Martínez-Esparza M, Jiménez-Cervantes C, Solano F, Lozano J A, García-Borrón J C
Department of Biochemistry and Molecular Biology, School of Medicine, University of Murcia, Spain.
Eur J Biochem. 1998 Jul 1;255(1):139-46. doi: 10.1046/j.1432-1327.1998.2550139.x.
The inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is present in the dermal and epidermal layers of normal skin [Kilgus, O., Payer, E., Schreiber, S., Elbe, A., Strohal, R. & Stingl, G. (1993) J. Invest. Dermatol. 100, 674-680]. Its local concentrations are modified by several stimuli, including wound healing and ultraviolet irradiation. Moreover, TNF-alpha inhibits melanogenesis in normal melanocytes [Swope, V., Abdel-Malek, Z., Kassem, L. & Norlund, J. (1991) J. Invest. Dermatol. 96, 180-185], and is, therefore, a potential autocrine/paracrine regulator of pigmentation. We have analyzed the mechanisms of this effect using B16/F10 melanoma cells as a model. Nanomolar concentrations of TNF-alpha inhibit the tyrosine hydroxylase and dopa oxidase activities of B16/F10 melanocytes, to less than 30% control levels, without effects on tyrosinase-related protein 2/dopachrome tautomerase (TRP2/DCT). The 50% inhibition was obtained at 1 nM TNF-alpha and 48 h treatment. The effect of TNF-alpha was noticeable after 6 h treatment, and maximal after 24 h. This inhibition is explained by decreased intracellular levels of tyrosinase and tyrosinase-related protein 1 (TRP1), but not of TRP2/DCT as detected by Western blotting. Northern-blot experiments showed that the inhibitory effect is partially explained by a reduced accumulation of the corresponding mRNAs, that dropped to about 50% of control values (48 h treatment, 5 nM TNF-alpha). Moreover, the tyrosine hydroxylase and dopa oxidase activities decreased more rapidly in TNF-alpha-treated cells than in control cells, under conditions of inhibition of protein synthesis. This suggests a TNF-mediated reduction of tyrosinase half-life. However, the possibility of an inhibitory post-translational modification of the enzyme induced by TNF cannot be ruled out. Therefore, the inhibitory effect of TNF-alpha on tyrosinase and TRP-1 results from combined effect on mRNA levels and enzymatic activity or protein stability.
炎症细胞因子肿瘤坏死因子-α（TNF-α）存在于正常皮肤的真皮层和表皮层中[Kilgus, O., Payer, E., Schreiber, S., Elbe, A., Strohal, R. & Stingl, G. (1993) 《皮肤病学研究杂志》100, 674 - 680]。其局部浓度会受到多种刺激的影响，包括伤口愈合和紫外线照射。此外，TNF-α可抑制正常黑素细胞中的黑素生成[Swope, V., Abdel-Malek, Z., Kassem, L. & Norlund, J. (1991) 《皮肤病学研究杂志》96, 180 - 185]，因此，它是一种潜在的色素沉着自分泌/旁分泌调节因子。我们以B16/F10黑色素瘤细胞为模型分析了这种作用的机制。纳摩尔浓度的TNF-α可将B16/F10黑素细胞的酪氨酸羟化酶和多巴氧化酶活性抑制至对照水平的30%以下，而对酪氨酸酶相关蛋白2/多巴色素互变异构酶（TRP2/DCT）无影响。在1 nM TNF-α处理48小时后可达到50%的抑制率。TNF-α处理6小时后效果明显，24小时后达到最大抑制效果。通过蛋白质印迹法检测发现，这种抑制作用是由于细胞内酪氨酸酶和酪氨酸酶相关蛋白1（TRP1）水平降低，但TRP2/DCT水平未降低所致。Northern印迹实验表明，这种抑制作用部分是由于相应mRNA积累减少，在48小时处理、5 nM TNF-α作用下，mRNA水平降至对照值的约50%。此外，在蛋白质合成受抑制的条件下，TNF-α处理的细胞中酪氨酸羟化酶和多巴氧化酶活性的下降速度比对照细胞更快。这表明TNF介导了酪氨酸酶半衰期的缩短。然而，不能排除TNF对该酶进行翻译后抑制修饰的可能性。因此，TNF-α对酪氨酸酶和TRP-1的抑制作用是对mRNA水平以及酶活性或蛋白质稳定性综合作用的结果。
