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The orphan receptor TR3 participates in angiotensin II-induced cardiac hypertrophy by controlling mTOR signalling.

Wang RH, He JP, Su ML, Luo J, Xu M, Du XD, Chen HZ, Wang WJ, Wang Y, Zhang N, Zhao BX, Zhao WX, Shan ZG, Han J, Chang C, Wu Q - EMBO Mol Med (2012)

Bottom Line: TR3 was shown to form a trimer with the TSC1/TSC2 complex that specifically promoted TSC2 degradation via a proteasome/ubiquitination pathway.As a result, mTORC1, but not mTORC2, was activated; this was accompanied by increased protein synthesis, enhanced production of reactive oxygen species and enlarged cell size, thereby resulting in cardiac hypertrophy.The elimination or reduction of TR3 may reduce cardiac hypertrophy; therefore, TR3 is a potential target for clinical therapy.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian Province, China.

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TR3 promoted mTORC1 activityCorrelation between TR3 expression and S6K1 phosphorylation after AngII treatment of H9C2 cells and NRCMs. Cells were treated with 500 nM AngII for the indicated durations. TR3 expression and S6K1 activity were measured by Western blotting using TR3 and phosphor-Thr389-S6K1 antibodies.Transfection of TR3 into H9C2 cells increases the phosphorylation of S6K1. The transfected cells were lysed, and the lysates were analysed using Western blotting. An empty vector was used as a control.Knockdown of TR3 decreases mTORC1 activity even in the presence of AngII. Endogenous TR3 in the NRCMs was knocked down using lentivirus-based RNA interference. NRCMs that were transfected with scrambled siRNA were used as a control. The cells were treated with 500 nM AngII for the indicated durations, and S6K1 phosphorylation was determined.TR3-deficient (TR3−/−) MEFs exhibited a lower response to AngII than wild-type (TR3+/+) MEFs. TR3+/+ and TR3−/− MEFs were obtained from WT and TR3-KO mice. The treatment was similar to that described above.TR3-KO mice and TR3-KD rats are not sensitive to AngII stimulation. WT and TR3-KO mice (left) or WT and TR3-KD rats, in which TR3 was knocked down in the left ventricles (right), were intraperitoneally injected with 500 µg/kg AngII at the indicated times. Total protein was prepared from the left ventricles of the mice or rats (n = 5 per group) and then subjected to Western blotting. Scrambled siRNA was used as a control.
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fig03: TR3 promoted mTORC1 activityCorrelation between TR3 expression and S6K1 phosphorylation after AngII treatment of H9C2 cells and NRCMs. Cells were treated with 500 nM AngII for the indicated durations. TR3 expression and S6K1 activity were measured by Western blotting using TR3 and phosphor-Thr389-S6K1 antibodies.Transfection of TR3 into H9C2 cells increases the phosphorylation of S6K1. The transfected cells were lysed, and the lysates were analysed using Western blotting. An empty vector was used as a control.Knockdown of TR3 decreases mTORC1 activity even in the presence of AngII. Endogenous TR3 in the NRCMs was knocked down using lentivirus-based RNA interference. NRCMs that were transfected with scrambled siRNA were used as a control. The cells were treated with 500 nM AngII for the indicated durations, and S6K1 phosphorylation was determined.TR3-deficient (TR3−/−) MEFs exhibited a lower response to AngII than wild-type (TR3+/+) MEFs. TR3+/+ and TR3−/− MEFs were obtained from WT and TR3-KO mice. The treatment was similar to that described above.TR3-KO mice and TR3-KD rats are not sensitive to AngII stimulation. WT and TR3-KO mice (left) or WT and TR3-KD rats, in which TR3 was knocked down in the left ventricles (right), were intraperitoneally injected with 500 µg/kg AngII at the indicated times. Total protein was prepared from the left ventricles of the mice or rats (n = 5 per group) and then subjected to Western blotting. Scrambled siRNA was used as a control.

Mentions: To further characterize the role of TR3, we determined whether AngII influences the transcriptional activity of TR3 in the cardiac myoblast cell line H9C2. Transfection of a reporter gene for NurRE (the response element of TR3) into cells revealed that TR3 transcriptional activity was not affected by AngII treatment (Supporting Information Fig S3A). To verify this finding, EMSA was performed; the result excluded the possibility that AngII affected endogenous TR3 targeting to DNA (Supporting Information Fig S3B). In addition, AngII elevated the expression of the TR3 protein but not the mRNA levels in H9C2 cells and neonatal rat cardiomyocytes (NRCMs) isolated from WT rats (Fig 3A and Supporting Information Fig S3C). This finding might have been the result of the extension of the half-life of TR3 by AngII because when cycloheximide (CHX) was used to block in vivo protein synthesis, the half-life (i.e., the time required for the degradation of 50% of the protein) of TR3 was prolonged by AngII (Supporting Information Fig S3D). These findings do not support the possibility that TR3 functions as a transcription factor to regulate cardiac hypertrophy; rather, the results suggest that TR3 is likely to be involved in regulation via protein–protein interactions.


