TRIM59 expression is regulated by Sp1 and Nrf1 in LPS-activated macrophages through JNK signaling pathway

Abstract

Activated macrophages play an important role in many inflammatory diseases including septic shock and atherosclerosis. TRIM59 has been showed to participate in many pathological processes, such as inflammation, cytotoxicity and tumorigenesis. However, the molecular mechanisms controlling its expression in activated macrophages are not fully understood. Here we report that TRIM59 expression is regulated by Sp1 and Nrf1 in LPS-activated macrophages. TRIM59 is highly expressed in macrophages, and markedly decreased by LPS stimuli in vivo and in vitro. TRIM59 promoter activity is also significantly suppressed by LPS and further analysis demonstrated that Sp1 and Nrf1 directly bound to the proximal promoter of TRIM59 gene. LPS treatment significantly decreased Sp1 expression, nuclear translocation and reduced its binding to the promoter, whereas increased Nrf1 expression, nuclear translocation and enhanced its binding to the promoter. Moreover, LPS-decreased TRIM59 expression was reversed by JNK inhibitor. Finally, TRIM59 level is significantly decreased during atherosclerosis progression. Taken together, our results demonstrated that TRIM59 expression was precisely regulated by Sp1 and Nrf1 in LPS-activated macrophages, which may be dependent on the activation of JNK signaling pathway and TRIM59 may be a potential therapeutic target for inflammatory diseases such as atherosclerosis.

Keywords: Atherosclerosis; Macrophage; Nrf1; Sp1; TRIM59.

Fig. 1.

TRIM59 expression in mouse tissues and cell lines. (A) Total proteins and RNA were extracted from mouse tissues (8-week old). (B) Total proteins and RNA were extracted from human different types of cells. (C) Total proteins and RNA were extracted from mouse different types of cells. TRIM59 protein (30 μg/sample) and mRNA levels were determined by Western blot and qPCR analysis and normalized to tubulin.

Fig. 2.

LPS inhibits TRIM59 expression in vivo. C57BL/6 J wild-type mice (~8-week-old) were i.p. injected with LPS (20 mg/kg). Tissues samples of spleen (A-B), liver (C-D), bone marrow (E-F) and aorta (G-H) were collected after 16 h of LPS injection and used to extract total proteins and RNA. Expression of TRIM59 protein or mRNA was determined by Western blot or qPCR analysis. Data are presented as mean ± SEM (n = 5); **p < 0.01, ***p < 0.001 vs. saline group in the corresponding groups by Student′s t-test.

Fig. 3.

LPS inhibits TRIM59 expression in hBMDMs.(A-D) Human blood monocyte-derived macrophages cells (hBMDMs) were treated with LPS at the indicated concentrations for 24 h, or with 0.2 μg/ml LPS for the indicated times. Expression of TRIM59 protein (A, B) or mRNA (C, D) was determined by Western blot or qPCR analysis. Data are presented as mean ± SEM (n = 3); *p < 0.05, ***p < 0.001 vs. Ctrl by one-way ANOVA.

Fig. 4.

LPS inhibits TRIM59 promoter activity. (A) TRIM59 promoter (from −600 to +40) which includes both potential Sp1 binding sites (GC box) and Nrf1 binding site (ARE) was cloned and named as p640TRIM59. (B) RAW264.7 cells in 24-well plates were transfected with DNA for p640TRIM59 and Renilla (for internal control). After 8 h of transfection, cells were treated with LPS at the indicated concentrations for 16 h, followed by determination of activities of firefly and Renilla luciferases using the Dual-Luciferase Reporter Assay system. Data are presented as mean ± SEM (n = 3); **p < 0.01, ***p < 0.001 by one-way ANOVA. (C-D) TRIM59 promoters with the indicated deletion or mutation were constructed separately. Same amount of DNA for these promoters was used to transfect HEK-293 T cells. After 4 h of transfection, cells were switched into complete medium for 20 h, followed by determination of activities of firefly and Renilla luciferases. Data are presented as mean ± SEM (n = 3); *p < 0.05, ***p < 0.001 by one-way ANOVA.

Fig. 5.

