Orlov S N, Thorin-Trescases N, Dulin N O, Dam T V, Fortuno M A, Tremblay J, Hamet P
Centre de Recherche du CHUM, Campus Hotel-Dieu, University of Montreal, Montreal, Quebec, Canada.
Cell Death Differ. 1999 Jul;6(7):661-72. doi: 10.1038/sj.cdd.4400539.
Intracellular signaling pathways that are involved in protection of vascular smooth muscle cells (VSMC) from apoptosis remain poorly understood. This study examines the effect of activators of cAMP/cGMP signaling on apoptosis in non-transfected VSMC and in VSMC transfected with c-myc (VSMC-MYC) or with its functional analogue, E1A-adenoviral protein (VSMC-E1A). Serum-deprived VSMC-E1A exhibited the highest apoptosis measured as the content of chromatin and low molecular weight DNA fragments, phosphatidylserine content in the outer surface of plasma membrane and caspase-3 activity (ten-, five-, four- and tenfold increase after 6 h of serum withdrawal, respectively). In VSMC-E1A, the addition of an activator of adenylate cyclase, forskolin, abolished chromatin cleavage, DNA laddering, caspase-3 activation and the appearance of morphologically-defined apoptotic cells triggered by 6 h of serum deprivation. In non-transfected VSMC and in VSMC-MYC, 6 h serum deprivation led to approximately six- and threefold activation of chromatin cleavage, respectively, that was also blocked by forskolin. In VSMC-E1A, inhibition of apoptosis was observed with other activators of cAMP signaling (cholera toxin, isoproterenol, adenosine, 8-Br-cAMP), whereas 6 h incubation with modulators of cGMP signaling (8-Br-cGMP, nitroprusside, atrial natriuretic peptide, L-NAME) did not affect the development of apoptotic machinery. The antiapoptotic effect of forskolin was abolished in 24 h of serum deprivation that was accompanied by normalization of intracellular cAMP content and protein kinase A (PKA) activity. Protection of VSMC-E1A from apoptosis by forskolin was blunted by PKA inhibitors (H-89 and KT5720), whereas transfection of cells with PKA catalytic subunit attenuated apoptosis triggered by serum withdrawal. The protection of VSMC-E1A by forskolin from apoptosis was insensitive to modulators of cytoskeleton assembly (cytochalasin B, colchicine). Neither acute (30 min) nor chronic (24 h) exposure of VSMC to forskolin modified basal and serum-induced phosphorylation of the MAP kinase ERK1/2. Thus, our results show that activation of cAMP signaling delays the development of apoptosis in serum-deprived VSMC at a site upstream of caspase-3 via activation of PKA and independently of cAMP-induced reorganization of the cytoskeleton network and the ERK1/2-terminated MAPK signaling cascade.
参与保护血管平滑肌细胞（VSMC）免于凋亡的细胞内信号通路仍未得到充分了解。本研究考察了cAMP/cGMP信号激活剂对未转染的VSMC以及转染了c-myc（VSMC-MYC）或其功能类似物E1A腺病毒蛋白（VSMC-E1A）的VSMC凋亡的影响。血清剥夺的VSMC-E1A表现出最高的凋亡水平，以染色质和低分子量DNA片段的含量、质膜外表面的磷脂酰丝氨酸含量以及caspase-3活性来衡量（血清撤除6小时后分别增加了10倍、5倍、4倍和10倍）。在VSMC-E1A中，添加腺苷酸环化酶激活剂福斯可林可消除血清剥夺6小时引发的染色质裂解、DNA梯状条带、caspase-3激活以及形态学上确定的凋亡细胞的出现。在未转染的VSMC和VSMC-MYC中，6小时血清剥夺分别导致染色质裂解激活约6倍和3倍，这也被福斯可林阻断。在VSMC-E1A中，用其他cAMP信号激活剂（霍乱毒素、异丙肾上腺素、腺苷、8-溴-cAMP）观察到凋亡受到抑制，而用cGMP信号调节剂（8-溴-cGMP、硝普钠、心房利钠肽、L-精氨酸甲酯）孵育6小时并不影响凋亡机制的发展。在血清剥夺24小时时，福斯可林的抗凋亡作用消失，同时细胞内cAMP含量和蛋白激酶A（PKA）活性恢复正常。福斯可林对VSMC-E1A的抗凋亡保护作用被PKA抑制剂（H-89和KT5720）减弱，而用PKA催化亚基转染细胞可减轻血清撤除引发的凋亡。福斯可林对VSMC-E1A的抗凋亡保护作用对细胞骨架组装调节剂（细胞松弛素B、秋水仙碱）不敏感。VSMC急性（30分钟）或慢性（24小时）暴露于福斯可林均未改变基础状态和血清诱导的丝裂原活化蛋白激酶ERK1/2的磷酸化。因此，我们的结果表明，cAMP信号的激活通过激活PKA并独立于cAMP诱导的细胞骨架网络重组和ERK1/2终止的丝裂原活化蛋白激酶信号级联反应，在caspase-3上游位点延迟血清剥夺的VSMC中凋亡的发展。
