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Sirt1 attenuates camptothecin-induced apoptosis through caspase-3 pathway in porcine preadipocytes
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Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/yexcr
Research Article
Sirt1 attenuates camptothecin-induced apoptosis through caspase-3 pathway in porcine preadipocytes Wei-jun Panga,, Yan Xionga, Yu Wanga, Qiang Tongb, Gong-she Yanga a
Laboratory of Animal Fat Deposition and Muscle Development, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China b Children’s Nutrition Research Center, Baylor College of Medicine, Houston, TX 77030, USA
article information
abstract
Article Chronology:
Adipose tissue is an important energy reservoir, and its over-development results in obesity in
Received 4 September 2012
humans or body fat over-deposition in livestock. Loss of preadipocytes through apoptosis has
Received in revised form
been proposed as an alternative way to reduce adipose tissue mass. At present, the effect and
7 December 2012
regulatory mechanism of Sirt1 and camptothecin on porcine preadipocyte apoptosis are still
Accepted 31 December 2012
largely unknown. Here, we evaluated whether Sirt1 had any role in the basal cellular and
Available online 9 January 2013
camptothecin-induced conditions in porcine preadipocytes. Flow cytometric analysis shows that
Keywords:
viable cells decrease as well as early apoptotic and late apoptotic cells increase after knockdown
Sirt1
of Sirt1 in porcine preadipocytes. Camptothecin induces porcine preadipocyte apoptosis in a
Camptothecin
dose-dependent manner, assessed with the Hoechst staining and western blot analysis.
Apoptosis
Interestingly, silencing of Sirt1 significantly enhances sensitivity of porcine preadipocytes to
Porcine preadipoyte
camptothecin, which may be due to upregulation of p53, acetylated p53, Bax, cleaved caspase-3
Caspase-3 pathway
and downregulation of Bcl-2. On the contrary, under the Sirt1 overexpression condition viable cells’ number significantly increases when challenging with camptothecin, and the protein levels of cleaved caspase-3, p53, acetylated p53 and Bax are downregulated. We also find that hyperacetylated p53 is the major effect of Sirt1 knockdown by overexpression of a mutated p53, whereas Sirt1 overexpression prevents camptothecin-induced apoptosis through p53 deacetylation in preadipocytes. Furthermore, repressing preadipocyte apoptosis of Sirt1 is mediated by direct interaction with cleaved caspase-3 using immunoprecipitation and inhibition of caspase3 transcriptional activity using luciferase reporter assays. & 2013 Elsevier Inc. All rights reserved.
Introduction Adipose tissue, which is determined by processes governing adipocyte size and number [1], is usually considered to be an inert mass for accumulating excess energy as fat [2]. Moderate fat is essential for animals, but more excess fat that deposits into adipose depots leads to obesity in humans or body fat over-deposition in meat n
animals. In humans, obesity is considered to be a risk factor associated with the genesis or development of various diseases, including type 2 diabetes mellitus, hypertension, coronary heart disease and cancer [3]. In meat animals, more excess fat declines the quality and palatability of meat. With regard to these serious health problems and quality of meat, the need to develop new and available strategies in reducing adipose mass has become more acute. Ways to
Corresponding author. Fax: þ86 29 87092430. E-mail addresses: [email protected], [email protected] (W.-j. Pang).
0014-4827/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yexcr.2012.12.025
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reduce adipose mass including decreased proliferation and differentiation of preadipocytes, increased lipolysis and also involve the loss of preadipocytes through apoptosis [4]. However, to decrease fat deposition through preadipocyte apoptosis is unclear. Apoptosis is a form of programmed cell death that results in removal of undesired cells without inflammation, and it plays a key role in maintenance of homeostasis in organisms [5]. Previous reports of adipose tissue apoptosis focused on mature adipocytes and in vivo studies. Esculetin decreased cell number by initiating mitochondria-mediated apoptosis in 3T3-L1 adipocytes [6]. The loss of adipose tissue in different anatomic locations in oleoyl– estrone treated rats mediated by apoptosis, either via the activation of death receptors or by the activation of cytochrome c from the mitochondria [7]. Evidence for apoptosis in preadipocytes has been obtained through the use of in vitro cell culture as well as in vivo studies in rodents and humans. Capsaicin-induced apoptosis and inhibited lipid accumulation in 3T3-L1 preadipocytes and adipocytes in a time- and dose-dependent manner [8]. Human preadipocytes isolated from adipose tissue were cultured and shown to undergo apoptosis in response to TNFa, but its molecular mechanism is unknown [9]. Compared with other animal models like mouse and rabbit, model of pig is more similar to human’s in the physiological characteristics, including cardiovascular anatomy and function, metabolism, size, tendency to obesity [10,11]. So it is a more ideal animal model to investigate body fat deposition of livestock as well as human obesity. Sirt1, the mammalian homologue of Sir2 (silent information regulator 2), is a NAD-dependent class III deacetylase [12]. A growing body of evidence has indicated that Sirt1 plays an important role in energy metabolism. SIRT1 interacts with PPAR-g and inhibits its transcriptional activity towards genes mediating fat storage through docking with its cofactors [13]. In addition to energy metabolism regulation, Sirt1 also plays an essential part in mediating survival of several types of cells by deacetylating other substrates, such as forkhead transcription factors (FoxOs) [14], tumor suppressor p53 [15], DNA repair factor Ku70 [16] and nuclear factor-kappa B (NF-kB) [17,18]. Kume et al. discovered that upregulation of Sirt1 prevented oxidative stress-induced mesangial cell apoptosis through the inactivation of p53 by deacetylation [19]. Sirt1 represses renal tubular cell apoptosis through the induction of anti-oxidative molecule catalase by a FoxO3a-dependent mechanism [20]. Based on these findings, we proposed that Sirt1 may have some relationship with the regulation of preadipocyte apoptosis. However, the effect of Sirt1 on apoptosis varies between cell types and species, and the regulatory mechanism of porcine preadipocyte apoptosis by Sirt1 is unknown. Therefore, we treated cells with Sirt1 shRNA, Sirt1 overexpression or the DNA topoisomerase I inhibitor camptothecin, which has been widely used to induce apoptosis under experimental conditions [21], and apoptosis was assessed to determine whether Sirt1 had any roles in basal condition and whether it exerted a protective action against camptothecin-induced apoptosis in porcine preadipocytes. Here we find that silencing of Sirt1 decreases cellular viability, induces apoptosis and sensitizes cells to camptothecin in porcine preadipocytes. However, under the Sirt1 overexpression condition, the number of viable cells significantly increases when challenging with camptothecin. Our study verified that Sirt1 really is a cellular protector, which maintains porcine preadipocyte survival under the basal or
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camptothecin-stimulated conditions. The regulatory mechanism of preadipocyte apoptosis is relative to the interaction between Sirt1 and cleaved caspase-3, the mediation of caspase-3 transcriptional activity and the acetylated p53 status by Sirt1.
Materials and methods Porcine primary preadipocyte culture Adipose tissue was removed under sterile conditions from the dorsal subcutaneous depot of the healthy piglets and washed with phosphate buffered saline (PBS). The adipose tissue mass was cut with scissors into approximately 1 mm3 sections and digested with collagenase type I (Invitrogen) at 37 1C for 1 h, followed by the filtration with a 200-mesh filter to remove undigested tissue residue. The pellet of stromal-vascular (SV) cells which contains the preadipocytes was collected by centrifugation at 1000 g for 10 min and then washed with serum-free medium (DMEM/F12 medium containing 15 mM NaHCO3, 100 U penicillin/ml and streptomycin). The SV cells was then incubated with an erythrocyte lysis buffer (0.154 M NH4CI,10 mM KHCO3 and 0.1 mM EDTA) at room temperature for 10 min. The collected pellets were used as preadipocyte [22]. After washing, preadipocytes were resuspended in DMEM/F12 medium (Gibco) containing with 10% fetal bovine serum (HyClone), 100 U/ml penicillin and 100 U/ml streptomycin. The cells were seeded in cell culture dishes at a density of 1 105 cells/well and cultured at 37 1C in humidified atmosphere with 5% CO2.
Lentiviral vectors Sirt1 shRNA expression vector was constructed with lentiviral plasmids pLentiHI. Sirt1 shRNA oligonucleotides (sense,50 -GATCCGGATGAAAGTGAGATTGAATCAAGAGTTCAATCTCACTTTCATCCTTTTTTC-30 ; antisense,50 -TCGAGAAAAAAGGATGAAAGTGAGATTGAACTCTTGATTCAATCTCACTTTCATCCG-30 ) corresponding to 2055–2074 of porcine Sirt1 mRNA (GenBank number was EU030283) were annealed and cloned into pLentiHI at BamHI and XhoI sites. In addition, Sirt1 overexpression, p53 and p53 mutant (Lys-382) constructs were from Dr. Q. Tong’s lab (Baylor College of Medicine, TX).
Lentiviral infection pLentiHI–Sirt1 shRNA transferred plasmid (3.3 mg), 2 mg D8.9 packaging plasmid and 3 mg VSV-G envelope protein plasmid were cotransfected into HEK293T packaging cells (2 105 cells/well) by using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction. Ditto, FG30-Sirt1 or FG30, packaging plasmid and envelope protein plasmid were contransfected into HEK293T packaging cells. After transfection (48 h), the supernatant containing viral particles was collected and passed through a 0.45 mm filter to remove cellular debris. Porcine preadipocytes were planted at a density of 1 105 cells/well and cultured in DMEM/F12 medium containing 10% FBS. On reaching 70–80% confluence, the viral suspension of Sirt1 shRNA, Sirt1 overexpression, their empty vectors, p53 and p53 mutant vectors containing 6 mg/ml polybrene were added, respectively. After infection (48 h), cells were harvested for analysis.
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Annexin V/PI flow cytometric analysis Annexin V-FITC in combination with propidium iodide (PI) was used to quantitatively determine the percentage of cells undergoing apoptosis. Briefly, porcine preadipocytes were planted at a density of 1 105 cells/well, on reaching 70–80% confluence, cells were infected with viral suspensions of Sirt1 shRNA, Sirt1 overexpression and their empty vectors, respectively. After 48 h, the cellular monolayer was released by using trypsin without EDTA. Cells were resuspended and incubated with Annexin V-FITC for 15 min at room temperature, in the dark, followed by PI staining. Cells were analyzed within 1 h by a FACS Caliber flow cytometer (Becton Dickinson, USA). Annexin V-FITC and PI double-negative cells were defined as normal cells, Annexin V-FITC-positive and PI-negative cells as early apoptotic cells, and Annexin V-FITC and PI double-positive cells as late apoptotic and necrotic cells. The Annexin V-FITC-PI binding assay was determined at least three times. Cell Quest software was used to calculate the percentages of distribution of normal, early apoptotic, late apoptotic, and necrotic cells. In addition, effect of p53 and p53 mutant on apoptosis under camptothecin- and Sirt1 shRNA-induced preadipocytes was evaluated by FACS analysis using Annexin/PI stained.
