Yan L, Moses M A, Huang S, Ingber D E
Departments of Surgery and Pathology, Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.
J Cell Sci. 2000 Nov;113 ( Pt 22):3979-87. doi: 10.1242/jcs.113.22.3979.
The growth and regression of capillary blood vessels during angiogenesis is greatly influenced by changes in the activity of matrix metalloproteinases (MMPs), which selectively degrade extracellular matrix (ECM) and thereby modulate capillary endothelial cell shape, growth and viability. However, changes in cell-ECM binding and cell spreading have also been reported to alter MMP secretion and activation. Studies were carried out to determine whether changes in integrin binding or cell shape feed back to alter MMP-2 processing in human capillary endothelial (HCE) cells. Catalytic processing of proMMP-2 to active MMP-2 progressively decreased when HCE cells were cultured on dishes coated with increasing densities of fibronectin (FN), which promote both integrin binding and cell spreading. Conversely, the highest levels of active MMP-2 were detected in round cells cultured on low FN. When measured 24 hours after plating, this increase in active MMP-2 was accompanied by a concomitant rise in mRNA and protein levels for the membrane-type 1 MMP (MT1-MMP), which catalyzes the cleavage of proMMP-2. To determine whether proMMP-2 processing was controlled directly by integrin binding or indirectly by associated changes in cell shape, round cells on low FN were allowed to bind to microbeads (4.5 microm diameter) coated with a synthetic RGD peptide or FN; these induce local integrin receptor clustering without altering cell shape. ProMMP-2 activation was significantly decreased within minutes after bead binding in these round cells, prior to any detectable changes in expression of MT1-MMP, whereas binding of beads coated with control ligands for other transmembrane receptors had no effect. This inhibitory effect was mimicked by microbeads coated with activating antibodies against alphaVbeta3 and beta1 integrins, suggesting a direct role for these cell-surface ECM receptors in modulating proMMP-2 activation. Similar inhibition of proMMP-2 processing by integrin binding, independent of cell spreading, was demonstrated in cells that were cultured on small, microfabricated adhesive islands that prevented cell spreading while presenting a high FN density directly beneath the cell. Interestingly, when spread cells were induced to round up from within by disrupting their actin cytoskeleton using cytochalasin D, proMMP-2 processing did not change at early times; however, increases in MT1-MMP mRNA levels and MMP-2 activation could be detected by 18 hours. Taken together, these results suggest the existence of two phases of MMP-2 regulation in HCE cells when they adhere to ECM: (1) a quick response, in which integrin clustering alone is sufficient to rapidly inhibit processing of proMMP-2 and (2) a slower response, in which subsequent cell spreading and changes in the actin cytoskeleton feed back to decrease expression of MT1-MMP mRNA and, thereby, further suppress cellular proteolytic activity.
血管生成过程中毛细血管的生长和消退受到基质金属蛋白酶（MMPs）活性变化的极大影响，MMPs可选择性降解细胞外基质（ECM），从而调节毛细血管内皮细胞的形状、生长和活力。然而，据报道细胞与ECM结合及细胞铺展的变化也会改变MMP的分泌和激活。开展了多项研究以确定整合素结合或细胞形状的变化是否会反馈改变人毛细血管内皮（HCE）细胞中MMP-2的加工过程。当将HCE细胞培养在包被有密度不断增加的纤连蛋白（FN）的培养皿上时，前MMP-2向活性MMP-2的催化加工过程逐渐减少，FN可促进整合素结合和细胞铺展。相反，在低FN上培养的圆形细胞中检测到活性MMP-2的水平最高。接种24小时后测量发现，活性MMP-2的这种增加伴随着膜型1 MMP（MT1-MMP）的mRNA和蛋白水平的同步升高，MT1-MMP可催化前MMP-2的裂解。为了确定前MMP-2的加工过程是直接受整合素结合控制还是间接受细胞形状相关变化的控制，将低FN上的圆形细胞与包被有合成RGD肽或FN的微珠（直径4.5微米）结合；这些微珠可诱导局部整合素受体聚集而不改变细胞形状。在这些圆形细胞中，微珠结合后数分钟内前MMP-2的激活就显著降低，此时MT1-MMP的表达尚无任何可检测到的变化，而包被有针对其他跨膜受体的对照配体的微珠则无影响。针对αVβ3和β1整合素的激活抗体包被的微珠可模拟这种抑制作用，表明这些细胞表面ECM受体在调节前MMP-2激活中具有直接作用。在培养于微小的、微加工的黏附岛（可防止细胞铺展但在细胞正下方呈现高FN密度）上的细胞中，也证实了整合素结合对前MMP-2加工的类似抑制作用，且与细胞铺展无关。有趣的是，当使用细胞松弛素D破坏铺展细胞的肌动蛋白细胞骨架从而诱导其从内部变圆时，早期前MMP-2的加工过程未发生变化；然而，18小时后可检测到MT1-MMP mRNA水平升高和MMP-2激活。综上所述，这些结果表明HCE细胞黏附于ECM时MMP-2的调节存在两个阶段：（1）快速反应，即仅整合素聚集就足以迅速抑制前MMP-2的加工；（2）较慢反应，即随后的细胞铺展和肌动蛋白细胞骨架的变化反馈降低MT1-MMP mRNA的表达，从而进一步抑制细胞的蛋白水解活性。
