Protein phosphatases: Possible bisphosphonate binding sites mediating stimulation of osteoblast proliferation

SUSANA MORELLI; PAOLA SCODELARO BILBAO; SEBASTIAN KATZ; VIRGINIA LEZCANO; EMILIO ROLDÁN; RICARDO BOLAND; GRACIELA SANTILLAN

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

ELSEVIER SCIENCE INC

Año: 2011 vol. 507 p. 248 - 253

0003-9861

We investigated the existence of a bisphosphonates (BP) target site in osteoblasts. Binding assays using [3H]-olpadronate (3[H]OPD) in whole cells showed the presence of specific, saturable and high affinity binding for OPD (Kd= 1.39 ± 0.33 µM) in osteoblasts. [3H]OPD was displaced from its binding site by micromolar concentrations of lidadronate, alendronate and etidronate (Kd =1.42 ± 0.15 µM, 2.00 ± 0.2 µM and 2.4 ± 0.4 µM, respectively), and by millimolar concentrations of the nonpermeant 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 andvpb(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.