Target gene profiles of 1,25(OH)2D3 and 25(OH)D3 in Cyp27b1 knockout mDCT cells.

Recently, it has been reported that 25(OH)D3 (25D3) has physiological bioactivity in certain tissues derived from the Cyp27b1 knockout mice. To investigate 25D3 function in the kidney as an informational crossroad of various calciotropic substances, we employed CRISPR-Cas9 system to knock out the Cyp27b1 gene in the mouse renal tubular cell line, mDCT cells. Unlike the previously reported mice targeted to the Cyp27b1 gene systemically, Cyp27b1 knockout mDCT cells did not produce any measurable 1a,25(OH)2D3 (1,25D3) after 25D3 administration. As was seen in the treatment with 10-8 M and higher dose of 1,25D3, we found that 10-7 M of 25D3 could translocate VDR into the nucleus and promoted expression of the representative 1,25D3-responsive Cyp24a1 gene in the Cyp27b1 knockout mDCT cells. The exhaustive target gene profiles of 25D3 showed results closely mimicking those of 1,25D3. Subsequently, we confirmed that 25D3 induced the expression of a calcium reabsorption-related gene, Calbindin-D9K gene, in a similar way to 1,25D3. As another example among others, we found that both 1,25D3 and 25D3 induced the expression of Megalin gene. Our ChIP assay identified that two VDRE sites at the upstream region of the Megalin gene contributed to such gene activation. Together, we surmise that the ability to stimulate VDR target genes may provide a novel perspective with 25D3 contribution in certain tissues. CRISPR-Cas9 mediated Cyp27b1 knockout mDCT cells were cultured as previously reported. Twenty-four hours before 1,25(OH)2D3 or 25(OH)D3 administration to the cells, culture medium was changed to the phenol red-free medium containing 2 % charcoal stripped fetal bovine serum (FBS), then the cells were exposed to the indicated hormones for another 24 h with the same medium. This study was analyzed 3 samples (N=1) described below. 128_siCT_V_1 was vehicle treated control sample, and 128_siCT_1_25D3_7_1 and 128_siCT_25D3_7_1 were 10-7 M of 1,25(OH)2D3 or 25(OH)D3 treated samples, respectively.