Biology Reference
In-Depth Information
Figure 3.2 Levels of the pro-
inflammatory markers COX-2 and
iNOS in astrocytes stimulated with
HMGB1.
Astrocyte proteins (20 g)
were subjected to Western blotting.
-Actin was used as a control for
protein loading. (A) Astrocytes were
left untreated (ctrl), or treated for 16
hours with the indicated additions;
(B) astrocytes were left untreated, or
pretreated for 15 minutes with the
indicated additions and then stimulated
with HMGB1 for 9 hours. Blots
are representative of three separate
experiments.
Source: Ref.
[46]
. Copyright 2007. The
American Association of Immunologists,
Inc.
HMGB1 (nM
)
ctrl CCM 400
40
4
COX-2
iNOS
β
-actin
(A)
-
+
+
+
40 nM HMGB1
-
-
+
-
mAb anti-RAGE
-
-
-
+
PD098059
COX-2
β
-actin
(B)
reported that up-regulation of COX-2 is also mediated by RAGE in microglia stimu-
lated by S100B, through activation of a Cdc42-Rac1-JNK and a Ras-Rac1-NF-B
pathway
[50]
. RAGE can interact with several protein ligands that are accumulated in
the extracellular matrix by dying cells or following tissue injuries and show the com-
mon characteristics of multiple -sheets
[51]
. Although the wide expression and the
ability to function as a sensor for the environmental cues suggest the implication of
RAGE in inflammatory tissue responses, the relevant downstream signal pathways
are not fully elucidated. Astrocytes stimulated with HMGB1 showed that the COX-2
immunoreactive signal disappeared in the presence of the mitogen-activated protein
kinase (MAPK)/extracellular signal-regulated kinase 1/2 (ERK) kinase (MEK) inhibi-
tor PD098059 (see
Figure 3.2(B)
), indicating that COX-2 induction requires activa-
tion of the MAPK/ERK1/2 pathway. A time-course analysis of ERK1/2 activation was
carried out on HMGB1-stimulated astrocytes by using an antibody that recognizes
phospho-42 ERK (p-ERK2) only when phosphorylated at Thr183 and Tyr185.
As shown in
Figure 3.3(A)
, HMGB1 promoted rapid activation of p-ERK2, reach-
ing a maximum at 10 minutes; this effect subsided almost completely within 2 hours.
This finding is in agreement with previous evidence showing that in neuron/astrocyte
cell cultures and organotypic hippocampal slice cultures, the activation of ERK1/2 is
necessary for the induction of COX-2
[52]
. In contrast, HMGB1, even at a concen-
tration up to 400 nM, did not trigger either activation of p38 MAPK or NF-B, or
degradation of IkB (data not shown), events that are often related to the generation
