25-Hydroxyvitamin D₃ 1α-Hydroxylase Splice Variants in Breast Cell Lines MCF-7 and MCF-10

Abstract

Background: It is known that 25(OH)D₃ can be metabolized to 1,25(OH)₂D₃ by 1α-OHase in breast tissue. This tissue-specific expression of 1α-OHase may act as the pivotal link between vitamin D status (25(OH)D₃ levels) and the anticancer effects of 1,25(OH)₂D₃. Alternative splicing frequently occurs in breast cancer cells; different splice variants of a given protein can display different biological functions and may cause tissue-specific variations. With this study it is the first time that expression and alternative splicing of 1α-OHase in the human breast cancer cell line MCF-7 and thebenign breast cell line MCF-10A are described. Materials and Methods: Expression of 1α-OHase RNA and protein was assessed using a real-time polymerase chain reaction (RT-PCR). The expression of 1α-OHase splice variants was detected by a highly specific PCR that combines nested and touchdown PCR. To determine which variants are translated in protein western blot analysis was carried out. Results: The expression of 1α-OHase was found to be 1.25-fold higher in MCF-7 compared to MCF-10A cells. In MCF-10A cells, at least 6 splice variants were detected whereas MCF-7 showed no or marginal expression levels of these variants. In MCF-7 cells the antibody detected a signal at 56 kDa corresponding to the size of normal 1α-OHase protein. In MCF-10A cells this signal was weaker. In western blot analysis at least two smaller variants at 45 kDa were found in MCF-7 cells. In MCF-10A cells at least 6 proteins between 37 and 56 kDa were detected with an only faint signal. Conclusion: We propose that alternative splicing of 1α-OHase can regulate the level of active enzyme. Splice variants may lead to a reduction of the protein. The significance of the smaller variants in MCF-7 cells has not been clarified either, but it is known that they are not able to use 25(OH)D₃ as a substrate to generate 1,25(OH)D₃. In MCF10A cells, more splice variants were identified, it may be that malignant cells contain inactive variants. How far they show a reduced activity remains unclear as no activity measurements were performed.