ApoM Suppresses TNF-α-induced Expression Of ICAM-1 And VCAM-1 Through Inhibiting The Activity Of NF-κB

BackgroundAtherosclerosis (AS), as a chronic inflammatory disease, is one of the most common diseases. Immune activation and inflammation are important aspects in the pathogenesis of AS. High density lipoprotein cholesterol (HDL-C) has anti-inflammatory and anti-oxidant properties in addition to mediating reverse cholesterol transport during atherosclerotic cardiovascular disease development. As an essential component of high-density lipoproteins (HDL), many studies have been performed to elucidate the biological function of apoM in lipid metabolism, diabetes, coronary artery diseases and atherosclerosis. In humans, the synthesis of apoM mainly occurs in liver and kidney proximal tubule cells. Besides lipid metabolism, apoM is also closely linked with inflammation as a negative acute response protein. Recent study reported that the messenger RNA (mRNA) levels of apoM in the liver were obviously decreased with the stimulation of lipopolysaccharide (LPS), zymosan, or turpentine administration, all of which could cause systemic inflammation. ApoM was also reported as a negative acute response protein, which decreased during infection and inflammation such as acute bacterial infections or chronic HIV infection. In addition, a recent research showed that the enhancing expression of apoM could markedly mediate the anti-inflammatory effect of propofol in LPS-stimulated THP-1 macrophages in a hepatocyte nuclear factor la (HNF-la) dependent manner. Recently, it has been reported that apoM could inhibit LPS-induced intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression in mice liver. By taking all of these results into consideration, it is trend to suggest that the apoM might play an important role in inflammatory responses. However, the mechanism of apoM involved in regulation of inflammatory reaction is not yet clear.As an important sequence-specific transcription factor, the nuclear factor of κB (NF-κB) could bind to an intronic enhancer of the immunoglobulin κ-light chain gene in many types of cells. The transcriptional activity of NF-κB is a key factor in regulating the expression of many genes (such as iNOS and IL-6) and is involved in many typical inflammatory responses (such as immune and inflammatory reactions, smooth muscle cell proliferation and angiogenesis). NF-KB-mediated chronic vascular inflammation was also mentioned as a critical participant in the initiation, progression of atherosclerosis and other inflammations. In the typical NF-κB signaling pathway, NF-κB was existed in inactive form in the cytoplasm, which was bounded to an inhibitor known as Inhibitor of NF-κB-a (IκBα). The IκBα could transport activated NF-κB from the nucleus to the cytoplasm, followed by releasing NF-κB into the nucleus via the phosphorylation or degradation of IκBα protein, and then caused inflammatory responses. Tumor necrosis factor-a (TNF-a) is the important pro-inflammatory cytokine and could trigger a strong vascular inflammatory response activation, which was believed as the eventual mechanism involved in the development of atherosclerosis. As one kind of cell adhesion molecules, ICAM-1 and VCAM-1 were highly expressed in human endothelial cells and could be markedly induced by the TNF-a in inflammation. Accumulating evidence suggested that the increasing levels of ICAM-1 and VCAM-1 could contribute to the recruitment of inflammatory cells at the sites of inflammation and exacerbate cell-mediated immune responses. Moreover, many investigations revealed that the activation of NF-κB was one of the main pathways that induce transcriptional regulation of TNF-a-induced adhesion molecules. However, the in vivo molecular mechanism of NF-κB in mediating the TNF-α-induced inflammatory responses was not fully clear. The anti-inflammatory effect of apoM on this progress whether through regulating NF-κB has not been reported.Hence, we accordingly hypothesized that apoM has a suppressive effect on the regulation of TNF-α-induced ICAM-1 and VCAM-1 expression. In this research, the levels of apoM,IκBα, ICAM-1 and VCAM-1 in HepG2 cells influenced by the treatment of TNF-α were measured by Western Blot analysis and RT-PCR technology. The effects of apoM on the expression of IκBα, ICAM-1 and VCAM-1 in HepG2 cells were also tested. Finally, these effects were observed once again by siRNA-mediated silencing of apoM (siRNA-apoM) in TNF-α treated HepG2 cells. In addition, the activity of NF-κB affected by siRNA-apoM was detected by ELISA assay in TNF-α treated HepG2 cells. A new insight into the anti-inflammatory effects of apoM was provided.Objective1). To investigate the effects