Identification of putative curcumin response element(s) in the lox-1 promoter
Additional experiments were conducted to elucidate the molecular mechanisms by which curcumin inhibited the lox-1 promoter activity in HSCs. To localize curcumin response element(s) in the lox-1 promoter, a group of luciferase reporter plasmids with various lengths of the lox-1 promoter region, including 2336 base pair (bp), 996 bp, 289 bp, 259 bp and 99 bp nucleotides, were used. Passaged HSCs were transfected with the group of luciferase reporter plasmids. After recovery, cells were treated with or without curcumin at 20 μM for 24 hr. As shown in Fig. 6A, compared to the corresponding untreated control, curcumin significantly reduced luciferase activities by approximately 50% in cells transfected with a plasmid containing a 5′-flanking fragment longer than -289 bp nucleotides of the lox-1 promoter. However, loss of the promoter region between -289 and -259 bp nucleotides in p259-Luc resulted in a marked reduction in luciferase activities and in response to curcumin. Although cells transfected with p259 or pp99 still showed the response to the inhibitory effect of curcumin, the difference in luciferase activities between cells with or without curcumin treatment was dramatically reduced from ∼50% to ∼30%. We could not exclude the presence of additional curcumin response elements (s) within the promoter region of -259 bp, even -99 bp. However, our results suggested that a major curcumin response element(s) might be located within the fragment between -289 and -259 bp, which also controlled the basal transcription activity of the promoter. We, therefore, paid our major attention to this region and studied the major curcumin response element(s) in the lox-1 promoter.
Computer-aided analyses found a putative T cell factor/lymphoid enhancer factor (TCF/LEF) binding site, i.e. CTTTGA, at -275bp to -269bp within the promoter region. TCF/LEF binding sites are targets of canonical Wnt signaling and bound by a complex of β-catenin with TCF/LEF, mediating the regulation of target gene transcription 42, 43. The plasmid pLOX-1(mut)-Luc with site-directed mutation in the TCF/LEF-1 binding site was generated from the parental plasmid pLOX-1-Luc. Passaged HSCs were transfected with pLOX-1-Luc or pLOX-1(mut)-Luc and subsequently treated with or without curcumin (20 μM) for 24 hr. Luciferase activity assays in Fig. 6B demonstrated that compared to the untreated control, curcumin significantly reduced, as expected, luciferase activities by 50.5% in cells transfected with wild-type pLOX-1-Luc. However, the site-directed mutation of the TCF/LEF-1 binding site in pLOX-1(mut)-Luc resulted in a significant reduction from ∼50% to ∼25% in the difference of luciferase activities between cells treated with or without curcumin, indicating a significant loss in response to curcumin. In addition, compared to pLOX-1-Luc, pLOX-1(mut)-Luc also led to an apparent reduction in luciferase activities in cells without curcumin treatment, suggesting an important role of the TCF/LEF-1 binding site in regulating the basal promoter activity of LOX-1 in HSCs. Taken together, these results suggested that the TCF/LEF-1 binding site might be responsible for both controlling the basal promoter activity, as well as responding to the curcumin treatment, i.e. a major curcumin response element, which played a critical role in the curcumin-caused inhibition of the lox-1 promoter activity.