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Mechanistic target of rapamycin complex 1 signaling regulates cell proliferation, cell survival, and differentiation in regenerating zebrafish fins.

Hirose K, Shiomi T, Hozumi S, Kikuchi Y - BMC Dev. Biol. (2014)

Bottom Line: Moreover, rapamycin treatment inhibits blastema and wound epidermal cell proliferation and survival during blastema formation and regenerative outgrowth, as well as osteoblast proliferation and differentiation during regenerative outgrowth.We further determined that mTORC1 signaling is regulated through IGF-1 receptor/phosphatidylinositol-3 kinase and Wnt pathways during fin regeneration.Taken together, our findings reveal that mTORC1 signaling regulates proliferation, survival, and differentiation of intra-ray cells, wound epidermis, blastema cells, and/or osteoblasts in various fin regeneration stages downstream of IGF and Wnt signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, Graduate School of Science, Hiroshima University, Kagamiyama 1-3-1, Higashi-Hiroshima, Hiroshima, 739-8526, Japan. hirosekentaro@hiroshima-u.ac.jp.

ABSTRACT

Background: The mechanistic target of rapamycin complex1 (mTORC1) signaling pathway has been implicated in functions of multicellular processes, including cell growth and metabolism. Although recent reports showed that many signaling pathways, including Activin, Bmp, Fgf, sonic hedgehog, Insulin-like growth factor (IGF), Notch, retinoic acid, and Wnt, are implicated in non-mammalian vertebrate regeneration, also known as epimorphic regeneration, mTORC1 function remains unknown.

Results: To investigate the role of mTORC1 signaling pathway in zebrafish caudal fin, we examined the activation and function of mTORC1 signaling using an antibody against phosphorylated S6 kinase and a specific inhibitor, rapamycin. mTORC1 signaling is activated in proliferative cells of intra-ray and wound epidermal cells before blastema formation, as well as in proliferative blastema cells, wound epidermal cells, and osteoblasts during regenerative outgrowth. Before blastema formation, proliferation of intra-ray and wound epidermal cells is suppressed, but cell death is not affected by mTORC1 signaling inhibition with rapamycin. Moreover, rapamycin treatment inhibits blastema and wound epidermal cell proliferation and survival during blastema formation and regenerative outgrowth, as well as osteoblast proliferation and differentiation during regenerative outgrowth. We further determined that mTORC1 signaling is regulated through IGF-1 receptor/phosphatidylinositol-3 kinase and Wnt pathways during fin regeneration.

Conclusion: Taken together, our findings reveal that mTORC1 signaling regulates proliferation, survival, and differentiation of intra-ray cells, wound epidermis, blastema cells, and/or osteoblasts in various fin regeneration stages downstream of IGF and Wnt signaling pathways.

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IGF-1R/PI3K and Wnt pathways regulate the activation of mTORC1 during fin regeneration. (A) Scheme of inhibitor treatments of rapamycin, LY294002 (a PI3K inhibitor), NVP-ADW742 (an IGF-1R inhibitor), or IWP-2 (a Wnt/β-catenin inhibitor) during fin regeneration. (B-K) Longitudinal sections of DMSO or inhibitors treated wild-type fin regenerates that were immunohistochemically stained with an antibody against p-S6K (green) at 24 (DMSO, n = 6; Rapa, n = 4; LY294002, n = 4; NVP-ADW742, n = 4; IWP-2, n = 5) and 48 hpa (DMSO, n = 6; Rapa, n = 4; LY294002, n = 4; NVP-ADW742, n = 4; IWP-2, n = 4). DAPI fluorescent signal (blue) indicates the presence of nuclei. The activation of S6K was blocked by LY294002, NVP-ADW742, or IWP-2 treatment. Dashed white lines indicate the amputation planes. Scale bars: 100 μm.
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Fig7: IGF-1R/PI3K and Wnt pathways regulate the activation of mTORC1 during fin regeneration. (A) Scheme of inhibitor treatments of rapamycin, LY294002 (a PI3K inhibitor), NVP-ADW742 (an IGF-1R inhibitor), or IWP-2 (a Wnt/β-catenin inhibitor) during fin regeneration. (B-K) Longitudinal sections of DMSO or inhibitors treated wild-type fin regenerates that were immunohistochemically stained with an antibody against p-S6K (green) at 24 (DMSO, n = 6; Rapa, n = 4; LY294002, n = 4; NVP-ADW742, n = 4; IWP-2, n = 5) and 48 hpa (DMSO, n = 6; Rapa, n = 4; LY294002, n = 4; NVP-ADW742, n = 4; IWP-2, n = 4). DAPI fluorescent signal (blue) indicates the presence of nuclei. The activation of S6K was blocked by LY294002, NVP-ADW742, or IWP-2 treatment. Dashed white lines indicate the amputation planes. Scale bars: 100 μm.

