Cardiovasc Toxicol DOI 10.1007/s12012-014-9287-6
SIRT4 Suppresses Inflammatory Responses in Human Umbilical Vein Endothelial Cells Yu Tao • Chunhua Huang • Yingmei Huang Lang Hong • Hong Wang • Zijie Zhou • Yun Qiu
Ó Springer Science+Business Media New York 2014
Abstract The most common feature of endothelial dysfunction is endothelial inflammation. A bunch of factors are associated with endothelial dysfunction. These include pro-inflammatory cytokines, cell adhesion molecules, and matrix degrading enzymes. SIRT4, a member of the sirtuin family, is a mitochondrial ADP-ribosyltransferase. The roles of SIRT4 in regulating inflammation in endothelial cells are unknown. In this study, we found that lipopolysaccharide treatment decreased the expression of SIRT4 in human umbilical vein endothelial cells. Silence of SIRT4 exacerbated the expression of pro-inflammatory cytokines (IL-1b, IL-6 and IL-8), COX-prostaglandin system (COX2), ECM remodeling enzymes MMP-9, and the adhesion molecule ICAM-1. The upregulation of these genes are involved in inflammation, vascular remodeling, and angiogenesis. In contrast, overexpression of SIRT4 attenuated the induction of these factors. Mechanistically, SIRT4 was found to interfere with the NF-jB signaling pathway by preventing NF-jB nuclear translocation and thereby has an anti-inflammatory function. Loss of SIRT4 increased the nuclear translocation as well as the transcriptional activity of NF-jB. However, overexpression of SIRT4 mitigated the nuclear translocation and the transcriptional activity of Yu Tao and Chunhua Huang are co-first authors. Y. Tao Y. Huang L. Hong H. Wang Z. Zhou Y. Qiu (&) Department of Cardiology, Jiangxi Provincial People’s Hospital, No, 92 Aiguo Road, Donghu District, Nanchang 330006, Jiangxi, People’s Republic of China e-mail: [email protected]
C. Huang Department of Neurology, Jiangxi Provincial Hospital of Chinese Medicine, Nanchang 330006, Jiangxi, People’s Republic of China
NF-jB. Our data suggested that SIRT4 might be a potential pharmacological target for inflammatory vascular diseases. Keywords SIRT4 Endothelial dysfunction NF-jB Pro-inflammatory cytokines
Introduction The endothelium is the thin layer of cells that lines the interior surface of blood vessels and lymphatic vessels . Endothelium participates in the regulation of cell–cell interactions during inflammatory and immunological processes. Endothelial dysfunction has been reported to play an initial role in certain chronic vascular inflammatory diseases and their pathogenesis . Efforts have been made to reduce or prevent the high incidence of morbidity and mortality associated with vascular complications in the past decades. It is essential to understand further the mechanism by which inflammation regulates endothelial function. A wide number of factors have been reported to participate in the inflammatory response and contribute to systemic inflammation. These factors include pro-inflammatory cytokines (IL-1b, IL-6, and IL-8) , extracellularmatrix (ECM) degrading enzymes matrix metalloproteinases (MMPs) , the cell adhesion molecule—intercellular adhesion molecule (ICAM-1)—and the cyclooxygenase 2 (COX2) . Multiple lines of evidence have shown that the sirtuin (SIRT) family can regulate multiple genes whose products are putatively involved in the regulation of endothelial cell function . There are seven members (SIRT1–SIRT7) in the sirtuin (SIRT) family. Among them, SIRT1 has been intensively studied . However, the physiological role of SIRT4 in endothelial function has not been reported before.
SIRT4 is one of the mitochondrial sirtuins which has been found to be specifically enriched in the heart, kidney, brain, and liver . In this study, we reported that the endotoxin lipopolysaccharide (LPS) inhibits the SIRT4 expression. In addition, we found that silence of SIRT4 by siRNA exacerbates the induction of pro-inflammatory cytokines (IL1b, IL-6, and IL-8), COX2, MMP-9, and ICAM-1 by LPS. However, overexpression of SIRT4 abolished the induction of these pro-inflammatory factors. Importantly, our data indicated that the anti-inflammatory effects of SIRT4 are mediated by NF-jB.
