Oxidized high-density lipoprotein promotes CD36 palmitoylation and increases lipid uptake in macrophages.

Oxidized high-density lipoprotein (oxHDL) reduces the ability of cells to mediate reverse cholesterol transport and also shows atherogenic properties. Palmitoylation of cluster of differentiation 36 (CD36), an important receptor mediating lipoprotein uptake, is required for fatty acid endocytosis. However, the relationship between oxHDL and CD36 has not been described in mechanistic detail. Here, we demonstrate using acyl-biotin exchange analysis that oxHDL activates CD36 by increasing CD36 palmitoylation, which promotes efficient uptake in macrophages. This modification increased CD36 incorporation into plasma lipid rafts and activated downstream signaling mediators, such as Lyn, Fyn, and c-Jun N-terminal kinase, which elicited enhanced oxHDL uptake and foam cell formation. Furthermore, blocking CD36 palmitoylation with the pharmacological inhibitor 2-bromopalmitate decreased cell surface translocation and lowered oxHDL uptake in oxHDL-treated macrophages. We verified these results by transfecting oxHDL-induced macrophages with vectors expressing wildtype or mutant CD36 (mCD36) in which the cytoplasmic palmitoylated cysteine residues were replaced. We show that cells containing mCD36 exhibited less palmitoylated CD36, disrupted plasma membrane trafficking, and reduced protein stability. Moreover, in ApoE-/-CD36-/- mice, lipid accumulation at the aortic root in mice receiving the mCD36 vector was decreased, suggesting that CD36 palmitoylation is responsible for lipid uptake in vivo. Finally, our data indicated that palmitoylation of CD36 was dependent on DHHC6 (Asp-His-His-Cys) acyltransferase and its cofactor selenoprotein K, which increased the CD36/caveolin-1 interaction and membrane targeting in cells exposed to oxHDL. Altogether, our study uncovers a causal link between oxHDL and CD36 palmitoylation and provides insight into foam cell formation and atherogenesis.

Up-regulation of thioredoxin system by puerarin inhibits lipid uptake in macrophages.

Cellular oxidative stress promotes lipid accumulation in macrophages during atherogenesis. Puerarin is a natural isoflavone with beneficial effects against oxidation and atherosclerosis. In this study, we investigated the effects of puerarin on lipid uptake and explored the underlying molecular regulation. We found puerarin up-regulated thioredoxin-1 (Trx1) and Trx reductase-1 (TrxR1) expression; it increased TrxR1 activity, cellular thiols contents and decreased oxidized form of Trx1, thus inhibiting cellular ROS generation. Confocal microscope and flow cytometry analysis showed fluorescence labeled Dil-oxLDL uptake was dramatically inhibited by puerarin in RAW264.7 cells as well as in primary bone marrow derived macrophages and peritoneal macrophages. The effects were reversed when Trx1 was inhibited by treatment with Trx1 inhibitor PX-12 or Trx1 siRNA. We also found scavenger receptors such as SR-A and Lox-1, but not CD36 were involved in the Trx1-mediated lipid uptake inhibition. Moreover, measurements of foam cell accumulation and ROS production in sections of aortic roots showed those were reduced by puerarin but raised when additional treatment with PX-12 or Trx1 siRNA in apoE-/- mice, which demonstrates the lipid uptake reduction by puerarin requires Trx1 inhibition in vivo. In addition, we analyzed the upstream regulation and found puerarin induced Nrf2 activity; cooperation between Nrf2 and ATF4 facilitated the puerarin effects. PERK phosphorylation was detected to be increased by puerarin, while PERK inhibition reduced cellular Trx1, TrxR1, nuclear Nrf2 and ATF4. Altogether, puerarin modulates PERK/Nrf2 that coordinates with ATF4 to active Trx1, which causes SR-A and Lox-1 reduction and lipid uptake inhibition in macrophages. This suggests Trx1 could be an effective target by puerarin in the prevention of atherosclerosis.

Isolation and identification of antioxidant peptides from tartary buckwheat albumin (Fagopyrum tataricum Gaertn.) and their antioxidant activities.

