Immune Netw.  2009 Jun;9(3):90-97. 10.4110/in.2009.9.3.90.

The Stimulation of CD147 Induces MMP-9 Expression through ERK and NF-kappaB in Macrophages: Implication for Atherosclerosis

• Kim JY
• Kim WJ
• Kim H
• Suk K
• Lee WH

• KMID: 2150647
• DOI: http://doi.org/10.4110/in.2009.9.3.90

Abstract

BACKGROUND: CD147, as a cellular receptor for cyclophilin A (CypA), is a multifunctional protein involved in tumor invasion, inflammation, tissue remodeling, neural function, and reproduction. Recent observations showing the expression of CD147 in leukocytes indicate that this molecule may have roles in inflammation. METHODS: In order to investigate the role of CD147 and its ligand in the pathogenesis of atherosclerosis, human atherosclerotic plaques were analyzed for the expression pattern of CD147 and CypA. The cellular responses and signaling molecules activated by the stimulation of CD147 were then investigated in the human macrophage cell line, THP-1, which expresses high basal level of CD147 on the cell surface. RESULTS: Staining of both CD147 and CypA was detected in endothelial cell layers facing the lumen and macrophage-rich areas. Stimulation of CD147 with its specific monoclonal antibody induced the expression of matrix metalloproteinase (MMP)-9 in THP-1 cells and it was suppressed by inhibitors of both ERK and NF-kappaB. Accordingly, the stimulation of CD147 was observed to induce phosphorylation of ERK, phosphorylation-associated degradation of IkappaB, and nuclear translocation of NF-kappaB p65 and p50 subunits. CONCLUSION: These results suggest that CD147 mediates the inflammatory activation of macrophages that leads to the induction of MMP-9 expression, which could play a role in the pathogenesis of inflammatory diseases such as atherosclerosis.

Keyword

macrophage; atherosclerosis; inflammation; CD147; cyclophilin A

MeSH Terms

Atherosclerosis
Cell Line
Cyclophilin A
Endothelial Cells
Humans
Inflammation
Leukocytes
Macrophages
NF-kappa B
Phosphorylation
Plaque, Atherosclerotic
Reproduction
Cyclophilin A
NF-kappa B

Figure

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  Figure 1 Endothelial cells and macrophages express CD147 and CypA in atherosclerotic plaques. Human carotid atherosclerotic plaques were sequentially sectioned and stained against CD68 (a macrophage marker), vWF (an endothelial cell marker), CD147, CypA. Mouse IgG (mIgG) was used for the staining as a negative control. Upper panel shows the plaques area facing the vessel lumen (L). Lower panel shows the shoulder area of a plaque which have macrophages and smooth muscle cells (SM). Note the low level staining of CypA in areas rich in SMCs.

• 

  Figure 2 Human monocyte/macrophage cell lines express high levels of CD147. (A) THP-1 and U937 cells were stained with anti-CD147 mAb (empty area) or isotype-matching mouse IgG (filled area). (B) THP-1 cells were stimulated with or without indicated amounts of CypA for 20 hr. Total cellular RNAs were collected and the expression levels of CD147 or GAPDH mRNA were measured using RT-PCR analysis.

• 

  Figure 3 The stimulation of CD147 induced the expression of MMP-9 in THP-1 cells. (A) THP-1 cells were stimulated with anti-CD147 mAb or isotype-matching mouse IgG (mIgG) that had been immobilized at indicated concentrations. LPS was used as a positive control. Some of the cells were also stimulated with antibodies that had been heat inactivated at 95℃ for 2 hr. Culture supernatants were collected 24 hr after activation and subjected to gelatin zymogram. (B) THP-1 cells were stimulated as in (A) and culture supernatants were concentrated (×10) and subjected to Western blot analysis using MMP-9 specific antibody. As loading control, the picture of the membrane used for Western blot analysis that had been stained with Coomassie Brilliant Blue is shown at the lower panel.

• 

  Figure 4 The stimulation of CD147 induces nuclear translocation of NF-κB p65 and p50 subunits. (A) THP-1 cells were stimulated with 1µg/ml of LPS or anti-CD147 mAb that had been immobilized at a concentration of 10µg/ml. Nuclear extracts were collected after indicated times and the Western blot analysis was performed using p65- or TFIIB-specific antibodies. TFIIB was used as a loading control for nuclear extracts. (B, C) THP-1 cells were stimulated with LPS, anti-CD147 mAb, or isotype-matching mouse IgG (mIgG) for 2 hr and analyzed with immunofluorescence analysis using an antibody specific for NF-κB p50 subunit. (B) shows representative pictures of cells in each samples and (C) shows the percentage of cells that had NF-κB p50 subunit at their nucleus.

• 

  Figure 5 CD147-induced expression of MMP-9 requires activation of NF-κB. (A) THP-1 cells were stimulated with 1µg/ml of LPS or immobilized anti-CD147 mAb (10µg/ml). Cell lysates were collected at indicated times and subjected to Western blot analysis using antibodies specific for phospho-IκB, IκB, and actin. (B) THP-1 cells were stimulated with indicated amounts of LPS or immobilized anti-CD147 mAb in the presence of indicated amounts of NF-κB inhibitors (sulfasalazine, TPCK, or ethyl pyruvate). Culture supernatants were collected 24 hr after activation and subjected to gelatin zymogram.

• 

  Figure 6 The activation of ERK is involved in the CD147-induced expression of MMP-9 and suppression of p38 and JNK activity augments the MMP-9 expression. (A) THP-1 cells were stimulated with 1µg/ml LPS or immobilized anti-CD147 mAb (10µg/ml) in the presence or absence or indicated amounts of PD98059 (PD), U0126 (U), SB203580 (SB), JNK inhibitor (JNK-I), or 0.2% DMSO as a vehicle control (VC). Culture supernatants were collected 24 hr after activation and subjected to gelatin zymogram. Numbers below each lane represent the band intensity which was normalized with band intensity of sample treated with only anti-CD147 in each gel. (B) THP-1 cells were stimulated with immobilized anti-CD147 mAb (10µg/ml). Cell lysates were collected at indicated times and the levels of phospho-ERK or ERK were analyzed using Western blot analysis. (C) THP-1 cells were stimulated with 0.1µM of CypA in the presence or absence of indicated amounts of PD98059 (PD), U0126 (U), SB203580 (SB), JNK inhibitor (JNK-I), negative control for JNK-I [J(-)], or 0.2% DMSO as a vehicle control (VC). Culture supernatants were collected 24 hr after activation and subjected to gelatin zymogram. (D) The bar graph shows the MMP-9 band intensities of each lane in panel (C) that was normalized with band intensity of sample treated with only CypA in each gel.

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