Abstract
Heme oxygenase-1 (HO-1) is known as a multiple functional enzyme. Its function is not only to catalyze heme degradation but also to contribute to cytoprotection against damages such as the ionizing radiation, oxidative stresses and the ischemia-reperfusion. We previously reported that several polyphenols such as caffeic acid phenethyl ester (CAPE) from propolis drastically induced HO-1 gene in the structure-dependent manner in mouse monocyte-macrophage cells. To reveal the drastic inductive pathway, the nucleotide sequence that is responsive to CAPE in HO-1 gene was assessed by reporter gene RNA assay. Five different regions of nucleotide sequences corresponding to HO-1 gene locus were cloned from mouse genome using PCR and were connected with a luciferase reporter plasmid. Series of the reporter plasmids were introduced into mouse monocyte-macrophage cells (RAW264.7) by electroporation together with a reference gene as an internal standard . The cells were incubated for 4 hours, treated with CAPE for 4 hours and harvested to isolate RNA. RNAs derived from the exogenously introduced reporter gene and reference gene were quantified by real-time RT-PCR using TaqMan(R) probe. The increase in the reporter gene RNA by CAPE was observed in only one reporter gene that possesses 4050 nucleotide-upstream to the 1st exon. This suggests the CAPE-responsive region is localized in the region spanning from 4050 nucleotide upstream to the 1st exon.
