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

GCR binding to  specific consensus double-stranded DNA binding sequences for GRE in nuclear extracts obtained from PBMCs  from GC-insensitive (n = 8),  GC-sensitive asthmatics (n = 6),  and normal subjects (n = 10).  (A) A representative EMSA for  GCR–GRE interactions using  freshly isolated nuclear extracts  from PBMCs of three normal  subjects, three GC-sensitive,  and four GC-insensitive asthmatics. (B) A Western blot of the  same PBMC nuclear extracts  probed with affinity-purified  rabbit anti–human GCR (Affinity BioReagents, Neshanic Station, NJ). Equal amounts of nuclear extract protein were assayed in each lane of A and B. (C) The average, relative GCR DNA binding in nuclear extracts from PBMCs of normal,  GC-sensitive, and GC-insensitive subjects determined by densitometric quantitation of multiple experiments. Levels are shown relative to the maximum  binding observed in normal subjects.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199113&req=5

Figure 2: GCR binding to specific consensus double-stranded DNA binding sequences for GRE in nuclear extracts obtained from PBMCs from GC-insensitive (n = 8), GC-sensitive asthmatics (n = 6), and normal subjects (n = 10). (A) A representative EMSA for GCR–GRE interactions using freshly isolated nuclear extracts from PBMCs of three normal subjects, three GC-sensitive, and four GC-insensitive asthmatics. (B) A Western blot of the same PBMC nuclear extracts probed with affinity-purified rabbit anti–human GCR (Affinity BioReagents, Neshanic Station, NJ). Equal amounts of nuclear extract protein were assayed in each lane of A and B. (C) The average, relative GCR DNA binding in nuclear extracts from PBMCs of normal, GC-sensitive, and GC-insensitive subjects determined by densitometric quantitation of multiple experiments. Levels are shown relative to the maximum binding observed in normal subjects.

Mentions: We next compared GCR DNA binding in nuclear extracts from 10 normal subjects, 6 GC-sensitive, and 8 GC-insensitive individuals (Fig. 2). In these experiments, PBMCs were treated with dexamethasone to translocate the GCRs to the nucleus. We found significant levels of GCR DNA binding in nuclear extracts from normal and GC-sensitive individuals (Fig. 2, A). When quantitated by densitometry of the autoradiographs, no significant difference in GCR DNA binding levels was noted between these two populations (Fig. 2, C). In contrast, nuclear extracts from GC-insensitive patients demonstrated significantly lower (P <0.001) levels of GCR DNA binding than normals or GC-sensitive individuals (Fig. 2, A and C). The decreased binding in these samples was not due to decreased GCR protein levels, as Western blot analysis of the nuclear extracts from normal, GC-sensitive, and GC-insensitive subjects revealed comparable levels of GCR protein (Fig. 2, B).


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)

GCR binding to  specific consensus double-stranded DNA binding sequences for GRE in nuclear extracts obtained from PBMCs  from GC-insensitive (n = 8),  GC-sensitive asthmatics (n = 6),  and normal subjects (n = 10).  (A) A representative EMSA for  GCR–GRE interactions using  freshly isolated nuclear extracts  from PBMCs of three normal  subjects, three GC-sensitive,  and four GC-insensitive asthmatics. (B) A Western blot of the  same PBMC nuclear extracts  probed with affinity-purified  rabbit anti–human GCR (Affinity BioReagents, Neshanic Station, NJ). Equal amounts of nuclear extract protein were assayed in each lane of A and B. (C) The average, relative GCR DNA binding in nuclear extracts from PBMCs of normal,  GC-sensitive, and GC-insensitive subjects determined by densitometric quantitation of multiple experiments. Levels are shown relative to the maximum  binding observed in normal subjects.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: GCR binding to specific consensus double-stranded DNA binding sequences for GRE in nuclear extracts obtained from PBMCs from GC-insensitive (n = 8), GC-sensitive asthmatics (n = 6), and normal subjects (n = 10). (A) A representative EMSA for GCR–GRE interactions using freshly isolated nuclear extracts from PBMCs of three normal subjects, three GC-sensitive, and four GC-insensitive asthmatics. (B) A Western blot of the same PBMC nuclear extracts probed with affinity-purified rabbit anti–human GCR (Affinity BioReagents, Neshanic Station, NJ). Equal amounts of nuclear extract protein were assayed in each lane of A and B. (C) The average, relative GCR DNA binding in nuclear extracts from PBMCs of normal, GC-sensitive, and GC-insensitive subjects determined by densitometric quantitation of multiple experiments. Levels are shown relative to the maximum binding observed in normal subjects.
Mentions: We next compared GCR DNA binding in nuclear extracts from 10 normal subjects, 6 GC-sensitive, and 8 GC-insensitive individuals (Fig. 2). In these experiments, PBMCs were treated with dexamethasone to translocate the GCRs to the nucleus. We found significant levels of GCR DNA binding in nuclear extracts from normal and GC-sensitive individuals (Fig. 2, A). When quantitated by densitometry of the autoradiographs, no significant difference in GCR DNA binding levels was noted between these two populations (Fig. 2, C). In contrast, nuclear extracts from GC-insensitive patients demonstrated significantly lower (P <0.001) levels of GCR DNA binding than normals or GC-sensitive individuals (Fig. 2, A and C). The decreased binding in these samples was not due to decreased GCR protein levels, as Western blot analysis of the nuclear extracts from normal, GC-sensitive, and GC-insensitive subjects revealed comparable levels of GCR protein (Fig. 2, B).

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