Interleukin-2 inhibits glucocorticoid receptor transcriptional activity through a mechanism involving STAT5 (signal transducer and activator of transcription 5) but not AP-1.

Cytokines and glucocorticoids (GCs) signaling pathways interfere with each other in the regulation of apoptosis and gene expression in the immune system. Interleukin-2 (IL-2), through the Janus kinase/signal transducers and activators of transcription (Jak/STAT) and mitogen-activated protein kinase (MAPK) pathways, activates STAT5 and activated protein-1 (AP-1) transcription factors, respectively, which are known to repress glucocorticoid receptor (GR) activity, at least in part, through protein-protein interactions. In this work, we have analyzed the mechanisms whereby IL-2 down-regulates the GC-induced transactivation of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) in murine CTLL-2 T lymphocytes. Mutagenesis studies revealed that the MMTV-LTR STAT5 binding site (-923/-914) was not required for IL-2-mediated inhibition but identified both glucocorticoid response elements (GREs) and the -104/+1 region as critical elements for this negative response. The DNA binding activities of transcription factors required for GC-mediated activation of the MMTV-LTR promoter and that bind to the -104/+1 region (nuclear factor-1, Oct-1) were not affected by IL-2 treatment. Overexpression of wild-type STAT5B enhanced the effect of IL-2 on MMTV-LTR activity, and a dominant negative form of STAT5B (Y699F) abolished the IL-2-mediated MMTV-LTR inhibition, whereas AP-1 activation had no effect in this system. Direct interaction between liganded GR and STAT5 was observed in CTLL-2 cells in a STAT5 phosphorylation-independent manner. Overexpression of nuclear coactivators CBP (CREB-binding protein) or SRC-1a (steroid receptor coactivator 1a) did not blunt IL-2 inhibitory effects. We suggest that the STAT5-repressive activity on the GC-dependent transcription may involve direct interaction of STAT5 with GR, is dependent on the promoter context and STAT5 activation level, and occurs independently of coactivators levels in T cells.

JAB1 interacts with both the progesterone receptor and SRC-1.

JAB1 (Jun activation domain-binding protein-1) has previously been described as a coactivator of AP1 transcription factor. We show here, by yeast and mammalian two-hybrid analyses and by pull-down experiments, that JAB1 also interacts with both the progesterone receptor (PR) and the steroid receptor coactivator 1 (SRC-1) and that it stabilizes PR-SRC-1 complexes. We also show that JAB1 potentiates the activity of a variety of transcription factors known to associate with SRC-1 (nuclear receptors, activator protein-1, and nuclear factor kappaB). This occurs without any modification of PR or SRC-1 concentration. JAB1 is a subunit of a large multiprotein complex that has been called the COP9 signalosome. The latter is present in plant and animal cells and has been shown to be involved in a variety of cellular mechanisms including transcription regulation, cell cycle control, and phosphorylation cascades. We now show that it is also involved in the mechanisms of action of nuclear receptors and of their coactivators.

Cellular stress and apoptosis.

The morphological characteristics of apoptosis are unique and imply a series of alterations including cell shrinkage, membrane blebbing, nuclear condensation and emergence of apoptotic bodies. Three phases can be determined during the process of apoptosis: these are an induction phase corresponding to the initiation of the apoptotic signal, an effector phase involving proteolysis of important substrates and a degradation phase where cell structures and functions are destroyed. Exposure to low doses of H(2)O(2) provokes apoptosis in a variety of cell types, whereas high doses of this oxidant leads to necrosis. Moreover, in addition to examples of chemically or physically induced apoptosis, physiological stimuli such as tumour necrosis factor-alpha, anti-Fas or growth factor withdrawal are accompanied under certain conditions by the production of free radicals. However, it is now well demonstrated that free radicals can activate the death programme but that they are not an essential part of apoptosis. DNA-damaging agents are able to block the cell cycle in G1 and to induce apoptosis. The DNA strand-breaks sensor will allow the expression of P53 leading to protease activation. However, this pathway is not solely responsible for apoptosis to occur, and ceramide production, stimulation of stress-activated protein kinases and subsequent induction of c-jun are also key events in this cascade.

Position 16 of the steroid nucleus modulates glucocorticoid-induced apoptosis at the transcriptional level in murine T-lymphocytes.

Synthetic glucocorticoids (GCs), which possess a different radical substituted in position 16 of the steroid nucleus structure, display various antiproliferative activities on activated lymphoid cells. We analysed this structure-function relationship between dexamethasone (DEX; methyl group in position 16 alpha) and beta-methasone (BM; methyl group in position 16 beta) with regard to two important aspects of GC activity, namely the activation of transcription and induction of apoptosis in IL-2-dependent murine lymphoid cells. DEX induced a higher percentage of apoptotic viable cells compared to BM. This structure-activity relationship was not related to differences in cytosolic glucocorticoid receptor (GR) affinity or kinetics of apoptosis. However, DEX was more efficient than BM in inducing transcriptional activation of an MMTV-CAT plasmid in transiently transfected CTLL-2 cells. In addition, DEX was more potent in inhibiting AP-1 DNA-binding activity compared to BM. These results suggest that the configuration in position 16 may influence the potency of GCs to induce apoptosis in lymphoid cells, mainly by modulating GR-induced transcription.

Mechanisms in interleukin-2 protection against glucocorticoid-induced apoptosis: regulation of AP-1 and glucocorticoid receptor transcriptional activities.

We have used the gibbon ape leukemia cell line MLA-144 and its corticoid-sensitive subclone MLA-E7T to analyze the mechanisms whereby interleukin-2 (IL-2) can protect T cells against dexamethasone-induced apoptosis. MLA cells are characterized by the constitutive expression of intermediate affinity receptors for IL-2, together with IL-4 receptors. MLA-144 cells secrete IL-2 and are insensitive to dexamethasone, whereas MLA-E7T cells do not constitutively produce significant amounts of IL-2 and undergo apoptotic cell death in the presence of dexamethasone. Exogenous IL-2 was shown to protect MLA-E7T cells against the apoptotic effect of dexamethasone and to increase both the DNA binding and transactivating functions of activator protein-1 (AP-1). The functional relationship between AP-1 and glucocorticoid receptors transcriptional activities was further investigated using transient expression of reporter gene constructs whose transcriptions are regulated by promoters containing TPA-responsive elements or glucocorticoid-responsive elements. The data reported here demonstrate that in MLA-144 cells, IL-2 or PMA stimulation antagonizes the glucocorticoid receptor, whereas in MLA-E7T, synergistic effects are observed between dexamethasone and IL-2 or PMA for transactivation of MMTV-CAT. Taken together with the finding that IL-2 but not PMA protects MLA-E7T from dexamethasone-induced apoptosis, our results indicate that IL-2 does not induce such a protection by repressing the transcriptional activity of the glucocorticoid receptor.

Structure-activity relationships in glucocorticoid-induced apoptosis in T lymphocytes.

Glucocorticoid-induced apoptosis in the murine interleukin-2-dependent T-cell line CTLL-2 and in freshly isolated thymocytes was studied. It was demonstrated here that in CTLL-2 cells, dexamethasone (methyl in position 16 alpha) was more efficient in inducing apoptosis than betamethasone (methyl in position 16 beta) or triamcinolone (hydroxyl in position 16). In contrast, no such difference between these three molecules was found in murine thymocytes. In addition, we showed that glucocorticoid-induced apoptosis on the two models was mediated through interaction with the glucocorticoid receptor and did not occur in the presence of inhibitors of transcription, translation or an endonuclease-inhibitor. Furthermore, in CTLL-2 cells, apoptosis took place in the presence of EGTA whereas it was prevented in murine thymocytes, thus indicating that calcium plays a different role in these two models. Finally, higher concentrations of interleukin-2 were needed to protect CTLL-2 cells against dexamethasone-induced apoptosis than that induced by betamethasone or triamcinolone. Thus, structural differences at position 16 of the steroid nucleus correlate with a different apoptosis-inducing activity by glucocorticoids which, however, is only evidenced in the calcium-independent CTLL-2 model.