Mentions: It is well known that the mTORC1/S6K pathway is regulated through the receptor tyrosine kinase/PI3K/Akt pathway in cell proliferation and metabolism [7,8]. To examine the upstream signaling pathway regulating mTORC1/S6K signaling in fin regeneration, various inhibitors for IGF, Wnt, Fgf, and ROS signaling pathways were tested from -12 h to 24 hpa or from 24 to 48 hpa, and S6K activation was determined; IGF signaling (LY294002 [25]: a PI3K inhibitor, NVP-ADW742 [26]: a IGF-1 receptor kinase inhibitor), Wnt signaling (IWP-2 [27]: a Wnt/β-catenin signaling inhibitor), Fgf signaling (SU5402 [28]: a Fgf receptor1 inhibitor), MAPK/Erk signaling (U0126 [29]: a MAPK/Erk inhibitor), and ROS signaling (VAS2870 [30]: an inhibitor of NADPH oxidase). No inhibitory effect for S6K activation was observed using SU5402, U0126, or VAS2870, even though fin regeneration was suppressed (Additional file 9: Figure S9). However, LY294002, NVP-ADW742, and IWP-2 inhibitor treatment markedly reduced the p-S6K signal in blastema and epidermal cells. The inhibitory effect of LY294002, NVP-ADW742, and IWP-2 on S6K activation was nearly similar to that of rapamycin during fin regeneration (Figure 7). These results suggest that both IGF-1R/PI3K and Wnt pathways activate mTORC1 in blastema and epidermal cells during zebrafish caudal fin regeneration.Figure 7


Mechanistic target of rapamycin complex 1 signaling regulates cell proliferation, cell survival, and differentiation in regenerating zebrafish fins.

Hirose K, Shiomi T, Hozumi S, Kikuchi Y - BMC Dev. Biol. (2014)

IGF-1R/PI3K and Wnt pathways regulate the activation of mTORC1 during fin regeneration. (A) Scheme of inhibitor treatments of rapamycin, LY294002 (a PI3K inhibitor), NVP-ADW742 (an IGF-1R inhibitor), or IWP-2 (a Wnt/β-catenin inhibitor) during fin regeneration. (B-K) Longitudinal sections of DMSO or inhibitors treated wild-type fin regenerates that were immunohistochemically stained with an antibody against p-S6K (green) at 24 (DMSO, n = 6; Rapa, n = 4; LY294002, n = 4; NVP-ADW742, n = 4; IWP-2, n = 5) and 48 hpa (DMSO, n = 6; Rapa, n = 4; LY294002, n = 4; NVP-ADW742, n = 4; IWP-2, n = 4). DAPI fluorescent signal (blue) indicates the presence of nuclei. The activation of S6K was blocked by LY294002, NVP-ADW742, or IWP-2 treatment. Dashed white lines indicate the amputation planes. Scale bars: 100 μm.
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Fig7: IGF-1R/PI3K and Wnt pathways regulate the activation of mTORC1 during fin regeneration. (A) Scheme of inhibitor treatments of rapamycin, LY294002 (a PI3K inhibitor), NVP-ADW742 (an IGF-1R inhibitor), or IWP-2 (a Wnt/β-catenin inhibitor) during fin regeneration. (B-K) Longitudinal sections of DMSO or inhibitors treated wild-type fin regenerates that were immunohistochemically stained with an antibody against p-S6K (green) at 24 (DMSO, n = 6; Rapa, n = 4; LY294002, n = 4; NVP-ADW742, n = 4; IWP-2, n = 5) and 48 hpa (DMSO, n = 6; Rapa, n = 4; LY294002, n = 4; NVP-ADW742, n = 4; IWP-2, n = 4). DAPI fluorescent signal (blue) indicates the presence of nuclei. The activation of S6K was blocked by LY294002, NVP-ADW742, or IWP-2 treatment. Dashed white lines indicate the amputation planes. Scale bars: 100 μm.
Mentions: It is well known that the mTORC1/S6K pathway is regulated through the receptor tyrosine kinase/PI3K/Akt pathway in cell proliferation and metabolism [7,8]. To examine the upstream signaling pathway regulating mTORC1/S6K signaling in fin regeneration, various inhibitors for IGF, Wnt, Fgf, and ROS signaling pathways were tested from -12 h to 24 hpa or from 24 to 48 hpa, and S6K activation was determined; IGF signaling (LY294002 [25]: a PI3K inhibitor, NVP-ADW742 [26]: a IGF-1 receptor kinase inhibitor), Wnt signaling (IWP-2 [27]: a Wnt/β-catenin signaling inhibitor), Fgf signaling (SU5402 [28]: a Fgf receptor1 inhibitor), MAPK/Erk signaling (U0126 [29]: a MAPK/Erk inhibitor), and ROS signaling (VAS2870 [30]: an inhibitor of NADPH oxidase). No inhibitory effect for S6K activation was observed using SU5402, U0126, or VAS2870, even though fin regeneration was suppressed (Additional file 9: Figure S9). However, LY294002, NVP-ADW742, and IWP-2 inhibitor treatment markedly reduced the p-S6K signal in blastema and epidermal cells. The inhibitory effect of LY294002, NVP-ADW742, and IWP-2 on S6K activation was nearly similar to that of rapamycin during fin regeneration (Figure 7). These results suggest that both IGF-1R/PI3K and Wnt pathways activate mTORC1 in blastema and epidermal cells during zebrafish caudal fin regeneration.Figure 7

