Fogh K, Sølvsten H, Jøhnke H, Kragballe K
Department of Dermatology, Marselisborg Hospital, University of Aarhus, Denmark.
Exp Dermatol. 1996 Feb;5(1):24-7. doi: 10.1111/j.1600-0625.1996.tb00089.x.
Psoriasis is characterized by hyperproliferation and impared differentiation of epidermal keratinocytes (KCs). Psoriasis can be treated with derivatives of retinoic acid (RA) and vitamin D3. Analogues of vitamin D3 are able to inhibit proliferation and stimulate differentiation of KCs. In contrast, RA inhibits terminal differentiation of KCs. Interactions are known to occur between RA and vitamin D3 signalling pathways. The purpose of the present study was to determine the effect of all-trans RA on the binding of 1,25-dihydroxy-vitamin D3 (1,25 (OH)2D3) to the vitamin D3 receptor (VDR) of cultured human KCs. Cultured KCs from normal adults were incubated with or without RA (10-9-10-7M) for 4-24 h. Cells were then harvested, homogenized and ultrasonicated. The extracted protein was incubated with 3H-1,25 (OH)2D3 (0.015-1.0 nM) with or without 250-fold excess nonradioactive 1,25 (OH)2D3 for 24 h and specific binding was determined by use of the dextran coated charcoal binding assay. Western blot analysis utilizing the monoclonal antibody 9A7 gamma to VDR was performed on protein extracted from the KCs. The bands resulting from Western blot analysis were visualized by enhanced chemiluminescence. From Scatchard analysis it was found that KCs bind 1,25 (OH)2D3 with high affinity (Kd = 0.175 nM). This binding was dose and time dependently inhibited by RA (60% inhibition at 10-7 M after 24 h of incubation). By Western blot analysis, RA had no effect on the amount of protein extracted from KCs at any of the RA concentrations tested. In conclusion, these results show that binding of vitamin D3 to its receptor of human KCs can be inhibited markedly by RA without effecting the amount of protein. These results are in contrast to results with other cell types in which RA upregulates binding of 1,25 (OH)2D3 to the VDR. Because interaction between retinoids and vitamin D3 may occur at different levels during signal transduction, it is not possible to predict from our results whether RA will inhibit the effects of vitamin D3 in vivo.
银屑病的特征是表皮角质形成细胞（KC）过度增殖和分化受损。银屑病可用维甲酸（RA）和维生素D3的衍生物治疗。维生素D3类似物能够抑制KC的增殖并刺激其分化。相反，RA抑制KC的终末分化。已知RA和维生素D3信号通路之间会发生相互作用。本研究的目的是确定全反式RA对1,25 - 二羟基维生素D3（1,25(OH)2D3）与培养的人KC的维生素D3受体（VDR）结合的影响。将来自正常成年人的培养KC在有或无RA（10-9 - 10-7M）的情况下孵育4 - 24小时。然后收获细胞，进行匀浆和超声处理。提取的蛋白质与3H - 1,25(OH)2D3（0.015 - 1.0 nM）在有或无250倍过量非放射性1,25(OH)2D3的情况下孵育24小时，并通过葡聚糖包被活性炭结合试验测定特异性结合。利用针对VDR的单克隆抗体9A7γ对从KC中提取的蛋白质进行蛋白质印迹分析。通过增强化学发光使蛋白质印迹分析产生的条带可视化。通过Scatchard分析发现，KC以高亲和力（Kd = 0.175 nM）结合1,25(OH)2D3。这种结合受到RA的剂量和时间依赖性抑制（孵育24小时后，在10-7M时抑制60%）。通过蛋白质印迹分析，在测试的任何RA浓度下，RA对从KC中提取的蛋白质量均无影响。总之，这些结果表明，RA可显著抑制维生素D3与其在人KC上的受体的结合，而不影响蛋白质量。这些结果与其他细胞类型的结果相反，在其他细胞类型中RA上调1,25(OH)2D3与VDR的结合。由于类视黄醇和维生素D3之间的相互作用可能在信号转导的不同水平发生，根据我们的结果无法预测RA在体内是否会抑制维生素D3的作用。
