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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

Formononetin induced the activations of ROCK and MMP2/9 pathways via ERα.(A) HUVECs were transfected with either non-specific siRNA as control of specific ERα siRNA for 2 days followed by 50 μM formononetin treatment for 30 or 60 min. The protein levels of ERα, 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 ERα 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 ERα siRNA in the presence of 50 μM formononetin. Results are expressed as percentages of controls (mean ± SD; n = 3), *p < 0.05, **p < 0.01 vs. control.
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f8: Formononetin induced the activations of ROCK and MMP2/9 pathways via ERα.(A) HUVECs were transfected with either non-specific siRNA as control of specific ERα siRNA for 2 days followed by 50 μM formononetin treatment for 30 or 60 min. The protein levels of ERα, 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 ERα 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 ERα siRNA in the presence of 50 μM formononetin. Results are expressed as percentages of controls (mean ± SD; n = 3), *p < 0.05, **p < 0.01 vs. control.

Mentions: The role of ERα in the activation of ROCK and MMP2/9 pathways by formononetin was further evaluated by using ERα siRNA. Figure 8 showed that HUVECs transfected with ERα siRNA resulted in a marked reduction in ERα expression along with marked reductions in the protein levels of MMP2 and MMP9, and the phosphorylation of MYPT1, LIMK1, cofilin and MLC2 upon exposure to formononetin. To further characterize the signaling partnership between ERα and ROCK, co-immunoprecipitation assays were performed. It was observed that in the presence of formononetin, there was significant increased in the interaction between ERα and ROCK-II (Fig. 9A–D). In order to dissect the functional relevance of the ERα/ROCK-II interaction, we further investigated if the ERα-associated ROCK-II is functionally activated by using the ER antagonist ICI 182,780. It was observed that the treatment of ICI 182,780 disrupted the interaction between ERα/ROCK-II induced by formononetin (Fig. 9E–H). These results suggested that formononetin induced direct association of ERα with ROCK-II that further led to the formation of an activated multi-protein complex.


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)

Formononetin induced the activations of ROCK and MMP2/9 pathways via ERα.(A) HUVECs were transfected with either non-specific siRNA as control of specific ERα siRNA for 2 days followed by 50 μM formononetin treatment for 30 or 60 min. The protein levels of ERα, 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 ERα 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 ERα siRNA in the presence of 50 μM formononetin. Results are expressed as percentages of controls (mean ± SD; n = 3), *p < 0.05, **p < 0.01 vs. control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4645220&req=5

f8: Formononetin induced the activations of ROCK and MMP2/9 pathways via ERα.(A) HUVECs were transfected with either non-specific siRNA as control of specific ERα siRNA for 2 days followed by 50 μM formononetin treatment for 30 or 60 min. The protein levels of ERα, 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 ERα 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 ERα siRNA in the presence of 50 μM formononetin. Results are expressed as percentages of controls (mean ± SD; n = 3), *p < 0.05, **p < 0.01 vs. control.
Mentions: The role of ERα in the activation of ROCK and MMP2/9 pathways by formononetin was further evaluated by using ERα siRNA. Figure 8 showed that HUVECs transfected with ERα siRNA resulted in a marked reduction in ERα expression along with marked reductions in the protein levels of MMP2 and MMP9, and the phosphorylation of MYPT1, LIMK1, cofilin and MLC2 upon exposure to formononetin. To further characterize the signaling partnership between ERα and ROCK, co-immunoprecipitation assays were performed. It was observed that in the presence of formononetin, there was significant increased in the interaction between ERα and ROCK-II (Fig. 9A–D). In order to dissect the functional relevance of the ERα/ROCK-II interaction, we further investigated if the ERα-associated ROCK-II is functionally activated by using the ER antagonist ICI 182,780. It was observed that the treatment of ICI 182,780 disrupted the interaction between ERα/ROCK-II induced by formononetin (Fig. 9E–H). These results suggested that formononetin induced direct association of ERα with ROCK-II that further led to the formation of an activated multi-protein complex.

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