Hoechst staining Cells treated with different concentrations (0, 1, 2, 4 mM) of camptothecin for 12 h and stained with Hoechst 33258 (Roche Diagnostics). Treated cells were fixed in 4% formaldehyde for 10 min at 4 1C, washed in cold PBS, and stained with Hoechst dye, followed by examination under a fluorescence microscope (Olympus, Japan). Apoptotic cells were defined as condensed pycnotic nuclei and chromosomal fragmentation. In addition to morphological changes of nuclei, apoptosis was also confirmed by the protein expression of activated caspase-3, which is the marker gene of apoptosis.
DNA fragmentation detection For assessment of apoptosis, the Apoptotic DNA Ladder Detection Kit (Roche Diagnostics) was used. This kit provides an easy and sensitive means for detecting DNA fragmentation in apoptotic cells. Porcine preadipocytes were seeded at 1 105 per well, on reaching 70–80% confluence, cells were infected with lentiviral supernatants contained Sirt1 shRNA or without. A Forty-eight hours after infection, cells were treated with or without 2 mM camptothecin for 12 h. The harvested cells were washed twice with PBS and subjected to lysed and then incubated with proteinase according to the manufacturer’s instructions. Internucleosomal DNA fragmentation in apoptotic cells were detected by a 1.2% agarose gel containing 0.5 mg/ml ethidium bromide at 5 V/cm for 1–2 h.
Western blot analysis Cells were lysed using the lysis buffer (pH 7.4) containing 50 mM Tris–HCl, 0.5% Triton X-100, 2 mM EDTA, 150 mM NaCl and 1 mM PMSF. The protein content was measured by the UV spectrophotometer. Protein samples were separated by 10% SDS-PAGE and electrotransfered to polyvinylidene fluoride
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membranes (Millipore). After being washed three times with a PBS buffer, the membranes were soaked in 5% nonfat dry milk for 2 h and incubated overnight at 4 1C with the primary antibodies against b-actin, Sirt1, cleaved caspase-3, p53, Bax, Bcl-2 or Tubulin (Santa Cruz Biotechnology), acetyl-lysine-382 p53 (Upstate). After incubation with anti-rabbit and anti-mouse horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) for 1 h at room temperature, the immune complexes were visualized by enhanced chemiluminescence methods; the band intensity was measured and quantitated. The resulting images were analyzed with Quantity One software (Bio-Rad, USA).
Immunoprecipitation The interaction between exogenous caspase-3 and Sirt1 was evaluated by the co-immunoprecipitation technique. HEK293T cells were cultured in high-glucose DMEM with 10% calf serum. For transfection, 1–5 mg of pcDNA–Sirt1–Flag, or pcDNA–caspase-3 or both were transfected into six-well plates by the calcium phosphate method. Similarly pcDNA–caspase-3–Flag, or pcDNA– Sirt1–3 or both were transfected. After transfection (24 h), cells were lysed in RIPA (50 mM Tris–Cl pH 7.4, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS) buffer. Cell lysate 200 ml was supplemented with a 300 ml lysis buffer to get the appropriate protein concentration and volume. Anti-Flag M2 agarose affinity gel (40 ml) (Sigma, A2220) was added to the lysate and rocked in 4 1C overnight. After washing for five times with a lysis buffer, protein was eluted with 2 loading buffer for the Western blot analysis. Furthermore, endogenous interactions between Sirt1, fulllength caspase-3 and cleaved caspase-3 were detected using the co-immunoprecipitation method. Porcine preadipocytes were incubated with camptothecin at 2 mM for 12 h. Then nuclear proteins and cytoplasmic proteins were extracted from preadipocytes using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce). An amount of 10 mg nuclear proteins or cytoplasmic proteins was respectively incubated with 4 mg antibodies against Sirt1 overnight and then captured by 1 ml goat anti-mouse IgG magnetic beads (NEB) for 4 h. After washing four times with a lysis buffer, proteins were eluted with 2 loading buffer for Western blot analysis using full-length caspase-3 antibody (Merck Millipore) and cleaved caspase-3 antibody (Merck Millipore), respectively. Histone H3.1 is the inner control. Histone H3.1 antibody was purchased from Signalway Antibody.
Luciferase reporter assays A luciferase reporter construct under the direction of a 300-bp fragment from human complement component C3 promoter has a high-affinity caspase3-binding site. Reporter construct (1 mg) was transfected into HEK293T cells along with pcDNA–Sirt1 using the calcium phosphate method. Renilla luciferase reporter plasmid (pRL-TK) was included as an internal control for transfection efficiency. After transfection (24 h), cells were lysed, and luciferase activity was measured with the dual luciferase kit (Promega) according to the manufacturer’s instruction. Briefly, 20 ml of cell lysate was mixed with 90 ml LAR II reagent, the fluorescence was recorded as firefly luciferase activity, and then 75 ml of stop and glow buffer was added to measure the Renilla
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luciferase. Firefly luciferase activity was normalized to Renilla luciferase activity.
Synergism of Sirt1 knockdown and camptothecin on apoptosis
Statistical analysis
To identify whether suppression of Sirt1 sensitizes porcine preadipocytes to camptothecin, porcine preadipocytes were infected with Sirt1 shRNA and then treated with 2 mM camptothecin. Twelve hours after treatment, cells were photographed under a light microscope. As shown in Fig. 3A, Sirt1 shRNA vector alone decreases cell population and declines the contact between adjacent cells significantly while we observed cells shrinked with condensed nucleus in camptothecin treatment alone. Additionally, camptothecin treated cells that were infected with Sirt1 shRNA have more distinguished apoptotic characteristics than that responses to Sirt1 shRNA or camptothecin alone. In comparison, the cells without any treatment showed extended fibroblast-like morphology, which had uniform cytoplasm and adherence to the culture dish tightly. To confirm the results from morphological assessment, DNA fragmentation analysis was performed. As shown in Fig. 3B, the electrophoretogram generated by agarose gel electrophoresis shows that silencing of Sirt1 causes slender degradation of cell DNA, which was similar to that of camptothecin treatment. In addition, the DNA extracted from camptothecin treated cells transfected with Sirt1 shRNA shows a noticeable clear-cut ladder of DNA fragments, accompanied by the decrease of large molecular weight DNA. In contrast, DNA isolated from the cells infected with empty vector remained totally intact and devoid of any fragmentation. This result implies that knockdown of Sirt1 increases the sensitivity of porcine preadipocytes to camptothecin involves the breakage of genomic DNA. FACS analysis (Fig. 3C) using Annexin/PI stained further confirmed our finding which is the synergism of both Sirt1 knockdown and camptothecin on apoptosis. Several observations supported that the role of Sirt1 is in the regulation of mammalian cell survival in response to DNA damage through the inactivation of p53 by deacetylation [23,24]. We therefore speculate that the enhanced camptothecin sensitization by knockdown of Sirt1 in porcine preadipocytes was associated with the acetylation of p53. As shown in Fig. 3D, the protein level of acetylated p53 in camptothecin treated and Sirt1 knockdown cells were elevated compared to empty vector treated cells which had little acetylated p53 protein. Moreover, the acetylated p53 level of camptothecintreated cells transfected with Sirt1 shRNA increased more significantly than that of silencing of Sirt1 or camptothecin treated along. The total p53 expression increased in parallel with acetylated p53. These findings strongly implicate that the acetylation status of p53, which relied on the deacetylized activity of Sirt1, may be crucial for camptothecin-induced porcine preadipocyte apoptosis. Acetylation of p53 activates transcription of downstream targets with proapoptotic properties thereby priming cells for apoptosis [25]. Consistent with previous report, Bax protein was also increased in Sirt1 shRNA infected cells combination with camptothecin treatment, compared with Sirt1 shRNA transfected or camptothecin treatment alone (Fig. 3D). These results indicate that inhibition of Sirt1 in preadipocytes caused an increase of Bax level, which in part could be due to the increased level of acelylated p53 in response to camptothecin. Expression patter of cleaved caspase-3 was similar to that of Bax. We also detected
Results are expressed as means7SEM. Statistical significance was determined by the Student’s t-test. Difference between groups were considered statistically significant if po0.05.
Results Sirt1 knockdown decreased viability and induced apoptosis To evaluate the function of Sirt1 in porcine preadipocyte, the knockdown experiments were performed by infecting porcine preadipocytes with pLentiHI–shSirt1. As shown in Fig. 1A, Sirt1 protein level is reduced by 57% compared to that of control cells. In contrast, the empty vector has only marginal effect on Sirt1 expression. This result indicates that Sirt1 gene is effectively suppressed by RNAi in porcine preadipocytes. Subsequently, the effect of Sirt1 knockdown on viability of porcine preadipocytes was studied. At 48 h after infection, porcine preadipocytes were analysed by Annexin V-FITC/PI apoptosis detection. Compared with control cells, pLentiHI–shSirt1 decreases the number of viable cells while increases necrotic cells and the cells in early apoptotic and late apoptotic stages (Fig. 1B). Viable cells distribution in pLentiHI–shSirt1 infected cells significantly decreases from 85 to 36% (po0.05). In addition, the early apoptotic and late apoptotic cell fractions increase from 4 to 30% (po0.01) and from 9 to 32% (po0.01), respectively. We also observed that the necrotic cells ratio in Sirt1 knockdown treated cells increased from 0.2 to about 3% (po0.05) (Fig. 1B). In contrast, the effect of empty vector on cell apoptosis was very low. Moreover, cleaved caspase-3 significantly increased in preadipocytes infected with pLentiHI–shSirt1 (Fig. 1C). Therefore, these results indicate that Sirt1 knockdown effectively decreases cell viability and induced cell apoptosis.
Camptothecin treatment caused DNA damage and induced cellular apoptosis To determine the function of Sirt1 under stimulated condition, we treated porcine preadipocytes with different concentrations (0, 1, 2, 4 mM) of camptothecin (Fig. 2A) to mimic DNA damage stress condition. Based on Hoechst staining, nuclear chromatin condensation and DNA fragmentation, which are the morphological changes characteristic of apoptosis, increase after 12 h incubation with camptothecin in a dose-dependent manner (Fig. 2B). Apoptosis is the result of an exquisite cascade of molecular events that ultimately result programmed cell death. Given the observations of nuclear morphologic changes, we also detected the cleaved caspase-3 and Sirt1 by Western blot analysis. Similar to Hoechst staining, the cleaved caspase-3 also increases dose dependently after 12 h incubation (Fig. 2C). But sirt1 expression has no change in preadipocytes induced with different concentrations of camptothecin. These results suggest that treatment with camptothecin caused DNA damage and induced preadipocyte apoptosis in a dose-dependent manner.
