Abstract
Calcium is a second messenger that controls a wide variety of cellular functions. Because of its multiple actions, there is a stringent requirement for calcium homeostasis, and this is achieved in part by a system of transport and storage proteins such as calreticulin located in the endoplasmic reticulum. Calreticulin is also found in the nucleus, suggesting that it may have a role in transcriptional regulation. It has been reported that calreticulin can inhibit steroid-regulated gene transcription by preventing receptor binding to DNA. Here we report that overexpression of the calreticulin gene in B16 mouse melanoma cells resulted in a decrease in retinoic acid (RA)-stimulated reporter gene expression. Gel shift analysis showed that purified calreticulin inhibited the binding of endogenous RAR to a beta-RA response element oligonucleotide, only if added prior to the addition of the oligonucleotide. Co-immunoprecipitation studies suggest a physical interaction between RAR and calreticulin. Transfection of the calreticulin gene into B16 cells inhibited the RA induction of protein kinase Calpha, a marker of RA-induced differentiation. We also found that cyclic AMP increased the expression of calreticulin. Cyclic AMP may act to antagonize RA action by both decreasing RAR expression (Y. Xiao, D. Desai, T. Quick, and R. M. Niles, J. Cell Physiol., in press) and stimulating calreticulin levels.
Highlights
Retinoic acid (RA),1 a metabolite of vitamin A, is essential for normal cell growth and differentiation [1, 2]
Transient Transfection of the Calreticulin Gene Inhibits RAdependent Reporter Gene Expression—Because calreticulin has been reported to inhibit the function of steroid receptors [26, 27], we determined whether overexpression of this gene in B16 melanoma cells would interfere with retinoic acid (RA)-dependent expression of a reporter gene containing the -RARE
Purified Calreticulin Inhibits the Binding of RAR from B16 Cells to a -RARE Oligonucleotide—Because it has been reported that calreticulin can inhibit steroid receptor activity by binding to a region within the DNA binding domain, we determined whether purified calreticulin would interfere with the ability of endogenous B16 RAR to bind to a -RARE oligonucleotide (Fig. 2)
Summary
RA is thought to achieve its biological action by altering the expression of genes through binding and activating nuclear receptors. These receptors (RAR) are similar to other steroid hormone receptors in that they have a ligand binding domain and a DNA binding domain [3, 4]. Calreticulin has been localized to the nucleus [18], which has led to speculation that the protein may influence gene expression This protein binds to the synthetic peptide KLGFFKR [22], which is similar to the amino acid sequence in the DNA binding domain of the superfamily of steroid hormone receptors [23,24,25]. We show that calreticulin expression is modulated by cyclic AMP
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