SIRT1 Inhibits Transforming Growth Factor β-Induced Apoptosis in Glomerular Mesangial Cells via Smad7 Deacetylation*

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 β (TGFβ)-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 TGFβ 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, TGFβ failed to induce apoptosis in Smad7 knockdown mesangial cells, indicating that Smad7 mainly mediates TGFβ-induced apoptosis of mesangial cells. Finally, SIRT1 overexpression attenuated both Smad7- and TGFβ-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 TGFβ-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 TGFβ-related kidney disease through its effect on cell survival.

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 ␤ (TGF␤)-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 TGF␤ 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, TGF␤ failed to induce apoptosis in Smad7 knockdown mesangial cells, indicating that Smad7 mainly mediates TGF␤-induced apoptosis of mesangial cells. Finally, SIRT1 overexpression attenuated both Smad7-and TGF␤-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 TGF␤-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 TGF␤-related kidney disease through its effect on cell survival.
Sir2 (silent information regulator 2) is a class III histone deacetylase with deacetylase activity, depending on intracellu-lar NAD ϩ concentrations (1)(2)(3). Sir2 functions, through its deacetylase activity, in a wide array of cellular processes including gene silencing, rDNA recombination, and life span extension under various stress conditions (1)(2)(3). Furthermore, mammalian Sir2 homolog (SIRT1) has recently been reported to deacetylate not only the lysine residues of histone proteins but also the lysine residues of some apoptosis-inducible nuclear proteins, such as p53 (4 -6) and forkhead family proteins (7,8). The deacetylation of these nuclear proteins by SIRT1 results in the inhibition of apoptosis (4 -8). Thus, it is conceivable that SIRT1 is a key regulator of cell defense and survival of mammalian cells through the interaction with apoptosis-inducible nuclear proteins. However, the target molecules of SIRT1 that are involved in apoptosis have been not fully elucidated.
Transforming growth factor ␤ (TGF␤) 2 is a multifunctional signaling cytokine that regulates apoptosis, cell cycle, differentiation, and extracellular matrix accumulation (9). Alternation of TGF␤-signaling has been implicated in the progression of kidney diseases (10). In TGF␤-transgenic mice, which exhibit progressive glomerulosclerosis, mesangial cell apoptosis is accelerated in the advanced stage glomerulosclerosis (11). TGF␤ signaling from the cell membrane to the nucleus is mediated and regulated by intracellular effector molecules, referred to as Smads (12). Among the Smads, Smad7 was originally recognized as an auto-inhibitory downstream molecule for TGF␤ signaling (13). In addition, Smad7 was also reported to modulate TGF␤-induced apoptosis (14 -19). However, the role of Smad7 in TGF␤-induced apoptosis depends on the cell type. Smad7 mediates TGF␤-induced apoptosis in podocytes (11) and prostatic carcinoma cells (17), whereas it inhibits TGF␤induced apoptosis in hematopoietic cells (16,19) and hepatocytes (14). In renal glomerular mesangial cells, TGF␤-induced apoptosis was facilitated by Smad7 overexpression and was inhibited by the antisense oligonucleotide to Smad7 (18). These reports support the notion that Smad7 mediates TGF␤-induced apoptosis in renal mesangial cells. Therefore, a more precise understanding of the regulation of Smad7 expression * This study was supported by a research grant from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to D. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1  in the kidney may lead to the prevention of TGF␤-related pathological changes, including mesangial cell apoptosis and glomerulosclerosis. It was recently reported that E3 ubiquitin ligase smurf1 (Smad ubiquitination regulatory factor 1)-mediated protein degradation through the ubiquitin-proteasome system regulates the level of Smad7 expression (20,21). The acetyl-transferase (p300)-mediated acetylation of two specific lysine residues (Lys-64 and -70) on Smad7 is one of its post-translational modifications and inhibits its degradation by interfering with the Smurf1-mediated ubiquitination, which mediates the stabilization of Smad7 (22). However, the precise mechanisms for the deacetylation of Smad7 and the correlation between its deacetylation and degradation are yet to be fully understood.
