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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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Visualization of the cytoskeleton architecture by EM replica technique.C6D8 cells were grown on glass coverslips, extracted with a non-ionic detergent, fixed with aldehyde, critical point dried, rotary shadowed with platinum/carbon (A) respectively carbon (B), detached from the glass, and transferred onto EM grids. The micrographs reveal maintenance of the 3D organization of the networks with this technique.
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pone-0006084-g008: Visualization of the cytoskeleton architecture by EM replica technique.C6D8 cells were grown on glass coverslips, extracted with a non-ionic detergent, fixed with aldehyde, critical point dried, rotary shadowed with platinum/carbon (A) respectively carbon (B), detached from the glass, and transferred onto EM grids. The micrographs reveal maintenance of the 3D organization of the networks with this technique.

Mentions: To proof unequivocally that GR is bound to individual IF filaments we assayed its localization at an ultrastructural level by electron microscopy. We did an immunogold labelling analysis on cultured C6D8 cells by replica EM. The cells were extracted with detergent under controlled conditions, fixed, labelled with antibodies, critical point dried and shadowed with platinum carbon or only carbon[37], [38]. The technique fills the gap between low resolution light microscopy and high resolution electron microscopy and is highly suited to understand changes in cytoskeleton architecture in relation to dynamic processes in the cell [39]. As shown in Figure 8 the procedure well preserved the 3D organization of the filament network. Immunogold labelling combined with carbon shadowing enabled us to distinguish vimentin filaments (10 nm immunogold particles) from other filament types, such as actin filaments (5 nm immunogold particles) (Figure 9A). Double labelling of GR (5 nm immunogold particles) and vimentin (10 nm immunogold particles) revealed an association of the GR label specifically with the vimentin containing filaments (Figure 9, B1–B3). The results suggest that GR is part of a larger complex bound to distinct sites at the vimentin/nestin filaments.


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)

Visualization of the cytoskeleton architecture by EM replica technique.C6D8 cells were grown on glass coverslips, extracted with a non-ionic detergent, fixed with aldehyde, critical point dried, rotary shadowed with platinum/carbon (A) respectively carbon (B), detached from the glass, and transferred onto EM grids. The micrographs reveal maintenance of the 3D organization of the networks with this technique.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006084-g008: Visualization of the cytoskeleton architecture by EM replica technique.C6D8 cells were grown on glass coverslips, extracted with a non-ionic detergent, fixed with aldehyde, critical point dried, rotary shadowed with platinum/carbon (A) respectively carbon (B), detached from the glass, and transferred onto EM grids. The micrographs reveal maintenance of the 3D organization of the networks with this technique.
Mentions: To proof unequivocally that GR is bound to individual IF filaments we assayed its localization at an ultrastructural level by electron microscopy. We did an immunogold labelling analysis on cultured C6D8 cells by replica EM. The cells were extracted with detergent under controlled conditions, fixed, labelled with antibodies, critical point dried and shadowed with platinum carbon or only carbon[37], [38]. The technique fills the gap between low resolution light microscopy and high resolution electron microscopy and is highly suited to understand changes in cytoskeleton architecture in relation to dynamic processes in the cell [39]. As shown in Figure 8 the procedure well preserved the 3D organization of the filament network. Immunogold labelling combined with carbon shadowing enabled us to distinguish vimentin filaments (10 nm immunogold particles) from other filament types, such as actin filaments (5 nm immunogold particles) (Figure 9A). Double labelling of GR (5 nm immunogold particles) and vimentin (10 nm immunogold particles) revealed an association of the GR label specifically with the vimentin containing filaments (Figure 9, B1–B3). The results suggest that GR is part of a larger complex bound to distinct sites at the vimentin/nestin filaments.

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