Our earlier study showed that oxidative stress in HLE cells induces transient
hTTase gene expression, which results in increased TTase enzyme activity and mRNA level, followed by a gradual downregulation of both when the oxidant in the culture media is totally detoxified.
16 Increased gene expression suggests that a certain transcription factor (or factors) was involved in this process. Analysis of the
hTTase gene’s 5′ region revealed several putative transcription factor–binding sites. Presence of a sequence that is similar to the AP-1 transcription factor–binding site
(Fig. 1A) prompted us to assume that it has a role in activation of the
hTTase gene. To check this hypothesis, we performed a GMSA with
32P-labeled double-stranded oligo containing a putative AP-1–binding site and nuclear lysates from normal and H
2O
2-treated HLE B3 cells
(Fig. 1B) . Our results demonstrated that binding of AP-1 to the putative binding site located in the
hTTase gene 5′ region occurred in an oxidation-dependent manner. The pattern of modulation of AP-1 binding shown in
Figure 1B was very similar to the previously observed changes in TTase mRNA level under the same experimental conditions.
16 Furthermore, the
hTTase gene promoter activity examined by the reporter assay demonstrated clearly that the plasmid containing either the intact
hTTase 5′ region or a fragment of it containing the AP-1–binding site could successfully express the
CAT gene in an oxidative stress–dependent manner, similar to the TTase expression in the H
2O
2-pretreated HLE B3 cells. On the contrary, the fragment devoid of the putative AP-1–binding site did not express the
CAT gene in a similar manner
(Figs. 5 6) . The mutation study also revealed that when the key sequence in the AP-1–binding site was substituted, the reporter gene’s expression no longer responded to oxidative stress, and thus activation of gene expression did not occur. All these findings strongly support our hypothesis that AP-1 may play a regulatory role in activation of the
hTTase gene. As far as we know, this is the first evidence of AP-1’s association with the expression of the
hTTase.