Curcumin inhibits EMMPRIN and MMP-9 expression through AMPK-MAPK and PKC signaling in PMA induced macrophages

Abstract

In coronary arteries, plaque disruption, the major acute clinical manifestations of atherosclerosis, leads to a subsequent cardiac event, such as acute myocardial infarction (AMI) and unstable angina pectoris (UA). Numerous reports have shown that high expression of MMP-9 (matrix metalloproteinase-9), MMP-13 (matrix metalloproteinase-13) and EMMPRIN (extracellular matrix metalloproteinase induce) in monocyte/macrophage results in the plaque progression and destabilization. Curcumin exerts well-known anti-inflammatory and antioxidant effects and probably has a protective role in the atherosclerosis. The purpose of our study was to investigate the molecular mechanisms by which curcumin affects MMP-9, MMP13 and EMMPRIN in PMA (phorbol 12-myristate 13-acetate) induced macrophages. Human monocytic cells (THP-1 cells) were pretreated with curcumin or compound C for 1 h, and then induced by PMA for 48 h. Total RNA and proteins were collected for real-time PCR and Western blot analysis, respectively. In the present study, the exposure to curcumin resulted in attenuated JNK, p38, and ERK activation and decreased expression of MMP-9, MMP-13 and EMMPRIN in PMA induced macrophages. Moreover, we demonstrated that AMPK (AMP-activated protein kinase) and PKC (Protein Kinase C) was activated by PMA during monocyte/macrophage differentiation. Furthermore, curcumin reversed PMA stimulated PKC activation and suppressed the chronic activation of AMPK, which in turn reduced the expression of MMP-9, MMP-13 and EMMPRIN. Therefore, it is suggested that curcumin by inhibiting AMPK-MAPK (mitogen activated protein kinase) and PKC pathway may led to down-regulated EMMPRIN, MMP-9 and MMP-13 expression in PMA-induced THP-1 cells.

Figure 1

Curcumin suppresses MMP-9, MMP13 and EMMPRIN expression in PMA induced macrophages. A. Effect of curcumin on cell viability. PMA-induced macrophages were treated with indicated concentrations of curcumin (5–100 μM) for 48 h, and cell viability was assessed using the CCK-8 assay. Cells incubated in a medium without curcumin were defined as a control and were considered 100% viable. B-D. Curcumin suppresses EMMPRIN, MMP-9 and MMP13 expression in PMA-induced macrophages. The mRNA level and protein level were measured by Real-time PCR and Western blot, respectively. *P < 0.05 vs PMA group, **P < 0.05 vs CTL group. CTL indicates that cells incubated in a medium without curcumin. 6.25 μM, 25 μM, 50 μM indicates that THP-1 cells were pretreated with indicated concentrations of curcumin before stimulating with 100 nM PMA for another 48 h. Cur: curcumin.

Figure 2

Curcumin suppresses the activation of MMP-9 and chronic AMPK activation. A-B. Effect of curcumin on the activity of MMP-9 from cultured supernatant. THP-1 cells were pretreated with curcumin before stimulating with PMA (100 nM) for another 48 h. MMP-9 activities were detected by gelatin zymography assay (A), and the corresponding densitometric measurement was shown in B. Cur: curcumin. C-D. Effect of curcumin on total AMPK and phospho-AMPKα (Thr172). Differentiated THP-1 cells were treated with indicated agents and assayed by Western blot using indicated antibodies (C), and the corresponding densitometric measurement was shown in D. *P < 0.05 vs PMA group, **P < 0.05 vs CTL group.

Figure 3

Curcumin inhibits JNK, ERK, and p38 phosphorylation and PKC pathway. A. Differentiated THP-1 cells were treated with indicated agents, and assayed by Western blot using indicated antibodies. Cells were pretreated with vehicle or curcumin at the indicated concentration (5–50 μM) for 1 h, followed by PMA for 48 h. B. Protein quantification was carried out by densitometric analysis. Normalized proteins of JNK, p-JNK, ERK, p-ERK, p38 and p-p38 were normalized based on the internal control β-actin. C. Expression of PKC-α and PKC-β1,cells were pretreated with vehicle or curcumin at the indicated concentration (5–50 μM) for 1 h, followed by PMA for 48 h. D. Densitometry measurements of protein analysis. The mean density values of PKC-αand PKC-β1 are expressed as ratios relative to that of β-actin. *P < 0.05 vs PMA group, **P < 0.05 vs CTL group.

Figure 4

PMA induced EMMPRIN, MMP-9 and MMP13 expression by macrophages is blocked by a specific ERK, p38 and/or JNK inhibitors. A-B. The addition of a specific ERK inhibitor (PD98059, 5 and 20 μM), p38 inhibitor (SB203580, 2.5 and 10 μM) completely blocks PMA-induced MMP-9 protein expression and activation, and p38-specific inhibitor showed weaker function. ERK1/2 and p38-specific inhibitor inhibitor significantly decreased EMMPRIN expression, whereas JNK specific inhibitor showed no inhibitory effect. C-D. ERK1/2, p38 and JNK-specific inhibitor at high dose showed remarkable inhibitory effect on MMP-13 expression. *P < 0.05 vs PMA group, #P >0.05 vs PMA group.

Figure 5

AMPK inhibitor mediated EMMPRIN, MMP-9 and MMP13 expression inhibition depends on the activation of MAPK pathway. A-C. Compound C, AMPK inhibitor, significantly inhibits EMMPRIN, MMP-9 and MMP13 expression in PMA induced THP-1 cells. Cells were pretreated with vehicle or Compound C (10 μM) for 1 h, followed by PMA for 48 h. The mRNA level of EMMPRIN, MMP-9 and MMP13 was determined by qPCR (A), and protein level was determined by Western blot (B) and quantified by densitometric analysis (C). Comp C indicates group treated with compound C; P + C indicates group treated with both PMA and compound C. D-E. Compound C inhibited the activation of MAPK pathway. Differentiated THP-1 cells were treated with indicated agents, and assayed by Western blot using indicated antibodies (D) and quantified by densitometric analysis (E). *P < 0.05 vs PMA group, **P < 0.05 vs CTL group.

Figure 6

Model for regulation of MMP-9, MMP-13 and EMMPRIN by curcumin. PMA induced EMMPRIN, MMP-9 and MMP13 expression by macrophages through AMPK and PKC pathway.Curcumin attenuates expression of MMP-9, MMP-13 and EMMPRIN by inhibiting the activation of AMPK and PKC pathway.