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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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Saturation binding  analysis of GCR DNA binding  affinity in nuclear extracts from  GC-sensitive and GC-insensitive  patients. Nuclear extracts were  prepared from PBMCs of a GC-sensitive and a GC-insensitive  asthmatic. Approximately 5 μg  of nuclear extract protein from  each subject was incubated in reactions with concentrations of  32P-labeled GRE probe from 0.1  to 100 ng. The reactions were  separated on polyacrylamide gels  and exposed to film. The resulting autoradiograms were subjected to densitometry to quantitate levels of  bound and free probe. Both shifted bands were included in the bound  probe levels. The figure shows the binding data plotted by the method of  Scatchard.
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Figure 3: Saturation binding analysis of GCR DNA binding affinity in nuclear extracts from GC-sensitive and GC-insensitive patients. Nuclear extracts were prepared from PBMCs of a GC-sensitive and a GC-insensitive asthmatic. Approximately 5 μg of nuclear extract protein from each subject was incubated in reactions with concentrations of 32P-labeled GRE probe from 0.1 to 100 ng. The reactions were separated on polyacrylamide gels and exposed to film. The resulting autoradiograms were subjected to densitometry to quantitate levels of bound and free probe. Both shifted bands were included in the bound probe levels. The figure shows the binding data plotted by the method of Scatchard.

Mentions: The low levels of GCR DNA binding in the GC-insensitive samples appears to be due to decreased GCR DNA binding affinity as compared to binding in normal and GC-sensitive individuals determined by saturation binding experiments. Fig. 3 shows a Scatchard plot of a representative saturation binding experiment comparing GCR DNA binding in a nuclear extract from a GC-sensitive subject to binding in a nuclear extract from a GC-sensitive patient. The slope of the curve derived from the GC-sensitive patient indicates a Kd of ∼10−9 M, whereas the curve obtained from the GC-insensitive patient indicated a lower affinity interaction with a Kd of 10−8 M.


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)

Saturation binding  analysis of GCR DNA binding  affinity in nuclear extracts from  GC-sensitive and GC-insensitive  patients. Nuclear extracts were  prepared from PBMCs of a GC-sensitive and a GC-insensitive  asthmatic. Approximately 5 μg  of nuclear extract protein from  each subject was incubated in reactions with concentrations of  32P-labeled GRE probe from 0.1  to 100 ng. The reactions were  separated on polyacrylamide gels  and exposed to film. The resulting autoradiograms were subjected to densitometry to quantitate levels of  bound and free probe. Both shifted bands were included in the bound  probe levels. The figure shows the binding data plotted by the method of  Scatchard.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Saturation binding analysis of GCR DNA binding affinity in nuclear extracts from GC-sensitive and GC-insensitive patients. Nuclear extracts were prepared from PBMCs of a GC-sensitive and a GC-insensitive asthmatic. Approximately 5 μg of nuclear extract protein from each subject was incubated in reactions with concentrations of 32P-labeled GRE probe from 0.1 to 100 ng. The reactions were separated on polyacrylamide gels and exposed to film. The resulting autoradiograms were subjected to densitometry to quantitate levels of bound and free probe. Both shifted bands were included in the bound probe levels. The figure shows the binding data plotted by the method of Scatchard.
Mentions: The low levels of GCR DNA binding in the GC-insensitive samples appears to be due to decreased GCR DNA binding affinity as compared to binding in normal and GC-sensitive individuals determined by saturation binding experiments. Fig. 3 shows a Scatchard plot of a representative saturation binding experiment comparing GCR DNA binding in a nuclear extract from a GC-sensitive subject to binding in a nuclear extract from a GC-sensitive patient. The slope of the curve derived from the GC-sensitive patient indicates a Kd of ∼10−9 M, whereas the curve obtained from the GC-insensitive patient indicated a lower affinity interaction with a Kd of 10−8 M.

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