|Budget Amount *help
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2002: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2001: ¥2,000,000 (Direct Cost: ¥2,000,000)
1. The differentiation represssing factor (DRF)-1 candidate (Dc) by one-hybrid method, which binds to KRE-M9 in the matrix metlloproteinase (MMP)-9 promoter was shown to exist as α or β with similarity to c-Jun molecule. 2. The DC gene-transfected 293T cells increased the amounts of KRE-M9-binding protein. 3. The secretion of MMP-9 from human cultured keratinocytes (Kc) was downregulated by the transfection of Dcβ. 4. In Kc, high Ca^<2+> concentration (hCa) for differentiation induced the MMP-9 expression and the trafficking of Dc into cytoplasm. In the dedifferentiated Kc after low Ca^<2+> back, DC was moved into nuclei with MMP-9 down-regulation. So far, phospho-c-Jun in the nuclei, DNA fragmentation, positive TUNEL staining, and the change in the concentrations of soluble Fas ligand have not been detected by hCa. 5. The addition of each cytokine, TGF-β, TNF-α or IL-1α, or the ultraviolet B (UVB) irradiation upregulated MMP-9 expression in Kc. Phospho-c-Jun was detected in the nuclei by TNF-α, IL-1α, or UVB. Luciferase assay showed that KRE-M9 was responsible for MMP-9 induction not only by hCa, but also by TNF-α or IL-1α. By UVB, the reduced number of live Kc, and annexin V positive Kc were observed. 6. By RT-PCR, APAF-1 was not detected with an ycytokine as described above. 7. DRF-1 was also observed in mesenchymal fibroblasts and HT-1080 cells. TGF-β or TNF-α also induced the MMP-9 expression from fibroblasts. 8. In situ zymography in the cases of squamous cell carcinoma detected gelatinolytic activity not only in the invasive lesions, but also in the dyskeratotic lesions, which was inhibited by MMP inhibitor, 1,10-phenanthroline. In conclusion, the involvement of HMP-9 in early apoptosis and the inhibition of MMP-9 expression by Dc were suggested.