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Formononetin promotes angiogenesis through the estrogen receptor alpha-enhanced ROCK pathway.

Li S, Dang Y, Zhou X, Huang B, Huang X, Zhang Z, Kwan YW, Chan SW, Leung GP, Lee SM, Hoi MP - Sci Rep (2015)

Bottom Line: In addition, results from co-immunoprecipitation suggested formononetin induced cell migration via recruiting of ERα/ROCK-II activated complex formation.More interestingly, in zebrafish embryo we observed that formononetin significantly promoted angiogenic sproutings in the subintestinal vessels (SIVs) that could be completely abolished by ROCK inhibitor.In this study, we elucidated the underlying mechanisms that formononetin produced proangiogenesis effects through an ERα-enhanced ROCK-II signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Macao, China.

ABSTRACT
Formononetin is an isoflavone that has been shown to display estrogenic properties and induce angiogenesis activities. However, the interrelationship between the estrogenic properties and angiogenesis activities of formononetin are not well defined. In the present study, docking and enzymatic assay demonstrated that formononetin displayed direct binding to the ligand-binding domain (LBD) of estrogen receptor alpha (ERα) with an agonistic property. Results from Human Umbilical Vein Endothelial Cells (HUVEC) by using real-time migration xCELLigence system, immunofluorescence and western blotting provided strong evidences of formononetin induced endothelial cell migration and dramatic actin cytoskeleton spatial modification through ERα-enhanced-ROCK-II/MMP2/9 signaling pathways. In addition, results from co-immunoprecipitation suggested formononetin induced cell migration via recruiting of ERα/ROCK-II activated complex formation. More interestingly, in zebrafish embryo we observed that formononetin significantly promoted angiogenic sproutings in the subintestinal vessels (SIVs) that could be completely abolished by ROCK inhibitor. In this study, we elucidated the underlying mechanisms that formononetin produced proangiogenesis effects through an ERα-enhanced ROCK-II signaling pathways. Results from the present study also expand our knowledge about the enigmatic underlying mechanisms of phytoestrogenic compounds in the promotion of angiogenesis in relation to ERα and ROCK interaction in endothelial cells and their relationship with actin assembly and cell migration.

No MeSH data available.


Related in: MedlinePlus

Inhibition of ROCK-II expression by ROCK-II siRNA abolished the activation of ROCK and MMP2/9 signaling pathways in HUVECs.(A) HUVECs were transfected with either non-specific siRNA as control or specific ROCK-II siRNA for 2 days followed by 50 μM formononetin treatment for 30 or 60 min. The protein levels of ROCK-II, MMP2, MMP9, P-MYPT1, P-LIMK1, P-cofilin and P-MLC2 were analyzed by using Western blotting. (B) Statistical analysis of the protein levels of MMP2 and MMP9 in HUVECs transfected with ROCK-II siRNA in the presence of 50 μM formononetin. (C) Statistical analysis of the protein levels of P-MYPT1, P-LIMK1, P-cofilin and P-MLC2 in HUVECs transfected with ROCK-II siRNA in the presence of 50 μM formononetin. Results are expressed as a percentage of control (means ± SD, n = 3), *p < 0.05, **p < 0.01 vs. control.
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f7: Inhibition of ROCK-II expression by ROCK-II siRNA abolished the activation of ROCK and MMP2/9 signaling pathways in HUVECs.(A) HUVECs were transfected with either non-specific siRNA as control or specific ROCK-II siRNA for 2 days followed by 50 μM formononetin treatment for 30 or 60 min. The protein levels of ROCK-II, MMP2, MMP9, P-MYPT1, P-LIMK1, P-cofilin and P-MLC2 were analyzed by using Western blotting. (B) Statistical analysis of the protein levels of MMP2 and MMP9 in HUVECs transfected with ROCK-II siRNA in the presence of 50 μM formononetin. (C) Statistical analysis of the protein levels of P-MYPT1, P-LIMK1, P-cofilin and P-MLC2 in HUVECs transfected with ROCK-II siRNA in the presence of 50 μM formononetin. Results are expressed as a percentage of control (means ± SD, n = 3), *p < 0.05, **p < 0.01 vs. control.

