Qingping Liu

7-ketocholesteryl-9-carboxynonanoate induced nuclear factor-kappa B activation in J774A.1 macrophages.

AIMS 7-Ketocholesteryl-9-carboxynonanoate (oxLig-1), a lipid moiety of oxidized low-density lipoprotein (oxLDL), has been reported to be a crucial ligand of beta2-glycoprotein I (β(2)-GPI), and plays a potential role in the development of atherosclerosis (AS), however, the role of the sole oxLig-1 in the development of AS remains unclear. MAIN METHODS Expression and phosphorylation levels of several proteins, such as nuclear factor-kappaB (NF-κB), protein kinase C (PKC), IκBα and inter-cellular adhesion molecule 1 (ICAM-1) were determined by Western blot; nuclear localization of NF-κB was studied by immunocytochemistry; NF-κB activation was assayed by electrophoretic mobility shift assay (EMSA); and expressions of genes associated with AS process were investigated by Mouse Atherosclerosis RT(2) Profiler PCR Array assay. KEY FINDINGS The present work indicated that oxLig-1 induced IκBα phosphorylation and results in the nuclear translocation of NF-κB in J774A.1 macrophages. Moreover, oxLig-1-induced NF-κB DNA binding activity was detected by EMSA. Indeed, oxLig-1 led to the activation of PKC prior to activating NF-κB. The treatment of oxLig-1 in J774A.1 macrophages up-regulates the expression of NF-κB target genes including ICAM-1. SIGNIFICANCE OxLig-1 on the oxLDL plays an important role in AS process, as evidenced by the NF-κB activation and up-regulation of genes involved in AS development in oxLig-1 challenged J774A.1 macrophages.

7-Ketocholesteryl-9-carboxynonanoate enhances the expression of ATP-binding cassette transporter A1 via CD36

BACKGROUND CD36 signal transductions have been reported by a variety of lipid moiety of oxidized low-density lipoprotein (oxLDL), however, CD36 signal induced by 7-ketocholesteryl-9-carboxynonanoate (oxLig-1), a lipid moiety of oxLDL has not been elucidated. METHODS AND RESULTS OxLig-1 leads to activation and recruitment of Src kinase Fyn, Lyn and caveolin-1 to CD36 in J774A.1 cells, but not in CD36 knockdown cells. The mitogen-activated protein (MAP) kinases c-Jun N-terminal kinase 2 (JNK2) and extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) are specifically phosphorylated in J774A.1 cells after treatment with oxLig-1 and inhibited by pretreatment of Src inhibitor, AG1879. The expression of ABCA1 is up-regulated by treatment with oxLig-1in J774A.1 cells, and the increased expression of ABCA1 is dramatically down-regulated by pretreatment with pharmacological inhibitors of ERK (PD98059) and JNK (SP600125). CONCLUSIONS The specific CD36 signal induced by oxLig-1 initiated the activation of Fyn, Lyn, caveolin-1, JNK2 and ERK1/2, and resulted in the up-regulation of ABCA1.

7-ketocholesteryl-9-carboxynonanoate enhances ATP binding cassette transporter A1 expression mediated by PPARγ in THP-1 macrophages.

BACKGROUND ATP binding cassette transporter A1 (ABCA1) is a member of the ATP-binding cassette transporter family. It plays an essential role in mediating the efflux of excess cholesterol. It is known that peroxisome proliferator-activated receptor gamma (PPARγ) promoted ABCA1 expression. We previously found 7-ketocholesteryl-9-carboxynonanoate (oxLig-1) upregulated ABCA1 partially through CD36 mediated signals. In the present study, we intended to test if PPARγ signally is involved in the upregulation mediated by oxLig-1. METHODS AND RESULTS First, we docked oxLig-1 and the ligand-binding domain (LBD) of PPARγ by using AutoDock 3.05 and subsequently confirmed the binding by ELISA assay. Western blotting analyses showed that oxLig-1 induces liver X receptor alpha (LXRα), PPARγ and consequently ABCA1 expression. Furthermore, oxLig-1 significantly enhanced ApoA-I-mediated cholesterol efflux. Pretreatment with an inhibitor for PPARγ (GW9662) or/and LXRα (GGPP) attenuated oxLig-1-induced ABCA1 expression. Under PPARγ knockdown by using PPARγ-shRNA, oxLig-1-induced ABCA1 expression and cholesterol efflux in THP-1 macrophages was blocked by 62% and 25% respectively. CONCLUSIONS These observations suggest that oxLig-1 is a novel PPARγ agonist, promoting ApoA-I-mediated cholesterol efflux from THP-1 macrophages by increasing ABCA1 expression via induction of PPARγ.