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Fig. 1 – Sirt1 knockdown decreases viability and induces apoptosis of porcine preadipocytes. (A) Sirt1 gene silencing by shRNA in porcine preadipocytes. Porcine preadipocytes were infected with lentiviral supernatants contained Sirt1 shRNA or scrambled shRNA. After infection (48 h), the levels of Sirt1 and cleaved caspase-3 proteins were determined by western blot analysis. (B) Downregulation of Sirt1 decreased viability and induced apoptosis in porcine preadipocytes by flow cytometric analysis. After infection (48 h), cells were incubated with Annexin V-FITC/PI double stain and determined by FACS Caliber flow cytometer and (C) Effect of Sirt1 knockdown on caspase-3 expression in porcine preadipocytes. After infection (48 h), cleaved caspase-3 was detected by Western blotting analysis. These results are represented as means7SEM of three independent experiments. po0.05 and po0.01.
the protein level of Bcl-2, which is an anti-apoptotic protein that can promote cell survival through interactions with other Bcl-2 related pro-apoptotic members, such as Bax. We found that the change of Bcl-2 protein is opposite to that of Bax and cleaved caspase-3.
Sirt1 knockdown increases acetylated p53 level in camptothecin-induced preadipocyte apoptosis To further investigate whether the deacetylated activity of Sirt1 may be crucial for camptothecin-induced porcine preadipocyte
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Fig. 2 – Camptothecin induces apoptosis of porcine preadipocytes. (A) Chemical structure of camptothecin. (B) Fluorescence microscopy of porcine preadipocytes after camptothecin exposure. Porcine preadipocytes were incubated with camptothecin at 0 lM, 1 lM, 2 lM, 4 lM for 12 h and cells were stained with fluorescent dye Hoechst 33258. After staining, cells were photographed under fluorescence microscopy at 100 magnification. Condensed nuclear chromatins were indicated using arrows and the condensed nuclear chromatin undergoing fragmentation in later stages of apoptosis are indicated using triangles and (C) Effect of camptothecin on caspase-3 expression and Sirt1 in porcine preadipocytes. Porcine preadipocytes were treated with camptothecin at the indicated concentrations for 12 h. Cleaved caspase-3 was detected by Western blotting analysis. These results are represented as means7SEM of three independent experiments. Different letters indicate significantly differences (po0.05). apoptosis, we performed the experiment of overexpression of a mutated p53 (Lys-382) which can mimic a hyperdeacetylated status of p53. Under camptothecin-, or Sirt1 shRNA-, or bothinduced preadipocytes, effect of p53 overexpression on apoptosis
is more significant than that of p53 mutant overexpression (Fig. 4A). Compared with only Sirt1 knockdown or camptothecin treatment, under the co-treatment condition of Sirt1 knockdown and camptothecin acetylated p53 level was significantly
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Fig. 3 – Sirt1 knockdown increases the sensitivity of porcine preadipocytes to camptothecin. (A) The morphological observation of porcine preadipocytes after treated with scrambled shRNA, Sirt1 shRNA, camptothecin, and Sirt1 shRNAþcamptothecin (SþC). After infection (36 h), the cells were treated with camptothecin (2 lM) for 12 h. (B) Detection of genomic DNA fragmentation in porcine preadipocytes transfected with scrambled shRNA, Sirt1 shRNA, camptothecin (2 lM), and Sirt1 shRNAþcamptothecin. DNA was extracted and subjected to pulsed field gel electrophoresis through a 1.2% agarose gel which was stained with ethidium bromide. (C) Synergism of treatment with both Sirt1 shRNA and camptothecin on preadipocyte apoptosis. The results of Cell Quest software calculated were expressed as bar graph to express the percentages of viable, early apoptotic, late apoptotic, and necrotic cells number to total number of cells and (D) Western blot analysis of Sirt1, p53, acetylated p53, Bax, Bcl-2 and cleaved caspase-3 protein expression in scrambled shRNA transfected cells (scrambled), Sirt1 shRNA transfected cells (Sirt1 shRNA), camptothecin and Sirt1 shRNA treated cells (SþC). This experiment was replicated in triplicate. Different letters indicate significantly differences (po0.05).
increased (Fig. 4B). The results show that hyperacetylated p53 is the major effect of Sirt1 knockdown, which increases camptothecin-induced porcine preadipocyte apoptosis.
Sirt1 overexpression attenuates apoptosis when challenging with camptothecin As shown in Fig. 5A, the effect of Sirt1 overexpression on viability of porcine preadipocytes treated with camptothecin was studied. At 48 h after treatment, porcine preadipocytes were analysed by Annexin V-FITC/PI apoptosis detection. Compared with the
treatment only with camptothecin, Sirt1 overexpressionþcamptothecin significantly increases the number of viable cells while decreases early apoptotic cells and late apoptotic cells. Viable cells distribution significantly increases from 28 to 49% (po0.05). In addition, the early apoptotic and late apoptotic cell fractions increase from 31 to 20% (po0.05) and from 33 to 23% (po0.05), respectively. We further examined levels of cleaved caspase-3, p53, acelylated p53 and Bax (Fig. 5B) and found that the protein levels in Sirt1 overexpressed cells significantly decreased compared with empty vector treated cells. Moreover, compared with the above protein levels under only
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Fig. 4 – Sirt1 knockdown acetylates p53 in camptothecin-induced preadipocytes. Preadipocytes were treated with p53 or p53 mutant infection, p53 or p53 mutant and Sirt1 shRNA co-infection, p53 or p53 mutant and Sirt1 shRNA co-infection induced with camptothecin (2 lM). After 48 h, on the one hand, FACS analysis using Annexin/PI stained was performed by the treated cells; on the other hand, total proteins were extracted for Western blot examination and analysis of p53, acetylated p53 and tubulin protein expressions. (A) Effect of p53 and p53 mutant on apoptosis under camptothecin- and Sirt1 shRNA-induced cells and (B) Sirt1 knockdown acetylates p53 in camptothecin-induced cells. These results are represented as means7SEM of 3 independent experiments. Different letters indicate significantly differences (po0.05). camptothecin, the protein levels under the treatment of Sirt1 overexpressedþcamptothecin significantly increase (po0.05). Therefore, these results indicate that Sirt1 overexpression effectively increases cell viability and inhibits cell apoptosis when challenging with camptothecin.
Molecular mechanism of Sirt1 repressing preadipocyte apoptosis To explore the molecular mechanism underlying Sirt1 inhibitory action on preadipocyte apoptosis, we focus on the effect of
exogenous Sirt1 on caspase-3 proteins, since caspase-3 plays important roles in promoting apoptosis. To investigate Sirt1 interaction with caspase-3, we transfected HEK293T cells with caspase-3 and Sirt1-Flag. Sirt1 proteins were immunoprecipitated with anti-Flag agarose beads. Caspase-3 interaction with Sirt1 protein was detected by immunoblot analysis with caspase-3 antibodies. As shown in Fig. 6A (upper), caspase-3 is coimmunoprecipitated with Sirt1-Flag. Ditto, as shown in Fig. 6A (lower), Sirt1 is co-immunoprecipitated with caspase-3-Flag. Here, we need point out that caspase-3 protein is full length in this experiment.
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Fig. 5 – Sirt1 overexpression attenuates apoptosis induced by camptothecin. (A) The results of Cell Quest software calculated were expressed as bar graph to express the percentages of viable, early apoptotic, late apoptotic, and necrotic cells number to total number of cells and (B) Western blot analysis of Sirt1, cleaved caspase-3, p53, acetylated p53, Bax and Tubulin protein expression in the control cells (Empty vector), Sirt1 overexpressed cells (Sirt1 overexpression), the cells treated with camptothecin, cells treated with camptothecin and Sirt1 overexpression (SþC). This experiment was replicated in triplicate. Different letters indicate significantly differences (po0.05).
The amino acid (aa) sequences of caspase-3 and Sirt1 from various species were aligned (Fig. 6B, Fig. S1B) and the phylogenetic tree was constructed by the Neighbor-Joining method (Fig. 6C, Fig. S1A). We find that aa sequences of caspase-3 and Sirt1 are highly conserved among Sus Scrofa, Homo Sapiens and Mus Musculus. Therefore, we further performed immunoprecipitation experiment to detect endogenous interaction between Sirt1 and caspase-3. The results show that endogenous interaction between Sirt1 and cleaved caspase-3 is in nuclear proteins under camptothecin stimulation condition (Fig. 6D). In addition, endogenous tenuous interaction between Sirt1 and full length caspase-3 are found in cytoplasmic proteins under camptothecin stimulation condition (Fig. 6D). To test whether Sirt1 inhibits adipocyte apoptosis through inhibiting caspase-3, a luciferase reporter construct containing
the caspase-3 binding site from human complement component C3 promoter was used. Caspase-3 activates this reporter construct, as shown in Fig. 6E; however, with the co-expression of Sirt1, caspase-3 transactivation activity is acutely reduced. Furthermore, the repression of caspase-3 transactivation by Sirt1 is dose dependent (Fig. 6E). The above results show that Sirt1 does not only directly inhibit the expression of caspase-3, also changes caspase-3 protein structure by protein–protein interaction.
