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.Monocyte chemoattractant protein-1 (MCP-1; 3 also known as CCL2) is a chemokine secreted by endothelial cells, vascular SMC, fibroblasts, and monocytes/macrophages (1). MCP-1 and its rodent analog, JE (2, 3), are not normally present in the arterial media or intima but have been found in human, primate, and rabbit atherosclerotic plaques (4 -7). In addition, MCP-1 mRNA and protein are induced within hours in the media and neointima in rat (8) and porcine (9) models of balloon arterial injury. Recent studies employing mice lacking MCP-1 or its receptor, CCR2, crossed into an atherosclerotic background (e.g. apoE Ϫ/Ϫ or LDLR Ϫ/Ϫ mice), have established that MCP-1 plays a dominant role in attracting monocyte/macrophages to developing atherosclerotic plaques (10 -12). Rupture of unstable atherosclerotic plaques, with exposure of thrombogenic material, plays a major role in acute coronary thrombotic events associated with unstable angina, myocardial infarction, and sudden death (13). In addition, MCP-1 and/or CCR2 appear to mediate intimal hyperplasia in rodent and primate models of arterial injury (14 -17). Therefore, MCP-1 appears to be an important target for attenuating arterial inflammation.Glucocorticoids are clinically important agents that possess a wide variety of anti-inflammatory and anti-proliferative properties. Glucocorticoid treatment is associated with decreased macrophage accumulation in a ...

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Monocyte chemoattractant protein 1 (MCP-1) plays a pivotal role in many inflammatory processes, including the progression of atherosclerosis and the response of the arterial wall to injury. We previously demonstrated that dexamethasone (Dex) inhibits MCP-1 mRNA accumulation in smooth muscle cells by decreasing its half-life. The effect of Dex was dependent upon the glucocorticoid receptor (GR) and independent of new transcription. Using RNA affinity and column chromatography, we have identified two proteins involved in regulating MCP-1 mRNA stability: Y-box binding protein 1 (YB-1), a multifunctional DNA/RNA-binding protein, and endoribonuclease UK114 (UK). By immunoprecipitation, YB and GR formed a complex present in equal amounts in extracts from untreated and Dex-treated cells. YB-1, UK, and GR small interfering RNA (siRNA) substantially inhibited the effect of Dex on MCP-1 mRNA accumulation. In addition, YB-1 antibody blocked the degradation of MCP-1 mRNA by cytoplasmic extracts from the Dex-treated cells. The degradative activity of extracts immunoprecipitated with antibodies to either YB-1 or GR was blocked with UK antibody. UK did not degrade MCP-1 mRNA; however, upon addition to nondegrading control extracts, it rapidly degraded MCP-1 mRNA. These studies define new roles for GR, YB-1, and UK in the formation of a molecular complex that degrades MCP-1 mRNA.

Successful blastocyst implantation depends on the interaction between cells of maternal endometrium and conceptus, as well as adequate blood supply to the site of blastocyst implantation. Nitric oxide (NO) generally plays a significant role in the local regulation of vascular physiology in a variety of mammalian tissue systems, however, its role in blastocyst implantation and placentation in the primate is not known. The aim of the present study was to examine: (i) NADH-diaphorase activity and expression of three isoforms of nitric oxide synthase (NOS), namely endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS) in pre-implantation stage monkey embryos, morula (n 5 4) and blastocyst (n 5 10), as well as, in different compartments of conceptus and maternal endometrium at primary implantation sites during lacunar (n 5 6) and villous (n 5 9) stages of placentation in the rhesus monkey, and (ii) the potential anti-nidatory effect of vaginal administration of NOS inhibitor during the peri-implantation period of conception cycles in rhesus monkeys. Pre-implantation stage blastocysts exhibited marked NADPH-diaphorase activity along with immunopositive iNOS mainly in the inner cell mass. During the lacunar stage, marked eNOS expression was observed in cytotrophoblast cells lining the embryonic cavity. However, cytotrophoblast cells lining villi, forming columns, and constituting anchoring villi expressed all the three isoforms of NOS in villous placenta stage tissue. During the lacunar stage, eNOS and iNOS protein expressions were observed in epithelial and decidual cells of endometrium. As gestation advanced, mRNAs for all three isoforms of NOS were observed to increase in epithelial and decidual cells, however, with no marked change in protein expression. Vaginal administration of a NOS inhibitor (N G -nitro-L-arginine methyl ester, L-NAME, 4, 6, and 8 mg/kg body weight or aminoguanidine, AG, 4 mg/kg body weight) during days 6 to 12 after ovulation resulted in pregnancy failure in a higher number of animals (L-NAME: 8 confirmed pregnancies in 25 animals; AG: 2 confirmed pregnancies in 8 animals) compared with control animals (5 pregnancies in 7 animals). It appears that NO may play an important role in the establishment of pregnancy in the rhesus monkey.Reproduction (2005) 130 321-332

Monocyte chemoattractant protein-1 (MCP-1) is an inflammatory chemokine that promotes atherosclerosis and is a mediator of the response to arterial injury. We previously demonstrated that platelet-derived growth factor (PDGF) and angiotensin II (Ang) induce the accumulation of MCP-1 mRNA in vascular smooth muscle cells mainly by increasing mRNA stability. In the present study, we have examined the signaling pathways involved in this stabilization of MCP-1 mRNA. The effect of PDGF (BB isoform) and Ang on MCP-1 mRNA stability was mediated by the PDGF β and angiotensin II receptor AT1R, respectively, and did not involve transactivation between the two receptors. The effect of PDGF-BB was blocked by inhibitors of protein kinase C (PKC), but not by inhibitors of phosphoinositol 3-kinase (PI3K), Src, or NADPH oxidase (NADPHox). In contrast, the effect of Ang was blocked by inhibitors of Src, and PKC, but not by inhibitors of PI3 K, or NADPHox. The effect of PDGF BB on MCP-1 mRNA stability was blocked by siRNA directed against PKCδ and protein kinase D (PKD), whereas the effect of Ang was blocked only by siRNA directed against PKCδ. These results suggest that the enhancement of MCP-1 mRNA stability by PDGF-BB and Ang are mediated by distinct "proximal" signaling pathways that converge on activation of PKCδ. This study identifies a novel role for PKCδ in mediating mRNA stability in smooth muscle cells.