Biology Reference
In-Depth Information
3.2.2 Pattern of Inflammatory Genes Induced by HMGB1
on Primary Astrocytes
The profile of astrocytes activated by HMGB1 was further defined by the semi-
quantitative reverse transcription-polymerase chain reaction reverse transcriptase-
polymerase chain reaction (RT-PCR) analysis of several genes that are up-regulated
in the fully inflammatory transcriptome of these cells
[43]
. As shown in
Figure 3.4
,
HMGB1 induced a significant increase in the levels of CCL5 and the ELR-
chemokines CXCL1 and CXCL2. However, the level of the monocyte chemoattrac-
tant protein CCL7 transcript was not affected, suggesting that HMGB1 is a specific,
rather than a general, activator of CC chemokine transcription in primary astrocytes.
This specificity is further supported by the fact that HMGB1 induced a twofold
increase in the level of the matrix metalloproteinase (MMP)-9 transcript, leaving
MMP-2, MMP-3, and the tissue inhibitor of the matrix metallo proteinase (TIMP)2
unaffected. Because MMP-3 transcription increases in rat brain astrocytes stimulated
with LPS
[59]
, the lack of MMP-3 induction by our recombinant HMGB1 also con-
firms the functional insignificance of possible contaminating LPS. Moreover, LPS
did not induce expression of the chemokines genes up-regulated by HMGB1, and the
increase of MMP-9 gene expression promoted by LPS was also significantly lower
than that obtained with HMGB1 (
Figure 3.4
).
As expected, HMGB1 induced a twofold increase in the level of the COX-2
transcript. However, this cytokine did not change the expression level of several
10
5h HMGB1
5h CCM
5h LPS
5
0
Figure 3.4 Effect of HMGB1 on the transcriptional regulation of genes related to
inflammatory activation of astrocytes.
Total RNA was isolated from astrocytes stimulated
with 40 nM HMGB1 (white bars) or CCM (gray bars) or 0.3 pg/ml LPS (checked bars) for
5 hours. Quantification of RT-PCR products relative to the indicated target genes was
carried out by the Quantity One Software. The expression of Glyceraldehyde 3-phosphate
dehydrogenase was used as an internal control. Three independent experiments were
performed. *
p
0.05; #
p
0.01 versus untreated cells (solid horizontal line).
Source: Ref. [46]. Copyright 2007. The American Association of Immunologists, Inc.
