JBIC Journal of Biological Inorganic Chemistry https://doi.org/10.1007/s00775-018-1563-7
ORIGINAL PAPER
Selenoprotein S inhibits inflammation‑induced vascular smooth muscle cell calcification Yali Ye1 · Weixia Bian1 · Fen Fu1 · Jian Hu2 · Hongmei Liu1 Received: 21 February 2018 / Accepted: 24 April 2018 © SBIC 2018
Abstract Vascular calcification is a prominent feature of many diseases including atherosclerotic cardiovascular disease (CVD), leading to high morbidity and mortality rates. A significant association of selenoprotein S (SelS) gene polymorphism with atherosclerotic CVD has been reported in epidemiologic studies, but the underlying mechanism is far from clear. To investigate the role of SelS in inflammation-induced vascular calcification, osteoblastic differentiation and calcification of vascular smooth muscle cells (VSMCs) induced by lipopolysaccharide (LPS) or tumor necrosis factor (TNF)-α were compared between the cells with and without SelS knockdown. LPS or TNF-α induced osteoblastic differentiation and calcification of VSMCs, as showed by the increases of runt-related transcription factor 2 (Runx2) protein levels, Runx2 and type I collagen mRNA levels, alkaline phosphatase activity, and calcium deposition content. These changes were aggravated when SelS was knocked down by small interfering RNA. Moreover, LPS activated both classical and alternative pathways of nuclear factor-κB (NFκB) signaling in calcifying VSMCs, which were further enhanced under SelS knockdown condition. SelS knockdown also exacerbated LPS-induced increases of proinflammatory cytokines TNF-α and interleukin-6 expression, as well as increases of endoplasmic reticulum (ER) stress markers glucose-regulated protein 78 and inositol-requiring enzyme 1α expression in calcifying VSMCs. In conclusion, the present study suggested that SelS might inhibit inflammation-induced VSMC calcification probably by suppressing activation of NF-κB signaling pathways and ER stress. Our findings provide new understanding of the role of SelS in vascular calcification, which will be potentially beneficial to the prevention of atherosclerotic CVD. Keywords Selenoprotein S · Vascular calcification · Inflammation · Nuclear factor-κB · Endoplasmic reticulum stress Abbreviations AAS Atomic absorption spectroscopy ALP Alkaline phosphatase BSA Bovine serum albumin Col I Type I collagen CVD Cardiovascular disease DMEM Dulbecco modified Eagle’s medium ECL Enhanced chemiluminescence ER Endoplasmic reticulum ERAD ER-associated protein degradation FCS Fetal calf serum * Hongmei Liu
[email protected] 1
Hubei Key Laboratory of Bioinorganic Chemistry and Materia Medica, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, People’s Republic of China
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA
2
GAPDH Glyceraldehyde 3-phosphate dehydrogenase GRP78 78-kDa glucose-regulated protein IL Interleukin IRE1α Inositol-requiring enzyme 1α LPS Lipopolysaccharide MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide NF-κB Nuclear factor-κB PCR Polymerase chain reaction RANKL Receptor activator of NF-κB ligand ROS Reactive oxygen species Runx2 Runt-related transcription factor 2 SelS Selenoprotein S siRNA Small interference RNA TNF-α Tumor necrosis factor-α VSMCs Vascular smooth muscle cells
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Introduction Vascular calcification refers to the deposition of the mineral calcium phosphate, most often in the form of hydroxyapatite, in the vessel wall, leading to increased stiffening and, therefore, decreased compliance of blood vessels. It is a prominent feature of many diseases, such as atherosclerotic cardiovascular disease (CVD), diabetes, and chronic renal disease, and has emerged as a powerful predictor of cardiovascular morbidity and mortality. Once considered only a passive deposition of hydroxyapatite in the arterial wall, it is now recognized that vascular calcification is a complex and highly regulated process similar to mineralization in bone tissue [1–3]. Vascular smooth muscle cells (VSMCs) play a critical role in mediating vascular calcification by undergoing two pathophysiological processes, osteoblastic differentiation and apoptosis. Apoptotic bodies derived from VSMCs can act as nucleation site for calcium crystal formation to initiate vascular calcification. Osteoblastic differentiation of VSMC is characterized by the expression of key osteogenic transcription factors such as Runt-related transcription factor 2 (Runx2), and many bone-related proteins such as type I collagen (Col I) and alkaline phosphatase (ALP), which results in the deposition of calcium phosphate mineral in extracellular matrix [1–3]. Recent studies provide compelling evidence that inflammation plays an important role in the pathogenesis of vascular calcification [1, 2, 4, 5]. Many proinflammatory cytokines are key contributors to the pathogenesis of vascular calcification, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-4, IL-6 [4, 5]. Among these proinflammatory cytokines, TNF-α is particularly important. TNF-α activated the Msx2/Wnt signaling pathway to promote arterial calcification in diabetic low density lipoprotein receptor knockout mice [6]. Furthermore, many in vitro studies showed that TNF-α