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Nestin modulates glucocorticoid receptor function by cytoplasmic anchoring.

Reimer R, Helmbold H, Szalay B, Hagel C, Hohenberg H, Deppert W, Bohn W - PLoS ONE (2009)

Bottom Line: The reaction pattern with phospho-GR specific antibodies and the presence of the chaperone HSC70 suggest that specifically the unliganded receptor is anchored to the IF system.Ligand addition releases GR from IFs and shifts the receptor into the nucleus.The data give evidence that nestin/vimentin specific anchoring modulates growth suppression by GR.

View Article: PubMed Central - PubMed

Affiliation: Heinrich-Pette-Institute for Experimental Virology and Immunology at the University of Hamburg, Hamburg, Germany.

ABSTRACT
Nestin is the characteristic intermediate filament (IF) protein of rapidly proliferating progenitor cells and regenerating tissue. Nestin copolymerizes with class III IF-proteins, mostly vimentin, into heteromeric filaments. Its expression is downregulated with differentiation. Here we show that a strong nestin expression in mouse embryo tissue coincides with a strong accumulation of the glucocorticoid receptor (GR), a key regulator of growth and differentiation in embryonic development. Microscopic studies on cultured cells show an association of GR with IFs composed of vimentin and nestin. Cells lacking nestin, but expressing vimentin, or cells expressing vimentin, but lacking nestin accumulate GR in the nucleus. Completing these networks with an exogenous nestin, respectively an exogenous vimentin restores cytoplasmic anchoring of GR to the IF system. Thus, heteromeric filaments provide the basis for anchoring of GR. The reaction pattern with phospho-GR specific antibodies and the presence of the chaperone HSC70 suggest that specifically the unliganded receptor is anchored to the IF system. Ligand addition releases GR from IFs and shifts the receptor into the nucleus. Suppression of nestin by specific shRNA abolishes anchoring of GR, induces its accumulation in the nucleus and provokes an irreversible G1/S cell cycle arrest. Suppression of GR prior to that of nestin prevents entry into the arrest. The data give evidence that nestin/vimentin specific anchoring modulates growth suppression by GR. We hypothesize that expression of nestin is a major determinant in suppression of anti-proliferative activity of GR in undifferentiated tissue and facilitates activation of this growth control in a precise tissue and differentiation dependent manner.

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GR colocalizes with nestin and vimentin in embryonic mouse fibroblasts.Fibroblasts were explanted from Balb/C embryos (E 14.5). (A1–A3) Coincident labelling of cytoplasmic GR and Vim is confined to a subpopulation of cells. (B1–B3) Cytoplasmic accumulation of GR identifies Nes expressing cells. (C1–C3) Incubation with the GR ligand dexamethasone (100 nM) abolishes co-staining of GR with Nes to the benefit of a nuclear GR staining. Bar in Fig. 3, A–C = 5 µm.
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pone-0006084-g003: GR colocalizes with nestin and vimentin in embryonic mouse fibroblasts.Fibroblasts were explanted from Balb/C embryos (E 14.5). (A1–A3) Coincident labelling of cytoplasmic GR and Vim is confined to a subpopulation of cells. (B1–B3) Cytoplasmic accumulation of GR identifies Nes expressing cells. (C1–C3) Incubation with the GR ligand dexamethasone (100 nM) abolishes co-staining of GR with Nes to the benefit of a nuclear GR staining. Bar in Fig. 3, A–C = 5 µm.

Mentions: We next asked if the coincident presence of nestin and GR is a stable phenotypic marker that is maintained after explantation of primary cells in tissue culture. Mouse embryo fibroblasts [29] were isolated from BALB/c embryos (E 14.5). At the 2nd passage after explantation about 5 to 20% of the cells in a culture showed a coincident staining of nestin and GR, and both colocalized with the vimentin filament system. (Figure 3, A and B). In nestin negative cells the receptor localized to the nucleus (Figure 3, B1, arrows). Addition of the GR ligand dexamethasone abolished the GR signal on the nestin/vimentin filament system and induced an accumulation of GR in the nucleus (Figure 3C). The proportion of nestin expressing cells declined gradually during passaging, decreasing to less than 1% nestin positive cells at the 6th passage, whereas vimentin expression was maintained. Concomitant with downregulation of nestin, the cells accumulated GR in the nucleus, adopted a senescent phenotype characterized by a flattened cell shape, positive staining for senescence associated β-galactosidase and entry into a sustained growth arrest (data not shown).


Nestin modulates glucocorticoid receptor function by cytoplasmic anchoring.

Reimer R, Helmbold H, Szalay B, Hagel C, Hohenberg H, Deppert W, Bohn W - PLoS ONE (2009)

GR colocalizes with nestin and vimentin in embryonic mouse fibroblasts.Fibroblasts were explanted from Balb/C embryos (E 14.5). (A1–A3) Coincident labelling of cytoplasmic GR and Vim is confined to a subpopulation of cells. (B1–B3) Cytoplasmic accumulation of GR identifies Nes expressing cells. (C1–C3) Incubation with the GR ligand dexamethasone (100 nM) abolishes co-staining of GR with Nes to the benefit of a nuclear GR staining. Bar in Fig. 3, A–C = 5 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2698154&req=5

pone-0006084-g003: GR colocalizes with nestin and vimentin in embryonic mouse fibroblasts.Fibroblasts were explanted from Balb/C embryos (E 14.5). (A1–A3) Coincident labelling of cytoplasmic GR and Vim is confined to a subpopulation of cells. (B1–B3) Cytoplasmic accumulation of GR identifies Nes expressing cells. (C1–C3) Incubation with the GR ligand dexamethasone (100 nM) abolishes co-staining of GR with Nes to the benefit of a nuclear GR staining. Bar in Fig. 3, A–C = 5 µm.
Mentions: We next asked if the coincident presence of nestin and GR is a stable phenotypic marker that is maintained after explantation of primary cells in tissue culture. Mouse embryo fibroblasts [29] were isolated from BALB/c embryos (E 14.5). At the 2nd passage after explantation about 5 to 20% of the cells in a culture showed a coincident staining of nestin and GR, and both colocalized with the vimentin filament system. (Figure 3, A and B). In nestin negative cells the receptor localized to the nucleus (Figure 3, B1, arrows). Addition of the GR ligand dexamethasone abolished the GR signal on the nestin/vimentin filament system and induced an accumulation of GR in the nucleus (Figure 3C). The proportion of nestin expressing cells declined gradually during passaging, decreasing to less than 1% nestin positive cells at the 6th passage, whereas vimentin expression was maintained. Concomitant with downregulation of nestin, the cells accumulated GR in the nucleus, adopted a senescent phenotype characterized by a flattened cell shape, positive staining for senescence associated β-galactosidase and entry into a sustained growth arrest (data not shown).

Bottom Line: The reaction pattern with phospho-GR specific antibodies and the presence of the chaperone HSC70 suggest that specifically the unliganded receptor is anchored to the IF system.Ligand addition releases GR from IFs and shifts the receptor into the nucleus.The data give evidence that nestin/vimentin specific anchoring modulates growth suppression by GR.

View Article: PubMed Central - PubMed

Affiliation: Heinrich-Pette-Institute for Experimental Virology and Immunology at the University of Hamburg, Hamburg, Germany.

ABSTRACT
Nestin is the characteristic intermediate filament (IF) protein of rapidly proliferating progenitor cells and regenerating tissue. Nestin copolymerizes with class III IF-proteins, mostly vimentin, into heteromeric filaments. Its expression is downregulated with differentiation. Here we show that a strong nestin expression in mouse embryo tissue coincides with a strong accumulation of the glucocorticoid receptor (GR), a key regulator of growth and differentiation in embryonic development. Microscopic studies on cultured cells show an association of GR with IFs composed of vimentin and nestin. Cells lacking nestin, but expressing vimentin, or cells expressing vimentin, but lacking nestin accumulate GR in the nucleus. Completing these networks with an exogenous nestin, respectively an exogenous vimentin restores cytoplasmic anchoring of GR to the IF system. Thus, heteromeric filaments provide the basis for anchoring of GR. The reaction pattern with phospho-GR specific antibodies and the presence of the chaperone HSC70 suggest that specifically the unliganded receptor is anchored to the IF system. Ligand addition releases GR from IFs and shifts the receptor into the nucleus. Suppression of nestin by specific shRNA abolishes anchoring of GR, induces its accumulation in the nucleus and provokes an irreversible G1/S cell cycle arrest. Suppression of GR prior to that of nestin prevents entry into the arrest. The data give evidence that nestin/vimentin specific anchoring modulates growth suppression by GR. We hypothesize that expression of nestin is a major determinant in suppression of anti-proliferative activity of GR in undifferentiated tissue and facilitates activation of this growth control in a precise tissue and differentiation dependent manner.

Show MeSH
Related in: MedlinePlus