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Splice Variants of the CYP27b1 Gene and the Regulation of 1,25-Dihydroxyvitamin D₃ Production

Burns and Allen Research Institute, Division of Endocrinology, Diabetes and Metabolism, Cedars-Sinai Medical Center, University of California, Los Angeles, School of Medicine, Los Angeles, California 90048

Address all correspondence and requests for reprints to: Martin Hewison, Division of Endocrinology, Diabetes and Metabolism, Cedars-Sinai Medical Center, Los Angeles, California 90048. E-mail: martin.hewison{at}cshs.org

The cytochrome P450 25-hydroxyvitamin D₃-1-hydroxylase (CYP27b1) plays a pivotal role in vitamin D physiology by catalyzing synthesis of active 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]. In common with other P450s, CYP27b1 is known to exhibit alternative splicing. Here we have cloned and sequenced several novel intron 2-containing, noncoding splice variant mRNAs for CYP27b1 in 1,25(OH)₂D₃-producing HKC-8 human proximal tubule and THP-1 monocytic cells. Regulation of 1,25(OH)₂D₃ synthesis in these cell lines by calciotropic and noncalciotropic factors was associated with altered expression of the CYP27b1 splice variants. To assess the functional significance of this, HKC-8 cells were transfected with short hairpin RNA (shRNA) to inhibit mRNAs containing sequences from intron 2. This resulted in a significant increase in the expression of CYP27b1 protein and synthesis of 1,25(OH)₂D₃ by HKC-8 cells compared with control cells for two different intron 2-containing shRNAs (both P < 0.001). shRNA to intron 2 had no significant effect on the levels of wild-type CYP27b1 mRNA, suggesting a posttranscriptional mechanism of action. By contrast, shRNA to wild-type CYP27b1 suppressed transcription and activity of the enzyme by 70 and 31%, respectively (both P < 0.01). These data indicate that noncoding splice variants of CYP27b1 are functionally active and may play a significant role in the regulation of 1,25(OH)₂D₃ synthesis during normal physiology.