Mitschele Tanja, Diesel Britta, Friedrich Michael, Meineke Viktor, Maas Ruth M, Gärtner Barbara C, Kamradt Jörn, Meese Eckart, Tilgen Wolfgang, Reichrath Jörg
Universitäts-Hautklinik, Universitätshautkliniken des Saarlandes, Homburg, Germany.
Lab Invest. 2004 Jun;84(6):693-702. doi: 10.1038/labinvest.3700096.
Using real-time PCR (LightCycler) and immunohistochemistry, we have analyzed expression of key components of the vitamin D system in basal cell carcinomas (BCCs) and normal human skin (NS). Increased VDR-immunoreactivity was demonstrated in BCCs using a streptavidin-peroxidase technique. RNA expression of vitamin D receptor (VDR) and of main enzymes involved in synthesis and metabolism of calcitriol (vitamin D-25-hydroxylase [25-OHase], 25-hydroxyvitamin D-1alpha-hydroxylase [1alpha-OHase], 1,25-dihydroxyvitamin D-24-hydroxylase [24-OHase]) was detected in BCCs and NS. Expression levels were determined as ratios between target genes (VDR, 1alpha-OHase, 25-OHase, 24-OHase) and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal control. Median of mRNA ratios for VDR/GAPDH (BCCs: 16.54; NS: 0.00021), 1alpha-OHase/GAPDH (BCCs: 0.739; NS 0.000803) and 24-OHase/GAPDH (BCCs: 0.00585; NS 0.000000366) was significantly (Wilcoxon-Mann-Whitney U-test) elevated in BCCs. In contrast, median of mRNA ratio for 25-OHase/GAPDH (BCCs: 0.17; NS: 0.016) was not significantly altered in BCCs as compared to NS. Additionally, we report for the first time expression of 1alpha-OHase splice variants in BCCs and NS, that were detected using conventional RT-PCR. In conclusion, our findings provide supportive evidence for the concept that endogeneous synthesis and metabolism of vitamin D metabolites as well as VDR expression may regulate growth characteristics of BCCs. New vitamin D analogs that exert little calcemic side effects, their precursors, or inhibitors of 24-OHase may offer a new approach for the prevention or therapy of BCCs. The function of alternative transcripts of 1alpha-OHase that we describe here for the first time in BCCs and NS and their effect on activity level has to be investigated in future experiments.
我们使用实时定量聚合酶链反应（LightCycler）和免疫组织化学方法，分析了基底细胞癌（BCC）和正常人皮肤（NS）中维生素D系统关键成分的表达情况。采用链霉亲和素 - 过氧化物酶技术显示，BCC中维生素D受体（VDR）免疫反应性增强。在BCC和NS中检测到了维生素D受体（VDR）以及参与骨化三醇合成和代谢的主要酶（维生素D - 25 - 羟化酶[25 - OHase]、25 - 羟基维生素D - 1α - 羟化酶[1α - OHase]、1,25 - 二羟基维生素D - 24 - 羟化酶[24 - OHase]）的RNA表达。表达水平通过目标基因（VDR、1α - OHase、25 - OHase、24 - OHase）与管家基因甘油醛 - 3 - 磷酸脱氢酶（GAPDH）的比值来确定，GAPDH作为内部对照。BCC中VDR/GAPDH（BCC：16.54；NS：0.00021）、1α - OHase/GAPDH（BCC：0.739；NS 0.000803）和24 - OHase/GAPDH（BCC：0.00585；NS 0.000000366）的mRNA比值中位数显著升高（Wilcoxon - Mann - Whitney U检验）。相比之下，BCC中25 - OHase/GAPDH的mRNA比值中位数（BCC：0.17；NS：0.016）与NS相比无显著变化。此外，我们首次报道了在BCC和NS中使用常规逆转录 - 聚合酶链反应检测到的1α - OHase剪接变体的表达。总之，我们的研究结果为维生素D代谢产物的内源性合成和代谢以及VDR表达可能调节BCC生长特性这一概念提供了支持性证据。具有低血钙副作用的新型维生素D类似物、其前体或24 - OHase抑制剂可能为BCC的预防或治疗提供新方法。我们首次在BCC和NS中描述的1α - OHase替代转录本的功能及其对活性水平的影响，有待未来实验进一步研究。
