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

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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.