Identification of the region in calponin h1 that suppresses proliferation and motility of human fibrosarcoma

为探讨人钙蛋白H1(CNH1)对人纤维肉瘤HT1080细胞增殖和运动能力的影响，将其导入人纤维肉瘤HT1080细胞中。CNH1转染组细胞形态扁平，肌动蛋白细丝排列整齐，细胞活力显著降低。为了确定CNh1抑制细胞增殖和运动的区域，将CNh1分为CHD、肌动蛋白结合位点(ABS)、重复序列(CNR)和C末端4个结构域，共12个片段。结果，CNR抑制了神经元的运动。由于CNR的重复序列1(R1)被报道为第二个肌动蛋白结合位点，并以蛋白激酶C(PKC)为靶标，因此对肌动蛋白的解聚和足体的形成进行了研究。细胞松弛素D可抑制CNH1细胞肌动蛋白解聚，PDBu(PKC刺激物)也可抑制足体的形成。将缺失ABS或CNR-R1的截短体和具有S175A和T184A突变的突变体分别转染。在所有三种转染体中，肌动蛋白横行纤维都从钙蛋白H1中分离出来，并促进了足体的形成。Calponin H1被认为通过与肿瘤细胞中的横向肌动蛋白纤维结合来抵抗细胞松弛素D或PDBu。

To investigate the effects of calponin h1(CNh1) on the cell proliferation and motility, human calponin h1 was transfected to human fibrosarcoma, HT1080. CNh1-transfected cells exhibited flattened morphology with organized actin filaments, significant decrease in cell motility. Anchorage-independent growth and tumorigenicity in nude mice were suppressed in CNh1-transfected cells.To identify the region of CNh1 that suppresses proliferation and motility, CNh1 consisted of four domains, CHD, actin binding site (ABS), CNrepeats (CNR) and C terminal, were divided to 12 truncates. As a result, the motility was suppressed by the CNR. Since Repeat 1 (R1) of CNR is reported as a second actin binding site, and has targets of protein kinase C (PKC), actin-depolymerization and podosome formation was investigated. Actin-depolymerization was suppressed by the stimulation with cytochalasin D, and formation of podosome was also suppressed with PDBu (PKC stimulator) in CNh1 transfected cells. Truncates that lack ABS or CNR-R1, and mutant that has mutation of S175A and T184A were transfected. In all three transfectants, transverse actin fiber was separated from calponin h1 and podosome formation was facilitated. Calponin h1 is considered to resist cytochalasin D or PDBu by binding to transverse actin fiber in tumor cells.