High glucose upregulates osteopontin expression by FoxO1 activation in macrophages

in Journal of Endocrinology
Correspondence should be addressed to B Lu or J Zheng: binlu@smmu.edu.cn or jyzheng@smmu.edu.cn

*(Q Zhang, C Wang and Y Tang contributed equally to this work)

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Hyperglycemia plays a major role in the development of diabetic macrovascular complications, including atherosclerosis and restenosis, which are responsible for the most of disability and mortality in diabetic patients. Osteopontin (OPN) is an important factor involved in atherogenesis, and hyperglycemia enhances the transcriptional activity of FoxO1 which is closely association with insulin resistance and diabetes. Here, we showed that plasma OPN levels were significantly elevated in type 2 diabetic patients and positively correlated with glycated albumin (GA). The more atherosclerotic lesions were observed in the aorta of diabetic ApoE−/− mice analyzed by Sudan IV staining. High glucose increased both the mRNA and protein expression levels of OPN and inhibited the phosphorylation of FoxO1 in RAW 264.7 cells. Overexpression of WT or constitutively active mutant FoxO1 promoted the expression levels of OPN, while the dominant-negative mutant FoxO1 decreased slightly the expression of OPN. Conversely, knockdown of FoxO1 reduced the expression of OPN. Luciferase reporter assay revealed that high glucose and overexpression of FoxO1 enhanced the activities of the OPN promoter region nt −1918 ~ −713. Furthermore, the interactions between FoxO1 and the OPN promoter were confirmed by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP). Our results suggest that high glucose upregulates OPN expression via FoxO1 activation, which would play a critical role in the development of diabetic atherogenesis.

 

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    The expression of OPN in type 2 diabetes patients with or without atherosclerosis. (A) Plasma OPN levels were detected with ELISA in 70 type 2 diabetes patients and 70 healthy volunteers. (B) Plasma OPN levels were detected with ELISA in atherosclerosis patients with or without type 2 diabetes. (C) The representative plaque samples from the carotid arteries of diabetic patients were analyzed by hematoxylin and eosin staining or immunohistochemical staining for OPN, FoxO1 and CD68. (×40 magnification). Values are mean ± s.e.m. *P < 0.05, **P < 0.01 (unpaired t test).

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    High glucose increases the expression of OPN and modulates FoxO1 phosphorylation in RAW 264.7 cells. (A) RAW264.7 cells were cultured in medium containing different concentration of glucose (5, 10, 25 mM) for an additional 48 h. The levels of OPN mRNA expression were determined by quantitative PCR and estimated by the ratio of OPN signal to GAPDH signal. (B) The protein lysated from (A) were analyzed for the expression of OPN, phosphorylated and total FoxO1 by Western blot. (C) The secreted OPN protein levels were detected by ELISA from cell culture supernatants. (D) RAW264.7 cells were stimulated by different concentration of insulin (1, 10, 100 nM) for 2 h, and detected for the expression levels of OPN mRNA by qPCR. (E) RAW264.7 cells were stimulated by different concentration of insulin (1, 10 nM) for 3 h and analyzed for the expression of OPN, phosphorylated and total FoxO1 by Western blot. (F) Immunofluorescence staining shows the distribution of FoxO1 in the cytoplasm and nucleus of RAW264.7 cultured in the media with 5 mM or 25 mM glucose by means of confocal microscopy. Each value represents mean ± s.e.m. obtained from three independent experiments. *P < 0.05, **P < 0.01 (unpaired t test).

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    Overexpression of FoxO1 upregulates OPN expression in RAW 264.7 cells. (A) RAW264.7 cells were infected with lentivirus encoding wild-type FoxO1 (FoxO1-WT), phosphorylation-defective mutant FoxO1 (FoxO1-ADA) or dominant-negative form of Foxo1 (FoxO1-Δ256) as indicated. The levels of OPN mRNA were determined by quantitative PCR and normalized to those of GAPDH. (B) The whole-cell extracts were assessed for the expression of FoxO1 and OPN by Western blot analysis. (C) The secreted OPN protein levels were detected by ELISA using cell culture supernatants. Each value represents mean ± s.e.m. obtained from three independent experiments. *P < 0.05, **P < 0.01 compared with LV control (unpaired t test).