Tartary buckwheat (Fagopyrum tataricum Gaertn.) albumin was hydrolyzed by alkaline protease, and three new antioxidant peptides (P1, P2, and P3) were successfully separated from the hydrolysate (TBAH). The sequences of the three antioxidant peptides were Gly-Glu-Val-Pro-Trp (GEVPW), Tyr-Met-Glu-Asn-Phe (YMENF), and Ala-Phe-Tyr-Arg-Trp (AFYRW), and their molecular weights were 586.65, 702.79, and 741.85 Da, respectively. All three peptides have a good antioxidant capacity, and P3 (AFYRW) demonstrates the best antioxidant activity of the three. The IC50 values of AFYRW for scavenging hydroxyl radicals (OH·) and (1,1-diphenyl-2-picrylhydrazyl) DPPH· free radicals were 0.65 and 0.64 mM, respectively. In addition, AFYRW exhibits the strongest lipid peroxidation inhibition ability and the highest reducing power. The results of this research indicate that the three isolated peptides can be used in the development of various antioxidant additives in the food and pharmaceutical industries.

[Preparation and application of mouse polyclonal antibodies against human Shisa like 1 (SSL1)].

Objective To prepare polyclonal antibodies against Shisa like 1 protein (SSL1) and study the localization of SSL1 in hepatocellular carcinoma SMMC-7721 cells. Methods Human SSL1 gene was cloned from HepG2 cells by reverse transcription PCR, and then inserted into prokaryotic expression vector pET-28a to generate the SSL1 expression vector. The recombinant plasmid pET28a-SSL1 was then transformed into E. coli BL21 (DE3) and induced to express by IPTG. Polyclonal antibody against SSL1 was generated by immunizing Kunming mouse with the purified protein by the routine method. The specificity of polyclonal antibody was verified by Western blot analysis. The expression of SSL1 in SMMC-7721 cells was detected by immunofluorescent cytochemistry. Golgi complexes were signed by Golgi-Tracker Red to analyze the subcellular localization of SSL1 protein in SMMC-7721 cells. Results The SSL1 gene was cloned and the recombinant vector pET28a-SSL1 was successfully constructed. Pure SSL1 protein expression in E. coli BL21 was confirmed and polyclonal antibodies against protein SSL1 was obtained in immunized Kunming mice. Immunofluorescent cytochemistry showed that SSL1 was expressed in the cytoplasm, and was co-localized with Golgi-Tracker Red in SMMC-7721 cells. Conclusion We have obtained SSL1 polyclonal antibodies with high specificity, which was proved situated in Golgi bodies of SMMC-7721 cells.

Enhanced cellular cholesterol efflux by naringenin is mediated through inhibiting endoplasmic reticulum stress - ATF6 activity in macrophages.

Naringenin improves lipoprotein profile and protects against cardiovascular disease. ATF6 is an endoplasmic reticulum (ER) stress sensor with the same activation processes with sterol regulator SREBPs. Clinical data revealed that ATF6 expression was associated with plasma cholesterol level. Here, we investigated whether naringenin was involved in the regulation of cholesterol efflux and tested the role of ER stress-ATF6 in the naringenin function. Results showed that naringenin increased cholesterol efflux to both apoA-I and HDL and gene expressions in ABCA1, ABCG1 and LXRα in RAW264.7 macrophages. Naringenin inhibited the cleaved ATF6 nuclear translocation and its target GRP78 and XBP-1 expressions. Naringenin-induced cholesterol efflux was modulated by treatment with ER stress inhibitor 4-phenylbutyric acid, inducer tunicamycin and ATF6 overexpression in RAW264.7 and/or THP-1 cells, which suggested the naringenin functions were mediated through inhibiting ER stress-ATF6 pathway. Next, we found high-fat diet (HFD) supplemented with naringenin increased by >1.2-fold in cholesterol efflux capacity in primary peritoneal macrophage in apoE-/- mice compared to only HFD-fed mice. The increase was significantly reduced by tunicamycin treatment. Naringenin decreased GRP78, XBP-1 and nuclear ATF6 levels in peritoneal macrophage and aorta and reduced atherosclerotic lesion at aortic root, but reversed by tunicamycin. These confirmed participation of ER stress-ATF6 in naringenin efficacy. Finally, we found naringenin promoted AKT phosphorylation; PI3K inhibitor LY294002 treatment increased nuclear ATF6 and reduced naringenin-enhanced ABCA1 expression and cholesterol efflux. We concluded naringenin as a regulator for cholesterol efflux, and the regulation was mediated by ATF6 branch of ER stress and PI3K/AKT pathway.

