Biomedical Engineering Reference
In-Depth Information
B. Defects in Ligand Binding
Using whole cell binding assays no significant changes in the dissociation
constant (K
d
) of monocyte and T-cell and receptor density (Bmax) of GR
in patients with CR asthma (59). GR
a
mRNA levels are reported to be
slightly lower in patients with COPD than normal (60), although this
has not been confirmed in other epithelial cells from smokers (healthy
smokers and COPD) (61). However, a statistically significant difference
was found between K
d
values in human bronchial epithelial cells from
smokers (healthy smokers and COPD) compared to nonsmokers. The
reduction of GR expression and altered affinity of GR may reflect either
an intrinsic defect in the GR in these patients or may relate to changes
in the receptor induced by inflammation or oxidative stress in more severe
COPD or asthma. Pretreatment with TNF
a
, which is a dominant proin-
flammatory cytokine in COPD, diminishes the ability of dexamethasone
to suppress IL-6 secretion in whole-blood cell cultures and to enhance
IL-1 receptor antagonist secretion by U937 cells. Tumor necrosis
factor-
a
resulted in a 60% decrease of the GR number without any change
in the binding affinity after 48 hr (62). Nitrosylation of GR by oxidative
stress is reported to decrease its affinity for corticosteroids (63). The rever-
sal of the reduced binding affinity is mimicked by incubation of cells with
high concentrations of IL-2
=
IL-4 or IL-13 treatment in peripheral blood
mononuclear cells (PBMCs) (2,64). Two explanations for the effect of
IL-2
=
IL-4 or TNF
a
on ligand binding characteristics have been proposed.
One theory is that steroid resistance is due to increased expression of the
dominant negative isoform of GR, GR
b
(2). As the result of alternative
splicing of the GR pre-mRNA, there are two homologous mRNAs and
protein isoforms, termed GR
a
and GR
b
. Both mRNAs contain the same
first eight exons of the GR gene. The remainder is derived by alternative
splicing of the last exon of the GR gene, resulting in either inclusion or
exclusion of exon 9
a
. The two protein isoforms have the same first 727
NH
2
-terminal amino acids. GR
b
differs from GR
a
only in its COOH ter-
minus with replacement of the last 50 amino acids of GR
a
with a unique
15 amino acids sequence lacking a steroid-binding domain. These differ-
ences render GR
b
unable to bind glucocorticoids, reduce its binding affi-
nity for DNA recognition sites, abolish its ability to transactivate
glucocorticoid-sensitive genes, and make it function as a dominant inhibi-
tor of GR
a
, possibly through formation of antagonistic GR
a
=
GR
b
hetero-
dimers. In COPD, GR
b
mRNA levels are reported to be increased in
patients with COPD compared with normal subjects (60), and also
increased numbers of cells expressing GR
b
have been reported in skin
biopsies from CR patients (65). In contrast, others have been unable to
detect enhanced GR
b
expression in PBMCs from CR asthmatics patients
(64,66).
