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Up-regulation of BMP-2 antagonizes TGF-β1/ROCK-enhanced cardiac fibrotic signalling through activation of Smurf1/Smad6 complex.

Wang S, Sun A, Li L, Zhao G, Jia J, Wang K, Ge J, Zou Y - J. Cell. Mol. Med. (2012)

Bottom Line: In addition, BMP-2 silencing abolished the effect of Y-27632, but not SB-431542 on suppression of TGF-β1 pathway.Further experiments showed that Smad6 Smurf1 interaction were required for BMP-2-evoked antagonizing effects.In conclusion, we propose that BMP-2, as a novel fibrosis antagonizing cytokine, may have potential beneficial effect in attenuating pressure overload-induced cardiac fibrosis.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital and Institutes of Biomedical Sciences, Fudan University, Shanghai, China.

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ROCK inhibition abrogated PKC-δ and Smad3 activation evoked by TGF-β1. (A) The effect of TGF-β1 (10 ng/ml) on the phosphorylation levels of PKC-δ and Smad3 in the presence or absence of Y-27632 or SB-431542, respectively for 24 hr, was determined by Western blotting, all the blots were normalized by total PKC-δ or Smad3 levels. (B) The effect of TGF-β1 (10 ng/ml) on the transcriptional levels of BMP-2 in the presence or absence of Y-27632 or SB-431542, respectively for 24 hr, was evaluated by quantitative PCR. (C) Western blotting analyses of TGF-β1 (10 ng/ml)-induced activation of PKC-δ and Smad3 with or without treatment of Y-27632 or SB-431542 for 24 hr in BMP-2 knock-down cardiomyocytes. Data were representative as means ± S.E.M. of n = 4 independent experiments. *P < 0.05, versus Control group; #P < 0.05, versus TGF-β1 (10 ng/ml) group.
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fig03: ROCK inhibition abrogated PKC-δ and Smad3 activation evoked by TGF-β1. (A) The effect of TGF-β1 (10 ng/ml) on the phosphorylation levels of PKC-δ and Smad3 in the presence or absence of Y-27632 or SB-431542, respectively for 24 hr, was determined by Western blotting, all the blots were normalized by total PKC-δ or Smad3 levels. (B) The effect of TGF-β1 (10 ng/ml) on the transcriptional levels of BMP-2 in the presence or absence of Y-27632 or SB-431542, respectively for 24 hr, was evaluated by quantitative PCR. (C) Western blotting analyses of TGF-β1 (10 ng/ml)-induced activation of PKC-δ and Smad3 with or without treatment of Y-27632 or SB-431542 for 24 hr in BMP-2 knock-down cardiomyocytes. Data were representative as means ± S.E.M. of n = 4 independent experiments. *P < 0.05, versus Control group; #P < 0.05, versus TGF-β1 (10 ng/ml) group.

Mentions: Considering that BMP-2 might be inhibited by ROCK elevation (Fig. 2D), we further analysed the role of ROCK kinase in TGF-β1-mediated PKC-δ and Smad3 signal transduction. As shown in Figure 3A, TGF-β1(10 ng/ml) induced a robust increase in phosphorylation levels of PKC-δ and Smad3, which were markedly reduced after inhibition of ROCK with treatment of Y-27632(10 μM) or SB431542(10 μM), respectively. Simultaneously, a significant increase in BMP-2 expression was observed in cardiomyocytes stimulated by Y-27632, but not by SB431542 (Fig. 3B). Next, we inhibited endogenous BMP-2 by transfecting lentivirus-BMP-2 siRNA, and determined the effect of Y-27632 or SB431542 on TGF-β1-mediated fibrotic signalling. Activated PKC-δ and Smad3 were not affected by Y-27632, but suppressed by SB431542 in BMP-2 knocked-down cardiomyocytes (Fig. 3C), which indicated that TGF-β1 mediated PKC-δ and Smad3 signal transduction via TGF-β type I receptor; these signalling cascades could be regulated by BMP-2 dependent ROCK inhibition. Furthermore, we explored the effects of PKC-δ and Smad3 on intercellular ROCK levels. Results indicated that TGF-β1-mediated ROCK elevation was not affected by inhibiting PKC-δ with GF109203X, or knocking-down with Smad3 siRNA (Fig. S2).