These previous results led us to investigate the possibility that apoptosis-associated nuclear protein Smad7 is a new target protein for SIRT1 and that the SIRT1-mediated deacetylation of Smad7 affects the degradation of Smad7. In the present study, we have investigated the capacity of SIRT1 to interact with Smad7 and inhibit TGF␤-induced apoptosis in renal glomerular mesangial cells. The results suggest that SIRT1 up-regulation is a potentially useful therapeutic target to prevent the progression of kidney disease.
DNA Constructs-We generated an expression vector for Myc-tagged wild-type (WT) Smad7 by subcloning the PCR products into the pCMV-Myc expression vector (Clontech, Palo Alto, CA) as previously reported (23). Point mutants of Myc-tagged Smad7 (K64R, K70R, and K64R/K70R, respectively) vectors and FLAG-SIRT1(H355A) vector were generated by site-directed mutagenesis (Stratagene, La Jolla, CA). The various fragments of Myc-tagged Smad7 (amino acids 1-812 (⌬CT) and 590 -1280 (⌬NT)) vectors were generated by PCR and cloned into the pCMV-Myc expression vector. FLAGtagged Smad7 vectors were generated by subcloning into the pFLAG-CMV-6a,b,c expression vector. FLAG-SIRT1 (mouse Sir2␣) vector was a kind gift from J. Luo (Columbia University, New York, NY), HA-tagged p300 vector was kindly provided by T. Nakajima (St. Marianna University school of Medicine, Kawasaki, Japan), 6ϫ Myc-tagged Smurf1 vector was a kind gift from K. Miyazono (University of Tokyo, Tokyo, Japan), and His-tagged ubiquitin vector was kindly gifted by D. Bohmann (University of Rochester Medical Center, Rochester, NY).
Immunoprecipitation and Immunoblotting-Cells were lysed in ice-cold lysis buffer (50 mM HEPES, pH 7.2, 150 mM NaCl, 1 mM EDTA, 20 mM NaF, 2 mM sodium orthovanadate, 1% (v/v) Triton X-100, 10% (v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 mM sodium butyrate, and 1% aprotinin) and cleared by centrifugation at 15,000 revolutions/min for 10 min at 4°C. For immunoprecipitation analysis using nuclear protein extractions of murine mesangial cells, cells were homogenized and lysed with hypotonic buffer (10 mmol/liter HEPES-KCl, pH 7.9, 1 mmol/liter EDTA, 15 mmol/liter KCl, 2 mmol/liter MgCl 2 , 1 mmol/liter dithiothreitol, and protease inhibitor mixture (Roche Molecular Biochemicals, Lewes, UK) with 0.8% Nonidet P-40, and the lysates were centrifuged at 3,000 ϫ g for 10 min. The pellets were resuspended in high salt buffer (hypotonic buffer with 420 mmol/liter NaCl and 25% glycerol), rotated for 30 min at 4°C, and centrifuged at 17,000 ϫ g for 30 min. The supernatants were used as nuclear extracts. Immunoprecipitations were carried out by adding the appropriate antibodies plus protein A/G-Sepharose beads followed by incubation at 4°C for 4 h. The immunoprecipitates were washed extensively, resolved by SDS-PAGE, and transferred to nitrocellulose membranes (Millipore, Bedford, MA). The membranes were incubated with the appropriate antibodies, washed, and incubated with horseradish peroxidase-coupled secondary antibodies (Amersham Biosciences). After washing, the blots were visualized by using an enhanced chemiluminescence detection system (PerkinElmer Life Sciences).
Glutathione S-Transferase (GST) Pulldown Experiments-GST fusion SIRT1 protein was expressed in Escherichia coli transformed with pGEX-SIRT1 and purified by using Mag-netGST protein purification according to the instructions provided by the manufacturer (Promega). GST pulldown assay using in vitro translated Myc-tagged Smad7 proteins was performed. Samples from the GST pulldown were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Smad7 was detected by using ␣Myc antibody.