enhanced osteoblastic differentiation and calcification of VSMCs via the cyclic AMP pathway [7], bone morphogenetic protein 2 [8], nuclear factor-κB (NF-κB) pathway [9], and the PERK-eIF2α-ATF4-CHOP axis of the endoplasmic reticulum (ER) stress response [10]. Selenium (Se) is an essential trace element that plays a crucial role on human health and disease [11]. The biological functions of Se are mainly carried out by selenoproteins, in which selenium is incorporated as the amino acid selenocysteine, the 21st naturally occurring amino acid. At present, 25 selenoproteins have been identified in humans. Selenoprotein S (SelS, also known as SEPS1, VIMP, Tanis, and SELENOS) is a selenoprotein that is localized to the ER membrane [12]. SelS has been identified as an component of ER-associated protein degradation (ERAD) complex,
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JBIC Journal of Biological Inorganic Chemistry
together with selenoprotein K, p97 ATPase, and Derlins [12, 13]. The ERAD complex mediates the retrotranslocation of unfolded and misfolded proteins from the ER lumen to cytosol for degradation. The function of SelS in ERAD process plays an important role in neutralizing ER stress. Thus, SelS has been identified as an ER stress-regulated protein protecting cells from apoptosis induced by ER stress [12]. In addition to its possible role in ERAD process, a large body of evidence implicated SelS in inflammation response. Two binding sites for NF-κB in the human SelS promoter have been identified and SelS expression was significantly increased by TNF-α, IL-1β, and lipopolysaccharide (LPS) in HepG2 cells and reactive astrocytes, suggesting that SelS is a target gene of NF-κB [14, 15]. SelS overexpression reduced the production of IL-1 and IL-6 probably through regulating ER stress in reactive astrocytes [15]. Moreover, variation in the SelS gene has been shown to be strongly associated with the circulating levels of IL-6, IL-1β, and TNF-α [16]. The genetic variation in SelS has also been associated with an increased risk for developing chronic inflammatory diseases, including atherosclerotic CVD [17, 18], preeclampsia [19], gastric [20] and colorectal [21] cancer, and other conditions that are associated with increased inflammation. The effect of SelS gene polymorphism on the risk of CVD was first investigated in two independent prospective Finnish cohorts by Alanne et al. [17]. They found a significant association of SelS gene polymorphism with CVD morbidity, especially in females. Further, the polymorphisms of SelS gene were observed to be associated with risk for subclinical CVD in European Americans enriched for type-2 diabetes [18]. These results suggest a potential role for SelS in the prevention of CVD, since SelS polymorphisms significantly decrease SelS expression [16]. However, the detailed molecular mechanism underlying these correlations is far from clear. Though vascular calcification is a prominent feature of atherosclerotic CVD, little is known about the effect of SelS on vascular calcification. Considering the critical role of inflammation in the pathogenesis of vascular calcification and the role of SelS in regulating inflammation, we hypothesized that SelS might inhibit vascular calcification by affecting inflammatory response. In the present work, we examined the effects of SelS on inflammation-induced vascular calcification and the corresponding mechanisms. LPS and TNF-α, two well-known inflammation stimuli, were used to induce osteoblastic differentiation and calcification of VSMCs. We found that SelS gene knockdown by small interference RNA (siRNA) aggravated osteoblastic differentiation and calcification of VSMCs induced by LPS or TNFα. Furthermore, SelS knockdown enhanced LPS-activated NF-κB signaling pathway, inflammatory response and ER stress in calcifying VSMCs. These results suggested that SelS suppresses inflammation-induced vascular calcification and thus plays a potential role in the prevention of CVD.
JBIC Journal of Biological Inorganic Chemistry
Materials and methods Materials Dulbecco modified Eagle’s medium (DMEM), fetal calf serum (FCS), Lipofectamine 2000 and Trizol were purchased from Invitrogen (Carlsbad, CA, USA). Sodium β-glycerophosphate, dexamethasone, LPS from E. coli, Alizarin Red S, and protease inhibitor cocktail were obtained from Sigma-Aldrich (St. Louis, MO, USA). Recombinant Rat TNF-α was obtained from R&D Systems (Minneapolis, MN, USA). SelS-specific polyclonal antibody was purchased from Abcam (Hong Kong, China). Runx2-specific antibody was obtained from Santa Cruz Technology. Antibodies against phosphorylated IκBα and p65, IκBα, p65, glucose-regulated protein 78 (GRP78), inositol-requiring enzyme 1α (IRE1α), β-actin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were obtained from Cell Signaling Technology (Beverly, MA, USA). M-MLV reverse transcriptase, SYBR Green PCR Master Mix kit, and all secondary antibodies were purchased from Thermo Scientific (Rockfora, IL, USA). The enhanced chemiluminescence (ECL) kit and PVDF membrane were purchased from Millipore (Billerica, MA, USA). All primers used in this study were synthesized by Sangon Biotech (Shanghai) Co., Ltd. All other reagents used were of analytical grade.
