Duann Pu, Ho Teh-Yuan, Desai Bijal D, Kapoian Toros, Cowen Daniel S, Lianos Elias A
Department of Medicine, Nephrology Division, Robert Wood Johnson Medical School, New Brunswick, NJ 0903-0019, USA.
J Investig Med. 2005 Jan;53(1):37-43. doi: 10.2310/6650.2005.00004.
Mesangial cell apoptosis has been proposed as a means of resolution of glomerular hypercellularity in proliferative forms of glomerular disease. We previously demonstrated that adenosine causes mesangial cell apoptosis by stimulating the A3-type adenosine receptor. This is a G protein-coupled receptor shown to activate kinases involved in apoptotic signaling. In this work, we assessed changes in phosphorylation of the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK)1/2 and in levels of specific pro- and antiapoptotic proteins following exposure of mesangial cells to the A3 adenosine receptor agonist N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA). Cultured mesangial cells were incubated with IB-MECA for 30 minutes and 6, 24, and 48 hours. IB-MECA was used at a concentration (30 microM) that induces a reproducible degree of mesangial cell apoptosis. Changes in ERK1/2 phosphorylation and in protein levels of Bcl-2, Bax, and caspase 3 were assessed by Western blot analysis. IB-MECA markedly increased phosphorylation of ERK1/2. This effect peaked at 5 minutes, dissipated by 20 minutes, and was abolished by the inhibitor of ERK phosphorylation, compound U0126, in a dose-dependent manner. This inhibitor had no effect on the extent of IB-MECA-induced apoptosis. Bcl-2 levels progressively declined, whereas those of Bax and activated caspase 3 increased. These observations indicate that stimulation of the A3-type adenosine receptor causes mesangial cell apoptosis via mechanisms independent of ERK activation. The observations also point to an imbalance in the expression of antiapoptotic (Bcl-2) and proapoptotic (Bax, caspase 3) proteins as a potential mechanism underlying adenosine-induced mesangial cell apoptosis.
系膜细胞凋亡被认为是增生性肾小球疾病中肾小球细胞增多症消退的一种方式。我们之前证明，腺苷通过刺激A3型腺苷受体导致系膜细胞凋亡。这是一种G蛋白偶联受体，已显示其可激活参与凋亡信号传导的激酶。在这项研究中，我们评估了系膜细胞暴露于A3腺苷受体激动剂N(6)-(3-碘苄基)-腺苷-5'-N-甲基脲苷(IB-MECA)后，丝裂原活化蛋白激酶细胞外信号调节激酶(ERK)1/2的磷酸化变化以及特定促凋亡和抗凋亡蛋白的水平变化。将培养的系膜细胞与IB-MECA孵育30分钟、6小时、24小时和48小时。使用浓度为30 microM的IB-MECA，该浓度可诱导可重复程度的系膜细胞凋亡。通过蛋白质印迹分析评估ERK1/2磷酸化以及Bcl-2、Bax和半胱天冬酶3蛋白水平的变化。IB-MECA显著增加ERK1/2的磷酸化水平。这种作用在5分钟时达到峰值，20分钟时消失，并被ERK磷酸化抑制剂化合物U0126以剂量依赖的方式消除。该抑制剂对IB-MECA诱导的凋亡程度没有影响。Bcl-2水平逐渐下降，而Bax和活化的半胱天冬酶3水平升高。这些观察结果表明A3型腺苷受体的刺激通过独立于ERK激活的机制导致系膜细胞凋亡。这些观察结果还表明抗凋亡蛋白(Bcl-2)和促凋亡蛋白(Bax、半胱天冬酶3)表达失衡是腺苷诱导系膜细胞凋亡的潜在机制之一。
