Department of Medicine, University of Alberta, Edmonton, Alberta, Canada.
Department of Medicine, University of Alberta, Edmonton, Alberta, Canada
Am J Physiol Renal Physiol. 2014 Sep 1;307(5):F623-33. doi: 10.1152/ajprenal.00070.2014. Epub 2014 Jul 9.
The function of TIMAP, an endothelial cell (EC)-predominant protein phosphatase 1-regulatory subunit, is poorly understood. We explored the potential role of TIMAP in the Akt-dependent regulation of glomerular EC proliferation, survival, and in vitro angiogenesis. To deplete TIMAP, the EC were transfected with TIMAP-specific or nonspecific small interfering (si) RNA. The rate of electrical impedance development across subconfluent EC monolayers, a measure of the time-dependent increase in EC number, was 93 ± 2% lower in TIMAP-depleted than in control EC. This effect on cell proliferation was associated with reduced DNA synthesis and increased apoptosis: TIMAP silencing reduced 5-ethynyl-2'-deoxyuridine incorporation by 38 ± 2% during the exponential phase of EC proliferation, and cleaved caspase 3 as well as caspase 3 activity increased in TIMAP-depleted relative to control cells. Furthermore, TIMAP depletion inhibited the formation of angiogenic sprouts by glomerular EC in three-dimensional culture. TIMAP depletion strongly diminished growth factor-stimulated Akt phosphorylation without altering ERK1/2 phosphorylation, suggesting a specific effect on the PI3K/Akt/PTEN pathway. Endogenous TIMAP and PTEN colocalized in EC and coimmunoprecipitated from EC lysates. The inhibitory PTEN phosphorylation on S370 was significantly reduced in TIMAP-depleted compared with control EC, while phosphorylation of PTEN on the S380/T382/T383 cluster remained unchanged. Finally, the PTEN inhibitor bpV(phen) fully reversed the suppressive effect of TIMAP depletion on Akt phosphorylation. The data indicate that in growing EC, TIMAP is necessary for Akt-dependent EC proliferation, survival, and angiogenic sprout formation and that this effect of TIMAP is mediated by inhibition of the tumor suppressor PTEN.
TIMAP 是一种主要存在于血管内皮细胞（EC）中的蛋白磷酸酶 1 调节亚基，其功能尚不清楚。我们探讨了 TIMAP 在 Akt 依赖性调节肾小球 EC 增殖、存活和体外血管生成中的潜在作用。为了耗尽 TIMAP，我们用 TIMAP 特异性或非特异性小干扰（si）RNA 转染 EC。在 TIMAP 耗尽的 EC 中，跨亚汇合 EC 单层的电阻抗发展率（衡量 EC 数量随时间的增加）比对照 EC 低 93±2%。这种对细胞增殖的影响与减少 DNA 合成和增加细胞凋亡有关：TIMAP 沉默使 EC 增殖指数期的 5-乙炔基-2'-脱氧尿苷掺入减少 38±2%，并且在 TIMAP 耗尽的细胞中 cleaved caspase 3 以及 caspase 3 活性增加。此外，TIMAP 耗尽抑制了三维培养中肾小球 EC 形成血管生成芽。TIMAP 耗尽强烈抑制生长因子刺激的 Akt 磷酸化，而不改变 ERK1/2 磷酸化，表明对 PI3K/Akt/PTEN 途径有特异性影响。内源性 TIMAP 和 PTEN 在 EC 中共定位，并从 EC 裂解物中共同免疫沉淀。与对照 EC 相比，TIMAP 耗尽的细胞中 PTEN 的 S370 抑制性磷酸化显著减少，而 PTEN 的 S380/T382/T383 簇的磷酸化保持不变。最后，PTEN 抑制剂 bpV(phen) 完全逆转了 TIMAP 耗尽对 Akt 磷酸化的抑制作用。数据表明，在生长的 EC 中，TIMAP 对于 Akt 依赖性 EC 增殖、存活和血管生成芽形成是必需的，而 TIMAP 的这种作用是通过抑制肿瘤抑制因子 PTEN 介导的。
