Efficacy of adenoviral TNF alpha antisense is enhanced by a macrophage specific promoter.

Macrophage-derived TNF alpha is a critical mediator of inflammation and destruction in diseases such as rheumatoid arthritis and Crohn's disease. These studies were undertaken to develop an effective adenovirus-based strategy to specifically suppress TNF alpha in primary human macrophages. A variety of promoters and LTRs were evaluated for effective expression in the macrophage cell line RAW 264.7. The CMV promoter and the Visna LTR were the most strongly expressed and were therefore used to drive the expression of TNF alpha antisense fragments. In transient transfection assays, the antisense fragment terminating at the 3' end of the first exon (216 bp) was superior to the others (70 and 750 bp), when expressed under the control of either the CMV promoter or the Visna LTR. Adenoviral vectors expressing the 216 bp TNF alpha antisense fragment, controlled by the CMV promoter or the Visna LTR, were both effective at suppressing LPS-induced TNF alpha secretion by primary human macrophages. However, the Visna LTR was more effective not only at suppressing LPS-induced TNF alpha secretion, but also IL-6, which is highly sensitive to TNF alpha secretion. These results demonstrate that effective, specific, suppression of TNF alpha in macrophages is possible, employing a directed antisense approach and a promoter system that is highly efficient in human macrophages.

Regulation of TNF-alpha expression in normal macrophages: the role of C/EBPbeta.

C/EBPbeta is present in monocytes and macrophages, binds to the proximal region of the TNF-alpha promoter, and contributes to its regulation. This study was performed to characterize the ability of C/EBPbeta to regulate the TNF-alpha gene in myelomonocytic cells and primary macrophages. In transient transfection assays, overexpression of wild type C/EBPbeta resulted in a 3-4-fold activation of a 120 base pair TNF-alpha promoter-reporter construct, while overexpression of a dominant negative (DN) C/EBPbeta inhibited LPS-induced activation. In vitro monocyte-differentiated macrophages, infected with an adenoviral vector expressing the DN C/EBPbeta (AdDNC/EBPbeta) or the control Adbetagal, expressed their transgenes weakly, however expression was greatly enhanced in the presence of PMA. Infection with AdDNC/EBPbeta resulted in 60% suppression of LPS induced TNFalpha secretion compared to Adbetagal infection (P<0.001) in PMA-treated macrophages. Northern blot analysis demonstrated approximately a 40% reduction of the TNF-alpha mRNA in the presence of the DN C/EBPbeta, suggesting that the effect of the DN C/EBPbeta was at the transcriptional level. In contrast, AdDNC/EBPbeta infection did not result in inhibition of LPS-induced TNF-alpha secretion in the absence of PMA. Further, DN versions of both C/EBPbeta and c-Jun, but not NF-kappaB p65, suppressed PMA-induced TNF-alpha secretion in macrophages. These observations demonstrate that, C/EBPbeta and c-Jun contribute to the regulation of the TNF-alpha gene in normal macrophages following treatment with PMA.

C/EBP beta in rheumatoid arthritis: correlation with inflammation, not disease specificity.

Rheumatoid arthritis synovial tissue was examined and compared with osteoarthritis tissue for the presence of the nuclear transcription factor C/EBP beta (NF-IL-6). The region (lining or sublining), cell type, and subcellular distribution (cytoplasmic or nuclear) of the expression of C/EBP beta was characterized. Rheumatoid arthritis synovial fluid and blood and normal peripheral blood were also examined. C/EBP beta was detected in the synovial lining and in sublining cells of synovial tissue from patients with both rheumatoid and osteoarthritis. A significant (P < 0.001 and < 0.05, respectively) increase in the percentage of cells with nuclear staining was seen in the lining layer, compared to cells in the sublining region, in rheumatoid and osteoarthritis. In both diseases a strong correlation (r = 0.79, P < 0.001) was observed between the percentage of cells in the synovial lining that were positive for nuclear C/EBP beta and lining cell depth. Two-color immunohistochemistry demonstrated that both macrophages and fibroblast-like synoviocytes were positive for nuclear C/EBP beta. The presence of C/EBP beta was confirmed by immunohistochemistry and Western blot analysis with isolated synovial fibroblasts. Nuclear C/EBP beta was also detected in rheumatoid synovial fluid monocytes/macrophages, but not in lymphocytes or neutrophils. Western blot analysis confirmed the presence of C/EBP beta in these cells. The intensity of C/EBP beta staining was greater (P < 0.001) in synovial fluid monocytes than in those from normal or rheumatoid peripheral blood. In conclusion, the enhanced nuclear staining for C/EBP beta in the synovial lining, compared to the sublining, suggesting activation in the lining, and the positive correlation of lining layer depth with the percentage of cells in the lining positive for nuclear C/EBP beta, suggest a potential role for C/EBP beta in chronic inflammation. The regulation of the production or activity of C/EBP beta, to inhibit inflammatory mediator expression by synovial macrophages and fibroblasts, offers a novel approach to therapeutic intervention.

Tumor necrosis factor alpha gene regulation: enhancement of C/EBPbeta-induced activation by c-Jun.

