The data demonstrate that treatment of mouse HT22 cells with mIFN-alpha inhibits GR function as manifested by decreased DEX-induced GR-mediated gene transcription and decreased GR-GRE binding. Under similar treatment conditions, mIFN-alpha had no effects on whole cell GR protein expression or GR nuclear translocation. Pharmacologic inhibition of Jak-STAT signaling pathways, but not p38 MAPK, reversed mIFN-alpha effects on GR function. Disrupting expression of the gene encoding for STAT5 with siRNA also reversed mIFN-alpha effects on GR function, while treatment of cells with siRNA directed against STAT1 and STAT2 failed to show significant effects. Of note, inhibition of STAT5 using siRNA had no effect on DEX-induced MMTV-luciferase activity in the absence of IFN-alpha. Co-immunoprecipitation of phospho-STAT5 and GR following treatment with mIFN-alpha plus DEX demonstrated nuclear phospho-STAT5-GR protein-protein interactions. Taken together, the data suggest that IFN-alpha inhibits GR function by activating STAT5, which then interacts with GR in the nucleus to prevent GR-GRE binding and reporter gene activation.
In the absence of ligand, GR exists in the cytoplasm associated with an assembly of chaperone molecules, notably heat shock proteins. Upon binding to hormone, GR dissociates from the chaperone complex allowing a conformational change that includes exposure of a nuclear localization signal. GR then associates with the cytoskeleton (Galigniana et al., 1998
) and translocates to the nucleus through nuclear pores. Once inside the nucleus, GR forms homodimers and binds to GREs on relevant glucocorticoid-sensitive genes or associates with other transcription factors through direct protein-protein interactions (Guiochon-Mantel et al., 1996
). Given the many steps in GR signaling, there are numerous points at which cytokines such as IFN-alpha could disrupt GR function, including effects on GR protein expression, hormone binding, nuclear translocation and GR interactions with its DNA response element as well as nuclear transcription factors (Pace et al., 2007
A number of studies have found that cytokines can alter GR expression (Miller et al., 1999
; Pace et al., 2007
). Relevant to IFN-alpha, treatment of hepatoblastoma (Huh7) and T cell leukemia-derived (Jurkat) cell lines with IFN-alpha for 72 hours was found to decrease GR mRNA and protein (Cai et al., 2005
). However, consistent with the observations reported herein, changes in GR expression were not apparent after 24 hours of IFN-alpha treatment (Cai et al., 2005
), suggesting that inhibition of GR function as a result of mIFN-alpha treatment for relatively short periods of time (e.g. 24 hours) does not involve decreased GR protein expression. Regarding hormone binding, plasma levels of IFN-alpha have been found to be inversely associated with monocyte GR binding affinity in AIDS patients (Norbiato et al., 1996
). Nevertheless, although binding affinity was not directly measured in the current study, data demonstrating intact GR nuclear translocation following IFN-alpha plus DEX suggest that ligand-dependent conformational changes and interaction with relevant cytoskeletal elements following hormone binding (indirect reflections of affinity for hormone) were not disrupted (Galigniana et al., 1998
Several studies have examined changes in nuclear translocation of GR as a result of cytokine exposure. For example, both IL-1 and IL-2 have been shown to inhibit DEX-induced GR translocation in vitro (Goleva et al., 2002
; Pariante et al., 1999
; Wang et al., 2004
). In addition, IL-1 receptor deficient mice fail to develop stress-induced glucocorticoid resistance, which has been shown to be secondary to decreased GR translocation in mouse splenocytes and is associated with increased susceptibility to endotoxic shock (Engler et al., 2008
; Quan et al., 2001
). Inhibition of GR translocation by IL-1 has been found to involve p38 MAPK signaling pathways (Wang et al., 2004
). Indeed, treatment with the pharmacological p38 inhibitor, SB203580, as well as antisense oligonucleotides targeting p38 reversed IL-1-induced inhibition of GR-mediated gene transcription in LMCAT cells (Wang et al., 2004
). Interestingly, p38 has also been implicated in the effects of IL-2 and IL-4 on GR binding affinity in peripheral blood mononuclear cells (Irusen et al., 2002
). Although IFN-alpha has been shown to activate p38 MAPK signaling pathways (Platanias, 2005
), IFN-alpha had no effects on GR nuclear translocation, and inhibition of p38 MAPK did not significantly attenuate the effects of IFN-alpha on GR function, indicating that Jak-STAT signaling pathways may be more relevant mediators of mIFN-alpha’s effect on GR.
