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Suppression of soluble adenylyl cyclase protects smooth muscle cells against oxidative stress-induced apoptosis

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Abstract

Apoptosis of vascular smooth muscle cells (VSMC) significantly contributes to the instability of advanced atherosclerotic plaques. Oxygen radicals are an important cause for VSMC death. However, the precise mechanism of oxidative stress-induced VSMC apoptosis is still poorly understood. Here, we aimed to analyse the role of soluble adenylyl cylclase (sAC). VSMC derived from rat aorta were treated with either H2O2 (300 µmol/L) or DMNQ (30 µmol/L) for 6 h. Oxidative stress-induced apoptosis was prevented either by treatment with 30 µmol/L KH7 (a specific inhibitor of sAC) or by stable sAC-knockdown (shRNA-transfection). A similar effect was found after inhibition of protein kinase A (PKA). Suppression of the sAC/PKA-axis led to a significant increase in phosphorylation of the p38 mitogen-activated protein kinase under oxidative stress accompanied by a p38-dependent phosphorylation/inactivation of the pro-apoptotic Bcl-2-family protein Bad. Pharmacological inhibition of p38 reversed these effects of sAC knockdown on apoptosis and Bad phosphorylation, suggesting p38 as a link between sAC and apoptosis. Analysis of the protein phosphatases 1 and 2A activities revealed an activation of phosphatase 1, but not phosphatase 2A, under oxidative stress in a sAC/PKA-dependent manner and its role in controlling the p38 phosphorylation. Inhibition of protein phosphatase 1, but not 2A, prevented the pro-apoptotic effect of oxidative stress. In conclusion, sAC/PKA-signaling plays a key role in the oxidative stress-induced apoptosis of VSMC. The cellular mechanism consists of the sAC-promoted and protein phosphatase 1-mediated suppression of p38 phosphorylation resulting to activation of the mitochondrial pathway of apoptosis.

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Abbreviations

DCF:

2′,7′-dichlorofluorescein

DMNQ:

2,3-dimethoxy-1,4-naphthoquinone

ERK1/2:

Extracellular signal-regulated kinases1/2

JNK:

c-Jun N-terminal kinases

LDH:

Lactate dehydrogenase

MAPK:

Mitogen-activated protein kinases

NAC:

N-acetyl-l-cysteine

OA:

Okadaic acid

PKA:

Protein kinase A

PP1:

Protein phosphatase 1

PP2A:

Protein phosphatase 2A

ROS:

Reactive oxygen species

Rp-cAMPS:

Rp-adenosine 3′,5′-cyclic monophosphorothioate

sAC:

Soluble adenylyl cyclase

tmAC:

Transmembrane adenylyl cyclases

TUNEL:

Terminal deoxynucleotidyl transferase dUTP nick end labeling

VSMC:

Vascular smooth muscle cells

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Acknowledgments

This work was supported by Grant LA 1159/7-1 of the Deutsche Forschungsgemeinschaft. S. Kumar was supported by fellowship from Deutsche Gesellschaft für Kardiologie. We also thank Dr. J. Buck (Cornell University, NY) for kindly providing the R21 antibodies and KH7.15. The technical help of G. Scheibel and K. Rezny is gratefully acknowledged.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Department of Clinical Pharmacology, Ruhr-University Bochum, Universitätsstrasse 150, 44801, Bochum, Germany

    Sanjeev Kumar, Avinash Appukuttan, H. Peter Reusch & Yury Ladilov

  2. Department of Molecular Gastroenterology-Oncology, Ruhr-University Bochum, Bochum, Germany

    Abdelouahid Maghnouj & Stephan Hahn

  3. Center for Cardiovascular Research, Charite, Berlin, Germany

    Yury Ladilov

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  1. Sanjeev Kumar
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  2. Avinash Appukuttan
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  3. Abdelouahid Maghnouj
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  4. Stephan Hahn
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  5. H. Peter Reusch
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  6. Yury Ladilov
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Correspondence to Yury Ladilov.

Additional information

Sanjeev Kumar and Avinash Appukuttan equally contributed to this article.

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Kumar, S., Appukuttan, A., Maghnouj, A. et al. Suppression of soluble adenylyl cyclase protects smooth muscle cells against oxidative stress-induced apoptosis. Apoptosis 19, 1069–1079 (2014). https://doi.org/10.1007/s10495-014-0989-9

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  • DOI: https://doi.org/10.1007/s10495-014-0989-9

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