Biology Reference
In-Depth Information
Time (min)
05 10
30
60
120
p-ERK 1/2
ERK 1/2
(A)
-
-
+
-
+
+
40 nM HMGB1
mAb anti-RAGE
p-ERK 1/2
1.0
2.4
1.5
ERK 1/2
(B)
Figure 3.3 Effect of HMGB1 on phosphorylation of ERK2.
Astrocyte proteins (20 g)
were subjected to Western blotting. (A) Upper panel: Time-course analysis of phospho-
ERK1/2 (p-ERK1/2) in astrocytes stimulated with 40 nM HMGB1. The total amounts of
ERK1/2 are shown in the lower panel. (B) Quantification of p-ERK1/2 in astrocytes untreated,
or treated for 10 minutes with HMGB1. Where indicated, a monoclonal RAGE antibody
(mAB anti-RAGE) was added 15 minutes before HMGB1 cell stimulation. Upper panel:
Numbers under the p-ERK2 bands refer to the relative quantification of the immunoreactive
signals. The total levels of ERK1/2 are shown in the lower panel. Blots are representative of
three separate experiments.
Source: Ref.
[46]
. Copyright 2007. The American Association of Immunologists, Inc.
of astrocyte inflammatory mediators
[43,53]
. It has been shown previously that the
expression of iNOS is dependent on p38 MAPK and NF-B activation in rat cor-
tical astrocytes
[54,55]
. The inability of HMGB1 to activate both p38 MAPK and
NF-B in these cells suggests a reason for its ineffectiveness as an inducer of iNOS
(see
Figure 3.2(A)
). Furthermore, pretreatment of astrocytes with an RAGE-blocking
antibody prevented the HMGB1-promoted ERK2 phosphorylation (
Figure 3.3(B)
).
Taken together, these results provide clear evidence of the involvement of the
MAPK/ERK1/2 cascade as a downstream transducer of HMGB1/RAGE signaling in
primary astrocytes. In keeping with this result, it has been demonstrated previously
that ERK-1/2 could be involved downstream of RAGE signal transduction
[56,57]
,
and that the binding of HMGB1 to the cell surface induced intracellular ERK-RAGE
interactions and enhanced ERK activity in the cell
[58]
.
