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Construction of a P450c27 fusion enzyme: a useful tool for analysis of vitamin D3 25-hydroxylase activity.

ABSTRACT: Liver mitochondrial P450c27, encoded by the CYP27 gene, can catalyse the 25-hydroxylation of vitamin D3 and the 27-hydroxylation of sterols. To facilitate the study of this enzyme in cell culture systems, we engineered a fusion protein consisting of P450c27 coupled to its electron-transport accessory proteins, ferredoxin and ferredoxin reductase, and assessed its enzyme activity by measuring the C-25 and C-27 (side-chain) hydroxylation of 1 alpha-hydroxyvitamin D3 (1 alpha-OH-D3). When incubated with 1 alpha-OH-D3, COS-1 cells transfected with a vector expressing the fusion protein produced 1 alpha,25-(OH)2D2 and 1 alpha,27-(OH)2D3 about four times more efficiently than did cells transfected with three individual components of the fusion. When incubated with the natural substrate, vitamin D3, the efficiency of hydroxylation was lower than that for 1 alpha-OH-D3 but still 1.7-fold higher for the fusion protein than for its individual components. The fusion protein was also able to reproduce qualitatively and quantitatively the activity shown by P450c27 in liver cells in situ. The P450c27-ferredoxin reductase-ferredoxin fusion construct represents a valuable tool for establishing the substrate specificity of this liver cytochrome and for evaluating its potential for activating pro-drug analogues of vitamin D.

SUBMITTER: Dilworth FJ

PROVIDER: S-EPMC1217927 | biostudies-other | 1996 Nov

REPOSITORIES: biostudies-other

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Project description:The cytochrome P450 mitochondrial enzyme 25-hydroxyvitamin D3 1alpha-hydroxylase (1alpha-hydroxylase) of renal tubule cells hydroxylates the major circulating form of vitamin D (25(OH)D3) to the active systemic hormone 1,25(OH)2D3. Local production of 1,25(OH)2D3 appears to occur also at other sites where 1alpha-hydroxylase is expressed for autocrine/paracrine regulation. To reduce risks of hypercalcemia during treatment with vitamin D, we have previously suggested use of non-1alpha-hydroxylated vitamin D analogues to target tissues where 1alpha-hydroxylase is expressed, including the parathyroid glands in secondary hyperparathyroidism. The present study was undertaken to examine expression of 1alpha-hydroxylase in breast cancer and to investigate whether a non-1alpha-hydroxylated vitamin D analogue displayed biological function. In addition, expression of the 25-hydroxyvitamin D3 24-hydroxylase (24-hydroxylase) and the vitamin D receptor (VDR) was investigated.The expression of 1alpha-hydroxylase, 24-hydroxylase and VDR was investigated in breast cancer specimens (n = 19) and normal breast tissues (n = 10) by immunohistochemistry and/or RT-PCR. Consecutive cryosections of 6 mum essentially free of immune cells were used in the analyses. The effect of vitamin D analogues on transcriptional activation was analyzed in transiently transfected MCF-7 breast cancer cells.1alpha-hydroxylase protein was demonstrated in 79% and 100% of breast cancer specimens and normal breast, respectively. The overall relative mRNA levels of 1alpha-hydroxylase and 24-hydroxylase in normal breast compared to breast tumors were: 1alpha-hydroxylase, 1 +/- 0.07 versus 0.7 +/- 0.05, respectively (p < 0.001); 24-hydroxylase, 1 +/- 0.08 verus 2.1 +/- 0.2, respectively (p < 0.001). The VDR was expressed in 95% of the tumors as expected, with mRNA levels of 1 +/- 0.09 and 1.4 +/- 0.12 (p < 0.05) in breast cancer and normal breast, respectively. The ketoconazole-sensitive transcription activation potential of the non-1alpha-hydroxylated vitamin D analogue prodrug of EB1089 (EB1285) was demonstrated in MCF-7 cells, which express 1alpha-hydroxylase. The activity of EB1285 was about 20% of 1,25(OH)2D3.These results demonstrate nearly normal expression levels of 1alpha-hydroxylase, 24-hydroxylase and VDR in the majority of investigated breast cancer specimens. A non-1alpha-hydroxylated vitamin D analogue displayed activity in breast cancer cells. Such analogues may present future therapeutic options for proliferative disorders where 1alpha-hydroxylase is expressed.

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Construction of a P450c27 fusion enzyme: a useful tool for analysis of vitamin D3 25-hydroxylase activity.

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