Characterization of a novel negative regulatory element in the human interleukin 4 promoter.

Interleukin 4 (IL-4) is a multifunctional cytokine that plays an important role in hematopoiesis, tumor cell growth, and cellular immune responses. Expression of the IL-4 gene is tightly controlled at the level of gene transcription, and many positive regulatory cis-elements have been identified in the proximal IL-4 promoter region. Relatively little is known about factors that downregulate IL-4 transcription. We performed a detailed deletional analysis of the proximal human IL-4 promoter and studied reporter gene activity in transiently transfected Jurkat T lymphoblasts. In this report, we characterize a novel negative regulatory element (termed P2 NRE) that is adjacent to a binding site for nuclear factor of activated T cells. Mutation of P2 NRE significantly enhanced the activity of a 175 base pair IL-4 promoter construct in transiently transfected Jurkat T lymphoblasts. Using nuclear extracts from Jurkat cells, we identify a candidate factor (termed Rep-1) that binds uniquely to the P2 NRE in DNA-binding assays. Rep-1 is not related to other factors previously shown to interact with the IL-4 promoter, and by UV cross-linking and SDS-PAGE analysis, we found that it migrates with a molecular mass of approximately 150 kDa. Characterizing the molecular mechanisms responsible for downregulating the IL-4 promoter should enhance our understanding of IL-4-gene dysregulation in disease states.

Glucocorticoids inhibit calcium- and calcineurin-dependent activation of the human IL-4 promoter.

The mechanism by which glucocorticoids (GC) inhibit IL-4 gene expression is currently unknown. In T lymphocytes, IL-4 gene expression is regulated at the level of transcription by increases in intracellular calcium concentration and by the calcium-activated phosphatase calcineurin. In this paper we report that dexamethasone (Dex) inhibits calcium ionophore-induced activation of the human IL-4 promoter in transiently transfected Jurkat T cells. Inhibition of the promoter by Dex is dependent on expression of the GC receptor (GR), because it does not occur in GR-deficient cells. Dex also represses activation of the promoter induced by cotransfecting cells with a constitutively active mutant of calcineurin. Using a series of deletion constructs, we show that the proximal 95 bp of the IL-4 promoter contain a Dex-sensitive regulatory element. This region contains the P1 sequence, a proximal binding site for NF-AT. A calcium-induced but Dex-inhibited nuclear complex containing NF-AT binds to the P1 element in EMSA. Using immunoprecipitation under nondenaturing conditions, we found that the GRalpha isoform coprecipitates with NF-ATc in nuclear extracts of calcium ionophore- and Dex-treated cells. Taken together, our results show that GC inhibit IL-4 gene expression by interfering with NF-AT-dependent transactivation of the proximal human IL-4 promoter.

Stat6 inhibits human interleukin-4 promoter activity in T cells.

The differentiation of naive T-helper (Th) cells into cytokine-secreting effector Th cells requires exposure to multiple signals, including exogenous cytokines. Interleukin-4 (IL-4) plays a major role in this process by promoting the differentiation of IL-4-secreting Th2 cells. In Th2 cells, IL-4 gene expression is tightly controlled at the level of transcription by the coordinated binding of multiple transcription factors to regulatory elements in the proximal promoter region. Nuclear factor of activated T cell (NFAT) family members play a critical role in regulating IL-4 transcription and interact with up to five sequences (termed P0 through P4) in the IL-4 promoter. The molecular mechanisms by which IL-4 induces expression of the IL-4 gene are not known, although the IL-4-activated transcription factor signal transducer and activator of transcription 6 (Stat6) is required for this effect. We report here that Stat6 interacts with three binding sites in the human IL-4 promoter by electrophoretic mobility shift assays. These sites overlap the P1, P2, and P4 NFAT elements. To investigate the role of Stat6 in regulating IL-4 transcription, we used Stat6-deficient Jurkat T cells with different intact IL-4 promoter constructs in cotransfection assays. We show that, whereas a multimerized response element from the germline IgE promoter was highly induced by IL-4 in Stat6-expressing Jurkat cells, the intact human IL-4 promoter was repressed under similar conditions. We conclude that the function of Stat6 is highly dependent on promoter context and that this factor promotes IL-4 gene expression in an indirect manner.

The role of the gut flora in the reduction of sulphinpyrazone in the rat.

Sulphinpyrazone underwent both reduction to a sulphide and oxidation to a sulphone after parenteral administration to normal Wistar rats. Oral administration was associated with a bioavailability of about 75% and with a 3-fold greater formation of the sulphide. However, no sulphide was detected in the plasma after oral administration of sulphinpyrazone to germ-free (BD/X) rats or normal rats treated with oral antibiotics. In vitro studies showed that the major site of reduction of sulphinpyrazone was the contents of the hind gut with little activity detected in the liver or other tissues. The sulphide was oxidised in vivo to sulphinpyrazone and small amounts of sulphone, while the latter underwent only slight reduction to sulphinpyrazone, but did not give detectable levels of the sulphide. These data suggest that the gut microflora are the main site of reduction of sulphinpyrazone in the rat in vivo.