Hansen C M, Madsen M W, Arensbak B, Skak-Nielsen T, Latini S, Binderup L
Department of Biochemistry, Leo Pharmaceutical Products, Ballerup, Denmark.
Cell Adhes Commun. 1998 Mar;5(2):109-20. doi: 10.3109/15419069809040285.
In the present investigation the effect of 1 alpha,25(OH)2D3 on the expression of the integrin laminin receptor on the melanoma cell line SK-MEL-28 has been examined. The SK-MEL-28 cells were shown to contain high-affinity receptors for 1 alpha,25(OH)2D3 and cell proliferation was found to be inhibited in a dose-dependent manner in response to the hormone. Using monoclonal antibodies against the alpha 6-sub-unit of the integrin laminin receptor, immunocytochemistry demonstrated that exposure of cells to 1 alpha,25(OH)2D3 for 5 days caused a reduced staining intensity. This observation was further confirmed by dot blot analysis, where a dose-dependent decline of alpha 6 expression was obtained after treatment of the cells with 1 alpha,25(OH)2D3 for 6 days. FACS-analysis was performed in order to quantify this decline, and it was found that the level of alpha 6-subunits on the cell surface was reduced by more than 40%. Additional investigations including Northern blot analyses of poly(A)+RNA extracts also showed a dose-dependent reduction of alpha 6 mRNA. Interestingly, the decrease of alpha 6 expression on the surface of SK-MEL-28 melanoma cells was accompanied by a reduced ability of the cells to adhere to an artificial basement membrane. In conclusion, the present investigation shows that besides having an antiproliferative effect on the SK-MEL-28 melanoma cells, 1 alpha,25(OH)2D3 is also able to inhibit the surface expression of the alpha 6-subunit of the integrin laminin receptor. Moreover, the results strongly indicate that 1 alpha,25(OH)2D3 exerts its regulatory effect on the alpha 6-subunit at the transcriptional level rather than at the protein level.
在本研究中，检测了1α,25(OH)₂D₃对黑色素瘤细胞系SK-MEL-28中整合素层粘连蛋白受体表达的影响。结果显示，SK-MEL-28细胞含有高亲和力的1α,25(OH)₂D₃受体，并且发现细胞增殖受到该激素的剂量依赖性抑制。使用针对整合素层粘连蛋白受体α6亚基的单克隆抗体，免疫细胞化学显示细胞暴露于1α,25(OH)₂D₃ 5天后染色强度降低。斑点印迹分析进一步证实了这一观察结果，在用1α,25(OH)₂D₃处理细胞6天后，α6表达呈剂量依赖性下降。进行流式细胞术分析以量化这种下降，发现细胞表面α6亚基的水平降低了40%以上。包括对poly(A)+RNA提取物进行Northern印迹分析在内的其他研究也显示α6 mRNA呈剂量依赖性减少。有趣的是，SK-MEL-28黑色素瘤细胞表面α6表达的降低伴随着细胞黏附于人工基底膜能力的下降。总之，本研究表明，1α,25(OH)₂D₃除了对SK-MEL-28黑色素瘤细胞具有抗增殖作用外，还能够抑制整合素层粘连蛋白受体α6亚基的表面表达。此外，结果强烈表明，1α,25(OH)₂D₃在转录水平而非蛋白质水平对α6亚基发挥调节作用。
