Y-box binding protein 1 and RNase UK114 mediate monocyte chemoattractant protein 1 mRNA stability in vascular smooth muscle cells.

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.

Protein kinase Cδ mediates MCP-1 mRNA stabilization in vascular smooth muscle cells.

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 ß 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.

A novel role for the glucocorticoid receptor in the regulation of monocyte chemoattractant protein-1 mRNA stability.

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.

Nitric oxide in blastocyst implantation in the rhesus monkey.

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 = 4) and blastocyst (n = 10), as well as, in different compartments of conceptus and maternal endometrium at primary implantation sites during lacunar (n = 6) and villous (n = 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.

A multiparametric study of the action of mifepristone used in emergency contraception using the Rhesus monkey as a primate model.

Mifepristone is a potent agent used in emergency contraception (EC). In the present study, we examined the contraceptive efficacy of mifepristone used in EC and then, using the model of mifepristone-based EC, we investigated its mechanism of action in the rhesus monkey. Sexually mature females were allowed to cohabitate with male animals from 1600 to 900 h of any one day of days 8-17 of cycle without (Group I; n = 6) and with a single dose of mifepristone (Group II, n = 31, 25 mg per animal, subcutaneous) 72 h postcoitus. Blood samples from all animals of Groups I and II were used to determine the concentrations of estradiol (E), progesterone (P) and chorionic gonadotrophin in peripheral circulation for retrospective analysis of the days of ovulation and blastocyst implantation. Four out of six animals (66.6%) in Group I became pregnant, while all 31 monkeys in Group II failed to establish pregnancy along with marginal changes in serum concentrations of E and P. In the second part of the study, animals were subjected to the same experimental protocol followed by collection of endometrial tissue samples on cycle day 22 from animals of both Group I (n = 6) and Group II (n = 24). Endometrial samples were subjected to morphological analysis including mitotic index, immunohistochemistry for vascular endothelial growth factor (VEGF), leukemia inhibitory factor (LIF), transforming growth factor beta1, estradiol receptor (ER), progesterone receptor (PR), proliferating cell nuclear antigen, placental protein 14 (PP 14) and detection of apoptosis by terminal nick end labeling method followed by histometric analysis. The results were retrospectively analyzed between the two groups on the basis of the day of treatment after ovulation: early luteal phase (days 0-3 postovulation) and mid-luteal phase (days 4-7 after ovulation). Mifepristone used in EC in the present study resulted in general loss of functional integrity of epithelial compartment characterized by loss of secretory maturation, increased apoptosis and higher degree of degeneration along with decreased expression of VEGF, LIF, PP14 and ER, while PR level increased as compared to control samples. The vascular compartment appeared to be compromised along with affected morphological features and decreased expression of VEGF, LIF, ER and PR following the administration of mifepristone. It appears that mifepristone used in EC alters the physiological homeostasis in epithelial and vascular compartments of implantation stage endometrium rendering it hostile to blastocyst implantation. Furthermore, the degree to which the endometrial function is affected largely depends on the day of mifepristone treatment in a parameter-specific manner resulting in a higher degree of degenerative changes in samples obtained from animals who received mifepristone during mid-luteal phase of cycles.

Immunohistochemical localization of leukemia inhibitory factor, interleukins 1 and 6 at the primary implantation site in the rhesus monkey.

Blastocyst implantation and placentation involve localized inflammatory type of responses at and around the site of nidation. In the present study, the likely involvement of inflammatory cytokines, namely, leukemia inhibitory factor (LIF), interleukins 1 alpha and 1 beta (IL-1alpha and IL-1beta) and IL-6 at the primary implantation site of the rhesus monkey was examined immunocytochemically during lacunar (n=6) and villous (n=8) stages of gestation. Trophoblast cells and extraembryonic mesenchymal cells were immunopositive for LIF and IL-1alpha. The distribution of IL-1beta and IL-6 in trophoblast cells was low in lacunar stage samples, however, a higher degree of immunopositivity for IL-6 was observed in villous stage samples. Decidual cells were immunopositive for all the cytokines studied. In lacunar stage samples, plaque cells adjacent to implanted nidus were immunopositive for all the cytokines examined, and the degree of their immunoprecipitation increased, except that of IL-1beta, during the villous stage. Luminal and glandular epithelial cells were immunopositive for LIF, IL-1alpha, IL-1beta and IL-6 in lacunar and in villous stage samples. LIF immunopositivity was detected in endothelial cells of blood vessels within and below chorionic plate and cytotrophoblast shell, while vascular smooth muscle cells were positive for all the cytokines studied. The temporo-spatial characteristics of LIF, IL-1alpha, IL-1beta and IL-6 protein expressions in primary implantation sites of the rhesus monkey suggest that these pro-inflammatory cytokines play specific roles in regulating trophoblast cell proliferation, differentiation, invasion and associated maternal tissue remodelling during early gestation.

Effect of vaginally administered fumagillin on immunohistochemical distribution of leukemia inhibitory factor, interleukin 6, transforming growth factor beta and vascular endothelial growth factor in implantation stage endometrium of the rhesus monkey.

Intravaginal administration of an anti-angiogenic agent, fumagillin, during blastocyst implantation inhibits pregnancy establishment in a dose-related manner in the rhesus monkey. In the present study, mated female rhesus monkeys were vaginally inserted with tampons containing vehicle (group 1; n = 5) and test agent (fumagillin, 4 mg/animal; group 2; n = 6) on cycle day 20, and endometrial tissue samples were collected on cycle day 24 from all monkeys and processed for histological examination and immunohistochemical localization for LIF, IL-6, TGF-beta and VEGF. Concentrations of estradiol-17 beta, progesterone and chorionic gonadotrophin in peripheral circulation were determined. From the serum profiles of the hormones, 2 monkeys in group 1, and 1 monkey in group 2 appeared pregnant. However, endometrial morphology revealed histological evidence of pregnancy in 3 out of 6 fumagillin-treated animals. Histometric analysis of immunohistochemical staining in epithelial, stromal and vascular compartments revealed that per cent areas occupied by immunoprecipitate for the cytokines studies did not change in epithelial and stromal compartments, except that for TGF-beta which was higher (P < 0.05) in epithelial compartment in group 2. No change was observed in immunoprecipitation areas for IL-6 in epithelial, stromal and vascular compartments. On the other hand, changes (P < 0.05) for LIF, TGF-beta and VEGF were evident in the vascular compartment. It is possible that disparate responses observed in glandular, stromal and vascular compartments in implantation stage endometrium following fumagillin treatment actually caused from associated decline in progesterone concentration in peripheral circulation. It is also possible that fumagillin, an angiostatic agent, affects the synthesis and secretion of cytokines primarily in the vascular compartment of implantation stage endometrium, and thereby manifests differential responses in epithelial, stromal and vascular compartments.