LPS inhibits TRIM59 promoter activity by regulating Sp1 and Nrf1 differently. HEK-293 T cells in 24-well plates were transfected with DNA for p640TRIM59 or plus Sp1 expression vector (A) or plus Nrf1 expression vector (D) as indicated and Renilla. After 4 h of transfection, cells were switched into complete medium for 20 h, followed by determination of activities of firefly and Renilla luciferases. *p < 0.05, **p < 0.01, ***p < 0.001 (n = 3). (B, E) HEK-293 T cells in 24-well plates were transfected with DNA for the indicated TRIM59 promoters and Sp1 or Nrf1 expression vector. Activities of firefly for the corresponding promoter and Renilla luciferases were determined. ***p < 0.001 (n = 3). (C, F) RAW264.7 cells in 24-well plates were transfected with DNA for the indicated TRIM59 promoters. After 8 h of transfection, cells were treated with LPS at the indicated concentrations for 16 h, followed by determination of activities of firefly and Renilla luciferases. *p < 0.05, ***p < 0.001 (n = 3).

Fig. 6.

LPS inhibits Sp1 but promotes Nrf1 expression in macrophages. (A-B) RAW264.7 cells were treated with LPS at the indicated concentrations overnight. Total and nuclear proteins were extracted and used to determine expression of Sp1 (A) or Nrf1 (B) protein by Western blot. Data are expressed as mean ± SEM (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA. (C-D) RAW264.7 cells were treated with LPS (0.5 μg/ml) overnight, followed by determination of Sp1 protein (C) or Nrf1 protein (D) expression and localization by immunofluorescent staining. Scale bar, 50 μm. (E-F) ChIP performed with an antibody against Sp1 or Nrf1 on chromatin isolated from RAW264.7 cells treated by LPS (0.2 μg/ml) overnight. The quantity of immunoprecipitated DNA was assessed by qPCR for the predicted GC box (E) or ARE (F) present in the TRIM59 promoter. The net quantities of enriched DNA were corrected with their corresponding input DNA. Data are expressed as mean ± SEM (n = 3); **p < 0.01 by Student′s t-test. qPCR products then conduct electrophoresis in 2% agarose gel stained with ethidium bromide and visualized under UV light.

Fig. 7.

LPS inhibits macrophage TRIM59 expression in Sp1- and Nrf1-dependent manners through JNK signaling pathway. (A) RAW264.7 cells in 6-well plates were transfected with scrambled siRNA (25 nM) or Sp1 siRNA (25 nM) followed by treatment with LPS (0.2 μg/ml) for 24 h, and then determined of Sp1 and TRIM59 protein expression by Western blot analysis. (B) Peritoneal macrophages in 6-well plates were transfected with DNA for Nrf1 expression vector or pEGFP-C2 vector followed by treatment with LPS (0.2 μg/ml) overnight. Total proteins were extracted from peritoneal macrophages and used to determine expression of Nrf1 and TRIM59 protein by Western blot analysis. Data are presented as mean ± SEM (n = 3); *p < 0.05, ***p < 0.001 by Student′s t-test or one-way ANOVA. (C-D) RAW264.7 cells were pretreated with a variety of inhibitors for TLR4 signaling pathway (JNK inhibitor: SP600125; ERK1/2 inhibitor: U0126; p38 inhibitor: SB203580; IKK/NF-κB inhibitor: Wedelolactone; PI3K inhibitor: LY294002) for 3 h and then were stimulated with 0.2 μg/ml LPS for 16 h. After treatment, total RNA and proteins were extracted and used to determine TRIM59 mRNA (C) and protein expression (D) by qPCR and Western blot analysis, respectively. Data are presented as mean ± SEM (n = 3); **p < 0.01, ***p < 0.001 by one-way ANOVA.

Fig. 8.

TRIM59 expression is decreased during atherosclerosis progression. (A) Western blot and qPCR analysis of TRIM59 protein and mRNA expression in the aortas of ApoE^−/− mice fed a western diet for the indicated time. Data are presented as mean ± SEM (n = 4); **p < 0.01, ***p < 0.001 by Student′s t-test. (B) Western blot and qPCR analysis of TRIM59 protein and mRNA expression in the peritoneal macrophages of ApoE^−/− mice fed a western diet for the indicated time. Data are presented as mean ± SEM (n = 4); **p < 0.01 by Student′s t-test. (C) qPCR analysis of TRIM59 mRNA level in peripheral blood monocytes of patients with hypercholesterolemia. Data are presented as mean ± SEM (n = 8 or 6); **p < 0.01 by Student′s t-test.

Fig. 9.

A proposed model of LPS/JNK/Sp1 or Nrf1 signaling pathways in regulation of TRIM59 expression. Sp1 promotes and Nrf1 inhibits TRIM59 expressions in LPS-activated macrophages. TRIM59 expression in LPS-activated macrophages may be dependent on the JNK signaling pathway, in which phosphorylation of JNK inhibits the expression, nuclear translocation and binding to TRIM59 promoter of Sp1, whereas increases the expression, nuclear translocation and binding to TRIM59 promoter of Nrf1.