Mentions: We further investigated the underlying mechanism of the formononetin-induced actin cytoskeleton rearrangement in HUVECs. Previous studies have demonstrated that the activation of ROCK signaling pathway is closely related to the regulation of stress fiber formation and cell migration in endothelial cells1314. Figure 5B,b showed that HUVECs transfected with non-specific siRNA when treated with formononetin (50 μM), actin stress fiber formation was increased significantly and the fibers were arranged longitudinally through the major axis of the endothelial cells. When HUVECs were transfected with ROCK-II siRNA, there was a reduction in actin stress fiber as well as a rapid modification of the stress fiber spatial organization with a progressive localization of actin towards the edge of the cell membrane to form cortical actin complexes (Fig. 5C,c). More interestingly, the inhibition of ROCK-II expression by siRNA abolished the actions of formononetin to increase stress fiber formation (Fig. 5D,d) as well as cell migration (Fig. 5E,F). Moreover, the cell migration of wounded HUVECs induced by formononetin treatment following scrape injury was completely abolished by the transfections of either ERα siRNA or ROCK-II siRNA (Supplementary Fig. 2). The effects of formononetin on the regulations of the downstream effectors of ROCK activation were further evaluated by using western blotting. The downstream substrates of ROCK proteins including MMP2/9 (matrix metalloproteinase 2/9), MYPT1 (myosin phosphatase target subunit 1), LIMK1 (Lim kinase 1), MLC2 (myosin light chain 2) and cofilin have been shown to regulate cell migration. Our data showed that formononetin increased the expressions of MMP2 and MMP9 in HUVECs significantly in a time-dependent manner (Fig. 6A,B). Formononetin also concurrently increased the phosphorylation of MYPT1, MLC2, LIMK1 and cofilin as shown in Fig. 6A,C. These effects induced by formononetin were abolished by the transfection of ROCK-II siRNA in HUVECs. Figure 7 showed that the transfection of ROCK-II siRNA resulted in marked reduction in the expression of ROCK-II protein along with similar decrease in the phosphorylation of MYPT1, MLC2, LIMK1, cofilin and the expression of MPP2 and MPP9 in formononetin treated HUVECs (Fig. 7).


Formononetin promotes angiogenesis through the estrogen receptor alpha-enhanced ROCK pathway.

Li S, Dang Y, Zhou X, Huang B, Huang X, Zhang Z, Kwan YW, Chan SW, Leung GP, Lee SM, Hoi MP - Sci Rep (2015)