Materials and Methods Cell Culture, Treatment, and Transfection Human umbilical vein endothelial cells (HUVECs) were from Lonza (Walkersville, USA). Cells were cultured in EBM-2 media with supplemental growth factors according to the manufacturer’s instructions . LPS was used to treat HUVECs for various periods of time with various doses as follows: To detect the alterations of SIRT4 expression in response to LPS in a dose-dependent manner, cells were treated with LPS for 24 h at the concentrations of 50, 100, and 200 ng/ml, respectively; to detect the alterations of SIRT4 expression in response to LPS in a time-dependent manner, cells were treated with 100 ng/ml LPS for 12, 24, and 48 h, respectively. Small interfering RNA (siRNA) for SIRT4 and negative control were from Invitrogen, USA. Cells were transfected with target siRNAs using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. Adenoviral SIRT4 Methods An AdEasybasic kit was used to construct the Ad-SIRT4 plasmid according to the manufacturer’s instructions (ATCC, USA). Briefly, in order to generate SIRT4/pAdTrack-CMV, we cloned a XhoI/NotI fragment of NmycPRC/pBSII into SalI/Not-digested pAdTrack-CMV. After linearized, adenoviral plasmids were transfected into human embryonic kidney 293 (HEK-293) using Lipofectamine 2000. The supernatant was collected and stored at -80 °C until use. HUVECs were infected by adenoviralSIRT4. Infection efficiency (95–100 %) was determined by GFP expression at 24 h after infection.
instructions . Two micrograms of target RNA was converted to cDNA by treatment with reverse transcriptase and oligo (dT) primer. Synthesized cDNA products were used for real-time PCR with SYBR GREEN PCR Master Mix (Applied Biosystems). The relative abundance of target mRNA was normalized to GAPDH. NF-jB Activity Assay NF-jB activity was determined by a luciferase assay. Briefly, HUVECs were cotransfected with 1 lg reporter (either pNF-jB-Luc or negative control; Qiagen) and 1 lg plasmid (empty vector or SIRT4 vector) transfected using Lipofectamine 2000 as described previously for 24 h . The cells were harvested in lysis buffer, and luminescence activity was measured using the Dual-Glo Luciferase Assay kit (Promega, USA). Relative luciferase activity was normalized by Renilla activity. Western Blot Analysis After indicated transfection and treatment, cells were lysed by a cell lysis buffer (Cell signaling, USA). For nuclear protein extraction, an NE-PER nuclear protein extraction kit was used following the manufacturer’s instructions (Thermo Scientific, USA) . Twenty micrograms of total protein extract was separated by 10 % SDS-PAGE and were electrotransfered onto Immobilon-P Membrane (Millipore, USA). Transferred membranes were blocked in TBS containing 10 % nonfat dry milk and 0.5 % Tween-20 for 2 h at RT, and the membrane was probed with the indicated primary antibodies for 3 h at room temperature (RT) and with the secondary antibodies for 2 h at RT. Immobilon Western Chemiluminescent HRP Substrate from Millipore was used for the blots development. Immunofluorescence For immunofluorescence, HUVECs were fixed by 4 % paraformaldehyde for 10 min at RT. Then cells were permeabilized with 0.4 % Triton X-100 in phosphate buffer saline in Tween-20 (PBST) for 15 min on ice, followed by blocked with 5 % BSA and 2.5 % FBS in PBST. Cells were then sequentially incubated with the anti-p65 antibody for 2 h at RT, and the Alexa-594 conjugated secondary antibody for 1 h at RT. Cytokine Assays In Vitro
Real-time Quantitative Polymerase Chain Reaction (QPCR) Total RNA from HUVECs was isolated using Trizol (Invitrogen, USA) in accordance with the manual
Cells (4 9 105 cells/mL) were seeded into 24-well plates. For the SIRT4 overexpression experiment, HUVECs were infected with Ad-SIRT4 for 24 h, followed by stimulation with 100 ng/ml LPS for another 24 h. For the knockdown
Fig. 1 Expression of SIRT4 in HUVECs after LPS treatment. HUVECs were stimulated with LPS at various concentrations for 24 h, and a mRNA levels of SIRT4 at various concentrations were determined by real-time PCR (*P \ 0.01 vs. non-treated control, n = 4). b Protein levels of SIRT4 at various concentrations were determined by western blot analysis (*P \ 0.01 vs. non-treated
control, n = 4). HUVECs were stimulated with LPS at indicated doses (100 ng/ml) for varying periods of time. c mRNA levels of SIRT4 at varying time periods were determined by real-time PCR (*P \ 0.01 vs. non-treated control, n = 4). d Protein levels of SIRT4 at varying time periods were determined by western blot analysis (*P \ 0.01 vs. non-treated control, n = 4)
experiment, HUVECs were transfected with siSIRT4 or NS for 24 h, followed by stimulation with 100 ng/ml LPS for another 24 h at 37 °C in an incubator containing 5 % CO2. Cell-free supernatants were collected and used in the assay for cytokines. The concentrations of the cytokines IL-1b, IL-6, and IL-8 in the supernatants were determined by using ELISA kits according to the manufacturer’s instructions.
