PGD2, produced by mast cells, has been detected in high concentrations at sites of allergic inflammation. It can stimulate vascular and other inflammatory responses by interaction with D prostanoid receptor (DP) and chemoattractant receptor-like molecule expressed on Th2 cells (CRTH2) receptors. A significant role for PGD2 in mediating allergic responses has been suggested based on the observation that enhanced eosinophilic lung inflammation and cytokine production is apparent in the allergen-challenged airways of transgenic mice overexpressing human PGD2 synthase, and PGD2 can enhance Th2 cytokine production in vitro from CD3/CD28-costimulated Th2 cells. In the present study, we investigated whether PGD2 has the ability to stimulate Th2 cytokine production in the absence of costimulation. At concentrations found at sites of allergic inflammation, PGD2 preferentially elicited the production of IL-4, IL-5, and IL-13 by human Th2 cells in a dose-dependent manner without affecting the level of the anti-inflammatory cytokine IL-10. Gene transcription peaked within 2 h, and protein release peaked ∼8 h after stimulation. The effect of PGD2 was mimicked by the selective CRTH2 agonist 13,14-dihydro-15-keto-PGD2 but not by the selective DP agonist BW245C, suggesting that the stimulation is mediated by CRTH2 and not DP. Ramatroban, a dual CRTH2/thromboxane-like prostanoid receptor antagonist, markedly inhibited Th2 cytokine production induced by PGD2, while the selective thromboxane-like prostanoid receptor antagonist SQ29548 was without effect. These data suggest that PGD2 preferentially up-regulates proinflammatory cytokine production in human Th2 cells through a CRTH2-dependent mechanism in the absence of any other costimulation and highlight the potential utility of CRTH2 antagonists in the treatment of allergic diseases.

Cyclic AMP-dependent expression of the steroidogenic acute regulatory (StAR) protein is thought to be the controlling step for steroid production, but the mechanisms through which external signals are translated into increased transcription of the StAR gene are unknown. We demonstrate that cyclic AMP-induced steroid synthesis is dependent upon the phosphorylation and activation of ERKs and that ERK activation results in enhanced phosphorylation of SF-1 and increased steroid production through increased transcription of the StAR gene. Adenylate cyclase activation with forskolin (FSK) caused a time-dependent increase in ERK activity and translocation from cytoplasm to nucleus, which correlated with an increase in StAR mRNA levels, StAR protein accumulation, and steroidogenesis. Similarly, ERK inhibition led to a reduction in the levels of FSKstimulated StAR mRNA, StAR protein, and steroid secretion. These effects were attributed to the finding that ERK activity is required for SF-1 phosphorylation, a transcription factor required for the regulation of StAR gene transcription. This conclusion was supported by our demonstration of an ERK-dependent increase in the binding of SF-1 from FSK-treated Y1 nuclei to three consensus double-stranded DNA sequences from the StAR promoter region. These observations suggest that the activation of ERK2/1 by increasing cAMP is an obligatory and regulated stage in the stimulation of steroid synthesis by cyclic AMP-generating stimuli.

The major prostanoid produced by mast cells is prostaglandin D 2 (PGD 2 ), which has been detected in high concentrations in the airways of asthmatics challenged with

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All Archaea catalyse the conversion of pyruvate to acetyl-CoA via a simple pyruvate oxidoreductase. This is in contrast to the Eukarya and most aerobic bacteria, which use the pyruvate dehydrogenase multienzyme complex [PDHC], consisting of multiple copies of three component enzymes : E1 (pyruvate decarboxylase), E2 (lipoate acetyl-transferase) and E3 (dihydrolipoamide dehydrogenase, DHLipDH). Until now no PDHC activity has been found in the Archaea, although DHLipDH has been discovered in the extremely halophilic Archaea and its gene sequence has been determined. In this paper, the discovery and sequencing of an operon containing the DHLipDH gene in the halophilic archaeon Haloferax volcanii are reported. Upstream of the DHLipDH gene are 3 ORFs which show highest sequence identities with the E1α, E1β and E2 genes of the PDHC from Gram-positive organisms. Structural predictions of the proposed protein product of the E2 gene show a domain structure characteristic of the E2 component in PDHCs, and catalytically important residues, including the lysine to which the lipoic acid cofactor is covalently bound, are conserved. Northern analyses indicate the transcription of the whole operon, but no PDHC enzymic activity could be detected in cell extracts. The presence in the E2 gene of an insertion (equivalent to approximately 100 aa) not found in bacterial or eukaryal E2 proteins, might be predicted to prevent multienzyme complex assembly. This is the first detailed report of the genes for a putative 2-oxoacid dehydrogenase complex in the Archaea, and the evolutionary and metabolic consequences of these findings are discussed.

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Prostaglandin D2 (PGD2) acting at the CRTH2 receptor (chemoattractant receptor-homologous molecule expressed on Th2 cells) has been linked with a variety of allergic and other inflammatory diseases. We describe a family of indole-1-sulfonyl-3-acetic acids that are potent and selective CRTH2 antagonists that possess good oral bioavailability. The compounds may serve as novel starting points for the development of treatments of inflammatory disease such as asthma, allergic rhinitis, and atopic dermatitis.

In addition to the well-documented role of protein kinases in the regulation of steroid production, phosphoprotein phosphatase (PP) activity is required for steroidogenesis. In the present study, we have used the mouse Y1 adrenocortical cell line to identify the site of action of PPs on steroid production by measuring the effects of PP inhibition on the expression of the steroidogenic acute regulatory (StAR) protein and on steroid production. Forskolin-induced activation of cyclic AMPdependent protein kinase (PKA) enhanced steroidogenesis and this was accompanied by an increased expression of StAR protein. Both steroidogenesis and StAR protein expression were inhibited by two structurally dissimilar inhibitors of PP1 and PP2A activities, okadaic acid and calyculin A. These results suggest that inhibition of PP1 and PP2A inhibits steroid production by preventing the expression of the StAR protein, implicating PP1/2A dephosphorylation reactions as important regulators of stimulus-dependent StAR protein expression, and thus of steroidogenesis.