Rai Shinya, Tanaka Hirokazu, Suzuki Mai, Ogoh Honami, Taniguchi Yasuhiro, Morita Yasuyoshi, Shimada Takahiro, Tanimura Akira, Matsui Keiko, Yokota Takafumi, Oritani Kenji, Tanabe Kenji, Watanabe Toshio, Kanakura Yuzuru, Matsumura Itaru
Department of Hematology and Rheumatology, Kinki University Faculty of Medicine, Osaka, Japan.
Division of Hematological Malignancy, National Cancer Center Research Institute, Tokyo, Japan; Department of Biological Science, Graduate School of Humanities and Sciences, Nara Women's University, Nara, Japan.
PLoS One. 2014 Oct 3;9(10):e109441. doi: 10.1371/journal.pone.0109441. eCollection 2014.
CALM is implicated in the formation of clathrin-coated vesicles, which mediate endocytosis and intracellular trafficking of growth factor receptors and nutrients. We previously found that CALM-deficient mice suffer from severe anemia due to the impaired clathrin-mediated endocytosis of transferrin receptor in immature erythroblast. However, CALM has been supposed to regulate the growth and survival of hematopoietic stem/progenitor cells. So, in this study, we focused on the function of CALM in these cells. We here show that the number of Linage-Sca-1+KIT+ (LSK) cells decreased in the fetal liver of CALM-/- mice. Also, colony forming activity was impaired in CALM-/- LSK cells. In addition, SCF, FLT3, and TPO-dependent growth was severely impaired in CALM-/- LSK cells, while they can normally proliferate in response to IL-3 and IL-6. We also examined the intracellular trafficking of KIT using CALM-/- murine embryonic fibroblasts (MEFs) engineered to express KIT. At first, we confirmed that endocytosis of SCF-bound KIT was not impaired in CALM-/- MEFs by the internalization assay. However, SCF-induced KIT trafficking from early to late endosome was severely impaired in CALM-/- MEFs. As a result, although intracellular KIT disappeared 30 min after SCF stimulation in wild-type (WT) MEFs, it was retained in CALM-/- MEFs. Furthermore, SCF-induced phosphorylation of cytosolic KIT was enhanced and prolonged in CALM-/- MEFs compared with that in WT MEFs, leading to the excessive activation of Akt. Similar hyperactivation of Akt was observed in CALM-/- KIT+ cells. These results indicate that CALM is essential for the intracellular trafficking of KIT and its normal functions. Also, our data demonstrate that KIT located in the early endosome can activate downstream molecules as a signaling endosome. Because KIT activation is involved in the pathogenesis of some malignancies, the manipulation of CALM function would be an attractive therapeutic strategy.
CALM与网格蛋白包被小泡的形成有关，网格蛋白包被小泡介导生长因子受体和营养物质的内吞作用及细胞内运输。我们之前发现，CALM缺陷小鼠因未成熟成红细胞中转铁蛋白受体的网格蛋白介导内吞作用受损而患有严重贫血。然而，CALM被认为可调节造血干/祖细胞的生长和存活。因此，在本研究中，我们聚焦于CALM在这些细胞中的功能。我们在此表明，CALM基因敲除小鼠胎肝中Linage-Sca-1+KIT+（LSK）细胞数量减少。此外，CALM基因敲除的LSK细胞集落形成活性受损。另外，CALM基因敲除的LSK细胞中SCF、FLT3和TPO依赖性生长严重受损，而它们能正常响应IL-3和IL-6进行增殖。我们还利用经基因工程改造表达KIT的CALM基因敲除小鼠胚胎成纤维细胞（MEF）研究了KIT的细胞内运输。首先，我们通过内化试验证实CALM基因敲除的MEF中与SCF结合的KIT的内吞作用未受损。然而，CALM基因敲除的MEF中SCF诱导的KIT从早期内体到晚期内体的运输严重受损。结果，虽然野生型（WT）MEF中SCF刺激30分钟后细胞内KIT消失，但它在CALM基因敲除的MEF中仍保留。此外，与WT MEF相比CALM基因敲除的MEF中SCF诱导的胞质KIT磷酸化增强且延长，导致Akt过度激活。在CALM基因敲除的KIT+细胞中观察到类似的Akt过度激活。这些结果表明CALM对KIT的细胞内运输及其正常功能至关重要。此外，我们的数据表明位于早期内体的KIT可作为信号内体激活下游分子。由于KIT激活参与某些恶性肿瘤的发病机制，操纵CALM功能可能是一种有吸引力的治疗策略。
