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Association of glucocorticoid insensitivity with increased expression of glucocorticoid receptor beta.

Leung DY, Hamid Q, Vottero A, Szefler SJ, Surs W, Minshall E, Chrousos GP, Klemm DJ - J. Exp. Med. (1997)

Bottom Line: Alternative splicing of the GC receptor (R) pre-messenger RNA generates a second GCR, termed GCR-beta, which does not bind GCs but antagonizes the transactivating activity of the classic GCR, termed GCR-alpha.These abnormalities can be reproduced by transfection of cell lines with the GCR-beta gene resulting in significant reduction of their GCR-alpha DNA binding capacity.We conclude that increased expression of GCR-beta is cytokine inducible and may account for GC insensitivity in this common inflammatory condition.

View Article: PubMed Central - PubMed

Affiliation: Division of Allergy-Immunology, National Jewish Medical and Research Center, Denver, CO 80206, USA. leungd@njc.org

ABSTRACT
In many chronic inflammatory disorders, glucocorticoid (GC) insensitivity is a challenging clinical problem associated with life-threatening disease progression. The molecular basis of GC insensitivity, however, is unknown. Alternative splicing of the GC receptor (R) pre-messenger RNA generates a second GCR, termed GCR-beta, which does not bind GCs but antagonizes the transactivating activity of the classic GCR, termed GCR-alpha. In the current study, we demonstrate that GC-insensitive asthma is associated with a significantly higher number of GCR-beta-immunoreactive cells in peripheral blood than GC-sensitive asthmatics or normal controls. Furthermore, we show that patients with GC-insensitive asthma have cytokine-induced abnormalities in the DNA binding capability of the GCR. These abnormalities can be reproduced by transfection of cell lines with the GCR-beta gene resulting in significant reduction of their GCR-alpha DNA binding capacity. We conclude that increased expression of GCR-beta is cytokine inducible and may account for GC insensitivity in this common inflammatory condition.

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Immunohistochemistry staining of PBMCs for expression of GCR-β. Low power magnification (×100) of cytospin preparations from PBMCs from (a) GC-insensitive and (b) GC-sensitive patients immunostained for GCR-β. Note the increase in the number of GCR-β–immunoreactive cells  in the GC-insensitive compared to GC-sensitive asthmatics. High power magnification (×400) of GCR-β immunoreactivity in PBMCs of (c) GC-insensitive and (d) GC-sensitive patients. Representative examples of PBMCs from GC-insensitive patients showing (e) immunoglobulin isotype control for  GCR-β and ( f  ) competitive inhibition of GCR-β immunoreactivity using preabsorption of the antibody with the GCR-β peptide.
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Figure 6: Immunohistochemistry staining of PBMCs for expression of GCR-β. Low power magnification (×100) of cytospin preparations from PBMCs from (a) GC-insensitive and (b) GC-sensitive patients immunostained for GCR-β. Note the increase in the number of GCR-β–immunoreactive cells in the GC-insensitive compared to GC-sensitive asthmatics. High power magnification (×400) of GCR-β immunoreactivity in PBMCs of (c) GC-insensitive and (d) GC-sensitive patients. Representative examples of PBMCs from GC-insensitive patients showing (e) immunoglobulin isotype control for GCR-β and ( f  ) competitive inhibition of GCR-β immunoreactivity using preabsorption of the antibody with the GCR-β peptide.

Mentions: These data suggest that the increased expression of GCR-β could account for GC-insensitive asthma. To test this hypothesis, PBMCs cytospun from seven GC-insensitive asthmatics, six GC-sensitive asthmatics, and seven normal subjects were stained by immunohistochemistry technique for the expression of GCR-β. Positive staining for GCR-β was observed on all samples stained with anti– GCR-β (see Fig. 6, a–d), but not the immunoglobulin isotype control (Fig. 6 e). This staining was blocked in the presence of purified GCR-β immunizing peptide indicating that the staining was specific for GCR-β (Fig. 6 f  ). As shown in Fig. 7, GC-insensitive asthma was associated with a significantly higher percentage of GCR-β+ cells (mean ± SEM = 20 ± 0.8%; P <0.001) than GC-sensitive asthmatics (9 ± 1%) or normal controls (6 ± 1%). This abnormal GCR-β expression in GC-insensitive PBMCs decreased significantly (P <0.01) toward normal after 48 h in culture media (Fig. 8). However, incubation with combination IL-2 and IL-4 sustained this abnormality. Furthermore, combination IL-2 and IL-4 induced significantly greater (P <0.001) GCR-β expression in normal PBMCs as compared to culture medium alone or baseline values (Fig. 8).


