Fang D, Setaluri V
Departments of Dermatology and Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, North Carolina, 27157, USA.
Biochem Biophys Res Commun. 1999 Mar 24;256(3):657-63. doi: 10.1006/bbrc.1999.0400.
Tyrosinase and a family of tyrosinase-related proteins (TRPs) are melanocyte differentiation gene products involved in melanin pigmentation. Members of the tyrosinase family share upstream transcriptional regulatory elements suggesting that expression of these genes is regulated by shared mechanisms. Microphthalmia transcription factor MITF, a melanocyte-specific basic helix-loop-helix protein, has been shown to transactivate tyrosinase and TRP-1 genes in vitro by binding to a shared regulatory sequence known as M box. The role of MITF in concomitant regulation of these genes in vivo is not clear. We showed earlier that in human melanoma cells TRP-1 can be regulated independently of tyrosinase and pigmentation. To investigate the role of MITF in TRP-1 regulation, we studied the effect of pharmacological agents that modulate transcription of tyrosinase and TRP-1 on MITF. In melanoma cells treated with hexamethylene bisacetamide (HMBA), transcription of TRP-1 gene was selectively and completely inhibited while steady state levels of tyrosinase, TRP-2, MITF mRNA and melanin content showed a modest increase. HMBA caused no detectable change in cellular MITF or its nuclear localization. This MITF-independent regulation of TRP-1 required continued synthesis of RNA and protein. Selective down-regulation of TRP-1 by HMBA occurred even in the presence of cholera toxin which up-regulates TRP-1 by cAMP-mediated pathways. These data show that TRP-1 gene can be down-regulated independently of MITF by de novo activation of negative regulatory factors. Thus, both activation of positive factors such as MITF and inactivation of negative regulatory factors may be required for TRP-1 gene expression during melanocytic differentiation.
酪氨酸酶和酪氨酸酶相关蛋白（TRPs）家族是参与黑色素沉着的黑素细胞分化基因产物。酪氨酸酶家族成员共享上游转录调控元件，这表明这些基因的表达受共同机制调控。小眼畸形转录因子MITF是一种黑素细胞特异性的碱性螺旋-环-螺旋蛋白，已证明它在体外通过结合一个称为M盒的共同调控序列来反式激活酪氨酸酶和TRP-1基因。MITF在体内对这些基因的协同调控作用尚不清楚。我们之前表明，在人类黑色素瘤细胞中，TRP-1可以独立于酪氨酸酶和色素沉着进行调控。为了研究MITF在TRP-1调控中的作用，我们研究了调节酪氨酸酶和TRP-1转录的药物对MITF的影响。在用六亚甲基双乙酰胺（HMBA）处理的黑色素瘤细胞中，TRP-1基因的转录被选择性地完全抑制，而酪氨酸酶、TRP-2、MITF mRNA的稳态水平和黑色素含量则适度增加。HMBA对细胞内的MITF及其核定位没有可检测到的变化。这种TRP-1的MITF非依赖性调控需要RNA和蛋白质的持续合成。即使在存在通过cAMP介导的途径上调TRP-1的霍乱毒素的情况下，HMBA也能选择性地下调TRP-1。这些数据表明，TRP-1基因可以通过新激活负调控因子而独立于MITF被下调。因此，在黑素细胞分化过程中，TRP-1基因表达可能既需要MITF等正调控因子的激活，也需要负调控因子的失活。
