Abstract
The proinflammatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is expressed in inflammatory and atherosclerotic lesions. GM-CSF is known to enhance monocytic expression of monocyte chemoattractant protein-1 (MCP-1). However, the molecular mechanism(s) by which GM-CSF up-regulates the MCP-1 expression remains to be clarified. Thus, in this study, we examined our hypothesis that GM-CSF up-regulates the MCP-1 expression via Jak2-Stat5 signaling pathway. In human monocytic cell line U937, GM-CSF increased MCP-1 expression in protein and mRNA levels. Furthermore, analysis of the GM-CSF promoter element revealed that the STAT5 (signal transducer and activator of transcription-5) transcription factor binding site, located between -152 and -144 upstream of the transcription start site, as well as Janus kinase-2-mediated Stat5 activation were necessary for the GM-CSF-induced transcriptional up-regulation of the MCP-1 gene. This GM-CSF-induced MCP-1 expression, measured as both protein and mRNA levels, was down-regulated by atorvastatin, a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor. However, this decrease in MCP-1 expression was not at the transcriptional level of MCP-1 gene but rather at the level of the stability of MCP-1 mRNA. These results indicate that GM-CSF regulates MCP-1 expression via Janus kinase-2-Stat5 pathway and by a novel regulatory mechanism of statins to reduce inflammatory reactions by down-regulating the expression of monocytic MCP-1, which promotes atherogenesis.
Highlights
Playing a central role in atherogenesis; their multiple functions include migration and production of growth factors, cytokines, and matrix-degrading enzymes as well as uptake of modified lipoproteins [4]
Together with the results obtained from the luciferase assay using mutations of the STAT5 motif, these results suggest that the STAT5 located at Ϫ152/Ϫ144 (STAT5p) is a potential granulocyte-macrophage colony-stimulating factor (GM-CSF)-responsive motif for Jak2- and Stat5a-mediated monocyte chemoattractant protein-1 (MCP-1) gene transcription in the U937 cells
We demonstrated that GM-CSF has multiple effects, enhancing monocytic migration via RhoA/integrin activation and via expression of matrix metalloproteinase (MMP) and MCP-1 [7, 9]
Summary
Chemicals—Mevalonate (Mev) and actinomycin D (AcD) were purchased from Sigma. Geranylgeranyltransferase inhibitor (GGTI-286), farnesyltransferase inhibitor (FTI-276), and Jak inhibitor (AG490) were from Merck. g) harboring cDNA of the MCP-1, -actin, and the plasmid cells were incubated with GM-CSF (10 ng/ml) for 8 h and (pGEM-Easy-T vector) without insert were linearized, denatured, incubated with AcD (10 g/ml) in the absence or presand spotted onto nylon membrane using a dot blot apparatus. The lysates from the U937 cells treated with structs used were the human MCP-1 promoter region up to GM-CSF in the presence or absence of Jak inhibitor AG490 Ϫ932 bp, including AP-1, NF-B, and STAT5 motifs, and its were applied to Western blotting. Electrophoretic Mobility Shift Assay (EMSA)—U937 cells transcriptional activity from the Ϫ932 bp construct in a dosewere incubated with GM-CSF (20 ng/ml, 15 min), and nuclear dependent manner (1–20 ng/ml for 8 h) (Fig. 1C). Nuclear extract (5 g) transfection, the GM-CSF response was decreased, but the and labeled probe (20 fmol) were incubated for 30 min at room basal activity was still preserved (Fig. 2A). STAT5d STAT5p AP-1d kB AP-1p TATA -306/-298 -152/-144 -97/-91 -89/-80 -69/-63 -37/-33
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