Qin Shao

AU-rich-element-dependent translation repression requires the cooperation of tristetraprolin and RCK/P54.

ABSTRACT AU-rich elements (AREs), residing in the 3′ untranslated region (UTR) of many labile mRNAs, are important cis-acting elements that modulate the stability of these mRNAs by collaborating with trans-acting factors such as tristetraprolin (TTP). AREs also regulate translation, but the underlying mechanism is not fully understood. Here we examined the function and mechanism of TTP in ARE-mRNA translation. Through a luciferase-based reporter system, we used knockdown, overexpression, and tethering assays in 293T cells to demonstrate that TTP represses ARE reporter mRNA translation. Polyribosome fractionation experiments showed that TTP shifts target mRNAs to lighter fractions. In murine RAW264.7 macrophages, knocking down TTP produces significantly more tumor necrosis factor alpha (TNF-α) than the control, while the corresponding mRNA level has a marginal change. Furthermore, knockdown of TTP increases the rate of biosynthesis of TNF-α, suggesting that TTP can exert effects at translational levels. Finally, we demonstrate that the general translational repressor RCK may cooperate with TTP to regulate ARE-mRNA translation. Collectively, our studies reveal a novel function of TTP in repressing ARE-mRNA translation and that RCK is a functional partner of TTP in promoting TTP-mediated translational repression.

In vivo enhancement of angiogenesis by adenoviral transfer of HIF-1α-modified endothelial progenitor cells (Ad-HIF-1α-modified EPC for angiogenesis)

Hypoxia inducible factor (HIF)-1alpha over-expression may have beneficial effects in cell therapy of hypoxia-induced pathophysiological processes, such as ischemic disease. Our previous study showed the feasibility of ex vivo modification of endothelial progenitor cells (EPCs) by HIF-1alpha transfection. In this study, we sought to determine if such ex vivo modified EPCs facilitated functional therapeutic neovascularization. Ad-HIF-1alpha was transduced in human EPC in vitro. HIF-1alpha-transduced EPCs were administered to nude mice with hindlimb ischemia. BrdU-labeling of these EPCs showed that they enhanced neovascularization in vivo. Limb and toe necrosis was significantly reduced in HIF-1alpha-EPC group compared to GFP-EPC group and medium control group at 14 days after transplantation (both P<0.05). A statistically significant difference was still observed in the HIF-1alpha group until 1 and 2 months of follow-up. Neovascularization was improved by both histological and physiological assessments. Exogenous EPC homing was observed. HIF-1alpha over-expression enhanced its mRNA and protein expression in the ischemia zone. The expression of genes downstream of HIF-1alpha was examined to explore the possible mechanism of EPC homing. In conclusion, HIF-1alpha-EPC gene transfer augments impaired neovascularization in experimentally induced mouse hindlimb ischemia in vivo.

Nuclear receptor Nur77 suppresses inflammatory response dependent on COX-2 in macrophages induced by oxLDL.

Oxidized low-density lipoprotein (oxLDL) cross-talks with macrophages, and both play a crucial role in the initiation and progression of atherosclerosis. Orphan nuclear receptor Nur77 is potently induced in macrophages by diverse stimuli, suggesting that it may be a key regulator of inflammation in vascular cells. The detailed mechanism of Nur77 activation and subsequent function in macrophages induced by oxLDL remains unclearly. In this study, we demonstrated that Nur77 is upregulated in a dose and time-dependent fashion by oxLDL stimulation in murine macrophages, as detected by real-time PCR and Western blotting. OxLDL activated the phosphorylation ERK1/2 and p38 MAPK, inhibition of p38 MAPK but not ERK1/2 attenuated Nur77 expression. Importantly, overexpression of Nur77 suppressed oxLDL-induced proinflammatory cytokines and chemokines secretion including tumor necrosis factor (TNF)alpha and monocyte chemoattractant protein-1(MCP-1). While knockdown Nur77 expression by specific small interfering RNA (siRNA) resulted in the enhancement of the secretion. Furthermore, exposure of macrophages to oxLDL significantly upregulated cyclooxygenase-2(COX-2) expression. However, this could be markedly inhibited by Nur77 overexpression. Also, Nur77 siRNA increased oxLDL-induced COX-2 expression and 6-mercaptopurine (6-MP) attenuated the increase. The results indicated that Nur77 is induced by oxLDL via p38 MAPK signal pathway and subsequently protects against inflammation by the inhibition of proinflammatory COX-2 pathway in activated macrophages. Specifically modifying transcription activity of Nur77 may represent a potential molecular target for the prevention and treatment of atherosclerosis.

