Apoptosis (2014) 19:1069–1079 DOI 10.1007/s10495-014-0989-9

ORIGINAL PAPER

Suppression of soluble adenylyl cyclase protects smooth muscle cells against oxidative stress-induced apoptosis Sanjeev Kumar • Avinash Appukuttan • Abdelouahid Maghnouj • Stephan Hahn H. Peter Reusch • Yury Ladilov



Published online: 30 April 2014 Ó Springer Science+Business Media New York 2014

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 lmol/L) or DMNQ (30 lmol/L) for 6 h. Oxidative stress-induced apoptosis was prevented either by treatment with 30 lmol/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

Sanjeev Kumar and Avinash Appukuttan equally contributed to this article.

Electronic supplementary material The online version of this article (doi:10.1007/s10495-014-0989-9) contains supplementary material, which is available to authorized users. S. Kumar  A. Appukuttan  H. Peter Reusch  Y. Ladilov (&) Department of Clinical Pharmacology, Ruhr-University Bochum, Universita¨tsstrasse 150, 44801 Bochum, Germany e-mail: [email protected] A. Maghnouj  S. Hahn Department of Molecular Gastroenterology-Oncology, Ruhr-University Bochum, Bochum, Germany Y. Ladilov Center for Cardiovascular Research, Charite, Berlin, Germany

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/PKAdependent 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. Keywords Soluble adenylyl cyclase  Apoptosis  Smooth muscle cells  Mitochondria  ROS  P38 Abbreviations DCF 20 ,70 -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 30 ,50 -cyclic monophosphorothioate sAC Soluble adenylyl cyclase tmAC Transmembrane adenylyl cyclases

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TUNEL VSMC

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Terminal deoxynucleotidyl dUTP nick end labeling Vascular smooth muscle cells

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of apoptosis was abolished by pharmacological inhibition or by knockdown of sAC.

Materials and methods Cell culture and treatments Introduction Increasing evidence suggests the contribution of the vascular smooth muscle cells (VSMC) apoptosis to the instability of advanced, symptomatic plaques [1–3]. VSMC apoptosis has also been associated with numerous other pathologies such as inflammation, calcification and thrombosis [4]. Several pro-apoptotic stimuli may contribute in VSMC apoptosis in atherosclerotic plaques, e.g. cytokines, hormones, and oxidative stress [5]. These stimuli may directly activate intrinsic (mitochondrial) or extrinsic (death receptors) apoptotic pathways. Nevertheless, the precise cellular pathways that govern VSMC apoptosis still have to be elucidated. Within several signaling pathways, the cAMP/protein kinase A (PKA) signaling plays an essential role in controlling apoptotic processes. Until recently the contribution of this pathway was exclusively attributed to the activation of the ß-receptor-coupled transmembrane adenylyl cyclase (tmAC). The subsequent diffusion of cAMP throughout the cytosol makes it difficult to selectively activate distally localized targets, e.g., within mitochondria, without also activating more proximal targets within the plasmalemma. A simple diffusion of cAMP would likely diminish specificity, selectivity and signal strength. This model is further complicated by the presence of phosphodiesterases that degrade cAMP and prevent its diffusion. Apart from tmAC, a second source of cAMP, the type 10 soluble adenylyl cyclase (sAC), has also been demonstrated for mammalian cells [6]. The cytosolic localization of sAC provides both specificity and selectivity by permitting the generation of cAMP in close proximity to intracellular targets. Whether sAC participates in apoptosis of VSMC is unknown. It has been shown that sAC co-localizes with mitochondria [7]. Furthermore, the translocation of sAC from the cytosol to the mitochondria has been demonstrated under extracellular acidosis or anoxia [8, 9]. Because mitochondria play a crucial role in apoptosis, we hypothesized that intracellular localization of sAC may contribute to VSMC apoptosis. Oxidative stress is a typical feature of advanced atherosclerotic plaques and an important trigger of apoptosis of VSMC [10]. Therefore, in the present study we aimed to prove the role sAC in apoptosis of VSMC induced by treatment with reactive oxygen species (ROS). We found that ROS-induced activation of the mitochondrial pathway

