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1α,25-Dihydroxyvitamin D 3  (1α,25(OH) 2 D 3 ) had earlier been regarded as the only active hormone. The newly identified actions of 25-hydroxyvitamin D 3  (25(OH)D 3 ) and 24R,25-dihydroxyvitamin D 3  (24R,25(OH) 2 D 3 ) broadened the vitamin D 3  endocrine system, however, the current data are fragmented and a systematic understanding is lacking. Here we performed the first systematic study of global gene expression to clarify their similarities and differences. Three metabolites at physiologically comparable levels were utilized to treat human and mouse fibroblasts prior to DNA microarray analyses. Human primary prostate stromal P29SN cells (hP29SN), which convert 25(OH)D 3  into 1α,25(OH) 2 D 3  by 1α-hydroxylase (encoded by the gene  CYP27B1 ), displayed regulation of 164, 171, and 175 genes by treatment with 1α,25(OH) 2 D 3 , 25(OH)D 3 , and 24R,25(OH) 2 D 3 , respectively. Mouse primary  Cyp27b1  knockout fibroblasts (m Cyp27b1   −/− ), which lack 1α-hydroxylation, displayed regulation of 619, 469, and 66 genes using the same respective treatments. The number of shared genes regulated by two metabolites is much lower in hP29SN than in m Cyp27b1   −/− . By using DAVID Functional Annotation Bioinformatics Microarray Analysis tools and Ingenuity Pathways Analysis, we identified the agonistic regulation of calcium homeostasis and bone remodeling between 1α,25(OH) 2 D 3  and 25(OH)D 3  and unique non-classical actions of each metabolite in physiological and pathological processes, including cell cycle, keratinocyte differentiation, amyotrophic lateral sclerosis signaling, gene transcription, immunomodulation, epigenetics, cell differentiation, and membrane protein expression. In conclusion, there are three distinct vitamin D 3  hormones with clearly different biological activities. This study presents a new conceptual insight into the vitamin D 3  endocrine system, which may guide the strategic use of vitamin D 3  in disease prevention and treatment.

Gene Expression Profiles in Human and Mouse Primary Cells Provide New Insights into the Differential Actions of Vitamin D3 Metabolites