Limits...
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.

Show MeSH

Related in: MedlinePlus

Reversibility and cytokine induction of GCR DNA binding  abnormalities in GC-insensitive asthmatics. In these experiments, nuclear  extracts were prepared from freshly isolated PBMCs or after incubation  with culture medium or the combination of IL-2 (50 U/ml) and IL-4 (50  U/ml) for 48 h at 37°C in 5% CO2. GCR DNA binding was then analyzed by EMSA as described in the Materials and Methods section.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199113&req=5

Figure 4: Reversibility and cytokine induction of GCR DNA binding abnormalities in GC-insensitive asthmatics. In these experiments, nuclear extracts were prepared from freshly isolated PBMCs or after incubation with culture medium or the combination of IL-2 (50 U/ml) and IL-4 (50 U/ml) for 48 h at 37°C in 5% CO2. GCR DNA binding was then analyzed by EMSA as described in the Materials and Methods section.

Mentions: We next tested the effect of combination IL-2 and IL-4 on GCR DNA binding in nuclear extracts from GC-insensitive individuals. For these experiments, nuclear extracts were prepared from freshly isolated PBMCs or from cells incubated in culture for 48 h in the presence or absence of IL-2 and IL-4. Fig. 4 shows the results of four experiments using PBMCs from four GC-insensitive patients. As expected, very little GCR DNA binding was present in freshly isolated PBMCs from any of the patients. However, after 48 h in the absence of IL-2 and IL-4, there was a significant (P <0.01) increase in GCR DNA binding in all four samples. Interestingly, this increase in GCR DNA binding was blocked or inhibited in the cells cultured in IL-2 and IL-4. These data suggest that the combination of these two cytokines plays a significant role in triggering and maintaining the GCR DNA binding defect.


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)

Reversibility and cytokine induction of GCR DNA binding  abnormalities in GC-insensitive asthmatics. In these experiments, nuclear  extracts were prepared from freshly isolated PBMCs or after incubation  with culture medium or the combination of IL-2 (50 U/ml) and IL-4 (50  U/ml) for 48 h at 37°C in 5% CO2. GCR DNA binding was then analyzed by EMSA as described in the Materials and Methods section.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199113&req=5

Figure 4: Reversibility and cytokine induction of GCR DNA binding abnormalities in GC-insensitive asthmatics. In these experiments, nuclear extracts were prepared from freshly isolated PBMCs or after incubation with culture medium or the combination of IL-2 (50 U/ml) and IL-4 (50 U/ml) for 48 h at 37°C in 5% CO2. GCR DNA binding was then analyzed by EMSA as described in the Materials and Methods section.
Mentions: We next tested the effect of combination IL-2 and IL-4 on GCR DNA binding in nuclear extracts from GC-insensitive individuals. For these experiments, nuclear extracts were prepared from freshly isolated PBMCs or from cells incubated in culture for 48 h in the presence or absence of IL-2 and IL-4. Fig. 4 shows the results of four experiments using PBMCs from four GC-insensitive patients. As expected, very little GCR DNA binding was present in freshly isolated PBMCs from any of the patients. However, after 48 h in the absence of IL-2 and IL-4, there was a significant (P <0.01) increase in GCR DNA binding in all four samples. Interestingly, this increase in GCR DNA binding was blocked or inhibited in the cells cultured in IL-2 and IL-4. These data suggest that the combination of these two cytokines plays a significant role in triggering and maintaining the GCR DNA binding defect.

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