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Rac1 activation induces tumour necrosis factor-α expression and cardiac dysfunction in endotoxemia.

Zhang T, Lu X, Beier F, Feng Q - J. Cell. Mol. Med. (2010)

Bottom Line: Furthermore, inhibition of PI3K and Rac1 activity decreased LPS-induced superoxide generation which was associated with a significant reduction in ERK1/2 phosphorylation.Deficiency in Rac1 significantly decreased myocardial TNF-α expression and improved cardiac function during endotoxemia.We conclude that PI3K-mediated Rac1 activation is required for induction of TNF-α expression in cardiomyocytes and cardiac dysfunction during endotoxemia.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada.

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Effects of PI3K and Rac1 on LPS-induced ERK1/2 phosphorylation in neonatal cardiomyocytes. (A) WT cardiomyocytes were treated with vehicle or LPS (1 μg/ml) for 15 min., 30 min., 1 and 2 hrs. ERK1/2 phosphorylation in these cells was measured by Western blot analysis. (B) WT cardiomyocytes were treated with LPS (1 μg/ml) for 30 min. with or without LY294002. ERK1/2 phosphorylation in these cells was measured. (C) Neonatal cardiomyocytes were infected with Ad-GFP or Ad-Rac1N17 for 24 hrs. Cardiomyocytes were treated with LPS (1 μg/ml) for 30 min. ERK1/2 phosphorylation was measured as described above. (D) and (E) Cardiomyocytes were treated with LPS with or without the ERK1/2 inhibitor U0126 (10 μM) for 3 and 5 hrs. TNF-α mRNA (D) and protein in culture medium (E) were measured by real-time RT-PCR and ELISA, respectively. Data are means ± S.E.M. from three to five independent experiments. *P < 0.05, **P < 0.01 versus control; †P < 0.05, ††P < 0.01 versus Ad-GFP+LPS and LPS.
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fig05: Effects of PI3K and Rac1 on LPS-induced ERK1/2 phosphorylation in neonatal cardiomyocytes. (A) WT cardiomyocytes were treated with vehicle or LPS (1 μg/ml) for 15 min., 30 min., 1 and 2 hrs. ERK1/2 phosphorylation in these cells was measured by Western blot analysis. (B) WT cardiomyocytes were treated with LPS (1 μg/ml) for 30 min. with or without LY294002. ERK1/2 phosphorylation in these cells was measured. (C) Neonatal cardiomyocytes were infected with Ad-GFP or Ad-Rac1N17 for 24 hrs. Cardiomyocytes were treated with LPS (1 μg/ml) for 30 min. ERK1/2 phosphorylation was measured as described above. (D) and (E) Cardiomyocytes were treated with LPS with or without the ERK1/2 inhibitor U0126 (10 μM) for 3 and 5 hrs. TNF-α mRNA (D) and protein in culture medium (E) were measured by real-time RT-PCR and ELISA, respectively. Data are means ± S.E.M. from three to five independent experiments. *P < 0.05, **P < 0.01 versus control; †P < 0.05, ††P < 0.01 versus Ad-GFP+LPS and LPS.

Mentions: We have previously shown that activation of ERK1/2 and p38 MAPK was essential for NADPH oxidase signalling and LPS-induced TNF-α expression in cardiomyocytes [23]. The effects of LPS on ERK1/2 activation were also determined in the present study. As shown in Figure 5A, LPS rapidly increased phosphorylation of ERK1/2 which peaked at 30 min. and returned to control levels after 2 hrs. LPS-induced ERK1/2 phosphorylation was completely blocked by a PI3K inhibitor, LY294002 (Fig. 5B), suggesting that LPS regulated ERK1/2 activity via PI3K. To examine whether Rac1 activity leads to ERK1/2 phosphorylation, ERK1/2 activation was measured in Ad-Rac1N17 infected cardiomyocytes. Overexpression of Rac1N17 significantly decreased LPS-induced phosphorylation of ERK1/2 compared with Ad-GFP infected group (Fig. 5C). Furthermore, U0126, a selective ERK1/2 inhibitor, decreased LPS-stimulated TNF-α mRNA and protein levels by 46% and 69%, respectively (Fig. 5D and E). Conversely, inhibition of Rac1 activation by Ad-Rac1N17 had no effect on p38 phosphorylation induced by LPS (Fig. 6). Taken together, these results suggest that the effects of PI3K and Rac1 on cardiomyocytes are mediated by ERK1/2 but not by p38 MAPK signalling.


