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Regulation of mTOR Signaling by Semaphorin 3F-Neuropilin 2 Interactions In Vitro and In Vivo.

Nakayama H, Bruneau S, Kochupurakkal N, Coma S, Briscoe DM, Klagsbrun M - Sci Rep (2015)

Bottom Line: Recent studies indicate that SEMA3F has biological effects in other cell types, however its mechanism(s) of function is poorly understood.Consistently, SEMA3F inhibits PI-3K and Akt activity, and responses are associated with the disruption of mTOR/rictor assembly and mTOR-dependent activation of the RhoA GTPase.In vivo, local and systemic overproduction of SEMA3F reduces tumor growth in NRP2-expressing xenografts.

View Article: PubMed Central - PubMed

Affiliation: 1] Vascular Biology Program, Boston Children's Hospital and Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115 [2] Transplant Research Program, Boston Children's Hospital and Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115 [3] Department of Surgery, Boston Children's Hospital and Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115 [4] Division of Cell Growth and Tumor Regulation, Proteo-Science Center, Ehime University, Toon, Ehime 791-0295, Japan.

ABSTRACT
Semaphorin 3F (SEMA3F) provides neuronal guidance cues via its ability to bind neuropilin 2 (NRP2) and Plexin A family molecules. Recent studies indicate that SEMA3F has biological effects in other cell types, however its mechanism(s) of function is poorly understood. Here, we analyze SEMA3F-NRP2 signaling responses in human endothelial, T cell and tumor cells using phosphokinase arrays, immunoprecipitation and Western blot analyses. Consistently, SEMA3F inhibits PI-3K and Akt activity, and responses are associated with the disruption of mTOR/rictor assembly and mTOR-dependent activation of the RhoA GTPase. We also find that the expression of vascular endothelial growth factor, as well as mTOR-inducible cellular activation responses and cytoskeleton stability are inhibited by SEMA3F-NRP2 interactions in vitro. In vivo, local and systemic overproduction of SEMA3F reduces tumor growth in NRP2-expressing xenografts. Taken together, SEMA3F regulates mTOR signaling in diverse human cell types, suggesting that it has broad therapeutic implications.

No MeSH data available.


Related in: MedlinePlus

SEMA3F disrupts both mTORC1 and mTORC2 complex formation.A, U87MG cells were treated with SEMA3F (640 ng/ml) for 30 minutes and were subjected to immunoprecipitation and Western blot analysis with anti-mTOR, -raptor and -rictor as illustrated. B, U87MG cells were treated with rapamycin (10 nM) or Torin 1 (10 nM) for 30 minutes, prior to SEMA3F (640 ng/ml) treatment for 60 minutes; lysates were analyzed by Western blot. C, The right bar graphs represent densitometric analysis of the illustrated blot showing the fold change in intensity (mean ± SD) relative to the untreated control (*p < 0.01; **p < 0.001 vs. untreated control). D, U87MG cells were transiently transfected with a pcDNA3.1 empty vector or with constitutively active Akt (2DAkt). Cells were treated with SEMA3F (640 ng/ml) and lysates were analyzed by Western blot. All data are representative of 3 independent experiments.
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f2: SEMA3F disrupts both mTORC1 and mTORC2 complex formation.A, U87MG cells were treated with SEMA3F (640 ng/ml) for 30 minutes and were subjected to immunoprecipitation and Western blot analysis with anti-mTOR, -raptor and -rictor as illustrated. B, U87MG cells were treated with rapamycin (10 nM) or Torin 1 (10 nM) for 30 minutes, prior to SEMA3F (640 ng/ml) treatment for 60 minutes; lysates were analyzed by Western blot. C, The right bar graphs represent densitometric analysis of the illustrated blot showing the fold change in intensity (mean ± SD) relative to the untreated control (*p < 0.01; **p < 0.001 vs. untreated control). D, U87MG cells were transiently transfected with a pcDNA3.1 empty vector or with constitutively active Akt (2DAkt). Cells were treated with SEMA3F (640 ng/ml) and lysates were analyzed by Western blot. All data are representative of 3 independent experiments.

