Previous results from our laboratory showed SLE T cells exhibit an increased sensitivity to estradiol [6
]. Administration of the ER antagonist Faslodex reduced disease activity in some SLE patients [23
]. We have extended those findings and used gene profiling to gain greater insight into signaling pathways that are stimulated by estradiol and may alter T cell function. We selected pathways that were uniquely controlled by estradiol in all six female SLE and all five control T cell samples obtained from healthy females. It is important to note that such an approach will not detect altered pathways in subsets of patients. Our rationale was to select those pathways differentially regulated by estradiol in all T cell samples in order to identify signaling pathways that were targets in the majority of SLE patient’s T cells. Confirming the validity of this approach was the identification of the T cell receptor signaling pathway as an estrogen target shown in earlier studies [24
] and known to be altered in SLE T cells [15
Estradiol increases signaling through PI3K/AKT, T Cell Receptor, Interferon, GM-CSF, Calcium and SAPK/JNK pathways () that are normally associated with regulating T cell function. Estradiol increases expression of the PI3K/AKT pathway in SLE and not in control T cells. In normal T cells, activation of the PI3K/AKT pathway stimulates cell survival, glucose metabolism and inducible transcription [30
]. It is important to note that alteration in the PI3K/AKT pathway is sufficient to promote autoimmunity [15
]. Estradiol enhances the T cell receptor signaling pathway which we [24
], and others [3
] have shown is altered in human SLE T cells. Estradiol increases GM-CSF signaling uniquely in activated SLE T cells (). GM-CSF is a critical hematopoietic growth factor that affects circulating leukocytes and is produced by activated T cells [32
]. GM-CSF can increase antigen-induced immune responses and alter the Th1/Th2 cytokine balance in T cells. Estradiol also augmented the stress-activated protein kinase (SAPK/JNK) pathway in SLE T cells. JNK1 and JNK2 stimulate the release of proinflammatory cytokines in activated T cells and modulate the differentiation of Th1 and Th2 cells [33
Calcium signaling is dysregulated in SLE T cells by several mechanisms (34–39). Engagement of the T cell receptor leads to a transient release of calcium that occurs earlier and is of greater magnitude in SLE compared with normal T cells [36
]. By contrast, long-term changes in calcium signaling is diminished in SLE T cells and may underlie abnormal T cell activation and/or the inability of SLE T cells to produce IL-2 [37
]. It is interesting to note that estradiol both stimulates and reduces calcium signaling in SLE T cells (compare and ). This finding suggests that estradiol plays a role in differentially affecting short term versus sustained calcium signaling in SLE T cells. Some clues regarding the molecular mechanisms involved in this apparent paradox are suggested by increased signaling through the pentose phosphate pathway in SLE T cells, which is increased by estradiol (). Previous studies have shown that the mitochondrial membrane in SLE T cells is hyperpolarized owing to reductions in ATP and glutathione [38
]. Mitochondrial hyperpolarization and persistent ATP depletion is a critical checkpoint that predispose SLE T cells to undergo cell death by necrosis rather than apoptosis. The mitochondrial transmembrane potential is regulated in part by the supply of NADPH produced by the pentose phosphate pathway [39
]. Since abnormal T cell activation and cell death underpin the pathology of SLE it is important to clarify the estrogen-dependent steps that contribute to alterations in this metabolic pathway and may change calcium signaling in SLE T cells.
Of the twenty signaling pathways that were uniquely downregulated by estradiol (), the decrease in IL-2 signaling is a central component of defective SLE T cell signaling [40
]. Local increases in nitric oxide at the site of T cell receptor engagement reduce IL-2 production [42
]. Nitric oxide is regulated by NADPH and, as revealed by our study, the pentose phosphate pathway is altered by estradiol in SLE T cells. Alternatively, IL-2 is regulated by a number of transcription factors including the cAMP response element-binding protein (CREB). Of relevance for IL-2 regulation in SLE T cells is the report from Katsiari et al. (43) showing that protein phosphatase 2A expression is higher in SLE than normal T cells leading to reduced transcription of IL-2. Protein kinase A (PKA) is a major target of cAMP activation. PKA signaling is maintained by a balance between kinase and phosphatase activity and the pathway is deficient in SLE T cells [44
]. Although additional studies are required to identify the targets for reduced cAMP signaling in response to estradiol, our data indicate that estradiol targets pathways known to be markers for abnormal signaling in SLE T cells.