The lipid moiety 7-ketocholesteryl-9-carboxynonanoate mediates binding interaction of oxLDL to LOX-1 and upregulates ABCA1 expression through PPARγ

Aim: Lectin‐like oxidized low‐density lipoprotein receptor‐1 (LOX‐1), a specific membrane receptor for oxidized low‐density lipoprotein (oxLDL), plays a crucial role in atherosclerosis progression. The aim of this study was to elucidate the role of 7‐ketocholesteryl‐9‐carboxynonanoate (oxLig‐1), a lipid component of oxLDL, in the binding of oxLDL to LOX‐1 and to determine whether oxLig‐1 binding to LOX‐1 is involved in the upregulation of ABCA1 expression. Main methods: OxLig‐1 binding to LOX‐1 was analysed by AutoDock 4.2.6 and confirmed by fluorescence immunocytochemistry and enzyme‐linked immunosorbent assays (ELISAs). LOX‐1, LXR&agr; and ABCA1 protein expression induced by oxLig‐1 or methylated oxLig‐1 was measured by western blotting. In addition, PPAR&ggr; activation was investigated using a dual‐luciferase reporter system. Furthermore, the signalling cascade involved in oxLig‐1‐induced ABCA1 expression was assessed using inhibitors for PPAR&ggr; and LXR&agr; and specific siRNAs against LOX‐1, PPAR&ggr; and LXR&agr;. Key findings: Docking, fluorescence immunocytochemistry and ELISA analyses showed that oxLig‐1 bound LOX‐1 and that the &ohgr;‐carboxyl group was critical for this binding. Moreover, oxLig‐1, but not methylated oxLig‐1, increased LOX‐1, LXR&agr;, and ABCA1 expression. Luciferase reporter assays indicated that oxLig‐1 activated PPAR&ggr; in the presence of LOX‐1. Additionally, the inhibitor and siRNA experiments showed that oxLig‐1 triggered the PPAR&ggr;‐LXR&agr; signalling pathway, leading to upregulation of ABCA1, and that this process required the participation of LOX‐1. Significance: Our observations indicate that oxLig‐1 is a critical epitope of oxLDL that mediates the binding of oxLDL to LOX‐1 and initiates PPAR&ggr; signal transduction to upregulate the expression of ABCA1.

Recombinant domain V of β2-glycoprotein I inhibits the formation of a 7-ketocholesteryl-9-carboxynonanoate and β2-glycoprotein I complex

Our prior study has been reported the formation of the oxidized low-density lipoprotein (oxLDL)/β(2)-glycoproteinI (β(2)-GPI)/autoantibody complex facilitated the antiphospholipid syndrome (APS) process. The domain V of β(2)-GPI binds to the negatively charged molecules, e.g. 7-ketochoresteryl-9-caboxynonanoate (oxLig-1) derived from the oxLDL and mediates the interaction between oxLDL and β(2)-GPI. In the present study, the oxLig-1/β(2)-GPI/anti-β(2)-GPI Ab (WB-CAL-1) model was established. The recombinant domain V of β(2)-GPI (rβ(2)-GPI DV) expressed in Escherichia coli competitively inhibits the interaction between β(2)-GPI and oxLig-1 in the enzyme-linked immunoassay. Moreover, the rβ(2)-GPI DV significantly inhibits the formation of the oxLig-1/β(2)-GPI/autoantibody complex in an APS patient. The present work suggests a novel possibility that rβ(2)-GPI DV could be used to inhibit the formation of oxLDL/β(2)-GPI/autoantibody complex, and give us a hint for the development of new therapeutic strategies to prevent the APS process.

