Cellular Repressor Of E1A Stimulated Genes Antagonize Inflammation In RAW 264.7 Cells Via Autophagy-lysosome Pathway

Object Atherosclerosis-related cardiovascular diseases are the leading cause of mortality worldwide. In addition to lipid dysfunction and arterial lipid accumulation, immune-inflammatory responses are major factors in directing the initiation and development of atherosclerosis[1, 2]. Macrophage inflammation is central to almost all aspects that contribute to the development of the atherosclerotic lesion, starting from the initiation of atherosclerosis up to plaque rupture that results in the induction of the coagulation cascade[3]. Therefore, finding an appropriate regulator of macrophage inflammation may be an effective pathway for atherosclerosis therapy.The cellular repressor of E1A-stimulated genes(CREG) is a secreted glycoprotein that has been shown to antagonize transcriptional activation and cellular transformation induced by the adenovirus E1 A oncoprotein[4]. Our preliminary work has discovered that the expression of CREG protein was decreased in both rabbit and human atherosclerosis artery compared with normal artery. In addition, the downregulation of CREG was time-dependent during atherosclerosis progression in rabbit artery. These findings raise the possibility that CREG may take part in the course of atherosclerosis.Our previous studies have demonstrated CREG had a potential protective effect on inflammation[5]. CREG mRNA profile in human and mouse tissue examined by mining EST databases on the NCBI-UniGene site shows that CREG expression is highest in lymph node in both species. It is known that lymph node is a tissue rich in macrophages. Therefore, we hypothesis that CREG may have a beneficial impact on macrophage imflammation which play an important role in the pathogenesis of atherosclerosis.In this study, we intended to examine whether CREG is involved in and can inhibit TNF-α induced inflammation in macrophages and to explore the potential mechanisms, consequently to assess whether CREG might be a therapeutic reagent for atherosclerosis.Materials and methods RAW 264.7 mouse macrophage-like cells were transfected with CREG siRNA to down-regulate the expression of CREG. The expression of CREG, cathepsin B, cathepsin L, LAMP1, IGFIIR, LC3, Beclin 1, p62 and Tubulin was identified by Western blot. The blots were quantified by Image-Pro Plus software. The amounts of IL-6 and MCP-1 secreted into the cell culture supernate and expressed in the tissue lysates were determined using the mouse IL-6 and MCP-1 ELISA kits. Colocalization of anti-His and anti-cathepsin B/ cathepsin L/ M6P/IGFIIR/ LAMP1 was determined by immunofluorescence analysis and confocal microscopy. Lysosomal visualization was carried out using the Lysotracker Red staining. Accumulation of autophagosomes and autolysosomes were detected in CREG-treated and CREG down-regulated cells using electron microscopy. The interaction between the exogenous recombinant CREG protein and cathepsin B, cathepsin L and IGFIIR were studied with immunoprecipitation analysis. Male ApoE-/- mice(n =40, age 10 weeks, 20–25 g) were fed a high-fat, high-cholesterol diet containing 21 % fat and 1.3 % cholesterol for 16 weeks. The mice underwent sham operation or were infused with CREG protein(Abcam, UK) at a rate of 30μg/kg/d during the 4-12 weeks by an osmotic mini-pump. Aortas were collected for oil red O and hematoxylin-eosin staining to assess atherosclerotic plaques. The expression of CD68 and MCP-1 in the atherosclerotic plaques was detected by the immunohistochemical staining.Results1. Involvement of CREG in inflammation of RAW 264.7 cells induced by TNF-αTNF-α is a pleiotropic cytokine with strong proinflammatory capacity. RAW 264.7 cells were cultured with medium in the presence of 20 ng/ml TNF-α for different times(0, 30 min, 2 h, 6 h,12 h,16 h and 24 h). ELISA analysis showed the secretion of pro-inflammatory cytokines including IL-6 and MCP-1 increased in a time-dependent manner. Meanwhile, the expression of CREG in RAW 264.7 cells was detected increasing firstly and then