Many of the effects of IFN-alpha are mediated by Jak-STAT signaling, including primary viral defense and immunosurveillance for malignant cells (Caraglia et al., 2005
; Platanias, 2005
; Platanias and Fish, 1999
). Upon activation, type I IFN-alpha receptors dimerize within the lipid bilayer, permitting transphosphorylation of these receptors. IFN-alpha receptors, in turn, phosphorylate key Jak tyrosine residues. Phosphorylated Jak tyrosine moieties then recruit inactive STAT monomers from the cytoplasm such as STAT1, STAT2, and STAT5. These STATs are phosphorylated by activated Jaks, leading to STAT dimerization and subsequent nuclear translocation where these complexes function as transcription factors (Platanias, 2005
; Rogatsky and Ivashkiv, 2006
). With respect to GR, the data indicate that IFN-alpha-induced activation of Jak-STAT pathways led to inhibition of GR function through STAT5-GR protein-protein interactions. These data are consistent with similar results obtained following treatment of cells with IL-2 (Biola et al., 2001
; Goleva et al., 2002
). Unlike IFN-alpha however, inhibition of GR function by IL-2 (as noted above) appears to involve both impairment of GR translocation as well as direct nuclear protein-protein interactions between GR and STAT5 (Goleva et al., 2002
). Of note, prolactin has also been shown to disrupt DEX-induced MMTV-luciferase activity through STAT5-GR protein-protein interactions. Indeed, co-immunoprecipitation studies have demonstrated that GR and STAT5 interact within nuclear extracts obtained from cells treated with both prolactin and DEX (Stocklin et al., 1996
). Interestingly, while STAT5 inhibits DEX-induced GR-mediated gene transcription, activation of GR may not, in turn, inhibit STAT-5-mediated gene transcription. Such is the case for the STAT5-regulated gene beta-casein. For example, treatment of HC11 mammary epithelial cell with both prolactin and glucocorticoids results in increased expression of beta-casein mRNA compared to treatment with prolactin alone (Stocklin et al., 1996
). This synergism between GR and STAT5, leading to increased STAT5-mediated gene transcription, has also been described in liver cells in response to growth hormone (Engblom et al., 2007
). Finally, it should be noted that IFN-alpha led to increased nuclear localization of the GR (), which paradoxically was associated with reduced DEX-induced MMTV-luciferase activity (). These data suggest that while STAT5-GR interactions may increase GR protein in the nucleus, these interactions inhibit GR transcriptional activity, possibly through the disruption of binding of steroid receptor co-factors relevant to GR transcription.
Interestingly, while STAT5 appears to inhibit GR function, there were no effects of STAT1 or STAT2 on the GR in the current study. Of note, STAT1 has been shown to play a critical role in the induction of IFN-alpha responsive genes in the brain of mice following IFN-alpha administration (Wang et al., 2008
). Nevertheless, activation of Jak-STAT pathways by IFN-alpha appears to lead to both genomic and non-genomic effects, including the interaction of STAT proteins with other transcription factors including the GR. Such effects, including specifically the interaction of STAT5 with GR, may in turn influence downstream expression of relevant glucocorticoid responsive genes.
Although it remains to be determined whether the effects of IFN-alpha on GR function generalize to primary hippocampal neurons or neurons located in other brain regions as well as other cell types throughout the body, inhibition of GR by STAT5 in a cell line of hippocampal origin is of interest, given the role that hippocampal GR play in the regulation of HPA axis function (Herman et al., 2005
). Alterations in hippocampal morphology and function are associated with major depression (Duman et al., 1997
), and these changes are believed to contribute to the HPA axis abnormalities seen in depressed patients. Of note, our group has previously reported that robust HPA axis responses to the initial injection of IFN-alpha are associated with vulnerability to IFN-alpha-induced depression (Capuron et al., 2003
). Moreover, chronic IFN-alpha administration has been associated with flattening of the diurnal cortisol curve and increased evening plasma cortisol concentrations, both of which were correlated with depressive symptoms (Raison et al., 2008
). To the extent that impaired hippocampal GR-mediated feedback inhibition of CRH and HPA axis function may be involved in these effects (as reflected by altered responses to DEX challenge tests in both patients with major depression and patients with flattened diurnal cortisol curves) (Pariante, 2004
; Raison et al., 2008
; Spiegel et al., 2006
), the findings presented herein may shed light on possible molecular mechanisms behind IFN-alpha-induced neuroendocrine changes and depression in relevant clinical populations. Because Jak-STAT signaling may underlie such effects, inhibition of STAT proteins including STAT5 and/or Jak-STAT signaling pathways may represent a possible therapeutic strategy for normalizing GR function and mood alterations in patients chronically exposed to IFN-alpha or other innate immune cytokines.
In conclusion, treatment of a mouse hippocampal cell line with mIFN-alpha disrupts DEX-induced activity of GR. Because IFN-alpha is widely used as immunotherapy for various diseases, and because studies have suggested that IFN-alpha-induced depression is associated with altered HPA axis function, the data provide potentially important insights into the pathophysiological mechanism(s) that mediate these effects. Disruption of Jak-STAT signaling upstream of STAT5, as well as disruption of STAT5 gene expression abrogated the effects of mIFN-alpha on GR function. Thus, STAT5, like p38 MAPK (Wang et al., 2004
) and Jun N-terminal kinase (Wang et al., 2005
), may be an important contributor to impaired GR signaling (and its relationship to mood disorders)(Raison and Miller, 2003
), and therefore may be a relevant factor in the pathophysiology of major depression that occurs in patients with increased inflammation.