Myocilin promotes substrate adhesion, spreading and formation of focal contacts in podocytes and mesangial cells
Myocilin promotes substrate adhesion, spreading and formation of focal contacts in podocytes and mesangial cells
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DOI：
10.1007/s00418-008-0518-4
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发表时间：
2009-02-01
影响因子：
2.3
通讯作者：
Tamm, Ernst R.
中科院分区：
文献类型：
--
作者：
Goldwich, Andreas;Scholz, Michael;Tamm, Ernst R.
肌动蛋白是嗅觉素家族的一种分泌的糖蛋白，在大鼠肾脏的足细胞中组成型表达，并在肾小球肾上腺素肾上腺炎期间在肾小球细胞中诱导。由于已经发现肌动蛋白与细胞外基质的原纤维成分相关，并且已经显示了绿色橄榄球素家族的其他成员的粘合剂特性，因此我们假设肌动蛋白可能在glomerulus中的细胞 - 矩阵相互作用中起作用。为了阐明肌动蛋白的功能特性，在293个EBNA细胞中表达重组肌动蛋白，并通过Ni-Chelate和肝素色谱法纯化。将培养板与肌动蛋白涂在一起，并在不朽的鼠足细胞细胞系中涂上原代大鼠的细胞和细胞，并在无血清条件下播种到板上。两种细胞类型均显示浓度依赖于肌动蛋白，这种作用具有统计学意义，可以用特定的抗体阻塞。与等量纤连蛋白或胶原蛋白1相比，肌动蛋白在促进底物粘附方面的有效性较小。当将肌动蛋白添加到低浓度的纤连蛋白时，观察到底物粘附的协同作用。在肌动蛋白底物上附着的细胞中，有25％的细胞显示出扩散和表达的局灶性接触，这些接触被Vinculin/phalloidin染色标记。当将人或鼠小梁网状工厂细胞播种在肌动蛋白底物上时，观察到可比的发现。肌动蛋白的粘合性特性需要多聚合物形成，并且当培养板与含有卵母蛋白结构域的肌动蛋白的C末端片段涂有培养板时，没有观察到。我们得出的结论是，肌动蛋白促进足细胞和膜细胞的底物粘附，并可能有助于两种细胞类型的体内细胞 - 基质粘附。
Myocilin, a secreted glycoprotein of the olfactomedin family, is constitutively expressed in podocytes of the rat kidney and induced in mesangial cells during mesangioproliferative glomerulonephritis. As myocilin has been found to be associated with fibrillar components of the extracellular matrix, and adhesive properties have been shown for other members of the olfactomedin family, we hypothesized that myocilin might play a role in cell-matrix interactions in the glomerulus. To elucidate functional properties of myocilin, recombinant myocilin was expressed in 293 EBNA cells and purified by Ni-chelate and heparin chromatography. Culture plates were coated with myocilin, and primary rat mesangial cells and cells from an immortal murine podocyte cell line were seeded onto the plates in serum free conditions. Both cell types showed concentration-dependant attachment to myocilin, an effect that was statistically significant and could be blocked with specific antibodies. When compared to equal amounts of fibronectin or collagen 1, myocilin was less effective in promoting substrate adhesion. Synergistic effects in substrate adhesion were observed when myocilin was added to low concentrations of fibronectin. Twenty-five percent of cells that had attached to myocilin substrates showed spreading and expressed focal contacts which were labeled by vinculin/phalloidin staining. Comparable findings were observed when human or murine trabecular meshwork cells were seeded on myocilin substrates. Adhesive properties of myocilin required multimer formation, and were not observed when culture plates were coated with a C-terminal fragment of myocilin, containing the olfactomedin domain. We conclude that myocilin promotes substrate adhesion of podocytes and mesangial cells, and might contribute to cell-matrix adhesion of both cell types in vivo.