Up-regulation of BMP-2 antagonizes TGF-β1/ROCK-enhanced cardiac fibrotic signalling through activation of Smurf1/Smad6 complex.

Wang S, Sun A, Li L, Zhao G, Jia J, Wang K, Ge J, Zou Y - J. Cell. Mol. Med. (2012)

ROCK inhibition abrogated PKC-δ and Smad3 activation evoked by TGF-β1. (A) The effect of TGF-β1 (10 ng/ml) on the phosphorylation levels of PKC-δ and Smad3 in the presence or absence of Y-27632 or SB-431542, respectively for 24 hr, was determined by Western blotting, all the blots were normalized by total PKC-δ or Smad3 levels. (B) The effect of TGF-β1 (10 ng/ml) on the transcriptional levels of BMP-2 in the presence or absence of Y-27632 or SB-431542, respectively for 24 hr, was evaluated by quantitative PCR. (C) Western blotting analyses of TGF-β1 (10 ng/ml)-induced activation of PKC-δ and Smad3 with or without treatment of Y-27632 or SB-431542 for 24 hr in BMP-2 knock-down cardiomyocytes. Data were representative as means ± S.E.M. of n = 4 independent experiments. *P < 0.05, versus Control group; #P < 0.05, versus TGF-β1 (10 ng/ml) group.
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fig03: ROCK inhibition abrogated PKC-δ and Smad3 activation evoked by TGF-β1. (A) The effect of TGF-β1 (10 ng/ml) on the phosphorylation levels of PKC-δ and Smad3 in the presence or absence of Y-27632 or SB-431542, respectively for 24 hr, was determined by Western blotting, all the blots were normalized by total PKC-δ or Smad3 levels. (B) The effect of TGF-β1 (10 ng/ml) on the transcriptional levels of BMP-2 in the presence or absence of Y-27632 or SB-431542, respectively for 24 hr, was evaluated by quantitative PCR. (C) Western blotting analyses of TGF-β1 (10 ng/ml)-induced activation of PKC-δ and Smad3 with or without treatment of Y-27632 or SB-431542 for 24 hr in BMP-2 knock-down cardiomyocytes. Data were representative as means ± S.E.M. of n = 4 independent experiments. *P < 0.05, versus Control group; #P < 0.05, versus TGF-β1 (10 ng/ml) group.
Mentions: Considering that BMP-2 might be inhibited by ROCK elevation (Fig. 2D), we further analysed the role of ROCK kinase in TGF-β1-mediated PKC-δ and Smad3 signal transduction. As shown in Figure 3A, TGF-β1(10 ng/ml) induced a robust increase in phosphorylation levels of PKC-δ and Smad3, which were markedly reduced after inhibition of ROCK with treatment of Y-27632(10 μM) or SB431542(10 μM), respectively. Simultaneously, a significant increase in BMP-2 expression was observed in cardiomyocytes stimulated by Y-27632, but not by SB431542 (Fig. 3B). Next, we inhibited endogenous BMP-2 by transfecting lentivirus-BMP-2 siRNA, and determined the effect of Y-27632 or SB431542 on TGF-β1-mediated fibrotic signalling. Activated PKC-δ and Smad3 were not affected by Y-27632, but suppressed by SB431542 in BMP-2 knocked-down cardiomyocytes (Fig. 3C), which indicated that TGF-β1 mediated PKC-δ and Smad3 signal transduction via TGF-β type I receptor; these signalling cascades could be regulated by BMP-2 dependent ROCK inhibition. Furthermore, we explored the effects of PKC-δ and Smad3 on intercellular ROCK levels. Results indicated that TGF-β1-mediated ROCK elevation was not affected by inhibiting PKC-δ with GF109203X, or knocking-down with Smad3 siRNA (Fig. S2).

Bottom Line: In addition, BMP-2 silencing abolished the effect of Y-27632, but not SB-431542 on suppression of TGF-β1 pathway.Further experiments showed that Smad6 Smurf1 interaction were required for BMP-2-evoked antagonizing effects.In conclusion, we propose that BMP-2, as a novel fibrosis antagonizing cytokine, may have potential beneficial effect in attenuating pressure overload-induced cardiac fibrosis.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital and Institutes of Biomedical Sciences, Fudan University, Shanghai, China.

Show MeSH
Related in: MedlinePlus