sustainable downregulation in both messenger RNA (mRNA) levels of SIRT4 (Fig. 1a) and in protein levels (Fig. 1b). HUVECs were incubated with 100 ng/ml LPS for various timespans. Results showed a sustainable reduction at both mRNA levels (Fig. 1c) and protein levels of SIRT4 in a time-dependent manner from 12 to 48 h (Fig. 1d). The reduced levels of SIRT4 induced by LPS suggested a potential role in LPS toxicity. We next determine the effect of SIRT4 knockdown on endothelial function in the presence of LPS. The successful knockdown of SIRT4 was shown in Fig. 2a. QPCR results indicated that treatment of HUVECs with LPS significantly increased the gene expression of IL-1b, IL-6, and IL-8, which has been exacerbated by knockdown of SIRT4 (Fig. 2b) at mRNA levels. Consistently, this finding was confirmed by Elisa assays at protein levels, as shown in Fig. 2c. COX-2 is an essential enzyme responsible for inflammation and pain. The induction of COX-2 by LPS was worsened by knockdown of SIRT4 (Fig. 2d). MMP-9 is an important enzyme involved in ECM degradation and ECM remodeling. Our results indicated that silence of SIRT4 also exacerbated the induction of MMP-9 induced by LPS (Fig. 2e). Endothelial ICAM-1 is responsible for the attachment of circulating monocytes to the endothelium. Indeed, it was found that SIRT4 knockdown
Statistical Analysis All data are presented as mean ± SEM from at least three separate experiments. One-way analysis of variance (ANOVA) was used to assess the statistical significance of differences among treatment groups. And differences between means were considered statistically significant at P \ 0.05.
