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Akt mediates 17beta-estradiol and/or estrogen receptor-alpha inhibition of LPS-induced tumor necresis factor-alpha expression and myocardial cell apoptosis by suppressing the JNK1/2-NFkappaB pathway.

Liu CJ, Lo JF, Kuo CH, Chu CH, Chen LM, Tsai FJ, Tsai CH, Tzang BS, Kuo WW, Huang CY - J. Cell. Mol. Med. (2009)

Bottom Line: We found that LPS challenge activated JNK1/2, and then induced IkappaB degradation, NFkappaB activation, TNF-alpha up-regulation and subsequent myocardial apoptotic responses.In addition, treatments involving E(2), membrane-impermeable BSA-E(2) and/or Dox, which induces ERalpha overexpression, significantly inhibited LPS-induced apoptosis by suppressing LPS-up-regulated JNK1/2 activity, IkappaB degradation, NFkappaB activation and pro-apoptotic proteins (e.g. TNF-alpha, active caspases-8, t-Bid, Bax, released cytochrome c, active caspase-9, active caspase-3) in myocardial cells.These findings suggest that E(2), BSA-E(2) and ERalpha expression exert their cardioprotective effects by inhibiting JNK1/2-mediated LPS-induced TNF-alpha expression and cardiomyocyte apoptosis through activation of Akt.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Chinese Medical Science, China Medical University, Taichung, Taiwan.

ABSTRACT
Evidence shows that women have lower tumour necrosis factor-alpha (TNF-alpha) levels and lower incidences of heart dysfunction and sepsis-related morbidity and mortality. To identify the cardioprotective effects and precise cellular/molecular mechanisms behind estrogen and estrogen receptors (ERs), we investigated the effects of 17beta-estradiol (E(2)) and estrogen receptor alpha (ERalpha) on LPS-induced apoptosis by analyzing the activation of survival and death signalling pathways in doxycycline (Dox)-inducible Tet-On/ERalpha H9c2 myocardial cells and ERalpha-transfected primary cardiomyocytes overexpressing ERalpha. We found that LPS challenge activated JNK1/2, and then induced IkappaB degradation, NFkappaB activation, TNF-alpha up-regulation and subsequent myocardial apoptotic responses. In addition, treatments involving E(2), membrane-impermeable BSA-E(2) and/or Dox, which induces ERalpha overexpression, significantly inhibited LPS-induced apoptosis by suppressing LPS-up-regulated JNK1/2 activity, IkappaB degradation, NFkappaB activation and pro-apoptotic proteins (e.g. TNF-alpha, active caspases-8, t-Bid, Bax, released cytochrome c, active caspase-9, active caspase-3) in myocardial cells. However, the cardioprotective properties of E(2), BSA-E(2) and ERalpha overexpression to inhibit LPS-induced apoptosis and promote cell survival were attenuated by applying LY294002 (PI3K inhibitor) and PI3K siRNA. These findings suggest that E(2), BSA-E(2) and ERalpha expression exert their cardioprotective effects by inhibiting JNK1/2-mediated LPS-induced TNF-alpha expression and cardiomyocyte apoptosis through activation of Akt.

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The apoptotic effects of LPS on Tet-On/ERα H9c2 cells. (A) Tet-On/ERα H9c2 cells were incubated for 24 hrs with the indicated concentrations of LPS (0–1000 ng/ml). LPS-induced apoptosis of Tet-On/ERαH9c2 cells were determined by TUNEL assay. (B) Tet-On/ERα H9c2 cells were treated with vehicle or LPS (0–5 μg/ml) for 24 hrs. Cell extracts were analyzed by immunoblotting with antibodies against proteins as indicated. *P < 0.05 versus control: **P < 0.01 versus control (mean ± S.E., n= 3).
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fig01: The apoptotic effects of LPS on Tet-On/ERα H9c2 cells. (A) Tet-On/ERα H9c2 cells were incubated for 24 hrs with the indicated concentrations of LPS (0–1000 ng/ml). LPS-induced apoptosis of Tet-On/ERαH9c2 cells were determined by TUNEL assay. (B) Tet-On/ERα H9c2 cells were treated with vehicle or LPS (0–5 μg/ml) for 24 hrs. Cell extracts were analyzed by immunoblotting with antibodies against proteins as indicated. *P < 0.05 versus control: **P < 0.01 versus control (mean ± S.E., n= 3).

Mentions: To identify the cytotoxic effect induced by LPS in myocardial cells, the inducible Tet-On/ERα H9c2 myocardial cells established in this study were incubated with LPS, a specific TLR4 agonist, and subjected to TUNEL analysis. After incubation with various concentrations of LPS (0–1000 ng/ml) for 24 hrs, we observed a significant dose-dependent reduction in cell viability. Few positively stained cells were noted in control cultures; however, in cultures treated with LPS (50–1000 ng/ml) for 24 hrs, there was a dose-dependent increase in the number of cells undergoing apoptosis (Fig. 1A). Immunoblotting assay was used to determine the expression levels of pro-apoptotic proteins, including TNF-α, active caspase-8, truncated Bid (tBid), released cytochrome c, active caspase-9 and active caspase-3 in Tet-On/ERα H9c2 myocardial cells after LPS (0–5 μg/ml) treatment. The results demonstrated that LPS dramatically induced the expression and activation of these pro-apoptotic proteins (Fig. 1B), suggesting that LPS indeed executed the apoptotic effects on myocardial cells by binding to TLR4.


