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Angiopoietin-1 (Ang-1) is one of a family of ligands for the Tie-2 receptor which has been demonstrated to be involved in angiogenesis. Little is known about the regulation of Ang-1 gene expression. We have previously demonstrated that TNF-alpha is able to up-regulate the expression of Ang-1 mRNA in synovial fibroblasts. This present study investigated the signal transduction pathways involved in the TNF-alpha induced expression of Ang-1. TNF-alpha signals primarily through the p38, JNK, MAP kinase, and IKK pathways resulting in the activation of the transcription factors AP-1 and NF-kappa B. Experiments with inhibitors and siRNA for these various signal transduction pathways revealed that TNF-alpha stimulation of Ang-1 expression occurs via the NF-kappa B signal transduction pathway.

There are at least four distinct families of enzymes that recognize and remove uracil from DNA. Family-3 (SMUG1) enzymes have recently been identified and have a preference for uracil in single-stranded DNA when assayed in vitro. Here we investigate the in vivo function of SMUG1 using the yeast Saccharomyces cerevisiae as a model system. These organisms lack a SMUG1 homologue and use a single enzyme, Ung1 to carry out uracil-repair. When a wild-type strain is treated with antifolate agents to induce uracil misincorporation into DNA, S-phase arrest and cellular toxicity occurs. The arrest is characteristic of checkpoint activation due to single-strand breaks caused by continuous uracil removal and self-defeating DNA repair. When uracil-DNA glycosylase is deleted (Δung1), cells continue through S-phase and arrest at G2/M, presumably due to the effects of stable uracil misincorporation in DNA. Pulsed field gel electrophoresis (PFGE) demonstrates that cells are able to complete DNA replication with uracil-substituted DNA and do not experience the extensive strand breakage attributed to uracil-DNA glycosylase-mediated repair. As a result, these cells experience early protection from antifolate-induced cytotoxicity. When either UNG1 or SMUG1 functions are reintroduced back into the null strain and then subjected to antifolate treatment, the cells revert back to the wild-type phenotype as shown by a restored sensitivity to drug and S-phase arrest. The arrest is accompanied by the accumulation of replication intermediates as determined by PFGE. Collectively, these data indicate that SMUG1 can act as a functional homolog of the family-1 uracil-DNA glycosylase enzymes.

CD23, the low-affinity IgE receptor, is up-regulated on interleukin (IL)-4-stimulated B cells and monocytes, with a concomitant increase in the release of soluble fragments of CD23 (sCD23) into the medium by proteolytic processing of the surface-bound intact CD23. The effect of inhibition of the processing of CD23 on IgE production in human and mouse cells and in a mouse model in vivo was evaluated. CD23 processing to sCD23 from RPMI 8866 (a human Epstein-Barr virus-transformed B cell line) cell membranes was inhibited by a broad-spectrum matrix-metalloprotease inhibitor, batimastat, with an IC50 of 0.15 microM. Batimastat also inhibited CD23 processing in whole RPMI 8866 cells as well as in IL-4-stimulated purified human monocytes with similar IC50. Batimastat inhibited IgE production from IL-4/anti-CD40-stimulated human tonsil B cells as well as mouse splenic B cells in a manner consistent with inhibition of CD23 processing. Release of soluble fragments of CD23 in the cell supernatants of tonsil B cells was inhibited over the concentration range of 1-10 microM batimastat and intact cell surface CD23 was increased on mouse splenic B cells in the presence of these concentrations of batimastat. IgE production of IL-4-stimulated human peripheral blood mononuclear cells was also blocked by 1-10 microM batimastat, again with comparable inhibition of sCD23 release over the same concentration range. Finally, in a mouse model of IgE production, batimastat inhibited IgE production in response to ovalbumin challenge as determined by serum IgE levels. Taken together, the data support a role of CD23 in IgE production and point to CD23 processing to sCD23 as a therapeutically relevant control point in the regulation of IgE synthesis.

