Effect of hyper- and microgravity on collagen post-translational controls of MC3T3-E1 osteoblasts.
We attempted to study the effects of microgravity (by clinostat) and hypergravity (using centrifugation) on collagen metabolism using murine MC3T3-E1 osteoblasts, especially focusing on collagen cross-link formation. We found that altered gravitational load affected the post-translational modification of collagen, particularly the collagen maturation pathway, through altered expression of enzymes involved in cross-link formation.


BACKGROUND
Gravitational loading plays important roles in the stimulation of differentiated osteoblast function and in the maintenance of skeletal tissues, whereas microgravity seems to result in osteopenia caused by impaired osteoblast differentiation. The aim of our study was to clarify the effects of altered gravitational environments on collagen metabolism, particularly the relationship between post-translational collagen quality and enzymes involved in cross-link formation, using murine osteoblastic MC3T3-E1 cells.


METHODS
Cells were cultured under vector-averaged microgravity (1 x 10(-3) g) using a clinostat or under conventional centrifugation techniques to generate hypergravity (20 g and 40 g) for 72 h. We then examined the expression patterns of lysyl oxidase and the two lysyl hydroxylase isoforms telopeptidyl lysyl hydroxylase (TLH; procollagen-lysine, 2-oxyglutarate, 5-dioxigenase 2 [PLOD2]) and helical lysyl hydroxylase (HLH; [PLOD1]) by quantitative real time polymerase chain reaction (PCR) analysis. Quantitative analysis of reducible immature (dihydroxylysinonorleucine, hydroxylysinonorleucine, and lysinonorleucine) and nonreducible mature (pyridinoline and deoxypyridinoline) cross-links, and maturation rate analysis of immature to mature cross-links by conventional metabolic labeling using tritium lysine were also performed.


RESULTS
Hypergravity upregulated both TLH mRNA expression and enzyme activity compared with stationary cultures, whereas microgravity stimulated both HLH mRNA expression and enzyme activity. These results were consistent with increased relative occupancy rates of telopeptidyl hydroxylysine-derived cross-links and helical hydroxylysine-derived forms observed under hypergravity and microgravity, respectively. Hypergravity stimulated not only lysyl oxidase mRNA expression but also increased enzyme activity and the sum of immature and mature cross-links. Furthermore, the conversion rate of immature cross-links to mature compounds was markedly increased under hypergravity but decreased under microgravity.


CONCLUSIONS
Altered gravitational loading may affect the post-translational modification of collagen through altered expression of enzymes involved in cross-link formation. These observations may be important in elucidating the mechanisms of osteopenia during space flight.
Saito
Mitsuru
M
Department of Orthopaedic Surgery, Jikei University School of Medicine, Tokyo, Japan. xlink67@gol.com
Soshi
Shigeru
S
Fujii
Katsuyuki
K
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation; D002455:Cell Division; D003094:Collagen / Q000737:chemistry / Q000378:metabolism*; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D000090602:Collagen Type I, alpha 1 Chain; D003432:Cross-Linking Reagents; D004247:DNA / Q000235:genetics; D006112:Gravitation; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D008249:Protein-Lysine 6-Oxidase / Q000235:genetics / Q000378:metabolism; D018474:Weightlessness Simulation
D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D003432:Cross-Linking Reagents; D003094:Collagen; D004247:DNA; C479138:PLOD2 protein, human; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase; D008249:Protein-Lysine 6-Oxidase