Basic study for the treatment of inflammatory skin diseases using the regulation of gelatinolytic activities.

明胶酶、基质金属蛋白酶(MMP2)和MMP9的明胶溶解活性以及金属蛋白酶组织抑制因子(TIMP)-1和TIMP-2的抑制作用在炎症性皮肤病中起重要作用。我们最近在基质金属蛋白酶-9启动子中发现了我们指定的分化抑制因子(DRF)-1结合的新的KRE-M9元件。在对基质金属蛋白酶-2和基质金属蛋白酶-9的RNA干扰后，对培养的细胞进行了对基质金属蛋白酶-2和基质金属蛋白酶-9半胱氨酸氨基转移酶3/7、8和9活性的干扰，这三种活性已被证明与炎症和凋亡有关。因此，caspase-3/7的活性在每一种干扰作用下都特别降低。KRE-M9元件对基质金属蛋白酶-9表达的调节利用诱骗作用诱导培养的细胞表达基质金属蛋白酶-9，表明KRE-M9元件具有抑制活性。用生物素标记的KRE-M9寡核苷酸和链霉亲和素-琼脂糖凝胶从核提取液中分离纯化DRF-1，并对其性质进行了分析。DRF-1被caspase-3/7活性断裂，caspase-3/7活性是由表皮角质形成细胞分化刺激诱导的。对DRF-1的RNA干扰不仅增强了基质金属蛋白酶-9的转录活性，而且还增强了角质形成细胞分化标志物总蛋白的转录活性。用被称为运输某些蛋白核输出的调节剂的软霉素B(LMB)处理培养的细胞时，加入LMB可减少基质金属蛋白酶-9的转录，而诱导TIMP-2的转录。小鼠局部应用LMB可抑制中波紫外线照射后的炎症反应，促进创面愈合。

Gelatinolytic activities by gelatinases, matrix metalloproteinase (MMP)-2 and MMP-9, with the inhibitions by tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2 have been shown to play important roles in the inflammatory skin diseases. We have recently found the novel KRE-M9 element to which our designated differentiation repressing factor (DRF)-1 binds in the MMP-9 promoter.1. RNA interference for MMP-2 and MMP-9Caspase 3/7, 8, and 9 activities, which have been shown to be responsible for inflammation as well as for apoptosis, were measured on cells in culture after the RNA interference for MMP-2 or for MMP-9. As a result, caspase-3/7 activity was especially reduced by each interference.2. Regulation of MMP-9 expression through KRE-M9 elementUsing decoy treatment for the KRE-M9 element on cells in culture, MMP-9 expression was induced, indicating the inhibitory activity of the KRE-M9 element. DRF-1 was purified from the nuclear extract by biotinylated KRE-M9 oligonucleotide and by streptavidine-Sepharose, and the nature of DRF-1 was analyzed. DRF-1 was fragmented by caspase-3/7 activity, which was induced by the stimulation for the differentiation of keratinocytes in the epidermis. RNA interference for DRF-1 enhanced the transcriptional activities not only for MMP-9, but also for involucrin, which is known to be the marker for the differentiation of keratinocyte.3. Treatment with leptomycin B (LMB) known as the regulater for trafficking the nuclear export of some proteinshe addition of LMB for the cells in culture reduced MMP-9 transcription, whereas it induced the TIMP-2 one. The topical application of LMB to mouse suppressed the inflammation after ultraviolet B irradiation, and it also showed the effect for the improvement of wound healing.