Abstract
Monocyte/macrophage infiltration to the subendothelial space of arterial wall is a critical initial step in atherogenesis, in which CC chemokine ligand 2 (CCL2)/monocyte chemoattractant protein-1 (MCP-1) is thought to play a key role. This study investigated the effectiveness of phosphodiesterase inhibitors, including the nonselective pentoxifylline (PTX) and the selective type III (cilostamide) and type IV (denbufylline) inhibitors, on cytokine-induced CCL2/MCP-1 production in cultured rat vascular smooth muscle cells (VSMCs), and the signal transduction mechanisms whereby they act. Our results showed that tumor necrosis factor (TNF)-α induced a marked increase in CCL2/MCP-1 production in dose- and time-dependent manners. 2-(2-Amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059), 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio) butadiene (U0126) [both inhibitors of p42/44 mitogen-activated protein kinase (MAPK) kinase], and anthra[1hyphen]9-cd]pyrazol-6(2H)-one (SP600125) [an inhibitor of c-Jun NH2-terminal kinases (JNKs)] attenuated TNF-α-induced CCL2/MCP-1 production, without affecting I-κBα degradation or p65/nuclear factor-κB (NF-κB) nuclear translocation. PD98059 abolished TNF-α-activated p42/44 MAPK phosphorylation and c-Fos up-regulation, whereas SP600125 inhibited TNF-α-activated JNK and c-Jun phosphorylation. The NF-κB inhibitor carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (MG132) attenuated TNF-α-induced CCL2/MCP-1 production in the presence of increased phospho-JNK and phospho-c-Jun levels. When SP600125 was added simultaneously, MG132 completely inhibited TNF-α-induced CCL2/MCP-1 production. Finally, the pretreatment of VSMCs with PTX or cilostamide, but not denbufylline, reduced TNF-α-induced CCL2/MCP-1 production, which was preceded by attenuation of p65/NF-κB nuclear translocation, p42/44 MAPK, and JNK-c-Jun phosphorylation, and c-Fos up-regulation. These data indicate that TNF-α-stimulated CCL2/MCP-1 production in rat VSMCs is dually regulated by activator protein-1 (AP-1) and NF-κB pathways, and inhibition of type III phosphodiesterase contributes substantially to the suppressive effect of PTX on CCL2/MCP-1 production via down-regulation of AP-1 and NF-κB signals.
Footnotes
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This work was supported by grants from the National Taiwan University Hospital 93S027 (to Y.-M.C.), the Ta-Tung Kidney Foundation, and the Mrs. Hsiu-Chin Lee Kidney Research Fund, Taipei, Taiwan.
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DOI: 10.1124/jpet.103.062620.
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ABBREVIATIONS: CCL2, CC chemokine ligand 2; MCP-1, monocyte chemoattractant protein-1; CCR2, chemokine receptor 2; VSMC, vascular smooth muscle cell; TNF, tumor necrosis factor; NF-κB, nuclear factor-κB; AP-1, activator protein-1; I-κB, inhibitory protein of NF-κB; MAPK, mitogen-activated protein kinase; JNK, c-Jun NH2-terminal kinase; PTX, pentoxifylline; PDE, phosphodiesterase; DMEM, Dulbecco's modified Eagle's media; FCS, fetal calf serum; PD98059, 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio) butadiene; SB203580, 4-[5-(4-fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyridine; MG132, carbobenzoxy-l-leucyl-l-leucyl-l-leucinal; SP600125, anthra[1-9-cd]pyrazol-6(2H)-one; PBS, phosphate-buffered saline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
- Received November 6, 2003.
- Accepted February 19, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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