Results Firstly, SIRT4 expression was examined in HUVECs incubated with LPS at various doses for various periods of time. LPS treatments administered on HUVECs at the concentrations of 50, 100, and 200 ng/ml for 24 h led to a
Fig. 2 Expression of inflammatory factors in SIRT4 knockdown HUVECs after incubation with LPS. HUVECs were transfected with siSIRT4 or NS for 24 h, followed by stimulation with 100 ng/ml LPS for another 24 h. siSIRT4, SIRT4 siRNA group; NS nonspecific siRNA group. a Successful knockdown of SIRT4 was determined by western blot analysis (*P \ 0.01 vs. NS group), b QPCR results revealed that SIRT4 knockdown cells have more expression of IL-1b, IL-6, and IL-8 in response to LPS [*P \ 0.01 vs. NS group; #P \ 0.01 vs. NS ? (LPS) group], c The mean concentration of IL-
1b, IL-6, and IL-8 in the culture medium of HUVECs was measured by ELISA [*P \ 0.01 vs. NS group; #P \ 0.01 vs. NS ? (LPS) group], d The expression of COX2 was determined by QPCR analysis [*P \ 0.01 vs. NS group; #P \ 0.01 vs. NS ? (LPS) group], e The expression of MMP9 was determined by real-time PCR analysis [*P \ 0.01 vs. NS group; #P \ 0.01 vs. NS ? (LPS) group], f The expression of ICAM-1 was determined by real-time PCR analysis [*P \ 0.01 vs. NS group; #P \ 0.01 vs. NS ? (LPS) group]
exacerbated the induction of ICAM-1 (Fig. 2f). In order to further confirm the effects of SIRT4 on endothelial activation in response to inflammatory cytokines, HUVECs were overexpressed with SIRT4 for 24 h, stimulated with 100 ng/ml LPS for an additional 24 h, and then assessed for the expression of pro-inflammatory factors. Successful overexpression of SIRT4 has been shown in Fig. 3a. As shown in Fig. 3b, overexpression of SIRT4 mitigated the expression of IL-1b, IL-6, and IL-8 induced by LPS at mRNA levels. Consistently, Elisa results indicated that overexpression of SIRT4 attenuated the expression of IL1b, IL-6, and IL-8 induced by LPS at protein levels (Fig. 3c). In addition, the induction of COX-2 by LPS was attenuated by overexpression of SIRT4 (Fig. 3d). Thirdly, overexpression of SIRT4 also partially inhibited the induction of MMP-9 by LPS (Fig. 3e). Lastly but importantly, the induction of ICAM-1 induced by LPS was tempered by overexpression of SIRT4 (Fig. 3f). These data demonstrate that SIRT4 can inhibit endothelial inflammation in response to LPS treatment. NF-jB has been considered as a critical factor in the endothelial activation in response to inflammatory stimuli . This factor is located in the cytoplasm as a heterodimer
of the p50 and p65 subunits under normal conditions. Activation of NF-jB is regulated by its inhibitor IjB, which can retain the entire complex in cytoplasm. However, phosphorylation and subsequent degradation of IjB occur in response to inflammatory cytokines, thereby leading to the liberation of NF-jB heterodimers, which can then translocate to the nucleus, and activate target inflammatory genes expression. Our results indicated that LPS-induced p65 nuclear translocation was blocked by overexpression of SIRT4 (Fig. 4a). Moreover, we assessed the inhibitory effect of SIRT4 on NF-jB by performing luciferase reporter assays. LPS drastically induced NF-jB luciferase activity, which was markedly suppressed by overexpression of SIRT4 (Fig. 4b). Immunofluorescence analysis revealed that LPS-induced p65 nuclear translocation was blocked by SIRT4 overexpression (Fig. 4c). In contrast, silence of SIRT4 exacerbated p65 nuclear translocation (Fig. 5a) as well as the NF-jB luciferase activity (Fig. 5b) induced by LPS. These results confirmed the inhibitory effects of SIRT4 in NF-jB activation. Also, immunostaining results revealed that LPS-induced p65 nuclear translocation was exacerbated by SIRT4 silencing, thereby confirming the inhibition effect of SIRT4 in NF-jB activation (Fig. 5c).
Fig. 3 Expression of inflammatory factors in SIRT4 overexpressing HUVECs after incubation with LPS. HUVECs were infected with AdSIRT4 for 24 h, followed by stimulation with 100 ng/ml LPS for another 24 h. Neo, null control group; SIRT4, SIRT4 overexpression group. a Western blot analysis revealed that SIRT4 was successfully overexpressed in HUVECs (*P \ 0.01 vs. Neo control), b After incubation with LPS for 24 h, QPCR results revealed that SIRT4 overexpression cells have less expression of IL-1b, IL-6, and IL-8 in response to LPS [*P \ 0.01 vs. Neo group; #P \ 0.01 vs.