of TNF-α on ICAM-1, VCAM-1, apoM and expression in HepG2 cells.2). To investigate the effects of apoM on ICAM-1, VCAM-1 and expression in HepG2 cells.3). To study whether the effects of apoM on ICAM-1 and VCAM-1 expression through regulating NF-κB activity in TNF-α treated HepG2 cells.Materials and methods1. MaterialsTNF-a was purchased from Sigma Aldrich (St. Louis, MO, USA). The PrimeScript(?) RT Reagent Kit (perfect real-time; catalog no. DRR037A) and SYBR(?) Premix Ex TaqTM II kit (Tli RNaseH Plus; catalog no. DRR820A) were obtained from TaKaRa Bio, Inc. (Shiga, Japan). TRIzol and Lipofectamine 2000 was purchased from Invitrogen.2. Cell cultureHuman hepatocytes (HepG2) were purchased from the American Type Culture Collection (Manassas, VA, USA). The HepG2 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and 1% penicillin/streptomycin. Cells were incubated at 37℃ in an atmosphere of 5% CO2.3. RNA isolation and real-time quantitative PCR analysisTotal RNA from cultured cells was extracted using TRIzol and other related reagents (Invitrogen Corporation, Carlsbad, CA, USA) according to the manufacturer’s instructions. Real-time quantitative PCR (RT-PCR) was performed on an ABI 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA), using SYBR Green detection chemistry. Melt curve analyses of all RT-PCR products were performed and found to produce a single DNA duplex. All samples were measured in triplicate, and the mean value was considered for comparative analysis. Quantitative measurements were determined using the ΔΔCt method, and glyceraldehyde 3-phosphate dehydrogenase expression was used as the internal control.4. Western Blot analysisProtein samples were extracted from cultured cells using the radio immunoprecipitation assay buffer (Biocolor Ltd., Belfast, Northern Ireland, UK) and quantified using the Bradford protein assay kit (KeyGen Biotechnologies, Nanjing, China). Then, protein samples were subjected to Western Blot analysis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis; 30μg protein per lane) using rabbit polyclonal anti-apoM antibodies, rabbit polyclonal anti-ICAM-1 antibodies, rabbit polyclonal anti-VCAM-1 antibodies, rabbit polyclonal anti-IκBα antibodies and rabbit polyclonal β-actin-specific antibodies (Abcam, Cambridge, MA, USA). Then, protein samples were incubated with HRP conjugated goat anti-rabbit antibody and were visualized by chemiluminescence method (ECL Plus Western Blot Detection System).5. Transfection with small interfering RNAThe small interfering RNAs (siRNAs) against apoM were purchased from Ribo Biotechnology (Guangzhou, China). Cells were seeded in 6 well plates or 60-mm dishes and grown to 30%-50% confluence before use. Cells were transfected with siRNA-apoM using Lipofectamine 2000 transfection reagent for 48h according to the manufacturer’s instructions. Cells were incubated at 37℃ in an atmosphere of 5% CO2. After 48h of transfection, Western Blot was performed.6. Lentivirus (LV) production and infectionPacked empty LV vectors with green fluorescent protein (GFP; LV-Mock), LV-mediated human apoM overexpression vector (LV-apoM) with GFP were prepared as previous report. Cells were seeded in 6 well plates or 60-mm dishes and grown to 30%-50% confluence before use. HepG2 cells were cultured in 25-cm2 vented flasks containing Dulbecco’s modified Eagle’s medium with 10% fetal calf serum under standard culture conditions (5%CO2,37℃). The cells were infected with the LV stock at a multiplicity of infection of 20 transducing units per cell in the presence of 4μL Polybrene (8mg/mL). Then cells were washed with fresh complete media after 6-24h of incubation. The GFP-positive cells were counted 96h post-transduction.7. Test the activity of NF-κBThe HepG2 cells were transfected with siRNA-apoM. Forty-eight hours after transfection, cells were stimulated with TNF-a (0 or lOng/mL), and the activity of NF-κB was analyzed after another 5h. Firstly, total nucleoprotein of cells was extracted and quantified using the Bradford protein assay kit. Then, NF-κB activity was used for normalization, and measured by ELISA Kit (Promega, Madison, WI, USA) in ELISA instrument (450nm).8. Statistical analysisData are expressed as means±standard deviations (SDs). Results were obtained by one-way analysis of variance and Student’s t test using SPSS v13.0 statistical software (SPSS Inc., Chicago, IL, USA). A two-tailed probability (p) value<0.05 was considered statistically significant.Results1. TNF-α down-regulates apoM and IκBα expression, up-regulates ICAM-1 and VCAM-1 expression in HepG2 cellsTNF-α is a key pro-inflammatory cytokine responsible for the expression of ICAM-1 and VCAM-1, which could cause the phosphorylation and degradation