Severe osteogenesis imperfecta in cyclophilin B-deficient mice.
Severe osteogenesis imperfecta in cyclophilin B-deficient mice.
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DOI：
10.1371/journal.pgen.1000750
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发表时间：
2009-12
期刊：
影响因子：
4.5
通讯作者：
Bram RJ
中科院分区：
文献类型：
--
作者：
Choi JW;Sutor SL;Lindquist L;Evans GL;Madden BJ;Bergen HR 3rd;Hefferan TE;Yaszemski MJ;Bram RJ
成骨不全(OI)是一种人类综合征，其特征是骨质疏松症导致骨骼极其脆弱。大多数常染色体显性遗传性OI病例是由I型胶原基因的点突变或剪接点突变引起的，这被认为是导致发育中的骨骼中异常的类骨质。OI也发生在Pro-3-羟基酶-1(LEPRE1)纯合子突变的人类中。虽然已知P3H1可以羟化I型胶原链中的单一残基(PRO-986)，但这种修饰如何促进胶原纤维的形成尚不清楚。P3H1存在于CRTAP和由ppiB基因编码的亲环素B(CypB)的复合体中。CRTAP的突变通过一种未知的机制导致小鼠和人类的OI，而CypB在这个复合体中的作用一直是一个完全的谜。为了研究哺乳动物CypB的作用，我们产生了缺乏这种蛋白的小鼠。在生命早期，Ppib-/-小鼠出现了脊柱后凸和严重的骨质疏松症。Ppib-/-小鼠的胶原纤维形态异常，进一步符合OI表型。体外研究表明，在CypB缺陷的成纤维细胞中，前胶原没有正确地定位在高尔基体上。我们发现，Ppib-/-细胞中P3H1的水平显著降低，而CRTAP不受CypB缺失的影响。相反，P3H1或CRTAP的敲除并不影响CypB的细胞水平，但在体外阻止了它与胶原的相互作用。此外，CRTAP基因敲除也导致了细胞P3H1的耗竭。与这些变化一致的是，在CypB基因敲除小鼠的细胞和组织中，P3H1对I型胶原的翻译后Pro-3-羟基化基本上是缺失的。这些数据为OI的病理生理学提供了重要的新的机制见解，并揭示了P3H1/CRTAP/CypB复合体的成员如何相互作用来指导胶原和骨的正确形成。成骨不全(OI)，也被称为“脆性骨病”，是一种遗传性疾病，含有胶原的结构中存在多种缺陷，包括骨骼、皮肤和其他结缔组织。OI患者的症状是身材矮小、脊柱侧弯、皮肤变薄、听力丧失，最值得注意的是骨质疏松症，很少或根本没有创伤。虽然许多病例是由于胶原基因的显性遗传点突变，但常染色体隐性形式已被描述为由于Pro-3-羟基酶-1(LEPRE1)和软骨相关蛋白(CRTAP)基因的缺陷，这两种蛋白质修改新合成的前胶原蛋白。一些OI患者在任何已知的疾病相关基因上都没有突变。在这里，通过利用新产生的基因敲除小鼠，我们鉴定了内质网驻留的Pro-异构酶亲环素B(CypB)是一种新的常染色体隐性遗传性OI基因。CypB、P3H1和CRTAP在维持它们各自的蛋白水平和与胶原结合的能力方面被证明具有相互关联的作用。这些研究加深了我们对胶原蛋白的理解，胶原蛋白是人体内含量最丰富的蛋白质，是如何正确组装成具有足够强度的骨骼的。
Osteogenesis Imperfecta (OI) is a human syndrome characterized by exquisitely fragile bones due to osteoporosis. The majority of autosomal dominant OI cases result from point or splice site mutations in the type I collagen genes, which are thought to lead to aberrant osteoid within developing bones. OI also occurs in humans with homozygous mutations in Prolyl-3-Hydroxylase-1 (LEPRE1). Although P3H1 is known to hydroxylate a single residue (pro-986) in type I collagen chains, it is unclear how this modification acts to facilitate collagen fibril formation. P3H1 exists in a complex with CRTAP and the peptidyl-prolyl isomerase cyclophilin B (CypB), encoded by the Ppib gene. Mutations in CRTAP cause OI in mice and humans, through an unknown mechanism, while the role of CypB in this complex has been a complete mystery. To study the role of mammalian CypB, we generated mice lacking this protein. Early in life, Ppib-/- mice developed kyphosis and severe osteoporosis. Collagen fibrils in Ppib-/- mice had abnormal morphology, further consistent with an OI phenotype. In vitro studies revealed that in CypB–deficient fibroblasts, procollagen did not localize properly to the golgi. We found that levels of P3H1 were substantially reduced in Ppib-/- cells, while CRTAP was unaffected by loss of CypB. Conversely, knockdown of either P3H1 or CRTAP did not affect cellular levels of CypB, but prevented its interaction with collagen in vitro. Furthermore, knockdown of CRTAP also caused depletion of cellular P3H1. Consistent with these changes, post translational prolyl-3-hydroxylation of type I collagen by P3H1 was essentially absent in CypB–deficient cells and tissues from CypB–knockout mice. These data provide significant new mechanistic insight into the pathophysiology of OI and reveal how the members of the P3H1/CRTAP/CypB complex interact to direct proper formation of collagen and bone. Osteogenesis Imperfecta (OI), also known as “brittle bone disease,” is an inherited condition with multiple defects in collagen-containing structures, including the bones, skin, and other connective tissues. Patients with OI suffer from short stature, scoliosis, thin skin, hearing loss, and, most notably, fragile bones that break with little or no trauma. Although many cases are due to dominantly inherited point mutations in the collagen genes, autosomal recessive forms have been described due to defects in the genes for Prolyl-3-Hydroxylase-1 (LEPRE1) and Cartilage-Associated Protein (CRTAP), proteins that modify newly synthesized procollagen. Some patients with OI do not have mutations in any of the known disease-related genes. Here, through the use of newly generated knockout mice, we identify the endoplasmic-reticulum resident prolyl-isomerase cyclophilin B (CypB) as a new autosomal recessive OI gene in mice. CypB, P3H1, and CRTAP were shown to have interrelated effects in maintaining their respective protein levels and ability to bind to collagen. These studies enhance our understanding about how collagen, the most abundant protein in the body, becomes properly assembled to form bones with adequate strength.
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