Mentions: mTORC1 and mTORC2 signaling is critical for cell metabolism3031 as well as the differentiation, proliferation and survival of many normal cell types2732333435. By immunoprecipitation, we found that SEMA3F inhibited the association between mTOR and both raptor and rictor (Fig. 2A), suggesting a biological effect on both mTORC1 and mTORC2 respectively. Consistent with this observation, cells treated with SEMA3F for 60 minutes had reduced levels of pAkt (T308 and S473) and pS6K (vs. untreated cells, Fig. 2B, lane 1 vs. 4). To determine if its primary mode of function relates to the inhibition of mTORC1 vs. mTORC2, we initially pretreated cells with rapamycin (10 nM for 30 minutes to inhibit mTORC1) and subsequently cultured cells in the absence or presence of SEMA3F and rapamycin for another 60 minutes. Treatment with rapamycin alone (for 90 minutes) as a control resulted in a marked inhibition of pS6K, but an induction in the levels of pAkt (T308 and S473) by 1.9-fold (p < 0.001) and 2.0-fold (p < 0.01), respectively (Fig. 2B, lanes 1 vs. 2 and Fig. 2C), as we and others previously reported363738. In contrast, U87MG cells that were treated with rapamycin for 30 minutes and subsequently treated with SEMA3F and rapamycin for an additional 60 minutes had reduced levels of both pS6K and pAkt (Fig. 2B, lane 2 vs. 5). Of note, this effect of SEMA3F on the inhibition of pAkt expression was similar to that observed when cells are treated with the mTORC1/C2 inhibitor Torin 1 (Fig. 2B, lane 3 vs. 4). SEMA3F also inhibited pSGK1 (S422) and pPKCα (S657, Fig. 1B and Supplementary Fig. 2A), other known targets of mTORC2 activity23.


Regulation of mTOR Signaling by Semaphorin 3F-Neuropilin 2 Interactions In Vitro and In Vivo.

Nakayama H, Bruneau S, Kochupurakkal N, Coma S, Briscoe DM, Klagsbrun M - Sci Rep (2015)

SEMA3F disrupts both mTORC1 and mTORC2 complex formation.A, U87MG cells were treated with SEMA3F (640 ng/ml) for 30 minutes and were subjected to immunoprecipitation and Western blot analysis with anti-mTOR, -raptor and -rictor as illustrated. B, U87MG cells were treated with rapamycin (10 nM) or Torin 1 (10 nM) for 30 minutes, prior to SEMA3F (640 ng/ml) treatment for 60 minutes; lysates were analyzed by Western blot. C, The right bar graphs represent densitometric analysis of the illustrated blot showing the fold change in intensity (mean ± SD) relative to the untreated control (*p < 0.01; **p < 0.001 vs. untreated control). D, U87MG cells were transiently transfected with a pcDNA3.1 empty vector or with constitutively active Akt (2DAkt). Cells were treated with SEMA3F (640 ng/ml) and lysates were analyzed by Western blot. All data are representative of 3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4496725&req=5

