Cytosolic 85-kDa Phospholipase A2-mediated Release of Arachidonic Acid Is Critical for Proliferation of Vascular Smooth Muscle Cells*
Cytosolic 85-kDa Phospholipase A2-mediated Release of Arachidonic Acid Is Critical for Proliferation of Vascular Smooth Muscle Cells*
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DOI：
10.1074/jbc.272.48.30504
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发表时间：
1997-11
期刊：
影响因子：
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通讯作者：
K. Anderson;A. Roshak;J. Winkler;M. McCord;L. Marshall
中科院分区：
文献类型：
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作者：
K. Anderson;A. Roshak;J. Winkler;M. McCord;L. Marshall
最近的证据表明，花生四烯酸（AA）可能参与调节细胞增殖。AA从细胞磷脂中释放的主要机制是通过磷脂酶A2（PLA 2）水解。本研究的目的是研究不同的14-kDa和85-kDa的PLA 2酶在人冠状动脉血管平滑肌细胞（hCAVSMC）增殖的作用。培养的hCAVSMC在生长培养基存在下增殖，典型的倍增时间为30-40小时，在达到汇合时以较慢的增殖速率生长（第8天），并最终经历生长的接触抑制（第10天）。在10天的培养期内，II型14-kDa PLA 2活性和质量均无变化。与此相反，85-kDa的PLA 2蛋白活性和mRNA随着培养时间的推移而下降。85-kDa PLA 2的减少与DNA合成的减少相关，表明85-kDa PLA 2与增殖之间可能存在关联。为了直接评价85-kDa PLA 2在增殖中的作用，我们检测了85-kDa PLA 2抑制剂（AACOCF 3）和85-kDa PLA 2反义寡核苷酸对增殖的影响。这两种试剂剂量依赖性地抑制增殖，而14 kDa的PLA 2抑制剂（SB 203347），钙非依赖性PLA 2抑制剂（HELSS），85 kDa的正义寡核苷酸，和一个无关的乱序控制寡核苷酸没有效果。85-kDa PLA 2影响细胞增殖的机制尚不清楚。抑制85-kDa PLA 2活性既不产生阶段特异性细胞周期停滞，也不产生细胞凋亡（荧光激活细胞分选仪分析）。添加AA（20 μm）减弱了AACOCF 3和85-kDa反义寡核苷酸的作用，表明AA是细胞增殖的关键介质。然而，虽然前列腺素E2（PGE 2）存在于培养基中，它在培养早期（第3天）达到峰值，吲哚美辛对细胞增殖没有影响，表明hCAVSMC增殖不是通过PGE 2介导的。这些数据提供了第一个直接的证据表明，PLA 2参与控制VSMC增殖，并表明85 kDa的PLA 2介导的AA的释放是至关重要的细胞增殖。
Recent evidence suggests that arachidonic acid (AA) may be involved in regulating cellular proliferation. The predominant mechanism of AA release from cellular phospholipids is via phospholipase A2 (PLA2) hydrolysis. The purpose of this study was to examine the roles of the distinct 14-kDa and 85-kDa PLA2 enzymes in human coronary artery vascular smooth muscle cell (hCAVSMC) proliferation. Cultured hCAVSMCs proliferate in the presence of growth medium with a typical doubling time of 30–40 h, grow at a slower proliferative rate upon reaching confluency (day 8), and eventually undergo contact inhibition of growth (day 10). Neither Type II 14-kDa PLA2activity nor mass changed over a 10-day culture period. In contrast, 85-kDa PLA2 protein activity and mRNA decreased as time in culture progressed. This reduction in 85-kDa PLA2correlated with reductions in DNA synthesis and suggested a possible association between 85-kDa PLA2 and proliferation. To directly evaluate the role of the 85-kDa PLA2 in proliferation we examined the effects of an 85-kDa PLA2inhibitor (AACOCF3) and 85-kDa PLA2 antisense oligonucleotides on proliferation. Both reagents dose dependently inhibited proliferation, whereas a 14-kDa PLA2 inhibitor (SB203347), a calcium-independent PLA2 inhibitor (HELSS), an 85-kDa sense oligonucleotide, and a nonrelevant scrambled control oligonucleotide had no effect. The mechanism by which 85-kDa PLA2 influences cellular proliferation remains unclear. Inhibition of 85-kDa PLA2 activity produced neither phase-specific cell cycle arrest nor apoptosis (fluorescence-activated cell sorter analysis). Addition of AA (20 μm) attenuated the effects of both AACOCF3 and 85-kDa antisense oligonucleotides implicating AA as a key mediator in cellular proliferation. However, although prostaglandin E2(PGE2) was present in the culture medium, it peaked early (day 3) in culture, and indomethacin had no effect on cellular proliferation indicating that hCAVSMC proliferation was not mediated through PGE2. These data provide the first direct evidence that PLA2 is involved in control of VSMC proliferation and indicate that 85-kDa PLA2-mediated liberation of AA is critical for cellular proliferation.