title: Protein phosphatases: possible bisphosphonate binding sites mediating stimulation of osteoblast proliferation.
abstract: We investigated the existence of a bisphosphonate (BP) target site in osteoblasts. Binding assays using [³H]-olpadronate ([³H]OPD) in whole cells showed the presence of specific, saturable and high affinity binding for OPD (K(d)=1.39 ± 0.33 μM) in osteoblasts. [³H]OPD was displaced from its binding site by micromolar concentrations of lidadronate, alendronate and etidronate (K(d)=1.42 ± 0.15 μM, 2.00 ± 0.2 μM and 2.4 ± 0.4 μM, respectively), and by millimolar concentrations of the non-permeant protein phosphatase (PP) substrates p-nitrophenylphosphate and α-naphtylphosphate. PP inhibitors orthovanadate, NaF or vpb(bipy) did not displace [³H]OPD. As expected, specific OPD binding was detected in the plasma membrane of ROS 17/2.8 cells, although significant BP binding was also found intracellularly. Moreover, OPD increased DNA synthesis in these cells with a temporal profile similar to the protein tyrosine phosphatase (PTP) inhibitors, Na₃VO₄ and vpb(bipy); but different from a general PP inhibitor (NaF). The stimulatory effect of OPD and PTP inhibitors on osteoblast proliferation was inhibited by the protein tyrosine kinase inhibitors genistein and geldanamycin. These results provide new evidence on the existence of a BP target in osteoblastic cells, presumably a PTP, which may be involved in the stimulatory action of BPs on osteoblast proliferation.
authors.affiliation: Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur. San Juan 670, 8000 Bahía Blanca, Argentina.
authors.forename: Susana
authors.initials: S
authors.lastname: Morelli
authors.forename: Paola Scodelaro
authors.initials: PS
authors.lastname: Bilbao
authors.forename: Sebastian
authors.initials: S
authors.lastname: Katz
authors.forename: Virginia
authors.initials: V
authors.lastname: Lezcano
authors.forename: Emilio
authors.initials: E
authors.lastname: Roldán
authors.forename: Ricardo
authors.initials: R
authors.lastname: Boland
authors.forename: Graciela
authors.initials: G
authors.lastname: Santillan
mesh_terms: D000818:Animals; D001665:Binding Sites; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004164:Diphosphonates / Q000378:metabolism* / Q000494:pharmacology; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D011485:Protein Binding; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
chemical_list: D004164:Diphosphonates; C055774:olpadronic acid; D017027:Protein Tyrosine Phosphatases