decreasing in the time course of TNF-α stimulation. These data indicates that the changes in CREG expression correlate with inflammation of RAW 264.7 cells induced by TNF-α and that CREG may play an important role in TNF-α induced inflammation.2. Changes of CREG expression modulate TNF-α-induced in?ammation in RAW 264.7 cellsTo determine whether CREG correlates with TNF-α induced inflammation, we evaluated the relationship between the expression of CREG and secretion of pro-inflammatory cytokines( IL-6 and MCP-1) using gain-of-function and loss-of-function approaches. Expression of CREG detected on immunoblotting demonstrated an increasing trend when RAW 264.7 cells were supplemented with exogenous recombinant CREG protein at different concentrations(0, 0.5, 2.5, 5, 10 and 20 μg/ml) for 24 h. Western analysis demonstrated 82.7% down-regulation of CREG expression in RAW 264.7 cells transfected with CREG siRNA(p < 0.001). Results of ELISA assays showed that the amount of IL-6 and MCP-1 secreted into the cell culture supernate decreased in a dose-dependent manner in the RAW 264.7 cells supplemented with exogenous recombinant CREG protein and increased in those knocking down of CREG expression by siRNA. The above results suggest that up-regulation of CREG attenuate and down-regulation of CREG promote the secretion of pro-inflammatory cytokines. Thus CREG may have the anti-inflammation effect on RAW 264.7 induced by TNF-α.3. CREG promotes autophagy which can attenuate the TNF-α-induced in?ammation in RAW 264.7 cellsIt has been reported that autophagy has a negative effect on inflammation[6, 7]. Thus autophagy may also play an important role in the inflammation of macrophages. To determine the effect of CREG on autophagy, accumulation of autophagosome and autolysosomes were detected in CREG-treated and CREG down-regulated cells using electron microscopy to obtain direct evidence of autophagy activation. Autophagic vesicles and autophagosomes were seen in the TNF-α-induced cells in the control groups. The numbers of autolysosomes increased in RAW 264.7 cells following treatment with CREG protein while less autophagosomes were seen in those transfected with CREG siRNA. Meanwhile, exogenous CREG protein induced autophagy with significant increased autophagosome-bound LC3-II and Beclin 1 abundance and decreased the level of p62, a protein that brings ubiquitinated aggregates into autophagosomes and gets consumed during autophagy. Increment of LC3-II and Beclin 1 and accumulation of p62 in CREG down-regulated cells suggested accumulation of autophagosomes and impairment of autophagy. To determine the contribution of autophagy to the anti-inflammation effect of CREG, 3-methyladenine(3-MA) and bafilomycin A(Sigma-Aldrich), both typical autophagy inhibitor were used following TNF-α stimulation. Inhibition of autophagy by 3-MA or bafilomycin A counteracted the anti-inflammation effect of CREG protein in RAW 264.7 cells. These results raise the possibility that autophagy might have dynamic effects on the TNF-α-induced inflammation in RAW 264.7 cells.4. Effects of CREG on lysosome biogenesis and maturation of cathepsin B and cathepsin LCREG has been reported to be a secretory glycoprotein predominantly localized within lysosomes[8]. To determine the role of CREG in lysosomes, we examined the effects of CREG on cathepsin B, cathepsin L and LAMP1 expression. Immunofluorescence analysis revealed that intense positivity for cathepsin B and cathepsin L was detectable in RAW 264.7 cells supplied with exogenous CREG protein(20 μg/ml) while the immunofluorescence intensity was obviously decreased in those transfected with siRNA to silence CREG expression. Western analysis demonstrated a similar trend to the immunocytofluorescent results in the expression of cathepsin B and cathepsin L. The protein expression of cathepsin B, cathepsin L and LAMP1 was significantly higher in the CREG protein-treated group while diminished in the CREG down-regulated group compared with those in the control groups. In addition, the activated form of cathepsin B(25 kDa) and cathepsin L(25 kDa) signals