Tumor necrosis factor alpha (TNF alpha) is a key regulatory cytokine whose expression is controlled by a complex set of stimuli in a variety of cell types. Previously, we found that the monocyte/macrophage-enriched nuclear transcription factor C/EBPbeta played an important role in the regulation of the TNF alpha gene in myelomonocytic cells. Abundant evidence suggests that other transcription factors participate as well. Here we have analyzed interactions between C/EBPbeta and c-Jun, a component of the ubiquitously expressed AP-1 complex. In phorbol myristate acetate (PMA)-treated Jurkat T cells, which did not possess endogenous C/EBPbeta, expression of c-Jun by itself had relatively little effect on TNF alpha promoter activity. However, the combination of C/EBPbeta and c-Jun was synergistic, resulting in greater than 130-fold activation. This effect required both the leucine zipper and DNA binding domains, but not the transactivation domain, of c-Jun, plus the AP-1 binding site centered 102/103 bp upstream of the transcription start site in the TNF alpha promoter. To determine if C/EBPbeta and c-Jun might cooperate to regulate the cellular TNF alpha gene in myelomonocytic cells, U937 cells that possess endogenous C/EBPbeta and were stably transfected with either wild-type c-Jun or the transactivation domain deletion mutant (TAM-67) were examined. U937 cells expressing ectopic wild-type c-Jun or TAM-67 secreted over threefold more TNF alpha than the control line in response to PMA plus lipopolysaccharide. Transient transfection of the U937 cells expressing TAM-67 suggested that TAM-67 binding to the -106/-99-bp AP-1 binding site cooperated with endogenous C/EBPbeta in the activation of the -120 TNF alpha promoter-reporter. DNA binding assays using oligonucleotides derived from the TNF alpha promoter suggested that C/EBPbeta and c-Jun interact in vitro and that the interaction may be DNA dependent. Our data demonstrate that the TNF alpha gene is regulated by the interaction of the ubiquitous AP-1 complex protein c-Jun and the monocyte/macrophage-enriched transcription factor C/EBPbeta and that this interaction contributes to the expression of the cellular TNF alpha gene in myelomonocytic cells. This interaction was unique in that it did not require the c-Jun transactivation domain, providing new insight into the cell-type-specific regulation of the TNF alpha gene.

Mutations which affect the inhibition of protein phosphatase 2A by simian virus 40 small-t antigen in vitro decrease viral transformation.

Three independent point mutations within residues 97 to 103 of the simian virus 40-small-t antigen (small-t) greatly reduced the ability of purified small-t to inhibit protein phosphatase 2A in vitro. These mutations affected the interaction of small-t antigen with the protein phosphatase 2A A subunit translated in vitro, and a peptide from the region identified by these mutations released the A subunit from immune complexes. When introduced into virus, the mutations eliminated the ability of small-t to enhance viral transformation of growth-arrested rat F111 cells. In contrast, the mutant small-t antigens were unimpaired in the transactivation of the adenovirus E2 promoter, an activity which was reduced by a double mutation in small-t residues 43 and 45.

Inhibition of the Na+/H(+)-antiporter by theophylline in growth-arrested monkey kidney cells.

Treatment of monkey kidney CV-1 cells with theophylline leads to growth arrest, with sensitivity to theophylline being greatly increased in the absence of bicarbonate. Theophylline, like amiloride, increases the sensitivity of cells to intracellular acidification and protects cells from acid shock. These observations and direct measurements of 22Na uptake suggest that growth-arresting effects of theophylline are mediated by the Na+/H(+)-antiporter.

Comparison of lysine and tryptophan catabolizing enzymes in rat and bovine tissues.

Earlier studies indicate that alpha-aminoadipate aminotransferase (AadAT) and kynurenine aminotransferase (KAT) activities from rat tissues are associated with a single protein. However, our recent studies indicate that AadAT activity from bovine liver and kidney is not associated with KAT activity. To test whether the lysine and tryptophan catabolism in bovine tissues differ from that in rat tissues, we compared the activities of enzymes involved in lysine and tryptophan pathways in rat and bovine tissues. The activities of lysine catabolizing enzymes such as AadAT, lysine alpha-ketoglutarate reductase and saccharopine dehydrogenase in the bovine tissues were significantly lower than those found in rat tissues. The activities of tryptophan catabolizing enzymes such as KAT and kynurenine hydroxylase in the bovine tissues were negligible as compared to those in rat tissues. The results suggest that lysine is degraded via the saccharopine pathway in the livers and kidneys of both species but the metabolism of tryptophan in bovine tissues may be different from that in rat tissues.

Purification and properties of 2-aminoadipate: 2-oxoglutarate aminotransferase from bovine kidney.

Previous studies with rat kidney preparations indicated that 2-aminoadipate aminotransferase (AadAT) and kynurenine aminotransferase (KAT) activities are properties of a single protein. We found that bovine kidney contains an appreciable amount of AadAT activity, but lacks KAT activity. AadAT from bovine and rat kidney extracts were purified to electrophoretic homogeneity. The purification procedure included fractionation with (NH1)2SO1, heat treatment, DEAE-cellulose chromatography and hydroxyapatite chromatography. Physical and kinetic properties, such as pH optima, Km for substrates, Mr, electrophoretic mobility and inhibition by dicarboxylic acids of bovine kidney AadAT, were similar to those of the rat kidney enzyme. However, bovine kidney AadAT differed from rat kidney AadAT in substrate specificity, amino acid composition and stability when stored. The titration curve of bovine kidney AadAT was also different from that of the rat kidney enzyme. The results suggest that bovine kidney AadAT may have some structural similarity to rat kidney AadAT and that the structural differences observed between the two enzymes may explain the absence of KAT activity in bovine kidney.