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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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Cytoplasmic localization of GR depends on the presence of nestin.(A) Suppression of Nes expression in C6D8 cells by transfection with nestin specific siRNA abolishes cytoplasmic GR staining; only nuclear GR staining is left. (C) GR is only nuclear in rat 1 cells which contain vimentin, but lack nestin. (D) Ectopic expression of nestin in rat1 cells induces cytoplasmic accumulation of GR. Bars in A, B, C, F, and G = 5 µm.
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pone-0006084-g006: Cytoplasmic localization of GR depends on the presence of nestin.(A) Suppression of Nes expression in C6D8 cells by transfection with nestin specific siRNA abolishes cytoplasmic GR staining; only nuclear GR staining is left. (C) GR is only nuclear in rat 1 cells which contain vimentin, but lack nestin. (D) Ectopic expression of nestin in rat1 cells induces cytoplasmic accumulation of GR. Bars in A, B, C, F, and G = 5 µm.

Mentions: To substantiate the relationship between presence of nestin and vimentin, and retention of GR in the cytoplasm we depleted nestin from C6D8 cells by use of specific siRNA. As shown in Figure 6A, siRNA transfected cultures showed areas where cells were nestin negative (marked by a white line). GR became exclusively nuclear in these nestin depleted cells and the cells flattened and enlarged. No such changes were obtained by transfection with a scrambled siRNA (Figure 6B).


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)

Cytoplasmic localization of GR depends on the presence of nestin.(A) Suppression of Nes expression in C6D8 cells by transfection with nestin specific siRNA abolishes cytoplasmic GR staining; only nuclear GR staining is left. (C) GR is only nuclear in rat 1 cells which contain vimentin, but lack nestin. (D) Ectopic expression of nestin in rat1 cells induces cytoplasmic accumulation of GR. Bars in A, B, C, F, and G = 5 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006084-g006: Cytoplasmic localization of GR depends on the presence of nestin.(A) Suppression of Nes expression in C6D8 cells by transfection with nestin specific siRNA abolishes cytoplasmic GR staining; only nuclear GR staining is left. (C) GR is only nuclear in rat 1 cells which contain vimentin, but lack nestin. (D) Ectopic expression of nestin in rat1 cells induces cytoplasmic accumulation of GR. Bars in A, B, C, F, and G = 5 µm.
Mentions: To substantiate the relationship between presence of nestin and vimentin, and retention of GR in the cytoplasm we depleted nestin from C6D8 cells by use of specific siRNA. As shown in Figure 6A, siRNA transfected cultures showed areas where cells were nestin negative (marked by a white line). GR became exclusively nuclear in these nestin depleted cells and the cells flattened and enlarged. No such changes were obtained by transfection with a scrambled siRNA (Figure 6B).

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