Functional Characterization of the HuR:CD83 mRNA Interaction

Maturation of dendritic cells (DC) is characterized by expression of CD83, a surface protein that appears to be necessary for the effective activation of naïve T-cells and T-helper cells by DC. Lately it was shown that CD83 expression is regulated on the posttranscriptional level by interaction of the shuttle protein HuR with a novel posttranscriptional regulatory RNA element (PRE), which is located in the coding region of the CD83 transcript. Interestingly, this interaction commits the CD83 mRNA to efficient nuclear export via the CRM1 pathway. To date, however, the structural basis of this interaction, which potentially involves three distinct RNA recognition motifs (RRM1–3) in HuR and a complex three-pronged RNA stem-loop element in CD83 mRNA, has not been investigated in detail. In the present work we analyzed this interaction in vitro and in vivo using various HuR- and CD83 mRNA mutants. We are able to demonstrate that both, RRM1 and RRM2 are crucial for binding, whereas RRM3 as well as the HuR hinge region contributed only marginally to this protein∶RNA interaction. Furthermore, mutation of uridine rich patches within the PRE did not disturb HuR:CD83 mRNA complex formation while, in contrast, the deletion of specific PRE subfragments from the CD83 mRNA prevented HuR binding in vitro and in vivo. Interestingly, the observed inhibition of HuR binding to CD83 mRNA does not lead to a nuclear trapping of the transcript but rather redirected this transcript from the CRM1- towards the NXF1/TAP-specific nuclear export pathway. Thus, the presence of a functional PRE permits nucleocytoplasmic trafficking of the CD83 transcript via the CRM1 pathway

Anti-tumor necrosis factor-alpha therapies attenuate adaptive arteriogenesis in the rabbit

The specific antagonists of tumor necrosis factor-alpha (TNF-alpha), infliximab and etanercept, are established therapeutic agents for inflammatory diseases such as rheumatoid arthritis and Crohn's disease. Although the importance of TNF-alpha in chronic inflammatory diseases is well established, little is known about its implications in the cardiovascular system. Because proliferation of arteriolar connections toward functional collateral arteries (arteriogenesis) is an inflammatory-like process, we tested in vivo the hypothesis that infliximab and etanercept have antiarteriogenic actions. Sixty-three New Zealand White rabbits underwent femoral artery occlusion and received infliximab, etanercept, or vehicle according to clinical dosage regimes. After 1 wk, collateral conductance, assessed with fluorescent microspheres, revealed significant inhibition of arteriogenesis (collateral conductance): 52.4 (SD 8.1), 35.2 (SD 7.7), and 33.3 (SD 10.1) ml.min(-1).100 mmHg(-1) with PBS, infliximab, and etanercept, respectively (P <0.001). High-resolution angiography showed no significant differences in number of collateral arteries, but immunohistochemical analysis demonstrated a decrease in mean collateral diameter, proliferation of vascular smooth muscle cells, and reduction of leukocyte accumulation around collateral arteries in treated groups. Infliximab and etanercept bound to infiltrating leukocytes, which are important mediators of arteriogenesis. Infliximab induced monocyte apoptosis, and neither substance affected monocyte expression of the adhesion molecule Mac-1. We demonstrated that TNF-alpha serves as a pivotal modulator of arteriogenesis, which is attenuated by treatment with TNF-alpha inhibitors. Reduction of collateral conductance is most likely due to inhibition of perivascular leukocyte infiltration and subsequent lower vascular smooth muscle cell proliferation. This is the first report showing a negative influence of TNF-alpha inhibitors on collateral artery growt

CRM1-mediated nuclear export of CD83 mRNA PRE.

<p><b>A.</b> COS7 cells were transiently transfected either with an expression vector encoding for human CD83 cDNA flanked by the homologous 5′- and 3′-UTR (wt; lane 1–6) or with a related vector in which essential PRE sequences were deleted (PREΔSubL1–3; lane 7–12). At day two posttransfection cultures were exposed to 10 nM of the CRM1 inhibitor LMB or DMSO (solvent control) for 8 hours. Total, cytoplasmic and nuclear RNA was isolated, subjected to CD83- and GAPDH-specific (negative control) PCR and analyzed by gel electrophoresis. <b>B.</b> Quantitative real-time PCR of the RNA probes shown in panel A. RNA ratios +LMB/−LMB are depicted. <b>C.</b> Total, cytoplasmic and nuclear RNA was isolated from cell cultures which were cotransfected with the CD83 PREΔSubL1–3 expression vector and either a construct expressing four tandem repeats of the MPMV CTE (4×CTE) or the respective parental vector (negative control). For NXF1/TAP-independent control, mitochondrial cytochrome C oxidase mRNA was detected in total and cytoplasmic RNA, while GAPDH-specific transcripts were detected in nuclear RNA. <b>D.</b> Quantitative real-time PCR of the RNA probes shown in panel C. RNA ratios +/− NXF1/TAP inactivation (+CTE/−CTE) are depicted.</p

RRM1 and RRM2 of HuR are necessary for efficient CD83 mRNA recognition.