The ω-carboxyl group of 7-ketocholesteryl-9-carboxynonanoate mediates the binding of oxLDL to CD36 receptor and enhances caveolin-1 expression in macrophages

CD36 signal transduction modulates the uptake of oxidized low-density lipoprotein (oxLDL) and foam cell formation. We previously observed that 7-ketocholesteryl-9-carboxynonanoate (oxLig-1), the lipid moiety of oxLDL, activates the CD36-Src-JNK/ERK1/2 signalling pathway. In this study, we assessed the role of the ω-carboxyl group in the binding of oxLig-1 to CD36 and investigated whether the binding of the ω-carboxyl group to CD36 triggers CD36-mediated signalling, thereby resulting in the upregulation of caveolin-1 expression. Our results showed that oxLig-1 bound to CD36 and that the ω-carboxyl group was critical for this binding. Furthermore, immunoprecipitation and Western blot analyses showed that interaction between the ω-carboxyl group of oxLig-1 and CD36 triggered intracellular Src-JNK/ERK1/2 signal transduction. Moreover, the binding of the ω-carboxyl group to CD36 induced caveolin-1 expression and translocation to the membrane in macrophages. Additionally, inhibitors of Src, JNK and ERK and siRNA targeting CD36 and NF-κB significantly suppressed the enhanced caveolin-1 expression induced by oxLig-1. In conclusion, these observations suggest that oxLig-1 is a critical epitope of oxLDL that mediates the binding of oxLDL to CD36 and activates downstream Src-JNK/ERK1/2-NF-κB signal transduction, resulting in upregulation of caveolin-1 expression in macrophages.

Recombinant domain V of beta(2)-Glycoprotein I inhibits oxldl-induced TF expression in macrophages

OBJECTIVE To investigate if lectin-like oxidised low density lipoprotein receptor is implicated in oxidised low density lipoprotein induced up regulation of tissue factor and whether recombinant domain V of beta (2)-Glycoprotein I expressed in Pichia pastoris inhibits the binding of oxidised and lectin-like low density lipoprotein. METHODS The expression of tissue factor and lectin-like oxidised low density lipoprotein receptor was detected using Western blot methods. Small interference ribonucleic acid of lectin-like oxidised low density lipoprotein receptor was used to block lectin-like oxidised low density lipoprotein receptor expression. Flow cytometry was used to test the effect of beta (2)-Glycoprotein I expressed in Pichia pastoris on the binding of oxidised low density lipoprotein with lectin-like oxidised low density lipoprotein receptor by using the lectin-like oxidised low density lipoprotein receptor-expressing 293T cells. RESULTS Oxidised low density lipoprotein at 5-10 g/mL increased tissue factor and lectin-like oxidised low density lipoprotein receptor expression, whereas 20-50 g/mL oxidised low density lipoprotein attenuated tissue factor expression. Inhibiting lectin-like oxidised low density lipoprotein receptor expression by small interference ribonucleic acid of lectin-like oxidised low density lipoprotein receptor impaired oxidised low density lipoprotein-induced tissue factor over expression in macrophages. Pretreatment with beta (2)-Glycoprotein I expressed in Pichia pastoris led to a strong inhibition of tissue factor and lectin-like oxidised low density lipoprotein receptor expression in a dose-dependent manner in macrophages. Flow cytometry analysis showed that beta (2)-Glycoprotein I expressed in Pichia pastoris attenuated the interaction of oxidised low density lipoprotein with lectin-like oxidised low density lipoprotein receptor in lectin-like oxidised low density lipoprotein receptor-expressing 293T cells. CONCLUSIONS Lectin-like oxidised low density lipoprotein receptor was implicated in the expression of tissue factor induced by oxidised low density lipoprotein, and beta (2)-Glycoprotein I expressed in Pichia pastoris inhibited oxidised low density lipoprotein-induced tissue factor and lectin-like oxidised low density lipoprotein receptor expression, at least in part, via inhibition of the interaction between oxidised low density lipoprotein and lectin-like oxidised low density lipoprotein receptor.