Galectin-3 Mediates Thrombin-Induced Vascular Smooth Muscle Cell Migration

Abstract

Vascular smooth muscle cell (VSMC) migration is an important step in the progression and development of vulnerable plaques. Thrombin is involved in both physiological and pathological processes of atherosclerosis. Therefore, the elucidation of the mechanisms underlying thrombin-induced VSMC migration is essential for devising effective treatments aimed at the prevention of plaque instability. In this study, we found that thrombin activated MAPK signaling pathways and increased the expression of galectin-3, which was also a well-known factor in atherosclerosis. Knockdown of galectin-3 by specific small interfering RNA (siRNA) blocked thrombin-induced activation of ERK1/2 and p38 MAPK, but not JNK MAPK. Src/FAK phosphorylation was also shown to be activated by thrombin. FAK autophosphorylation at Y397 was most significantly inhibited by galectin-3 siRNA. Galectin-3 siRNA or specific inhibitor (P38 MAPK inhibitor and ERK1/2 inhibitor) effectively prevented thrombin-induced VSMC migration via reducing paxillin expression. These findings demonstrate, for the first time, that thrombin stimulation of VSMC migration and paxillin expression are regulated by galectin-3, and ERK1/2, p38 MAPK, and Src/FAK signaling pathways are involved in this process. These results are beneficial to clarify the role of galectin-3 in thrombin-induced advanced lesions in atherosclerosis and shed new insights into the regulatory mechanism of VSMC migration in combating plaque rupture.

Keywords: VSMCs; galectin-3; migration; signaling pathway; thrombin.

Figure 1

Thrombin increased gal-3 expression in VSMCs and cell migration. Cells were treated with thrombin over a range of concentrations (0, 0.25, 0.5, 1, and 2 U/ml) for different times (0, 0.5, 1, 6, 12, 24, and 48 h) and gal-3 expression was measured by Western blot (A,D) or qRT-PCR (C,F). Quantification of Western blot results is shown (B,E). Band density of native VSMCs was defined as a control and considered to 1. *p < 0.05 compared with control.

Figure 2

Gal-3 mediated thrombin-induced VSMC migration. VSMCs were transfected with gal-3 siRNA for 24 h. The mRNA and protein expression levels of gal-3 were measured by qRT-PCR and Western blotting separately. Western blot and qRT-PCR results of gal-3 are shown (A,C,D). Band density of VSMCs transfected with scramble siRNA was chosen as a reference and set to 1. *p < 0.05 vs. the control. After transfection with either control or gal-3 siRNA for 24 h, VSMCs were added in the upper chamber. After treatment with thrombin (2 U/ml) for 24 h in the lower chamber, migrated cells from the upper chamber to the lower surface were counted in five non-overlapping fields under a microscope (×100) (B,E). These blue stains are the nucleus of VSMCs that were stained with DAPI. Control siRNA transfected VSMCs were chosen as a reference and set to 1. *p < 0.05 vs. control; ^#p < 0.05 vs. thrombin.

Figure 3

ERK/P38 signaling pathway mediates thrombin effects in VSMCs. Time dependence of thrombin-mediated MAPK signaling pathway activation in VSMCs. Cells were treated with 2 U/ml thrombin over a range of times (0–60 min), and the expression of JNK, p-JNK, ERK, p-ERK, p38, and p-p38 was measured by Western blot (A,B). After transfection with either control or gal-3 siRNA for 24 h, VSMCs were incubated for 30 min in the absence or presence of thrombin (2 U/ml), and the expression of JNK, p-JNK, ERK, p-ERK, p38, and p-p38 was measured by Western blot again. Western blot results are shown (D,E). Quantification of the results is given in the right panel. Band density of native VSMCs was defined as a control and considered to 1. *p < 0.05 compared with control. After incubated with either SB203580 or PD98059, VSMCs were added in the upper chamber. After treatment with thrombin (2 U/ml) for 24 h in the lower chamber, migrated cells from the upper chamber to the lower surface were counted in five non-overlapping fields under a microscope (×100) (C,F). Native VSMCs were chosen as a reference and set to 1. *p < 0.05 vs. control; ^#p < 0.05 vs. thrombin.

Figure 4

Thrombin promoted paxillin phosphorylation via ERK/P38 signaling pathway. VSMCs were treated with thrombin (2 U/ml) for different times (0, 10, 20, 30, and 60 min) and the expression of p-paxillin was measured by Western blot (A,B). Cells were treated with gal-3 siRNA, PD98059, or SB203580, and then incubated in the absence or presence of thrombin (2 U/ml). The expression of p-paxillin was measured by Western blot (C,D). Quantification of the results is shown in the right panel. Band density of native VSMCs was defined as a control and considered to 1. *p < 0.05 vs. control; ^#p < 0.05 vs. thrombin.

Figure 5

Thrombin activated distinct FAK signalings via gal-3. VSMCs were treated with thrombin over a range of concentrations (2 U/ml) for different times (0, 10, 30, and 60 min), and the expression of p-src and src, the phosphorylation of FAK at Tyr-397,−576/577, and−925, and FAK were measured by Western blot (A,C). After transfection with either control or gal-3 siRNA for 24 h, VSMCs were incubated for 30 min in the absence or presence of thrombin (2 U/ml), and the expression of p-src and src, the phosphorylation of FAK at Tyr-397,−576/577, and−925, and FAK were measured by Western blot (B,D). Quantification of the results is shown in the right panel. Band density of native VSMCs was defined as a control and considered to 1. *p < 0.05 vs. control; ^#p < 0.05 vs. thrombin.

Figure 6

Galectin-3 mediated thrombin-induced VSMC migration. Thrombin induced activation of p38, ERK, Src, and FAK signaling pathways via gal-3, leading to paxillin phosphorylation and cell migration in VSMCs.