Fluvastatin inhibits AGE-induced cell proliferation and migration via an ERK5-dependent Nrf2 pathway in vascular smooth muscle cells

Abstract

Advanced glycation endproduct (AGE)-induced vascular smooth muscle cell (VSMC) proliferation and reactive oxygen species (ROS) production are emerging as important mechanisms of diabetic vasculopathy, but little is known about the molecular mechanism responsible for the antioxidative effects of statins on AGEs. It has been reported that statins exert pleiotropic effects on the cardiovascular system due to decreases in AGE-induced cell proliferation, migration, and vascular inflammation. Thus, in the present study, the authors investigated the molecular mechanism by which statins decrease AGE-induced cell proliferation and VSMC migration. In cultured VSMCs, statins upregulated Nrf2-related antioxidant gene, NQO1 and HO-1, via an ERK5-dependent Nrf2 pathway. Inhibition of ERK5 by siRNA or BIX02189 (a specific ERK5 inhibitor) reduced the statin-induced upregulations of Nrf2, NQO1, and HO-1. Furthermore, fluvastatin was found to significantly increase ARE promoter activity through ERK5 signaling, and to inhibit AGE-induced VSMC proliferation and migration as determined by MTT assay, cell counting, FACS analysis, a wound scratch assay, and a migration chamber assay. In addition, AGE-induced proliferation was diminished in the presence of Ad-CA-MEK5α encoding a constitutively active mutant form of MEK5α (an upstream kinase of ERK5), whereas depletion of Nrf2 restored statin-mediated reduction of AGE-induced cell proliferation. Moreover, fluvastatin suppressed the protein expressions of cyclin D1 and Cdk4, but induced p27, and blocked VSMC proliferation by regulating cell cycle. These results suggest statin-induced activation of an ERK5-dependent Nrf2 pathway reduces VSMC proliferation and migration induced by AGEs, and that the ERK5-Nrf2 signal module be viewed as a potential therapeutic target of vasculopathy in patients with diabetes and complications of the disease.

Fig 1. The involvement of ERK5 in statin-induced Nrf2 signaling in VSMCs.

(A-B) Western blot analysis of Nrf2, NQO1, and HO-1 in statin-treated VSMCs. Cells were exposed to fluvastatin and pitavastatin for 24 h at the indicated dosages. In addition, protein samples were refined from cultured VSMCs treated with fluvastatin (5 μM) or pitavastatin (5 μM) for the indicated times. (C) Western blot analysis of ERK5, NQO1, HO-1, phospho-ERK1/2, and phospho-ERK5 in BIX02189 treated VSMCs. Protein samples were obtained from cultured VSMCs treated with 5 μM fluvastatin or 5 μM pitavastatin for 24 h. (D) VSMCs were transfected with control or ERK5 siRNA (50 pM) for 30 h and then treated with 5 μM fluvastatin for 24 h. Protein levels of NQO1, HO-1, ERK5 and tubulin were determined by Western blotting with specific antibodies. In addition, phosphorylation levels of ERK1/2 and ERK5 were determined by immunoblotting with specific antibodies. Bar graphs present the densitometric quantification of Western blot bands. Results are representative of three independent experiments. *, #, †, p<0.05; **, ##, ††, p<0.01 compared with control. NS indicates not significant.

Fig 2. The involvement of ERK5 in fluvastatin-induced Nrf2 signaling in VSMCs.

(A-C) Quantitative RT-PCR analysis of the mRNA expressions of Nrf2, NQO1, and HO-1 in VSMCs treated with fluvastatin. VSMCs were treated with BIX02189 (2 μM) for 1 h and then incubated with 5 μM fluvastatin for 6 h. qRT-PCR analysis was performed in triplicate. Results are representative of three independent experiments. *, p < 0.05. (D) VSMCs were co-transfected with pARE and pRL-tk and then stimulated with 5 μM fluvastatin for 24 h in the presence or absence of BIX02189 (2 μM). Promoter activity was measured by using a Dual-Luciferase reporter assay kit and a GloMax 20/20 luminometer. Transfection efficiency was normalized versus Renilla luciferase activity derived from pRL-tk construct. Reporter assay was performed in triplicate. Results are presented as the means ± SDs of three independent experiments. **p < 0.01.

