We have previously reported that transcriptional induction of cyclooxygenase-2 (COX-2) isoenzyme occurs early after T cell receptor triggering, suggesting functional implications of cyclooxygenase activity in this process. Here, we identify the cis-acting elements responsible for the transcriptional activation of this gene in human T lymphocytes. COX-2 promoter activity was induced upon T cell activation both in primary resting T lymphocytes and in Jurkat cells. This induction was abrogated by inhibition of calcineurin phosphatase with the immunosuppressive drug cyclosporin A, whereas expression of an active calcineurin catalytic subunit enhanced COX-2 transcriptional activation. Moreover, cotransfection of nuclear factor of activated T cells (NFAT) wild type protein transactivated COX-2 promoter activity. Conversely, dominant negative mutants of NFATc or c-Jun proteins inhibited COX-2 induction. Electrophoretic mobility shift assays and site-directed mutagenesis allowed the identification of two regions of DNA located in the positions ؊117 and ؊58 relative to the transcriptional start site that serves as NFAT recognition sequences. These results emphasize the central role that the Ca 2؉ /calcineurin pathway plays in COX-2 transcriptional regulation in T lymphocytes pointing to NFAT/activator protein-1 transcription factors as essential for COX-2 promoter regulation in these cells. Prostaglandin endoperoxide synthase or cyclooxygenase (COX)1 is the enzyme responsible for the conversion of arachidonic acid to prostaglandin H 2 , the main step in the prostaglandin synthesis pathway. Two forms of the enzyme, named COX-1 and COX-2, have been shown to be expressed in mammalian tissues. COX-1 is considered a housekeeping enzyme constitutively expressed in most tissues, whereas the COX-2 isoform is induced by several stimuli including cytokines and mitogens and is thought to be responsible for the increased production of prostaglandins in pathologic processes (reviewed in Refs. 1-3). Promoter regions of the COX-2 gene of mouse (4), rat (5), chicken (6), and humans (7-9) have been cloned. Regardless of the animal species, these promoters contain a classical TATA box, an E-box, and binding sites for transcription factors such as nuclear factor B, nuclear factor-IL6/CCAATenhancer binding protein), and cyclic AMP-response element (CRE) -binding proteins. These sequences have been shown to act as positive regulatory elements for the COX gene transcription in different cell types (5, 10 -14). We have recently shown that COX-2 expression is induced in T cells upon T cell receptor (TCR) activation playing an important role in controlling this process (15). However, no studies about COX-2 promoter regulation in these cells have been reported so far.Activation of T cells triggers a complex regulatory cascade of events that culminates in the induced transcription of a variety of activation-associated genes (16 -18). Many of them are cytokines that in turn regulate cell proliferation, differentiation, and acquisition of effector f...

We have previously described that IFN-γ induces cyclooxygenase 2 and inducible NO synthase expression by a mechanism that involved endogenously produced TNF-α. In this study, we report that TNF-α production is induced by IFN-γ treatment in the murine macrophage cell line RAW 264.7. TNF-α mRNA levels are increased in cells treated with IFN-γ in a time-dependent manner and IFN-γ also increased human TNF-α promoter-dependent transcription. Two regions in the TNF-α promoter seem to be responsible for the IFN-γ response: a distal region between −1311 and −615 bp of the human TNF-α promoter, and a proximal region located between −95 and −36 bp upstream of the transcriptional start. In contrast, IFN-γ stimulation induces the expression of the transcription factors IRF-1 and IRF-8. Overexpression of these transcription factors produces an increase in the transcriptional activity of the human TNF-α promoter. There is a correlation between the regions of the TNF-α promoter responsible of the transcriptional activation elicited by IRF-1 and IRF-8 and those required for IFN-γ response. In addition, IRF-1 and IRF-8 are recruited to the TNF-α promoter in IFN-γ-treated RAW 264.7 cells, as demonstrated by chromatin immunoprecipitation assays. Moreover, overexpression of IRF-1 and IRF-8 induces TNF-α production in unstimulated RAW 264.7 macrophages, comparable to the production of TNF-α elicited by IFN-γ stimulation, and silencing of IRF-1 and/or IRF-8 with specific small interfering RNAs, decreases IFN-γ-elicited TNF-α production. In summary, IFN-γ treatment induces TNF-α expression at transcriptional level requiring the coordinate action of IRF-1 and IRF-8.