<p><b>A.</b> Schematic diagram of the domain structure of HuR. The positions of the three RNA recognition motifs (RRM1–3) and the hinge region (H) in the human 326 amino acid (aa) HuR protein are indicated. Recombinant proteins (indicated by probe #) which were subsequently analyzed for CD83 PRE binding are depicted. Probe numbers correspond to gel lanes in panel B and C. <b>B.</b> Coomassie-stained SDS-PAGE of the recombinantly expressed and purified proteins (indicated by arrowheads). M, marker proteins. <b>C.</b> Radiolabelled CD83wt PRE coding sequence RNA was incubated either with GST (negative control) or with various HuR-derived GST-fusion proteins. Lane 1: GST; lane 2: full-length HuR; lane 3: HuR aa 1–244 (RRM1-RRM2-H); lane 4: HuR aa 27–93 (RRM1); lane 5: HuR aa 108–174 (RRM2); lane 6: HuR aa 246–317 (RRM3); lane 7: HuR aa 103–244 (RRM2-H); lane 8: HuR aa 190–328 (H-RRM3); HuR aa 175–245 (H). CD83 PRE RNA:protein interaction was analyzed by gel retardation assay as before.</p

HuR binds in a specific manner to the CD83 PRE RNA region.

<p><b>A.</b> Increasing amounts of bacterial expressed GST-HuR protein was incubated either with the radiolabelled CD83 mRNA wildtype (wt) coding sequence (CD83wt; lane 1–7) or the respective probe lacking the HuR target sequence PRE (CD83ΔPRE; lane 8–14). Complex formation was visualized by gel retardation assay and autoradiography. Lane 1: CD83wt RNA alone; lane 2: GST negative control; lane 3–7 increasing amounts of GST-HuR (0.095–0.475 µM); lane 8: CD83ΔPRE alone; lane 9: GST negative control; lane 10–14: increasing amounts of GST-HuR (0.095–0.475 µM). <b>B.</b> Analysis of PRE binding specificity by competition experiments. GST-HuR protein (lane 2–4 and lane 11–13: 0.119 µM, 0.238 µM, 0.475 µM, respectively; lane 5–9 and 14–18: 0.475 µM, respectively) or GST (1 µM) for negative control (lane 1 and 10) was incubated together with radiolabelled CD83wt PRE mRNA and analyzed as before. Increasing amounts (1–5 fold excess over CD83wt PRE mRNA) of either unlabelled HIV-1 RRE RNA (lane 5–9) or unlabelled TNFα ARE RNA (lane 14–18) were added to individual binding reactions.</p

Leukocyte subpopulations and arteriogenesis: Specific role of monocytes, lymphocytes and granulocytes

Objective: Circulating leukocytes play a crucial role during arteriogenesis. However, known pro-arteriogenic compounds (MCP-1, GM-CSF) acting via monocytic pathways also exert positive effects on granulocytes and lymphocytes. The role of these two cell types in arteriogenesis remains yet to be clarified, which was the aim of the current study. Methods: Ninety New Zealand White Rabbits received either phosphate buffered saline (PBS), monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8). neutrophil activating protein-2 (NAP-2) or lymphotactin (Ltn) via osmotic minipumps after unilateral femoral artery ligation. In vitro stimulation and in vivo assessment of chemoattraction confirmed cell-specific action of the compounds in rabbits. Arteriogenesis was evaluated by angiography and collateral conductance measurements using fluorescent microspheres. Quantitative immunohistology was used to quantify transmigrated leukocyte subtypes after infusion of the factors. Results: MCP-1 infusion attracts monocytes and granulocytes, whereas IL-8 attracts all three cell types albeit monocytes to a significantly lower degree than MCP-1. NAP-2 and lymphotactin selectively attract granulocytes, respectively, lymphocytes. Of the tested cytokines, only MCP-1 stimulates arteriogenesis, as assessed by collateral conductance measurements ((ml/(min 100 mm/Hg)): PBS, 50.70 +/- 5.15; MCP-1, 216.30 +/- 12.30: IL-8. 58.91 +/- 5.56; NAP-2, 66.83 +/- 8.72; Ltn, 52.80 +/- 5.37) and angiographic findings. Conclusion: This study for the first time provides evidence that not granulocytes or T-lymphocytes but monocytes are the key mediators of arteriogenesis. (c) 2005 Elsevier Ireland Ltd. All rights reserve

Deletion of individual CD83 PRE substructures impairs binding to HuR.