Discussion In this study, we find that silencing of Sirt1 induces porcine preadipocytes apoptosis and significantly enhances the sensitivity
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Fig. 6 – Sirt1 interacts with caspase-3 and inhibits caspase-3 activity. (A) Exogenous Sirt1 interacts with caspase-3 in 293T cells. HEK293 cells were transiently transfected with caspase-3, Sirt1-Flag, or both by the calcium phosphate method. After transfection (40 h), cells were lysed, and Sirt1-Flag proteins were immunoprecipitated with agarose beads and conjugated with anti-Flag antibodies. The precipitated proteins were washed 4 times and analyzed by Western blotting as indicated. Vice versa. IP, immunoprecipitation. IB, immunoblotting. (B) The amino acid (aa) sequences of caspase-3 from various species were aligned, with conserved residues shown . Full length caspase-3 is cleaved at Asp28Ser29 and Asp175Ser176, with cleaved position shown blue letters in red line box. Caspase-3 protein Accession No.: Sus Scrofa (BAB55544.1), Homo Sapiens (NP_004337.2) and Mus Musculus (NP_033940.1). (C) Phylogenetic tree of the caspase-3 proteins. The phylogenetic tree is constructed by the Neighbor-Joining method. The numbers by the branches indicate bootstrap values based on 1000 replications. Branch lengths are relative to the degree of divergence. Caspase-3 protein Accession No. and length: Bos Taurus (NP_001071308.1; 275 aa), Gallus gallus (NP_990056.1; 283 aa), Xenopus laevis (NP_001081225.1; 282 aa), Danio rerio (NP_571952.1; 282 aa). (D) Interaction of endogenous Sirt1 and caspase-3 or cleaved caspase-3 in cytoplasm and nucleus of preadipocytes under Camptothecin stimulation and (E) The suppression of caspase-3 transactivation activity by Sirt1. HEK293T cells were transfected with a caspase3 responsive luciferase reporter (pC3-Luc) and a Renilla luciferase construct (pRL-TK), together with a caspase-3 expressing construct. Relative luciferase activity is presented to demonstrate the effect of Sirt1 on caspase-3. Different amounts of Sirt1expressing plasmid (2–8 lg) were used to test the dosage effect of Sirt1 on caspase-3 transactivation. The results are presented as means7SEM of 3 independent experiments. po0.05 and po0.01.
of cells to camptothecin. It provides new and important information that Sirt1 functions as a cell survival factor in porcine preadipocytes under basal and stimulated conditions. Some evidence suggested that downregulation of Sirt1 could induce apoptosis in mammalian cells. In glioma CD133-positive cells, downregulation of Sirt1 enhanced radiosensitivity and radiationinduced apoptosis [26]. In prostate cancer PC3 and DU145 cells, silencing of Sirt1 inhibited cell growth and increased sensitivity to camptothecin and cisplatin [27]. Consider together with the above results, our findings in porcine preadipocytes indicate that
Sirt1 universally functions as a cell survival factor in mammalian cells. As preadipocytes are the initial formation of adipose depots and possess the ability to undergo complete differentiation into mature adipocytes [28], to decrease adipose tissue mass by induction of preadipocytes apoptosis to control the number of preadipocytes may be more useful. Consequently, identifying key factors that control this process is important for the development of new approaches to decrease fat deposition. Therefore, we investigated whether Sirt1 had effects on the apoptosis of
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preadipocyte. Here, we find that Sirt1 overexpression attenuates fat cell apoptosis induced with camptothecin. These results indicate that Sirt1 is a critical factor on apoptotic regulation of porcine preadipocytes and suitable to define Sirt1 as a cellular protector. However, we must point out to explore the mechanisms to reduce adipose mass through preadipocyte apoptosis involved the impairment of Sirt1’s function in preadipocyte under differentiation-induced condition would not be promising because since 2004 it has been reported that RNA interference of Sirt1 enhances adipogenesis [13]. Here, our research provided a new idea to reduce adipose mass through apoptosis regulated by other genes. Importantly, under normal (non-differentiationinduced) condition, inhibition of Sirt1 results in decreasing the number of preadipocytes by apoptosis. Camptothecin is a strong DNA-damaging compound. In this study, we incubated porcine preadipocytes with different concentrations of camptothecin, indicating that camptothecin caused DNA damage and induced apoptosis in porcine preadipocytes in a dose-dependent manner. Moreover, silencing of Sirt1 significantly increases the sensitivity of porcine preadipocytes to camptothecin compared to either cells treated with camptothecin or cells treated with Sirt1 shRNA alone because of increasing cleaved casepase-3. Interestingly, Sirt1 expression has no change in preadipocytes treated with different concentrations of camptothecin. At present, the reason is unclear. The p53 tumor suppressor protein plays a central role in the chemosensitivity and radiosensitivity of cells by inducing cell cycle arrest and apoptosis [29,30]. Acetylated p53 (lysine-382) is one of the activated states of p53, which has been reported to increase transcriptional activity of p53 in response to various stressors. In line with Sirt1 function in p53 regulation, Sirt1 tightly binds to and specifically deacetylates p53, thereby impairing its DNA binding activity and causes reduction of p53-mediated apoptosis in cultured cells in response to DNA damage [24]. A recent study showed that acelylated-p53 mediated camptothecin induced apoptosis. RSV, a small molecule polyphenolic Sirt1 activator, markedly decreased CPT induced p53 acetylation in wild-type neurons [31]. Herein, we observed that acetylated-p53 appeared after Sirt1 knockdown compared to empty vector transfection, suggesting that the increased acetylated p53 is associated with silencing of Sirt1, which is consistent with the previous report. Importantly, Sirt1 knockdown combination with camptothecin treatment significantly upregulates the protein level of acetylated-p53 more than the responses to the individual treatment alone and more than the calculated additive response, suggesting that Sirt1 is an intrinsic regulator of the acetylation status of p53 to promote porcine preadipocyte survival against camptothecin-induced action. Through overexpression of a mutated p53, we further affirmed that the hyperacetylated p53 is the major effect of Sirt1 knockdown, which increases camptothecin-induced porcine preadipocyte apoptosis. Bax is a proapoptotic Bcl-2 family protein that resides in the cytosol and translocates to mitochondria upon induction of apoptosis, which can be upregulated and transcriptional activated by p53. Chipuk et al. reported p53 directly activated Bax to promote mitochondrial outer-membrane permeabilization (MOMP) and to engage the apoptotic program [32]. Here, we found that Sirt1 shRNA and camptothecin individual increased Bax protein and combinations of Sirt1 shRNA and camptothecin
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future increased Bax protein more than any one treatment alone, suggesting that silencing of Sirt1 in porcine preadipocytes increased the level of Bax, which in part could be due to the increased level of acelylated p53 in response to camptothecin. Interestingly, the changed tendency of Bcl-2 protein is opposite to Bax, and we speculate that it may be resulted from Bax heterodimerized with Bcl-2 and counteracted Bcl-2 expression. Sirt1 has also been reported to deacetylate other transcriptional factors in addition to p53, including Ku70, FoxOs and E2F1 [33]. Moreover, we found that expression patter of caspase-3 was similar to p53 and Bax in our experiments. It was reported that H2O2 and TNFa increased acetylation of p53 lysine-382, as well as caspase-3 cleavage, an indicator of apoptotic signaling [34]. Therefore, it is reasonable to conclude that Sirt1-regulated and camptothecin-induced porcine preadipocyte apoptosis is via the p53–caspase-3 mediated pathway. Caspase-3 is a key terminal molecular regulated apoptosis in cellular apoptosis pathways. However, it is unclear whether that Sirt1 physically interacts with caspase-3, so we explored molecular mechanism of Sirt1 repressing preadipocyte apoptosis by the experiments of immunoprecipitation and luciferase reporter assays. Expression patter and subcellular location of caspase-3 and Sirt1 had been reported [35–38]. Caspase-3 and Sirt1 expressed in porcine preadipocytes (data not shown). It was reported that exogenous administration of Sirt1 protein or administration of resveratrol that is known to activate Sirt1 in the presence of elevated D-glucose fostered the translocation of endogenous Sirt1 from the cytoplasm to the nucleus [39,40]. Caspase-3 likewise translocated from the cytoplasm to the nucleus [41]. Therefore, the two proteins exist in both cytosol and nucleus, and have potential physical interaction. To further verify the hypothesis, endogenous Sirt1 in nuclear and cytoplasmic proteins of porcine preadipocytes were respectively immunoprecipitated with anti-Sirt1 antibody, and co-precipitation of caspase-3 or cleaved caspase-3 was respectively detected by Western blot assay. Fortunately, the results show that endogenous interaction between Sirt1 and cleaved caspase-3 is prominent in nuclear proteins, although endogenous interaction between Sirt1 and full length caspase-3 is found in cytoplasmic proteins under camptothecin-stimulated condition. Interestingly, endogenous interaction between Sirt1 and cleaved caspase-3 or full length caspase-3 is not found under normal condition. Because of high conservation of caspase-3 and Sirt1 protein aa sequences among Sus Scrofa, Homo Sapiens and Mus Musculus, this endogenous interaction may exit in human preadipocyes. Under differentiation-induced or non-differentiation-induced conditions, we think that inhibition of Sirt1 results in different physiological changes of preadipocytes. Under non-differentiation-induced condition, inhibition of Sirt1 decreases the number of preadipocytes by apoptosis. Under differentiation-induced condition, inhibition of Sirt1 improves, whereas activation of Sirt1 inhibits adipogenesis preadipocytes. At present, many previous reports have shown that activation of Sirt1 should have therapeutic potency to improve obesity and diabetes through regulation of preadipocyte differentiation [42–44]. Expansion of adipose tissue occurs through the formation of new adipocytes, via the differentiation of preadipocytes, and the enlargement of existing adipocytes [45]. Recently, researchers reported a novel hopeful therapic approach for obesity and diabetes through preadipocyte apoptosis regulated via mast cells.
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Fig. 7 – Model for the regulation of preadipocyte apoptosis by Sirt1 and Camptothecin. Under the stimulations of Camptothecin, Sirt1 shRNA, Sirt1 overexpression, both Camptothecin and Sirt1 shRNA, both Camptothecin and Sirt1 overexpression, cleaved caspase-3 significantly increases or decreases to trigger or inhibit cellular apoptosis. Sirt1 overexpression attenuates apoptosis induced with camptothecin, whereas Sirt1 knockdown aggravates apoptosis induced with camptothecin through caspase-3 pathway.
Macrophages may alter preadipocyte fate through apoptosis [46]. In both murine cell lines and human primary cells, proinflammatory activation of macrophages inhibits their prosurvival activity, favoring preadipocyte death [47]. White adipose tissue (WAT) from obese humans contains large numbers of mast cells which participate in the relative metabolic disorders of obesity and diabetes by affecting energy expenditure, glucose and insulin sensitivities, apoptosis, and preadipocyte differentiation [48]. These findings open a new era of basic research regarding mast cells in WAT and offer hope to patients suffering from obesity and diabetes. Based on above results, we think that it may be hopeful to decrease adipose tissue mass by induction of preadipocyte apoptosis to control its number. Based on our findings, model for the regulation of preadipocyte apoptosis by Sirt1 and Camptothecin was established (Fig. 7). In conclusion, our study indicates that Sirt1 overexpression inhibits apoptosis and attenuates camptothecin-induced apoptosis through caspase-3 pathway, whereas silencing of Sirt1 leads to apoptosis and enhances the sensitivity of porcine preadipocytes to camptothecin. Our findings may provide a new clue that regulation of Sirt1 could potentially be a plausible method for improvement of porcine meat quality and the treatment of human obesity.
Conflict of interest statement The authors declare that there are no conflicts of interest.
Acknowledgment This study was supported by the Natural Sciences Foundation of China (No. 30600437), NWAFU Young Research Programme (No. 0114030) and NWAFU Basic Science Research Programme (No. QN2009021). Moreover, we thank the reviewer’s comments very much.
