The role of geranylgeranylated proteins in human mesangial cell proliferation
The role of geranylgeranylated proteins in human mesangial cell proliferation
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DOI：
10.1038/sj.ki.5001713
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发表时间：
2006-10-01
影响因子：
19.6
通讯作者：
Hendry, B. M.
中科院分区：
文献类型：
--
作者：
Khwaja, A.;Sharpe, C. C.;Hendry, B. M.
鸟嘌呤 5'-三磷酸酶 (GTPase) 的 Rho 家族在调节细胞增殖、肾小管间质纤维化和肾小球血流动力学方面发挥着关键作用。通过添加香叶基香叶基部分进行的 RhoGTPase 翻译后异戊二烯化对于细胞定位和信号传导活性至关重要。本研究使用 GGTI 298（一种 GG 的特异性抑制剂）和 (ii) 洛伐他汀（一种 HMG-辅乙酰基 A 还原酶抑制剂）来研究 (i) 抑制香叶基香叶基化 (GG) 对人系膜细胞 (HMC) 增殖和凋亡的影响，洛伐他汀会耗尽异戊二烯化底物的可用性。使用活细胞数测定和测量溴脱氧尿苷 (BrdU) 掺入来评估 HMC 增殖。 Hoechst 33342染色用于测定细胞凋亡。通过蛋白质印迹分析细胞外信号调节蛋白激酶 (Erk) 1/2 和 Akt 激活。使用 Rhotekin Pull-down 测定法测定 Rho 激活。进行免疫细胞化学来研究对肌动蛋白细胞骨架和 RhoA 定位的影响。 GGTI 298 (10-20 μM) 和洛伐他汀 (5-10 μM) 有效抑制血小板衍生生长因子和血清刺激的 HMC 增殖并诱导细胞凋亡。洛伐他汀的这些作用通过与香叶基香叶基焦磷酸盐共同孵育而减弱。 C3 外切酶是一种专门针对 Rho 的梭菌毒素，也能抑制 BrdU 掺入并促进细胞凋亡。 GGTI 298 增加 RhoA 的胞质表达，阻止 RhoA 激活，并抑制 Erk1/2 和存活蛋白 Akt 的激活。 GGTI 298、洛伐他汀和 C3 外切酶抑制 HMC 增殖并促进细胞凋亡。抑制 GG 会增加胞质 RhoA 表达，破坏肌动蛋白细胞骨架，并抑制 RhoA 激活。这些结果表明，用他汀类药物或 GGTI 298 靶向香叶基香叶基化蛋白是治疗人类系膜增生性肾病的一种有前途的治疗策略。
The Rho family of guanine 5'-triphosphatases (GTPases) play a key role in regulating cell proliferation, tubulointerstitial fibrosis, and glomerular hemodynamics. The post-translational prenylation of RhoGTPases by the addition of a geranylgeranyl moiety is critical for cellular localization and signaling activity. This study investigates the effects of (i) inhibiting geranylgeranylation (GG) in human mesangial cell (HMC) proliferation and apoptosis, using GGTI 298, a specific inhibitor of GG and (ii) lovastatin, an HMG-coacetyl A-reductase inhibitor, which depletes the availability of prenylation substrates. HMC proliferation was assessed using an assay of viable cell number and measuring bromodeoxyuridine (BrdU) incorporation. Hoechst 33342 staining was used to determine apoptosis. Extracellular signal-regulated protein kinase (Erk) 1/2 and Akt activation were analysed by Western blotting. Rho activation was determined using the Rhotekin pull-down assay. Immunocytochemistry was performed to study the effects on the actin cytoskeleton and RhoA localization. GGTI 298 (10-20 mu M) and lovastatin (5-10 mu M) potently inhibited platelet-derived growth factor and serum-stimulated HMC proliferation and induced apoptosis. These effects of lovastatin were attenuated by co-incubation with geranylgeranylpyrophosphate. C3 exoenzyme, a clostridial toxin that specifically targets Rho also inhibited BrdU incorporation and promoted apoptosis. GGTI 298 increased cytosolic expression of RhoA, prevented RhoA activation, and inhibited the activation of Erk1/2 and the survival protein Akt. GGTI 298, lovastatin, and C3 exoenzyme inhibit HMC proliferation and promote apoptosis. Inhibiting GG increases cytosolic RhoA expression, disrupts the actin cytoskeleton, and inhibits RhoA activation. These results suggest that targeting geranylgeranylated proteins with statins or GGTI 298 is a promising therapeutic strategy in human mesangioproliferative renal disease.