Nuclear factor of activated T cells regulates osteopontin expression in arterial smooth muscle in response to diabetes-induced hyperglycemia

Abstract

Objective: Hyperglycemia is a recognized risk factor for cardiovascular disease in diabetes. Recently, we reported that high glucose activates the Ca(2+)/calcineurin-dependent transcription factor nuclear factor of activated T cells (NFAT) in arteries ex vivo. Here, we sought to determine whether hyperglycemia activates NFAT in vivo and whether this leads to vascular complications.

Methods and results: An intraperitoneal glucose-tolerance test in mice increased NFATc3 nuclear accumulation in vascular smooth muscle. Streptozotocin-induced diabetes resulted in increased NFATc3 transcriptional activity in arteries of NFAT-luciferase transgenic mice. Two NFAT-responsive sequences in the osteopontin (OPN) promoter were identified. This proinflammatory cytokine has been shown to exacerbate atherosclerosis and restenosis. Activation of NFAT resulted in increased OPN mRNA and protein in native arteries. Glucose-induced OPN expression was prevented by the ectonucleotidase apyrase, suggesting a mechanism involving the release of extracellular nucleotides. The calcineurin inhibitor cyclosporin A or the novel NFAT blocker A-285222 prevented glucose-induced OPN expression. Furthermore, diabetes resulted in higher OPN expression, which was significantly decreased by in vivo treatment with A-285222 for 4 weeks or prevented in arteries from NFATc3(-/-) mice.

Conclusions: These results identify a glucose-sensitive transcription pathway in vivo, revealing a novel molecular mechanism that may underlie vascular complications of diabetes.

Figure 1. Hyperglycemia increases NFATc3 nuclear accumulation in vivo

A. Confocal images showing NFATc3 nuclear accumulation in cerebral arteries from hyperglycemic (IP-GTT) and normoglycemic (control) C57Bl/6 mice. Upper panels are pseudo colored images showing nuclear NFATc3 in white; middle and lower panels are original images showing the DNA-binding dye SYTOX Green (green) and NFATc3 (red), respectively. Bar=20 μm. B. Summarized data from experiments in A showing fluorescence intensity of nuclear NFATc3 (N=6 in each group, 147 and 181 images per group, *P < 0.05). C. Glucose levels before and after IP-GTT, corresponding to experiments in A and B (***P < 0.001 vs. all groups).

Figure 2. Streptozotocin-induced hyperglycemia increases NFAT-dependent transcriptional activity

A. Blood glucose levels in NFAT-luc transgenic mice treated with STZ or vehicle. *P < 0.05 and ***P < 0.001 vs. vehicle. B. Correlation between luciferase activity (RLU μg^-1) and blood glucose levels at day 16 (N=12). C. NFAT- luciferase activity in aortas treated as in A, measured at day 8, 12 and 16 after the first STZ injection, normalized to vehicle. Luciferase activity in vehicle mice did not differ between days 8, 12 and 16. N=12-16 for each time point. *P < 0.05 vs. vehicle. D. Luciferase activity in aortas from diabetic NFAT-luc/NFATc3^+/+, NFAT-luc/NFATc3^+/- and NFAT-luc/NFATc3^-/- mice, measured at day 12 after the first STZ injection. Values are normalized to non-diabetic NFAT-luc/NFATc3^+/+ vehicle (dotted line). N=6-9 mice in each group. *P < 0.05 vs. vehicle.

Figure 3. Regulation of NFAT activity leads to changes in OPN protein expression in intact arteries

A. Confocal images showing expression of OPN (red, upper panels) in cerebral arteries and nuclear staining with SYTOX Green (lower panels). Arteries were stained immediately after dissection (fresh) or after 2d organ culture in the presence or absence of UTP (10 μmol/L, supplemented daily). Bars=50 μm. B. Summarized confocal data showing OPN expression in cerebral vessels cultured for 2d in 5 mmol/L and 25 mmol/L glucose (90.1 and 450.5 mg/dL respectively) with or without A-285222 (1 μmol/L) or apyrase (0.32 U/mL), or in 25 mmol/L mannitol. Values are normalized to 25 mmol/L glucose (N=13, 26-126 images analyzed in each group, ***P < 0.001 for 25 mmol/L glucose vs. all other bars). C. Summarized confocal data showing OPN expression in cerebral arteries cultured for 2d with or without UTP (10 μmol/L) in the presence or absence of A-285222 (1 μmol/L). Values are normalized to UTP (N=6, 26 to 75 images analyzed in each group, ***P < 0.001 for UTP vs. all other bars).

Figure 4. Involvement of NFATc3 in the regulation of OPN expression

A & B. Summarized data from confocal immunofluorescence experiments showing increased OPN expression upon UTP stimulation for 3 d in intact cerebral arteries and aortas from NFATc3^+/+ mice, but no effect in arteries from NFATc3^-/- mice. Aortas were divided in halves and cultured either with or without UTP (100 μmol/L, N=3, 45-57 images analyzed in each group, ***P < 0.001 vs. all other bars). For the cerebral arteries, half of the arteries from each mouse were cultured with UTP and the other half without (10 μmol/L, N=3, 15-21 images in each group, ***P < 0.001 vs. all other bars). C. VSMCs and intact aortas were incubated for 3 hours in 5 mmol/L glucose or 25 mmol/L glucose with or without 1 μmol/L A-285222. NFATc3 enrichment by ChIP was performed and associated DNA complexes analyzed by quantitative PCR (in triplicates). Primers specific to NFAT binding site 3 of the OPN promoter were used. Aortas from 3-4 mice were used for each IP reaction, experiments were performed 3-5 times (for VSMCs and aortas, respectively), ***P < 0.001. D. OPN mRNA expression in fresh and cultured aortas (2 days in 5 mmol/L or 25 mmol/L glucose containing media). Results are shown as ratio of OPN expression to cyclophilin B, normalized to 5 mmol/L glucose. N=2, 10, 18 for fresh, 5 mmol/L and 25 mmol/L glucose respectively, in triplicate, *P < 0.05; **P < 0.01 and ***P < 0.001.

Figure 5. Lack of NFATc3 protein or in vivo treatment with A-285222 prevents OPN induction

A. Blood glucose levels in BalB/c mice treated with STZ or vehicle. Mice received i.p. injections once a day of A-285222 or equal volumes of saline (control). B. Summarized data and representative immunoblot from western blot experiments showing OPN expression normalized to β-actin in the aorta of mice treated as in A (N=7-8 in each group). **P < 0.01 and ***P < 0.001 vs. untreated control. C. Blood glucose levels in NFATc3^+/+ and NFATc3^-/- mice treated with STZ or vehicle. D. Summarized data and representative immunoblot from western blot experiments showing OPN expression normalized to β-actin in the aorta of mice treated as in C (N=6-8 in each group).