and the ratios of mature form(25 kDa, 25 kDa)/ pro-enzyme form(40 kDa, 42 kDa) were enhanced in the CREG protein-treated group and opposite results were presented in the CREG down-regulated group. Lyso Tracker Red is a weakly-basic cell-permeablefluorescent amine dye that accumulates in lysosomes; as such it can be used to assess lysosomal numbers[9]. Fluorescent and bright field images of RAW 264.7 cells stained with this dye showed more intense red fluorescence in cells incubated with exogenous CREG protein and less in those transfected with CREG siRNA. Therefore, CREG protein stimulates the expression and maturity of cathepsin B and cathepsin L and induces the biogenesis of lysosomes. Since lysosomal activity is essential to autophagy[10], we deduce that CREG may mediate the regulation of autophagy via its effects on lysosomes.5. The exogenous CREG protein is localized in lysosomes, interacts with lysosomes via the mannose 6-phosphate/insulin-like growth factor II receptor(M6P/IGFIIR) and affects the distribution of M6P/IGFIIRIt has been found that CREG is a lysosomal protein binding directly to the M6P/IGFIIR and depends on the interaction with M6 P receptors for efficient delivery to lysosomes[8]. Whereas it is unknown if exogenous CREG protein exhibits the same characteristics as the endogenous CREG and if CREG has any effect on M6P/IGFIIR in RAW 264.7 cells. The exogenous recombinant CREG protein is His-tag protein purified with Ni-NTA column. When RAW 264.7 cells were double-stained with antibodies to His and cathepsin B, cathepsin L, the lysosomal membrane protein LAMP-1 and M6P/IGFIIR respectively, extensive co-localization of the two antigens was observed. Furthermore, coimmunoprecipitation analyses also confirmed the interactions between the His-tag CREG protein and cathepsin B, cathepsin L and M6P/IGFIIR. Western blot showed that there was no significant difference in the expression of M6P/IGFIIR in RAW 264.7 cells with CREG up-regulated and down-regulated. The immunofluorescence staining revealed that the M6P/IGFIIR presented widely in cytoplasm and on cell membrane and colocalized with LAMP1, the structural component of lysosomes in CREG-treated RAW 264.7 cells. Whereas the localization of M6P/IGFIIR was predominantly near the perinuclear structure and co-localized partially with LAMP1 in the CREG down-regulated cells. These findings provide evidence that exogenous CREG protein is located in lysosomes and it plays a critical role not in the expression but in the distribution of M6P/IGFIIR which is involved in binding and trafficking of lysosomal enzymes.6. CREG protein alleviates aortic atherosclerosis development and affectes inflammation and autophagy in aortas of ApoE-/- miceTo confirm the effect of CREG protein on atherosclerosis, we fed the ApoE-/- mice a high-fat, high-cholesterol diet for 16 weeks. The mice underwent sham operation or were infused with CREG protein during the 4-12 weeks by an osmotic mini-pump. En face oil red O-stained aortas from CREG-treated ApoE-/- mice showed a significant reduction in atheromatous plaques in the luminal surface of the aortas compared with control group. Haematoxylin-eosin staining showed that the aortic lesion area was significantly reduced in the CREG-treated mice compared with those in the control group. The expression of MCP-1 in arterial tissue detected by ELISA assays and CD68, a marker of active macrophages and MCP-1 in atherosclerotic lesions of aortic root sections determined by immunohistochemical staining were all decreased in CREG-treated group. Furthermore, Western analysis of aorta lysates showed increased autophagosome-bound LC3-II abundance and p62 consumption in CREG-treated group. In conclusion, CREG protein shows significant effects on alleviating development of aortic atherosclerosis, suppressing inflammation and enhancing autophagy in aortas of ApoE-/- mice.Conclusion CREG can inhibit macrophage inflammation and promotes autophagy mediated by lysosome biogenensis which is related to the distribution of M6P/IGFIIR. CREG may have a potential effect on atherosclerosis therapy.