RETINOIC ACID REGULATES THE EXPRESSION OF THE CALCIUM-BINDING PROTEIN, CALBINDIN-D28K

It is a well established fact that the calcium-binding protein, calbindin-D-28k, is influenced by Vitamin D in intestine and kidney, However, very little is known concerning the regulation of calbindin-D-28k in brain, Although few genes that are regulated by retinoic acid (RA) have been identified in the nervous system, we now report that the human medulloblastoma cell line D283 (which is derived from cerebellum and has a distinctly neuronal phenotype) contains calbindin-D-28k endogenously and that calbindin protein and mRNA can be induced 10- to 15-fold in these cells by 10(-7) M RA. These findings are the first evidence of RA-mediated regulation of calbindin, The time course of response, as determined by Northern blot analysis, indicated that the first significant increase in calbindin-D-28k mRNA is at 12 h with a plateau of calbindin mRNA induction at 72 h after RA treatment. The induction of calbindin mRNA by RA was preceded by an induction of retinoic acid receptor-alpha mRNA and was accompanied by an induction of retinoid X receptor-alpha mRNA, Calbindin-D-28k mRNA levels in D283 medulloblastoma cells as well as the induction of calbindin mRNA by RA were not significantly affected by 1,25-dihydroxyvitamin D-3 treatment, Deletion mutant analysis of the native calbindin-D-28k promoter and cotransfection of CV-1 or D283 medulloblastoma cells in the presence of retinoic acid receptor-alpha and/or retinoid X receptor-alpha expression vectors as well as results of nuclear transcription assays did not indicate transcriptional regulation of calbindin-D-28k by RA. Studies of calbindin-D-28k mRNA in control and RA-pretreated D283 medulloblastoma cells at various times (3-24 h) after treatment with 4 mu g/ml actinomycin D indicated that the half-life of calbindin-D-28k mRNA was significantly increased in the presence of RA, suggesting regulation of calbindin-D-28k mRNA stability by RA, Thus, calbindin-D-28k is one of the few known targets of RA action in cells that express a neuronal phenotype. In addition, our findings present further evidence of an interrelationship between the actions of 1,25-dihydroxyvitamin D-3 and the active metabolites of vitamin A.