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    Silencing expression of FoxO1 decreases OPN expression in RAW 264.7 cells. (A) RAW264.7 cells were infected with lentivirus containing three distinct short hairpin RNA (shRNA) targeting FoxO1 or scrambled shRNA (shNon). The levels of FoxO1 were normalized to those of GAPDH after qRT-PCR. (B) The levels of OPN mRNA were detected by qRT-PCR. (C) The whole-cell extracts were assessed for the expression of FoxO1 and OPN by Western blot analysis. (D) The secreted OPN protein levels were detected by ELISA using cell culture supernatants. (E) RAW264.7 cells infected with LV-shFoxO1 or LV-shNon were cultured in media with 5 mM or 25 mM glucose for 48 h, then were detected for OPN mRNA levels by qPCR. Each value represents mean ± s.e.m. obtained from three independent experiments. *P < 0.05, **P < 0.01 compared with LV-shNon (unpaired t test).

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    High glucose promotes the binding of FoxO1 with the OPN promoter in RAW 264.7 cells. (A) The RAW 264.7 cells stabled transfected with luciferase reporter gene containing different region of OPN promoter as indicated. Forks represent the mutant of putative FBEs in OPN promoter. The activities of luciferase were normalized by cells counting. (B) The cells were cultured in medium containing different concentration of glucose (5 mM, 10 mM and 25 mM) for 48 h and harvested to measure the activities of luciferase as described in (A). (C) The cells were infected with lentivirus encoding wild-type FoxO1 (FoxO1-WT), phosphorylation-defective mutant FoxO1 (FoxO1-ADA) or dominant-negative form of Foxo1 (FoxO1-Δ256) as indicated and harvested to measure the activities of luciferase as described in (A). (D) The cells were transfected with scrambled shRNA (shNon) or three distinct FoxO1-targeting short hairpin RNA (shFoxO1), and harvested to measure the activities of luciferase as described in (A). Each value represents mean ± s.e.m. obtained from three independent experiments. *P < 0.05, **P < 0.01 compared with LV-shNon (unpaired t test).

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    Direct binding of FoxO1 to FoxO1-binding sites in the promoter of OPN. (A) Analysis of protein–DNA complexes formed between oligonucleotides containing FoxO1-binding sites from OPN promoter and nuclear extracts (NE) from RAW 264.7 cells by EMSA. For oligonucleotide competition experiments, 200-fold excess of unlabeled oligonucleotides were added before the addition of the labeled probe. Specific shifts are marked with an arrow. Gel shown is representative of three independent experiments. (B) ChIP assays were performed to detect the binding of FoxO1 with OPN promoter in RAW 264.7 cells. The sheared DNA fragments were immunoprecipitated with antibody against FoxO1, and subjected to PCR using primer pairs located in the region nt −1918~−1569 and nt −349~−102 of the OPN promoter, respectively. The PCR results were separated in a 2% agarose gel. Gel shown is representative of three independent experiments. (C) RAW 264.7 cells were cultured in media with different concentration of glucose (5, 10, 25 mM) for 48 h, and then ChIP assays were performed to detect the binding of FoxO1 with OPN promoter as described in (B). Gel shown is representative of three independent experiments.

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    High glucose accelerates atherogenesis in diabetic mice. Male ApoE-deficient mice and littermate control of C57BL/6J mice were injected with 50 mg/kg STZ or equal volume of vehicle (0.05 M citrate buffer) intraperitoneally for continuous 5 days. (A) Ten days after the first STZ injection, the blood glucose levels of C57BL/6 wild-type mice (WT), ApoE−/− mice (ApoE−/−), STZ-induced diabetic wild-type mice (WT+STZ) and ApoE−/− mice (ApoE−/−+STZ), were detected by glucometers (n = 6/group). (B) Six weeks after the first STZ injection, the plasmic OPN levels of four groups of mice were detected by ELISA. (C) The representative images of mouse aortae analyzed by Sudan IV staining to visualize accumulation of lipids present in atherosclerotic lesions. (D) Mean atherosclerotic lesion areas of the mouse aorta were measured by Image-Pro Plus 6.0 software. Values are mean ± s.e.m. *P < 0.05, **P < 0.01 (unpaired t test).

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