Estradiol uniquely downregulates 28 signal transduction pathways in activated control T cells (). Several of these pathways are known to be defective in SLE T cells including cell receptor signaling [45
], ERK/MAPK signaling [46
], IL-4 [48
], NF-κB [49
] and apoptosis signaling [50
]. Although nothing is known about estradiol-dependent signaling in normal T cells, our results suggest that estradiol downregulates key signaling pathways in activated normal T cells. Estrogen is considered a promoter of the immune response (51). Our results indicate that estradiol downregulates numerous pathways in activated T cells and suggests that failure to downregulate these same pathways in SLE T cells could contribute to SLE T cell hyperactivation. The concept now requires further investigation because it suggests downregulation is an important mechanism that is defective in the SLE T cell response to estradiol.
In this report, we chose to further study the interferon-α pathway owing to its importance in SLE and other autoimmune diseases [17
]. Seven of thirteen genes identified in interferon-α signaling showed greater than a 1.5 mean-fold increase in the SLE T cell samples (). If regulation of this pathway is typical for estradiol targets, it suggests that estrogen-dependent upregulation of signaling will occur by increased expression of multiple genes within a pathway rather than a large change in the magnitude of expression of a single gene. We found that expression of IFIT1 in SLE T cell samples was bimodal in that some patient’s T cells responded strongly to estradiol while others were less responsive to the hormone. Changes in IFIT1 expression correlated moderately with SLE disease activity. Interestingly, T cells from patients with renal disease showed the greatest sensitivity to estradiol. Other studies have found significant differences in IFIT1 expression in SLE compared with control T cells [28
] and increased expression correlated with renal disease [53
]. It is likely that our study underestimates estradiol effects on IFIT1 expression since the T cells were cultured for 16 h in serum free medium without added interferon-α.
The interferon stimulated gene factor 3 (ISGF3) is a heterotrimeric complex central to the regulation of IFN gene transcription [54
]. Binding of IFN to cell surface receptors activates STAT1 and STAT2 proteins which complex with interferon regulatory factor 9 (IRF9) forming the ISGF3 activation complex. The ISGF3 complex enters the nucleus where it binds to specific DNA sequences and increases the rates of transcription of interferon gene targets. While many of the signaling events downstream of interferon signaling are known [22
], the mechanisms involved in regulating IFN-dependent gene transcription are less well understood.
Transcriptional coactivators are important modulators of gene regulation. Coactivators regulate gene transcription by modifying chromatin structure and enhancing promoter accessibility. In addition, coactivators can recruit other factors to the promoter of responsive genes that enhance the transcriptional response. DRIP150 is a member of a multi-protein complex that shares several subunits with the mammalian Mediator complex [29
]. Mediator complexes function as a bridge between distal transcription activators and RNA polymerase II. The Mediator complex is essential for the regulation of NF-κB, SP1 and most nuclear hormone receptor signaling pathways including the ER [13
]. DRIP150 interacts with both ER-α and ER-β [56
]. ISGF3-mediated transcription is dependent on STAT2 interactions with DRIP150 [55
]. Interestingly, one of the DRIP coactivators, TRAP220 showed preference for interacting with ER-β over ER-α [56
Our data indicate that DRIP150 expression is altered in SLE compared with normal T cells in response to estradiol. These results are the first to suggest that abnormal cofactor recruitment to the promoter of genes involved in cell signaling could lead to enhanced pathway activation and disrupt normal T cell function. Over and or under expression of key signaling pathways is expected to contribute to the development of autoimmune disease. It may be that abnormal coactivator expression recruits ERs to the promoters of genes that are not normally regulated by estradiol. Alternatively, inappropriate cofactor recruitment could occur by differential expression of ER subtypes such that ratio of ER-α/ER-β is altered in SLE T cells [57
] leading to abnormal signal transduction. It is now clear that the time has come to explore the role of coactivators and corepressors as possible mechanisms underlying the deregulation of numerous genes in SLE T cells.