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

Specificity of electrophoretic mobility shift blot for GCR– GRE interactions using nuclear extracts from a normal subject. The specific monomeric and dimeric GCR–GRE retarded bands are indicated by  arrows. The effect of dexamethasone (1 μM) on the density of the GCR– GRE band is shown at 60 min. Specificity is shown by the inhibitory effect of 100-fold excess of unlabeled oligonucleotide and anti-GCR antibody  on GCR–GRE binding.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199113&req=5

Figure 1: Specificity of electrophoretic mobility shift blot for GCR– GRE interactions using nuclear extracts from a normal subject. The specific monomeric and dimeric GCR–GRE retarded bands are indicated by arrows. The effect of dexamethasone (1 μM) on the density of the GCR– GRE band is shown at 60 min. Specificity is shown by the inhibitory effect of 100-fold excess of unlabeled oligonucleotide and anti-GCR antibody on GCR–GRE binding.

Mentions: An EMSA was used to measure the interaction between GCR and its specific DNA recognition sequence or GRE. The first step in these studies was to confirm that the DNA binding activity observed in our EMSAs was due to GCR DNA binding. As shown in Fig. 1, two retarded bands, corresponding to monomeric and dimeric GCRs, were observed in EMSAs performed with a 30-bp oligonucleotide containing the consensus GRE. Although these two bands were observed in reactions performed with nuclear extracts from untreated cells, their intensity was much higher in reactions containing extracts from dexamethasone-treated cells. A 100-fold excess of cold nonspecific oligonucleotide containing the consensus CCAAT/enhancer binding protein binding sequence had no effect on the DNA binding activity, but excess cold GRE oligonucleotide completely inhibited formation of the two retarded bands. Likewise, antibodies to the GCR completely blocked complex formation, whereas antibodies to other transcription factors had no effect on DNA binding. The ability of dexamethasone to enhance complex formation and the ability of GCR specific antibodies and oligonucleotides to inhibit their formation clearly indicates that the DNA binding activity observed in these reactions is due to the GCR.


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)

Specificity of electrophoretic mobility shift blot for GCR– GRE interactions using nuclear extracts from a normal subject. The specific monomeric and dimeric GCR–GRE retarded bands are indicated by  arrows. The effect of dexamethasone (1 μM) on the density of the GCR– GRE band is shown at 60 min. Specificity is shown by the inhibitory effect of 100-fold excess of unlabeled oligonucleotide and anti-GCR antibody  on GCR–GRE binding.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Specificity of electrophoretic mobility shift blot for GCR– GRE interactions using nuclear extracts from a normal subject. The specific monomeric and dimeric GCR–GRE retarded bands are indicated by arrows. The effect of dexamethasone (1 μM) on the density of the GCR– GRE band is shown at 60 min. Specificity is shown by the inhibitory effect of 100-fold excess of unlabeled oligonucleotide and anti-GCR antibody on GCR–GRE binding.
Mentions: An EMSA was used to measure the interaction between GCR and its specific DNA recognition sequence or GRE. The first step in these studies was to confirm that the DNA binding activity observed in our EMSAs was due to GCR DNA binding. As shown in Fig. 1, two retarded bands, corresponding to monomeric and dimeric GCRs, were observed in EMSAs performed with a 30-bp oligonucleotide containing the consensus GRE. Although these two bands were observed in reactions performed with nuclear extracts from untreated cells, their intensity was much higher in reactions containing extracts from dexamethasone-treated cells. A 100-fold excess of cold nonspecific oligonucleotide containing the consensus CCAAT/enhancer binding protein binding sequence had no effect on the DNA binding activity, but excess cold GRE oligonucleotide completely inhibited formation of the two retarded bands. Likewise, antibodies to the GCR completely blocked complex formation, whereas antibodies to other transcription factors had no effect on DNA binding. The ability of dexamethasone to enhance complex formation and the ability of GCR specific antibodies and oligonucleotides to inhibit their formation clearly indicates that the DNA binding activity observed in these reactions is due to the GCR.

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