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Spontaneously immortalized aortic VSMC were derived from newborn rats (a gift from Peter Jones, University of Southern California, CA). The cells were grown in minimum essential Eagle’s medium supplemented with 10 % fetal calf serum, 2 % tryptose phosphate broth, 50 U/mL penicillin, and 50 lg/mL streptomycin. The fetal calf serum concentration was reduced to 2 % 24 h before the experiments. The smooth muscle phenotype was confirmed by the expression of typical SMC markers, i.e., immunocytostaining with antibodies against alpha smooth muscle actin (Sigma Aldrich) and smoothelin (Abcam). All cells express these markers. To induce oxidative stress VSMC were treated with 2,3dimethoxy-1,4-naphthoquinone (DMNQ, 30 lmol/L, 6 h) or H2O2 (300 lmol/L, 6 h). KH7 (30 lmol/L, Cayman) and KH7.15 (30 lmol/L, kindly provided by Dr. J. Buck, Cornell University, NY), N-acetyl-L-cysteine (NAC, 1 mmol/L, Sigma Aldrich), p38 inhibitor (SB202190, 15 lmol/L, Sigma Aldrich), okadaic acid (0.01–10 lmol/L, Calbiochem) and Rp-adenosine 30 ,50 -cyclic monophosphorothioate (RpcAMP, 100 lmol/L, Biolog) were applied as indicated. Detection of cellular reactive oxygen species To examine ROS formation, VSMC were loaded 30 min prior to analysis with DCF (20 ,70 -dichlorodihydrofluorescein diacetate, succinimidyl ester, 10 lmol/L, Invitrogen), a nonfluorescent dye that is converted into a highly fluorescent DCF in the presence of free radicals. Cells were lysed with buffer containing 100 mmol/L NaCl, 100 mmol/L EDTA, 1 % SDS, 50 mmol/L Tris (pH 8.0). DCF fluorescence was analyzed by excitation at 485 ± 10 nm and emission at 530 ± 10 nm using an ELISA reader. Additionally, in some experiments cells were loaded 60 min prior to the end of experiment with 5 lmol/L MitoSOX red (Invitrogen) (15 min at 37 °C), a fluorescent dye specifically targeted to mitochondria and selectively oxidized by superoxide anions leading to red fluorescence. The cells were fixed with 2 % paraformaldehyde and stored in 70 % ethanol. Analysis of red fluorescence was performed using a FACS Calibur (BD Biosciences) at absorption/emission maxima *510/580 nm. Data are expressed as a percentage of MitoSOX positive cells, i.e. cells exhibiting markedly higher fluorescence compared to the control cells.

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TUNEL assay The cells were fixed with 2 % paraformaldehyde and stored in 70 % ethanol. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed using an ApoDirect kit (Calbiochem) according to the manufacturer’s instructions. Analyses of the TUNEL positive cells were performed using a FACS Calibur (BD Biosciences). Lactate dehydrogenase (LDH) release assay LDH activity in the cell culture medium was used as an indicator for necrosis and determined by using a Cytotoxicity Detection kit (Roche Applied Science). After each experiment, the culture medium was centrifuged at 600 g for 5 min (4 °C), and the supernatant was used for the LDH analysis by applying an ELISA reader.

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using puromycin selection. The extent of sAC knockdown was determined using western blot analysis with antibodies against sAC. The shRNA oligonucleotide sequences were as follows: shRNA-1

shRNA-2

shRNA-3

cAMP determination Analysis of total cellular cAMP content was performed using a cAMP (direct) Enzyme Immunoassay kit (Assay Designs). The measured absorbance at 405 nm was used to calculate the concentration of cAMP by applying a calibration curve. Subcellular fractionation The cells were lysed by mechanical homogenization using a Dounce homogenizer. Subcellular fractionation was performed using a Mitochondria Isolation kit (Sigma Aldrich) according to the manufacturer’s instructions. Briefly, the lysed cells were centrifuged at 600 g for 5 min (4 °C), and the resulting supernatant was centrifuged at 11,000 g for 10 min (4 °C). After this centrifugation step, the pellet was defined as the mitochondrial fraction and the supernatant as the cytosolic, mitochondria-free fraction. The purity of the cytosolic fraction was confirmed by the absence of Tim23 (BD Biosciences) applying western blot analysis. Stable sAC knockdown The sAC-targeted shRNA and scrambled shRNA expression plasmids were constructed using a pLKO.1-puro vector (Addgene). Pairs of sense and antisense hairpin oligonucleotides were annealed to form the shRNA cassette and subcloned into the EcoRI/AgeI sites of the pLKO.1-puro vector. Recombinant lentiviral particles were prepared by transfecting HEK293T cells with the appropriate pLKO.1puro plasmid plus the pCMVDR8.2 and pHIT G plasmids. The cells stably transfected with the shRNA were purified