Inhibition of ROCK-II expression by ROCK-II siRNA abolished the activation of ROCK and MMP2/9 signaling pathways in HUVECs.(A) HUVECs were transfected with either non-specific siRNA as control or specific ROCK-II siRNA for 2 days followed by 50 μM formononetin treatment for 30 or 60 min. The protein levels of ROCK-II, MMP2, MMP9, P-MYPT1, P-LIMK1, P-cofilin and P-MLC2 were analyzed by using Western blotting. (B) Statistical analysis of the protein levels of MMP2 and MMP9 in HUVECs transfected with ROCK-II siRNA in the presence of 50 μM formononetin. (C) Statistical analysis of the protein levels of P-MYPT1, P-LIMK1, P-cofilin and P-MLC2 in HUVECs transfected with ROCK-II siRNA in the presence of 50 μM formononetin. Results are expressed as a percentage of control (means ± SD, n = 3), *p < 0.05, **p < 0.01 vs. control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f7: Inhibition of ROCK-II expression by ROCK-II siRNA abolished the activation of ROCK and MMP2/9 signaling pathways in HUVECs.(A) HUVECs were transfected with either non-specific siRNA as control or specific ROCK-II siRNA for 2 days followed by 50 μM formononetin treatment for 30 or 60 min. The protein levels of ROCK-II, MMP2, MMP9, P-MYPT1, P-LIMK1, P-cofilin and P-MLC2 were analyzed by using Western blotting. (B) Statistical analysis of the protein levels of MMP2 and MMP9 in HUVECs transfected with ROCK-II siRNA in the presence of 50 μM formononetin. (C) Statistical analysis of the protein levels of P-MYPT1, P-LIMK1, P-cofilin and P-MLC2 in HUVECs transfected with ROCK-II siRNA in the presence of 50 μM formononetin. Results are expressed as a percentage of control (means ± SD, n = 3), *p < 0.05, **p < 0.01 vs. control.
Mentions: We further investigated the underlying mechanism of the formononetin-induced actin cytoskeleton rearrangement in HUVECs. Previous studies have demonstrated that the activation of ROCK signaling pathway is closely related to the regulation of stress fiber formation and cell migration in endothelial cells1314. Figure 5B,b showed that HUVECs transfected with non-specific siRNA when treated with formononetin (50 μM), actin stress fiber formation was increased significantly and the fibers were arranged longitudinally through the major axis of the endothelial cells. When HUVECs were transfected with ROCK-II siRNA, there was a reduction in actin stress fiber as well as a rapid modification of the stress fiber spatial organization with a progressive localization of actin towards the edge of the cell membrane to form cortical actin complexes (Fig. 5C,c). More interestingly, the inhibition of ROCK-II expression by siRNA abolished the actions of formononetin to increase stress fiber formation (Fig. 5D,d) as well as cell migration (Fig. 5E,F). Moreover, the cell migration of wounded HUVECs induced by formononetin treatment following scrape injury was completely abolished by the transfections of either ERα siRNA or ROCK-II siRNA (Supplementary Fig. 2). The effects of formononetin on the regulations of the downstream effectors of ROCK activation were further evaluated by using western blotting. The downstream substrates of ROCK proteins including MMP2/9 (matrix metalloproteinase 2/9), MYPT1 (myosin phosphatase target subunit 1), LIMK1 (Lim kinase 1), MLC2 (myosin light chain 2) and cofilin have been shown to regulate cell migration. Our data showed that formononetin increased the expressions of MMP2 and MMP9 in HUVECs significantly in a time-dependent manner (Fig. 6A,B). Formononetin also concurrently increased the phosphorylation of MYPT1, MLC2, LIMK1 and cofilin as shown in Fig. 6A,C. These effects induced by formononetin were abolished by the transfection of ROCK-II siRNA in HUVECs. Figure 7 showed that the transfection of ROCK-II siRNA resulted in marked reduction in the expression of ROCK-II protein along with similar decrease in the phosphorylation of MYPT1, MLC2, LIMK1, cofilin and the expression of MPP2 and MPP9 in formononetin treated HUVECs (Fig. 7).

Bottom Line: In addition, results from co-immunoprecipitation suggested formononetin induced cell migration via recruiting of ERα/ROCK-II activated complex formation.More interestingly, in zebrafish embryo we observed that formononetin significantly promoted angiogenic sproutings in the subintestinal vessels (SIVs) that could be completely abolished by ROCK inhibitor.In this study, we elucidated the underlying mechanisms that formononetin produced proangiogenesis effects through an ERα-enhanced ROCK-II signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Macao, China.

ABSTRACT
Formononetin is an isoflavone that has been shown to display estrogenic properties and induce angiogenesis activities. However, the interrelationship between the estrogenic properties and angiogenesis activities of formononetin are not well defined. In the present study, docking and enzymatic assay demonstrated that formononetin displayed direct binding to the ligand-binding domain (LBD) of estrogen receptor alpha (ERα) with an agonistic property. Results from Human Umbilical Vein Endothelial Cells (HUVEC) by using real-time migration xCELLigence system, immunofluorescence and western blotting provided strong evidences of formononetin induced endothelial cell migration and dramatic actin cytoskeleton spatial modification through ERα-enhanced-ROCK-II/MMP2/9 signaling pathways. In addition, results from co-immunoprecipitation suggested formononetin induced cell migration via recruiting of ERα/ROCK-II activated complex formation. More interestingly, in zebrafish embryo we observed that formononetin significantly promoted angiogenic sproutings in the subintestinal vessels (SIVs) that could be completely abolished by ROCK inhibitor. In this study, we elucidated the underlying mechanisms that formononetin produced proangiogenesis effects through an ERα-enhanced ROCK-II signaling pathways. Results from the present study also expand our knowledge about the enigmatic underlying mechanisms of phytoestrogenic compounds in the promotion of angiogenesis in relation to ERα and ROCK interaction in endothelial cells and their relationship with actin assembly and cell migration.

No MeSH data available.


Related in: MedlinePlus