Uchida Masashi, Ishii Itsuko, Hirata Kaori, Yamamoto Fumiko, Tashiro Kaori, Suzuki Takayoshi, Nakayama Yuji, Ariyoshi Noritaka, Kitada Mitsukazu
Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, Japan.
Cell Physiol Biochem. 2011;27(6):669-80. doi: 10.1159/000330076. Epub 2011 Jun 17.
Dedifferentiated rabbit vascular smooth muscle cells (SMCs) exhibit similar features to differentiated SMCs when cultured in three-dimensional matrices of type-I collagen called "honeycombs," but the mechanism is unknown. The role of filamin, an actin-binding protein that links actin filaments in SMCs, was investigated.
Filamin and other related proteins were detected by western blot analysis and immunofluorescence staining. Honeycomb size was measured to confirm the contraction of SMCs.
Full-length filamin was expressed in subconfluent SMCs cultured on plates; however, degradation of filamin, which might be regulated by calpain, was observed in confluent SMCs cultured on plates and in honeycombs. While filamin was co-localized with β-actin in subconfluent SMCs grown on plates, filamin was detected in the cytoplasm in SMCs cultured in honeycombs, and degraded filamin was mainly detected in the cytoplasmic fraction of these cells. In addition, β-actin expression was low in the cytoskeletal fraction of SMCs cultured in honeycombs compared with cells cultured on plates, and the size of the honeycombs used for culturing SMCs was significantly reduced.
These data suggest that degradation of filamin in SMCs cultured in honeycombs induces structural weakness of β-non-muscle actin filaments, thereby permitting SMCs in honeycombs to achieve contractility.
去分化的兔血管平滑肌细胞（SMCs）在称为“蜂巢”的I型胶原蛋白三维基质中培养时，表现出与分化的SMCs相似的特征，但其机制尚不清楚。研究了细丝蛋白（一种在SMCs中连接肌动蛋白丝的肌动蛋白结合蛋白）的作用。
通过蛋白质免疫印迹分析和免疫荧光染色检测细丝蛋白及其他相关蛋白。测量蜂巢大小以确认SMCs的收缩情况。
全长细丝蛋白在平板上培养的亚汇合SMCs中表达；然而，在平板上和蜂巢中培养的汇合SMCs中观察到细丝蛋白的降解，这可能受钙蛋白酶调节。当细丝蛋白在平板上生长的亚汇合SMCs中与β-肌动蛋白共定位时，在蜂巢中培养的SMCs的细胞质中检测到细丝蛋白，并且降解的细丝蛋白主要在这些细胞的细胞质部分中检测到。此外，与在平板上培养的细胞相比，在蜂巢中培养的SMCs的细胞骨架部分中β-肌动蛋白表达较低，并且用于培养SMCs的蜂巢大小显著减小。
这些数据表明，在蜂巢中培养的SMCs中细丝蛋白的降解会导致β-非肌肉肌动蛋白丝的结构弱化，从而使蜂巢中的SMCs实现收缩性。
