FLRT2 Interacts With Fibronectin in the ATDC5 Chondroprogenitor Cells
FLRT2 Interacts With Fibronectin in the ATDC5 Chondroprogenitor Cells
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DOI：
10.1002/jcp.24597
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发表时间：
2014-10-01
影响因子：
5.6
通讯作者：
Gong, Siew-Ging
中科院分区：
文献类型：
--
作者：
Flintoff, K. A.;Arudchelvan, Y.;Gong, Siew-Ging
表达研究表明FLRT 2参与颅面骨骼发生过程中颅神经嵴细胞迁移和前软骨细胞凝聚。我们的目的是确定FLRT 2是否参与介导ATDC 5软骨祖细胞系中的细胞-基质相互作用。ATDC 5细胞的免疫定位实验表明，FLRT 2存在于细胞膜上以及细胞外，在那里它与纤连蛋白（Fn）共定位。在基质的细胞提取后，在ATDC 5衍生的细胞外基质（ECM）中鉴定了FLRT 2，并且进一步发现FLRT 2与细胞培养物中的Fn包被的珠相关。通过阻断肽阻断Fn原纤维形成导致细胞外FLRT 2积累的同时减少。在向培养物补充Fn阻断肽后的7天内，Fn原纤维形成出现部分反弹，并伴随着FLRT 2共表达的再次出现。免疫共沉淀证实FLRT 2和Fn直接或间接相互作用。免疫沉淀和蛋白质印迹分析与抗体识别位于FLRT 2的胞外和胞内结构域上的表位进一步揭示了不同大小的条带的存在，表明FLRT 2可能以膜结合和脱落形式存在。因此，我们的数据提供了FLRT 2和/或其裂解产物可能与Fn和其他ECM蛋白协同调节关键细胞事件的证据。进一步的研究将是必要的，在更精确地描绘FLRT 2在介导细胞和细胞基质的相互作用在正常发育过程中的作用。(C)2014 Wiley Periodicals，Inc.
Expression studies have implicated FLRT2 in cranial neural crest cell migration and prechondrogenic cell condensation during craniofacial skeletogenesis. We aimed to determine whether FLRT2 was involved in mediating cell-matrix interactions in the ATDC5 chondroprogenitor cell line. Immunolocalization experiments of ATDC5 cells revealed that FLRT2 was present on the cell membrane as well as extracellularly, where it colocalized with Fibronectin (Fn). After cell extraction of the matrix, FLRT2 was identified in the ATDC5-derived extracellular matrix (ECM) and was further found to be associated with Fn-coated beads in cell cultures. Blockage of Fn fibril formation via a blocking peptide resulted in a concomitant decrease in extracellular FLRT2 accumulation. Over a 7-day period following the replenishment of the Fn blocking peptide to the cultures, there was a partial rebound in Fn fibril formation that was accompanied by a concomitant reappearance of FLRT2 co-expression. Co-immunoprecipitation confirmed that FLRT2 and Fn interacted, either directly or indirectly. Immunoprecipitation and Western blot analyses with antibodies recognizing epitopes located on the extra- and intracellular domains of FLRT2 further revealed the presence of different sized bands, suggesting that FLRT2 may exist in both membrane-bound and shed forms. Our data therefore provide evidence that FLRT2 and/or its cleavage products may be cooperating with Fn and other ECM proteins to regulate critical cellular events. Further studies will be necessary in delineate more precisely the roles of FLRT2 in mediating cell- and cell-matrix interactions during normal development. (C) 2014 Wiley Periodicals, Inc.