Scramble

ADCY10-2468-s: CCG GGA GAG CTT GAC TCG TAC CTG GGC TCG AGG CCA GGT ACG AGT CAA GCT CTC TTT TTG; ADCY10-2468-as: AAT TCA AAA AGA GAG CTT GAC TCG TAC CTG GCC TCG AGC CCA GGT ACG AGT CAA GCT CTC. ADCY10-618-s: CCG GGT GGA AAG TGG AAC GAA AGC AGC TCG AGG TGC TTT CGT TCC ACT TTC CAC TTT TTG; ADCY10-618-as: AAT TCA AAA AGT GGA AAG TGG AAC GAA AGC ACC TCG AGC TGC TTT CGT TCC ACT TTC CAC. ADCY10-1983-s: CCG GGA TAC TCA TGG CTA ACG TAC TGC TCG AGG AGT ACG TTA GCC ATG AGT ATC TTT TTG; ADCY10-1983-as: AAT TCA AAA AGA TAC TCA TGG CTA ACG TAC TCC TCG AGC AGT ACG TTA GCC ATG AGT ATC. Scramble-s: CCG GTG GTT TAC ATG TCG ACA ATT TGC TCG AGG AAA TTG TCG ACA TGT AAA CCA TTT TTG; Scramble-as: AAT TCA AAA ATG GTT TAC ATG TCG ACA ATT TCC TCG AGC AAA TTG TCG ACA TGT AAA CCA.

Western blotting VSMC were lysed in Laemmli buffer. Equal amounts of total proteins (20–40 lg/well) were separated by SDSPAGE and transferred onto nitrocellulose membranes. The primary antibodies used were sAC (clone R21, kindly provided by Dr. J. Buck, Cornell University, USA), cytochrome c (Sigma), cytochrome oxidase IV (Molecular Probes), actin (Chemicon International), caspase-3, Tim23 (BD Biosciences). All other antibodies were from Cell Signaling. Specific bands were visualized after incubation using peroxidase-linked/horseradish peroxidase-labeled secondary antibodies by chemiluminescence using the ECL?kit (Amersham Biosciences). Equal sample loading was confirmed by staining with antibodies against actin. Analysis of protein phosphatase 2A (PP2A) activity Activity of PP2A was quantitated using an immunocapture ELISA-based assay (Human/Mouse/Rat Active PP2A DuoSet IC R&D Systems) applying a PP2A specific antibody according to the manufacturer’s protocol. Cells were lysed with a buffer containing 120 mmol/L NaCl,

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0.1 mmol/L EGTA, 0.1 mmol/L EDTA, 0.5 % NP-40, a protease inhibitors mixture, 1 mmol/L PMSF and 50 mmol/L HEPES, pH 7.5. At the end of assay the free phosphate was detected by a sensitive dye-binding assay using malachite green and molybdic acid. By calculating the rate of phosphate release, the activity of PP2A was determined and normalized to the protein concentration in lysats.

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(Fig. 1c). Treatment with 30 lmol/L KH7 prevented the pro-apoptotic effects of DMNQ (Fig. 1a, b). To exclude unspecific side-effects of KH7, experiments with the inactive analogue KH7.15 [12] were performed. In contrast to KH7, treatment with KH7.15 had no effect on the cellular cAMP level (Fig. 1c) and apoptosis (Fig. 1a, b). Mitochondria play an essential role in sAC-dependent apoptosis in VSMC

Analysis of protein phosphatase 1 (PP1) activity

Data are given as mean ± SEM. Comparisons of the means between the groups were performed using a oneway analysis of variance followed by the Holm-Sidak post hoc test. Statistical significance was accepted when P \ 0.05.

Previous reports emphasized the importance of mitochondria in ROS-induced apoptosis of various cell types [13]. Similarly, a marked rise of cytosolic cytochrome c and a cleavage of caspase-9 were found in VSMC during treatment with DMNQ or H2O2, which were significantly reduced by treatment with KH7 (Fig. 1d, e). Oxidative stress had no effect on sAC expression (Fig. 1f). To further substantiate the role of sAC in ROS-induced, mitochondria-dependent apoptosis of VSMC, expression of sAC was suppressed by transfection with shRNA, which reduced the expression of the 50 kDa sAC protein by C85 % compared to scrambled shRNA or empty vector when targeted shRNA-1 was applied (Fig. 2a). Therefore, VSMC clone transfected with shRNA-1 were used for further analyses. The ROS-induced rise of apoptotic cell number, cleavage of caspase-3 and mitochondrial cytochrome c release were attenuated by sAC knockdown (Fig. 2b–e). Interestingly, the analysis of the subcellular distribution of sAC demonstrated a marked co-localization of the cyclase with mitochondria during oxidative stress (Online Resource Fig. 2). Although this finding does not necessarily indicate a causal role of the sAC translocation in inducing the mitochondrial cytochrome c release, it nevertheless supports a potential role of the cyclase in the mitochondrial pathway of apoptosis. Altogether, these data suggest a key role of sAC in the ROS-induced activation of the mitochondrial pathway of apoptosis in VSMC.

Results

Role of p38-Bad pathway in sAC-dependent apoptosis in VSMC

To determine the specific PP1 activity, the unspecific protein phosphatase activity in cell lysates (similar buffer as described above) was measured by a Phosphatase Assay kit (G Biosciences) according to the manufacturer’s protocol. Simultaneously, in separated aliquots, okadaic acid (OA) was added at concentrations of 1 nmol/L (to suppress the activity of PP2A) and 5 lmol/L (to suppress the activity of PP2A and PP1). Pilot experiments applying immunocapture ELISA-based assay for active PP2A (see above) demonstrated that 1 nmol/L OA nearly completely suppresses the PP2A activity (Online Resource Fig. 4). The specific activity of PP1 was calculated according to the following equation: PP1activity ¼ TPA½OA1  TPA½OA5; where TPA[OA1] is total phosphatase activity at the presence of 1 nmol/L OA; TPA[OA5] is total phosphatase activity at the presence of 5 lmol/L OA. Statistical analysis

Inhibition of sAC prevents ROS-induced apoptosis of VSMC Treatment of VSMC with 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) for 4 h led to a ROS formation (Online Resource Fig. 1) and significantly increased the number of apoptotic cells and caspase-3 cleavage after 6-h treatment, but has no effect on LDH-release (Fig. 1a, b). To examine the role of sAC in VSMC apoptosis, treatment with the selective sAC-inhibitor KH7 [11] was applied. Preliminary test demonstrated that KH7 dose-dependently reduced cellular cAMP-content with a maximal effect at 30 lmol/L

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To further investigate the underlying cellular mechanism(s) involved in the sAC-dependent control of the ROSinduced apoptosis, the potential contribution of Akt and mitogen activated protein kinases (MAPK) was examined. We found no sAC-dependent effects of H2O2 treatment on JNK, ERK1/2 and Akt phosphorylation (data not shown). In contrast, phosphorylation of p38 was significantly altered by sAC knockdown. Particularly, in control cells (scrambled) phosphorylation of p38 increased after 2 h of H2O2 treatment and disappeared after 4 h (Fig. 3a). sAC knockdown completely prevented this down-regulation of the phosphorylated p38 form.

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Fig. 1 Oxidative stress leads to VSMC apoptosis in a sAC-dependent manner. a, b Analyses of the apoptotic cell number (TUNEL-positive cells, percent of the total cell number), lactate dehydrogenase (LDH) activity in the cell culture medium (presented as a ratio to individual protein content, relative units) and cleaved caspase-3 (western blot) performed with untreated, control VSMC (Con) or with cells after the following 6-h treatments: 30 lmol/L KH7, 30 lmol/L KH7.15 (inactive analogue of KH7) or 30 lmol/L DMNQ. Values are mean ± SEM (n = 6–8). *P \ 0.05 versus DMNQ or DMNQ?KH7.15. c Analysis of total cellular cAMP content after 6-h treatment with different concentrations of KH7. White square indicates the effects of 6-h treatment with 30 lmol/L KH7.15. Values

are mean ± SEM (n = 4). d, e Western blot analysis of cytochrome c and cleaved caspase-9 (39 kDa) followed by statistical analysis of the optical band density (relative units). Values are mean ± SEM (n = 4–5). *P \ 0.05 versus Control, #P \ 0.05 versus DMNQ or H2O2.The conditions were similar to those described for Fig. 1a, b with addition of the 6-h treatment with 300 lmol/L H2O2. f Western blot analysis of sAC (50 kDa) performed with lysates of control VSMC or with cells treated for 4 h either with DMNQ (30 lmol/L) or with H2O2 (300 lmol/L). All western blot data are representative of 4–5 independent experiments that yielded similar results

Increasing evidence suggests a key role of Bcl-2 family proteins in controlling the mitochondrial pathway of apoptosis. Within several pro-apoptotic members of this family, the activity of Bad protein is regulated by a p38-

dependent phosphorylation, which in turn leads to a Bad inactivation due to its sequestration by 14–3–3 protein [14]. In the present study we found a marked, sAC-dependent alteration of Bad phosphorylation. Time course analysis in

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Fig. 2 Knockdown of sAC prevents the oxidative stress-induced apoptosis. a Western blot analysis of sAC (50 kDa) performed with lysates of VSMC transfected with empty vector, with scrambled shRNA (scrambled) or with different sAC-targeted shRNA (shRNA). Note that transfection with shRNA-1 produced maximal sAC knockdown. b, c Analyses of the apoptotic cell number (TUNELpositive cells, percent of the total cell number), lactate dehydrogenase (LDH) activity in the cell culture medium (presented as a ratio to individual protein content, relative units) and cleaved caspase-3 (western blot) performed with VSMC transfected with scrambled shRNA (scrambled) or with sAC-targeted shRNA (shRNA) without

treatment or after 6-h treatment with 30 lmol/L DMNQ. Values are mean ± SEM (n = 5–7). *P \ 0.05 versus scrambled?DMNQ. d, e Western blot analysis of cytosolic cytochrome c followed by statistical analysis of the optical bad density (relative units) performed with lysates of VSMC transfected with scrambled shRNA (scrambled) or with sAC-targeted shRNA (shRNA) without treatment or after 6-h treatment with 30 lmol/L DMNQ or 300 lmol/L H2O2. Values are mean ± SEM (n = 3). *P \ 0.05 versus scrambled?DMNQ and scrambled?H2O2. All western blot data are representative of 3–6 independent experiments that yielded similar results

scrambled cells demonstrated that H2O2 treatment led to an initial phosphorylation of Bad at Ser112. However, after 4 h, the Bad phosphorylation was markedly reduced and returned to its initial level after 6 h (Fig. 3a). In contrast, in sAC-knockdowned cells Bad phosphorylation remained stable even after 6 h of ROS treatment. Thus, both phosphorylation/activation of p38 and phosphorylation/inactivation of Bad were markedly preserved by sACknockdown.

To support a causal role of p38 in Bad phosphorylation and apoptosis, treatment with the specific p38 inhibitor SB202190 was performed in sAC-knockdowned cells. SB202190 dose-dependently reduced p38 phosphorylation during the 4-h treatment with H2O2, which was accompanied by a dose-dependent rise of caspases-9 cleavage (Online Resource Fig. 3). In contrast, a 4-h treatment with SB202190 had no effect on caspase-9 cleavage in control cells (Fig. 3b), which excludes the possible side effects of

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Fig. 3 Role of p38 and Bad in sAC-dependent apoptosis. a Western blot analysis of phosphorylated p38-MAP kinase (phos-p38) and phosphorylated Bad (phos-Bad) performed with lysates of VSMC transfected with scrambled shRNA (scrambled) or with sAC-targeted shRNA (sAC-knockdown) without treatment or after 2, 4, or 6 h treatment with 300 lmol/L H2O2. b Western blot analysis of

phosphorylated Bad (phos-Bad), phosphorylated p38-MAP kinase (phos-p38) or cleaved caspase-9 performed with lysates of sACknockdowned VSMC without treatment (Con) or after treatment for 4 h with 300 lmol/L H2O2 in the absence or presence of p38 inhibitor SB202190 (SB, 15 lmol/L). All western blot data are representative of three to four independent experiments that yielded similar results

this compound on apoptosis. Furthermore, inhibition of p38 prevented the H2O2-induced phosphorylation of Bad in sAC-knockdowned cells (Fig. 3b). Thus, p38 seems to be an upstream kinase controlling Bad phosphorylation and apoptosis in our model.

cleavage, suggesting that PP1 is an upstream enzyme governing the p38/Bad/caspase-9 pathway of apoptosis. Further analysis of the phosphatase activity revealed a significant rise of PP1 activity under H2O2 treatment, which was prevented by sAC knockdown (Fig. 4b). In contrast, no ROS-induced PP2A activation could be found. Thus, PP1 seems to be an important link between sAC and p38.

Role of protein phosphatase 1 (PP1) and 2A (PP2A) in sAC-dependent apoptosis in VSMC Protein phosphatases substantially contribute to the p38 phosphorylation status. Because in our model p38 phosphorylation was vanished after 4 h of ROS treatment, we supposed that protein phosphatases may be involved in this effect. To challenge this hypothesis, the role of two serine/ threonine protein phosphatases, i.e. PP1 and PP2A, was examined by applying okadaic acid, a specific inhibitor of PP1 and PP2A. This inhibitor used in nanomolar concentrations selectively suppresses the activity of PP2A (IC50 0.1–1.0 nmol/L), whereas at micromolar concentrations inhibits both PP1 (IC50 20–100 nmol/L) and PP2A [15, 16]. In our model okadaic acid nearly completely suppressed PP2A activity at 1 nmol/L (Online Resource Fig. 4), however had no effect on p38 and Bad phosphorylation as well as on caspase-9 cleavage during H2O2 treatment up to 5 nmol/L (Fig. 4a). In contrast, a marked increase of p38 phosphorylation was observed during treatment with okadaic acid at 0.5 lmol/L, suggesting the involvement of PP1 in dephosphorylation of p38. Interestingly, this effect was accompanied by an increase in Bad phosphorylation as well as by reduction of caspases-9

Role of PKA in sAC-dependent apoptosis in VSMC Protein kinase A (PKA) is a direct downstream target for sAC. Therefore, we investigated the potential contribution of this kinase in ROS-induced apoptosis and PP1 activation. Since only control cells transfected with scrambled shRNA, but not sAC-knockdowned cells, show activation of PP1 (Fig. 4b) under H2O2 treatment, these control cells were used for the experiments. Application of the specific cAMP-binding site inhibitor adenosine Rp-adenosine 30 ,50 cyclic monophosphorothioate (Rp-cAMPS, 100 lmol/L) during H2O2 treatment prevented activation of PP1 (Fig. 4d) and increased expression of phosphorylated forms of p38 and Bad VSMC. These effects were accompanied by suppression of caspases-9 cleavage (Fig. 4c).

Discussion The aim of the present study was to examine whether sAC may contribute to the oxidative stress-induced apoptosis of

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Fig. 4 Role of protein phosphatases in sAC-dependent apoptosis. a Western blot analysis of phosphorylated p38-MAP kinase (phosp38), phosphorylated Bad (phos-Bad), or cleaved caspase-9 performed with lysates of VSMC treated for 4 h with 300 lmol/L H2O2 in the absence or presence of okadaic acid at low (1.0 and 5.0 nmol/L) or high (0.5 lmol/L) concentrations. b Statistical analyses of the protein phosphatase 1 (PP1) and 2A (PP2A) activity (presented as a ratio to individual protein content, relative units) performed with VSMC transfected with scrambled shRNA (scrambled) or with sACtargeted shRNA (sAC-knockdown) without treatment (Con) or after 4-h treatment with 300 lmol/L H2O2. Values are mean ± SEM (n = 6). *P \ 0.05 versus Con (PP1). c Western blot analysis of

phosphorylated p38-MAP kinase (phos-p38), phosphorylated Bad (phos-Bad) or cleaved caspase-9 performed with lysates of VSMC treated for 4 h with 300 lmol/L H2O2 in the absence or presence of PKA inhibitor (Rp-cAMP, 100 lmol/L). All western blot data are representative of four independent experiments that yielded similar results. d Statistical analyses of the protein phosphatase 1 (PP1) and 2A (PP2A) activity (presented as a ratio to individual protein content, relative units) performed with untreated VSMC or with cells after 4-h treatment with 300 lmol/L H2O2 in the absence or presence of PKA inhibitor (Rp-cAMP, 100 lmol/L). Values are mean ± SEM (n = 5–6). *P \ 0.05 versus Con (PP1)

