Nelimarkka L O, Nikkari S T, Ravanti L S, Kähäri V M, Järveläinen H T
Department of Medical Biochemistry, University of Turku and Turku University Central Hospital, Finland.
Matrix Biol. 1998 Aug;17(4):293-304. doi: 10.1016/s0945-053x(98)90082-8.
Recently, we have shown that the tumor necrosis factor-alpha (TNF-alpha)-induced morphological change of EA.hy 926 human endothelial cells is associated with a decrease in the net synthesis of two proteoglycans (PGs), biglycan and syndecan-1, both of which have been suggested to play a role in cell adhesion. Here we have examined whether this phenotypic modulation of EA.hy 926 cells also involves altered expression of matrix metalloproteinases (MMPs) or their tissue inhibitors (TIMPs). We demonstrate that, when forming cobblestone-like monolayer cultures, these cells express and synthesize collagenase-1 (MMP-1), stromelysin-1 (MMP-3) and 72 kDa (MMP-2) and 92 kDa (MMP-9) gelatinases, all of which have previously been found in either normal or pathological human vascular wall. EA.hy 926 cells also express membrane-typel MMP (MT1-MMP), but not matrilysin (MMP-7) and collagenase-3 (MMP-13). As regards TIMPs, we show that these cells express TIMP-1 and TIMP-2, but not TIMP-3 or TIMP-4. Exposure of the cells to TNF-alpha changed the cell morphology from a polygonal shape into a spindle shape and also increased the mRNA levels of MMP-1, MMP-3 and MMP-9, but slightly decreased the MMP-2 mRNA level. No change at the mRNA level of MT1-MMP was observed. Similarly to unstimulated cultures, no mRNA for MMP-7 or MMP-13 was detected in the TNF-alpha treated cultures. TNF-alpha had no effect on the TIMP-1 and TIMP-2 mRNA levels and did not induce TIMP-3 or TIMP-4 expression. Gelatin zymography and Western blot analysis revealed that the increase observed at the mRNA level of MMP-3 and MMP-9 was similar to that of their net protein level; furthermore, the active form of MMP-1 was induced. Our results indicate that the TNF-alpha-induced morphological change of EA.hy 926 cells is associated not only with specific changes in the expression of PGs by the cells, but also with specific changes in the expression of MMPs.
最近，我们已经表明，肿瘤坏死因子-α（TNF-α）诱导的EA.hy 926人内皮细胞形态变化与两种蛋白聚糖（PGs），双糖链蛋白聚糖和syndecan-1的净合成减少有关，这两种蛋白聚糖均被认为在细胞粘附中起作用。在此，我们研究了EA.hy 926细胞的这种表型调节是否也涉及基质金属蛋白酶（MMPs）或其组织抑制剂（TIMPs）表达的改变。我们证明，当形成鹅卵石样单层培养物时，这些细胞表达并合成胶原酶-1（MMP-1）、基质溶解素-1（MMP-3）以及72 kDa（MMP-2）和92 kDa（MMP-9）明胶酶，所有这些酶先前在正常或病理性人类血管壁中均有发现。EA.hy 926细胞还表达膜型1 MMP（MT1-MMP），但不表达基质溶素（MMP-7）和胶原酶-3（MMP-13）。关于TIMPs，我们表明这些细胞表达TIMP-1和TIMP-2，但不表达TIMP-3或TIMP-4。将细胞暴露于TNF-α会使细胞形态从多边形变为纺锤形，同时也会增加MMP-1、MMP-3和MMP-9的mRNA水平，但会略微降低MMP-2的mRNA水平。未观察到MT1-MMP的mRNA水平有变化。与未刺激的培养物类似，在TNF-α处理的培养物中未检测到MMP-7或MMP-13的mRNA。TNF-α对TIMP-1和TIMP-2的mRNA水平没有影响，也未诱导TIMP-3或TIMP-4的表达。明胶酶谱分析和蛋白质印迹分析表明，在MMP-3和MMP-9的mRNA水平上观察到的增加与其净蛋白水平的增加相似；此外，还诱导了MMP-1的活性形式。我们的结果表明，TNF-α诱导的EA.hy 926细胞形态变化不仅与细胞中PGs表达的特定变化有关，还与MMPs表达的特定变化有关。