Atorvastatin improves plaque stability in ApoE-knockout mice by regulating chemokines and chemokine receptors.

It is well documented that statins protect atherosclerotic patients from inflammatory changes and plaque instability in coronary arteries. However, the underlying mechanisms are not fully understood. Using a previously established mouse model for vulnerable atherosclerotic plaque, we investigated the effect of atorvastatin (10 mg/kg/day) on plaque morphology. Atorvastatin did not lower plasma total cholesterol levels or affect plaque progression at this dosage; however, vulnerable plaque numbers were significantly reduced in the atorvastatin-treated group compared to control. Detailed examinations revealed that atorvastatin significantly decreased macrophage infiltration and subendothelial lipid deposition, reduced intimal collagen content, and elevated collagenase activity and expression of matrix metalloproteinases (MMPs). Because vascular inflammation is largely driven by changes in monocyte/macrophage numbers in the vessel wall, we speculated that the anti-inflammatory effect of atorvastatin may partially result from decreased monocyte recruitment to the endothelium. Further experiments showed that atorvastatin downregulated expression of the chemokines monocyte chemoattractant protein (MCP)-1, chemokine (C-X3-C motif) ligand 1 (CX3CL1) and their receptors CCR2 and, CX3CR1, which are mainly responsible for monocyte recruitment. In addition, levels of the plasma inflammatory markers C-reactive protein (CRP) and tumor necrosis factor (TNF)-α were also significantly decrease in atorvastatin-treated mice. Collectively, our results demonstrate that atorvastatin can improve plaque stability in mice independent of plasma cholesterol levels. Given the profound inhibition of macrophage infiltration into atherosclerotic plaques, we propose that statins may partly exert protective effects by modulating levels of chemokines and their receptors. These findings elucidate yet another atheroprotective mechanism of statins.

Retinoid X receptor agonists inhibit phorbol-12-myristate-13-acetate (PMA)-induced differentiation of monocytic THP-1 cells into macrophages

Monocyte/macrophage differentiation is an essential process during atherosclerosis development. The retinoid X receptor (RXR) is a member of the nuclear hormone receptor superfamily, which plays an important regulatory role in many metabolic disorders, including atherosclerosis. The purpose of this study was to investigate the effect of RXR agonist on monocyte/macrophage differentiation in vitro. The THP-1 cell line was differentiated into a macrophage-like phenotype by incubation with phorbol-12-myristate-13-acetate (PMA) in the presence or absence of RXR agonist. The viability of adherent differentiated THP-1 cells was determined by MTT assay. Macrophage surface marker CD11b and CD36 was analyzed by flow cytometry. Phagocytosis was measured by fluorescence-labeled latex beads. The production of Cytokine Tunlornecrosisfactor-α (TNF-α), Interlaken-12p70 (IL-12p70), and Matrix metalloproteinase-9 (MMP-9), each of which was analyzed by ELISA. In the presence of the RXR agonists 9-cis retinoic acid or SR11237, PMA-induced THP-1 cells became less adherent, showed decreased macrophage-like morphological changes, decreased cell surface antigen CD11b and CD36 expression, and down regulated the phagocytosis of latex beads and the production of TNF-α and MMP-9. These data suggest that RXR agonists inhibit PMA-induced THP-1 cell differentiation into macrophage-like cells, which may be helpful in understanding the anti-atherosclerotic effect of RXR and its agonists.

