Ratnam A V, Bikle D D, Cho J K
Department of Medicine, VAMC/University of California, San Francisco 94121, USA.
J Cell Physiol. 1999 Feb;178(2):188-96. doi: 10.1002/(SICI)1097-4652(199902)178:2<188::AID-JCP8>3.0.CO;2-4.
The steroid hormone 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) regulates cell proliferation and differentiation. Intracellular calcium (Cai) concentrations play a crucial role in these events. From our previous studies, we have demonstrated a calcium receptor (CaR) in keratinocytes which appears to regulate the initial release of Cai from intracellular stores in response to extracellular calcium (Cao) and so is likely to participate in the differentiation process. In this study, we determined whether the ability of 1,25(OH)2D3 to enhance Ca++ -induced differentiation was mediated at least in part through changes in the CaR. Keratinocytes were grown in keratinocyte growth medium (KGM) with 0.03 mM, 0.1 mM, or 1.2 mM Ca and treated with 10(-8) M 1,25(OH)2D3 till harvest after 5, 7, 14, and 21 days. CaR mRNA levels were quantitated by polymerase chain reaction. The results were compared to the ability of 1,25(OH)2D3 to enhance calcium-stimulated increases in Cai. In cells grown in 0.03 mM Ca, the CaR mRNA levels decreased with time. 1,25(OH)2D3 stimulated the levels at 5 days and prevented the falloff over the subsequent 16 days. On the other hand, in cells grown in 0.1 or 1.2 mM Ca, the message levels remained high, and 1,25(OH)2D3 had no further effect. To study the functional relationship, we harvested cells after 5 and 7 days in culture following a 24 h treatment with 1,25(OH)2D3 or vehicle to measure the Cai response to 2 mM Cao. The preconfluent cells grown in 0.03 mM Ca showed a nearly twofold increase in the Cai response to Cao when pretreated with 1,25(OH)2D3, whereas the confluent cells and those grown in 1.2 mM Ca showed no enhancement by 1,25(OH)2D3. Studies with 45Ca influx into keratinocytes revealed that 1,25(OH)2D3 enhanced the influx in preconfluent and confluent cells when grown in KGM containing 0.03 mM Ca but not in cells grown in 1.2 mM calcium. We conclude that 1,25(OH)2D3 maintains the CaR mRNA levels in cells grown in 0.03 mM Ca, thus maintaining their responsiveness to Cao and so ensuring their ability to differentiate in response to the calcium signal.
类固醇激素1,25 - 二羟基维生素D3（1,25(OH)2D3）调节细胞增殖和分化。细胞内钙（Cai）浓度在这些过程中起关键作用。根据我们之前的研究，我们已在角质形成细胞中证实一种钙受体（CaR），它似乎可调节细胞内钙库对细胞外钙（Cao）的反应而导致的Cai的初始释放，因此可能参与分化过程。在本研究中，我们确定1,25(OH)2D3增强钙离子诱导的分化的能力是否至少部分是通过钙受体的变化介导的。角质形成细胞在含有0.03 mM、0.1 mM或1.2 mM钙的角质形成细胞生长培养基（KGM）中培养，并用10(-8) M 1,25(OH)2D3处理，直至在5、7、14和21天后收获。通过聚合酶链反应定量CaR mRNA水平。将结果与1,25(OH)2D3增强钙刺激的Cai增加的能力进行比较。在0.03 mM钙中培养的细胞中，CaR mRNA水平随时间下降。1,25(OH)2D3在第5天刺激其水平，并防止在随后的16天中下降。另一方面，在0.1或1.2 mM钙中培养的细胞中，mRNA水平保持较高，1,25(OH)2D3没有进一步影响。为了研究功能关系，在用1,25(OH)2D3或赋形剂处理24小时后，在培养5天和7天后收获细胞，以测量Cai对2 mM Cao的反应。在0.03 mM钙中生长的未汇合细胞在用1,25(OH)2D3预处理后，对Cao的反应增加了近两倍，而汇合细胞和在1.2 mM钙中生长的细胞未显示出1,25(OH)2D3的增强作用。对45Ca流入角质形成细胞的研究表明，当在含有0.03 mM钙的KGM中生长时，1,25(OH)2D3增强了未汇合和汇合细胞中的流入，但在1.2 mM钙中生长的细胞中未增强。我们得出结论，1,25(OH)2D3维持在0.03 mM钙中生长的细胞中的CaR mRNA水平，从而维持它们对Cao的反应性，因此确保它们响应钙信号而分化的能力。
