Li L, Kruszewski F H, Punnonen K, Tucker R W, Yuspa S H, Hennings H
Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bethesda, Maryland 20892.
J Cell Physiol. 1993 Mar;154(3):643-53. doi: 10.1002/jcp.1041540324.
Primary mouse keratinocytes in culture are induced to terminally differentiate by increasing extracellular Ca2+ concentrations (Cao) from 0.05 mM to > or = 0.1 mM. The addition of Sr2+ (> or = 2.5 mM) to medium containing 0.05 mM Ca2+ induces focal stratification and terminal differentiation, which are similar to that found after increasing the Cao to 0.12 mM. Sr2+ in 0.05 mM Ca2+ medium induces the expression of the differentiation-specific keratins, keratin 1 (K1), keratin 10 (K10), and the granular cell marker, filaggrin, as determined by both immunoblotting and immunofluorescence. Sr2+ induces the expression of those differentiation markers in a dose dependent manner, with an optimal concentration of 5 mM. In the absence of Ca2+ in the medium, the Sr2+ effects are reduced, and Sr2+ is ineffective when both Ca2+ and serum are deleted from the medium. Sr2+ treatment increases the ratio of fluorescence intensity of the intracellular Ca2+ sensitive probe, fura-2, indicating an associated rise in the level of intracellular free Ca2+ and/or Sr2+. At doses sufficient to induce differentiation, Sr2+ also increases the level of inositol phosphates in primary keratinocytes within 30 min. The uptake curves of 85Sr2+ by primary keratinocytes are similar to those of 45Ca2+. At low concentrations, the initial uptake of both 45Ca2+ and 85Sr2+ reaches a plateau within 1 hr; at higher concentrations, the uptake of both 45Ca2+ and 85Sr2+ increases continuously for 12 hr. In keratinocytes pre-equilibrated with 45Ca2+ in 0.05 mM Ca2+ medium, Sr2+ causes an increase of 45Ca2+ uptake, which is dependent on the presence of serum. These results suggest that Sr2+ utilizes the same signalling pathway as Ca2+ to induce keratinocyte terminal differentiation and that Ca2+ may be required to exert these effects.
培养中的原代小鼠角质形成细胞通过将细胞外Ca2+浓度（Cao）从0.05 mM提高到≥0.1 mM而被诱导终末分化。向含有0.05 mM Ca2+的培养基中添加Sr2+（≥2.5 mM）可诱导局灶性分层和终末分化，这与将Cao提高到0.12 mM后观察到的情况相似。通过免疫印迹和免疫荧光测定，0.05 mM Ca2+培养基中的Sr2+可诱导分化特异性角蛋白角蛋白1（K1）、角蛋白10（K10）以及颗粒细胞标志物丝聚蛋白的表达。Sr2+以剂量依赖性方式诱导这些分化标志物的表达，最佳浓度为5 mM。在培养基中不存在Ca2+的情况下，Sr2+的作用减弱，而当培养基中同时去除Ca2+和血清时，Sr2+则无效。Sr2+处理增加了细胞内Ca2+敏感探针fura-2的荧光强度比值，表明细胞内游离Ca2+和/或Sr2+水平相应升高。在足以诱导分化的剂量下，Sr2+还可在30分钟内增加原代角质形成细胞中肌醇磷酸的水平。原代角质形成细胞对85Sr2+的摄取曲线与对45Ca2+的摄取曲线相似。在低浓度下，45Ca2+和85Sr2+的初始摄取在1小时内达到平台期；在较高浓度下，45Ca2+和85Sr2+的摄取持续增加12小时。在用45Ca2+在0.05 mM Ca2+培养基中预平衡的角质形成细胞中，Sr2+导致45Ca2+摄取增加，这依赖于血清的存在。这些结果表明，Sr2+利用与Ca2+相同的信号通路来诱导角质形成细胞终末分化，并且可能需要Ca2+来发挥这些作用。