The orphan receptor TR3 participates in angiotensin II-induced cardiac hypertrophy by controlling mTOR signalling.

Wang RH, He JP, Su ML, Luo J, Xu M, Du XD, Chen HZ, Wang WJ, Wang Y, Zhang N, Zhao BX, Zhao WX, Shan ZG, Han J, Chang C, Wu Q - EMBO Mol Med (2012)

TR3 promoted mTORC1 activityCorrelation between TR3 expression and S6K1 phosphorylation after AngII treatment of H9C2 cells and NRCMs. Cells were treated with 500 nM AngII for the indicated durations. TR3 expression and S6K1 activity were measured by Western blotting using TR3 and phosphor-Thr389-S6K1 antibodies.Transfection of TR3 into H9C2 cells increases the phosphorylation of S6K1. The transfected cells were lysed, and the lysates were analysed using Western blotting. An empty vector was used as a control.Knockdown of TR3 decreases mTORC1 activity even in the presence of AngII. Endogenous TR3 in the NRCMs was knocked down using lentivirus-based RNA interference. NRCMs that were transfected with scrambled siRNA were used as a control. The cells were treated with 500 nM AngII for the indicated durations, and S6K1 phosphorylation was determined.TR3-deficient (TR3−/−) MEFs exhibited a lower response to AngII than wild-type (TR3+/+) MEFs. TR3+/+ and TR3−/− MEFs were obtained from WT and TR3-KO mice. The treatment was similar to that described above.TR3-KO mice and TR3-KD rats are not sensitive to AngII stimulation. WT and TR3-KO mice (left) or WT and TR3-KD rats, in which TR3 was knocked down in the left ventricles (right), were intraperitoneally injected with 500 µg/kg AngII at the indicated times. Total protein was prepared from the left ventricles of the mice or rats (n = 5 per group) and then subjected to Western blotting. Scrambled siRNA was used as a control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig03: TR3 promoted mTORC1 activityCorrelation between TR3 expression and S6K1 phosphorylation after AngII treatment of H9C2 cells and NRCMs. Cells were treated with 500 nM AngII for the indicated durations. TR3 expression and S6K1 activity were measured by Western blotting using TR3 and phosphor-Thr389-S6K1 antibodies.Transfection of TR3 into H9C2 cells increases the phosphorylation of S6K1. The transfected cells were lysed, and the lysates were analysed using Western blotting. An empty vector was used as a control.Knockdown of TR3 decreases mTORC1 activity even in the presence of AngII. Endogenous TR3 in the NRCMs was knocked down using lentivirus-based RNA interference. NRCMs that were transfected with scrambled siRNA were used as a control. The cells were treated with 500 nM AngII for the indicated durations, and S6K1 phosphorylation was determined.TR3-deficient (TR3−/−) MEFs exhibited a lower response to AngII than wild-type (TR3+/+) MEFs. TR3+/+ and TR3−/− MEFs were obtained from WT and TR3-KO mice. The treatment was similar to that described above.TR3-KO mice and TR3-KD rats are not sensitive to AngII stimulation. WT and TR3-KO mice (left) or WT and TR3-KD rats, in which TR3 was knocked down in the left ventricles (right), were intraperitoneally injected with 500 µg/kg AngII at the indicated times. Total protein was prepared from the left ventricles of the mice or rats (n = 5 per group) and then subjected to Western blotting. Scrambled siRNA was used as a control.
Mentions: To further characterize the role of TR3, we determined whether AngII influences the transcriptional activity of TR3 in the cardiac myoblast cell line H9C2. Transfection of a reporter gene for NurRE (the response element of TR3) into cells revealed that TR3 transcriptional activity was not affected by AngII treatment (Supporting Information Fig S3A). To verify this finding, EMSA was performed; the result excluded the possibility that AngII affected endogenous TR3 targeting to DNA (Supporting Information Fig S3B). In addition, AngII elevated the expression of the TR3 protein but not the mRNA levels in H9C2 cells and neonatal rat cardiomyocytes (NRCMs) isolated from WT rats (Fig 3A and Supporting Information Fig S3C). This finding might have been the result of the extension of the half-life of TR3 by AngII because when cycloheximide (CHX) was used to block in vivo protein synthesis, the half-life (i.e., the time required for the degradation of 50% of the protein) of TR3 was prolonged by AngII (Supporting Information Fig S3D). These findings do not support the possibility that TR3 functions as a transcription factor to regulate cardiac hypertrophy; rather, the results suggest that TR3 is likely to be involved in regulation via protein–protein interactions.

Bottom Line: TR3 was shown to form a trimer with the TSC1/TSC2 complex that specifically promoted TSC2 degradation via a proteasome/ubiquitination pathway.As a result, mTORC1, but not mTORC2, was activated; this was accompanied by increased protein synthesis, enhanced production of reactive oxygen species and enlarged cell size, thereby resulting in cardiac hypertrophy.The elimination or reduction of TR3 may reduce cardiac hypertrophy; therefore, TR3 is a potential target for clinical therapy.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian Province, China.

Show MeSH
Related in: MedlinePlus