title: The Functional co-operativity of Tissue-Nonspecific Alkaline Phosphatase (TNAP) and PHOSPHO1 during initiation of Skeletal Mineralization.
abstract: Phosphatases are recognised to have important functions in the initiation of skeletal mineralization. Tissue-nonspecific alkaline phosphatase (TNAP) and PHOSPHO1 are indispensable for bone and cartilage mineralization but their functional relationship in the mineralization process remains unclear. In this study, we have used osteoblast andex-vivometatarsal cultures to obtain biochemical evidence for co-operativity and cross-talk between PHOSPHO1 and TNAP in the initiation of mineralization. Clones 14 and 24 of the MC3T3-E1 cell line were used in the initial studies. Clone 14 cells expressed high levels of PHOSPHO1 and low levels of TNAP and in the presence of β-glycerol phosphate (BGP) or phosphocholine (P-Cho) as substrates and they mineralized their matrix strongly. In contrast clone 24 cells expressed high levels of TNAP and low levels of PHOSPHO1 and mineralized their matrix poorly. LentiviralPhospho1overexpression in clone 24 cells resulted in higher PHOSPHO1 and TNAP protein expression and increased levels of matrix mineralization. To uncouple the roles of PHOSPHO1 and TNAP in promoting matrix mineralization we used PHOSPHO1 (MLS-0263839) and TNAP (MLS-0038949) specific inhibitors, which individually reduced mineralization levels ofPhospho1overexpressing C24 cells, whereas the simultaneous addition of both inhibitors essentially abolished matrix mineralization (85 %; P<0.001). Using metatarsals from E15 mice as a physiologicalex vivomodel of mineralization, the response to both TNAP and PHOSPHO1 inhibitors appeared to be substrate dependent. Nevertheless, in the presence of BGP, mineralization was reduced by the TNAP inhibitor alone and almost completely eliminated by the co-incubation of both inhibitors. These data suggest critical non-redundant roles for PHOSPHO1 and TNAP during the initiation of osteoblast and chondrocyte mineralization.
authors.affiliation: The Roslin Institute and R(D)SVS, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG.
authors.forename: Carmen
authors.initials: C
authors.lastname: Huesa
authors.affiliation: The Roslin Institute and R(D)SVS, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG.
authors.forename: Dean
authors.initials: D
authors.lastname: Houston
authors.affiliation: Sanford Children's Health Research Center, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA.
authors.forename: Tina
authors.initials: T
authors.lastname: Kiffer-Moreira
authors.affiliation: Sanford Children's Health Research Center, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA.
authors.forename: Manisha M
authors.initials: MM
authors.lastname: Yadav
authors.affiliation: Sanford Children's Health Research Center, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA.
authors.forename: Jose Luis
authors.initials: JL
authors.lastname: Millan
authors.affiliation: The Roslin Institute and R(D)SVS, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG.
authors.forename: Colin
authors.initials: C
authors.lastname: Farquharson
keywords: PHOSPHO1; alkaline phosphatase; chondrocytes; metatarsals; mineralization; osteoblasts