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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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Suppression of nestin by RNA interference induces a G1/S cell cycle arrest.(A, A1, A2) Human A172 cells transduced with nestin specific shRNA (Nes-shRNA) show loss of nestin expression (A) and increased nuclear GR staining (A1); (B, B1; B2) localization of Nes (B) and GR (B1) in A172 cells transduced with scrambled shRNA (scr-shRNA). (C) Cells expressing Nes-shRNA are arrested in G1/S transition, indicated by the altered cell cycle profile, the decrease in cyclin A expression, and maintenance of cyclin E expression (C, lane 3). (D) Suppression of GR in advance impairs activation of a G1/S arrest by Nes-shRNA. Cyclin A is detectable, cyclin E does not accumulate to high levels (D, lane 3). (E) Proportions of cell cycle phases in A172 cultures expressing scrambled, Nes specific, or in a sequential manner GR and Nes specific shRNA. Mean values and SDs are of three independent experiments, counting 10,000 cells in each experiment; bar in A and B = 10 µm.
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pone-0006084-g011: Suppression of nestin by RNA interference induces a G1/S cell cycle arrest.(A, A1, A2) Human A172 cells transduced with nestin specific shRNA (Nes-shRNA) show loss of nestin expression (A) and increased nuclear GR staining (A1); (B, B1; B2) localization of Nes (B) and GR (B1) in A172 cells transduced with scrambled shRNA (scr-shRNA). (C) Cells expressing Nes-shRNA are arrested in G1/S transition, indicated by the altered cell cycle profile, the decrease in cyclin A expression, and maintenance of cyclin E expression (C, lane 3). (D) Suppression of GR in advance impairs activation of a G1/S arrest by Nes-shRNA. Cyclin A is detectable, cyclin E does not accumulate to high levels (D, lane 3). (E) Proportions of cell cycle phases in A172 cultures expressing scrambled, Nes specific, or in a sequential manner GR and Nes specific shRNA. Mean values and SDs are of three independent experiments, counting 10,000 cells in each experiment; bar in A and B = 10 µm.

Mentions: In late passages of MEFs, when the cells had ceased proliferation and adopted a senescence like phenotype, nestin positive cells were hardly detectable. This let us ask whether loss of nestin could be causally related to activation of growth suppression. To evaluate this hypothesis we assayed the impact of nestin loss on cell growth in A172 cultures, a human cell line expressing nestin and vimentin and showing cytoplasmic anchoring of GR to this filament system. Nestin was suppressed by transduction of the cells with nestin specific shRNA (compare Figure 11, A and B). Suppression was obvious in Western-blot already 24 h post transduction (data not shown). Concomitantly, in immunofluorescence the nuclear GR signal increased relative to the cytoplasmic signal (compare Figure 11, A1 and B1). Flow cytometry analysis showed an increase in the proportion of G1 cells in cultures transduced with nestin shRNA, whereas the proportion of S-phase and G2 phase cells declined (Figure 11, C and E). The cyclin pattern revealed a decrease in the cyclin A level in nestin shRNA transduced cells, whereas the cyclin E level was maintained or even increased (Figure 11C, lane 3). Nestin shRNA transduced cells could not be subcultivated in contrast to cells transduced with a scrambled (scr) shRNA. It indicates that suppression of nestin arrested A172 cells irreversibly in G1/S.


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)

Suppression of nestin by RNA interference induces a G1/S cell cycle arrest.(A, A1, A2) Human A172 cells transduced with nestin specific shRNA (Nes-shRNA) show loss of nestin expression (A) and increased nuclear GR staining (A1); (B, B1; B2) localization of Nes (B) and GR (B1) in A172 cells transduced with scrambled shRNA (scr-shRNA). (C) Cells expressing Nes-shRNA are arrested in G1/S transition, indicated by the altered cell cycle profile, the decrease in cyclin A expression, and maintenance of cyclin E expression (C, lane 3). (D) Suppression of GR in advance impairs activation of a G1/S arrest by Nes-shRNA. Cyclin A is detectable, cyclin E does not accumulate to high levels (D, lane 3). (E) Proportions of cell cycle phases in A172 cultures expressing scrambled, Nes specific, or in a sequential manner GR and Nes specific shRNA. Mean values and SDs are of three independent experiments, counting 10,000 cells in each experiment; bar in A and B = 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2698154&req=5

pone-0006084-g011: Suppression of nestin by RNA interference induces a G1/S cell cycle arrest.(A, A1, A2) Human A172 cells transduced with nestin specific shRNA (Nes-shRNA) show loss of nestin expression (A) and increased nuclear GR staining (A1); (B, B1; B2) localization of Nes (B) and GR (B1) in A172 cells transduced with scrambled shRNA (scr-shRNA). (C) Cells expressing Nes-shRNA are arrested in G1/S transition, indicated by the altered cell cycle profile, the decrease in cyclin A expression, and maintenance of cyclin E expression (C, lane 3). (D) Suppression of GR in advance impairs activation of a G1/S arrest by Nes-shRNA. Cyclin A is detectable, cyclin E does not accumulate to high levels (D, lane 3). (E) Proportions of cell cycle phases in A172 cultures expressing scrambled, Nes specific, or in a sequential manner GR and Nes specific shRNA. Mean values and SDs are of three independent experiments, counting 10,000 cells in each experiment; bar in A and B = 10 µm.
Mentions: In late passages of MEFs, when the cells had ceased proliferation and adopted a senescence like phenotype, nestin positive cells were hardly detectable. This let us ask whether loss of nestin could be causally related to activation of growth suppression. To evaluate this hypothesis we assayed the impact of nestin loss on cell growth in A172 cultures, a human cell line expressing nestin and vimentin and showing cytoplasmic anchoring of GR to this filament system. Nestin was suppressed by transduction of the cells with nestin specific shRNA (compare Figure 11, A and B). Suppression was obvious in Western-blot already 24 h post transduction (data not shown). Concomitantly, in immunofluorescence the nuclear GR signal increased relative to the cytoplasmic signal (compare Figure 11, A1 and B1). Flow cytometry analysis showed an increase in the proportion of G1 cells in cultures transduced with nestin shRNA, whereas the proportion of S-phase and G2 phase cells declined (Figure 11, C and E). The cyclin pattern revealed a decrease in the cyclin A level in nestin shRNA transduced cells, whereas the cyclin E level was maintained or even increased (Figure 11C, lane 3). Nestin shRNA transduced cells could not be subcultivated in contrast to cells transduced with a scrambled (scr) shRNA. It indicates that suppression of nestin arrested A172 cells irreversibly in G1/S.

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