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Papers In Press, published online ahead of print October 17, 2000
Clinical Biochemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Tsukui, Kanagawa 199-0195
Corresponding Author: shorie-v{at}pharm.teikyo-u.ac.jp
The interactions between retinoic acid- (RA-) dependent transcriptional regulatory sequences of the 5'-untranslated region of the thrombomodulin (TM) gene and nuclear RA-responsive proteins were studied using human pancreas BxPC-3 cells. Deletion mutants of pTM-CAT plasmid revealed the presence of distal and proximal RA-responsive regions containing direct repeat with 4 spaces (DR4) and three of four Sp1 sites, respectively. Cotransfection of a pTM-CAT plasmid with expression plasmids of RA receptors (RARa, RARb and RARg) augmented the promoter activity under the condition of lower retinoid X receptor-a (RXRa) expression, whereas the activity was greatly diminished when RXRa was highly expressed. An electrophoretic mobility shift assay with cDNA containing the DR4 indicated that heterodimers of RAR and RXRa interacted with the DR4 site, though the interaction gradually disappeared with increase in the ratio of RXRa/RAR. On the other hand, Sp1 protein interacted especially with the tandem Sp1 site corresponding to the first and second Sp1 sequences of the four Sp1 sites in the proximal RA-responsive region. The binding of Sp1 to Sp1 sites was independent of RAR-RXR heterodimer, but increased with increase in Sp1 concentration in the presence of unknown factor(s) of reticulocyte lysate. Upon treatment of the cells with RA, time-dependent increases in the ratio of RARb to RXRa and the phosphorylated form of Sp1 were observed. We concluded that two genomic DNA regions, the DR4 site (-1531 to -1516) and the first and second Sp1 binding sites (-145 to -121), were involved in the RA-dependent augmentation of TM gene expression through increased interactions of the two regions with heterodimer of RAR-RXR and nuclear Sp1, respectively.
J. Biol. Chem, 10.1074/jbc.M004942200
Submitted on June 7, 2000
Revised on October 13, 2000
Accepted on October 17, 2000
Acceleration of thrombomodulin gene transcription by retinoic acid:retinoic acid receptors and Sp1 regulate the promoter activity through Interactions with two different sequences in the 5'-flanking region of human gene
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