Fig 3. Fluvastatin inhibited AGEs-induced cell proliferation through the ERK5-Nrf2 pathway in VSMCs.

(A) Serum starved VSMCs were exposed to the indicated concentrations of AGEs for 24 h. (B) Serum starved VSMCs were pretreated with 5 μM fluvastatin in the presence or absence of BIX02189 (2 μM) for 1 h and then exposed to AGEs (10 μg/ml) for 24 h. Cell viability was determined using a MTT assay. (C) Cell proliferation was determined by using cell counting with same condition. Results are representative of three independent experiments. *, p < 0.05; **, p < 0.01.

Fig 4. The effect of Nrf2 siRNA on the ERK5 activation-mediated inhibition of AGE-induced VSMC proliferation.

(A) VSMC cells were infected with adenovirus encoding LacZ or CA-MEK5α for 48 h, and then exposed to AGEs for 24 h. Cell viability was determined using a MTT assay. Results are presented as the means±SEs of three independent experiments. *, p < 0.05; **, p < 0.01. (B) Cell proliferation was determined by using cell counting with same condition. Results are representative of three independent experiments. **, p < 0.01. Amounts of protein expression were determined by immunoblotting with specific antibodies for HA and Nrf2.

Fig 5. Fluvastatin regulated AGE-induced cell cycle progression through an ERK5 activating pathway in VSMCs.

(A) Serum starved VSMCs were pretreated with 5 μM fluvasatin in the presence or absence of BIX02189 (2 μM), and then exposed to AGEs 10 μg/ml AGEs for 24 h. Protein expressions were determined by immunoblotting with anti-cyclin D1, anti-CDK4, anti-p27, and anti-tubulin. Results are representative of three independent experiments. Asterisk indicates a nonspecific band. Bar graphs present the densitometric quantification of Western blot bands. (B) VSMCs were seeded onto 6 well plates at a density of 1×10⁴ cells/ml, pretreated with fluvastatin (5 μM) in the presence or absence of BIX02189 (2 μM), and then exposed to AGEs (10 μg/ml) for 24 h. For cell cycle analysis, cells were then detached, stained with PI, and subjected to flow cytometry. *, p < 0.05; **, p < 0.01 (n = 5).

Fig 6. The effect of Nrf2 siRNA on the ERK5 activation-mediated inhibition of AGE-induced cell cycle progression.

VSMCs infected with Ad-LacZ or Ad-CA-MEK5α were transfected with siNrf2 for 48 h and then immunoblotted using anti-cyclin D1, anti-CDK4, anti-p27, anti-Nrf2, anti-HA, and anti-tubulin. Results are representative of three independent experiments. Asterisk indicates a nonspecific band. Bar graphs present the densitometric quantification of Western blot bands.

Fig 7. Fluvastatin regulated AGE-induced cell migration through an ERK5-dependent pathway in VSMCs.

(A) VSMC cells were cultured until near confluent in 6 well dishes. Cell migration was assessed using a modified scratch assay. VSMCs were pretreated with 5 μM fluvastatin in the presence or absence of BIX02189 (2 μM), and then exposed to 10 μg/ml AGEs for 24 h. Wound areas were visualized using a phase contrast microscope. Results are representative of three independent experiments. (B) For selective migration assay, transwell system was used. Cells were seeded in the inner chamber and pretreated with BIX02189 (2 μM) in the presence or absence of fluvastatin (5 μM). AGE (10 μg/ml) were added to the lower for 12 h. After fixing, cells were visualized by crystal violet staining. Unmigrated cells were scraped off and then migrated cells were counted under a light microscope. Results are representative of three independent experiments. *, p < 0.05; **, p < 0.01.