Appendix A.
Supporting information
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.yexcr.2012.12.025.
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Research Article
Sirt1 attenuates camptothecin-induced apoptosis through caspase-3 pathway in porcine preadipocytes Wei-jun Panga,, Yan Xionga, Yu Wanga, Qiang Tongb, Gong-she Yanga a
Laboratory of Animal Fat Deposition and Muscle Development, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China b Children’s Nutrition Research Center, Baylor College of Medicine, Houston, TX 77030, USA
article information
abstract
Article Chronology:
Adipose tissue is an important energy reservoir, and its over-development results in obesity in
Received 4 September 2012
humans or body fat over-deposition in livestock. Loss of preadipocytes through apoptosis has
Received in revised form
been proposed as an alternative way to reduce adipose tissue mass. At present, the effect and
7 December 2012
regulatory mechanism of Sirt1 and camptothecin on porcine preadipocyte apoptosis are still
Accepted 31 December 2012
largely unknown. Here, we evaluated whether Sirt1 had any role in the basal cellular and
Available online 9 January 2013
camptothecin-induced conditions in porcine preadipocytes. Flow cytometric analysis shows that
Keywords:
viable cells decrease as well as early apoptotic and late apoptotic cells increase after knockdown
Sirt1
of Sirt1 in porcine preadipocytes. Camptothecin induces porcine preadipocyte apoptosis in a
Camptothecin
dose-dependent manner, assessed with the Hoechst staining and western blot analysis.
Apoptosis
Interestingly, silencing of Sirt1 significantly enhances sensitivity of porcine preadipocytes to
Porcine preadipoyte
camptothecin, which may be due to upregulation of p53, acetylated p53, Bax, cleaved caspase-3
Caspase-3 pathway
and downregulation of Bcl-2. On the contrary, under the Sirt1 overexpression condition viable cells’ number significantly increases when challenging with camptothecin, and the protein levels of cleaved caspase-3, p53, acetylated p53 and Bax are downregulated. We also find that hyperacetylated p53 is the major effect of Sirt1 knockdown by overexpression of a mutated p53, whereas Sirt1 overexpression prevents camptothecin-induced apoptosis through p53 deacetylation in preadipocytes. Furthermore, repressing preadipocyte apoptosis of Sirt1 is mediated by direct interaction with cleaved caspase-3 using immunoprecipitation and inhibition of caspase3 transcriptional activity using luciferase reporter assays. & 2013 Elsevier Inc. All rights reserved.
Introduction Adipose tissue, which is determined by processes governing adipocyte size and number [1], is usually considered to be an inert mass for accumulating excess energy as fat [2]. Moderate fat is essential for animals, but more excess fat that deposits into adipose depots leads to obesity in humans or body fat over-deposition in meat n
animals. In humans, obesity is considered to be a risk factor associated with the genesis or development of various diseases, including type 2 diabetes mellitus, hypertension, coronary heart disease and cancer [3]. In meat animals, more excess fat declines the quality and palatability of meat. With regard to these serious health problems and quality of meat, the need to develop new and available strategies in reducing adipose mass has become more acute. Ways to
Corresponding author. Fax: þ86 29 87092430. E-mail addresses: [email protected], [email protected] (W.-j. Pang).
0014-4827/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yexcr.2012.12.025
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reduce adipose mass including decreased proliferation and differentiation of preadipocytes, increased lipolysis and also involve the loss of preadipocytes through apoptosis [4]. However, to decrease fat deposition through preadipocyte apoptosis is unclear. Apoptosis is a form of programmed cell death that results in removal of undesired cells without inflammation, and it plays a key role in maintenance of homeostasis in organisms [5]. Previous reports of adipose tissue apoptosis focused on mature adipocytes and in vivo studies. Esculetin decreased cell number by initiating mitochondria-mediated apoptosis in 3T3-L1 adipocytes [6]. The loss of adipose tissue in different anatomic locations in oleoyl– estrone treated rats mediated by apoptosis, either via the activation of death receptors or by the activation of cytochrome c from the mitochondria [7]. Evidence for apoptosis in preadipocytes has been obtained through the use of in vitro cell culture as well as in vivo studies in rodents and humans. Capsaicin-induced apoptosis and inhibited lipid accumulation in 3T3-L1 preadipocytes and adipocytes in a time- and dose-dependent manner [8]. Human preadipocytes isolated from adipose tissue were cultured and shown to undergo apoptosis in response to TNFa, but its molecular mechanism is unknown [9]. Compared with other animal models like mouse and rabbit, model of pig is more similar to human’s in the physiological characteristics, including cardiovascular anatomy and function, metabolism, size, tendency to obesity [10,11]. So it is a more ideal animal model to investigate body fat deposition of livestock as well as human obesity. Sirt1, the mammalian homologue of Sir2 (silent information regulator 2), is a NAD-dependent class III deacetylase [12]. A growing body of evidence has indicated that Sirt1 plays an important role in energy metabolism. SIRT1 interacts with PPAR-g and inhibits its transcriptional activity towards genes mediating fat storage through docking with its cofactors [13]. In addition to energy metabolism regulation, Sirt1 also plays an essential part in mediating survival of several types of cells by deacetylating other substrates, such as forkhead transcription factors (FoxOs) [14], tumor suppressor p53 [15], DNA repair factor Ku70 [16] and nuclear factor-kappa B (NF-kB) [17,18]. Kume et al. discovered that upregulation of Sirt1 prevented oxidative stress-induced mesangial cell apoptosis through the inactivation of p53 by deacetylation [19]. Sirt1 represses renal tubular cell apoptosis through the induction of anti-oxidative molecule catalase by a FoxO3a-dependent mechanism [20]. Based on these findings, we proposed that Sirt1 may have some relationship with the regulation of preadipocyte apoptosis. However, the effect of Sirt1 on apoptosis varies between cell types and species, and the regulatory mechanism of porcine preadipocyte apoptosis by Sirt1 is unknown. Therefore, we treated cells with Sirt1 shRNA, Sirt1 overexpression or the DNA topoisomerase I inhibitor camptothecin, which has been widely used to induce apoptosis under experimental conditions [21], and apoptosis was assessed to determine whether Sirt1 had any roles in basal condition and whether it exerted a protective action against camptothecin-induced apoptosis in porcine preadipocytes. Here we find that silencing of Sirt1 decreases cellular viability, induces apoptosis and sensitizes cells to camptothecin in porcine preadipocytes. However, under the Sirt1 overexpression condition, the number of viable cells significantly increases when challenging with camptothecin. Our study verified that Sirt1 really is a cellular protector, which maintains porcine preadipocyte survival under the basal or
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camptothecin-stimulated conditions. The regulatory mechanism of preadipocyte apoptosis is relative to the interaction between Sirt1 and cleaved caspase-3, the mediation of caspase-3 transcriptional activity and the acetylated p53 status by Sirt1.
Materials and methods Porcine primary preadipocyte culture Adipose tissue was removed under sterile conditions from the dorsal subcutaneous depot of the healthy piglets and washed with phosphate buffered saline (PBS). The adipose tissue mass was cut with scissors into approximately 1 mm3 sections and digested with collagenase type I (Invitrogen) at 37 1C for 1 h, followed by the filtration with a 200-mesh filter to remove undigested tissue residue. The pellet of stromal-vascular (SV) cells which contains the preadipocytes was collected by centrifugation at 1000 g for 10 min and then washed with serum-free medium (DMEM/F12 medium containing 15 mM NaHCO3, 100 U penicillin/ml and streptomycin). The SV cells was then incubated with an erythrocyte lysis buffer (0.154 M NH4CI,10 mM KHCO3 and 0.1 mM EDTA) at room temperature for 10 min. The collected pellets were used as preadipocyte [22]. After washing, preadipocytes were resuspended in DMEM/F12 medium (Gibco) containing with 10% fetal bovine serum (HyClone), 100 U/ml penicillin and 100 U/ml streptomycin. The cells were seeded in cell culture dishes at a density of 1 105 cells/well and cultured at 37 1C in humidified atmosphere with 5% CO2.
Lentiviral vectors Sirt1 shRNA expression vector was constructed with lentiviral plasmids pLentiHI. Sirt1 shRNA oligonucleotides (sense,50 -GATCCGGATGAAAGTGAGATTGAATCAAGAGTTCAATCTCACTTTCATCCTTTTTTC-30 ; antisense,50 -TCGAGAAAAAAGGATGAAAGTGAGATTGAACTCTTGATTCAATCTCACTTTCATCCG-30 ) corresponding to 2055–2074 of porcine Sirt1 mRNA (GenBank number was EU030283) were annealed and cloned into pLentiHI at BamHI and XhoI sites. In addition, Sirt1 overexpression, p53 and p53 mutant (Lys-382) constructs were from Dr. Q. Tong’s lab (Baylor College of Medicine, TX).
Lentiviral infection pLentiHI–Sirt1 shRNA transferred plasmid (3.3 mg), 2 mg D8.9 packaging plasmid and 3 mg VSV-G envelope protein plasmid were cotransfected into HEK293T packaging cells (2 105 cells/well) by using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction. Ditto, FG30-Sirt1 or FG30, packaging plasmid and envelope protein plasmid were contransfected into HEK293T packaging cells. After transfection (48 h), the supernatant containing viral particles was collected and passed through a 0.45 mm filter to remove cellular debris. Porcine preadipocytes were planted at a density of 1 105 cells/well and cultured in DMEM/F12 medium containing 10% FBS. On reaching 70–80% confluence, the viral suspension of Sirt1 shRNA, Sirt1 overexpression, their empty vectors, p53 and p53 mutant vectors containing 6 mg/ml polybrene were added, respectively. After infection (48 h), cells were harvested for analysis.
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Annexin V/PI flow cytometric analysis Annexin V-FITC in combination with propidium iodide (PI) was used to quantitatively determine the percentage of cells undergoing apoptosis. Briefly, porcine preadipocytes were planted at a density of 1 105 cells/well, on reaching 70–80% confluence, cells were infected with viral suspensions of Sirt1 shRNA, Sirt1 overexpression and their empty vectors, respectively. After 48 h, the cellular monolayer was released by using trypsin without EDTA. Cells were resuspended and incubated with Annexin V-FITC for 15 min at room temperature, in the dark, followed by PI staining. Cells were analyzed within 1 h by a FACS Caliber flow cytometer (Becton Dickinson, USA). Annexin V-FITC and PI double-negative cells were defined as normal cells, Annexin V-FITC-positive and PI-negative cells as early apoptotic cells, and Annexin V-FITC and PI double-positive cells as late apoptotic and necrotic cells. The Annexin V-FITC-PI binding assay was determined at least three times. Cell Quest software was used to calculate the percentages of distribution of normal, early apoptotic, late apoptotic, and necrotic cells. In addition, effect of p53 and p53 mutant on apoptosis under camptothecin- and Sirt1 shRNA-induced preadipocytes was evaluated by FACS analysis using Annexin/PI stained.
