Matrix metalloproteinases mediate β-adrenergic receptor-stimulated apoptosis in adult rat ventricular myocytes
Matrix metalloproteinases mediate β-adrenergic receptor-stimulated apoptosis in adult rat ventricular myocytes
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DOI：
10.1152/ajpcell.00606.2004
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发表时间：
2005-07-01
影响因子：
5.5
通讯作者：
Singh, K
中科院分区：
文献类型：
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作者：
Menon, B;Singh, M;Singh, K
基质金属蛋白酶（MMPs）及其抑制剂（TIMPs）的合成和活性变化与心肌重塑有关。在这里，我们测量了MMP和TIMP的表达和活性，并检验了MMP活性增加在β-肾上腺素能受体（β-AR）刺激的成年大鼠心室肌细胞（ARVMs）凋亡中起促凋亡作用的假设。通过实时PCR分析，β-AR刺激（异丙肾上腺素，24 h）增加MMP-2和TIMP-1的mRNA水平，而其降低TIMP-2 mRNA水平。Western印迹分析、免疫细胞化学分析、凝胶内酶谱和MMP-2活性测定证实了β-AR刺激的MMP-2蛋白水平和活性增加。通过TdT介导的dUTP缺口末端标记染色测定，使用GM-6001（MMPs的广谱抑制剂）、SB 3CT（MMP-2的抑制剂）和纯化的TIMP-2抑制MMPs可抑制β-AR刺激的细胞凋亡。单独用活性MMP-2处理增加了凋亡细胞的数量。GM-6001和SB 3CT预处理可抑制MMP-2介导的细胞凋亡。免疫共沉淀研究表明，在β-AR刺激后，MMP-2与β（1）-整合素的物理结合增加。使用SB 3CT抑制MMP-2或使用层粘连蛋白刺激β（1）-整联蛋白信号传导抑制MMP-2与β（1）-整联蛋白的增加的缔合。β-AR刺激增加了多聚-DP-核糖-聚合酶的裂解，这被MMP-2的抑制所抑制。这些数据表明：1）β-AR刺激增加MMP-2表达和活性并抑制TIMP-2表达; 2）MMP（最可能是MMP-2）的抑制抑制β-AR刺激的细胞凋亡;和3）MMP-2的细胞凋亡作用可以至少部分地通过其与β（1）整联蛋白和聚ADP-核糖聚合酶切割的相互作用来介导。
Changes in the synthesis and activity of matrix metalloproteinases ( MMPs) and their inhibitors (TIMPs) are associated with myocardial remodeling. Here we measured the expression and activity of MMPs and TIMPs, and tested the hypothesis that increased MMP activity plays a proapoptotic role in beta-adrenergic receptor (beta-AR)-stimulated apoptosis of adult rat ventricular myocytes (ARVMs). beta-AR stimulation ( isoproterenol, 24 h) increased mRNA levels of MMP-2 and TIMP-1 while it decreased TIMP-2 mRNA levels as analyzed by real-time PCR. Western blot analysis, immunocytochemical analysis, in-gel zymography, and MMP-2 activity assay confirmed beta-AR-stimulated increases in MMP-2 protein levels and activity. Inhibition of MMPs using GM-6001 ( a broad-spectrum inhibitor of MMPs), SB3CT ( inhibitor of MMP-2), and purified TIMP-2 inhibited beta-AR-stimulated apoptosis as determined by TdT-mediated dUTP nick end labeling staining. Treatment with active MMP-2 alone increased the number of apoptotic cells. This increase in MMP-2-mediated apoptosis was inhibited by GM-6001 and SB3CT pretreatment. Coimmunoprecipitation studies indicated increased physical association of MMP-2 with beta(1)-integrins after beta-AR stimulation. Inhibition of MMP-2 using SB3CT or stimulation of beta(1)-integrin signaling using laminin inhibited the increased association of MMP-2 with beta(1)-integrins. beta-AR stimulation increased poly-DP- ribose-polymerase cleavage, which was inhibited by inhibition of MMP-2. These data suggest the following: 1) beta-AR stimulation increases MMP-2 expression and activity and inhibits TIMP-2 expression; 2) inhibition of MMPs, most likely MMP-2, inhibits beta-AR-timulated apoptosis; and 3) the apoptotic effects of MMP-2 may be mediated, at least in part, via its interaction with beta(1) integrins and poly-ADP-ribose-polymerase cleavage.