Acceleration of Thrombomodulin Gene Transcription by Retinoic Acid

Abstract

The interactions between retinoic acid-(RA)-dependent transcriptional regulatory sequences of the 5-untranslated region of the thrombomodulin 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 (RAR␣, RAR␤, and RAR␥) augmented the promoter activity under the condition of lower retinoid X receptor-␣ (RXR␣) expression, whereas the activity was greatly diminished when RXR␣ was highly expressed. An electrophoretic mobility shift assay with cDNA containing the DR4 indicated that heterodimers of RAR and RXR␣ interacted with the DR4 site, although the interaction gradually disappeared with the increase in the ratio of RXR␣/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 the 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 RAR␤ to RXR␣ 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 thrombomodulin gene expression through increased interactions of the two regions with heterodimer of RAR-RXR␣ and nuclear Sp1, respectively. Thrombomodulin (TM)1 is an essential cofactor for activation of protein C by thrombin on vascular endothelial cells (1-3). TM expression of human endothelial cells is decreased by tumor necrosis factor-␣ (4 -6), interleukin-1 (6, 7), endotoxin (8), and phorbol ester (5, 6) and oxidized LDL (9, 10), but we have found that it is increased by all-trans-retinoic acid (t-RA) (11, 12) and/or cAMP (11, 13) in human umbilical vein endothelial (HUVE) cells, through the acceleration of transcriptional activity. Several studies on the regulatory region of human TM gene have been reported (14 -17), and Dittman et al. (17) showed the existence of an RA response element (RARE) in the 5Ј-flanking region of the gene. The direct effects of retinol and its derivatives (retinoids), such as t-RA and 9-cis-RA (9C-RA), on the expressions of many genes are apparently mediated by nuclear receptor proteins that are members of the steroid and thyroid hormone receptor (TR) superfamily of transcriptional regulators (18, 19). Nuclear retinoid receptor dimers, of which retinoid X receptor (RXR) is a mandatory constituent, are required for effective activation of the t-RA and/or 9C-RA response pathways (20 -25). However, the direct repeats of RARE separated by 4 base sequences (DR4) at Ϫ1531 to Ϫ1516 fro...

The t-PA -7351C>T enhancer polymorphism decreases Sp1 and Sp3 protein binding affinity and transcriptional responsiveness to retinoic acid

“…This could be due to a RA-induced change in the phosphorylation state of Sp1. 37,38 Similar results have been reported with GC-box motifs derived from promoters of other genes expressed in the endothelium. 37,39,40 We also identified RAR␤ and RAR␥ as DR5 binding proteins.…”

“…Despite this, several attempts to detect a DNA-protein complex containing both RAR/RXR and Sp1 have failed. [38][39][40] However, Husmann et al, 44 who studied a GC-box in the interleukin 1B promoter, detected a RAR-Sp1-GCbox complex by EMSA when using RAR fusion protein and recombinant Sp1. On the basis of the results of our study, we speculate that the polymorphic GC-box and the DR5 site may be a functionally coupled transcriptional unit that is quantitatively influenced by the SNP at position Ϫ7351.…”

A Novel Retinoic Acid-Responsive Element Regulates Retinoic Acid-Induced BLR1 Expression

“…An Sp1 binding motif within the GT box-containing element was predicted by sequence analysis using the Transfac database. Sp1 was found to interact with the heterodimer RAR-RXR in the regulation of thrombomodulin gene transcription by retinoid acid (28). This motivated a test of whether an Sp1 consensus sequence oligonucleotide would compete with the GT box-containing element.…”

Transcriptional Regulation of the Membrane-associated Prostaglandin E2 Synthase Gene

“…It has been reported that tandem GC box sequences exist in several gene promoters, such as prostacyclin synthase (35), thromboxane receptor (36), hemopoietic cell kinase (37), Nmethyl-D-aspartate receptor 1 (38), and thrombomodulin (39,40). These tandem GC boxes represent binding sites for members of the Sp1 transcription factor family (41) that play an important role in the control of transcription of the aforementioned genes.…”