| Objective:Lipopolysaccharide(LPS), also endotoxin, is the component of cell wall of gram-negative bacteria. LPS is toxic when it is released from death bacteria. CD36, as a pattern recognition receptor, can recognize various antigens including LPS and mediate signaling pathway and regulate function of cells based on its structure. The ubiquitination of CD36 can acutely regulate its level and function in cell membrane. So based on site mutation we can investigate the influence of ubiqutination on the function of CD36. Macrophages, a kind of immunocyte, participate inflammation significantly. Macrophages secrete TNF-α and IL-6, and then mediate autophagy against infection under the stimulation of LPS. CD36 is expressed in macrophages, but it is still unclear to elucidate the influence of CD36 on autophagy and inflammatory cytokines secretation as well as related mechanisms. It is needed to investigate the influence of CD36 ubiqutination on the signaling pathways and the function of cells.CHO cell express no CD36, but has the capability of high amplification and expression. Firstly, therefore, the three stably transfected cell lines including CHO/Vector, CHO/CD36 WT and CHO/CD36 K/A were stimulated by LPS to investigate the effect of CD36 and its ubiquitination on MAPK signaling pathway and autophagy, in order to research the role of CD36 and the molecular mechanisms in inflammation and autophagy. Secondly, we establish a model that LPS stimulates RAW264.7 macrophages pretreated with various conditions, including si RNA and ERK inhibitor, to examine effects of CD36 and ERK signaling pathway on secretion of inflammation-related cytokines and autophagy. The results will provide theoretical and experimental evidence for elucidating molecular mechanisms underlying secretion of inflammation-related cytokines and autophagy induced by LPS.Methods:1. The influences of CD36 and its ubiquitination on MAPK signaling pathway and autophagy(1) Determine the suitable concentration and time of LPS stimulating CHO cellsCHO/CD36 WT cells were stimulated by different concentrations of LPS, namely 0, 5, 10, 20, 50, 100, 200, 500, 1000 ng/ml, for same time and lysed. CHO/CD36 WT cells were stimulated by 100 ng/ml of LPS for 0, 10, 30, 60, 120, 240 min and lysed. The level of ERK phosphorylation were then detected by western blot to determine the suitable concentration and time of LPS.(2) The influences of CD36 and its ubiquitination on MAPK signaling pathwaysThe role of MAPK signaling pathways are significant in various physiological and pathological processes. Among them extracellular regulated protein kinase(ERK), c-Jun N-terminal kinase(JNK) and p38 are the most important and widely studied. LPS stimulated CHO/Vector, CHO/CD36 WT and CHO/CD36 K/A cells, at the same groups that cells without LPS stimulation were set up, divided into 6 groups. Following the working condition of LPS above, 100 ng/ml LPS stimulated cells for 2 h, then WB detected the level of ERK, JNK and p38 phosphorylation, that is, the activation of ERK, JNK and p38.(3) The influences of CD36 and its ubiquitination on autophagy induced by LPSProtein kinases C(PKC) plays a conserved role in autophagy regulation, diacylglycerol(DAG) binds C1 domain of PKC isoforms to active the function, so using the C1 domain from PKC as a probe, we visualized DAG. CHO/Vector, CHO/CD36 WT and CHO/CD36 K/A cells were co-transfected with RFP-LC3 and GFP-PKC-C1 plasmids, then stimulated with LPS or not for 2 h. The colocalization of autophagosomes with DAG were detected by confocol laser scanning microscope(CLSM).2. To investigate the influence of CD36 on LPS-induced secretion of inflammatory cytokines and autophagy in macrophages and related molecular mechanisms(1) Determination of the suitable concentration and incubation time of LPS stimulating RAW264.7 cellsRAW264.7 cells were stimulated by a series of concentrations of LPS from 0, 5, 10, 20, 50, 100, 500 to 1000 ng/ml, for the same time and lysed. Then 100 ng/ml LPS stimulated RAW264.7 cells for 0, 0.5, 1, 2, 4, 8, 16, 24 h and cells were lysed. The Methods: expression of microtubule-associated protein 1 light chain 3-Ⅱ(MAP1-LC3-Ⅱ) were detected by WB to determine the suitable concentration and incubation time of LPS(2) The activation of MAPK and expression of LC3-Ⅱ and Beclin-1 in RAW264.7 cells stimulated by LPSRAW264.7 cells were stimulated by 100 ng/ml LPS or vehicle for 16 h and then lysed. The level of ERK, JNK and p38 phosphorylation and expression of LC3-Ⅱ and Beclin-1 were detected by WB.(3) The influence of CD36 on the activation of ERK, autophagy and inflammatory cytokines in macrophage induced by LPSThe expression of CD36 was blocked by si RNA. Macrophages with or without CD36 knockdown were stimulated with 100 ng/ml LPS or vehicle, respectively. After 16 h incubation, cells were collected, lysed and detected in the following experiments: ① The level of ERK phosphorylation and expression of LC3-Ⅱand Beclin-1 was detected by WB. ②Immunofluorescence detected the quantities of autophagosomes, the aggregation of LC3 dots. Cytokines such as tumor necrosis factor-α(TNF-α), interleukin-6(IL-6) and interleukin-10(IL-10) were examined by enzyme-linked immunosorbent assay(ELISA).