Incadronate and etidronate accelerate phosphate-primed mineralization of MC4 cells via ERK1/2-Cbfal signaling pathway in a Ras-independent manner: Further involvement of mevalonate-pathway blockade for incadronate

Two types of bisphosphonates (BPs), incadronate (INC) and etidronate (ETI) accelerated phosphate (Pi)-primed mineralization of MC4 cells in a subnanom olar dose range. Intracellular signaling pathways involved were examined. 1 ) The effect of INC but not ETI was partially suppressed by two mevalonate (MVA) pathway metabolites, farnesylpyrophosphate (FPP) and geranylgeranylpy rophosphate (GGPP). 2) The BP-like accelerating effect was produced by stat ins and also by Toxin B, a Rho GTPases-specific inhibitor. 3) INC induced C bfa1-nuclear localization within hours; and in an in vivo experiment using ovariectomized mice, its 3 weeks dosing exhibited the same effect in tibial extracts. 4) BPs promoted luciferase expression in murine p1.3-osteocalcin gene 2-luc and p6-osteoblast specific element 2-luc transfected cells, jus t as MVA, FPP and GGPP did independently and additively to INC. 5) BPs acti vated extracellular signal-regulated kinase (ERK1/2) in a Pas-independent m anner within 5 min, and Pi was found to sensitize MC4 cells to BPs. MVA and its metabolites also activated ERKs but in a Pas-dependent manner and addi tively to INC. Ras dependency was determined using N17Ras-transfected cells . A MEK (MAP kinase-ERK kinase)-specific inhibitor PD98059 alone partly and with FPP completely blocked INC-induced mineralization. The results sugges t that BPs act on Pi-sensitized MC4 cells to accelerate mineralization via nonRas-MEK-ERK1/2-Cbfa1 transactivation pathway and INC additionally acts b y inhibiting the MVA pathway.