Hoechst staining Cells treated with different concentrations (0, 1, 2, 4 mM) of camptothecin for 12 h and stained with Hoechst 33258 (Roche Diagnostics). Treated cells were fixed in 4% formaldehyde for 10 min at 4 1C, washed in cold PBS, and stained with Hoechst dye, followed by examination under a fluorescence microscope (Olympus, Japan). Apoptotic cells were defined as condensed pycnotic nuclei and chromosomal fragmentation. In addition to morphological changes of nuclei, apoptosis was also confirmed by the protein expression of activated caspase-3, which is the marker gene of apoptosis.
DNA fragmentation detection For assessment of apoptosis, the Apoptotic DNA Ladder Detection Kit (Roche Diagnostics) was used. This kit provides an easy and sensitive means for detecting DNA fragmentation in apoptotic cells. Porcine preadipocytes were seeded at 1 105 per well, on reaching 70–80% confluence, cells were infected with lentiviral supernatants contained Sirt1 shRNA or without. A Forty-eight hours after infection, cells were treated with or without 2 mM camptothecin for 12 h. The harvested cells were washed twice with PBS and subjected to lysed and then incubated with proteinase according to the manufacturer’s instructions. Internucleosomal DNA fragmentation in apoptotic cells were detected by a 1.2% agarose gel containing 0.5 mg/ml ethidium bromide at 5 V/cm for 1–2 h.
Western blot analysis Cells were lysed using the lysis buffer (pH 7.4) containing 50 mM Tris–HCl, 0.5% Triton X-100, 2 mM EDTA, 150 mM NaCl and 1 mM PMSF. The protein content was measured by the UV spectrophotometer. Protein samples were separated by 10% SDS-PAGE and electrotransfered to polyvinylidene fluoride
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membranes (Millipore). After being washed three times with a PBS buffer, the membranes were soaked in 5% nonfat dry milk for 2 h and incubated overnight at 4 1C with the primary antibodies against b-actin, Sirt1, cleaved caspase-3, p53, Bax, Bcl-2 or Tubulin (Santa Cruz Biotechnology), acetyl-lysine-382 p53 (Upstate). After incubation with anti-rabbit and anti-mouse horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) for 1 h at room temperature, the immune complexes were visualized by enhanced chemiluminescence methods; the band intensity was measured and quantitated. The resulting images were analyzed with Quantity One software (Bio-Rad, USA).
Immunoprecipitation The interaction between exogenous caspase-3 and Sirt1 was evaluated by the co-immunoprecipitation technique. HEK293T cells were cultured in high-glucose DMEM with 10% calf serum. For transfection, 1–5 mg of pcDNA–Sirt1–Flag, or pcDNA–caspase-3 or both were transfected into six-well plates by the calcium phosphate method. Similarly pcDNA–caspase-3–Flag, or pcDNA– Sirt1–3 or both were transfected. After transfection (24 h), cells were lysed in RIPA (50 mM Tris–Cl pH 7.4, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS) buffer. Cell lysate 200 ml was supplemented with a 300 ml lysis buffer to get the appropriate protein concentration and volume. Anti-Flag M2 agarose affinity gel (40 ml) (Sigma, A2220) was added to the lysate and rocked in 4 1C overnight. After washing for five times with a lysis buffer, protein was eluted with 2 loading buffer for the Western blot analysis. Furthermore, endogenous interactions between Sirt1, fulllength caspase-3 and cleaved caspase-3 were detected using the co-immunoprecipitation method. Porcine preadipocytes were incubated with camptothecin at 2 mM for 12 h. Then nuclear proteins and cytoplasmic proteins were extracted from preadipocytes using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce). An amount of 10 mg nuclear proteins or cytoplasmic proteins was respectively incubated with 4 mg antibodies against Sirt1 overnight and then captured by 1 ml goat anti-mouse IgG magnetic beads (NEB) for 4 h. After washing four times with a lysis buffer, proteins were eluted with 2 loading buffer for Western blot analysis using full-length caspase-3 antibody (Merck Millipore) and cleaved caspase-3 antibody (Merck Millipore), respectively. Histone H3.1 is the inner control. Histone H3.1 antibody was purchased from Signalway Antibody.
Luciferase reporter assays A luciferase reporter construct under the direction of a 300-bp fragment from human complement component C3 promoter has a high-affinity caspase3-binding site. Reporter construct (1 mg) was transfected into HEK293T cells along with pcDNA–Sirt1 using the calcium phosphate method. Renilla luciferase reporter plasmid (pRL-TK) was included as an internal control for transfection efficiency. After transfection (24 h), cells were lysed, and luciferase activity was measured with the dual luciferase kit (Promega) according to the manufacturer’s instruction. Briefly, 20 ml of cell lysate was mixed with 90 ml LAR II reagent, the fluorescence was recorded as firefly luciferase activity, and then 75 ml of stop and glow buffer was added to measure the Renilla
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luciferase. Firefly luciferase activity was normalized to Renilla luciferase activity.
Synergism of Sirt1 knockdown and camptothecin on apoptosis
Statistical analysis
To identify whether suppression of Sirt1 sensitizes porcine preadipocytes to camptothecin, porcine preadipocytes were infected with Sirt1 shRNA and then treated with 2 mM camptothecin. Twelve hours after treatment, cells were photographed under a light microscope. As shown in Fig. 3A, Sirt1 shRNA vector alone decreases cell population and declines the contact between adjacent cells significantly while we observed cells shrinked with condensed nucleus in camptothecin treatment alone. Additionally, camptothecin treated cells that were infected with Sirt1 shRNA have more distinguished apoptotic characteristics than that responses to Sirt1 shRNA or camptothecin alone. In comparison, the cells without any treatment showed extended fibroblast-like morphology, which had uniform cytoplasm and adherence to the culture dish tightly. To confirm the results from morphological assessment, DNA fragmentation analysis was performed. As shown in Fig. 3B, the electrophoretogram generated by agarose gel electrophoresis shows that silencing of Sirt1 causes slender degradation of cell DNA, which was similar to that of camptothecin treatment. In addition, the DNA extracted from camptothecin treated cells transfected with Sirt1 shRNA shows a noticeable clear-cut ladder of DNA fragments, accompanied by the decrease of large molecular weight DNA. In contrast, DNA isolated from the cells infected with empty vector remained totally intact and devoid of any fragmentation. This result implies that knockdown of Sirt1 increases the sensitivity of porcine preadipocytes to camptothecin involves the breakage of genomic DNA. FACS analysis (Fig. 3C) using Annexin/PI stained further confirmed our finding which is the synergism of both Sirt1 knockdown and camptothecin on apoptosis. Several observations supported that the role of Sirt1 is in the regulation of mammalian cell survival in response to DNA damage through the inactivation of p53 by deacetylation [23,24]. We therefore speculate that the enhanced camptothecin sensitization by knockdown of Sirt1 in porcine preadipocytes was associated with the acetylation of p53. As shown in Fig. 3D, the protein level of acetylated p53 in camptothecin treated and Sirt1 knockdown cells were elevated compared to empty vector treated cells which had little acetylated p53 protein. Moreover, the acetylated p53 level of camptothecintreated cells transfected with Sirt1 shRNA increased more significantly than that of silencing of Sirt1 or camptothecin treated along. The total p53 expression increased in parallel with acetylated p53. These findings strongly implicate that the acetylation status of p53, which relied on the deacetylized activity of Sirt1, may be crucial for camptothecin-induced porcine preadipocyte apoptosis. Acetylation of p53 activates transcription of downstream targets with proapoptotic properties thereby priming cells for apoptosis [25]. Consistent with previous report, Bax protein was also increased in Sirt1 shRNA infected cells combination with camptothecin treatment, compared with Sirt1 shRNA transfected or camptothecin treatment alone (Fig. 3D). These results indicate that inhibition of Sirt1 in preadipocytes caused an increase of Bax level, which in part could be due to the increased level of acelylated p53 in response to camptothecin. Expression patter of cleaved caspase-3 was similar to that of Bax. We also detected
Results are expressed as means7SEM. Statistical significance was determined by the Student’s t-test. Difference between groups were considered statistically significant if po0.05.
Results Sirt1 knockdown decreased viability and induced apoptosis To evaluate the function of Sirt1 in porcine preadipocyte, the knockdown experiments were performed by infecting porcine preadipocytes with pLentiHI–shSirt1. As shown in Fig. 1A, Sirt1 protein level is reduced by 57% compared to that of control cells. In contrast, the empty vector has only marginal effect on Sirt1 expression. This result indicates that Sirt1 gene is effectively suppressed by RNAi in porcine preadipocytes. Subsequently, the effect of Sirt1 knockdown on viability of porcine preadipocytes was studied. At 48 h after infection, porcine preadipocytes were analysed by Annexin V-FITC/PI apoptosis detection. Compared with control cells, pLentiHI–shSirt1 decreases the number of viable cells while increases necrotic cells and the cells in early apoptotic and late apoptotic stages (Fig. 1B). Viable cells distribution in pLentiHI–shSirt1 infected cells significantly decreases from 85 to 36% (po0.05). In addition, the early apoptotic and late apoptotic cell fractions increase from 4 to 30% (po0.01) and from 9 to 32% (po0.01), respectively. We also observed that the necrotic cells ratio in Sirt1 knockdown treated cells increased from 0.2 to about 3% (po0.05) (Fig. 1B). In contrast, the effect of empty vector on cell apoptosis was very low. Moreover, cleaved caspase-3 significantly increased in preadipocytes infected with pLentiHI–shSirt1 (Fig. 1C). Therefore, these results indicate that Sirt1 knockdown effectively decreases cell viability and induced cell apoptosis.
Camptothecin treatment caused DNA damage and induced cellular apoptosis To determine the function of Sirt1 under stimulated condition, we treated porcine preadipocytes with different concentrations (0, 1, 2, 4 mM) of camptothecin (Fig. 2A) to mimic DNA damage stress condition. Based on Hoechst staining, nuclear chromatin condensation and DNA fragmentation, which are the morphological changes characteristic of apoptosis, increase after 12 h incubation with camptothecin in a dose-dependent manner (Fig. 2B). Apoptosis is the result of an exquisite cascade of molecular events that ultimately result programmed cell death. Given the observations of nuclear morphologic changes, we also detected the cleaved caspase-3 and Sirt1 by Western blot analysis. Similar to Hoechst staining, the cleaved caspase-3 also increases dose dependently after 12 h incubation (Fig. 2C). But sirt1 expression has no change in preadipocytes induced with different concentrations of camptothecin. These results suggest that treatment with camptothecin caused DNA damage and induced preadipocyte apoptosis in a dose-dependent manner.
