Possible Mechanisms of Di(2-ethylhexyl) Phthalate-Induced MMP-2 and MMP-9 Expression in A7r5 Rat Vascular Smooth Muscle Cells

Abstract

Proliferation and migration of vascular smooth muscle cells (VSMC) are important in the development and/or progression of many cardiovascular diseases, including atherosclerosis. Evidence shows that matrix metalloproteinase (MMP)-2 and MMP-9 are related to the pathogenesis of atherosclerosis. The expressions of MMP-2 and MMP-9 in atherosclerosis are regulated via various pathways, such as p38 mitogen activated protein kinase (MAPK), extracellular signal regulated kinase 1 and 2 (ERK1/2), Akt, and nuclear factor kappa (NF-κB). Di(2-ethylhexyl) phthalate (DEHP) has been shown to induce atherosclerosis by increasing tumor necrosis factor (TNF)-α, interleukin (IL)-6, and intercellular adhesion molecule (ICAM) productions. However, whether DEHP poses any effects on MMP-2 or MMP-9 expression in VSMC has not yet been answered. In our studies, rat aorta VSMC was treated with DEHP (between 2 and 17.5 ppm) and p38 MAPK, ERK1/2, Akt, NF-κB, and MMP-2 and MMP-9 proteins and activities were measured. Results showed that the presence of DEHP can induce higher MMP-2 and MMP-9 expression than the controls. Similar results on MMP-regulating proteins, i.e., p38 MAPK, ERK1/2, Akt, and NF-κB, were also observed. In summary, our current results have showed that DEHP can be a potent inducer of atherosclerosis by increasing MMP-2 and MMP-9 expression at least through the regulations of p38 MAPK, ERK1/2, Akt, and NF-κB.

Keywords: Akt; ERK1/2; MMP-2; MMP-9; NF-κB; di(2-ethylhexyl) phthalate (DEHP); p38 MAPK; vascular smooth muscle cells (VSMC).

Figure 1

Effects of di(2-ethylhexyl) phthalate (DEHP) on cell viability in vascular smooth muscle cells (VSMC). VSMC (n ≥ 8) were treated with DEHP (concentrations between 2 and 17.5 ppm) for 24 h prior to cell viability being measured.

Figure 2

(a) Effects of DEHP on MMP-2 and MMP-9 protein expression. VSMC (n ≥ 3) were treated with DEHP (concentrations between 2 and 17.5 ppm) for 24 h prior to MMP-2 and MMP-9 protein extraction and expression. Statistics are shown for DEHP-treated cells * p < 0.05 and *** p < 0.005, compared to the respective controls; (b) Effects of DEHP on MMP-2 activity. VSMC were treated (n ≥ 8) with DEHP (concentrations between 2 and 17.5 ppm) for 24 h prior to MMP-2 activities being measured. Statistics are shown for DEHP-treated cells *** p < 0.005, compared to the control; and (c) Effects of DEHP on MMP-9 activity. VSMC were treated (n ≥ 8) with DEHP (concentrations between 2 and 17.5 ppm) for 24 h prior to MMP-9 activities being measured. Statistics are shown for DEHP-treated cells *** p < 0.005, compared to the control.

Figure 2

(a) Effects of DEHP on MMP-2 and MMP-9 protein expression. VSMC (n ≥ 3) were treated with DEHP (concentrations between 2 and 17.5 ppm) for 24 h prior to MMP-2 and MMP-9 protein extraction and expression. Statistics are shown for DEHP-treated cells * p < 0.05 and *** p < 0.005, compared to the respective controls; (b) Effects of DEHP on MMP-2 activity. VSMC were treated (n ≥ 8) with DEHP (concentrations between 2 and 17.5 ppm) for 24 h prior to MMP-2 activities being measured. Statistics are shown for DEHP-treated cells *** p < 0.005, compared to the control; and (c) Effects of DEHP on MMP-9 activity. VSMC were treated (n ≥ 8) with DEHP (concentrations between 2 and 17.5 ppm) for 24 h prior to MMP-9 activities being measured. Statistics are shown for DEHP-treated cells *** p < 0.005, compared to the control.

Scheme 1

Possible mechanisms of DEHP induced MMP-2 and MMP-9 activities and protein expression. Pathways with question marks are clarified in this study. White arrows indicate an increase in activity or expression of proteins. Lighting signs indicate that proteins are activated (phosphorylated).

Figure 3

Effects of DEHP on phosphorylated p38 MAPK, phosphorylated ERK1/2, phosphorylated Akt, and NF-κB (p65) protein expression. VSMC (n ≥ 3) were treated with DEHP (concentrations between 2 and 17.5 ppm) for 20 min (p38 MAPK, ERK1/2, and Akt) or 12 h (NF-κB) prior to protein extraction. Phosphorylated p38 MAPK, phosphorylated ERK1/2, phosphorylated Akt, and NF-κB (p65) were expressed by Western blotting. Statistics are shown for DEHP-treated cells * p < 0.05, ** p < 0.01, and *** p < 0.005, compared to the respective control groups.

Figure 4

(a) Effects of p38 MAPK, ERK1/2, Akt, and NF-κB inhibitors on DEHP-induced MMP-2 expression. VSMC (n ≥ 3) were pre-treated with SB203580 (10 µM), UO126 (10 µM), SH-5 (10 µM), and Helenalin (10 µM) for 2 h, DEHP (concentrations between 2 and 17.5 ppm) was then added and incubated for further 24 h prior to protein extraction. MMP-2 was expressed by Western blotting; and (b) effects of p38 MAPK, ERK1/2, Akt, and NF-κB inhibitors on DEHP-induced MMP-2 activity. VSMC (n ≥ 3) were pre-treated with SB203580 (10 µM), UO126 (10 µM), SH-5 (10 µM), and Helenalin (10 µM) for 2 h, DEHP (7 or 10.5 ppm) was then added and incubated for further 24 h prior to MMP-2 activity being measured. Statistics are shown for all inhibitors-treated cells * p < 0.05, ** p < 0.01, and *** p < 0.005, compared to the DEHP-treated group.

Figure 5

(a) Effects of p38 MAPK, ERK1/2, Akt, and NF-κB inhibitors on DEHP-induced MMP-9 expression. VSMC (n ≥ 3) were pre-treated with SB203580 (10 µM), UO126 (10 µM), SH-5 (10 µM), and Helenalin (10 µM) for 2 h, DEHP (between 2 and 17.5 ppm) was then added and incubated for further 24 h prior to protein extraction. MMP-9 was expressed by Western blotting; and (b) effects of p38 MAPK, ERK1/2, Akt, and NF-κB inhibitors on DEHP-induced MMP-9 activity. VSMC (n ≥ 3) were pre-treated with SB203580 (10 µM), UO126 (10 µM), SH-5 (10 µM), and Helenalin (10 µM) for 2 h, DEHP (7 or 10.5 ppm) was then added and incubated for further 24 h prior to MMP-9 activities being measured. Statistics are shown for SB203580-, SH-5-, and Helenalin-treated cells *** p < 0.005, compared to the DEHP-treated group; for UO126-treated cells (UO/7); ** p < 0.01, compared to the 7 ppm of DEHP-treated group; and for UO126-treated cells (UO/10.5); * p < 0.01, compared to the 10.5 ppm of DEHP-treated group.

Figure 6

Effects of DEHP on cell migration. VSMC (n ≥ 3) were treated with DEHP (between 2 and 17.5 ppm) after the wound being created. Cell migration was observed under a light microscope (200×) at 24 h and 48 h after initial wound creation.