ERK5/KLF2 activation is involved in the reducing effects of puerarin on monocyte adhesion to endothelial cells and atherosclerotic lesion in apolipoprotein E-deficient mice.

Puerarin has properties of anti-oxidation and anti-inflammation, which has been demonstrated protective effects in atherosclerosis and other cardiovascular diseases. However, the detail molecular mechanism still remains unclear. Here, we determined whether the atheroprotective effect of puerarin was by reducing monocyte adhesion and explored the underlying mechanism. The results showed that puerarin dose- and time-dependently reduced oxLDL-induced monocyte THP-1 adhesion to HUVECs and the expression of adhesion-related genes such as VCAM-1, ICAM-1, MCP-1 and IL-8 in HUVECs. Puerarin activated ERK5 phosphorylation and up-regulated expressions of downstream KLF2 and its targeted genes endothelial nitric oxide synthase and thrombomodulin. However, the protective effects were reversed by ERK5/KLF2 pathway inhibitor XDM8-92, BIX02189 or KLF2 siRNA suggesting the pathway involved in the function. The ex vivo assay, in which THP-1 adhesion to endothelium isolated from apoE-/- mice received various treatments further confirmed the results from HUVECs. Finally, we found that the atherosclerotic lesions in both cross sections at aortic root and whole aorta were significantly reduced in high fat-diet (HFD) mice with puerarin treatment compared with the HFD-only mice, but were increased respectively by 76% and 71% in XMD8-92 group, and 82% and 73% in BIX02189 group. Altogether, the data revealed that puerarin inhibited the monocyte adhesion in vitro and in vivo and thus reduced atherosclerotic lesions in apoE-/- mice; the protective effects were mediated by activation of ERK5/KLF2 signaling pathway. Our findings advance the understanding of puerarin function in atherosclerosis and point out a way to prevent the disease.

The influence of duckweed species diversity on ecophysiological tolerance to copper exposure.

In excess, copper is toxic to plants. In the plants, Landoltia punctata and Lemna minor grown in mixed and monoculture, the effects of exposure to varying concentrations of copper (0.01, 0.1, 0.5 and 1mgL(-1) Cu) for seven days were assessed by measuring changes in the chlorophyll, protein and malondialdehyde (MDA) content, catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX) activity. According to results, Cu levels in plants increased with increasing Cu concentration. The level of photosynthetic pigments and crude proteins decreased only upon exposure to high Cu concentrations. However, the starch and malondialdehyde (MDA) content increased. These results suggested a stress alleviation that was possibly the result of antioxidants such as CAT and SOD, the activities of which increased with increasing Cu levels. APX activity increased in L. punctata, but decreased in L. minor, under monoculture or mixed culture conditions. In addition, the duckweed in mixed culture exhibited increased antioxidant enzyme activities which provide increased resistance to copper in moderate copper concentrations. As the copper concentration increased, the duckweed in the mixed culture limited the uptake of copper to avoid toxicity.

4-Hydroxytamoxifen-stimulated processing of cyclin E is mediated via G protein-coupled receptor 30 (GPR30) and accompanied by enhanced migration in MCF-7 breast cancer cells.

Over-expression of cleaved cyclin E in breast tumors is closely associated with tumor progression and resistance to antiestrogens. 17ß-Estradiol (E2) has been recently shown to induce cyclin E processing in breast cancer cells. Tamoxifen has been used in patients with estrogen-sensitive breast cancer, yet resistance to antiestrogens and recurrence will appear in some of the patients after its continued use. We therefore addressed possible effects of tamoxifen on the generation of cleaved cyclin E and its signal mechanism(s) in estrogen-responsive MCF-7 breast cancer cells that express both G protein-coupled protein (GPR) 30 and estrogen receptor α (ERα). 4-Hydroxytamoxifen (OHT, tamoxifen's active form) failed to prevent E2-induced proteolysis of cyclin E and migration, but rather triggered cyclin E cleavage coincident with augmented migration. OHT-induced cyclin E truncation also occurred in SK-BR-3 cells that express GPR30 and lack ERα, but not in MDA-MB-231 cells that express neither GPR30 nor ERα. G1, a specific GPR 30 agonist, caused dramatic proteolysis of cyclin E and enhanced migration. Furthermore, OHT-stimulated cleavage of cyclin E and migration were tremendously attenuated by G15, a GPR30 antagonist, or siRNA against GPR30. In addition, inhibitors for EGFR or ERK1/2 remarkably suppressed OHT-induced truncation of cyclin E, suggesting involvement of EGFR signaling. Collectively, our data indicate that OHT contributes to the production of proteolyzed cyclin E via GPR30 with augmented migration in MCF-7 cells.

