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Soluble adenylyl cyclase controls mitochondria-dependent apoptosis in coronary endothelial cells.

ABSTRACT: The cAMP signaling pathway plays an essential role in modulating the apoptotic response to various stress stimuli. Until now, it was attributed exclusively to the activity of the G-protein-responsive transmembrane adenylyl cyclase. In addition to transmembrane AC, mammalian cells possess a second source of cAMP, the ubiquitously expressed soluble adenylyl cyclase (sAC). However, the role of this cyclase in apoptosis was unknown. A mitochondrial localization of this cyclase has recently been demonstrated, which led us to the hypothesis that sAC may play a role in apoptosis through modulation of mitochondria-dependent apoptosis. To prove this hypothesis, apoptosis was induced by simulated in vitro ischemia or by acidosis, which is an important component of ischemia. Suppression of sAC activity with the selective inhibitor KH7 or sAC knockdown by small interfering RNA transfection abolished endothelial apoptosis. Furthermore, pharmacological inhibition or knockdown of protein kinase A, an important cAMP target, demonstrated a significant anti-apoptotic effect. Analysis of the underlying mechanisms revealed (i) the translocation of sAC to mitochondria under acidic stress and (ii) activation of the mitochondrial pathway of apoptosis, i.e. cytochrome c release and caspase-9 cleavage. sAC inhibition or knockdown abolished the activation of the mitochondrial pathway of apoptosis. Analysis of mitochondrial co-localization of Bcl-2 family proteins demonstrated sAC- and protein kinase A-dependent translocation of Bax to mitochondria. Taken together, these results suggest the important role of sAC in modulating the mitochondria-dependent pathway of apoptosis in endothelial cells.

SUBMITTER: Kumar S

PROVIDER: S-EPMC2685657 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Soluble adenylyl cyclase controls mitochondria-dependent apoptosis in coronary endothelial cells.

Kumar Sanjeev S Kostin Sawa S Flacke Jan-Paul JP Reusch H Peter HP Ladilov Yury Y

The Journal of biological chemistry 20090331 22

The cAMP signaling pathway plays an essential role in modulating the apoptotic response to various stress stimuli. Until now, it was attributed exclusively to the activity of the G-protein-responsive transmembrane adenylyl cyclase. In addition to transmembrane AC, mammalian cells possess a second source of cAMP, the ubiquitously expressed soluble adenylyl cyclase (sAC). However, the role of this cyclase in apoptosis was unknown. A mitochondrial localization of this cyclase has recently been demo ...[more]

PMID: 19336406

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Project description:UnlabelledAnion exchanger 2 (AE2), the principal bicarbonate secretor in the human biliary tree, is down-regulated in primary biliary cholangitis. AE2 creates a "bicarbonate umbrella" that protects cholangiocytes from the proapoptotic effects of bile salts by maintaining them deprotonated. We observed that knockdown of AE2 sensitized immortalized H69 human cholangiocytes to not only bile salt-induced apoptosis (BSIA) but also etoposide-induced apoptosis. Because the toxicity of etoposide is pH-independent, there could be a more general mechanism for sensitization of AE2-depleted cholangiocytes to apoptotic stimuli. We found that AE2 deficiency led to intracellular bicarbonate accumulation and increased expression and activity of soluble adenylyl cyclase (sAC), an evolutionarily conserved bicarbonate sensor. Thus, we hypothesized that sAC regulates BSIA. H69 cholangiocytes and primary mouse cholangiocytes were used as models. The sAC-specific inhibitor KH7 not only reversed sensitization to BSIA in AE2-depleted H69 cholangiocytes but even completely prevented BSIA. sAC knockdown by tetracycline-inducible short hairpin RNA also prevented BSIA. In addition, sAC inhibition reversed BSIA membrane blebbing, nuclear condensation, and DNA fragmentation. Furthermore, sAC inhibition also prevented BSIA in primary mouse cholangiocytes. Mechanistically, sAC inhibition prevented Bax phosphorylation at Thr167 and mitochondrial translocation of Bax and cytochrome c release but not c-Jun N-terminal kinase activation during BSIA. Finally, BSIA in H69 cholangiocytes was inhibited by intracellular Ca(2+) chelation, aggravated by thapsigargin, and unaffected by removal of extracellular calcium.ConclusionsBSIA is regulated by sAC, depends on intracellular Ca(2+) stores, and is mediated by the intrinsic apoptotic pathway; down-regulation of AE2 in primary biliary cholangitis sensitizes cholangiocytes to apoptotic insults by activating sAC, which may play a crucial role in disease pathogenesis. (Hepatology 2016;64:522-534).

Dataset Information

Soluble adenylyl cyclase controls mitochondria-dependent apoptosis in coronary endothelial cells.

Publications

Soluble adenylyl cyclase controls mitochondria-dependent apoptosis in coronary endothelial cells.

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