Evidence is presented on two forms of uracil-DNA glycosylase (UDG1 and UDG2) that exist in human cells. We have developed an affinity technique to isolate uracil-DNA glycosylases from HeLa cells. This technique relies on the use of a uracil-DNA glycosylase inhibitor (Ugi) produced by the Bacillus subtilis bacteriophage, PBS2. Affinity-purified preparations of uracil-DNA glycosylase, derived from total HeLa cell extracts, reveal a group of bands in the 36,000 molecular weight range and a single 30,000 molecular weight band when analyzed by SDS-PAGE and silver staining. In contrast, only the 30,000 molecular weight band is seen in HeLa mitochondrial preparations. Separation of HeLa cell nuclei from the postnuclear supernatant reveals that uracil-DNA glycosylase activity is evenly distributed between the nuclear compartment and the postnuclear components of the cell. Immunostaining of a nuclear extract with antisera to UDG1 indicates that the nuclear associated uracil-DNA glycosylase activity is not associated with the highly conserved uracil-DNA glycosylase, UDG1. With the use of Ugi-Sepharose affinity chromatography, we show that a second and distinct uracil-DNA glycosylase is associated with the nuclear compartment. Immunoblot analysis, utilizing antisera generated against UDG1, reveals that the 30,000 molecular weight protein and a protein in the 36,000 range share common epitopes. Cycloheximide treatment of HeLa cells indicates that upon inhibition of protein synthesis, the higher molecular weight species disappears and is apparently post-translationally processed into a lower molecular weight form. This is substantiated by mitochondrial import studies which reveal that in vitro expressed UDG1 becomes resistant to trypsin treatment within 15 min of incubation with mitochondria. Within this time frame, a lower molecular weight form of uracil-DNA glycosylase appears and is associated with the mitochondria. Antibodies generated against peptides from specific regions of the cyclin-like uracil-DNA glycosylase (UDG2), demonstrate that this nuclear glycosylase is a phosphoprotein with a molecular weight in the range of 36,000. SDS-PAGE analysis of Ugi affinity-purified and immunoprecipitated UDG2 reveals two closely migrating phosphate-containing species, indicating that UDG2 either contains multiple phosphorylation sites (resulting in heterogeneous migration) or that two distinct forms of UDG2 exist in the cell. Cell staining of various cultured human cell lines corroborates the finding that UDG1 is largely excluded from the nucleus and that UDG2 resides mainly in the nucleus. Our results indicate that UDG1 is targeted to the mitochondria and undergoes proteolytic processing typical of resident mitochondrial proteins that are encoded by nuclear DNA. These results also indicate that the cyclin-like uracil-DNA glycosylase (UDG2) may be a likely candidate for the nuclear located base-excision repair enzyme.

Inhibitors of human methionine aminopeptidase type 2 (hMetAP2) are of interest as potential treatments for cancer. A new class of small molecule reversible inhibitors of hMetAP2 was discovered and optimized, the 4-aryl-1,2,3-triazoles. Compound 24, a potent inhibitor of cobalt-activated hMetAP2, also inhibits human and mouse endothelial cell growth. Using a mouse matrigel model, this reversible hMetAP2 inhibitor was also shown to inhibit angiogenesis in vivo.

Angiopoietin- I (Ang-1) and Ang-2 are ligands for the receptor tyrosine kinase, Tie-2. Ang-1, a Tie-2 agonist, may have a vascular stabilizing role in angiogenesis, while Ang-2, an endogenous antagonist of Tie-2, may have an early role in angiogenesis, destabilizing existing vasculature. We show that these ligands are expressed by rheumatoid synovial fibroblasts (RSF) and investigate whether their expression was modulated by proinflammatory cytokines present in the joint in rheumatoid arthritis (RA).Using quantitative PCR we determined the level of expression of these 2 ligands in RSF and chronic inflamed synovial tissue. The level of expression of these ligands after treatment with proinflammatory cytokines and hypoxia was also determined.We observed constitutive expression of Ang-1 and Ang-2 in RSF and chronic inflamed synovial tissue. Ang-1 was the most highly expressed ligand in late stage RA synovial fibroblasts; however, in chronic inflamed synovial tissue, Ang-2 was predominant and was expressed at strikingly high levels (70 to 120-fold increase). We observed that tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta), but not interleukin 1beta or hypoxia, stimulated Ang-1 gene expression in RSE This was confirmed at the protein level as media from TNF-alpha treated RSF resulted in increased autophosphorylation of Tie-2. In contrast, TNF-alpha and TGF-beta had no effect on Ang-2 expression in RSF, but augmented expression of Ang-2 in normal synovial fibroblasts.The angiopoietins are important angiogenic factors constitutively present in RA, and their expression is modulated by certain cytokines. Ang-2 may have an important role in rheumatoid tissue where vigorous angiogenesis is occurring.