Inhibition of retinoic acid receptor function and retinoic acid-regulated gene expression in mouse melanoma cells by calreticulin. A potential pathway for cyclic AMP regulation of retinoid action.
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.
Desai
D
D
Department of Biochemistry and Molecular Biology, Marshall University School of Medicine, Huntington, West Virginia 25755, USA.
Michalak
M
M
Singh
N K
NK
Niles
R M
RM
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000502:physiology*; D037282:Calreticulin; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D004790:Enzyme Induction; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D007527:Isoenzymes / Q000096:biosynthesis; D007700:Kinetics; D008546:Melanoma, Experimental / Q000378:metabolism*; D051379:Mice; D018832:Molecular Chaperones / Q000502:physiology; D011493:Protein Kinase C / Q000096:biosynthesis; D051571:Protein Kinase C-alpha; D018168:Receptors, Retinoic Acid / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012261:Ribonucleoproteins / Q000096:biosynthesis / Q000502:physiology*; D014162:Transfection; D014212:Tretinoin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D007527:Isoenzymes; D018832:Molecular Chaperones; D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; C082925:retinoic acid receptor beta; D015124:8-Bromo Cyclic Adenosine Monophosphate; D014212:Tretinoin; D000242:Cyclic AMP; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha