Abstract
We have investigated the mechanisms underlying cell-specific glucocorticoid repression of calcitonin/calcitonin gene-related peptide (CGRP) gene expression. Treatment with the synthetic glucocorticoid dexamethasone has been shown to decrease mRNA levels in the 44-2C thyroid C cell line. Nuclear run-on assays showed that dexamethasone repressed transcription 2-3-fold in 44-2C cells. In contrast, dexamethasone stimulated calcitonin/CGRP transcription 4-6-fold in the CA77 thyroid C cell line. Transient transfection assays were used to map repression of reporter gene activity in 44-2C cells to a neuroendocrine cell-specific enhancer located between -920 and -1125 base pairs (bp). Within this region, an 18-bp element was found that conferred both full basal enhancer activity and dexamethasone-dependent repression in 44-2C cells. The 18-bp region contains possible binding sites for AP-1 and helix-loop-helix transcription factors as well as a glucocorticoid receptor half-site. Colocalization of repression and enhancer activity was then investigated in other cell lines. In CA77 cells, while the 920-1125 region strongly enhanced transcription, the 18-bp region conferred only partial activation and dexamethasone had little effect on reporter gene activity. Dexamethasone did not repress the calcitonin/CGRP activity in the heterologous HeLa and Rat1 fibroblast cell lines. These results suggest that glucocorticoids repress transcription of the calcitonin/CGRP gene by inhibiting cell-specific transcription factor activity.
Highlights
We haveinvestigatedthemechanismsunderlying kinase gene is stimulated by glucocorticoids in the liver and cell-specific glucocorticoid repression of calcitonin/ kidney but repressed in adipose tissue ( 2 )
An 18-bp element was found tin (4), corticotropin-releasing hormone(5),and thyrotropinreleasing hormone genes (6).While in general the mechanisms underlying cell-specific glucocorticoid regulation arenot known, recent work with the proliferin gene enhancer has provided some insights (7). These studies indicated that selective interactions between the glucocorticoid receptor and theAP-1transcriptionfactorattheenhancercaneither that conferred both full basal enhancer activity and stimulate or repress transcriptioonf a reporter gene
AS a starting point to understanding the cell-specific glucocorticoid regulation of the calcitonin/CGRP gene, we have investigated the mechanismof repression of the gene in the 44-2C medullary thyroid carcinoma cell line
Summary
(10 mM TES, pH 7.4, 10 mM EDTA, 0.2% sodium dodecyl sulfate, 300 mM NaC1). Nitrocellulose filters containingimmobilized plasmid. 6-Actin was included transcription across both calcitonin aCnGdRP-specific exons as a hybridization control and pBKS plasmid DNA as a control for background hybridization. We foundthatdexamethasonetreatment of 44-2C cells decreased the calcitonin/CGRP gene transcription rate to a level consistent with th3e-4-fold decrease seen insteady state harvested 24 h later. 20-50 p1 of supernatant cell extract was assayedfor luciferase activity as previously described (18)using a luminometer To demonstrate that transcriptional repression occurs across both the calcitonin and CGRP exons in 44-2C cells, DNA fragments specific for calcitonin (exon4)orCGRP(exons 5 and 6) were used.
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