Subnuclear Trafficking of Glucocorticoid Receptors In Vitro: Chromatin Recycling and Nuclear Export

Yang, Jun; Liu, Jimin; DeFranco, Donald B.

doi:10.1083/jcb.137.3.523

Cited by 96 publications

(62 citation statements)

References 92 publications

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“…52,53 It is noteworthy that tyrosine phosphatase inhibitors stimulated chromatin-released GR export from nuclei, which suggests the involvement of a phosphotyrosine system in the regulation of nuclear GR export. 54 In agreement with these data, it has been reported that treatment of A549 cells with TNF-␣ targeted GR, possibly by phosphorylation, to remain in cytoplasm. 55 However, TNF-␣ increased the GR number and glucocorticoidinduced transcriptional activity of the GR in L-929 mouse fibroblast, 56 which is in contradiction with the above data.…”

Section: Discussionsupporting

confidence: 77%

Glucocorticoid receptor down-Regulates c-jun amino terminal kinases induced by tumor necrosis factor ? in fetal rat hepatocyte primary cultures

et al. 1999

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The effect of dexamethasone on Jun N-terminal kinase (JNK) activity was assayed by using fetal hepatocytes in primary culture. The addition of tumor necrosis factor ␣ (TNF-␣) caused an increase in JNK in a dose-and timedependent manner. We show that activation of JNK by this extracellular signal is inhibited by dexamethasone in a dose-dependent fashion. This inhibitory effect was observed in cells treated for 10 minutes with dexamethasone in the presence of protein phosphatase inhibitors such as orthovanadate or okadaic acid, or in cells previously treated with actinomycin D. Glucocorticoid receptor (GR) can be precipitated with the fusion protein, GST-c-Jun (1-79), bound to agarose beads. However, the inhibitory effect of glucocorticoids on JNK activity was also observed using ATF-2 as substrate. In addition, dexamethasone inhibits JNK phosphorylation induced by TNF-␣. Finally, we show that GR can also be phosphorylated in tyrosine residues in response to TNF-␣ and epidermal growth factor (EGF) upon ligand-binding. Our results suggest that the antiinflammatory effect of glucocorticoids on the inflammatory pathways induced by TNF-␣ can be explained, at least in part, by modulating JNK activity through a direct proteinprotein interaction; the JNK phosphorylation and tyrosinephosphorylation state of GR may be regulatory steps also involved in that effect. (HEPATOLOGY 1999;29:849-857.)

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Section: Discussionsupporting

confidence: 77%

Glucocorticoid receptor down-Regulates c-jun amino terminal kinases induced by tumor necrosis factor ? in fetal rat hepatocyte primary cultures

et al. 1999

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“…Thus, the seemingly oppositional effects of vanadate on GR that we report are not unique to our cells or receptor, suggesting that the signal mechanism which mediates the vanadate stimulus is conserved, at least between human, simian and murine cells. That vanadate can actually stimulate tyrosine phosphorylation while modulating GR action has been shown by DeFranco and co-workers [34]. In this work, molybdate or vanadate treatment of permeabilized cells caused both an increase in overall tyrosine phosphorylation of proteins within the nucleus and stimulation of nuclear export of hormone-free receptors.…”

Section: Discussionsupporting

confidence: 65%

Vanadate increases glucocorticoid receptor-mediated gene expression: a novel mechanism for potentiation of a steroid receptor

Calzi

Periyasamy

et al. 2002

The Journal of Steroid Biochemistry and Molecular Biology

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Transition metal oxyanions, such as molybdate, tungstate and vandadate, have been shown to prevent in vitro hormone-induced activation of the glucocorticoid receptor (GR) by blocking dissociation of the GR/heat shock protein heterocomplex. In this work, we report a novel effect of vanadate: in vivo potentiation of GR-mediated gene expression. In cells stably-transfected with complex (mouse mammary tumor virus (MMTV)) or minimal GR-regulated CAT reporters, treatment with 500 M vanadate caused CAT gene expression to dramatically increase, even at saturating concentrations of dexamethasone; while no such effect was seen in response to RU486 antagonist. Similar treatment with molybdate had no effect on GR activity, suggesting that the response to vanadate was not a general property of transition metal oxyanions. Treatment with vanadate after hormone-induced nuclear translocation of the GR also caused potentiation, demonstrating that vanadate was acting on a post-transformation event, perhaps by affecting the transactivation function of DNA-bound GR. Paradoxically, vanadate caused an apparent but temporary "loss" of GR protein immediately after treatment (as measured by loss of reactivity to BuGR2 antibody and of hormone-binding capacity) that returned to normal at approximately 8 h post-treatment, suggesting that potentiation of GR transactivation function (as measured by our CAT assays) was probably occurring during the later stages (8-24 h) of this assay. However, gel shift analyses revealed that vanadate could induce binding of the hormone-free GR to glucocorticoid response element (GRE)-containing oligonucleotides immediately after treatment. Thus, the rapid vanadate-induced "loss" of GR was not due to degradation of GR protein. Yet, vanadate in the absence of hormone had no effect on CAT reporter expression, demonstrating that this form of the GR still requires agonist for its enhanced transcriptional activity. As an indication of the potential mechanism of vanadate action, vanadate was found to dramatically stimulate the mitogen-activated protein kinases, ERK-1 and ERK-2. In addition, vanadate potentiation of GR reporter gene expression was completely blocked by the tyrosine kinase inhibitor herbimycin A. Taken as a whole, our results suggest that vanadate can have dramatic and complex effects on GR structure and function, resulting in hormone-free activation of GR DNA-binding function, as well as alterations to the BuGR2 epitope and hormone-binding domains-while at the same time stimulating tyrosine phosphorylation pathways controlling GR-mediated gene transcription.