In Vitro Translation-[ 35 S]Methionine-labeled, in vitro translated FLAG-tagged SIRT1 and each Myc-tagged Smad7 (WT, ⌬CT, or ⌬NT) were generated with the rabbit reticulocyte lysate in vitro translation system (Promega), and these pro-

SIRT1 Attenuates TGF␤-induced Apoptosis
teins were used to perform immunoprecipitation analysis as described previously (25). Immunoprecipitation results were detected by autoradiography after 16 h at Ϫ80°C.
Pulse-Chase Analysis-Myc-tagged Smad7 vector was transfected in the MMCs infected with either pBABE or pBABE-SIRT1 retroviral vector. Twenty-four hours after transfection, these cells were incubated for 1 h with 35 S-labeled cysteine and methionine (Amersham Biosciences) in cysteine-and methionine-free Dulbecco's modified Eagle's medium. The cells were washed and incubated in Dulbecco's modified Eagle's medium supplemented with 50 g/ml cysteine and methionine for the indicated time periods (0, 30, 60, and 120 min) followed by immunoprecipitation using ␣Myc. Immunoprecipitated samples were separated by SDS-PAGE, and the gels were dried and analyzed by autoradiography after 16 h at Ϫ80°C. The amount of 35 S-labeled Myc-Smad7 at each point was plotted as the percent of the amount at the start of the chase.
RNA Interference-The small interference RNA to mouse Smad7 (5Ј-CAUCAAGGCUUUUGACUAUGAGAAA-3Ј) was designed by and bought from iGENE Therapeutics (Tsukuba, Japan). MMCs on a 6-well plate were transfected with Smad7 RNAi using the TransIT-TKO reagent (Madison, WI). After 24 h, cells were treated with TGF␤ (5 ng/ml) for 48 h. Levels of Smad7 expression were determined by immunoblot analysis 72 h after transfection.
Analysis and Quantitation of Apoptosis-MMCs treated with various concentrations of TGF␤ for the indicated time periods (0 -48 h) were stained with DAPI for 5 min at room temperature and then examined under a fluorescence microscope (Olympus BX61; Tokyo, Japan). The staining was performed in five independent experiments for each group, and 30 random fields (ϳ600 nuclei) were studied in each experiment. Apoptosis was defined as nuclear condensation, and the results were expressed as the percent of apoptotic cell number to total number of nuclei/field. Smad7-or Smad7(K64R/K70R)-induced apoptosis was detected as described below. MMCs were transfected with either Myc-tagged Smad7 or Smad7(K64R/ K70R) followed by immunostaining using anti-Myc antibody and DAPI staining. The percentage of Myc-positive cells with apoptotic nuclei was scored 36 h after transfection. Apoptosis was also confirmed by assessing the amount of cleaved caspase-3 and cleaved PARP, as biological apoptotic markers, by immunoblot analysis.
Statistical Analysis-Results are expressed as the mean Ϯ S.E. Analysis of variance with subsequent Scheffe's test was used to determine the significance of differences in multiple comparisons. p Ͻ 0.05 was considered statistically significant.

