The Growth-dependent Expression of Angiotensin II Type 2 Receptor Is Regulated by Transcription Factors Interferon Regulatory Factor-1 and −2

Angiotensin II type 2 (AT2) receptor is abundantly and widely expressed in fetal tissues but present only in restricted tissues in the adult such as brain and atretic ovary. This receptor is speculated to be involved in tissue growth and/or differentiation. To elucidate the molecular mechanism of growth-regulated AT2 receptor expression, we cloned the mouse AT2 receptor genomic DNA and studied its promoter function in mouse fibroblast-derived R3T3 cells, which express AT2 receptor in the confluent, quiescent state but very low levels of the receptor in actively growing state. Promoter/luciferase reporter deletion analysis of AT2 receptor in R3T3 cells showed that the putative negative regulatory region is located between the positions -453 and -225, which plays an important role in the transcriptional control of AT2 receptor gene expression along with the cell growth. We identified the interferon regulatory factor (IRF) binding motif in this region using DNase foot-printing analysis and demonstrated that IRF binding oligonucleotide treatment increased the AT2 receptor expression in growing R3T3 cells but not in confluent cells. Furthermore, by antisense treatment, we demonstrated that IRF-2 attenuated the AT2 receptor expression in both growing and confluent R3T3 cells, whereas IRF-1 enhanced AT2 receptor expression in the confluent cells only. Consistent with this result, gel mobility shift assay demonstrated that growing R3T3 cells exhibited only IRF-2 binding, whereas confluent cells exhibited both IRF-1 and IRF-2 binding. Furthermore, we observed using reverse transcription-polymerase chain reaction that the IRF-1 mRNA expression was more abundant in confluent cells than growing cells, whereas IRF-2 expression did not change with R3T3 cell growth. We conclude that, in confluent cells, the enhanced expression of IRF-1 antagonizes the IRF-2 effect and increases the AT2 receptor expression. We speculate that these transcriptional factors influence cell growth in part by regulating AT2 receptor expression.