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“…Digitonin-permeabilization and hypotonic buffer extraction of 3617.4 cells were performed essentially as described (19). Briefly, cells were rinsed with PBS and then transport buffer (20 mM Hepes-KOH, pH 7.8͞110 mM potassium acetate͞5 mM sodium acetate͞2 mM magnesium acetate͞1 mM EGTA).…”

Section: Methodsmentioning

confidence: 99%

“…We set out to develop an in situ assay for nuclear protein mobility that would maintain the transcriptional competence of isolated nuclei yet provide a system where the effects of specific subnuclear trafficking factors could be assessed. Previously, we used a modified hypotonic buffer extraction of digitonin-permeabilized cells (24) to reveal differential nuclear affinities of ligand-bound vs. unliganded nuclear GR (19). This technique was applied herein to mouse 3617.4 and 5953 cell lines, which contain an integrated copy of a GFP-GR and -PR chimera, respectively, under the control of a tet-regulated promoter (17,25).…”

Section: In Situ Assay To Assess Gr Mobility In Transcriptionally Commentioning

confidence: 99%

Molecular chaperones function as steroid receptor nuclear mobility factors

Elbi

Walker

Romero

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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137

118

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Live cell imaging has revealed the rapid mobility of steroid hormone receptors within nuclei and their dynamic exchange at transcriptionally active target sites. Although a number of other proteins have been shown to be highly mobile within nuclei, the identity of soluble factors responsible for orchestrating nuclear trafficking remains unknown. We have developed a previously undescribed in situ subnuclear trafficking assay that generates transcriptionally active nuclei, which are depleted of soluble factors required for the nuclear mobility of glucocorticoid (GR) and progesterone receptors (PR). Using this system and a fluorescence recovery after photobleaching technique, we demonstrate that nuclear mobility of GR recovered on incubation with reticulocyte lysate was inhibited by geldanamycin, a drug that blocks the chaperone activity of heat-shock protein 90. Direct proof of molecular chaperone involvement in steroid receptor subnuclear trafficking was provided by the ATP-dependent recovery of nuclear mobility of GR and PR on incubation with various combinations of purified chaperone and͞or cochaperone proteins. Additionally, for both receptors, the inclusion of hormone during the recovery period leads to a retardation of nuclear mobility. Thus, our results provide a description of soluble nuclear mobility factors and furthermore demonstrate a previously unrecognized role for molecular chaperones in the regulation of steroid receptor function within the nucleus.O ur understanding of the mechanisms of protein trafficking has been greatly aided by the advent of technologies that allow the movement of individual proteins to be visualized in live cells (1, 2). When applied to the assessment of nuclear protein trafficking, such techniques have uniquely revealed details of protein movement within a single subcellular compartment (3-6). Although protein mobility within the nucleus can be diffusion-limited, low-or high-affinity interactions with specific soluble or solid-state targets can reduce the kinetics of nuclear protein trafficking (7). Nonetheless, nuclear proteins that are components of large macromolecular complexes are highly mobile and possess fast diffusion coefficients (7).The nuclear receptor superfamily of proteins act as transcription factors with activities regulated by ligand binding (8,9). Once associated with their target genes through direct interactions with specific DNA sequences or tethering to other DNA-bound factors, nuclear receptors recruit large coregulator complexes to linked promoters (10, 11). These coregulators include coactivator and corepressor complexes that modulate RNA polymerase II activity in part through direct modification of histone proteins within core nucleosomes (12)(13)(14). The interaction of specific components (e.g., SRC-1) of coactivator complexes with a ligand-bound nuclear receptor (e.g., estrogen receptor) reduces their mobility within live cell nuclei, perhaps reflecting the assembly of functional coactivator͞nuclear receptor complexes on active target genes (15)....

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Subnuclear Trafficking of Glucocorticoid Receptors In Vitro: Chromatin Recycling and Nuclear Export

Cited by 96 publications

References 92 publications

Glucocorticoid receptor down-Regulates c-jun amino terminal kinases induced by tumor necrosis factor ? in fetal rat hepatocyte primary cultures

Glucocorticoid receptor down-Regulates c-jun amino terminal kinases induced by tumor necrosis factor ? in fetal rat hepatocyte primary cultures

Vanadate increases glucocorticoid receptor-mediated gene expression: a novel mechanism for potentiation of a steroid receptor

Molecular chaperones function as steroid receptor nuclear mobility factors

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