Khwaja Arif, Sharpe Claire C, Noor Mazhar, Kloog Yoel, Hendry Bruce M
Department of Renal Medicine, GKT School of Medicine, King's College London, London, United Kingdom.
Kidney Int. 2005 Aug;68(2):474-86. doi: 10.1111/j.1523-1755.2005.00425.x.
Many of the proliferative cytokines implicated in human mesangial cell (HMC) proliferation signal through the superfamily of Ras GTPases. The Ras antagonist, S-trans, trans- farnesylthiosalicylic acid (FTS), was used to investigate the effects of the inhibition of Ras signaling on HMC proliferation.
Ras expression and membrane localization, MAPK, and Akt activation were analyzed by Western blotting. Ras activation was determined with a pull-down assay using the Ras-binding domain of Raf. HMC growth curves were assessed using the MTS assay of viable cell number, while DNA synthesis was measured with BrdU incorporation. Hoechst 33342 staining was used to determine apoptosis.
FTS reduced the membrane localization of Ras in both serum and platelet-derived growth factor (PDGF). FTS (7.5-20 micromol/L) potently inhibited PDGF-induced HMC proliferation but had no effect on serum-induced proliferation. FTS (10-20 micromol/L) inhibited both Ras and phospho-MAPK activation by serum and PDGF. Furthermore, FTS (10-20 micromol/L) increased HMC apoptosis in the presence of PDGF but not in serum. Moreover, PDGF-stimulated activation of the survival protein Akt was inhibited by FTS. In contrast, serum-stimulated activation of Akt was unaffected by FTS.
FTS (5-20 micromol/L) inhibits PDGF-induced but not serum-induced HMC proliferation. FTS (10-20 micromol/L) also promotes HMC apoptosis in the presence of PDGF but not serum. These effects appear to be mediated by inhibitory effects on Ras-dependent signaling that occur as a result of the dislodgment of Ras from its membrane-anchorage sites by FTS. The selectivity of FTS toward PDGF-driven HMC proliferation suggests that FTS may be a valuable therapeutic in mesangioproliferative renal disease.
许多参与人系膜细胞（HMC）增殖的促增殖细胞因子通过Ras GTP酶超家族进行信号传导。使用Ras拮抗剂S-反式，反式-法尼基硫代水杨酸（FTS）来研究抑制Ras信号传导对HMC增殖的影响。
通过蛋白质免疫印迹分析Ras表达、膜定位、丝裂原活化蛋白激酶（MAPK）和Akt激活。使用Raf的Ras结合结构域通过下拉试验测定Ras激活。使用活细胞数量的MTS试验评估HMC生长曲线，同时用溴脱氧尿苷（BrdU）掺入法测量DNA合成。使用Hoechst 33342染色来确定细胞凋亡。
FTS降低了血清和血小板衍生生长因子（PDGF）中Ras的膜定位。FTS（7.5 - 20微摩尔/升）有效抑制PDGF诱导的HMC增殖，但对血清诱导的增殖没有影响。FTS（10 - 20微摩尔/升）抑制血清和PDGF诱导 的Ras和磷酸化MAPK激活。此外，FTS（10 - 20微摩尔/升）在存在PDGF的情况下增加HMC凋亡，但在血清中则不然。此外，FTS抑制PDGF刺激的存活蛋白Akt的激活。相比之下，血清刺激的Akt激活不受FTS影响。
FTS（5 - 20微摩尔/升）抑制PDGF诱导的而非血清诱导的HMC增殖。FTS（10 - 20微摩尔/升）在存在PDGF而非血清的情况下也促进HMC凋亡。这些作用似乎是由对Ras依赖性信号传导的抑制作用介导的，这是由于FTS使Ras从其膜锚定位点脱离所致。FTS对PDGF驱动的HMC增殖的选择性表明FTS可能是系膜增生性肾病中有价值的治疗药物。
