Intracellular distribution of the lysyl oxidase propeptide in osteoblastic cells.
Lysyl oxidase plays a critical role in the formation of the extracellular matrix, and its activity is required for the normal maturation and cross-linking of collagen and elastin. An 18-kDa lysyl oxidase propeptide (LOPP) is generated from 50-kDa prolysyl oxidase by extracellular proteolytic cleavage during the biosynthesis of active 30-kDa lysyl oxidase enzyme. The fate and the functions of the LOPP are largely unknown, although intact LOPP was previously observed in osteoblast cultures. We investigated the spatial localization of molecular forms of lysyl oxidase, including LOPP in proliferating and differentiating osteoblasts, by using confocal immunofluorescence microscopy and Western blots of cytoplasmic and nuclear extracts. In the present study, a stage-dependent intracellular distribution of LOPP in the osteoblastic cell was observed. In proliferating osteoblasts, LOPP epitopes were principally associated with the Golgi and endoplasmic reticulum, and mature lysyl oxidase epitopes were found principally in the nucleus and perinuclear region. In differentiating cells, LOPP and mature lysyl oxidase immunostaining showed clear colocalization with the microtubule network. The subcellular distribution of LOPP and its temporal and physical association with microtubules were confirmed by Western blot and far Western blot studies. We also report that N-glycosylated and nonglycosylated LOPP are present in MC3T3-E1 cell cultures. We conclude that LOPP has a stage-dependent intracellular distribution in osteoblastic cells. Future studies are needed to investigate whether the LOPP associations with microtubules or the osteoblast nucleus have functional effects for osteoblast differentiation and bone formation.
Guo
Ying
Y
Division of Oral Biology, Boston University Goldman School of Dental Medicine, Boston, MA 02118, USA.
Pischon
Nicole
N
Palamakumbura
Amitha H
AH
Trackman
Philip C
PC
D016475:3T3 Cells; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D051379:Mice; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism*; D021381:Protein Transport; D008249:Protein-Lysine 6-Oxidase / Q000235:genetics / Q000378:metabolism*; D014404:Tubulin
D014404:Tubulin; D008249:Protein-Lysine 6-Oxidase