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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

mTORC2 participates in SEMA3F-induced RhoA inactivation and loss of stress fibers. A, U87MG cells were treated with SEMA3F (640 ng/ml), rapamycin (10 nM) or Torin 1 (10 nM) for 30 minutes. Subsequently, cells were stained with Alexa Fluor 488 phalloidin and Hoechst 33342 to identify F-actin cytoskeleton stress fibers and cellular nuclei, respectively. Representative cellular staining of is shown in each panel; the bar graph shows the mean ± SD number of fibers/cell in an average of 3 independent experiments. The scale bar indicates 20 μm. B, U87MG cells were transiently transfected with a pcDNA3.1 empty vector or with a wild type (WT) mTOR plasmid and after 18 hours were treated with SEMA3F (640 ng/ml) for 30 minutes. Cells were stained as described above in A. Representative cellular staining is shown; bar graph represents the number of fibers/cell (mean ± SD) from 3 independent experiments. C, U87MG cells were transfected with control siRNA or with raptor- or rictor-specific siRNAs (20 nM). After 48 hours, they were treated with SEMA3F for 30 minutes and stained with Alexa Fluor 488 phalloidin and Hoechst 33342 as above. The number of stress fibers was evaluated in 3 independent experiments and shown as the mean ± SD. D, U87MG cells were transiently transfected with pcDNA3.1 empty vector or with our WT mTOR plasmid. After 18 hours, the cells were treated with SEMA3F (640 ng/ml) for 10 minutes and RhoA activity was evaluated. E, U87MG cells were transfected with control siRNA or with raptor- or rictor-specific siRNAs (20 nM), were treated with SEMA3F (640 ng/ml) for 10 minutes and RhoA activity was analyzed. In Panels D–E, the intensity of active RhoA was normalized to respective total RhoA; the numbers below each gel lane represent the fold-change in intensity relative to control. Panels D–E are representative of 3 independent experiments.
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f3: mTORC2 participates in SEMA3F-induced RhoA inactivation and loss of stress fibers. A, U87MG cells were treated with SEMA3F (640 ng/ml), rapamycin (10 nM) or Torin 1 (10 nM) for 30 minutes. Subsequently, cells were stained with Alexa Fluor 488 phalloidin and Hoechst 33342 to identify F-actin cytoskeleton stress fibers and cellular nuclei, respectively. Representative cellular staining of is shown in each panel; the bar graph shows the mean ± SD number of fibers/cell in an average of 3 independent experiments. The scale bar indicates 20 μm. B, U87MG cells were transiently transfected with a pcDNA3.1 empty vector or with a wild type (WT) mTOR plasmid and after 18 hours were treated with SEMA3F (640 ng/ml) for 30 minutes. Cells were stained as described above in A. Representative cellular staining is shown; bar graph represents the number of fibers/cell (mean ± SD) from 3 independent experiments. C, U87MG cells were transfected with control siRNA or with raptor- or rictor-specific siRNAs (20 nM). After 48 hours, they were treated with SEMA3F for 30 minutes and stained with Alexa Fluor 488 phalloidin and Hoechst 33342 as above. The number of stress fibers was evaluated in 3 independent experiments and shown as the mean ± SD. D, U87MG cells were transiently transfected with pcDNA3.1 empty vector or with our WT mTOR plasmid. After 18 hours, the cells were treated with SEMA3F (640 ng/ml) for 10 minutes and RhoA activity was evaluated. E, U87MG cells were transfected with control siRNA or with raptor- or rictor-specific siRNAs (20 nM), were treated with SEMA3F (640 ng/ml) for 10 minutes and RhoA activity was analyzed. In Panels D–E, the intensity of active RhoA was normalized to respective total RhoA; the numbers below each gel lane represent the fold-change in intensity relative to control. Panels D–E are representative of 3 independent experiments.

Mentions: We next wished to determine whether the inhibition of mTORC2 serves as an intermediary response to link SEMA3F activity with cytoskeletal collapse. U87MG cells were treated either with SEMA3F, rapamycin or Torin 1 for 30 minutes and the actin cytoskeleton was visualized using phalloidin staining. As we previously reported6, SEMA3F markedly inhibited stress fiber formation and cytoskeletal arrangement compared to untreated cells (Fig. 3A, p = 0.002). Moreover, a similar effect was observed in cells treated with the mTORC1/C2 inhibitor Torin 1 (p = 0.01). In contrast, treatment with the mTORC1 inhibitor rapamycin failed to elicit any cytoskeletal changes (Fig. 3A; Supplementary Fig. 3). Also, while SEMA3F inhibited stress fiber formation by 90% in pcDNA3.1 control vector transfected cells, it had minimal effects in U87MG cells transfected with an mTOR overexpression construct (Fig. 3B). We interpret these findings to suggest that the effect of SEMA3F on stress fiber formation is not mediated via mTORC1. Consistent with this interpretation, knockdown of raptor also had minimal effects on stress fiber formation and cytoskeleton collapse (Fig. 3C). However, SEMA3F reduced stress fiber formation in raptor-siRNA treated cells (Fig. 3C); and notably, siRNA knockdown of rictor alone was sufficient to elicit collapse (p = 0.02). These data suggest that mTORC2 serves as an intermediary to modulate SEMA3F-inducible cytoskeletal collapse.