Association of glucocorticoid insensitivity with increased expression of glucocorticoid receptor beta.

Leung DY, Hamid Q, Vottero A, Szefler SJ, Surs W, Minshall E, Chrousos GP, Klemm DJ - J. Exp. Med. (1997)

Immunohistochemistry staining of PBMCs for expression of GCR-β. Low power magnification (×100) of cytospin preparations from PBMCs from (a) GC-insensitive and (b) GC-sensitive patients immunostained for GCR-β. Note the increase in the number of GCR-β–immunoreactive cells  in the GC-insensitive compared to GC-sensitive asthmatics. High power magnification (×400) of GCR-β immunoreactivity in PBMCs of (c) GC-insensitive and (d) GC-sensitive patients. Representative examples of PBMCs from GC-insensitive patients showing (e) immunoglobulin isotype control for  GCR-β and ( f  ) competitive inhibition of GCR-β immunoreactivity using preabsorption of the antibody with the GCR-β peptide.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199113&req=5

Figure 6: Immunohistochemistry staining of PBMCs for expression of GCR-β. Low power magnification (×100) of cytospin preparations from PBMCs from (a) GC-insensitive and (b) GC-sensitive patients immunostained for GCR-β. Note the increase in the number of GCR-β–immunoreactive cells in the GC-insensitive compared to GC-sensitive asthmatics. High power magnification (×400) of GCR-β immunoreactivity in PBMCs of (c) GC-insensitive and (d) GC-sensitive patients. Representative examples of PBMCs from GC-insensitive patients showing (e) immunoglobulin isotype control for GCR-β and ( f  ) competitive inhibition of GCR-β immunoreactivity using preabsorption of the antibody with the GCR-β peptide.
Mentions: These data suggest that the increased expression of GCR-β could account for GC-insensitive asthma. To test this hypothesis, PBMCs cytospun from seven GC-insensitive asthmatics, six GC-sensitive asthmatics, and seven normal subjects were stained by immunohistochemistry technique for the expression of GCR-β. Positive staining for GCR-β was observed on all samples stained with anti– GCR-β (see Fig. 6, a–d), but not the immunoglobulin isotype control (Fig. 6 e). This staining was blocked in the presence of purified GCR-β immunizing peptide indicating that the staining was specific for GCR-β (Fig. 6 f  ). As shown in Fig. 7, GC-insensitive asthma was associated with a significantly higher percentage of GCR-β+ cells (mean ± SEM = 20 ± 0.8%; P <0.001) than GC-sensitive asthmatics (9 ± 1%) or normal controls (6 ± 1%). This abnormal GCR-β expression in GC-insensitive PBMCs decreased significantly (P <0.01) toward normal after 48 h in culture media (Fig. 8). However, incubation with combination IL-2 and IL-4 sustained this abnormality. Furthermore, combination IL-2 and IL-4 induced significantly greater (P <0.001) GCR-β expression in normal PBMCs as compared to culture medium alone or baseline values (Fig. 8).

Bottom Line: Alternative splicing of the GC receptor (R) pre-messenger RNA generates a second GCR, termed GCR-beta, which does not bind GCs but antagonizes the transactivating activity of the classic GCR, termed GCR-alpha.These abnormalities can be reproduced by transfection of cell lines with the GCR-beta gene resulting in significant reduction of their GCR-alpha DNA binding capacity.We conclude that increased expression of GCR-beta is cytokine inducible and may account for GC insensitivity in this common inflammatory condition.

View Article: PubMed Central - PubMed

Affiliation: Division of Allergy-Immunology, National Jewish Medical and Research Center, Denver, CO 80206, USA. leungd@njc.org

ABSTRACT
In many chronic inflammatory disorders, glucocorticoid (GC) insensitivity is a challenging clinical problem associated with life-threatening disease progression. The molecular basis of GC insensitivity, however, is unknown. Alternative splicing of the GC receptor (R) pre-messenger RNA generates a second GCR, termed GCR-beta, which does not bind GCs but antagonizes the transactivating activity of the classic GCR, termed GCR-alpha. In the current study, we demonstrate that GC-insensitive asthma is associated with a significantly higher number of GCR-beta-immunoreactive cells in peripheral blood than GC-sensitive asthmatics or normal controls. Furthermore, we show that patients with GC-insensitive asthma have cytokine-induced abnormalities in the DNA binding capability of the GCR. These abnormalities can be reproduced by transfection of cell lines with the GCR-beta gene resulting in significant reduction of their GCR-alpha DNA binding capacity. We conclude that increased expression of GCR-beta is cytokine inducible and may account for GC insensitivity in this common inflammatory condition.

Show MeSH
Related in: MedlinePlus