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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

Schematic cartoon showing regulatory signaling pathways mediated by SEMA3F-NRP2/Plexin A1 interactions.SEMA3F binds to the NRP2-Plexin A1 complex and associates with PTEN to inactivate PI-3K and mTORC2/Akt-dependent signaling. Receptor-mediated signals may also inactivate mTORC2/Akt signaling via PTEN-independent mechanisms in tumor cell lines. Functionally, these regulatory/pro-resolution signals suppress cell proliferation, migration, cytoskeletal stress fiber rearrangement and cell survival. Our previous findings demonstrate that SEMA3F also inhibits cytoskeleton structure in part by inactivating RhoA through both the ABL2 kinase and p190RhoGAP6; the current studies show that the inactivation of RhoA and cytoskeletal stress fiber rearrangement is also mediated via the inhibition of mTORC2.
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f6: Schematic cartoon showing regulatory signaling pathways mediated by SEMA3F-NRP2/Plexin A1 interactions.SEMA3F binds to the NRP2-Plexin A1 complex and associates with PTEN to inactivate PI-3K and mTORC2/Akt-dependent signaling. Receptor-mediated signals may also inactivate mTORC2/Akt signaling via PTEN-independent mechanisms in tumor cell lines. Functionally, these regulatory/pro-resolution signals suppress cell proliferation, migration, cytoskeletal stress fiber rearrangement and cell survival. Our previous findings demonstrate that SEMA3F also inhibits cytoskeleton structure in part by inactivating RhoA through both the ABL2 kinase and p190RhoGAP6; the current studies show that the inactivation of RhoA and cytoskeletal stress fiber rearrangement is also mediated via the inhibition of mTORC2.

Mentions: The semaphorin family of axonal guidance molecules, including SEMA3F, are well established to promote neuronal growth cone collapse that results from concomitant rearrangement of actin cytoskeletal stress fibers. SEMA3F is a potent inhibitor of tumor cell and EC adhesion, spreading and motility in vitro and in vivo56. In addition, SEMA3F does not induce apoptosis in U87MG cells within 24 hours6. In tumor and vascular endothelial cells, we previously observed that SEMA3F inactivates RhoA, thereby inhibiting cytoskeletal stress fiber formation612. In this report, we extend our signaling studies and demonstrate that mTORC2 is an intermediary in this response, and is indispensable for RhoA inactivation. For example, we show that SEMA3F treatment results in cell collapse following transfection of cells with mTOR (to induce mTORC1), suggesting that this effect is either mTOR-independent and/or is associated with targeting of mTORC2. Consistent with an effect on mTORC2, siRNA knockdown of rictor or the treatment of U87MG cells with the mTORC2 inhibitor Torin 1 consistently reduced the number of stress fibers as observed following treatment with SEMA3F. Importantly, we also noted that SEMA3F further reduced stress fibers in rictor-siRNA treated cells, indicating that SEMA3F likely inactivates RhoA in part via mTORC2 and in part via another mechanism such as the ABL2/p190RhoGAP pathway as we previously described6 (Fig. 6). Although mTORC2 is reported to interact with the Rho GTPase family and mediate F-actin cytoskeleton re-organization1855, our new findings indicate that this effect can be targeted through stimulation of NRP2-induced signals.


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)

Schematic cartoon showing regulatory signaling pathways mediated by SEMA3F-NRP2/Plexin A1 interactions.SEMA3F binds to the NRP2-Plexin A1 complex and associates with PTEN to inactivate PI-3K and mTORC2/Akt-dependent signaling. Receptor-mediated signals may also inactivate mTORC2/Akt signaling via PTEN-independent mechanisms in tumor cell lines. Functionally, these regulatory/pro-resolution signals suppress cell proliferation, migration, cytoskeletal stress fiber rearrangement and cell survival. Our previous findings demonstrate that SEMA3F also inhibits cytoskeleton structure in part by inactivating RhoA through both the ABL2 kinase and p190RhoGAP6; the current studies show that the inactivation of RhoA and cytoskeletal stress fiber rearrangement is also mediated via the inhibition of mTORC2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496725&req=5

f6: Schematic cartoon showing regulatory signaling pathways mediated by SEMA3F-NRP2/Plexin A1 interactions.SEMA3F binds to the NRP2-Plexin A1 complex and associates with PTEN to inactivate PI-3K and mTORC2/Akt-dependent signaling. Receptor-mediated signals may also inactivate mTORC2/Akt signaling via PTEN-independent mechanisms in tumor cell lines. Functionally, these regulatory/pro-resolution signals suppress cell proliferation, migration, cytoskeletal stress fiber rearrangement and cell survival. Our previous findings demonstrate that SEMA3F also inhibits cytoskeleton structure in part by inactivating RhoA through both the ABL2 kinase and p190RhoGAP6; the current studies show that the inactivation of RhoA and cytoskeletal stress fiber rearrangement is also mediated via the inhibition of mTORC2.
Mentions: The semaphorin family of axonal guidance molecules, including SEMA3F, are well established to promote neuronal growth cone collapse that results from concomitant rearrangement of actin cytoskeletal stress fibers. SEMA3F is a potent inhibitor of tumor cell and EC adhesion, spreading and motility in vitro and in vivo56. In addition, SEMA3F does not induce apoptosis in U87MG cells within 24 hours6. In tumor and vascular endothelial cells, we previously observed that SEMA3F inactivates RhoA, thereby inhibiting cytoskeletal stress fiber formation612. In this report, we extend our signaling studies and demonstrate that mTORC2 is an intermediary in this response, and is indispensable for RhoA inactivation. For example, we show that SEMA3F treatment results in cell collapse following transfection of cells with mTOR (to induce mTORC1), suggesting that this effect is either mTOR-independent and/or is associated with targeting of mTORC2. Consistent with an effect on mTORC2, siRNA knockdown of rictor or the treatment of U87MG cells with the mTORC2 inhibitor Torin 1 consistently reduced the number of stress fibers as observed following treatment with SEMA3F. Importantly, we also noted that SEMA3F further reduced stress fibers in rictor-siRNA treated cells, indicating that SEMA3F likely inactivates RhoA in part via mTORC2 and in part via another mechanism such as the ABL2/p190RhoGAP pathway as we previously described6 (Fig. 6). Although mTORC2 is reported to interact with the Rho GTPase family and mediate F-actin cytoskeleton re-organization1855, our new findings indicate that this effect can be targeted through stimulation of NRP2-induced signals.

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