f2: SEMA3F disrupts both mTORC1 and mTORC2 complex formation.A, U87MG cells were treated with SEMA3F (640 ng/ml) for 30 minutes and were subjected to immunoprecipitation and Western blot analysis with anti-mTOR, -raptor and -rictor as illustrated. B, U87MG cells were treated with rapamycin (10 nM) or Torin 1 (10 nM) for 30 minutes, prior to SEMA3F (640 ng/ml) treatment for 60 minutes; lysates were analyzed by Western blot. C, The right bar graphs represent densitometric analysis of the illustrated blot showing the fold change in intensity (mean ± SD) relative to the untreated control (*p < 0.01; **p < 0.001 vs. untreated control). D, U87MG cells were transiently transfected with a pcDNA3.1 empty vector or with constitutively active Akt (2DAkt). Cells were treated with SEMA3F (640 ng/ml) and lysates were analyzed by Western blot. All data are representative of 3 independent experiments.
Mentions: mTORC1 and mTORC2 signaling is critical for cell metabolism3031 as well as the differentiation, proliferation and survival of many normal cell types2732333435. By immunoprecipitation, we found that SEMA3F inhibited the association between mTOR and both raptor and rictor (Fig. 2A), suggesting a biological effect on both mTORC1 and mTORC2 respectively. Consistent with this observation, cells treated with SEMA3F for 60 minutes had reduced levels of pAkt (T308 and S473) and pS6K (vs. untreated cells, Fig. 2B, lane 1 vs. 4). To determine if its primary mode of function relates to the inhibition of mTORC1 vs. mTORC2, we initially pretreated cells with rapamycin (10 nM for 30 minutes to inhibit mTORC1) and subsequently cultured cells in the absence or presence of SEMA3F and rapamycin for another 60 minutes. Treatment with rapamycin alone (for 90 minutes) as a control resulted in a marked inhibition of pS6K, but an induction in the levels of pAkt (T308 and S473) by 1.9-fold (p < 0.001) and 2.0-fold (p < 0.01), respectively (Fig. 2B, lanes 1 vs. 2 and Fig. 2C), as we and others previously reported363738. In contrast, U87MG cells that were treated with rapamycin for 30 minutes and subsequently treated with SEMA3F and rapamycin for an additional 60 minutes had reduced levels of both pS6K and pAkt (Fig. 2B, lane 2 vs. 5). Of note, this effect of SEMA3F on the inhibition of pAkt expression was similar to that observed when cells are treated with the mTORC1/C2 inhibitor Torin 1 (Fig. 2B, lane 3 vs. 4). SEMA3F also inhibited pSGK1 (S422) and pPKCα (S657, Fig. 1B and Supplementary Fig. 2A), other known targets of mTORC2 activity23.

Bottom Line: Recent studies indicate that SEMA3F has biological effects in other cell types, however its mechanism(s) of function is poorly understood.Consistently, SEMA3F inhibits PI-3K and Akt activity, and responses are associated with the disruption of mTOR/rictor assembly and mTOR-dependent activation of the RhoA GTPase.In vivo, local and systemic overproduction of SEMA3F reduces tumor growth in NRP2-expressing xenografts.

View Article: PubMed Central - PubMed

Affiliation: 1] Vascular Biology Program, Boston Children's Hospital and Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115 [2] Transplant Research Program, Boston Children's Hospital and Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115 [3] Department of Surgery, Boston Children's Hospital and Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115 [4] Division of Cell Growth and Tumor Regulation, Proteo-Science Center, Ehime University, Toon, Ehime 791-0295, Japan.

ABSTRACT
Semaphorin 3F (SEMA3F) provides neuronal guidance cues via its ability to bind neuropilin 2 (NRP2) and Plexin A family molecules. Recent studies indicate that SEMA3F has biological effects in other cell types, however its mechanism(s) of function is poorly understood. Here, we analyze SEMA3F-NRP2 signaling responses in human endothelial, T cell and tumor cells using phosphokinase arrays, immunoprecipitation and Western blot analyses. Consistently, SEMA3F inhibits PI-3K and Akt activity, and responses are associated with the disruption of mTOR/rictor assembly and mTOR-dependent activation of the RhoA GTPase. We also find that the expression of vascular endothelial growth factor, as well as mTOR-inducible cellular activation responses and cytoskeleton stability are inhibited by SEMA3F-NRP2 interactions in vitro. In vivo, local and systemic overproduction of SEMA3F reduces tumor growth in NRP2-expressing xenografts. Taken together, SEMA3F regulates mTOR signaling in diverse human cell types, suggesting that it has broad therapeutic implications.

No MeSH data available.


Related in: MedlinePlus