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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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Human PBMCs anchoring GR express nestin and the stem cell marker CD133.(A–D) Confocal images of PBMCs isolated from cord blood; (A1–A3) cells were attached to glass coverslips, fixed with acetone, and double labelled with antibodies to GR and Nes. The proportion of Nes positive cells is less than 1%. (B) Nes positive cells are negative for the hematopoietic lineage marker CD34.(C) Nes positive cells express the stem cell marker CD133 (merged image). (D) Merged image of cells extracted with a non-ionic detergent, fixed with paraformaldehyde, double labelled with antibodies to GR and Nes and incubated with the DNA stain DRAQ5 [79]. Cytoplasmic GR in Nes expressing cells resists extraction with a non-ionic detergent, whereas Nes negative cells retain GR only in the nucleus (arrow). Bars in B, C, and D = 5 µm.
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pone-0006084-g002: Human PBMCs anchoring GR express nestin and the stem cell marker CD133.(A–D) Confocal images of PBMCs isolated from cord blood; (A1–A3) cells were attached to glass coverslips, fixed with acetone, and double labelled with antibodies to GR and Nes. The proportion of Nes positive cells is less than 1%. (B) Nes positive cells are negative for the hematopoietic lineage marker CD34.(C) Nes positive cells express the stem cell marker CD133 (merged image). (D) Merged image of cells extracted with a non-ionic detergent, fixed with paraformaldehyde, double labelled with antibodies to GR and Nes and incubated with the DNA stain DRAQ5 [79]. Cytoplasmic GR in Nes expressing cells resists extraction with a non-ionic detergent, whereas Nes negative cells retain GR only in the nucleus (arrow). Bars in B, C, and D = 5 µm.

Mentions: Individual steps in blood cell differentiation are well defined by specific markers, making it an ideal system to determine the relationship between nestin/GR colocalization and the status of cell differentiation. Nestin expression is known to identify blood precursor or stem cells [28]. Here we assayed peripheral blood mononuclear cells (PBMCs) isolated from human cord blood for this phenotype. CD34 and CD133 were used as markers of early stages of blood cell differentiation. Only a small proportion (less than 1%) of human cord blood cells was nestin positive (Figure 2, A1–A3). Nestin positive cord blood cells were negative for CD34, an early marker of the hematopoietic lineage and vice versa (Figure 2B), but they expressed the stem cell marker CD133 (Figure 2C). When cord blood cells were extracted with a nonionic detergent, only those cells which were positive for nestin and CD133 retained GR in the cytoplasm (Figure 2D). Cells which lacked nestin showed a nuclear GR signal (Figure 2D, arrow). The data indicate that cytoplasmic retention of GR in nestin expressing PBMCs defines adult stem or progenitor cells.


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)

Human PBMCs anchoring GR express nestin and the stem cell marker CD133.(A–D) Confocal images of PBMCs isolated from cord blood; (A1–A3) cells were attached to glass coverslips, fixed with acetone, and double labelled with antibodies to GR and Nes. The proportion of Nes positive cells is less than 1%. (B) Nes positive cells are negative for the hematopoietic lineage marker CD34.(C) Nes positive cells express the stem cell marker CD133 (merged image). (D) Merged image of cells extracted with a non-ionic detergent, fixed with paraformaldehyde, double labelled with antibodies to GR and Nes and incubated with the DNA stain DRAQ5 [79]. Cytoplasmic GR in Nes expressing cells resists extraction with a non-ionic detergent, whereas Nes negative cells retain GR only in the nucleus (arrow). Bars in B, C, and D = 5 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2698154&req=5

pone-0006084-g002: Human PBMCs anchoring GR express nestin and the stem cell marker CD133.(A–D) Confocal images of PBMCs isolated from cord blood; (A1–A3) cells were attached to glass coverslips, fixed with acetone, and double labelled with antibodies to GR and Nes. The proportion of Nes positive cells is less than 1%. (B) Nes positive cells are negative for the hematopoietic lineage marker CD34.(C) Nes positive cells express the stem cell marker CD133 (merged image). (D) Merged image of cells extracted with a non-ionic detergent, fixed with paraformaldehyde, double labelled with antibodies to GR and Nes and incubated with the DNA stain DRAQ5 [79]. Cytoplasmic GR in Nes expressing cells resists extraction with a non-ionic detergent, whereas Nes negative cells retain GR only in the nucleus (arrow). Bars in B, C, and D = 5 µm.
Mentions: Individual steps in blood cell differentiation are well defined by specific markers, making it an ideal system to determine the relationship between nestin/GR colocalization and the status of cell differentiation. Nestin expression is known to identify blood precursor or stem cells [28]. Here we assayed peripheral blood mononuclear cells (PBMCs) isolated from human cord blood for this phenotype. CD34 and CD133 were used as markers of early stages of blood cell differentiation. Only a small proportion (less than 1%) of human cord blood cells was nestin positive (Figure 2, A1–A3). Nestin positive cord blood cells were negative for CD34, an early marker of the hematopoietic lineage and vice versa (Figure 2B), but they expressed the stem cell marker CD133 (Figure 2C). When cord blood cells were extracted with a nonionic detergent, only those cells which were positive for nestin and CD133 retained GR in the cytoplasm (Figure 2D). Cells which lacked nestin showed a nuclear GR signal (Figure 2D, arrow). The data indicate that cytoplasmic retention of GR in nestin expressing PBMCs defines adult stem or progenitor cells.

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