Matsumoto K, Azuma Y, Kiyoki M, Okumura H, Hashimoto K, Yoshikawa K
Department of Dermatology, Osaka University School of Medicine, Japan.
Biochim Biophys Acta. 1991 May 17;1092(3):311-8. doi: 10.1016/s0167-4889(97)90006-9.
In this study, we investigated the possibility that cultured keratinocytes from normal human adult skin produce 1,25-dihydroxyvitamin D-3 (1,25(OH)2D3, a biologically active form of vitamin D-3) from 25-hydroxyvitamin D-3 [25(OH)D3], and that 1,25(OH)2D3 endogenously produced by keratinocytes is involved in the self regulation of their growth and differentiation. To determine whether 1,25(OH)2D3 is produced from 25(OH)D3 by skin keratinocytes, 25(OH)[3H]D3 was added to keratinocyte cultures and incubated for 1 h and 5 h. The intracellular and extracellular metabolites were analyzed by three chromatographic systems. The three chromatograms revealed that the major metabolite produced from 25(OH)D3 was 1,25(OH)2D3. Most of the 1,25(OH)2D3 endogenously produced from 25(OH)D3 remained within the cells. To examine the time course of 1,25(OH)2D3 production, the amount of 1,25(OH)[3H]D3 was measured at 15 min, 1 h, 5 h and 10 h, being at a maximum 1 h after the addition of 25(OH)D3. These data indicate that keratinocytes rapidly convert 25(OH)D3 to 1,25(OH)2D3 and that 1,25(OH)2D3 is not released into the medium. To determine whether endogenously produced 1,25(OH)2D3 is involved in the regulation of growth and differentiation of normal human keratinocytes, we examined the effects of 1,25(OH)2D3 and 25(OH)D3 on their growth and differentiation. Keratinocyte growth was inhibited to 52.6% and 23.4% by 10(-8) M and 10(-7) M 1,25(OH)2D3 and to 80.5% and 23.9% by 10(-8) M and 10(-7) M 25(OH)D3, respectively. Differentiation of these cells was evaluated by quantifying the number which express involucrin, a precursor protein of cornified envelope. The population of involucrin expressing cells (differentiated cells) increased from 6.2% to 14.5% by 2.5.10(-7) M 1,25(OH)2D3, and to 11.8% by 2.5.10(-7) M 25(OH)D3. These results clearly indicate that 25(OH)D3 is as effective on human keratinocytes as 1,25(OH)2D3 in inhibiting growth and inducing differentiation, although to a slightly lesser extent than 1,25(OH)2D3. The possibility that the effect of 25(OH)D3 is mediated through binding to the 1,25(OH)2D3 receptor can be excluded, since a competitive binding assay revealed that the affinity of 25(OH)D3 for the 1,25(OH)2D3 receptor in a cytosolic extract of keratinocytes was 100-times lower than that of 1,25(OH)2D3. Thus, these results suggest that 1,25(OH)2D3 endogenously produced in keratinocytes from 25(OH)D3 is involved in the regulation of their growth and differentiation in vitro.
在本研究中，我们探究了来自正常成人皮肤的培养角质形成细胞能否将25-羟基维生素D-3[25(OH)D3]转化为1,25-二羟基维生素D-3（1,25(OH)2D3，维生素D-3的一种生物活性形式），以及角质形成细胞内源性产生的1,25(OH)2D3是否参与其生长和分化的自我调节。为了确定皮肤角质形成细胞能否将25(OH)D3转化为1,25(OH)2D3，将25(OH)[3H]D3添加到角质形成细胞培养物中，并孵育1小时和5小时。通过三种色谱系统分析细胞内和细胞外代谢产物。这三种色谱图显示，由25(OH)D3产生的主要代谢产物是1,25(OH)2D3。从25(OH)D3内源性产生的1,25(OH)2D3大部分保留在细胞内。为了检测1,25(OH)2D3产生的时间进程，在添加25(OH)D3后的15分钟、1小时、5小时和10小时测量1,25(OH)[3H]D3的量，在添加25(OH)D3后1小时达到最大值。这些数据表明角质形成细胞能迅速将25(OH)D3转化为1,25(OH)2D3，且1,25(OH)2D3不会释放到培养基中。为了确定内源性产生的1,25(OH)2D3是否参与正常人角质形成细胞的生长和分化调节，我们检测了1,25(OH)2D3和25(OH)D3对其生长和分化的影响。10(-8)M和10(-7)M的1,25(OH)2D3分别将角质形成细胞的生长抑制至52.6%和23.4%，10(-8)M和10(-7)M的25(OH)D3分别将其抑制至80.5%和23.9%。通过定量表达兜甲蛋白（一种角质包膜前体蛋白）的细胞数量来评估这些细胞的分化。2.5×10(-7)M的1,25(OH)2D3使表达兜甲蛋白的细胞群体（分化细胞）从6.2%增加到14.5%，2.5×10(-7)M的25(OH)D3使其增加到11.8%。这些结果清楚地表明，25(OH)D3在抑制生长和诱导分化方面对人角质形成细胞的作用与1,25(OH)2D3相当，尽管程度略低于1,25(OH)2D3。25(OH)D3的作用是通过与1,25(OH)2D3受体结合介导的这一可能性可以排除，因为竞争性结合试验表明，25(OH)D3对角质形成细胞胞质提取物中1,25(OH)2D3受体的亲和力比1,25(OH)2D3低100倍。因此，这些结果表明，角质形成细胞从25(OH)D3内源性产生的1,25(OH)2D3在体外参与其生长和分化的调节。