A novel strategy for conditional gene knockout based on ΦC31 integrase and Gal4/UAS system in Drosophila.

The Gal4/upstream activating sequences (UAS) system offers great advantages in target gene expression by separating the responder line and diverse tissue-specific expressed Gal4 driver lines in Drosophila. The bipartite system is commonly used in gain-of-function analysis, and by combining with the RNA interference technology, it can also be applied in loss-of-function analysis. However, the off-target effect caused by this strategy has not been well solved so far. Furthermore, it can only partially knockdown a specific gene expression. In this study, a novel conditional gene knockout method that combined the use of ϕC31 integrase and Gal4/UAS system was described. The target gene was preliminarily flanked by ϕC31 integrase recognition sites attB and attP, followed by conditional expressed Gal4 lines to drive the recombinase that were under UAS control to achieve spatial and temporal gene deletion. We found the strategy performed well in Drosophila, and the efficiency was higher than 82% in gene knockout by self-excision. Our strategy takes advantage of exiting Gal4 library to drive the recombinase, rather than conventionally used method which the recombinase was droved directly by specific promoters, thereby providing a more flexible and versatile tool for gene function analysis in Drosophila.

[Effects of Rhaponticum uniflorum on activity and mRNA expression of CYP3A1 in rats hepatocytes].

OBJECTIVE: To study effects of Rhaponticum uniflorum on the activities and mRNA expression of cytochrome P4503A1 in rats hepatocytes. METHODS: Rat hepatocytes sandwich cultures were treated by Rhaponticum uniflorum in different concentrations (15.6, 31.2, 62.4 mg/ml) for 48 hours. The enzymic activities of CYP3A1 were reflected by the activities of liver microsomal erythromyeine N-demethylase (ERD). The level change in the mRNA expression were tested by RT-PCR. RESULTS: After treatment with the medicine, there was concentration-dependent inhibition in both enzymic activity and mRNA level of CYP3A1 compared with the control. The rates of the inhibitory effect to CYP3A1 activity were 26.4%, 44.2% and 65.4% respectively, while those of the inhibitory effect to mRNA level were 29.4%, 49.8% and 56.0%. CONCLUSION: Rhaponticum uniflorum inhibits the enzymic activity of CYP3A1 and lowers CYP3A1 expression at the transcriptive levels.

S-phase delay in human hepatocellular carcinoma cells induced by overexpression of integrin beta1.

AIM: To clarify the mechanisms of integrin overexpression in negatively regulating the cell cycle control of hepatocellular carcinoma cells SMMC-7721. METHODS: The cell cycle pattern was determined by flow cytometry. The mRNA and protein expression levels were assayed by RT-PCR and Western blot, respectively. Stable transfection was performed by Lipofectamine 2000 reagent, and cells were screened by G418. RESULTS: Overexpression of alpha5beta1 or beta1 integrin induced S-phase delay in SMMC-7721 cells, and this delay was possibly due to the accumulation of cyclin-dependent kinase inhibitors (CKIs) p21(cip1) and p27(kip1). The decrease of protein kinase B (PKB) phosphorylation was present in this signaling pathway, but focal adhesion kinase (FAK) was not involved. When phosphorylation of PKB was solely blocked by wortmannin, p27(kip1) protein level was increased. Moreover, S-phase delay was recurred when attachment of the parental SMMC-7721 cells was inhibited by the preparation of poly-HEME, and this cell cycle pattern was similar to that of beta1-7721 or alpha5beta1-7721 cells. CONCLUSION: S-phase delay induced by overexpression of integrin beta1 subunit is attributed to the decrease of PKB phosphorylation and subsequent increases of p21(cip1) and p27(kip1) proteins, and may be involved in the unoccupied alpha5beta1 because of lack of its ligands.