Absence of the ER Cation Channel TMEM38B/TRIC-B Disrupts Intracellular Calcium Homeostasis and Dysregulates Collagen Synthesis in Recessive Osteogenesis Imperfecta.
Absence of the ER Cation Channel TMEM38B/TRIC-B Disrupts Intracellular Calcium Homeostasis and Dysregulates Collagen Synthesis in Recessive Osteogenesis Imperfecta.
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DOI：
10.1371/journal.pgen.1006156
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发表时间：
2016-07
期刊：
影响因子：
4.5
通讯作者：
Marini JC
中科院分区：
文献类型：
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作者：
Cabral WA;Ishikawa M;Garten M;Makareeva EN;Sargent BM;Weis M;Barnes AM;Webb EA;Shaw NJ;Ala-Kokko L;Lacbawan FL;Högler W;Leikin S;Blank PS;Zimmerberg J;Eyre DR;Yamada Y;Marini JC
隐性成骨不全症（OI）是由参与翻译后与I型胶原相互作用的蛋白质缺陷引起的。最近，发现了一种由TMEM38B零突变引起的新型中重度OI。TMEM38B编码内质网膜一价阳离子通道tricc - b，被认为可以平衡ip3r介导的细胞内Ca2+释放。TMEM38B突变导致成骨不全的分子机制尚不清楚。我们在TMEM38B中发现了3个具有隐性缺陷的先证者。TRIC-B蛋白在先证者成纤维细胞和成骨细胞中检测不到，尽管TMEM38B转录物减少。尽管SERCA和IP3R具有正常的稳定性，但tricc - b缺乏导致内质网腔内Ca2+释放受损，与储存操作的钙进入不足有关。值得注意的是，稳态内质网Ca2+在trici - b缺乏症中没有变化，这支持了trici - b在内质网钙消耗和恢复动力学中的作用。Ca2+通量紊乱导致内质网应激和BiP升高，并在多个步骤中失调先证型I型胶原的合成。胶原螺旋赖氨酸羟基化减少，而端肽羟基化增加，尽管LH1增加，Ca2+依赖性FKBP65减少。虽然PDI水平维持，但前胶原链组装在先证细胞中被延迟。由此产生的错误折叠的胶原在tricc - b无细胞中基本保留，与分泌的胶原减少50-70%一致。不被蛋白酶体降解的低稳定性的胶原蛋白不被纳入细胞外基质，细胞外基质只含有正常稳定性的胶原蛋白，导致基质不足。这些数据支持tricc - b在细胞内Ca2+稳态中的作用，并证明TMEM38B的缺失通过内质网钙通量动力学失调导致成骨不全，影响多种胶原特异性伴侣和修饰酶。成骨不全症（OI）是一种遗传性结缔组织疾病，以骨折易感性和生长缺陷为特征。大多数OI病例是由编码I型胶原蛋白、COL1A1和COL1A2基因的常染色体显性突变引起的。对导致显性和隐性成骨不全的新基因缺陷的描述使人们认识到，骨病理不仅是由I型胶原数量和初级结构的异常引起的，而且是由翻译后修饰、折叠、细胞内运输和细胞外基质结合的缺陷引起的。最近，编码内质网K+通道TRIC-B的TMEM38B突变被确定为OI表型的病因。然而，TRIC-B缺失导致成骨不全的机制尚未报道。利用三个独立先证者建立的细胞系，我们已经证明，尽管内质网稳态Ca2+是正常的，但缺乏tricc - b会导致内质网Ca2+通量和储存操作钙进入（SOCE）异常。细胞内钙动力学的破坏改变了内质网内多种胶原蛋白相互作用伴侣和修饰酶的表达和活性。因此，tricc - b缺乏通过损害内质网内钙依赖性基因表达和蛋白-蛋白相互作用，导致胶原合成失调，从而导致成骨不全。
Recessive osteogenesis imperfecta (OI) is caused by defects in proteins involved in post-translational interactions with type I collagen. Recently, a novel form of moderately severe OI caused by null mutations in TMEM38B was identified. TMEM38B encodes the ER membrane monovalent cation channel, TRIC-B, proposed to counterbalance IP3R-mediated Ca2+ release from intracellular stores. The molecular mechanisms by which TMEM38B mutations cause OI are unknown. We identified 3 probands with recessive defects in TMEM38B. TRIC-B protein is undetectable in proband fibroblasts and osteoblasts, although reduced TMEM38B transcripts are present. TRIC-B deficiency causes impaired release of ER luminal Ca2+, associated with deficient store-operated calcium entry, although SERCA and IP3R have normal stability. Notably, steady state ER Ca2+ is unchanged in TRIC-B deficiency, supporting a role for TRIC-B in the kinetics of ER calcium depletion and recovery. The disturbed Ca2+ flux causes ER stress and increased BiP, and dysregulates synthesis of proband type I collagen at multiple steps. Collagen helical lysine hydroxylation is reduced, while telopeptide hydroxylation is increased, despite increased LH1 and decreased Ca2+-dependent FKBP65, respectively. Although PDI levels are maintained, procollagen chain assembly is delayed in proband cells. The resulting misfolded collagen is substantially retained in TRIC-B null cells, consistent with a 50–70% reduction in secreted collagen. Lower-stability forms of collagen that elude proteasomal degradation are not incorporated into extracellular matrix, which contains only normal stability collagen, resulting in matrix insufficiency. These data support a role for TRIC-B in intracellular Ca2+ homeostasis, and demonstrate that absence of TMEM38B causes OI by dysregulation of calcium flux kinetics in the ER, impacting multiple collagen-specific chaperones and modifying enzymes. Osteogenesis imperfecta (OI) is a heritable disorder of connective tissues characterized by fracture susceptibility and growth deficiency. Most OI cases are caused by autosomal dominant mutations in the genes encoding type I collagen, COL1A1 and COL1A2. Delineation of novel gene defects causing dominant and recessive forms of OI has led to the understanding that the bone pathology results not only from abnormalities in type I collagen quantity and primary structure, but also from defects in post-translational modification, folding, intracellular transport and extracellular matrix incorporation. Recently, mutations in TMEM38B, which encodes the integral ER membrane K+ channel TRIC-B, have been identified as causative for the OI phenotype. However, the mechanism by which absence of TRIC-B causes OI has not been reported. Using cell lines established from three independent probands, we have demonstrated that absence of TRIC-B leads to abnormal ER Ca2+ flux and store-operated calcium entry (SOCE), although ER steady state Ca2+ is normal. Disruption of intracellular calcium dynamics alters the expression and activity of multiple collagen interacting chaperones and modifying enzymes within the ER. Thus TRIC-B deficiency causes OI by dysregulation of collagen synthesis, through the impairment of calcium-dependent gene expression and protein-protein interactions within the ER.