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Fig. 1 – Sirt1 knockdown decreases viability and induces apoptosis of porcine preadipocytes. (A) Sirt1 gene silencing by shRNA in porcine preadipocytes. Porcine preadipocytes were infected with lentiviral supernatants contained Sirt1 shRNA or scrambled shRNA. After infection (48 h), the levels of Sirt1 and cleaved caspase-3 proteins were determined by western blot analysis. (B) Downregulation of Sirt1 decreased viability and induced apoptosis in porcine preadipocytes by flow cytometric analysis. After infection (48 h), cells were incubated with Annexin V-FITC/PI double stain and determined by FACS Caliber flow cytometer and (C) Effect of Sirt1 knockdown on caspase-3 expression in porcine preadipocytes. After infection (48 h), cleaved caspase-3 was detected by Western blotting analysis. These results are represented as means7SEM of three independent experiments. po0.05 and po0.01.
the protein level of Bcl-2, which is an anti-apoptotic protein that can promote cell survival through interactions with other Bcl-2 related pro-apoptotic members, such as Bax. We found that the change of Bcl-2 protein is opposite to that of Bax and cleaved caspase-3.
Sirt1 knockdown increases acetylated p53 level in camptothecin-induced preadipocyte apoptosis To further investigate whether the deacetylated activity of Sirt1 may be crucial for camptothecin-induced porcine preadipocyte
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Fig. 2 – Camptothecin induces apoptosis of porcine preadipocytes. (A) Chemical structure of camptothecin. (B) Fluorescence microscopy of porcine preadipocytes after camptothecin exposure. Porcine preadipocytes were incubated with camptothecin at 0 lM, 1 lM, 2 lM, 4 lM for 12 h and cells were stained with fluorescent dye Hoechst 33258. After staining, cells were photographed under fluorescence microscopy at 100 magnification. Condensed nuclear chromatins were indicated using arrows and the condensed nuclear chromatin undergoing fragmentation in later stages of apoptosis are indicated using triangles and (C) Effect of camptothecin on caspase-3 expression and Sirt1 in porcine preadipocytes. Porcine preadipocytes were treated with camptothecin at the indicated concentrations for 12 h. Cleaved caspase-3 was detected by Western blotting analysis. These results are represented as means7SEM of three independent experiments. Different letters indicate significantly differences (po0.05). apoptosis, we performed the experiment of overexpression of a mutated p53 (Lys-382) which can mimic a hyperdeacetylated status of p53. Under camptothecin-, or Sirt1 shRNA-, or bothinduced preadipocytes, effect of p53 overexpression on apoptosis
is more significant than that of p53 mutant overexpression (Fig. 4A). Compared with only Sirt1 knockdown or camptothecin treatment, under the co-treatment condition of Sirt1 knockdown and camptothecin acetylated p53 level was significantly
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Fig. 3 – Sirt1 knockdown increases the sensitivity of porcine preadipocytes to camptothecin. (A) The morphological observation of porcine preadipocytes after treated with scrambled shRNA, Sirt1 shRNA, camptothecin, and Sirt1 shRNAþcamptothecin (SþC). After infection (36 h), the cells were treated with camptothecin (2 lM) for 12 h. (B) Detection of genomic DNA fragmentation in porcine preadipocytes transfected with scrambled shRNA, Sirt1 shRNA, camptothecin (2 lM), and Sirt1 shRNAþcamptothecin. DNA was extracted and subjected to pulsed field gel electrophoresis through a 1.2% agarose gel which was stained with ethidium bromide. (C) Synergism of treatment with both Sirt1 shRNA and camptothecin on preadipocyte apoptosis. The results of Cell Quest software calculated were expressed as bar graph to express the percentages of viable, early apoptotic, late apoptotic, and necrotic cells number to total number of cells and (D) Western blot analysis of Sirt1, p53, acetylated p53, Bax, Bcl-2 and cleaved caspase-3 protein expression in scrambled shRNA transfected cells (scrambled), Sirt1 shRNA transfected cells (Sirt1 shRNA), camptothecin and Sirt1 shRNA treated cells (SþC). This experiment was replicated in triplicate. Different letters indicate significantly differences (po0.05).
increased (Fig. 4B). The results show that hyperacetylated p53 is the major effect of Sirt1 knockdown, which increases camptothecin-induced porcine preadipocyte apoptosis.
Sirt1 overexpression attenuates apoptosis when challenging with camptothecin As shown in Fig. 5A, the effect of Sirt1 overexpression on viability of porcine preadipocytes treated with camptothecin was studied. At 48 h after treatment, porcine preadipocytes were analysed by Annexin V-FITC/PI apoptosis detection. Compared with the
treatment only with camptothecin, Sirt1 overexpressionþcamptothecin significantly increases the number of viable cells while decreases early apoptotic cells and late apoptotic cells. Viable cells distribution significantly increases from 28 to 49% (po0.05). In addition, the early apoptotic and late apoptotic cell fractions increase from 31 to 20% (po0.05) and from 33 to 23% (po0.05), respectively. We further examined levels of cleaved caspase-3, p53, acelylated p53 and Bax (Fig. 5B) and found that the protein levels in Sirt1 overexpressed cells significantly decreased compared with empty vector treated cells. Moreover, compared with the above protein levels under only
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Fig. 4 – Sirt1 knockdown acetylates p53 in camptothecin-induced preadipocytes. Preadipocytes were treated with p53 or p53 mutant infection, p53 or p53 mutant and Sirt1 shRNA co-infection, p53 or p53 mutant and Sirt1 shRNA co-infection induced with camptothecin (2 lM). After 48 h, on the one hand, FACS analysis using Annexin/PI stained was performed by the treated cells; on the other hand, total proteins were extracted for Western blot examination and analysis of p53, acetylated p53 and tubulin protein expressions. (A) Effect of p53 and p53 mutant on apoptosis under camptothecin- and Sirt1 shRNA-induced cells and (B) Sirt1 knockdown acetylates p53 in camptothecin-induced cells. These results are represented as means7SEM of 3 independent experiments. Different letters indicate significantly differences (po0.05). camptothecin, the protein levels under the treatment of Sirt1 overexpressedþcamptothecin significantly increase (po0.05). Therefore, these results indicate that Sirt1 overexpression effectively increases cell viability and inhibits cell apoptosis when challenging with camptothecin.
Molecular mechanism of Sirt1 repressing preadipocyte apoptosis To explore the molecular mechanism underlying Sirt1 inhibitory action on preadipocyte apoptosis, we focus on the effect of
exogenous Sirt1 on caspase-3 proteins, since caspase-3 plays important roles in promoting apoptosis. To investigate Sirt1 interaction with caspase-3, we transfected HEK293T cells with caspase-3 and Sirt1-Flag. Sirt1 proteins were immunoprecipitated with anti-Flag agarose beads. Caspase-3 interaction with Sirt1 protein was detected by immunoblot analysis with caspase-3 antibodies. As shown in Fig. 6A (upper), caspase-3 is coimmunoprecipitated with Sirt1-Flag. Ditto, as shown in Fig. 6A (lower), Sirt1 is co-immunoprecipitated with caspase-3-Flag. Here, we need point out that caspase-3 protein is full length in this experiment.
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Fig. 5 – Sirt1 overexpression attenuates apoptosis induced by camptothecin. (A) The results of Cell Quest software calculated were expressed as bar graph to express the percentages of viable, early apoptotic, late apoptotic, and necrotic cells number to total number of cells and (B) Western blot analysis of Sirt1, cleaved caspase-3, p53, acetylated p53, Bax and Tubulin protein expression in the control cells (Empty vector), Sirt1 overexpressed cells (Sirt1 overexpression), the cells treated with camptothecin, cells treated with camptothecin and Sirt1 overexpression (SþC). This experiment was replicated in triplicate. Different letters indicate significantly differences (po0.05).
The amino acid (aa) sequences of caspase-3 and Sirt1 from various species were aligned (Fig. 6B, Fig. S1B) and the phylogenetic tree was constructed by the Neighbor-Joining method (Fig. 6C, Fig. S1A). We find that aa sequences of caspase-3 and Sirt1 are highly conserved among Sus Scrofa, Homo Sapiens and Mus Musculus. Therefore, we further performed immunoprecipitation experiment to detect endogenous interaction between Sirt1 and caspase-3. The results show that endogenous interaction between Sirt1 and cleaved caspase-3 is in nuclear proteins under camptothecin stimulation condition (Fig. 6D). In addition, endogenous tenuous interaction between Sirt1 and full length caspase-3 are found in cytoplasmic proteins under camptothecin stimulation condition (Fig. 6D). To test whether Sirt1 inhibits adipocyte apoptosis through inhibiting caspase-3, a luciferase reporter construct containing
the caspase-3 binding site from human complement component C3 promoter was used. Caspase-3 activates this reporter construct, as shown in Fig. 6E; however, with the co-expression of Sirt1, caspase-3 transactivation activity is acutely reduced. Furthermore, the repression of caspase-3 transactivation by Sirt1 is dose dependent (Fig. 6E). The above results show that Sirt1 does not only directly inhibit the expression of caspase-3, also changes caspase-3 protein structure by protein–protein interaction.
