Role of scarf and its binding target proteins in epidermal calcium homeostasis
Role of scarf and its binding target proteins in epidermal calcium homeostasis
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DOI：
10.1074/jbc.m702035200
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发表时间：
2007-06-22
影响因子：
4.8
通讯作者：
Morasso, Maria I.
中科院分区：
文献类型：
--
作者：
Hwang, Joonsung;Kalinin, Alexandr;Morasso, Maria I.
新的钙离子结合蛋白，Scarf（皮肤钙调素相关因子）属于钙调素样蛋白家族，并在表皮的分化层中表达。为了确定Scarf在分层过程中的作用，我们着手通过亲和色谱法和随后的质谱分析来鉴定结合靶蛋白。鉴定了几种结合因子，包括14-3-3s、膜联蛋白、钙网蛋白、ERp 72（内质网蛋白72）和核仁素，并通过免疫共沉淀和共定位分析证实了它们与Scarf的相互作用。为了进一步了解Scarf在体内表皮中的功能，我们通过急性屏障破坏来改变表皮Ca 2+梯度。免疫组化和Western blot分析Scarf及其结合靶蛋白表达水平的变化。Ca 2+梯度破坏可上调Scarf、膜联蛋白、钙网蛋白和ERp 72的表达，而下调14-3-3s和核仁素的表达。由于膜联蛋白，钙网蛋白和ERp 72已牵连在钙离子诱导的细胞运输，包括分泌板层体和钙稳态，我们建议，这些蛋白质的相互作用的围巾可能是至关重要的屏障恢复的过程中。另一方面，14-3-3s和核仁素的下调可能参与角质形成细胞分化和生长抑制的过程。在离子霉素处理的小鼠角质形成细胞和创伤愈合过程中，研究了Scarf及其结合靶蛋白的钙依赖性定位和上调。我们发现在创伤后第4天和第7天，伤口边缘的表皮中Scarf的表达和核存在增加，从而导致Scarf在屏障恢复中的作用。我们的研究结果表明，Scarf作为一个Ca 2+传感器起着至关重要的作用，潜在地调节其结合靶蛋白的功能，在恢复表皮Ca 2+梯度的过程中，以及在表皮屏障形成的Ca 2+依赖的方式。
The novel Ca2+-binding protein, Scarf ( skin calmodulin-related factor) belongs to the calmodulin-like protein family and is expressed in the differentiated layers of the epidermis. To determine the roles of Scarf during stratification, we set out to identify the binding target proteins by affinity chromatography and subsequent analysis by mass spectrometry. Several binding factors, including 14-3-3s, annexins, calreticulin, ERp72 ( endoplasmic reticulum protein 72), and nucleolin, were identified, and their interactions with Scarf were corroborated by co-immunoprecipitation and co-localization analyses. To further understand the functions of Scarf in epidermis in vivo, we altered the epidermal Ca2+ gradient by acute barrier disruption. The change in the expression levels of Scarf and its binding target proteins were determined by immunohistochemistry and Western blot analysis. The expression of Scarf, annexins, calreticulin, and ERp72 were up-regulated by Ca2+ gradient disruption, whereas the expression of 14-3-3s and nucleolin was reduced. Because annexins, calreticulin, and ERp72 have been implicated in Ca2+-induced cellular trafficking, including the secretion of lamellar bodies and Ca2+ homeostasis, we propose that the interaction of Scarf with these proteins might be crucial in the process of barrier restoration. On the other hand, down-regulation of 14-3-3s and nucleolin is potentially involved in the process of keratinocyte differentiation and growth inhibition. The calcium-dependent localization and up-regulation of Scarf and its binding target proteins were studied in mouse keratinocytes treated with ionomycin and during the wound-healing process. We found increased expression and nuclear presence of Scarf in the epidermis of the wound edge 4 and 7 days post-wounding, entailing the role of Scarf in barrier restoration. Our results suggest that Scarf plays a critical role as a Ca2+ sensor, potentially regulating the function of its binding target proteins in a Ca2+-dependent manner in the process of restoration of epidermal Ca2+ gradient as well as during epidermal barrier formation.