Atorvastatin suppresses inflammatory response induced by oxLDL through inhibition of ERK phosphorylation, IκBα degradation, and COX-2 expression in murine macrophages

Macrophages crosstalk with oxidized low‐density lipoprotein (oxLDL), play a critical role in the initiation, progression, and subsequently stability of atherosclerotic plaques. Statins, inhibitors of HMG CoA (3‐hydroxy‐3‐methylglutaryl coenzyme A) reductase, reduce the expression of inflammatory proteins in addition to their lipid‐lowering action. However, the effect and detailed anti‐inflammation mechanisms of statins in macrophages induced by oxLDL remain unclearly. In the present study, we investigated the effect of atorvastatin on inflammatory response upon oxLDL stimulation in murine macrophages and analyzed the underlying mechanisms. Tumor necrosis factor (TNF)α and monocyte chemoattractant protein‐1 (MCP‐1) mRNA levels were assayed by real‐time PCR. The expression of cyclooxygenases‐2 (COX‐2) was detected by real‐time PCR and Western blotting. While mitogen‐activated protein kinase (MAPK) phosphorylation and IκBα degradation were determined by Western blotting. Our results showed that exposure of RAW264.7 cells to oxLDL, substantially changed the morphology of the cells and increased TNFα and MCP‐1 secretion. While pretreatment with atorvastatin resulted in a significant inhibition of oxLDL‐induced morphological alteration and inflammatory cytokines expression in a dose‐dependent fashion. Further investigation of the molecular mechanism revealed that oxLDL upregulated the transcription and protein expression of COX‐2 in a time‐dependent manner. Whereas, pretreatment with atorvastatin suppressed COX‐2 expression, MAPK activation and IκBα degradation. Thus, we conclude that the anti‐inflammatory effect of atorvastatin is mediated through the inhibition of proinflammatory COX‐2. Furthermore, suppression of ERK phosphorylation and IκBα degradation is involved in this regulation. Our findings provide a novel evidence that statins suppress inflammatory response, exert its anti‐atherogenic actions via against inflammation beyond cholesterol‐lowing effect. J. Cell. Biochem. 113: 611–618, 2012.

Atorvastatin suppresses oxidized LDL-induced dendritic cell-like differentiation of RAW264.7 cells regulated by the p38 MAPK pathway

Dendritic cells (DCs) are the most potent professional antigen-presenting cells and are involved in the initiation and progression of atherosclerosis. Recent data suggest that mature macrophages differentiate into dendritic-like cells when exposed to oxidized low-density lipoprotein (oxLDL). The purpose of the present study was to determine the effect of atorvastatin on the differentiation of macrophages to DCs and the molecular mechanisms of this transition. Mouse macrophage-like RAW264.7 cell was differentiated into a dendritic-like phenotype by incubation with oxLDL in the absence or presence of atorvastatin. The results showed that atorvastatin suppressed DC-like morphologic changes in vitro as assessed by decreased expression of DC maturation markers (CD83, CD11c, CD86, major histocompatibility complex class II, and CD1d). Atorvastatin also inhibited other oxLDL-induced functional changes including endocytic activity, ability to induce T cell proliferation, and cytokine secretion. Western blot analysis showed that oxLDL treatment of RAW264.7 cells induced phosphorylation of p38 mitogen-activated protein kinase (MAPK). However, blocking p38 MAPK with SB203580 significantly downregulated the expression of DC maturation markers, accompanied by decreased cytokine secretion. The findings of the present work demonstrate that that atorvastatin suppresses the oxLDL-induced DC-like differentiation of RAW264.7 cells by inactivating the p38 MAPK signaling pathway.

Atorvastatin ameliorates LPS-induced inflammatory response by autophagy via AKT/mTOR signaling pathway.

