Cell Cycle Phase Regulates Glucocorticoid Receptor Function

Matthews, Laura; Johnson, James R.; Berry, Andrew; Trebble, Peter; Cookson, Ann; Spiller, David G.; Rivers, Caroline A; Norman, M. R.; White, Mike; Ray, David

doi:10.1371/journal.pone.0022289

Cited by 30 publications

(37 citation statements)

References 45 publications

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“…Immunoblot analysis and reporter gene assays were performed as described by Matthews et al (11). Purification of mitotic spindle fractions was as described by Sauer et al (17), real-time fluorescent cell imaging was performed as described by Trebble et al (49), and quantitative PCR analysis was as described by Poolman et al (50).…”

Section: Methodsmentioning

confidence: 99%

“…In mitosis, GR is phosphorylated on both S203 and S211, but in a ligand-independent manner (11), and more comprehensive phosphoproteomic analyses identify the presence of multiple N-terminal GR phosphoforms in purified mitotic spindle fractions (12). Although altered GR function in mitosis has been shown (11,13,14), the kinases responsible and cellular consequences have not been defined.…”

mentioning

confidence: 99%

“…Although altered GR function in mitosis has been shown (11,13,14), the kinases responsible and cellular consequences have not been defined. The role of GR in cancer has received little attention beyond acute lymphoblastic leukemia, in which GR is powerfully proapoptotic (15).…”

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Glucocorticoid receptor regulates accurate chromosome segregation and is associated with malignancy

Matthews

Berry

Morgan

et al. 2015

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

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The glucocorticoid receptor (GR) is a member of the nuclear receptor superfamily, which controls programs regulating cell proliferation, differentiation, and apoptosis. We have identified an unexpected role for GR in mitosis. We discovered that specifically modified GR species accumulate at the mitotic spindle during mitosis in a distribution that overlaps with Aurora kinases. We found that Aurora A was required to mediate mitosis-driven GR phosphorylation, but not recruitment of GR to the spindle. GR was necessary for mitotic progression, with increased time to complete mitosis, frequency of mitotic aberrations, and death in mitosis observed following GR knockdown. Complementation studies revealed an essential role for the GR ligand-binding domain, but no clear requirement for ligand binding in regulating chromosome segregation. The GR N-terminal domain, and specifically phosphosites S203 and S211, were not required. Reduced GR expression results in a cell cycle phenotype, with isolated cells from mouse and human subjects showing changes in chromosome content over prolonged passage. Furthermore, GR haploinsufficient mice have an increased incidence of tumor formation, and, strikingly, these tumors are further depleted for GR, implying additional GR loss as a consequence of cell transformation. We identified reduced GR expression in a panel of human liver, lung, prostate, colon, and breast cancers. We therefore reveal an unexpected role for the GR in promoting accurate chromosome segregation during mitosis, which is causally linked to tumorigenesis, making GR an authentic tumor suppressor gene.glucocorticoid receptor | mitosis | aneuploidy | DNA damage | cancer G lucocorticoids (Gcs) act through the glucocorticoid receptor (GR), a member of the nuclear hormone receptor superfamily, and a ligand-activated transcription factor (1-4). The GR is ubiquitously expressed and regulates energy metabolism, immunity, and cell fate decisions. The quiescent GR resides in the cytoplasm in a complex with heat shock proteins and immunophilins, attached to the microtubule architecture of the cell in a heat shock protein 90-dependent manner (5). Ligand binding drives GR transformation, involving N-terminal phosphorylation on S203 and S211 and rapid translocation to the nucleus requiring attachment to dynein by heat shock protein 90, immunophilins, and dynamitin (6). Once in the nucleus, GR binds directly to DNA to regulate transcription, or tethers to other DNA-bound transcription factors, such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and activator protein 1 (AP1), to regulate their function (7-10).In mitosis, GR is phosphorylated on both S203 and S211, but in a ligand-independent manner (11), and more comprehensive phosphoproteomic analyses identify the presence of multiple N-terminal GR phosphoforms in purified mitotic spindle fractions (12). Although altered GR function in mitosis has been shown (11, 13, 14), the kinases responsible and cellular consequences have not been defined. The role of G...

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Glucocorticoid receptor regulates accurate chromosome segregation and is associated with malignancy

Matthews

Berry

Morgan

et al. 2015

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

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“…It is excluded from the nucleus upon the initiation of mitosis through early G 1 . This is likely due to changes in GR phosphorylation in the AF-1 domain by CDK proteins, whose activity changes based on the cell cycle (55). Moreover, these cells have been shown to be resistant to dexamethasone immediately following cell division, and as a result GR failed to translocate (41,55).…”

Section: Discussionmentioning

confidence: 99%

Endothelial nuclear lamina is not required for glucocorticoid receptor nuclear import but does affect receptor-mediated transcription activation

Nayebosadri

2013

American Journal of Physiology-Cell Physiology

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Nayebosadri A, Ji JY. Endothelial nuclear lamina is not required for glucocorticoid receptor nuclear import but does affect receptor-mediated transcription activation. Am J Physiol Cell Physiol 305: C309 -C322, 2013. First published May 22, 2013; doi:10.1152/ajpcell.00293.2012The lamina serves to maintain the nuclear structure and stiffness while acting as a scaffold for heterochromatin and many transcriptional proteins. Its role in endothelial mechanotransduction, specifically how nuclear mechanics impact gene regulation under shear stress, is not fully understood. In this study, we successfully silenced lamin A/C in bovine aortic endothelial cells to determine its role in both glucocorticoid receptor (GR) nuclear translocation and glucocorticoid response element (GRE) transcriptional activation in response to dexamethasone and shear stress. Nuclear translocation of GR, an antiinflammatory nuclear receptor, in response to dexamethasone or shear stress (5, 10, and 25 dyn/cm 2 ) was observed via time-lapse cell imaging and quantified using a Bayesian image analysis algorithm. Transcriptional activity of the GRE promoter was assessed using a dual-luciferase reporter plasmid. We found no dependence on nuclear lamina for GR translocation from the cytoplasm into the nucleus. However, the absence of lamin A/C led to significantly increased expression of luciferase under dexamethasone and shear stress induction as well as changes in histone protein function. PCR results for NF-B inhibitor alpha (NF-BIA) and dual specificity phosphatase 1 (DUSP1) genes further supported our luciferase data with increased expression in the absence of lamin. Our results suggest that absence of lamin A/C does not hinder passage of GR into the nucleus, but nuclear lamina is important to properly regulate GRE transcription. Nuclear lamina, rather than histone deacetylase (HDAC), is a more significant mediator of shear stress-induced transcriptional activity, while dexamethasone-initiated transcription is more HDAC dependent. Our findings provide more insights into the molecular pathways involved in nuclear mechanotransduction. glucocorticoid receptor; nuclear lamina; shear stress; dexamethasone; endothelial cells CARDIOVASCULAR DISEASES SUCH as atherosclerosis are the leading cause of death in the United States. The initiation and development of atherosclerosis are typically associated with endothelial dysfunction (20,51). Endothelial cells at the lumen of blood vessels are continuously exposed to both hemodynamic shear stress and cyclic stretch due to blood flow (16). The distinct local hemodynamic profiles influence endothelial behaviors in the vasculature. Atherosclerotic lesions tend to develop at regions of disturbed flow and low shear stress at bifurcation sites and curvatures, while regions with uniform high laminar shear stress are protected (10,48,66). Endothelial responses to shear stress can be categorized as immediate [rapid release of nitric oxide (NO); (49)] as well as short-term

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“…The process of translocation to the nucleus post ligand binding occurs rapidly, with the majority of cellular GR being nuclear 30 minutes after treatment with 100 nM Dex (Nishi et al, 1999). In addition cell cycle phase is able to regulate the subcellular localisation of unliganded GR, but with far slower kinetics of nuclear accumulation (Matthews et al, 2011). In the nucleus GR binds to cis-elements to activate or repress target gene expression, recruiting co-modulator proteins from distinct classes to effect chromatin remodelling, and recruitment of the basal transcriptional machinery (Ford et al, 1997;Jones and Shi, 2003;Ito et al, 2006;Johnson et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

A ligand-specific kinetic switch regulates glucocorticoid receptor trafficking and function

Trebble

Woolven

Saunders

et al. 2013

Journal of Cell Science

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SummaryThe ubiquitously expressed glucocorticoid receptor (GR) is a major drug target for inflammatory disease, but issues of specificity and target tissue sensitivity remain. We now identify high potency, non-steroidal GR ligands, GSK47867A and GSK47869A, which induce a novel conformation of the GR ligand-binding domain (LBD) and augment the efficacy of cellular action. Despite their high potency, GSK47867A and GSK47869A both induce surprisingly slow GR nuclear translocation, followed by prolonged nuclear GR retention, and transcriptional activity following washout. We reveal that GSK47867A and GSK47869A specifically alter the GR LBD structure at the HSP90-binding site. The alteration in the HSP90-binding site was accompanied by resistance to HSP90 antagonism, with persisting transactivation seen after geldanamycin treatment. Taken together, our studies reveal a new mechanism governing GR intracellular trafficking regulated by ligand binding that relies on a specific surface charge patch within the LBD. This conformational change permits extended GR action, probably because of altered GR-HSP90 interaction. This chemical series may offer anti-inflammatory drugs with prolonged duration of action due to altered pharmacodynamics rather than altered pharmacokinetics.

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Cell Cycle Phase Regulates Glucocorticoid Receptor Function

Cited by 30 publications

References 45 publications

Glucocorticoid receptor regulates accurate chromosome segregation and is associated with malignancy

Glucocorticoid receptor regulates accurate chromosome segregation and is associated with malignancy

Endothelial nuclear lamina is not required for glucocorticoid receptor nuclear import but does affect receptor-mediated transcription activation

A ligand-specific kinetic switch regulates glucocorticoid receptor trafficking and function

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