Inhibition of vascular endothelial growth factor cotranslational translocation by the cyclopeptolide CAM741.

The cyclopeptolide CAM741 inhibits cotranslational translocation of vascular cell adhesion molecule 1 (VCAM1), which is dependent on its signal peptide. We now describe the identification of the signal peptide of vascular endothelial growth factor (VEGF) as the second target of CAM741. The mechanism by which the compound inhibits translocation of VEGF is very similar or identical to that of VCAM1, although the signal peptides share no obvious sequence similarities. By mutagenesis of the VEGF signal peptide, two important regions, located in the N-terminal and hydrophobic segments, were identified as critical for compound sensitivity. CAM741 alters positioning of the VEGF signal peptide at the translocon, and increasing hydrophobicity in the h-region reduces compound sensitivity and causes a different, possibly more efficient, interaction with the translocon. Although CAM741 is effective against translocation of both VEGF and VCAM1, the derivative NFI028 is able to inhibit only VCAM1, suggesting that chemical derivatization can alter not only potency, but also the specificity of the compounds.

The translocation inhibitor CAM741 interferes with vascular cell adhesion molecule 1 signal peptide insertion at the translocon.

The cyclopeptolide CAM741 selectively inhibits cotranslational translocation of vascular cell adhesion molecule 1 (VCAM1), a process that is dependent on its signal peptide. In this study we identified the C-terminal (C-) region upstream of the cleavage site of the VCAM1 signal peptide as most critical for inhibition of translocation by CAM741, but full sensitivity to the compound also requires residues of the hydrophobic (h-) region and the first amino acid of the VCAM1 mature domain. The murine VCAM1 signal peptide, which is less susceptible to translocation inhibition by CAM741, can be converted into a fully sensitive signal peptide by two amino acid substitutions identified as critical for compound sensitivity of the human VCAM1 signal peptide. Using cysteine substitutions of non-critical residues in the human VCAM1 signal peptide and chemical cross-linking of targeted short nascent chains we show that, in the presence of CAM741, the N- and C-terminal segments of the VCAM1 signal peptide could be cross-linked to the cytoplasmic tail of Sec61beta, indicating altered positioning of the VCAM1 signal peptide relative to this translocon component. Moreover, translocation of a tag fused N-terminal to the VCAM1 signal peptide is selectively inhibited by CAM741. Our data indicate that the compound inhibits translocation of VCAM1 by interfering with correct insertion of its signal peptide into the translocon.

Serpina1 is a potent inhibitor of IL-8-induced hematopoietic stem cell mobilization.

Here, we report that cytokine-induced (granulocyte colony-stimulating factor and IL-8) hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) mobilization is completely inhibited after low-dose (0.5 Gy) total-body irradiation (TBI). Because neutrophil granular proteases are regulatory mediators in cytokine-induced HSC/HPC mobilization, we considered a possible role for protease inhibitors in the induction of HSC/HPC mobilization. Bone marrow (BM) extracellular extracts that were obtained from murine femurs after 0.5 Gy of TBI contained an inhibitor of elastase. Also, after low-dose TBI, both Serpina1 mRNA and protein concentrations were increased in BM extracts, compared with extracts that were obtained from controls. The inhibitory activity in BM extracts of irradiated mice was reversed by addition of an Ab directed against Serpina1. To further study a possible in vivo role of Serpina1 in HSC/HPC mobilization, we administered Serpina1 before IL-8 injection. This administration resulted in an almost complete inhibition of HSC/HPC mobilization, whereas heat-inactivated Serpina1 had no effect. These results indicate that low-dose TBI inhibits cytokine-induced HSC/HPC mobilization and induces Serpina1 in the BM. Because exogenous administration of Serpina1 inhibits mobilization, we propose that radiation-induced Serpina1 is responsible for the inhibition of HSC/HPC mobilization. Also, we hypothesize that cytokine-induced HSC/HPC mobilization is determined by a critical balance between serine proteases and serine protease inhibitors.

Selective inhibition of cotranslational translocation of vascular cell adhesion molecule 1.

Increased expression of vascular cell adhesion molecule 1 (VCAM1) is associated with a variety of chronic inflammatory conditions, making its expression and function a target for therapeutic intervention. We have recently identified CAM741, a derivative of a fungus-derived cyclopeptolide that acts as a selective inhibitor of VCAM1 synthesis in endothelial cells. Here we show that the compound represses the biosynthesis of VCAM1 in cells by blocking the process of cotranslational translocation, which is dependent on the signal peptide of VCAM1. CAM741 does not inhibit targeting of the VCAM1 nascent chains to the translocon channel but prevents translocation to the luminal side of the endoplasmic reticulum (ER), through a process that involves the translocon component Sec61beta. Consequently, the VCAM1 precursor protein is synthesized towards the cytosolic compartment of the cells, where it is degraded. Our results indicate that the inhibition of cotranslational translocation with low-molecular-mass compounds, using specificity conferred by signal peptides, can modulate the biosynthesis of certain secreted and/or membrane proteins. In addition, they highlight cotranslational translocation at the ER membrane as a potential target for drug discovery.

