GC and ERα cooperate to upregulate CRTh2 expression
Total daily dose of ICS correlated with whole blood levels ofCRTh2 mRNA in women, but not men (Fig. 1E, 1F). Therefore, we
next examined whether GC and ERα signaling directly influences CRTh2
expression. Th2 cells treated with both DEX and PPT (24 hours) exhibited
significantly more CRTh2 mRNA compared to vehicle or DEX alone (Fig.
3A). Western blot analysis showed higher abundance of total CRTh2
protein following co-treatment with PPT and DEX (Fig. 3B). CRTh2 is a
plasma membrane receptor and so we assessed surface levels by flow
cytometry and found CRTh2 was significantly increased following
co-treatment with DEX and PPT (Fig. 3C) or DEX and E2 (Fig. 3D).
Upregulation of CRTh2 was likely due to a transcriptional effect since
the CRTh2 promoter construct contains putative GC response elements
(GRE) and activity was increased by DEX treatment alone and further
enhanced with both DEX and PPT (24 hours, Fig. 3E). Together, these
experiments show that concomitant activation of GR and ERα increases
CRTh2 expression.
GC
and ERα enhance type 2 cytokine release following CRTh2 activation
Since concomitant DEX and PPT treatment increased CRTh2 expression (Fig.
3), we considered whether this exposure primed Th2 cells for heightened
responsiveness to PGD2. Th2 cells were pre-treated with
DEX alone or DEX and PPT (24 hours), washed and stimulated with
PGD2 (24 hours) to activate the cells through CRTh2. We
found that while pre-treatment with DEX alone reduced both cytokines,
Th2 cells pre-treated with DEX and PPT released significantly more IL-5
(Fig. 4A) and IL-13 (Fig. 4B) in response to PGD2compared to DEX alone or no pre-treatment.
These
results suggest that the cooperative effect of GC and ERα on CRTh2
levels enhances the Th2 cell response to PGD2,
increasing type 2 cytokine release.