Overexpression of lysyl hydroxylase-2b leads to defective collagen fibrillogenesis and matrix mineralization
Overexpression of lysyl hydroxylase-2b leads to defective collagen fibrillogenesis and matrix mineralization
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DOI：
10.1359/jbmr.041026
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发表时间：
2005-01-01
影响因子：
6.2
通讯作者：
Yamauchi, M
中科院分区：
文献类型：
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作者：
Pornprasertsuk, S;Duarte, WR;Yamauchi, M
我们分析了几种表达高水平LH2b的MC3T3-E1细胞衍生克隆，分析了它们形成胶原原纤维和矿化的能力。这些克隆在体外和体内均表现出更小的胶原原纤维和缺陷的基质矿化，表明lh2b催化的胶原翻译后修饰在骨基质形成和矿化中起关键作用。我们最近发现，赖氨酸羟化酶（LH） 2b通过作用于胶原的末端肽基赖氨酸残基，调节成骨细胞系MC3T3-E1中的胶原交联途径。为了进一步阐明LH2b在骨生理中的作用，我们研究了过表达LH2b对胶原纤维形成和基质矿化的影响。材料和方法：培养几个表达高水平LH2b的mc3tt - e1来源的成骨细胞克隆（S克隆）和两个对照（即MC3T3-E1细胞和空载体转染的细胞）。采用MALDI-TOF质谱法对LH2b进行鉴定。透射电镜观察了胶原纤维的形成，体外和体内矿化实验分析了这些克隆和细胞形成矿化基质的能力。结果：S克隆培养的胶原原纤维直径明显小于对照组。在S无性系中，基质矿化的发生明显延迟，与对照相比，其培养物中形成的矿化结节明显减少。当移植到免疫缺陷小鼠体内时，S克隆在体内不能形成矿化基质，而在对照组中形成了骨样矿化基质。胶原原纤维直径和体外基质矿化时间/程度与LH2b水平呈负相关。体外细胞分化不受LH2b过表达的影响。结论：这些结果表明LH2b催化的胶原翻译后修饰（即端肽基赖氨酸羟基化和随后的交联）在骨中胶原基质的形成和矿化中起关键作用。
Several MC3T3-E1 cell-derived clones expressing higher levels of LH2b were analyzed for their abilities to form collagen fibrils and mineralization. The clones all exhibited smaller collagen fibrils and defective matrix mineralization in vitro and in vivo, indicating a critical role of LH2b-catalyzed post-translational modifications of collagen in bone matrix formation and mineralization.Introduction: We have recently shown that lysyl hydroxylase (LH) 2b, through its action on the telopeptidyl lysine residues of collagen, regulates collagen cross-linking pathway in the osteoblastic cell line, MC3T3-E1. To further elucidate the roles of LH2b in bone physiology, the effects of overexpression of LH2b on collagen fibrillogenesis and matrix mineralization were investigated.Materials and Methods: Several MCT3Tt-E1-derived osteoblastic cell clones expressing higher levels of LH2b (S clones) and two controls (i.e., MC3T3-E1 cells and those transfected with an empty vector) were cultured. MALDI-TOF mass spectrometry was used to identify the LH2b. The collagen fibrillogenesis in the cultures was characterized by transmission electron microscopy, and the ability of these clones and cells to form mineralized matrix was analyzed by both in vitro and in vivo mineralization assays.Results: The diameter of collagen fibrils in the S clone cultures was markedly smaller than that of the controls. The onset of matrix mineralization in the S clones was significantly delayed, and considerably fewer mineralized nodules were formed in their cultures in comparison with the controls. When transplanted into immuno-deficient mice, the S clones failed to form mineralized matrices in vivo, whereas a bone-like mineralized matrix was well formed by the controls. The diameter of the collagen fibrils and the timing/extent of matrix mineralization in vitro were inversely correlated with the level of LH2b. In vitro cell differentiation was unaffected by the LH2b overexpression.Conclusions: These results indicate a critical role of LH2b catalyzed post-translational modification of collagen (i.e., telopeptidyl lysine hydroxylation and subsequent cross-linking) in collagen matrix formation and mineralization in bone.