Cell culture Primary VSMCs used in the present studies were isolated from the thoracic aortas of male Sprague–Dawley rats and identified as previously described [22]. The procedure was approved by the local Ethics Committee of Huazhong University of Science and Technology. VSMCs were cultured in the growing media (DMEM containing 20% FCS, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 μg/ml streptomycin) with media changes every 2–3 days. Cells between passages 3 and 7 were used for all experiments.
In vitro calcification and cell treatment Calcification of VSMCs was induced in osteogenic media (containing 0.25 mM l -ascorbic acid, 10 mM β-glycerophosphate, and 1 0 −8 M dexamethasone) with media changes every 2–3 days [23]. After 11 days, the cells became calcifying, and were used as calcifying VSMCs for the subsequent siRNA and LPS or TNF-α treatment in osteogenic media. Alternatively, after transfection with siRNA for 24 h, VSMCs were incubated with LPS for 6 days in osteogenic media with media changes every 2–3 days. This
cell treatment procedure was repeated according the experimental requirements.
RNA interference experiments An effective siRNA to target rat SelS (GeneBank number NM_173120.2) as previously described [24] and a scramble siRNA served as a negative control were synthesized by RiboBio (Guangzhou, China). VSMCs were transfected with control or SelS siRNA using Lipofectamine 2000 according to the manufacturer’s instructions. To evaluate the efficiency of SelS gene knockdown, the mRNA and protein levels of SelS were determined by quantitative real-time polymerase chain reaction (PCR) and Western blotting, respectively, after transfection for 48 h.
Quantitative real‑time PCR The gene expressions of SelS [24], Runx2, Col I [22, 25], TNF-α, and IL-6 [26] were determined by quantitative realtime PCR as previously described. Briefly, total RNA was isolated from calcifying VSMCs using Trizol and reversetranscribed into cDNA. SYBR Green-based real-time PCR was performed on a DNA Engine Opticon 2 (MJ Research, Boston, MA) using the SYBR Green PCR Master Mix kit, according to the vendor’s protocol. Quantification was performed using the ΔΔCT method [27] with GAPDH gene as internal control. The relative abundance of target mRNA in each sample was presented as the fold increase relative to the control group (experimental/control).
Western blotting analysis Whole cell extracts were prepared in lysis buffer (50 mM HEPES, pH 7.4, containing 150 mM NaCl, 1% Triton X-100, 0.4% SDS, protease inhibitor cocktail, 1 mM N a3VO4, and 1 mM NaF). After centrifugation at 12,000 rpm for 15 min at 4 °C, the supernatants were collected for Western blotting analysis. The total protein concentration of the supernatants was determined with the Lowry method [28] using bovine serum albumin (BSA) as the standard. Equal amounts of protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and electrically transferred to a PVDF membrane. Non-specific binding of the membrane was blocked by using 5% BSA for 1 h at room temperature. The membranes were incubated with the indicated primary antibodies overnight at 4 °C, and then with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. The blots were visualized using an ECL detection system. Relative levels of target proteins were quantified using Quantity One software (Bio-Rad Laboratories).
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JBIC Journal of Biological Inorganic Chemistry
ALP activity assay ALP activity was determined as previously described [22], and the values were normalized to the total protein content of the cell layer as determined with the Lowry method [28] using BSA as the standard.
Measurement of calcium deposition in extracellular matrix Calcium deposition in extracellular matrix was viewed by Alizarin red staining as previously described [29]. Briefly, VSMCs were fixed in 4% formaldehyde in phosphatebuffered saline for 45 min at 4 °C. The cultures were then washed with double distilled water and exposed to Alizarin Red S solution (2% aqueous) for 5 min. After being washed again with double distilled water, the cultures were observed under a microscope at a magnification of 100× in at least five randomly selected fields. Quantification of calcium deposition in extracellular matrix was determined by atomic absorption spectroscopy (AAS) (AA-300, Perkin Elmer) as described previously [25]. The calcium content of the cell layer was normalized to protein content as determined with the Lowry method [28] using BSA as the standard.
Statistical analysis All experiments were performed at least three times, and the results from a representative experiment are shown as mean ± SD. Differences between groups were compared by one-way analysis of variance (ANOVA), followed by the least significant difference (LSD) or a Dunnett T3 multiple comparison test. A value of P