Neo ? (LPS) group], c The mean concentration of IL-1b, IL-6, and IL-8 in the culture medium of HUVECs was measured by ELISA [*P \ 0.01 vs. Neo group; #P \ 0.01 vs. Neo ? (LPS) group], d The expression of COX2 was determined by QPCR analysis [*P \ 0.01 vs. Neo group; #P \ 0.01 vs. Neo ? (LPS) group], e The expression of MMP9 was determined by real-time PCR [*P \ 0.01 vs. Neo group; #P \ 0.01 vs. Neo ? (LPS) group], f The expression of ICAM-1 was determined by real-time PCR analysis [*P \ 0.01 vs. Neo group; #P \ 0.01 vs. Neo ? (LPS) group]
Fig. 4 Overexpression of SIRT4 inhibits transcriptional activity of NF-jB. HUVECs were infected with Ad-SIRT4 for 24 h, followed by stimulation with 100 ng/ml LPS for another 24 h. a Effects of SIRT4 on p65 nuclear translocation. Nuclei were extracted for western blot analysis (*P \ 0.01 vs. control group; #P \ 0.01 vs. LPS group), b NF-jB luciferase reporter assays. HUVECs transfected with pNF-jB-Luc reporter were treated with 100 ng/ml LPS as indicated for 24 h before measuring luciferase activity (*P \ 0.01 vs. control group; #P \ 0.01 vs. LPS group), c HUVECs were immunostained with anti-p65 antibodies
Cardiovasc Toxicol Fig. 5 Inhibition of SIRT4 promotes transcriptional activity of NF-jB. HUVECs were transfected with siSIRT4 or NS for 24 h, followed by stimulation with 100 ng/ml LPS for another 24 h. a Effects of SIRT4 on p65 nuclear translocation. Nuclei were extracted for western blot analysis (*P \ 0.01 vs. NS group; #P \ 0.01 vs. NS ? LPS group), b NF-jB luciferase reporter assays. HUVECs transfected with pNFjB-Luc reporter were treated with 100 ng/ml LPS as indicated for 24 h before measuring luciferase activity (*P \ 0.01 vs. NS group; #P \ 0.01 vs. NS ? LPS group), c HUVECs were immunostained with anti-p65 antibodies
Discussion This is the first study to report that LPS can decrease SIRT4 expression in HUVECs. This led to the hypothesis that SIRT4 may also play an important role in infection induced endothelial dysfunction. In SIRT4 silent cells, in the presence of LPS, there was an increase in the expression of the pro-inflammatory cytokines IL-1b, IL-6, and IL8, ECM degrading enzymes MMP9, COX-prostaglandin system COX-2, and the cell adhesion molecular ICAM-1. However, overexpression of SIRT4 attenuated the induction of these factors by LPS. Systemic inflammation causes an upregulation of a wide number of factors that lead to severe injury of vascular endothelial cells. The inhibition effect of SIRT4 on a wide number of factors which lead to severe injury of vascular endothelial cells suggests that it can exert beneficial effects on systemic inflammation. SIRT6 is another member in the sirtuins family with an ADP-ribosyltransferase activity. In consistence with our current findings, a recent study has reported that LPS decreased expression of SIRT6 in HUVECs. Importantly, loss of SIRT6 in endothelial cells is associated with
upregulation of genes involved in inflammation, vascular remodeling, and angiogenesis . In addition, an important protective role for SIRT1 has been highlighted in vascular dysfunction. On one hand, infection decreases SIRT1 expression and activity . On the other hand, activation of SIRT1 exerts beneficial effects on endothelial cell dysfunction . This study also highlights the relevance of SIRT4 as an important inhibitor of NF-jB. NF-jB has been considered as a critical factor in the activation of endothelial dysfunction related genes . SIRT4 is mainly located in the mitochondrial matrix in mammalian cells . NF-jB subunits RelA and p50, the IjBa inhibitor as well as the upstream kinases IKKa, IKKb, and IKKc have been identified in mitochondria . Thus, we speculate that SIRT4 regulates NF-jB activity by interacting with NF-jB or its regulators in mitochondria. Different from other sirtuins, SIRT4 has a weak ADP-ribosyltransferase activity . Whether its ADP-ribosyltransferase activity accounts for its inhibitory effects on NF-jB is still unknown. Further study will provide a complete picture of the underlying mechanisms. Taken together, our study suggested that
SIRT4 may be a potential pharmacological target for inflammatory vascular diseases.
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