of IκBα protein. As stimulus of systemic inflammation, LPS, zymosan and turpentine could markedly decrease apoM mRNA levels. We first explored the possible changes of apoM, IκBα, VCAM-1 and ICAM-1 expression caused by TNF-α treatment in HepG2 cells by RT-PCR and Western Blot analysis. We found that the levels of apoM mRNA and its corresponding protein were obviously decreased by TNF-α treatment in HepG2 cells, while the levels of ICAM-1 and VCAM-1 mRNA and their corresponding proteins were increased, the protein levels of IκBα were also markedly decreased (P<0.05). These results further verified the pro-inflammatory effect of TNF-a in HepG2 cells. Moreover, an antagonism between apoM with TNF-α was also observed.2. Effects of apoM on IκBα, ICAM-1 and VCAM-1 expression in HepG2 cellsIt has been widely reported that the apoM was an important participant in the lipid metabolism. However, the role of apoM in regulating inflammatory cytokines expression in HepG2 cells was still unclear. To fill this gap, Lentivirus-mediated human apoM overexpression vector (LV-apoM) was infected into HepG2 cells, followed by investigating the changes of IκBα, ICAM-1 and VCAM-1 expression in the cells. With the treatment of LV-apoM, the protein levels of apoM in the HepG2 cells were increased about 9.9-fold compared to the control group (P<0.05). Western Blot data revealed that the protein levels of ICAM-1 and VCAM-1 were significantly decreased by 57.8% and 52.8% respectively with the treatment of LV-apoM (P<0.05), which indicated that apoM was involved in the inhibition of ICAM-1 and VCAM-1 expression in the HepG2 cells. It is interesting to observe that the protein levels of IκBα were also enhanced by the treatment of LV-apoM (P<0.05), which demonstrated that the IκBα protein could be a mediator in the apoM involved inhibition of ICAM-1 and VCAM-1 expression in the HepG2 cells.3. ApoM is involved in TNF-a-induced up-regulation of ICAM-1 and VCAM-1 expression through inhibiting activity of NF-κB in HepG2 cellsTo further investigate whether apoM was involved in TNF-a-induced inflammatory processes, we first performed transfection of small interfering RNA (siRNA) against apoM into HepG2 cells. HepG2 cells were then incubated with siRNA-NC+Ong/mL TNF-a (Control), siRNA-apoM+Ong/mL TNF-a (siRNA-apoM), siRNA-NC+1 Ong/mL TNF-a (TNF-a), or siRNA-apoM+1 Ong/mL TNF-a (siRNA-apoM+TNF-a) for 24h, respectively. We found the expression of apoM protein was markedly decreased by apoM siRNA treatment in HepG2 cells compared to the control siRNA group (P<0.05). Then, we examined the effects of apoM and TNF-a on the expression of IκBα, ICAM-1 and VCAM-1 in HepG2 cells by Western Blot analysis, respectively. As shown in data, the treatment with TNF-a increased ICAM-1 and VCAM-1 protein expression and these effects were markedly enhanced by siRNA-mediated silencing of apoM in HepG2 cells (P<0.05). With the treatment of TNF-a, the protein levels of ICAM-1 and VCAM-1 were dramatically increased by 200.8% and 190.9% respectively with the presence of siRNA-apoM (P<0.05). However, without the treatment of TNF-a, the protein levels of ICAM-1and VCAM-1 were only increased by 93.3% and 110.6% respectively with the presence of siRNA-apoM (P<0.05), which indicated that the apoM could be involved in TNF-a-induced up-regulation of ICAM-1 and VCAM-1 expression in the HepG2 cells. We observed that the IκBα, an inhibitor of NF-κB-α which transported activated NF-κB from the nucleus to the cytoplasm, was obviously decreased by TNF-a and this effect was also dramatically enhanced by additional siRNA-mediated silencing of apoM in HepG2 cells. So we next investigated whether apoM suppressed the expression of ICAM-1 and VCAM-1 through inhibiting the activity of NF-κB in HepG2 cells by ELISA assay. We found that the activity of NF-κB was obviously increased with the treatment of TNF-a or siRNA-apoM in HepG2 cells. With the treatment of TNF-a, the activity of NF-κB could increase by additional 475.4% with the presence of siRNA-apoM in HepG2 cells (P<0.05). In comparison, the treatment of TNF-a in HepG2 cells could only increase by about 160% of the activity of NF-κB (P<0.05). These results demonstrated that apoM was involved in TNF-a-induced up-regulation of ICAM-1 and VCAM-1 expression mainly through inhibiting activity of NF-κB in HepG2 cells.Conclusion1). TNF-a down-regulated apoM and IκBα expression, up-regulated ICAM-1 and VCAM-1 expression in HepG2 cells.2). Expression of ICAM-1 and VCAM-lwere downregulated by apoM, while IκBα expression was upregulated by apoM in HepG2 cells.3). ApoM significantly downregulated TNF-a-induced expression of VCAM-1 and ICAM-1 through inhibiting the activity of NF-κB in HepG2 cells...