ANALYSIS FOR THE MODULATION OF MATRIX METALLOPROTEINASE-9 EXPRESSION IN APOPTOSIS

1.用单杂交方法筛选出与基质金属蛋白酶（MMP）-9启动子中的KRE-M9结合的分化抑制因子（DRF）-1候选物（Dc），该候选物与c-Jun分子相似，以α或β形式存在。2. DC基因转染的293 T细胞增加了KRE-M9结合蛋白的量。3.转染Dcβ后，人角质形成细胞（Kc）分泌MMP-9的能力下降。4.在Kc中，高浓度的Ca^<2+>诱导MMP-9的表达和Dc向细胞质的运输。在低Ca^2+返流后的去分化Kc中，随着MMP-9的下调，DC向核内移动。迄今为止，hCa未检测到细胞核中磷酸化c-Jun、DNA断裂、TUNEL阳性染色和可溶性Fas配体浓度的变化。5.加入TGF-β、TNF-α或IL-1α或紫外线B（UVB）照射可上调KC中MMP-9的表达。用TNF-α、IL-1α或UVB在细胞核中检测到磷酸化c-Jun。荧光素酶检测显示KRE-M9不仅参与hCa对MMP-9的诱导，而且参与TNF-α和IL-1α对MMP-9的诱导。UVB照射后，Kc细胞存活率降低，Annexin V阳性Kc细胞数减少。6.通过RT-PCR，如上所述用γ细胞因子未检测到APAF-1。7.在间充质成纤维细胞和HT-1080细胞中也观察到DRF-1。TGF-β或TNF-α也诱导成纤维细胞表达MMP-9。8.在鳞状细胞癌的情况下，原位酶谱检测明胶分解活性不仅在浸润性病变，但也在角化不良病变，这是抑制MMP抑制剂，1，10-菲咯啉。结论：HMP-9参与了早期细胞凋亡，Dc可抑制MMP-9的表达。

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.