title: Extracellular matrix mineralization promotes E11/gp38 glycoprotein expression and drives osteocytic differentiation.
abstract: Osteocytes are terminally differentiated osteoblasts which reside in a mineralized extracellular matrix (ECM). The factors that regulate this differentiation process are unknown. We have investigated whether ECM mineralization could promote osteocyte formation. To do this we have utilised MLO-A5 pre-osteocyte-like cells and western blotting and comparative RT-PCR to examine whether the expression of osteocyte-selective markers is elevated concurrently with the onset of ECM mineralization. Secondly, if mineralization of the ECM is indeed a driver of osteocyte formation, we reasoned that impairment of ECM mineralization would result in a reversible inhibition of osteocyte formation. Supplementation of MLO-A5 cell cultures with ascorbic acid and phosphate promoted progressive ECM mineralization as well as temporally associated increases in expression of the osteocyte-selective markers, E11/gp38 glycoprotein and sclerostin. Consistent with a primary role for ECM mineralization in osteocyte formation, we also found that inhibition of ECM mineralization, by omitting phosphate or adding sodium pyrophosphate, a recognized inhibitor of hydroxyapatite formation, resulted in a 15-fold decrease in mineral deposition that was closely accompanied by lower expression of E11 and other osteocyte markers such as Dmp1, Cd44 and Sost whilst expression of osteoblast markers Ocn and Col1a increased. To rule out the possibility that such restriction of ECM mineralization may produce an irreversible modification in osteoblast behaviour to limit E11 expression and osteocytogenesis, we also measured the capacity of MLO-A5 cells to re-enter the osteocyte differentiation programme. We found that the mineralisation process was re-initiated and closely allied to increased expression of E11 protein after re-administration of phosphate or omission of sodium pyrophosphate, indicating an ECM mineralization-induced restoration in osteocyte formation. These results emphasise the importance of cell-ECM interactions in regulating osteoblast behaviour and, more importantly, suggest that ECM mineralization exerts pivotal control during terminal osteoblast differentiation and acquisition of the osteocyte phenotype.
authors.affiliation: Division of Developmental Biology, The Roslin Institute, The Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh, United Kingdom. prideauxm@umkc.edu
authors.forename: Matthew
authors.initials: M
authors.lastname: Prideaux
authors.forename: Nigel
authors.initials: N
authors.lastname: Loveridge
authors.forename: Andrew A
authors.initials: AA
authors.lastname: Pitsillides
authors.forename: Colin
authors.initials: C
authors.lastname: Farquharson
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002113:Calcification, Physiologic / Q000187:drug effects / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D011756:Diphosphates / Q000494:pharmacology; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D006023:Glycoproteins / Q000378:metabolism; D018960:Hyaluronan Receptors / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism; D010011:Osteocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D053402:PHEX Phosphate Regulating Neutral Endopeptidase / Q000378:metabolism; D012886:Skull / Q000166:cytology / Q000254:growth & development; D000098853:Podoplanin
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D011756:Diphosphates; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; D018960:Hyaluronan Receptors; D036341:Intercellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D053402:PHEX Phosphate Regulating Neutral Endopeptidase; C476399:Cd44 protein, mouse; C513308:Dmp1 protein, mouse; C490441:Pdpn protein, mouse; D000098853:Podoplanin; C486030:Sost protein, mouse; C506232:Phex protein, mouse; C003319:sodium pyrophosphate