Macrophages play crucial roles in immune response and atherosclerosis‐related cardiovascular disease. Recent evidence of macrophage autophagy has demonstrated a novel pathway through which contributes to vascular inflammation. The aim of this study was to elucidate the role of autophagy in the inhibition of inflammatory response in macrophages by atorvastatin. We found that atorvastatin promoted autophagy flow determined by up‐regulating the expression of autophagy‐related protein microtubule‐associated protein light chain (LC3B), inducing the formation of autophagosomes and down‐regulating the expression of SQSTM1/P62, which is consumed during autophagy. Atorvastatin also inhibited the expression of inflammatory factors IL‐1β and TNFα induced by LPS in RAW264.7 cells. Furthermore, pretreatment with an autophagy inhibitor 3MA or LY294002 attenuated the suppressive effect of atorvastatin on LPS‐induced IL‐1β and TNFα expression. Additionally, knockdown autophagy‐related gene 5(Atg5) with a special siRNA also prevented the role of atorvastatin in decreasing IL‐1β and TNFα release induced by LPS. Finally, we detected that AKT/mTOR/P70S6K signaling pathway was involved in atorvastatin‐induced autophagy in macrophages. These data suggest that atorvastatin attenuates LPS‐induced inflammatory factors secretion, at least in part, through enhancing autophagy by AKT/mTOR signaling pathway. Our findings provide a novel evidence that statins exert anti‐inflammatory effect in atherosclerosis by autophagy activation.

Orphan nuclear receptor Nur77 Inhibits Oxidized LDL-induced differentiation of RAW264.7 murine macrophage cell line into dendritic like cells

BackgroundNur77 is an orphan nuclear receptor expressed in human atheroma. In vascular cells in vitro, Nur77 expression is induced by pro-inflammatory factors, such as oxidized LDL (oxLDL).MethodsWe analyze the role of Nur77 in the oxLDL-induced differentiation of macrophages into dendritic cells (DC). The murine RAW264.7 macrophage cell line was stably transfected with expression plasmids encoding either GFP or GFP fusions with either full-length Nur77 (GFP-Nur77), Nur77 lacking the DNA binding domain (GFP-Nur77-ΔDBD) or Nur77 lacking the transactivation domain (GFP-Nur77-ΔTAD).ResultsGFP-Nur77 overexpression significantly suppressed the effect of oxLDL treatment on DC morphologic changes, expression of DC maturation markers, endocytic activity, allogeneic activation of T cell proliferation, and the activity and secretion of pro-inflammatory cytokines. Analysis of GFP-Nur77-ΔTAD and GFP-Nur77-ΔDBD indicated that the Nur77 DNA binding and transactivation domains were both required for this effect. GFP-Nur77-ΔDBD consistently had the opposite effect to GFP-Nur77, increasing DC-type differentiation in all assays. Interestingly, GFP-Nur77-ΔDBD protein was cytosolic, whereas GFP-Nur77 and GFP-Nur77-ΔTAD were both nuclear.ConclusionsThese data show that GFP-Nur77 inhibited differentiation of oxLDL-treated macrophages into DC. The effects of Nur77 on the macrophage phenotype may involve changes in its subcellular distribution.

Selective activation of CB2 receptor improves efferocytosis in cultured macrophages

AIMS Recent evidence indicates that the defective ability to clear apoptotic cells by macrophages (efferocytosis) and the resultant apoptotic cells accumulation in atherosclerotic plaques play an important role during the progression of unstable plaques. The cannabinoid type 2 receptor (CB2), has recently been emerging as a new target to reduce vulnerability and promote stability of plaques, however, the underlying mechanisms have not been studied in detail. In the present study, we investigated whether selective activation of CB2 improves efferocytosis of macrophages. MAIN METHODS RAW264.7 macrophage line and primary-isolated peritoneal lavage macrophages from C57bl/6J mice were cultured. The efferocytosis of macrophages was analyzed by using flow cytometry or confocal microscopy; and the possible mechanisms involved in regulation of efferocytosis were also explored by using molecular biology methods. KEY FINDINGS We found that JWH-133 and HU-308, selective agonists of CB2 receptor, concentration-dependently increased the phagocytosis of apoptotic cells in normal-cultured and oxidative low density lipoprotein (OxLDL) -loaded RAW264.7 and primary macrophages. JWH-133 and HU-308 also up-regulated expressions of tyrosine kinase family phagocytic receptors MerTK, Tyro3 and Axl, reduced levels of TNF-alpha and reactive oxygen species (ROS) induced by OxLDL, and inhibited activation of RhoA GTPase. SIGNIFICANCE The selective activation of CB2 improves efferosytosis of normal-cultured and OxLDL-loaded macrophages, which might provide a novel mechanism on how CB2 activation reduces vulnerability and promotes stability of atherosclerotic plaques.