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. , 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.
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 (more ...)
We next compared GCR DNA binding in nuclear extracts from 10 normal subjects, 6 GC-sensitive, and 8 GC-insensitive individuals (Fig. ). 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. , A). When quantitated by densitometry of the autoradiographs, no significant difference in GCR DNA binding levels was noted between these two populations (Fig. , 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. , 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. , B).
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 (more ...)
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. 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.
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 (more ...)
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. 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.
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 (more ...)
The actual mechanisms by which cytokines or immune activation might induce a decrease in GCR-GRE binding are unknown. Cloning of the human GCR cDNA and gene revealed that alternative splicing of the GCR pre–messenger RNA (mRNA) gives rise to an additional homologous mRNA and protein isoform, termed GCR-β, that is distinct from the ligand-activated classical GCR, GCR-α. Both mRNAs contain the first eight exons of the GCR gene (15
). The remainder is derived by alternative splicing of the nucleotide sequence encoded by the last exon of the GCR gene, corresponding to either exon 9α or 9β. The two protein isoforms have the same first 727 NH2
-terminal amino acids. GCR-β differs from GCR-α only in its COOH terminus with replacement of the last 50 amino acids of GCR-α with a unique 15 amino acid sequence. These differences render GCR-β unable to bind GC hormones, thereby antagonizing the transactivating activity of the classic GCR-α molecule (16
). Recent studies also indicate that, in the presence of GC, GCR-β heterodimerizes with ligand-bound GCR-α and translocates into the nucleus where it acts as a dominant negative inhibitor of the classic GCR (13
To determine whether overexpression of GCR-β might also result in reduced GCR DNA binding activity, HepG2 cell lines were transfected with increasing amounts of a plasmid, pRSV–GCR-β, from which the full-length GCR-β protein was expressed. The cells were cotransfected with the plasmid, pHook-1, which allowed us to separate the transfected cells from untransfected cells by using the Capture-Tec System (Invitrogen, Carlsbad, CA). Nuclear extracts from the transfected, GCR-β–expressing cells were subjected to EMSA using the labeled GRE probe. As shown in Fig. , the introduction of GCR-β into HepG2 cells inhibits GCR DNA binding activity in nuclear extracts. The ability of GCR-β to block GCR DNA binding exhibited concentration dependence as very little inhibition of binding was noted in nuclear extracts from cells transfected with 1 or 5 μg of pRSV–GCR-β. However, 10 μg of GCR-β plasmid decreased GCR DNA binding by ~25%, and 50 μg of plasmid blocked binding by 70%. Thus, the overexpression of GCR-β in HepG2 cells inhibited GCR DNA binding activity.
Figure 5 Overexpression of GCR-β reduces GCR DNA binding affinity. HepG2 cells grown in Dulbecco's modified Eagles medium containing 10% fetal calf serum cotransfected with the indicated amounts of the GCR-β expression plasmid, pRSV–GCR-β, (more ...)
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. , a–d
), but not the immunoglobulin isotype control (Fig. e
). This staining was blocked in the presence of purified GCR-β immunizing peptide indicating that the staining was specific for GCR-β (Fig. f
). As shown in Fig. , 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. ). 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. ).
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 (more ...)
Figure 7 Percent of GCR-β+ cells in freshly isolated PBMCs from GC-insensitive asthmatics versus GC-sensitive asthmatics and control subjects. PBMCs were processed and stained for GCR-β immunoreactivity as described in Materials and Methods. (more ...)
Figure 8 Effect of combination IL-2 and IL-4 on percent GCR-β– immunoreactive cells in PBMCs from normal subjects and GC-insensitive asthmatics. PBMC from four normal donors and three GC-insensitive asthma patients were analyzed for GCR-β (more ...)