A Pivotal Role for AP-1-Mediated Osteopontin Expression in the Increased Migration of Vascular Smooth Muscle Cells Stimulated With HMGB1

Abstract

Migration of vascular smooth muscle cells (VSMCs) plays an essential role in the development of vascular remodeling in the injured vasculatures. Previous studies have identified high-mobility group box 1 (HMGB1) as a principal effector mediating vascular remodeling; however, the mechanisms involved have not been fully elucidated. Thus, this study investigated the role of HMGB1 on VSMC migration and the underlying molecular mechanisms involved. VSMCs were ex plant cultured using rat thoracic aorta, and the cellular migration was measured using wound-healing assay. Osteopontin (OPN) mRNA and protein were determined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. The OPN promoter was cloned into pGL3 basic to generate a pLuc-OPN-2284 construct. Migration of VSMCs stimulated with HMGB1 (100ng/ml) was markedly increased, which was significantly attenuated in cells pretreated with MPIIIB10 (100-300ng/ml), a neutralizing monoclonal antibody for OPN as well as in cells deficient of OPN. In VSMCs stimulated with HMGB1, OPN mRNA and protein levels were significantly increased in association with an increased promotor activity of OPN gene. Putative-binding sites for activator protein 1 (AP-1) and CCAAT/enhancer-binding protein beta (C/EBPβ) in the indicated promoter region were suggested by TF Search, and the HMGB1-induced expression of OPN was markedly attenuated in cells transfected with siRNA for AP-1. VSMC stimulated with HMGB1 also showed an increased expression of AP-1. Results of this study suggest a pivotal role for AP-1-induced OPN expression in VSMC migration induced by HMGB1. Thus, the AP-1-OPN signaling axis in VSMC might serve as a potential therapeutic target for vascular remodeling in the injured vasculatures.

Keywords: activator protein 1; cell migration; high-mobility group box 1; osteopontin; vascular smooth muscle cells.

Figure 1

The effects of HMGB1 on VSMC migration. Rat aortic VSMCs were treated with HMGB1 (10 to 100ng/ml) for 48h. Cell migration was determined by wound-healing assay. Representative photomicrographs of cell monolayers at 48h post-wounding. Cell migration area (%) was calculated from the ratio of changes in wound area. Scale bar: 500μM. Data were quantified, expressed as the mean±SEM of six independent experiments. ^*p<0.05 and ^**p<0.01 vs. value in control.

Figure 2

Characteristics of OPN mRNA and protein expression in VSMCs stimulated with HMGB1. (A) VSMCs were treated with HMGB1 (10 to 100ng/ml) for 3h or 100ng/ml of HMGB1 for 0 to 48h, and then, mRNA levels of OPN were determined by RT-PCR. GAPDH was used as a control. Relative intensities were expressed as the mean±SEM of 3–4 independent experiments. ^*p<0.05 and ^**p<0.01 vs. corresponding value in control or 0h. (B) VSMCs were stimulated with HMGB1 (10 to 100ng/ml) for 48h or 100ng/ml of HMGB1 for 0 to 48h, and then, protein levels of OPN were determined by Western blot. β-actin was used as a control. Relative intensities were expressed as the mean±SEM of 4–6 independent experiments. ^*p<0.05 and ^**p<0.01 vs. corresponding value in control or 0h.

Figure 3

The role of OPN on HMGB1-induced VSMC migration. (A) VSMCs were pretreated with MPIIIB10 (30 to 300ng/ml) or IgG (1μg/ml) for 1h and then stimulated with HMGB1 (100ng/ml) for 48h. Cell migration was determined by wound-healing assay. Cell migration area (%) was calculated from the ratio of changes in wound area. Scale bar: 500μm. Data were quantified, expressed as the mean±SEM of five independent experiments. ^**p<0.01 vs. value in control and ^#p<0.05 vs. corresponding value in vehicle. (B) VSMCs were transfected with negative control siRNA or OPN siRNA (200nM) for 48h and then stimulated with HMGB1 (100ng/ml) for 48h. Cell migration was determined by wound-healing assay. Cell migration area (%) was calculated from the ratio of changes in wound area. Scale bar: 500μm. Data were quantified, expressed as the mean±SEM of four independent experiments. ^**p<0.01 vs. value in negative control in vehicle and ^#p<0.05 vs. corresponding value in negative control in HMGB1.

Figure 4

Identification of the transcription factors mediating OPN expression in HMGB1-stimulated VSMCs. (A) VSMCs were transfected with various promoter constructs or an empty luciferase vector (pGL3) for 48h and then stimulated with HMGB1 (100ng/ml) for 3h. Relative luciferase activities were expressed as the mean±SEM of four independent experiments. ^**p<0.01 vs. corresponding value in control. (B) Nucleotide sequence of the –538~–234 promoter region of the OPN gene is shown. The sequence of potential binding sites for AP-1 and C/EBPβ in pLuc-OPN-538 were underlined. The transcription factor-binding sites were identified using TF search software. (C) VSMCs were transfected with AP-1 and C/EBPβ siRNAs (200nM) for 48h and then stimulated with HMGB1 (100ng/ml) for 48h. The expression of OPN was determined by Western blot. β-actin was used as a control. Relative intensities were expressed as the mean±SEM of five independent experiments. ^**p<0.01 vs. corresponding value in negative control in vehicle and ^##p<0.01 vs. value in negative control in HMGB1.

Figure 5

Role of AP-1 in HMGB1-induced VSMC migration. VSMCs were transfected with AP-1 siRNA (200nM) for 48h and then stimulated with HMGB1 (100ng/ml) 48h. Cell migration was determined by wound-healing assay. Representative photomicrographs of cell monolayers at 48h post-wounding. Cell migration area (%) was calculated from the ratio of changes in wound area. Scale bar: 500μm. Data were quantified, expressed as the mean±SEM of five independent experiments. ^**p<0.01 vs. value in negative control in vehicle and ^##p<0.01 vs. corresponding value in negative control in HMGB1.