Effect of cyclic adenosine 3',5'-monophosphate and protein kinase A on ligand-dependent transactivation via the vitamin D receptor.
We examined the effects of cyclic adenosine 3',5'-monophosphate (cAMP) and protein kinase A (PKA) on the ligand-dependent transactivation mediated via the 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) receptor (VDR). A human VDR expression plasmid was transfected into HeLa, Saos-2 and MG63 cells with a luciferase reporter gene construct containing the vitamin D responsive element. With the addition of 0.5 mM 8 bromo-cAMP, the response to 1,25(OH)2D3 was suppressed to 61 and 78% in the HeLa and Saos-2 cells, respectively. The suppressive effect of 8 bromo-cAMP was observed without the introduction of the VDR expression plasmid in the MG63 cells. In the HeLa cells the co-expression of PKA reduced the ligand-inducible transactivation to 61% and the fold induction by 1,25(OH)2D3 to 89% of that without PKA. The CREB binding protein (CBP) was recently reported to integrate the intracellular signals via the cAMP/PKA cascade and nuclear hormone receptors. However, the suppressive effect of cAMP was not influenced by the co-expression of CBP. Lastly, we introduced point mutations at possible PKA phosphorylation sites into the VDR expression vector at serine-172 and threonine-175, but both mutant receptors still exhibited reduced transactivation with the co-expression of PKA. These results indicate that the phosphorylation of proteins other than the VDR may also be involved in the inhibitory effect mediated by the cAMP/PKA cascade.
Nakajima
S
S
Department of Environmental Medicine, Research Institute, Osaka Medical Center for Maternal and Child Health, Izumi, Japan.
Yamagata
M
M
Sakai
N
N
Ozono
K
K
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D050882:CREB-Binding Protein; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D017354:Point Mutation; D018167:Receptors, Calcitriol / Q000235:genetics* / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection
D008024:Ligands; D009687:Nuclear Proteins; D018167:Receptors, Calcitriol; D015534:Trans-Activators; D015124:8-Bromo Cyclic Adenosine Monophosphate; D004247:DNA; D000242:Cyclic AMP; D050882:CREB-Binding Protein; C494363:CREBBP protein, human; D017868:Cyclic AMP-Dependent Protein Kinases