title: Overexpression of lysyl hydroxylase-2b leads to defective collagen fibrillogenesis and matrix mineralization.
abstract: 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.
  
  
  BACKGROUND
  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.
  
  
  METHODS
  Several MC3T3-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 immunodeficient 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.
authors.affiliation: Dental Research Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7455, USA.
authors.forename: Suchaya
authors.initials: S
authors.lastname: Pornprasertsuk
authors.forename: Wagner R
authors.initials: WR
authors.lastname: Duarte
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Mochida
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Yamauchi
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002113:Calcification, Physiologic / Q000235:genetics / Q000502:physiology*; D002478:Cells, Cultured; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D024022:Fibrillar Collagens / Q000378:metabolism* / Q000648:ultrastructure; D015870:Gene Expression; D058574:Integrin-Binding Sialoprotein; D051379:Mice; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D010006:Osteoblasts / Q000378:metabolism; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011499:Protein Processing, Post-Translational / Q000235:genetics / Q000502:physiology*; D012795:Sialoglycoproteins / Q000235:genetics / Q000378:metabolism; D000076182:Sp7 Transcription Factor; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation
chemical_list: D015415:Biomarkers; D024042:Collagen Type I; D024022:Fibrillar Collagens; C546872:Ibsp protein, mouse; D058574:Integrin-Binding Sialoprotein; D009363:Neoplasm Proteins; D012795:Sialoglycoproteins; D000076182:Sp7 Transcription Factor; C479466:Sp7 protein, mouse; D014157:Transcription Factors; D015675:Osteocalcin; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase