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. 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. Increasing evidence suggests that apoptosis of endothelial cells (EC) 3The abbreviations used are: EC, endothelial cell(s); tmAC, transmembrane adenylyl cyclase; sAC, soluble adenylyl cyclase; siRNA, small interfering RNA; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; PKA, protein kinase A; ddAdo, 2′,5′-dideoxyadenosine. may be responsible for acute and chronic vascular diseases, e.g. through atherogenesis (1Chen J. Mehta J.L. Haider N. Zhang X. Narula J. Li D. Circ. Res. 2004; 94: 370-376Crossref PubMed Scopus (241) Google Scholar), endothelial dysfunction (2Werner N. Wassmann S. Ahlers P. Kosiol S. Nickenig G. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 112-116Crossref PubMed Scopus (270) Google Scholar), or thrombosis (3Bombeli T. Karsan A. Tait J.F. Harlan J.M. Blood. 1997; 89: 2429-2442Crossref PubMed Google Scholar). Within several signaling mechanisms, a cAMP-dependent signaling pathway plays a substantial role in mediating apoptotic cell death induced by various stress factors. Elevation of the cellular cAMP either by forskolin-induced stimulation of the G-protein-responsive transmembrane adenylyl cyclase (tmAC) or by treatment with cAMP analogs has been shown to lead to both induction and suppression of apoptosis in different cell types (4Kwak H.J. Park K.M. Choi H.E. Chung K.S. Lim H.J. Park H.Y. Cell. Signal. 2008; 20: 803-814Crossref PubMed Scopus (71) Google Scholar, 5Smith P.G. Wang F. Wilkinson K.N. Savage K.J. Klein U. Neuberg D.S. Bollag G. Shipp M.A. Aguiar R. Blood. 2005; 105: 308-316Crossref PubMed Scopus (125) Google Scholar, 6Zhang L. Insel P.A. J. Biol. Chem. 2004; 279: 20858-20865Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 7Rudolph J.A. Poccia J.L. Cohen M.B. J. Biol. Chem. 2004; 279: 14828-14834Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). This discrepancy may be due to differences in cell types and experimental models. Alternatively, a lack of specificity of tmAC-induced signals, especially directed to distant intracellular targets like mitochondria, may be a cause of the discrepancy. Indeed, the classical model of cAMP signaling requires the diffusion of cAMP from plasma membrane-localized tmAC to targets localized throughout the cell. Diffusion of cAMP throughout the cytosol makes it difficult to selectively activate distally localized targets without also activating more proximal targets. Therefore, such diffusion of cAMP would likely diminish specificity, selectivity, and signal strength. This model is further complicated by the presence of phosphodiesterases, which degrade cAMP, thus preventing its diffusion. In addition to tmAC, a second source of cAMP, soluble adenylyl cyclase (sAC), was demonstrated for mammalian cells (8Buck J. Sinclair M.L. Schapal L. Cann M.J. Levin L.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 79-84Crossref PubMed Scopus (426) Google Scholar, 9Chen Y. Cann M.J. Litvin T.N. Iourgenko V. Sinclair M.L. Levin L.R. Buck J. Science. 2000; 289: 625-628Crossref PubMed Scopus (685) Google Scholar). Cytosolic localization of sAC provides both specificity and selectivity by permitting generation of cAMP proximal to intracellular targets. Furthermore, this model for cAMP action incorporates phosphodiesterases, which would act to limit diffusion and prevent nonspecific effector activation. Whether sAC participates in apoptosis was unknown. A previous report demonstrated that sAC is co-localized with mitochondria (10Zippin J.H. Chen Y. Nahirney P. Kamenetsky M. Wuttke M.S. Fischman D.A. Levin L.R. Buck J. FASEB J. 2003; 17: 82-84Crossref PubMed Scopus (240) Google Scholar). Because mitochondria play a fundamental role in apoptosis (11Vander Heiden M.G. Thompson C.B. Nat. Cell Biol. 1999; 1: E209-E216Crossref PubMed Scopus (602) Google Scholar), we hypothesized that sAC may influence the development of apoptosis by modulating the mitochondrial pathway of apoptosis. Therefore, we aimed to examine the role of sAC in apoptotic cell death, especially its role in the modulation of the mitochondria-dependent pathway of apoptosis. For this purpose, apoptosis was induced in rat coronary EC by simulated in vitro ischemia or by acidosis. By applying pharmacological inhibition of sAC or small interfering RNA (siRNA)-mediated sAC knockdown, we found that sAC activity is required for the induction of apoptosis by ischemia or acidosis. Additionally, translocation of sAC to mitochondria and the sAC-dependent release of cytochrome c suggest that this cyclase specifically regulates the mitochondrial pathway of apoptosis. Cell Culture—Coronary EC were isolated from 250–300-g male Wistar rats and maintained in Earle's minimal essential medium 199 supplemented with 10% fetal calf serum and 10% newborn calf serum (12Piper H.M. Spahr R. Mertens S. Krützfeldt A. Watanabe H. Piper H.M. Cell Culture Techniques in Heart and Vessel Research. Springer, Heidelberg1990: 158-177Crossref Google Scholar). The purity of the cell culture (>98% EC) was confirmed by immunochemical staining with antibodies against von Willebrand factor and by uptake of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate-labeled acetylated low density lipoprotein as described previously (13Ando H. Kubin T. Schaper W. Schaper J. Am. J. Physiol. Heart Circ. Physiol. 1999; 276: 1755-1768Crossref PubMed Google Scholar). Experiments were performed with monolayers reaching 90% confluence. Experimental Protocols—To induce apoptosis, cells were exposed to acidosis (pH 6.4) in M199 medium supplemented with 2% serum for 3 h at 37 °C. Analysis of the buffer pH after acidic treatment did not reveal any significant alteration. Alternatively, apoptosis was induced by treatment with simulated ischemia (glucose-free anoxia at pH 6.4) for 2 h at 37 °C as described previously (14Kumar S. Kasseckert S. Kostin S. Abdallah Y. Piper H.M. Steinhoff G. Reusch H.P. Ladilov Y. J. Cell. Mol. Med. 2007; 11: 798-809Crossref PubMed Scopus (9) Google Scholar). KH7 (10 μmol/liter; ChemDiv Inc.) (15Hess K.C. Jones B.H. Marquez B. Chen Y. Ord T.S. Kamenetsky M. Miyamoto C. Zippin J.H. Kopf G.S. Suarez S.S. Levin L.R. Williams C.J. Buck J. Moss S.B. Dev. Cell. 2005; 9: 249-259Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar), 2′,5′-dideoxyadenosine (50 μmol/liter; Calbiochem), H-89 (3 μmol/liter; Calbiochem), or adenosine cyclic 3′:5′-monophosphorothioate (30 μmol/liter; Calbiochem) was applied simultaneously with acidosis or simulated ischemia. At the end of the experiments, floating cells were collected and used together with attached cells for further analyses. We found that ≤5% of the cells were floating at the end of the experiments. Caspase-3 Activity Assay—Caspase-3 activity in cell extracts was a cellular activity the of the to the of cell extracts and of activity were performed to the The of was as at The activity of was as the in cells and apoptotic and were with and as described previously C. J. J. M. Am. J. Physiol. Cell. Mol. Physiol. PubMed Scopus Google Scholar). For a of from to was were as apoptotic with due to staining the in cell death from was performed to the were with a culture with were for were in buffer (pH and inhibitor were a protein of proteins were proteins were to The antibodies used were sAC by J. (10Zippin J.H. Chen Y. Nahirney P. Kamenetsky M. Wuttke M.S. Fischman D.A. Levin L.R. Buck J. FASEB J. 2003; 17: 82-84Crossref PubMed Scopus (240) Google cytochrome c cytochrome and protein kinase and were after with antibodies by an was confirmed by with buffer by treatment with antibodies against was performed as described by Heiden Heiden M.G. Thompson C.B. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). cells were by in buffer (pH inhibitor and To and cellular cell were at for at °C. The was at for at °C. this the was as the mitochondrial and the as the The purity of the was confirmed by the of cytochrome of sAC was by treatment of EC with to the sAC RNA or to the of In the EC were with were to the cells were transfection in M199 medium 10% fetal serum without or was with in for at and to the culture medium at a of of either were at 37 °C for was by a for sAC, which revealed of sAC and of and attached to culture were with and with For mitochondrial cells were for with with the cells were for with with and with by treatment with The cells were with a of were at a was and of For of co-localization of sAC with mitochondria, were The been for experimental to that the collected demonstrated a of from to and were for of in with cells were were as to a to the of and to the of co-localization in the and cAMP of the cellular cAMP was performed after 2 h of acidic treatment the cAMP from of cell extracts and cAMP were performed to the The at was used to the of cAMP, applying a as of the was performed by of by the was was sAC in or of i.e. of EC in cell culture medium with 2% serum for a apoptotic cells and significant activity were to simulated in vitro i.e. anoxia in with acidosis (pH for 3 h led to a significant in the of apoptotic cells and activity To examine the role of sAC in EC apoptosis, treatment with the selective sAC inhibitor KH7 (10 was performed (15Hess K.C. Jones B.H. Marquez B. Chen Y. Ord T.S. Kamenetsky M. Miyamoto C. Zippin J.H. Kopf G.S. Suarez S.S. Levin L.R. Williams C.J. Buck J. Moss S.B. Dev. Cell. 2005; 9: 249-259Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). demonstrated that KH7 at a of sAC in various cell types (15Hess K.C. Jones B.H. Marquez B. Chen Y. Ord T.S. Kamenetsky M. Miyamoto C. Zippin J.H. Kopf G.S. Suarez S.S. Levin L.R. Williams C.J. Buck J. Moss S.B. Dev. Cell. 2005; 9: 249-259Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, H. A. J. N. Kamenetsky M. N. Buck J. Levin L.R. C. J. Med. 2005; PubMed Scopus Google Scholar). In KH7 applied at of revealed a anti-apoptotic and at not of sAC with KH7 treatment abolished the in the activity and the of apoptotic cells treatment with the inhibitor simulated ischemia EC apoptosis, the apoptosis in EC induced by Because acidosis is an important component of ischemia and apoptosis in various cell types S. B. J. 1999; PubMed Google Scholar, P. Y. H. J. 2005; PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), we further sAC may play a role in apoptosis. to the inhibition of sAC with KH7 abolished the in activity and the of apoptotic cells To the anti-apoptotic of KH7 may be by an of cAMP, treatment with the cAMP (30 was acidosis. The of KH7 was abolished To the of nonspecific of KH7 we performed a in which KH7 was applied together with in the activity The demonstrated significant of KH7 activity not of sAC of further a role of sAC in apoptosis of coronary EC, of sAC was by with of EC with the sAC protein by In significant was found of which is the tmAC expressed in cells in H.J. PubMed Scopus (22) Google Scholar). Analysis of apoptosis revealed that the activation of and the in apoptotic cell were by treatment with these The of the apoptosis suppression induced by sAC knockdown was with pharmacological inhibition of sAC by KH7 treatment Therefore, sAC knockdown and the pharmacological inhibition the of sAC in apoptosis of EC under acidic To the apoptosis staining of the was performed This apoptosis confirmed by staining and activity and demonstrated of EC apoptosis tmAC in of tmAC may in apoptosis, treatment with 2′,5′-dideoxyadenosine a inhibitor of L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), was significant in apoptotic under or under acidic stress was after this treatment in cAMP were found treatment with In treatment with a forskolin-induced in cAMP a of to Therefore, these suggest a role of tmAC under acidic stimulation of tmAC acidic i.e. apoptosis of EC of the pathway in EC apoptosis. A, knockdown or inhibition of sAC with KH7 (10 treatment with acidosis the cellular of were treatment with or after inhibition of tmAC by (50 cells. treatment with 3 2 cAMP This was abolished by tmAC inhibition with (50 not by inhibition of sAC with knockdown of the of or inhibition of with H-89 (3 or with acidic apoptosis of the acidosis. shown is a of the of from extracts of EC after treatment with for of with The in of of to cAMP is the of adenylyl Therefore, we inhibition of sAC to of cellular cAMP with acidosis the cellular cAMP with the of sAC or inhibition by KH7 treatment the cellular cAMP acidic stress The of KH7 was not due to the nonspecific action tmAC KH7 forskolin-induced cAMP This is in with previous that KH7 has tmAC applied at H. A. J. N. Kamenetsky M. N. Buck J. Levin L.R. C. J. Med. 2005; PubMed Scopus Google Scholar). Because is a intracellular for cAMP, we inhibition of may the apoptosis in either with the inhibitor H-89 (3 or with the inhibitor (30 acidic activity and the of apoptotic cells Furthermore, knockdown of the of apoptosis of EC to play an essential role in the of apoptosis of an in sAC-dependent mitochondrial localization of sAC was demonstrated previously in several cell types (10Zippin J.H. Chen Y. Nahirney P. Kamenetsky M. Wuttke M.S. Fischman D.A. Levin L.R. Buck J. FASEB J. 2003; 17: 82-84Crossref PubMed Scopus (240) Google Scholar), which the of mitochondria in the action of To this hypothesis, we the mitochondrial co-localization of sAC in For this purpose, and were a low of mitochondrial localization of sAC was found in coronary with acidosis the mitochondrial localization of sAC, the mitochondrial translocation of In the release of cytochrome c from mitochondria was acidic cytochrome c was by of i.e. caspase-9 and with KH7 abolished the cytochrome c release and cleavage. To more the underlying cellular mechanisms of the sAC-dependent of the mitochondrial pathway of apoptosis, the mitochondrial co-localization of the and anti-apoptotic Bcl-2 family i.e. and was A mitochondrial translocation of and was found acidic However, Bax translocation was by inhibition of sAC with KH7 treatment significant in mitochondrial of and Bcl-2 was found in not to KH7 knockdown of sAC also abolished activation of mitochondrial apoptosis and Bax translocation has been shown previously that may Bax translocation to mitochondria by at H. H. G. N. S. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, we the sAC-dependent mitochondrial translocation of Bax also requires the We found that inhibition of by treatment with 3 H-89 or adenosine cyclic 3′:5′-monophosphorothioate the mitochondrial translocation of Bax under acidic stress To further the of mitochondrial Bax translocation for apoptosis in a was which revealed a significant the of mitochondrial Bax and the of apoptosis activity and the of apoptotic under different experimental of density of Bax in mitochondrial by and of apoptosis. acidosis acidosis acidosis acidosis sAC acidosis The of this was to sAC plays a role in apoptosis of coronary EC induced by or acidic treatment and the signaling that be The as (i) sAC plays a role in simulated or EC apoptosis. (ii) The signaling pathway of the translocation of sAC to mitochondria and the activation of the mitochondrial pathway of apoptosis, i.e. translocation of Bax to mitochondria, release of cytochrome and of To examine the role of sAC, we i.e. ischemia and acidosis. has been shown to be an important of apoptosis A. L. R. D.S. Circ. Res. PubMed Scopus Google Scholar, V. A. R. J. Mol. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), and acidosis is its essential component J.H. V. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). is to several e.g. D. Res. Google and H.P. J. Am. J. Full Text PDF PubMed Scopus Google Scholar). The of acidosis was demonstrated in various cell types S. B. J. 1999; PubMed Google Scholar, P. Y. H. J. 2005; PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google as as in the J. M. C. J. 2008; Full Text Full Text PDF PubMed Scopus Google and in from J.H. 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Insel P.A. J. Biol. Chem. 2004; 279: 20858-20865Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The for this discrepancy is and may be due to differences in cell types or experimental models. from the nonspecific treatment with cAMP the of these tmAC Whether the of cAMP may also play a role in apoptosis unknown. Therefore, in this we the hypothesis that sAC may be in apoptosis induced by an different i.e. pharmacological inhibition of sAC with KH7 and sAC knockdown by we demonstrated that sAC plays a role in or apoptosis. In tmAC not to be in apoptosis in coronary EC inhibition of this cyclase with cAMP and apoptotic 3 and pharmacological stimulation of tmAC acidic stress by treatment with cellular cAMP and apoptosis, an anti-apoptotic of tmAC in This the role of The for the of these is Increasing evidence suggests that specificity of signal is of the second and its signaling cAMP, such either to the plasma or in the of intracellular i.e. mitochondria and (10Zippin J.H. Chen Y. Nahirney P. Kamenetsky M. Wuttke M.S. Fischman D.A. Levin L.R. Buck J. FASEB J. 2003; 17: 82-84Crossref PubMed Scopus (240) Google Scholar), cAMP to various and cellular Indeed, a has demonstrated that cAMP by tmAC and sAC to different cell A. Levin L.R. Buck J. J.A. J. Res. 2008; PubMed Scopus Google Scholar). Furthermore, EC, M. T. Circ. Res. 2006; PubMed Scopus Google of tmAC and sAC activation the EC In with these the results of this further the of the cellular of the cAMP for or preventing apoptosis. cAMP a of the cell plays a significant role in the of cAMP signal signaling is by which and at in to J.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Google Scholar). Because of the different localization of sAC and tmAC of cAMP, of to different targets may lead to i.e. or The activity of mammalian sAC has been shown to be by and i.e. and Y. Cann M.J. Litvin T.N. Iourgenko V. Sinclair M.L. Levin L.R. Buck J. Science. 2000; 289: 625-628Crossref PubMed Scopus (685) Google Scholar, W. Wang Zhang J. Am. J. Physiol. Cell Physiol. 2005; PubMed Scopus Google Scholar). The of sAC activation to model the in the medium was not has been shown previously that of coronary EC at pH to S. Kasseckert S. Kostin S. Abdallah Y. C. A. Reusch H.P. Piper H.M. Steinhoff G. Ladilov Y. Res. 2007; PubMed Scopus Google Scholar, Y. C. A. M. T. Piper H.M. Res. 2000; PubMed Scopus Google Scholar), which may to activation of we did not the activity of sAC, of cellular cAMP revealed a significant sAC-dependent of this under acidic to be an important in sAC-dependent apoptosis. The role of mitochondria in or apoptosis was demonstrated previously J.H. V. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, P. H.M. P. Circ. Res. 2003; PubMed Scopus Google Scholar). In with these in this we activation of the mitochondrial pathway of apoptosis. Furthermore, (i) mitochondrial translocation of sAC and (ii) of cytochrome c caspase-9 and mitochondrial Bax translocation by inhibition of sAC that activation of the mitochondrial pathway is under the of in model the of cells demonstrated a of mitochondrial co-localization with sAC under acidic of the cells sAC translocation may in activation of the mitochondrial pathway of apoptosis, it not lead to cell sAC may the mitochondrial pathway of apoptosis is Increasing evidence suggests that translocation of of the Bcl-2 protein e.g. from the cytosol to mitochondria, by of the mitochondrial is an important for the release of mitochondrial cytochrome c L. P. S. 2006; PubMed Scopus Google Scholar). that inhibition of sAC translocation of Bax to mitochondria may suggest that sAC participates in the activation and translocation of Bax to mitochondria. In with this previous demonstrated several mechanisms for cAMP-dependent Bax cAMP-dependent activation may L. J. F. J. Biol. Chem. 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Buck for antibodies for We M. of for the The of G. and is with