Lack of TRIC-B dysregulates cytoskeleton assembly, trapping β-catenin at osteoblast adhesion sites.

Barbara Maria Contento, Nadia Garibaldi, Alessandra Sala, Erika Palladino, Amanda Oldani, Alessandra Carriero, Antonella Forlino, Roberta Besio
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Abstract

The trimeric intracellular cation channel B (TRIC-B), encoded by TMEM38B, is a potassium (K+) channel present in the endoplasmic reticulum membrane, where it counterbalances calcium (Ca2+) exit. Lack of TRIC-B activity causes a recessive form of the skeletal disease osteogenesis imperfecta (OI), namely OI type XIV, characterized by impaired intracellular Ca2+ flux and defects in osteoblast (OB) differentiation and activity. Taking advantage of the OB-specific Tmem38b knockout mouse (Runx2Cre;Tmem38bfl/fl; cKO), we investigated how the ion imbalance affects the osteogenetic process. We found an abnormal cytoskeleton in the cKO OBs, with actin accumulation at OB adhesion sites. The reduced amount of active Ca2+-dependent actin-binding proteins myristoylated alanine-rich C-kinase substrate (MARCKS) and fascin, which modulate cytoskeletal actin dynamics, explains the altered cytoskeletal assembly. The actin clusters at adhesion sites trap β-catenin, a key structural protein at cell-cell junction sites, that abnormally accumulates despite the significant reduction in both N- and E-cadherins. Besides its structural fuction at cell borders, β-catenin also has a pivotal role as a transcription factor for proper osteoblastogenesis. Immunofluorescence of cKO nuclei revealed impaired nuclear β-catenin translocation, further validated in human fetal OB knocked out for TMEM38B, which was not rescued by specifically stimulating the canonical Wnt pathway. Thus, we demonstrated in vitro that alterations of intracellular Ca2+ homeostasis, as a consequence of lack of TRIC-B, cause cytoskeleton disorganization in cKO OBs, resulting in abnormal β-catenin accumulation at cell adhesion sites and reduced nuclear β-catenin translocation, contributing to impaired osteoblastogenesis.

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缺乏tricc - b会失调细胞骨架组装,在成骨细胞粘附位点捕获β-连环蛋白。
由TMEM38B编码的三聚体细胞内阳离子通道B (tricc -B)是存在于内质网膜上的钾(K+)通道,在那里它平衡钙(Ca2+)的出口。tricc - b活性的缺乏导致隐性形式的骨骼疾病成骨不全症(OI),即OI型XIV,其特征是细胞内Ca2+通量受损和成骨细胞(OB)分化和活性缺陷。利用ob特异性Tmem38b敲除小鼠(Runx2Cre;Tmem38bfl/fl;cKO),我们研究了离子失衡如何影响成骨过程。我们在cKO OB中发现异常的细胞骨架,在OB粘附部位有肌动蛋白积累。活性Ca2+依赖性肌动蛋白结合蛋白肉豆蔻酰化富丙氨酸c激酶底物(MARCKS)和束状蛋白的减少,其调节细胞骨架肌动蛋白动力学,解释了细胞骨架组装的改变。尽管N-钙粘蛋白和e -钙粘蛋白显著减少,但粘附位点的肌动蛋白簇捕获了细胞-细胞连接位点的关键结构蛋白β-连环蛋白。除了其在细胞边界的结构功能外,β-连环蛋白还作为一种转录因子在成骨细胞正常发生中起着关键作用。cKO细胞核的免疫荧光显示细胞核β-catenin易位受损,这在TMEM38B敲除的人胎儿OB中得到进一步验证,TMEM38B不能通过特异性刺激典型Wnt通路来拯救。因此,我们在体外证明,由于缺乏trici - b,细胞内Ca2+稳态的改变会导致cKO OBs细胞骨架紊乱,导致细胞粘附位点的β-连环蛋白异常积聚和细胞核β-连环蛋白易位减少,从而导致成骨细胞形成受损。
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