(4) The influence of ERK inhibitor on the activation of ERK, autophagy and inflammatory cytokines in macrophage induced by LPSRAW264.7 cells were stimulated for 24 h with a series of concentrations of ERK inhibitor(PD 0325901) varied from 0, 5, 10, 20, 50, to 100 n M. WB examined the phosphorylation of ERK and the survival rate of cells by methyl thiazolyl tetrazolium(MTT), to determine the working concentration of ERK inhibitor. To further investigate the influence of the activation of ERK on autophagy and inflammatory cytokines, we established four cell models. Cells incubated with or without ERK inhibitor were stimulated by 100 ng/ml LPS or vehicle. After 16 h incubation, cells were collected for examination of the different indexes following(3)。Results:1. The influences of CD36 and its ubiquitination on MAPK signaling pathway and autophagy(1) Determine the suitable concentration and incubation time of LPS stimulating CHO cells: WB results showed that the activation of ERK reached highest when cells were stimulated with 100 ng/ml LPS. Compared with other time points, when 100 ng/ml LPS stimulated CHO/CD36 WT cells for 2 h, the phosphorylation of ERK was highest. So 100ng/ml LPS were used to stimulate CHO cells for 2 h in the following experiments.(2) The influences of CD36 and its ubiquitination on MAPK signaling pathways: WB analysis showed that p ERK/t ERK and p JNK/t JNK increased in CHO/CD36 WT cells upon LPS stimulation, while p38 activation changed little. In contrast, the activation of ERK, JNK and p38 had no changes in CHO/Vector and CHO/CD36 K/A cells after LPS treatment.(3) The influences of CD36 and its ubiquitination on autophagy induced by LPS: CLSM experiments showed that LPS induced the colocalization of LC3 dots and DAG with autophagosomes in CHO/CD36 WT and CHO/CD36 K/A cells. The quantities of the above two colocalizations were higher than those without LPS treatment and CHO/Vector cells treated with LPS. But quantities of the colocalization of DAG with autophagosomes in CHO/CD36 WT and CHO/CD36 K/A cells had no difference.2. To investigate the influence of CD36 on LPS-induced secretion of inflammatory cytokines and autophagy in macrophage and related molecular mechanisms.(1) Determination of the concentration and incubation time of LPS stimulating RAW264.7 cells: WB analysis showed that cells were stimulated with increasing concentrations of LPS. Under the condition of 100 ng/ml LPS the level of LC3-Ⅱ reached saturation degree. The expression of LC3-Ⅱincreased gradually, and reached saturation degree at 16 h when cells were treated with 100 ng/ml LPS for different times. Then considering the reference and results, 100 ng/ml LPS were used to stimulate RAW264.7 cells for 16 h in the following experiments.(2) The activation of MAPK and expression of LC3-Ⅱ and Beclin-1 in RAW264.7 cells stimulated by LPS: Analysis of WB showed that p ERK/t ERK and p JNK/t JNK increased in RAW264.7 cells upon LPS, while p38 activation changed little, the expression of LC3-Ⅱ/Tubulin and Beclin-1/Tubulin increased significantly.(3) CD36 affects the activation of ERK, autophagy and inflammatory cytokines in macrophage induced by LPS: Analysis of WB and CLSM showed that after cells were stimulated with LPS, phosphorylation of ERK increased significantly, LC3-Ⅱ/Tubulin and Beclin-1/Tubulin and LC3 dots were higher than cells without LPS treatment. When cells with CD36 downregulated by si RNA were treated, ERK activation decreased while LC3-Ⅱ/Tubulin and Beclin-1/Tubulin as well as LC3 dots further increased. ELISA results showed that secretion of TNF-α and IL-6 increased significantly but the level of IL-10 changed little in macrophages stimulated with LPS. In contract, CD36 knockdown decreased the secretion of TNF-α and IL-6 while IL-10 increased significantly upon LPS treatment.(4) The influence of ERK inhibitor on the activation of ERK, autophagy and inflammatory cytokines induced by LPS: MTT assay showed that the survival ratio was above 50% when cells were incubated with 20 n M ERK inhibitor. The ERK activation was the lowest in cells cultured with 20 n M and 50 n M inhibitor, so 20 n M was the working concentration of ERK inhibitor.WB and CLSM analysis showed that after cells were stimulated with LPS, the activation of ERK increased significantly. LC3-Ⅱ/GAPDH, Beclin-1/GAPDH and LC3 dots were higher than cells without LPS treatment. Macrophages pre-treated with or without ERK inhibitor, pharmaceutical inhibition of ERK, decreased ERK activation significantly, while enhanced LC3-Ⅱ/GAPDH and LC3 dots upon LPS treatment. ELISA showed that cytokines secretion in macrophages pre-treated with or without ERK inhibitor decreased TNF-α and IL-6, while enhanced IL-10 secretion upon LPS treatment.Conclusion:1. Studies on three CHO cells stimulated by LPS showed that CD36 mediated the activation of ERK and JNK and is essential for autophagy induced by LPS. If the ubiquitination of CD36 is blocked, upon stimulation of cells by LPS, the activation of ERK and JNK had no significantly difference compared with groups without LPS treatment. Analysis of CLSM showed that, mutant of ubiquitination had no impact on formation of autophagosomes mediated by CD36, which DAG was involved in. However, further researches for definite mechanisms whether ubiquitination of CD36 influenced autophagy were needed.2. ERK signaling pathway participated in the autophagy and cytokines response in macrophages upon LPS stimulation. CD36 may regulate autophagy and anti-inflammatory cytokines responses negatively and mediate pro-inflammatory cytokines responses positively through ERK signaling pathway in macrophages stimulated with LPS. |
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