<p><b>A.</b> GST alone (negative control, 2.2 µM, lane 1, 6, 11, 16) or increasing amounts of recombinant GST-HuR (0.19 µM, 0.38 µM, 0.57 µM, 0.95 µM) were incubated with radiolabelled CD83wt PRE RNA or several combinations of uridine-rich element (URE) mutants as indicated at the top of the panels. Formation of protein-RNA interaction (indicated by arrowhead) was subsequently detected by gel retardation analysis. <b>B.</b> GST alone (negative control; 2.2 µM, lane 1, 5, 9, 13) or increasing amounts of recombinant GST-HuR (0.237 µM, 0.475 µM, 0.95 µM; lanes 2–4, 6–8, 10–12, 14–16, respectively) were incubated together with radiolabelled full-length CD83wt PRE RNA or various PRE subloop (SubL) deletions (depicted at the top of the panels). Complex formation (indicated by arrowhead) was subsequently visualized by gel retardation assay as before. <b>C.</b> COS7 cells were transfected with expression vectors encoding for human CD83 cDNA flanked by the entire homologous 5′- and 3′-UTR (lane 1 and 2) or derivatives thereof (lane 3: ΔSubL1; lane 4: ΔSubL2; lane 5: ΔSubL3). Cellular lysates were subjected to immuno-PCR using anti-HuR antiserum (lane 2–5) or rabbit IgG for negative control (lane 1). CD83-specific RNA was detected by PCR followed by gel electrophoresis. A mock reaction without template served as additional negative control (nc; lane 6). This experiment has been reproduced at least three times with the same results.</p

Differential effects of MCP-1 and leptin on collateral flow and arteriogenesis

Objective: Strategies to therapeutically stimulate collateral artery growth in experimental models have been studied intensively in the last decades. However, the experimental methods to detect collateral artery growth are discussed controversially and vary significantly. We compared different methods in a model of arteriogenesis in the rabbit hind limb and determined the effects on collateral flow of a known proarteriogenic factor, monocyte chemoattractant protein-1 (MCP-1), and a cytokine not previously evaluated for its arteriogenic efficacy, the adipocytokine leptin. Methods and results: Forty-two New Zealand White rabbits received either MCP-1, leptin or PBS after ligation of the right femoral artery. The pro-arteriogenic effect of MCP-1 was confirmed by flow measurements during reactive hyperemia, as demonstrated by increased flow ratio (PBS 0.56+/-0.07 vs. MCP-1 0.77+/-0.06, no unit, p <0.0001), ankle-brachial index and microsphere-based conductance measurements (PBS 50.8+/-2.1 vs. MCP-1 225.8+/-8.8 ml/min/100 mm Hg, p <0.001). Biological activity of leptin on rabbit monocytes was shown by a dose dependent increase in Mac-1 expression. In-vivo administration of leptin also led to an increase in hyperemic flow and flow ratio (leptin 0.69+/-0.03, p <0.05 vs. PBS), but not to an increase in collateral conductance (leptin 54.7+/-4.1 ml/min/100 mm Hg, p=ns vs. PBS) or proliferation of vascular smooth muscle cells (Ki-67 staining: PBS 24.7+/-3.9%, leptin 22.7%+/-0.8% (p=ns), MCP-1 32.0+/-1.9% (p <0.01)). Ki-67 mRNA measured by real-time polymerase chain reaction increased (8.8+/-3.1-fold, p <0.01) during natural arteriogenesis, and was further enhanced (25.5+/-8.1-fold, p <0.005) after stimulation with MCP-1. Conclusion: MCP-1 and leptin increase collateral flow in the rabbit bind limb model. In contrast to MCP-1, leptin does not enhance direct markers of vascular proliferation such as collateral conductance under maximal vasodilation and proliferation indices. The observed increase in hyperemic collateral flow thus most probably can be attributed to the well-documented vasodilatory effects of leptin. These data stress the necessity of the use of proliferation markers and microsphere-based conductance measurements under maximal vasodilation in order to separate effects of substances on vascular proliferation from effects on vasodilation. (C) 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserve