9-cis retinoic acid induces insulin-like growth factor binding protein-3 through DR8 retinoic acid responsive elements Free

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IGFBP-3, which is the most abundant IGFBP in circulation and reach 150 kD when it binds with IGF-I, cannot penetrate endothelial cells and is implicated in homeostasing the content of IGFs in the circulation. IGFBP-3 regulates IGF-1 bioactivity by sequestering IGF-1 away from its receptor in the extracellular milieu and thereby inhibiting the critical role of IGFs in cellular growth, differentiation, transformation, and apoptosis. Besides its IGF-I_dependent function, IGFBP-3, like IGFBP-1 and IGFBP-5, has IGF-I_independent antiproliferative and pro-apoptotic effects. Previously authors showed that in non-small cell lung cancer (NSCLC) cells, IGFBP-3 gene expression is regulated either by epigenetical change or genetic difference in the promoter. In the present study, we analyzed the influence and mechanisms of 9-cis retinoic acid (9cRA) on the expression of IGFBP-3. Treatment of 9cRA does not influence of NSCLC cell lines in clinically achievable concentration and induced IGFBP-3 and retinoic acid receptor-β (RAR-β) expression in dose and time dependent manner, reached peak level at 500 nM in 24∼48 hr treatment. Transient transfection of NSCLC cell lines with a reporter construct driven by the human IGFBP-3 gene promoter indicated that 9cRA induce its gene expression via the -358∼-245 region (relative to mRNA cap site) of the IGFBP-3 promoter. Unilateral deletion and site-directed mutagenesis identified retinoic-acid responsive element (RARE), which interestingly correspond to a direct repeat of two GGGTCA-related hexanucleotides separated by an 8 bp only (DR8-type response element). Cotransfection assay with RAR-β expression vector potentiated and with siRNA against RAR-β abolishes effect 9cRA on IGFBP-3 expression. Electric mobility shift assay further confirmed specific binding of RAR-β DR8-type response elements. In this study, we show that IGFBP-3 gene expression by 9cRA is mediated by a distinct DR8 responsive element located in the proximal region of the IGFBP promoter and involves the RAR-β which is a putative tumor suppressor gene.