PPARγ binds to the promoter region to suppress the expression ofHeparanase
Chronic high glucose stimulation caused increased heparanase and
declined PPARγ expression, which suggested that PPARγ acted as a
transcription repressor for Heparanase . As shown in Fig. 7A, we
found three predicted binding sites of PPARγ in the promoter region
(defined as 2000 bases forward transcriptional start site (TSS) of gene)
of the murine Heparanase gene from the JASPAR database
(http://jaspar.genereg.net/) according to the scanning
score. These binding sites were identified as peroxisome proliferator
responsive elements (PPRE), including PPRE1 (5’‐GGAGGTGACAGGTGA‐3’,
-1567/-1552), PPRE2 (5’‐CCTGGGCAGAGGCA‐3’, -442/-427), and PPRE3
(5’‐TTAAGGCAGAAGGGA‐3’, -384/-369). The predicted sequence of PPRE in
the JASPAR database was shown in Fig. 7B. We then performed luciferase
reporter gene tests to detect whether PPARγ could bind to these promoter
regions of the Heparanase gene. As shown
in Fig.
7C, the whole construct (2000 bases forward TSS, -2000/0) was able to
significantly drive expression of the luciferase reporter gene in 293T
cells (Fig. 7C). By contrast, the activity of luciferase was
dose-dependently inhibited by a PPARγ agonist rosiglitazone (ROSI, Fig.
7C). The region of these three predicted binding sites that could bind
PPARγ were further investigated by determining the sequential
5’-deletion constructs from p-1000 (-1000/0). A higher expression of the
luciferase reporter gene suggested an enhanced expression after deletion
of PPRE1. However, fluorescence could be also inhibited by rosiglitazone
(Fig. 7C). After the promoter region was cut to 300 bases length
(-300/0), the transcriptional activity was abolished (Fig. 7C),
suggesting a cooperation of PPRE1, PPRE2, and PPRE3 in the regulation ofHeparanase gene expression. Similarly, chromatin
immunoprecipitation (CHIP) confirmed that the PCR product from the
chromatin pulled down by an anti-PPARγ antibody corresponded to the
predicted Heparanase gene promoter region (Fig. 7D).