Fold change in mRNA levels was calculated after normalization against β-tubulin mRNA (internal control). **P<0.01 and *P<0.05 (Student's t-test). (B) DNA methylation status of FoxO3 CpG islands in p18KO, p18Rev, and WT cells was analyzed by bisulfite sequencing. The methylation status in the gray boxed region was determined for ten clones obtained from each cell line. Individual clones are indicated by lines with circles. Methylated and non-methylated cytosines are indicated by closed and open circles, respectively. (C) A series of fragments from the intronic region of the mouse FoxO3a gene were subcloned upstream of a luciferase reporter gene. Each construct was transfected into MEFs, and luciferase activity was measured and normalized against pRLTK activity. Normalized luciferase activity is expressed as means±SD (n = 3). ***P<0.001 and **P<0.01 (Student's t-test).
Mentions: Because the upregulation of FoxO3a was a chronic event, we investigated the role of the epigenetic mechanisms for FoxO3a upregulation in p18KO cells. Treatment of WT cells with 5-aza-deoxycytidine (5-aza-dCA), an inhibitor of DNA methyltransferase, significantly upregulated the transcription of FoxO3a; trichostatin A (TSA), an inhibitor of histone deacetylase, also upregulated Foxo3 transcription, albeit to a lesser extent (Figure 4A). This observation suggests that the expression of the Foxo3 gene is affected by its DNA methylation status. Bisulfite sequencing analyses of Foxo3 CpG islands revealed that DNA demethylation of a specific region at the 5′ end of the second intron occurred specifically in p18KO cells. Luciferase reporter assays revealed that the enhancer activity was located close to the 3′ end of the methylated region (Figure 4C). Thus, it is possible that DNA methylation in the adjacent region would interfere with enhancer activity, and hence inactivate the expression of Foxo3 gene. These observations suggest that mTORC1 is involved in the regulation of gene expression by controlling the DNA methylation status of the Foxo3 gene.

Bottom Line: Although Akt was activated by mTORC1 inactivation, FoxO3a was upregulated via an epigenetic mechanism and hypophosphorylated at Ser314, which resulted in its nuclear accumulation.Consistently, mTORC1 inactivation induced downregulation of serum- and glucocorticoid-inducible kinase 1 (SGK1), the kinase responsible for Ser314 phosphorylation.SGK1 overexpression suppressed CDKI expression in p18-deficient cells, whereas SGK1 knockdown induced CDKI expression in wild-type cells, resulting in the suppression of cell proliferation.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka, Japan.

ABSTRACT
The mechanistic target of rapamycin (mTOR) functions as a component of two large complexes, mTORC1 and mTORC2, which play crucial roles in regulating cell growth and homeostasis. However, the molecular mechanisms by which mTOR controls cell proliferation remain elusive. Here we show that the FoxO3a transcription factor is coordinately regulated by mTORC1 and mTORC2, and plays a crucial role in controlling cell proliferation. To dissect mTOR signaling, mTORC1 was specifically inactivated by depleting p18, an essential anchor of mTORC1 on lysosomes. mTORC1 inactivation caused a marked retardation of cell proliferation, which was associated with upregulation of cyclin-dependent kinase inhibitors (CDKIs). Although Akt was activated by mTORC1 inactivation, FoxO3a was upregulated via an epigenetic mechanism and hypophosphorylated at Ser314, which resulted in its nuclear accumulation. Consistently, mTORC1 inactivation induced downregulation of serum- and glucocorticoid-inducible kinase 1 (SGK1), the kinase responsible for Ser314 phosphorylation. Expression of FoxO3a mutated at Ser314 suppressed cell proliferation by inducing CDKI expression. SGK1 overexpression suppressed CDKI expression in p18-deficient cells, whereas SGK1 knockdown induced CDKI expression in wild-type cells, resulting in the suppression of cell proliferation. These results suggest that mTORC1, in coordination with mTORC2, controls cell proliferation by regulating FoxO3a gene expression and SGK1-mediated phosphorylation of FoxO3a at Ser314.

Show MeSH
Related in: MedlinePlus