Kume Shinji, Haneda Masakazu, Kanasaki Keizo, Sugimoto Toshiro, Araki Shin-ichi, Isshiki Keiji, Isono Motohide, Uzu Takashi, Guarente Leonard, Kashiwagi Atsunori, Koya Daisuke
Department of Medicine, Shiga University of Medical Science, Otsu, Shiga 520-2192, Japan.
J Biol Chem. 2007 Jan 5;282(1):151-8. doi: 10.1074/jbc.M605904200. Epub 2006 Nov 10.
SIRT1, a class III histone deacetylase, is considered a key regulator of cell survival and apoptosis through its interaction with nuclear proteins. In this study, we have examined the likelihood and role of the interaction between SIRT1 and Smad7, which mediates transforming growth factor beta (TGFbeta)-induced apoptosis in renal glomerular mesangial cells. Immunoprecipitation analysis revealed that SIRT1 directly interacts with the N terminus of Smad7. Furthermore, SIRT1 reversed acetyl-transferase (p300)-mediated acetylation of two lysine residues (Lys-64 and -70) on Smad7. In mesangial cells, the Smad7 expression level was reduced by SIRT1 overexpression and increased by SIRT1 knockdown. SIRT1-mediated deacetylation of Smad7 enhanced Smad ubiquitination regulatory factor 1 (Smurf1)-mediated ubiquitin proteasome degradation, which contributed to the low expression of Smad7 in SIRT1-overexpressing mesangial cells. Stimulation by TGFbeta or overexpression of Smad7 induced mesangial cell apoptosis, as assessed by morphological apoptotic changes (nuclear condensation) and biological apoptotic markers (cleavages of caspase3 and poly(ADP-ribose) polymerase). However, TGFbeta failed to induce apoptosis in Smad7 knockdown mesangial cells, indicating that Smad7 mainly mediates TGFbeta-induced apoptosis of mesangial cells. Finally, SIRT1 overexpression attenuated both Smad7- and TGFbeta-induced mesangial cell apoptosis, whereas SIRT1 knockdown enhanced this apoptosis. We have concluded that Smad7 is a new target molecule for SIRT1 and SIRT1 attenuates TGFbeta-induced mesangial cell apoptosis through acceleration of Smad7 degradation. Our results suggest that up-regulation of SIRT1 deacetylase activity is a potentially useful therapeutic strategy for prevention of TGFbeta-related kidney disease through its effect on cell survival.
沉默信息调节因子1（SIRT1）是一种III类组蛋白去乙酰化酶，通过与核蛋白相互作用，被认为是细胞存活和凋亡的关键调节因子。在本研究中，我们检测了SIRT1与Smad7相互作用的可能性及其作用，Smad7介导转化生长因子β（TGFβ）诱导的肾小球系膜细胞凋亡。免疫沉淀分析显示，SIRT1直接与Smad7的N端相互作用。此外，SIRT1逆转了乙酰转移酶（p300）介导的Smad7上两个赖氨酸残基（Lys-64和-70）的乙酰化。在系膜细胞中，SIRT1过表达降低了Smad7的表达水平，而SIRT1敲低则增加了Smad7的表达水平。SIRT1介导的Smad7去乙酰化增强了Smad泛素化调节因子1（Smurf1）介导的泛素蛋白酶体降解，这导致了SIRT1过表达的系膜细胞中Smad7的低表达。通过形态学凋亡变化（核浓缩）和生物学凋亡标志物（caspase3和聚（ADP-核糖）聚合酶的裂解）评估，TGFβ刺激或Smad7过表达诱导系膜细胞凋亡。然而，TGFβ未能在Smad7敲低的系膜细胞中诱导凋亡，表明Smad7主要介导TGFβ诱导的系膜细胞凋亡。最后，SIRT1过表达减弱了Smad7和TGFβ诱导的系膜细胞凋亡，而SIRT1敲低则增强了这种凋亡。我们得出结论，Smad7是SIRT1的新靶分子，SIRT1通过加速Smad7降解减弱TGFβ诱导的系膜细胞凋亡。我们的结果表明，上调SIRT1去乙酰化酶活性可能是一种通过影响细胞存活来预防TGFβ相关肾病的有用治疗策略。