Bottom Line: Moreover, rapamycin treatment inhibits blastema and wound epidermal cell proliferation and survival during blastema formation and regenerative outgrowth, as well as osteoblast proliferation and differentiation during regenerative outgrowth.We further determined that mTORC1 signaling is regulated through IGF-1 receptor/phosphatidylinositol-3 kinase and Wnt pathways during fin regeneration.Taken together, our findings reveal that mTORC1 signaling regulates proliferation, survival, and differentiation of intra-ray cells, wound epidermis, blastema cells, and/or osteoblasts in various fin regeneration stages downstream of IGF and Wnt signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, Graduate School of Science, Hiroshima University, Kagamiyama 1-3-1, Higashi-Hiroshima, Hiroshima, 739-8526, Japan. hirosekentaro@hiroshima-u.ac.jp.

ABSTRACT

Background: The mechanistic target of rapamycin complex1 (mTORC1) signaling pathway has been implicated in functions of multicellular processes, including cell growth and metabolism. Although recent reports showed that many signaling pathways, including Activin, Bmp, Fgf, sonic hedgehog, Insulin-like growth factor (IGF), Notch, retinoic acid, and Wnt, are implicated in non-mammalian vertebrate regeneration, also known as epimorphic regeneration, mTORC1 function remains unknown.

Results: To investigate the role of mTORC1 signaling pathway in zebrafish caudal fin, we examined the activation and function of mTORC1 signaling using an antibody against phosphorylated S6 kinase and a specific inhibitor, rapamycin. mTORC1 signaling is activated in proliferative cells of intra-ray and wound epidermal cells before blastema formation, as well as in proliferative blastema cells, wound epidermal cells, and osteoblasts during regenerative outgrowth. Before blastema formation, proliferation of intra-ray and wound epidermal cells is suppressed, but cell death is not affected by mTORC1 signaling inhibition with rapamycin. Moreover, rapamycin treatment inhibits blastema and wound epidermal cell proliferation and survival during blastema formation and regenerative outgrowth, as well as osteoblast proliferation and differentiation during regenerative outgrowth. We further determined that mTORC1 signaling is regulated through IGF-1 receptor/phosphatidylinositol-3 kinase and Wnt pathways during fin regeneration.

Conclusion: Taken together, our findings reveal that mTORC1 signaling regulates proliferation, survival, and differentiation of intra-ray cells, wound epidermis, blastema cells, and/or osteoblasts in various fin regeneration stages downstream of IGF and Wnt signaling pathways.

Show MeSH
Related in: MedlinePlus