Abstract:
Compound A (CpdA), a dissociated glucocorticoid receptor
modulator, decreases corticosteroid-binding globulin (CBG),
adrenocorticotropic hormone (ACTH), and luteneinizing
hormone levels in rats. Whether this is due to transcriptional
regulation by CpdA is not known. Using promoter reporter
assays we show that CpdA, like dexamethasone (Dex), directly
transrepresses these genes. Results using a rat Cbg
proximal-promoter reporter construct in BWTG3 and
HepG2 cell lines support a glucocorticoid receptor (GR)-dependent
transrepression mechanism for CpdA. However,
CpdA, unlike Dex, does not result in transactivation via
glucocorticoid-responsive elements within a promoter
reporter construct even when GR is co-transfected. The
inability of CpdA to result in transactivation via glucocorticoid-
responsive elements is confirmed on the endogenous
tyrosine aminotransferase gene, whereas transrepression
ability is confirmed on the endogenous CBG gene. Consistent
with a role for CpdA in modulating GR activity, whole cell
binding assays revealed that CpdA binds reversibly to the GR,
but with lower affinity than Dex, and influences association of
[3H]Dex, but has no effect on dissociation. In addition, like
Dex, CpdA causes nuclear translocation of the GR, albeit to a
lesser degree. Several lines of evidence, including fluorescence
resonance energy transfer, co-immunoprecipitation,
and nuclear immunofluorescence studies of nuclear localization-
deficient GR show that CpdA, unlike Dex, does not elicit
ligand-induced GR dimerization. Comparison of the behavior
of CpdA in the presence of wild type GR to that of Dex with
a dimerization-deficient GR mutant (GRdim) strongly supports
the conclusion that loss of dimerization is responsible
for the dissociated behavior of CpdA.