Akt mediates 17beta-estradiol and/or estrogen receptor-alpha inhibition of LPS-induced tumor necresis factor-alpha expression and myocardial cell apoptosis by suppressing the JNK1/2-NFkappaB pathway.

Liu CJ, Lo JF, Kuo CH, Chu CH, Chen LM, Tsai FJ, Tsai CH, Tzang BS, Kuo WW, Huang CY - J. Cell. Mol. Med. (2009)

The apoptotic effects of LPS on Tet-On/ERα H9c2 cells. (A) Tet-On/ERα H9c2 cells were incubated for 24 hrs with the indicated concentrations of LPS (0–1000 ng/ml). LPS-induced apoptosis of Tet-On/ERαH9c2 cells were determined by TUNEL assay. (B) Tet-On/ERα H9c2 cells were treated with vehicle or LPS (0–5 μg/ml) for 24 hrs. Cell extracts were analyzed by immunoblotting with antibodies against proteins as indicated. *P < 0.05 versus control: **P < 0.01 versus control (mean ± S.E., n= 3).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4516514&req=5

fig01: The apoptotic effects of LPS on Tet-On/ERα H9c2 cells. (A) Tet-On/ERα H9c2 cells were incubated for 24 hrs with the indicated concentrations of LPS (0–1000 ng/ml). LPS-induced apoptosis of Tet-On/ERαH9c2 cells were determined by TUNEL assay. (B) Tet-On/ERα H9c2 cells were treated with vehicle or LPS (0–5 μg/ml) for 24 hrs. Cell extracts were analyzed by immunoblotting with antibodies against proteins as indicated. *P < 0.05 versus control: **P < 0.01 versus control (mean ± S.E., n= 3).
Mentions: To identify the cytotoxic effect induced by LPS in myocardial cells, the inducible Tet-On/ERα H9c2 myocardial cells established in this study were incubated with LPS, a specific TLR4 agonist, and subjected to TUNEL analysis. After incubation with various concentrations of LPS (0–1000 ng/ml) for 24 hrs, we observed a significant dose-dependent reduction in cell viability. Few positively stained cells were noted in control cultures; however, in cultures treated with LPS (50–1000 ng/ml) for 24 hrs, there was a dose-dependent increase in the number of cells undergoing apoptosis (Fig. 1A). Immunoblotting assay was used to determine the expression levels of pro-apoptotic proteins, including TNF-α, active caspase-8, truncated Bid (tBid), released cytochrome c, active caspase-9 and active caspase-3 in Tet-On/ERα H9c2 myocardial cells after LPS (0–5 μg/ml) treatment. The results demonstrated that LPS dramatically induced the expression and activation of these pro-apoptotic proteins (Fig. 1B), suggesting that LPS indeed executed the apoptotic effects on myocardial cells by binding to TLR4.

Bottom Line: We found that LPS challenge activated JNK1/2, and then induced IkappaB degradation, NFkappaB activation, TNF-alpha up-regulation and subsequent myocardial apoptotic responses.In addition, treatments involving E(2), membrane-impermeable BSA-E(2) and/or Dox, which induces ERalpha overexpression, significantly inhibited LPS-induced apoptosis by suppressing LPS-up-regulated JNK1/2 activity, IkappaB degradation, NFkappaB activation and pro-apoptotic proteins (e.g. TNF-alpha, active caspases-8, t-Bid, Bax, released cytochrome c, active caspase-9, active caspase-3) in myocardial cells.These findings suggest that E(2), BSA-E(2) and ERalpha expression exert their cardioprotective effects by inhibiting JNK1/2-mediated LPS-induced TNF-alpha expression and cardiomyocyte apoptosis through activation of Akt.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Chinese Medical Science, China Medical University, Taichung, Taiwan.

ABSTRACT
Evidence shows that women have lower tumour necrosis factor-alpha (TNF-alpha) levels and lower incidences of heart dysfunction and sepsis-related morbidity and mortality. To identify the cardioprotective effects and precise cellular/molecular mechanisms behind estrogen and estrogen receptors (ERs), we investigated the effects of 17beta-estradiol (E(2)) and estrogen receptor alpha (ERalpha) on LPS-induced apoptosis by analyzing the activation of survival and death signalling pathways in doxycycline (Dox)-inducible Tet-On/ERalpha H9c2 myocardial cells and ERalpha-transfected primary cardiomyocytes overexpressing ERalpha. We found that LPS challenge activated JNK1/2, and then induced IkappaB degradation, NFkappaB activation, TNF-alpha up-regulation and subsequent myocardial apoptotic responses. In addition, treatments involving E(2), membrane-impermeable BSA-E(2) and/or Dox, which induces ERalpha overexpression, significantly inhibited LPS-induced apoptosis by suppressing LPS-up-regulated JNK1/2 activity, IkappaB degradation, NFkappaB activation and pro-apoptotic proteins (e.g. TNF-alpha, active caspases-8, t-Bid, Bax, released cytochrome c, active caspase-9, active caspase-3) in myocardial cells. However, the cardioprotective properties of E(2), BSA-E(2) and ERalpha overexpression to inhibit LPS-induced apoptosis and promote cell survival were attenuated by applying LY294002 (PI3K inhibitor) and PI3K siRNA. These findings suggest that E(2), BSA-E(2) and ERalpha expression exert their cardioprotective effects by inhibiting JNK1/2-mediated LPS-induced TNF-alpha expression and cardiomyocyte apoptosis through activation of Akt.

Show MeSH
Related in: MedlinePlus