VSMC, and which signaling pathways might be involved. The principal findings are the following: (i) sAC plays an important role in the ROS-induced VSMC apoptosis. (ii) The underlying signaling pathway consists of the sAC/ PKA-dependent activation of PP1 under oxidative stress leading to dephosphorylation/inactivation of p38 followed by dephosphorylation/activation of the pro-apoptotic protein Bad (Fig. 5). To induce oxidative stress two tools were applied in the present study, i.e. H2O2 and DMNQ treatment. Although

H2O2 treatment is a widely used tool to induce oxidative stress it has some disadvantages for in vitro models. For example, H2O2 treatment even for short time increases cell fragility excluding analyses requiring cell disattachment procedure, i.e., trypsin treatment, due to increased membrane permeability (own observations). A number of alternative tools have been used to induce oxidative stress in cultured cells. The quinones are of particular interest. Quinones are ubiquitous in nature and are also formed as metabolites from a variety of drugs,

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Apoptosis

Fig. 5 Schematic diagram demonstrating the sAC-dependent pathway controlling apoptosis in VSMC under oxidative stress. Increase of ROS formation leads to the sAC- and PKA-dependent activation of PP1. PP1 dephosphorylates/inactivates the p38-MAP kinase. The loss of p38 activity attenuates p38-dependent phosphorylation/inactivation of pro-apoptotic Bad, thus promoting the mitochondrial pathway of apoptosis. Symbol ? indicates activation. Symbol p indicates phosphorylated form of protein. A dotted line shows reduced effect of p38 on Bad phosphorylation

environmental pollutants and food derivatives by the action of cytochrome P450 system [17]. The redox-cycling agent DMNQ leading to intracellular superoxide anion formation has been proposed as a model to study the role of ROS in cell toxicity and apoptosis [18]. In our study treatment with 30 lmol/L DMNQ produced oxidation of the ROS-sensitive dye DCF comparable with H2O2 treatment (Online Resource Fig. 1). Furthermore, a similar activation of the mitochondrial pathway of apoptosis, i.e., cytochrome c release and caspases-9 cleavage, was observed under H2O2 or DMNQ treatment after 6 h. Nevertheless, analyzing underlying cellular mechanisms at earlier time points we found that treatment with H2O2 provides more reproducible results. Therefore, this model of oxidative stress was used in some sets of experiments. Many efforts have been undertaken to understand the cellular mechanisms of ROS-induced apoptosis. In the present study the contribution of cAMP-dependent signaling has been investigated. Although the role of this pathway in mediating apoptosis was demonstrated in several reports [19, 20], the majority of the studies investigated the

role of cAMP-signaling pathway through activation of tmAC, e.g. by forskolin or catecholamines, whereas the role of the intracellular localized sAC-pool of cAMP was not considered. Our previous studies suggested the involvement of sAC in ischemic stress-induced apoptosis in coronary endothelial cells and cardiomyocytes [8, 9]. In the present study we focused on the role of this intracellular localized cyclase in the ROS-induced VSMC apoptosis. For this purpose treatment with the selective sAC inhibitor KH7 [12] or sAC knockdown were applied. Both approaches to inhibit sAC led to significant suppression of ROSinduced apoptosis. It has been shown that mitochondria participate in oxidative stress-induced apoptosis [13]. Injury of mitochondria due to a permeabilisation of its outer and inner membrane followed by depolarization and release of proapoptotic proteins, e.g. cytochrome c, is a widely accepted scenario of the mitochondria-dependent apoptosis [13]. In agreement with these findings, activation of the mitochondrial pathway of apoptosis was found in our model, which was abolished by inhibition of sAC during ROS treatment. In our recent study we found that oxysterol-induced apoptosis in VSMC is sAC-dependent and involves a mitochondrial ROS formation [21]. In this study the oxidative stress induced by oxysterol treatment is downstream to sAC and is rather a result then a cause of sAC activity. Indeed, suppression of the mitochondrial Bax binding (a sAC-dependent event) by treatment with the Bax-inhibiting peptide prevented the ROS release. This signaling is, therefore, different from the signaling described in the present study. Here ROS is a direct trigger of the sACdependent apoptosis, i.e. ROS is upstream to sAC. Thus, sAC may play a role in oxidative stress-induced apoptosis either as a target or as a cause of ROS formation. To further understand the sAC-dependent signaling leading to an activation of the mitochondrial pathway of apoptosis, the role of PKA, a major down-stream target of sAC, was investigated in the present study. Previous reports demonstrated a compartmentalization of PKA within mitochondrial microdomains [22, 23] and its role in mitochondrial apoptosis [22, 24]. Mitochondrial translocation of sAC observed in the present study during oxidative stress (Online Resource Fig. 2) argues for the selective activation of PKA localized within the mitochondrial microdomains. Applying treatment with specific PKA inhibitor we found that PKA plays an essential role in the activation of the mitochondrial pathway of apoptosis. To find out the signaling link between sAC/PKA-axis and apoptosis, a contribution of Akt and MAP kinases was examined. We found that p38 MAP kinase plays a causal role in sAC-dependent, ROS-induced apoptosis. Particularly, the initial rise in p38-phosphorylation was followed