Rac1 activation induces tumour necrosis factor-α expression and cardiac dysfunction in endotoxemia.

Zhang T, Lu X, Beier F, Feng Q - J. Cell. Mol. Med. (2010)

Effects of PI3K and Rac1 on LPS-induced ERK1/2 phosphorylation in neonatal cardiomyocytes. (A) WT cardiomyocytes were treated with vehicle or LPS (1 μg/ml) for 15 min., 30 min., 1 and 2 hrs. ERK1/2 phosphorylation in these cells was measured by Western blot analysis. (B) WT cardiomyocytes were treated with LPS (1 μg/ml) for 30 min. with or without LY294002. ERK1/2 phosphorylation in these cells was measured. (C) Neonatal cardiomyocytes were infected with Ad-GFP or Ad-Rac1N17 for 24 hrs. Cardiomyocytes were treated with LPS (1 μg/ml) for 30 min. ERK1/2 phosphorylation was measured as described above. (D) and (E) Cardiomyocytes were treated with LPS with or without the ERK1/2 inhibitor U0126 (10 μM) for 3 and 5 hrs. TNF-α mRNA (D) and protein in culture medium (E) were measured by real-time RT-PCR and ELISA, respectively. Data are means ± S.E.M. from three to five independent experiments. *P < 0.05, **P < 0.01 versus control; †P < 0.05, ††P < 0.01 versus Ad-GFP+LPS and LPS.
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fig05: Effects of PI3K and Rac1 on LPS-induced ERK1/2 phosphorylation in neonatal cardiomyocytes. (A) WT cardiomyocytes were treated with vehicle or LPS (1 μg/ml) for 15 min., 30 min., 1 and 2 hrs. ERK1/2 phosphorylation in these cells was measured by Western blot analysis. (B) WT cardiomyocytes were treated with LPS (1 μg/ml) for 30 min. with or without LY294002. ERK1/2 phosphorylation in these cells was measured. (C) Neonatal cardiomyocytes were infected with Ad-GFP or Ad-Rac1N17 for 24 hrs. Cardiomyocytes were treated with LPS (1 μg/ml) for 30 min. ERK1/2 phosphorylation was measured as described above. (D) and (E) Cardiomyocytes were treated with LPS with or without the ERK1/2 inhibitor U0126 (10 μM) for 3 and 5 hrs. TNF-α mRNA (D) and protein in culture medium (E) were measured by real-time RT-PCR and ELISA, respectively. Data are means ± S.E.M. from three to five independent experiments. *P < 0.05, **P < 0.01 versus control; †P < 0.05, ††P < 0.01 versus Ad-GFP+LPS and LPS.
Mentions: We have previously shown that activation of ERK1/2 and p38 MAPK was essential for NADPH oxidase signalling and LPS-induced TNF-α expression in cardiomyocytes [23]. The effects of LPS on ERK1/2 activation were also determined in the present study. As shown in Figure 5A, LPS rapidly increased phosphorylation of ERK1/2 which peaked at 30 min. and returned to control levels after 2 hrs. LPS-induced ERK1/2 phosphorylation was completely blocked by a PI3K inhibitor, LY294002 (Fig. 5B), suggesting that LPS regulated ERK1/2 activity via PI3K. To examine whether Rac1 activity leads to ERK1/2 phosphorylation, ERK1/2 activation was measured in Ad-Rac1N17 infected cardiomyocytes. Overexpression of Rac1N17 significantly decreased LPS-induced phosphorylation of ERK1/2 compared with Ad-GFP infected group (Fig. 5C). Furthermore, U0126, a selective ERK1/2 inhibitor, decreased LPS-stimulated TNF-α mRNA and protein levels by 46% and 69%, respectively (Fig. 5D and E). Conversely, inhibition of Rac1 activation by Ad-Rac1N17 had no effect on p38 phosphorylation induced by LPS (Fig. 6). Taken together, these results suggest that the effects of PI3K and Rac1 on cardiomyocytes are mediated by ERK1/2 but not by p38 MAPK signalling.

Bottom Line: Furthermore, inhibition of PI3K and Rac1 activity decreased LPS-induced superoxide generation which was associated with a significant reduction in ERK1/2 phosphorylation.Deficiency in Rac1 significantly decreased myocardial TNF-α expression and improved cardiac function during endotoxemia.We conclude that PI3K-mediated Rac1 activation is required for induction of TNF-α expression in cardiomyocytes and cardiac dysfunction during endotoxemia.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada.

Show MeSH
Related in: MedlinePlus