SIRT1 Interacts with Smad7 Both in Vivo and in Vitro-To
investigate the general interaction between SIRT1 and Smad7 in vivo, we performed co-immunoprecipitation analysis in transiently transfected COS7 cells using either Myc-tagged Smad7 vector, FLAG-tagged SIRT1 vector, or both of these vectors (Fig. 1, A and B). Myc-tagged Smad7 was detected in the immunoprecipitates with ␣FLAG of co-transfection (Fig. 1A), and FLAG-tagged SIRT1 was detected in the immunoprecipitates with ␣Myc of co-transfection (Fig. 1B), indicating that SIRT1 interacts with Smad7 in vivo. We next performed immunoprecipitation experiments in vitro using either in vitro translated FLAG-tagged SIRT1 proteins, Myc-tagged Smad7 proteins, or both of these proteins (Fig. 1C), which provided additional evidence for an interaction between SIRT1 and Smad7 (Fig. 1C). In further immunoprecipitation analysis using the deletion  1 and 2) and FLAG-tagged SIRT1 vector (lanes 2 and 3) in COS7 cells. Immunoprecipitation (IP) was performed using ␣FLAG (A) or ␣Myc (B). Whole cell lysates (input) and immunoprecipitates were estimated by immunoblotting (IB) with ␣FLAG or ␣Myc. C, immunoprecipitation analysis in vitro. Either 35 Slabeled FLAG-tagged SIRT1 (lanes 1, 4, and 7), Myc-tagged Smad7 (lanes 2, 5, and 8), or both proteins (lanes 3, 6, and 9) were produced in the rabbit reticulocyte lysate translation system. Immunoprecipitation was performed with ␣FLAG (lanes 4 -6) or ␣Myc (lanes 7-9). In vitro translated proteins (input) and the immunoprecipitates were resolved by SDS-PAGE and estimated by autoradiography.  1-8) were generated in vitro. Immunoprecipitation was performed using ␣Myc (lanes 5-8). F, GST pulldown assay. Direct interaction of Smad7 with GST-SIRT1 was determined by GST pulldown assay with in vitro translated Myc-tagged Smad7. JANUARY 5, 2007 • VOLUME 282 • NUMBER 1 mutants of Myc-tagged Smad7 vectors, SIRT1 was detected in the immunoprecipitates of both Smad7(WT) and Smad7(⌬CT) but not Smad7(⌬NT), indicating that SIRT1 interacts with the N terminus of Smad7 (Fig. 1, D and E). We confirmed a direct interaction between SIRT1 and Smad7 by using GST pulldown assay (Fig. 1F). These findings show that SIRT1 directly interacts with Smad7.

SIRT1 Attenuates TGF␤-induced Apoptosis
SIRT1 Deacetylates the Lysine Residues of Smad7-To confirm that two specific lysine residues (Lys-64 and -70) of Smad7 are acetylated by acetyltransferase p300 (22), we generated two single-mutated and a double-mutated Smad7 vector in which these lysine residues were mutated to arginine residues (K64R, K70R and K64R/K70R, respectively). In vivo acetylation assays revealed that wild-type Smad7 was acetylated by p300 ( Fig. 2A). However, the acetylation of the lysine residues was partially decreased in the single-mutated Smad7(K64R and K70R), and it was completely inhibited in the double-mutated Smad7(K64R/ K70R) (Fig. 2A). These results indicated that two specific lysine residues (Lys-64 and -70) of Smad7 were acetylated by p300 as previously reported (22). We next investigated whether SIRT1 could deacetylate the lysine residues of Smad7. The p300-mediated acetylation of the lysine residues of Smad7 was decreased in SIRT1-overexpressing cells (Fig. 2B, lane 3). SIRT1(H355A) was reported to function as a dominant negative mutant and to inhibit the endogenous SIRT1-mediated deacetylation (4). SIRT1(H355A) inhibited SIRT1-mediated decreases in the p300-induced acetylation of the lysine residues of Smad7 in a dose-dependent manner (Fig. 2B, lanes 4 and 5). These results indicate that SIRT1 functionally interacts with Smad7, resulting in the deacetylation of the lysine residues of Smad7.