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by its dephosphorylation 4 h after H2O2 treatment. This delayed dephosphorylation was abrogated by knockdown of sAC or by inhibition of the sAC downstream target PKA. Interestingly, a similar pattern of Bad phosphorylation at Ser112 during ROS-treatment in scrambled and sAC-knockdown cells was found suggesting the causal role of p38 in Bad phosphorylation at Ser112. Application of the specific p38 inhibitor SB202190 has proved this hypothesis. In agreement with our study numerous reports suggest Bad as a principal link in suppression of apoptosis by several pro-survival kinases, like Akt, p70S6K and MAPK [25–27]. Particularly, phosphorylation of Bad at Ser112 directly by activated p38 has been demonstrated [28]. Loss of Bad phosphorylation due to inhibition of p38 in our study was accompanied by increased caspases-9 cleavage suggesting an anti-apoptotic role of p38 signaling in ROS-induced cell death. The initial activation of p38, i.e. 2 h after H2O2 treatment, seems to be sAC-independent, since it was observed in both scrambled and sAC-knockdowned cells. In opposite, disappearance of the p38 phosphorylation was in a strong dependence on the sAC expression. Since this effect was abolished by treatment with okadaic acid at micromolar (specific for PP1 and P2A), but not at nanomolar (specific only for PP2A [15, 16] concentration, we suppose that PP1, rather than PP2A, may be involved. Indeed, a significant rise in activity of PP1, but not PP2A, was found during H2O2 treatment, which was prevented by sAC knockdown. Since this PP1 activation was also abolished by inhibition of PKA, we suggest that PKA is the downstream target of cAMP generated by sAC responsible for the activation of PP1 in our study. The signaling link between PKA and PP1 remains still unresolved in this study. Regulation of PP1 activity is complex and under control of several inhibitor proteins, i.e., inhibitor-1, inhibitor-2, inhibitor-5, dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP-32), and nuclear inhibitor of protein phosphatase-1 (NIPP-1). Excepting inhibitor-2, activity of all inhibitors may be affected by PKA-dependent phosphorylation [29]. However, only phosphorylation of NIPP-1 by PKA leads to suppression of its inhibitory potential [29]. Therefore, sAC/ PKA-dependent inhibition of NIPP1 may be responsible for the ROS-induced activation of PP1. In conclusion, the present study described a novel signaling pathway controlling the mitochondria-dependent apoptosis induced by oxidative stress in VSMC (Fig. 5). During oxidative stress sAC supports the PKA-dependent activation of PP1 followed by p38 dephosphorylation. The loss of p38 activity attenuates phosphorylation/inactivation of pro-apoptotic Bad and leads to activation of the mitochondrial pathway of apoptosis.

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Apoptosis (2014) 19:1069–1079 Acknowledgments This work was supported by Grant LA 1159/7-1 of the Deutsche Forschungsgemeinschaft. S. Kumar was supported by fellowship from Deutsche Gesellschaft fu¨r 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 of interest.

The authors declare that they have no conflict

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

Apoptosis of vascular smooth muscle cells (VSMC) significantly contributes to the instability of advanced atherosclerotic plaques. Oxygen radicals are...
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