Abstract
Monocyte chemoattractant protein-1 (MCP-1) plays an important role in attracting monocytes to sites of inflammation and is the dominant mediator of macrophage accumulation in atherosclerotic plaques. We have previously shown that glucocorticoids inhibit the secretion of MCP-1 in arterial smooth muscle cells (SMC) by markedly decreasing MCP-1 mRNA stability. We now report that the destabilization of MCP-1 mRNA is mediated by the glucocorticoid receptor (GR). The GR antagonist, RU486, blocked the effect of the glucocorticoid dexamethasone (Dex) on MCP-1 mRNA stability in SMC culture. Using a previously reported in vitro mRNA gel shift and stability assay, antibodies to the GR blocked the ability of cytoplasmic extracts from Dex-treated SMC to decay MCP-1 mRNA. Recombinant human GR (rhGR) bound in a concentration-dependent manner to in vitro transcribed MCP-1 mRNA, whereas other members of the steroid hormone receptor family did not. Binding of GR to MCP-1 mRNA was specific as it was not found to bind other mRNAs. Immunoprecipitation of GR in extracts from Dex-treated SMC followed by real-time reverse transcription-PCR demonstrated that endogenous GR was bound specifically to MCP-1 mRNA. The addition of exogenous rhGR blocked the ability of extracts from Dex-treated SMC to degrade MCP-1 mRNA, suggesting that exogenous rhGR can compete with an endogenous GR-containing degradative complex. These data suggest a novel role for the GR in binding to and facilitating mRNA degradation. These results provide novel insights into GR function and may provide a new approach to attenuate the inflammatory response mediated by MCP-1.
Highlights
smooth muscle cells (SMC), fibroblasts, and monocytes/macrophages [1]
To elucidate the role of the glucocorticoid receptor (GR) in this process, SMC were treated for 3 h with 20 ng/ml PDGF alone or in the presence of 1 M Dex, and Monocyte chemoattractant protein-1 (MCP-1) mRNA abundance was analyzed by blot hybridization
We observed at very early incubation times (e.g. 5 min) that the gel shift with Dex extract was higher than the gel shift seen with control extracts, suggesting that additional proteins bind to the MCP-1 mRNA in the Dex extract, perhaps facilitating its degradation (Fig. 1C)
Summary
Reagents—rhGR (product number G1542), recombinant human estrogen receptor (rhER; E1528), and RU486 (M8046) were purchased from Sigma-Aldrich. Linearized cDNAs were transcribed in vitro with T3 RNA polymerase (Roche Applied Science) in the presence of [␣-32P]UTP (800 Ci/mmol, PerkinElmer Life Sciences) as described previously [26]. In vitro RNA gel shifts and decay assays were performed and analyzed on polyacrylamide gels as described previously [26]. Beads (100 l) were incubated for 16 h at 4 °C with 6 mg of antibody, washed five times with NT2 buffer, and resuspended in 850 l of NT2 buffer containing 200 units of Rnase OUT (Roche Applied Science), 1 mM vanadyl ribonucleoside complexes, 1 mM dithiothreitol, and 15 mM EDTA. RNA Blot Analysis—Isolation of total RNA and RNA blot hybridization with the full-length rat JE cDNA were performed as described previously [24]. Equal loading of total RNA was verified by ethidium bromide staining of the 18 S and 28 S ribosomal RNA
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