SIRT1 Accelerates Degradation of Smad7 in Mesangial Cells-We confirmed that endogenous SIRT1 interacted with endogenous Smad7 in the nuclei of MMCs (Fig. 3A). To examine the physiological effects of SIRT1 on Smad7 in MMCs, SIRT1 expression in MMCs was modified through retroviral infection with either pBABE-SIRT1 or pSUPER-SIRT1 RNAi for overexpression (10-fold) or knockdown (7-fold), respectively, of the SIRT1 gene (Fig. 3B). The acetylated lysine residues of Smad7 have been reported to protect it against Smurf1mediated ubiquitination and degradation (22). We therefore investigated the role of SIRT1 in the regulation of Smad7 degradation. The levels of Smad7 expression were significantly decreased in the SIRT1-overexpressing MMCs and were increased in the SIRT1 knockdown MMCs (Fig. 3B). However, SIRT1(H355A) significantly attenuated the decreased Smad7 expression in the SIRT1-overexpressing MMCs in a dose-dependent manner (Fig. 3, C and D). To confirm that the decreased amount of Smad7 expression in the SIRT1-overexpressing MMCs was caused by the stimulation of degradation, we performed degradation assays using methionine pulsechase analysis. This analysis revealed that the degradation of Smad7 was significantly accelerated in the SIRT1-overexpressing MMCs (Fig. 4A). To test the effect of SIRT1 on the ubiquitin-proteasome system in MMCs, we performed ubiquitination assays in the SIRT1-overexpressing and knockdown MMCs. Ubiquitination assays revealed that the ubiquitination of Smad7 was notably increased in the SIRT1-overexpressing MMCs and was decreased in the SIRT1 knockdown MMCs (Fig. 4B). SIRT1(H355A) inhibited the acceleration of Smad7 ubiquitination in the SIRT1-overexpressing MMCs (Fig. 4C). Furthermore, the decreased amount of Smad7 expression in the SIRT1-overexpressing MMCs was significantly reversed by lactacystin, known as a proteasome inhibitor (Fig. 4, D and E). These findings suggest that SIRT1 enhances the smurf1-mediated ubiquitination and degradation of Smad7 in MMCs.
induced apoptosis in MMCs through interaction with the specific lysine residues of Smad7.
SIRT1 Inhibits TGF␤-induced Mesangial Cell Apoptosis-Smad7 has been reported to accelerate TGF␤-induced mesangial cell apoptosis (18). We examined the effects of SIRT1 on TGF␤-induced apoptosis in MMCs. The number of apoptotic cells was significantly increased by TGF␤-stimulation after 48 h of incubation in a dose-dependent manner (Fig. 6, A and C). Similarly, the amounts of cleaved caspase-3 and cleaved PARP were also markedly increased by TGF␤ stimulation in MMCs (Fig. 6, B and D). In the Smad7 knockdown MMCs using Smad7 RNAi, TGF␤ failed to increase the amount of cleaved caspase-3 and cleaved PARP (Fig. 6E). These findings suggest that Smad7 is required for TGF␤-induced apoptosis in MMCs. We finally examined the effects of SIRT1 on TGF␤-induced mesangial cell apoptosis using the SIRT1-overexpressing and knockdown MMCs. TGF␤-induced apoptotic cells, cleavage of caspase-3, and PARP were decreased in the SIRT1-overexpressing MMCs, whereas these apoptotic markers were additionally increased in the SIRT1-knockdown MMCs (Fig. 6, F and G). In the similar way to Smad7-induced apoptosis, SIRT1 also acts a negative regulator of TGF␤-induced apoptosis in MMCs.

DISCUSSION
In this study, we have presented the first evidence that SIRT1 can modulate TGF␤-induced apoptosis in renal glomerular mesangial cells through direct interaction with Smad7. To date, SIRT1 has been reported to interact with some nuclear proteins, such as p53 (4 -6) or forkhead family proteins (7,8), which could modulate the functions of these proteins through the deacetylation. Here, we show (a) that Smad7 is a new sub-strate for SIRT1, (b) that the SIRT1mediated deacetylation of Smad7 stimulates the smurf1-mediated degradation of Smad7 through the ubiquitin-proteasome system, and (c) that SIRT1 inhibits TGF␤and Smad7-induced apoptosis in MMCs.
The acetylation/deacetylation of lysine residues on nuclear proteins is thought to affect multiple protein functions, such as transcriptional activity, DNA binding, protein binding, protein stability, and translocation. Recently, SIRT1-mediated deacetylation of apoptosis-inducible nuclear proteins, such as p53 (4 -6), FOXO (7,8), and Ku70 (26), has been reported to promote cell survival. Consistent with this evidence, our results indicating that the SIRT1-mediated deacetylation of two lysine residues (Lys-64 and -70) on Smad7 suggest that SIRT1 plays an important role in renal glomerular mesangial cell survival under stimulation of TGF␤. This evidence strongly supports the contribution of the SIRT1-mediated deacetylation of the nuclear proteins to cell survival.
