Gene regulation by 3-D structure of extracellular matrix : a model for hepatic stellate cells

细胞与细胞外基质(ECM)之间的相互作用对细胞的形态、增殖、运动、分化和基因表达等功能至关重要。已知I型胶原纤维的天然形式影响这些细胞功能和几种细胞类型中的ECM降解酶，如基质金属蛋白酶-2(MMP-2，明胶酶A)和基质金属蛋白酶-13(胶原酶-3)。在这项研究中，我们研究了包括I型胶原在内的细胞外基质成分在调节培养的肝星状细胞(HSCs)的形态和功能中的作用。HSC在I型胶原涂层表面培养时呈肌成纤维样细胞形态，应力纤维发达，而在Matrigel表面培养时呈圆形，提示HSC识别细胞外I型胶原纤维的天然结构并改变其形态和功能。然后我们研究了细胞内信号的参与，包括蛋白激酶、PI-3激酶、小G蛋白、微管相关…在I型胶原凝胶上培养的HSCs中有更多的ED蛋白(MAP2)和细胞骨架重组。细胞外I型胶原对SP1、BCRP、dysttin、KIF1和KAP3B等几种基因的表达有不同的调节作用。这些结果表明，细胞表面与细胞外间质胶原结合可能触发细胞内信号传递和细胞骨架相关蛋白基因表达的改变，最终诱导细胞骨架重组的过程延长。条件培养液的明胶酶谱显示，在I型胶原凝胶上培养的HSCs中，MMP2的前体和活性形式增加，而在明胶、IV型胶原涂层表面或Matrigel上培养的HSCs中MMP2的活性增加，提示I型胶原纤维的天然形式在PMP2激活中的重要性。RT-PCR分析显示，在I型胶原凝胶上培养的HSCs中，MMP2、MT1-MMP1和TIMP-2mRNA的表达水平升高。因此，天然的纤维状而非单体的I型胶原通过MT1-MMPs和TIMP-2诱导HSC产生和激活原-MMP2。较少

Interaction between cells and extracellular matrix (ECM) is essential for cell functions such as morphology, proliferation, motility, differentiation, and gene expression. The native form of type I collagen fibrils is known to affect these cell functions and ECM degrading enzymes such as matrix metalloprotease-2 (MMP-2, gelatinase A) and MMP-13 (collageanase-3) in several cell types. In this study we examined the roles of ECM components including type I collagen in the regulation of morphology and function of cultured hepatic stellate cells (HSCs). HSCs showed myofibroblast-like cell shapes with well-developed stress fibers when cultured on type I collagen coated surface, whereas rounded shapes when cultured on Matrigel, suggesting that HSCs recognize native structure of extracellular type I collagen fibrils and change their morphology and function. Then we examined the involvement of intracellular signaling including protein kinases, PI-3 kinase, small G-proteins, microtubule-associat … More ed protein (MAP2), and cytoskeleton reorganization in HSCs cultured on type I collagen gel. Several mRNA species including SP1, BCRP, dystonin, KIF1, and KAP3B were differentially regulated by extracellular type I collagen. These results indicated that cell surface-binding to extracellular interstitial collagen may trigger intracellular signaling and alteration in gene expression of cytoskeleton-related proteins, and finally induced cytoskeleton reorganization of process elongation. Gelatin zymography of the conditioned media revealed that pro and active forms of MMP-2 was increased in the HSCs cultured on type I collagen gel but not gelatin-, or type IV collagen-coated surface or Matrigel, suggesting the importance of the native form of type I collagen fibrils in pro-MMP-2 activation. RT-PCR analysis indicated that MMP-2, MT1-MMP and TIMP-2 mRNA levels were elevated in HSCs cultured on type I collagen gel. Therefore, the native fibrillar but not monomeric form of type I collagen induced pro-MMP-2 production and activation through MT1-MMP and TIMP-2 in cultured HSCs. Less