Discussion In this study, we find that silencing of Sirt1 induces porcine preadipocytes apoptosis and significantly enhances the sensitivity
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Fig. 6 – Sirt1 interacts with caspase-3 and inhibits caspase-3 activity. (A) Exogenous Sirt1 interacts with caspase-3 in 293T cells. HEK293 cells were transiently transfected with caspase-3, Sirt1-Flag, or both by the calcium phosphate method. After transfection (40 h), cells were lysed, and Sirt1-Flag proteins were immunoprecipitated with agarose beads and conjugated with anti-Flag antibodies. The precipitated proteins were washed 4 times and analyzed by Western blotting as indicated. Vice versa. IP, immunoprecipitation. IB, immunoblotting. (B) The amino acid (aa) sequences of caspase-3 from various species were aligned, with conserved residues shown . Full length caspase-3 is cleaved at Asp28Ser29 and Asp175Ser176, with cleaved position shown blue letters in red line box. Caspase-3 protein Accession No.: Sus Scrofa (BAB55544.1), Homo Sapiens (NP_004337.2) and Mus Musculus (NP_033940.1). (C) Phylogenetic tree of the caspase-3 proteins. The phylogenetic tree is constructed by the Neighbor-Joining method. The numbers by the branches indicate bootstrap values based on 1000 replications. Branch lengths are relative to the degree of divergence. Caspase-3 protein Accession No. and length: Bos Taurus (NP_001071308.1; 275 aa), Gallus gallus (NP_990056.1; 283 aa), Xenopus laevis (NP_001081225.1; 282 aa), Danio rerio (NP_571952.1; 282 aa). (D) Interaction of endogenous Sirt1 and caspase-3 or cleaved caspase-3 in cytoplasm and nucleus of preadipocytes under Camptothecin stimulation and (E) The suppression of caspase-3 transactivation activity by Sirt1. HEK293T cells were transfected with a caspase3 responsive luciferase reporter (pC3-Luc) and a Renilla luciferase construct (pRL-TK), together with a caspase-3 expressing construct. Relative luciferase activity is presented to demonstrate the effect of Sirt1 on caspase-3. Different amounts of Sirt1expressing plasmid (2–8 lg) were used to test the dosage effect of Sirt1 on caspase-3 transactivation. The results are presented as means7SEM of 3 independent experiments. po0.05 and po0.01.
of cells to camptothecin. It provides new and important information that Sirt1 functions as a cell survival factor in porcine preadipocytes under basal and stimulated conditions. Some evidence suggested that downregulation of Sirt1 could induce apoptosis in mammalian cells. In glioma CD133-positive cells, downregulation of Sirt1 enhanced radiosensitivity and radiationinduced apoptosis [26]. In prostate cancer PC3 and DU145 cells, silencing of Sirt1 inhibited cell growth and increased sensitivity to camptothecin and cisplatin [27]. Consider together with the above results, our findings in porcine preadipocytes indicate that
Sirt1 universally functions as a cell survival factor in mammalian cells. As preadipocytes are the initial formation of adipose depots and possess the ability to undergo complete differentiation into mature adipocytes [28], to decrease adipose tissue mass by induction of preadipocytes apoptosis to control the number of preadipocytes may be more useful. Consequently, identifying key factors that control this process is important for the development of new approaches to decrease fat deposition. Therefore, we investigated whether Sirt1 had effects on the apoptosis of
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preadipocyte. Here, we find that Sirt1 overexpression attenuates fat cell apoptosis induced with camptothecin. These results indicate that Sirt1 is a critical factor on apoptotic regulation of porcine preadipocytes and suitable to define Sirt1 as a cellular protector. However, we must point out to explore the mechanisms to reduce adipose mass through preadipocyte apoptosis involved the impairment of Sirt1’s function in preadipocyte under differentiation-induced condition would not be promising because since 2004 it has been reported that RNA interference of Sirt1 enhances adipogenesis [13]. Here, our research provided a new idea to reduce adipose mass through apoptosis regulated by other genes. Importantly, under normal (non-differentiationinduced) condition, inhibition of Sirt1 results in decreasing the number of preadipocytes by apoptosis. Camptothecin is a strong DNA-damaging compound. In this study, we incubated porcine preadipocytes with different concentrations of camptothecin, indicating that camptothecin caused DNA damage and induced apoptosis in porcine preadipocytes in a dose-dependent manner. Moreover, silencing of Sirt1 significantly increases the sensitivity of porcine preadipocytes to camptothecin compared to either cells treated with camptothecin or cells treated with Sirt1 shRNA alone because of increasing cleaved casepase-3. Interestingly, Sirt1 expression has no change in preadipocytes treated with different concentrations of camptothecin. At present, the reason is unclear. The p53 tumor suppressor protein plays a central role in the chemosensitivity and radiosensitivity of cells by inducing cell cycle arrest and apoptosis [29,30]. Acetylated p53 (lysine-382) is one of the activated states of p53, which has been reported to increase transcriptional activity of p53 in response to various stressors. In line with Sirt1 function in p53 regulation, Sirt1 tightly binds to and specifically deacetylates p53, thereby impairing its DNA binding activity and causes reduction of p53-mediated apoptosis in cultured cells in response to DNA damage [24]. A recent study showed that acelylated-p53 mediated camptothecin induced apoptosis. RSV, a small molecule polyphenolic Sirt1 activator, markedly decreased CPT induced p53 acetylation in wild-type neurons [31]. Herein, we observed that acetylated-p53 appeared after Sirt1 knockdown compared to empty vector transfection, suggesting that the increased acetylated p53 is associated with silencing of Sirt1, which is consistent with the previous report. Importantly, Sirt1 knockdown combination with camptothecin treatment significantly upregulates the protein level of acetylated-p53 more than the responses to the individual treatment alone and more than the calculated additive response, suggesting that Sirt1 is an intrinsic regulator of the acetylation status of p53 to promote porcine preadipocyte survival against camptothecin-induced action. Through overexpression of a mutated p53, we further affirmed that the hyperacetylated p53 is the major effect of Sirt1 knockdown, which increases camptothecin-induced porcine preadipocyte apoptosis. Bax is a proapoptotic Bcl-2 family protein that resides in the cytosol and translocates to mitochondria upon induction of apoptosis, which can be upregulated and transcriptional activated by p53. Chipuk et al. reported p53 directly activated Bax to promote mitochondrial outer-membrane permeabilization (MOMP) and to engage the apoptotic program [32]. Here, we found that Sirt1 shRNA and camptothecin individual increased Bax protein and combinations of Sirt1 shRNA and camptothecin
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future increased Bax protein more than any one treatment alone, suggesting that silencing of Sirt1 in porcine preadipocytes increased the level of Bax, which in part could be due to the increased level of acelylated p53 in response to camptothecin. Interestingly, the changed tendency of Bcl-2 protein is opposite to Bax, and we speculate that it may be resulted from Bax heterodimerized with Bcl-2 and counteracted Bcl-2 expression. Sirt1 has also been reported to deacetylate other transcriptional factors in addition to p53, including Ku70, FoxOs and E2F1 [33]. Moreover, we found that expression patter of caspase-3 was similar to p53 and Bax in our experiments. It was reported that H2O2 and TNFa increased acetylation of p53 lysine-382, as well as caspase-3 cleavage, an indicator of apoptotic signaling [34]. Therefore, it is reasonable to conclude that Sirt1-regulated and camptothecin-induced porcine preadipocyte apoptosis is via the p53–caspase-3 mediated pathway. Caspase-3 is a key terminal molecular regulated apoptosis in cellular apoptosis pathways. However, it is unclear whether that Sirt1 physically interacts with caspase-3, so we explored molecular mechanism of Sirt1 repressing preadipocyte apoptosis by the experiments of immunoprecipitation and luciferase reporter assays. Expression patter and subcellular location of caspase-3 and Sirt1 had been reported [35–38]. Caspase-3 and Sirt1 expressed in porcine preadipocytes (data not shown). It was reported that exogenous administration of Sirt1 protein or administration of resveratrol that is known to activate Sirt1 in the presence of elevated D-glucose fostered the translocation of endogenous Sirt1 from the cytoplasm to the nucleus [39,40]. Caspase-3 likewise translocated from the cytoplasm to the nucleus [41]. Therefore, the two proteins exist in both cytosol and nucleus, and have potential physical interaction. To further verify the hypothesis, endogenous Sirt1 in nuclear and cytoplasmic proteins of porcine preadipocytes were respectively immunoprecipitated with anti-Sirt1 antibody, and co-precipitation of caspase-3 or cleaved caspase-3 was respectively detected by Western blot assay. Fortunately, the results show that endogenous interaction between Sirt1 and cleaved caspase-3 is prominent in nuclear proteins, although endogenous interaction between Sirt1 and full length caspase-3 is found in cytoplasmic proteins under camptothecin-stimulated condition. Interestingly, endogenous interaction between Sirt1 and cleaved caspase-3 or full length caspase-3 is not found under normal condition. Because of high conservation of caspase-3 and Sirt1 protein aa sequences among Sus Scrofa, Homo Sapiens and Mus Musculus, this endogenous interaction may exit in human preadipocyes. Under differentiation-induced or non-differentiation-induced conditions, we think that inhibition of Sirt1 results in different physiological changes of preadipocytes. Under non-differentiation-induced condition, inhibition of Sirt1 decreases the number of preadipocytes by apoptosis. Under differentiation-induced condition, inhibition of Sirt1 improves, whereas activation of Sirt1 inhibits adipogenesis preadipocytes. At present, many previous reports have shown that activation of Sirt1 should have therapeutic potency to improve obesity and diabetes through regulation of preadipocyte differentiation [42–44]. Expansion of adipose tissue occurs through the formation of new adipocytes, via the differentiation of preadipocytes, and the enlargement of existing adipocytes [45]. Recently, researchers reported a novel hopeful therapic approach for obesity and diabetes through preadipocyte apoptosis regulated via mast cells.
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Fig. 7 – Model for the regulation of preadipocyte apoptosis by Sirt1 and Camptothecin. Under the stimulations of Camptothecin, Sirt1 shRNA, Sirt1 overexpression, both Camptothecin and Sirt1 shRNA, both Camptothecin and Sirt1 overexpression, cleaved caspase-3 significantly increases or decreases to trigger or inhibit cellular apoptosis. Sirt1 overexpression attenuates apoptosis induced with camptothecin, whereas Sirt1 knockdown aggravates apoptosis induced with camptothecin through caspase-3 pathway.
Macrophages may alter preadipocyte fate through apoptosis [46]. In both murine cell lines and human primary cells, proinflammatory activation of macrophages inhibits their prosurvival activity, favoring preadipocyte death [47]. White adipose tissue (WAT) from obese humans contains large numbers of mast cells which participate in the relative metabolic disorders of obesity and diabetes by affecting energy expenditure, glucose and insulin sensitivities, apoptosis, and preadipocyte differentiation [48]. These findings open a new era of basic research regarding mast cells in WAT and offer hope to patients suffering from obesity and diabetes. Based on above results, we think that it may be hopeful to decrease adipose tissue mass by induction of preadipocyte apoptosis to control its number. Based on our findings, model for the regulation of preadipocyte apoptosis by Sirt1 and Camptothecin was established (Fig. 7). In conclusion, our study indicates that Sirt1 overexpression inhibits apoptosis and attenuates camptothecin-induced apoptosis through caspase-3 pathway, whereas silencing of Sirt1 leads to apoptosis and enhances the sensitivity of porcine preadipocytes to camptothecin. Our findings may provide a new clue that regulation of Sirt1 could potentially be a plausible method for improvement of porcine meat quality and the treatment of human obesity.
Conflict of interest statement The authors declare that there are no conflicts of interest.
Acknowledgment This study was supported by the Natural Sciences Foundation of China (No. 30600437), NWAFU Young Research Programme (No. 0114030) and NWAFU Basic Science Research Programme (No. QN2009021). Moreover, we thank the reviewer’s comments very much.
Appendix A.
Supporting information
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.yexcr.2012.12.025.
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