Negative cross-talk between the human orphan nuclear receptor Nur77/NAK-1/TR3 and nuclear factor-kappaB.

The effect of orphan nuclear receptor Nur77 overexpression on activation of an interleukin-2 (IL-2) promoter-luciferase construct was analyzed in the human leukemic cell line Jurkat. Cotransfection of the IL-2 promoter construct together with the Nur77 expression plasmid resulted in a significant repression of IL-2 promoter activation compared to control cells. The repression by Nur77 requires the N-terminal activation function-1 domain. The repressive effect of Nur77 on IL-2 promoter activation is mediated through inhibition of the transcription factor complex nuclear factor-kappaB (NF-kappaB), since blocking or alteration of the IL-2 NF-kappaB binding sites resulted in abrogation of the repressive effect of Nur77. Moreover, further examination of a reporter gene construct containing multiple copies of the IL-2 CD28 response element (CD28RE) showed that Nur77 can inhibit transactivation mediated by the NF-kappaB components p65 and c-Rel. However, no effect of Nur77 was seen on p65-mediated transactivation of a construct containing multiple NF-kappaB binding sites of the HIV LTR. Our data suggest that Nur77 is able to block activation through NF-kappaB when bound to low-affinity NF-kappaB binding sites, such as those located in the IL-2 promoter.

Reduced stem cell mobilization in mice receiving antibiotic modulation of the intestinal flora: involvement of endotoxins as cofactors in mobilization.

Since endotoxins are potent inducers of stem cell mobilization, we hypothesized that their presence in the gut may play a role in cytokine-induced mobilization. To address this possibility we added ciprofloxacin and polymyxin B to the drinking water of Balb/c mice mobilized with either interleukin-8 (IL-8), granulocyte colony-stimulating factor (G-CSF), or flt3 ligand (FL). The yield of colony-forming units (CFUs) was significantly reduced in all mice treated with these antibiotics when compared with controls (IL-8: 192 +/- 61 vs 290 +/- 64, P <.05; G-CSF: 1925 +/- 1216 vs 3371 +/- 1214, P <.05; FL: 562 +/- 213 vs 1068 +/- 528, P <.05). Treatment with ciprofloxacin eliminated only aerobic Gram-negative bacteria from the feces without effect on mobilization. Polymyxin B treatment did not result in decontamination but significantly reduced the number of mobilized hematopoietic progenitor cells (HPCs) most likely due to the endotoxin binding capacity of polymyxin B. More than 90% of the gastrointestinal flora consists of anaerobic bacteria. Elimination of the anaerobic flora by metronidazol led to a significantly reduced number of mobilized HPCs when compared with controls (IL-8: 55 +/- 66 vs 538 +/- 216, P <.05). Germ-free OF1 mice showed a significantly reduced mobilization compared with their wild-type controls (IL-8 controls: 378 +/- 182, IL-8 germ free: 157 +/- 53, P <.05). Finally, we performed reconstitution experiments adding Escherichia coli-derived endotoxins to the drinking water of decontaminated mice. This resulted in partial restoration of the IL-8-induced mobilization (67 +/- 28 vs 190 +/- 98.1, P <.01). Our results indicate that endotoxins serve as cofactors in cytokine-induced mobilization. Modification of the endotoxin content by antibiotic treatment may affect the yield of cytokine-induced mobilization.

Neutrophils are indispensable for hematopoietic stem cell mobilization induced by interleukin-8 in mice.

The CXC chemokine interleukin-8 (IL-8/CXCL8) induces rapid mobilization of hematopoietic progenitor cells (HPCs). Previously we showed that mobilization could be prevented completely in mice by pretreatment with neutralizing antibodies against the beta2-integrin LFA-1 (CD11a). In addition, murine HPCs do not express LFA-1, indicating that mobilization requires a population of accessory cells. Here we show that polymorphonuclear cells (PMNs) serve as key regulators in IL-8-induced HPC mobilization. The role of PMNs was studied in mice rendered neutropenic by administration of a single injection of antineutrophil antibodies. Absolute neutropenia was observed up to 3-5 days with a rebound neutrophilia at day 7. The IL-8-induced mobilizing capacity was reduced significantly during the neutropenic phase, reappeared with recurrence of the PMNs, and was increased proportionally during the neutrophilic phase. In neutropenic mice, the IL-8-induced mobilizing capacity was restored by the infusion of purified PMNs but not by infusion of mononuclear cells. Circulating metalloproteinase gelatinase B (MMP-9) levels were detectable only in neutropenic animals treated with PMNs in combination with IL-8, showing that in vivo activated PMNs are required for the restoration of mobilization. However, IL-8-induced mobilization was not affected in MMP-9-deficient mice, indicating that MMP-9 is not indispensable for mobilization. These data demonstrate that IL-8-induced mobilization of HPCs requires the in vivo activation of circulating PMNs.