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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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Retention of GR on the cytoskeleton requires vimentin and nestin.(A1–A2) The Vim expressing subclone C6D8 accumulates GR in the cytoplasm. Cytoplasmic GR colocalizes with Nes. (B1) GR in the Vim deficient subclone C6D10 is only nuclear. (B2) A proportion of the Vim negative C6D10 cells expresses Nes. (C) Expression of an exogenous mouse Vim protein in Vim deficient cells restores cytoplasmic accumulation of GR and colocalization of GR, Nes, and Vim. Bars in A–C = 5 µm.
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pone-0006084-g004: Retention of GR on the cytoskeleton requires vimentin and nestin.(A1–A2) The Vim expressing subclone C6D8 accumulates GR in the cytoplasm. Cytoplasmic GR colocalizes with Nes. (B1) GR in the Vim deficient subclone C6D10 is only nuclear. (B2) A proportion of the Vim negative C6D10 cells expresses Nes. (C) Expression of an exogenous mouse Vim protein in Vim deficient cells restores cytoplasmic accumulation of GR and colocalization of GR, Nes, and Vim. Bars in A–C = 5 µm.

Mentions: To determine the need of nestin and vimentin for GR anchoring we analyzed cell lines varying in IF-protein composition. Rat C6 subclones which either express vimentin or completely lack cytoplasmic IFs [31], [32] were used to determine this relationship in detail. GR was present in the cytoplasm of C6D8 cells, a subclone which contains an IF network composed of vimentin and nestin (Figure 4, A1). In contrast, GR was strictly nuclear in C6D10 cells, which lack cytoplasmic IFs (Figure 4, B1). About 40% of the cells in a C6D10 culture displayed a diffuse nestin staining in the cytoplasm (Figure 4, B2). C6D10 cells contained significantly lower amounts of nestin than vimentin positive cells (Figure 5A). To determine an association of GR with the cytoskeleton the cells were lysed in situ by use of the detergent Triton-X100. GR staining was retained on cytoskeletons of extracted C6D8 cells (Figure 4, A2). Confocal microscopy and colocalization analysis pointed to coincident staining patterns of GR and vimentin in these cells (Figure S1, A1–A3). In contrast, neither tubulin (Figure S1, B1–B2) nor actin (Figure S1, C1–C2) colocalized with GR, as indicated by the broad scatter diagrams. We subjected C6D8 cells to treatment with cytochalasin B, a drug which disrupts the actin filament system, but leaves the vimentin filament system intact. In these cells the GR label still colocalized with vimentin filaments and did not redistribute with actin into patches (Figure S2).


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)

Retention of GR on the cytoskeleton requires vimentin and nestin.(A1–A2) The Vim expressing subclone C6D8 accumulates GR in the cytoplasm. Cytoplasmic GR colocalizes with Nes. (B1) GR in the Vim deficient subclone C6D10 is only nuclear. (B2) A proportion of the Vim negative C6D10 cells expresses Nes. (C) Expression of an exogenous mouse Vim protein in Vim deficient cells restores cytoplasmic accumulation of GR and colocalization of GR, Nes, and Vim. Bars in A–C = 5 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006084-g004: Retention of GR on the cytoskeleton requires vimentin and nestin.(A1–A2) The Vim expressing subclone C6D8 accumulates GR in the cytoplasm. Cytoplasmic GR colocalizes with Nes. (B1) GR in the Vim deficient subclone C6D10 is only nuclear. (B2) A proportion of the Vim negative C6D10 cells expresses Nes. (C) Expression of an exogenous mouse Vim protein in Vim deficient cells restores cytoplasmic accumulation of GR and colocalization of GR, Nes, and Vim. Bars in A–C = 5 µm.
Mentions: To determine the need of nestin and vimentin for GR anchoring we analyzed cell lines varying in IF-protein composition. Rat C6 subclones which either express vimentin or completely lack cytoplasmic IFs [31], [32] were used to determine this relationship in detail. GR was present in the cytoplasm of C6D8 cells, a subclone which contains an IF network composed of vimentin and nestin (Figure 4, A1). In contrast, GR was strictly nuclear in C6D10 cells, which lack cytoplasmic IFs (Figure 4, B1). About 40% of the cells in a C6D10 culture displayed a diffuse nestin staining in the cytoplasm (Figure 4, B2). C6D10 cells contained significantly lower amounts of nestin than vimentin positive cells (Figure 5A). To determine an association of GR with the cytoskeleton the cells were lysed in situ by use of the detergent Triton-X100. GR staining was retained on cytoskeletons of extracted C6D8 cells (Figure 4, A2). Confocal microscopy and colocalization analysis pointed to coincident staining patterns of GR and vimentin in these cells (Figure S1, A1–A3). In contrast, neither tubulin (Figure S1, B1–B2) nor actin (Figure S1, C1–C2) colocalized with GR, as indicated by the broad scatter diagrams. We subjected C6D8 cells to treatment with cytochalasin B, a drug which disrupts the actin filament system, but leaves the vimentin filament system intact. In these cells the GR label still colocalized with vimentin filaments and did not redistribute with actin into patches (Figure S2).

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