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)

mTORC2 participates in SEMA3F-induced RhoA inactivation and loss of stress fibers. A, U87MG cells were treated with SEMA3F (640 ng/ml), rapamycin (10 nM) or Torin 1 (10 nM) for 30 minutes. Subsequently, cells were stained with Alexa Fluor 488 phalloidin and Hoechst 33342 to identify F-actin cytoskeleton stress fibers and cellular nuclei, respectively. Representative cellular staining of is shown in each panel; the bar graph shows the mean ± SD number of fibers/cell in an average of 3 independent experiments. The scale bar indicates 20 μm. B, U87MG cells were transiently transfected with a pcDNA3.1 empty vector or with a wild type (WT) mTOR plasmid and after 18 hours were treated with SEMA3F (640 ng/ml) for 30 minutes. Cells were stained as described above in A. Representative cellular staining is shown; bar graph represents the number of fibers/cell (mean ± SD) from 3 independent experiments. C, U87MG cells were transfected with control siRNA or with raptor- or rictor-specific siRNAs (20 nM). After 48 hours, they were treated with SEMA3F for 30 minutes and stained with Alexa Fluor 488 phalloidin and Hoechst 33342 as above. The number of stress fibers was evaluated in 3 independent experiments and shown as the mean ± SD. D, U87MG cells were transiently transfected with pcDNA3.1 empty vector or with our WT mTOR plasmid. After 18 hours, the cells were treated with SEMA3F (640 ng/ml) for 10 minutes and RhoA activity was evaluated. E, U87MG cells were transfected with control siRNA or with raptor- or rictor-specific siRNAs (20 nM), were treated with SEMA3F (640 ng/ml) for 10 minutes and RhoA activity was analyzed. In Panels D–E, the intensity of active RhoA was normalized to respective total RhoA; the numbers below each gel lane represent the fold-change in intensity relative to control. Panels D–E are representative of 3 independent experiments.
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Related In: Results  -  Collection

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Show All Figures
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f3: mTORC2 participates in SEMA3F-induced RhoA inactivation and loss of stress fibers. A, U87MG cells were treated with SEMA3F (640 ng/ml), rapamycin (10 nM) or Torin 1 (10 nM) for 30 minutes. Subsequently, cells were stained with Alexa Fluor 488 phalloidin and Hoechst 33342 to identify F-actin cytoskeleton stress fibers and cellular nuclei, respectively. Representative cellular staining of is shown in each panel; the bar graph shows the mean ± SD number of fibers/cell in an average of 3 independent experiments. The scale bar indicates 20 μm. B, U87MG cells were transiently transfected with a pcDNA3.1 empty vector or with a wild type (WT) mTOR plasmid and after 18 hours were treated with SEMA3F (640 ng/ml) for 30 minutes. Cells were stained as described above in A. Representative cellular staining is shown; bar graph represents the number of fibers/cell (mean ± SD) from 3 independent experiments. C, U87MG cells were transfected with control siRNA or with raptor- or rictor-specific siRNAs (20 nM). After 48 hours, they were treated with SEMA3F for 30 minutes and stained with Alexa Fluor 488 phalloidin and Hoechst 33342 as above. The number of stress fibers was evaluated in 3 independent experiments and shown as the mean ± SD. D, U87MG cells were transiently transfected with pcDNA3.1 empty vector or with our WT mTOR plasmid. After 18 hours, the cells were treated with SEMA3F (640 ng/ml) for 10 minutes and RhoA activity was evaluated. E, U87MG cells were transfected with control siRNA or with raptor- or rictor-specific siRNAs (20 nM), were treated with SEMA3F (640 ng/ml) for 10 minutes and RhoA activity was analyzed. In Panels D–E, the intensity of active RhoA was normalized to respective total RhoA; the numbers below each gel lane represent the fold-change in intensity relative to control. Panels D–E are representative of 3 independent experiments.
Mentions: We next wished to determine whether the inhibition of mTORC2 serves as an intermediary response to link SEMA3F activity with cytoskeletal collapse. U87MG cells were treated either with SEMA3F, rapamycin or Torin 1 for 30 minutes and the actin cytoskeleton was visualized using phalloidin staining. As we previously reported6, SEMA3F markedly inhibited stress fiber formation and cytoskeletal arrangement compared to untreated cells (Fig. 3A, p = 0.002). Moreover, a similar effect was observed in cells treated with the mTORC1/C2 inhibitor Torin 1 (p = 0.01). In contrast, treatment with the mTORC1 inhibitor rapamycin failed to elicit any cytoskeletal changes (Fig. 3A; Supplementary Fig. 3). Also, while SEMA3F inhibited stress fiber formation by 90% in pcDNA3.1 control vector transfected cells, it had minimal effects in U87MG cells transfected with an mTOR overexpression construct (Fig. 3B). We interpret these findings to suggest that the effect of SEMA3F on stress fiber formation is not mediated via mTORC1. Consistent with this interpretation, knockdown of raptor also had minimal effects on stress fiber formation and cytoskeleton collapse (Fig. 3C). However, SEMA3F reduced stress fiber formation in raptor-siRNA treated cells (Fig. 3C); and notably, siRNA knockdown of rictor alone was sufficient to elicit collapse (p = 0.02). These data suggest that mTORC2 serves as an intermediary to modulate SEMA3F-inducible cytoskeletal collapse.

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