The present study has demonstrated that SIRT1 directly interacts with Smad7, resulting in the deacetylation of Smad7. Furthermore, our study has revealed that SIRT1 accelerates the Smurf1-mediated ubiquitination and degradation of Smad7, which causes the instability of Smad7. A previous report shows that both acetylated Smad7 and mutated Smad7(K64R/K70R) fails to bind to ubiquitin ligase and smurf-1, and subsequently both expressions were protected from the ubiquitin-proteasome-mediated degradation, resulting in the increased stability of Smad7 expression (22). In addition to these results, our results indicating that SIRT1 accelerates the ubiquitin-proteasome-mediated degradation of Smad7 through the deacetylation of Smad7 provided further evidence that unacetylated/ deacetylated lysine residues (Lys-64 and -70) of Smad7 were required for the binding to smurf1. With respect to the regulation of Smad7 acetylation, it has been recently reported that some class I and II histone deacetylase interact with Smad7 (27). This interaction results in the deacetylation of Smad7 with subsequent enhancement of degradation by ubiquitination; however, the direct interaction between SIRT1 and Smad7 was not fully examined (27). Our results clearly show that class III histone deacetylase and SIRT1 directly interact with Smad7, resulting in the acceleration of Smad7 degradation. Several nuclear proteins, including p53 (4 -6,28) and NFB (RelA/ p65) (29), are also deacetylated both by class I histone deacetylase and SIRT1, which modulate the functional effects of these nuclear proteins. At present, the differential roles between class I histone deacetylase-and SIRT1-mediated deacetylation of these nuclear proteins remain unclear. Our results provided the possibility that SIRT1 inhibits p300mediated acetylation of lysine residues of Smad7 through direct and functional interaction. A recent report suggests that SIRT1 deacetylates lysine residues of p300, resulting in the inhibition of acetyltransferase activity of p300 (30). Therefore, it is possible that SIRT1 may decrease acetylation of lysine residues of Smad7 through both direct interaction and the inhibition of transferase activity of p300, although this study did not reveal the precise mechanism by which the acetylation of Smad7 was decreased. However, we did show that SIRT1 directly interacts with Smad7 and inhibits the p300-mediated acetylation of the lysine residues of Smad7. These results suggest that Smad7 is a new target for SIRT1 deacetylase activity.
In the kidney, Smad7 is considered to play an important role in the regulation of TGF␤-induced apoptosis besides the inhibition of TGF␤ signaling (11,15,18). A recent report shows that overexpression of Smad7, but not regulatory Smads (Smad2/3), enhances apoptosis in mesangial cells (18). In addition, antisense oligonucleotides to Smad7 prevent TGF␤-induced apoptosis in mesangial cells (18). Our results also confirmed the role of Smad7 on TGF␤-induced mesangial cell apoptosis. Furthermore, we showed that SIRT1 could inhibit Smad7-and TGF␤-induced mesangial cell apoptosis through the enhancement of Smad7 degradation and inhibition of caspase-3 and PARP activation. Other roles of Smad7, except for the inhibition of TGF␤-signaling, may be involved in TGF␤-induced apoptosis. Interactions between Smad7 and some pro-apoptotic molecules, such as the activation of p38 (31,32) or NF-B (11,31), have been reported to be important for TGF␤-induced apoptosis. The relationship between these pro-apoptotic molecules, Smad7 and SIRT1, has not been clarified in our study, although we have demonstrated that SIRT1 inhibits TGF␤-induced mesangial cell apoptosis through Smad7 degradation. To clarify further the role of SIRT1 and Smad7 in TGF␤-induced apoptosis in all cell types including mesangial cells, more investigation is required.
In conclusion, our study has provided new information regarding the functional significance of the interaction between SIRT1 and Smad7 in TGF␤-induced mesangial cell apoptosis. We suggest that up-regulation of SIRT1 is a potentially useful therapeutic strategy for prevention of glomerular diseases through its effect on cell survival.