Identification of Novel Glucocorticoid-Response Elements in Human Elastin Promoter and Demonstration of Nucleotide Sequence Specificity of the Receptor Binding

Abstract

Glucocorticoids exert their action on gene expression through activation of cytoplasmic glucocorticoid receptors (GRs) that bind to glucocorticoid response elements (GREs). The consensus GRE consists of two half sites (underlined), AGAACANNNTGTTCT. We have recently cloned the entire human elastin gene. Nucleotide sequencing of the promoter region disclosed the presence of three putative GREs with the downstream half-site sequence TGTTCC that has homology with the consensus GRE, although the upstream half site showed no homology. To examine the functionality of these putative GREs in binding to the GRs, we performed gel mobility shift and supershift assays with synthetic oligomers containing the putative GREs and a recombinant GR protein, expressed in a baculovirus system. All three GREs identified in the elastin promoter bound the receptor. A chimeric oligonucleotide containing the upstream consensus GRE half site and the downstream elastin promoter GRE half site was capable of binding the receptor, and this binding could be competed with the elastin promoter GRE. Nonconservative substitution of single nucleotides (positions 1-6) in the elastin GRE indicated that mutations in the positions 1-3 and 6 had relatively little effect, but substitutions in positions 4 and 5 rendered the oligomer less effective in competing for the binding. These observations suggest that the downstream half site of GREs in the human elastin promoter is sufficient for receptor binding and certain nucleotides are critical for the efficient binding. The results also imply that the three GREs within the human elastin promoter are active and mediate the glucocorticoid-induced up-regulation of human elastin promoter activity.

ALY Is a Common Coactivator of RUNX1 and c-Myb on the Type B Leukemogenic VirusEnhancer

“…6A). c-Myb binds the consensus sequence NNCNTAACGGTTTT based on a binding site selection assay for yeast (7), whereas GR binds to the consensus sequence AGAACANNNTG TTCT (19). Our previous results have shown that the ϩ586 site is more important for enhancer function in Jurkat CD4 ϩ T cells than in CD4 ϩ CD8 ϩ T cells (35).…”

Effect of Carnitine on Cutaneous Wound Healing in Immunosuppressed Rats

“…Glucocorticoids exert their effects by activating cytoplasmic glucocorticoid receptors, which result in gene expression [11]. The most well known receptor for glucocorticoids is the intracellular glucocorticoid receptor-␣ (GR␣).…”

“…In a study by Del Monaco et al, glucocorticoids were shown to induce up-regulation of human elastin promoter activity and regulate gene expression at the transcriptional level [11]. In wound healing, the extracellular matrix has an important role and elastin is an extracellular matrix protein whose secretion is enhanced by glucocorticoids [11].…”

Extracellular Matrix Molecules: Potential Targets in Pharmacotherapy

“…As summarized in Table 2, besides collagens, several other ECM molecules are also targeted by glucocorticoids. These include elastin (Del Monaco et al, 1997); proteoglycans decorin and biglycan (Kähä ri et al, 1995); hyaluronan (Saarni and Hopsu-Havu, 1978); noncollagenous glycoproteins, particularly fibronectin, laminin, (Stahn et al, 2007) Modulation of the expression of various types of collagen (Oikarinen et al, 1987;Russell et al, 1989;Oishi et al, 2002), elastin (Del Monaco et al, 1997), proteoglycans decorin and biglycan (Kähäri et al, 1995), HA (Saarni and Hopsu-Havu, 1978), and various noncollagenous matrix glycoproteins such as fibronectin, laminin, and tenascin (Dean et al, 1988;Simo et al, 1992;Ekblom et al, 1993); suppression of MMP synthesis and activity (DiBattista et al, 1991;Sadowski and Steinmeyer, 2001); influence on TIMP expression and activity (Sadowski and Steinmeyer, 2001) NSAIDs e.g., indomethacin, diclofenac, naproxen, and -coxibs such as celecoxib…”