PHOSPHOCITRATE INHIBITS A BASIC CALCIUM-PHOSPHATE AND CALCIUM PYROPHOSPHATE DIHYDRATE CRYSTAL-INDUCED MITOGEN-ACTIVATED PROTEIN-KINASE CASCADE SIGNAL-TRANSDUCTION PATHWAY

Calcium deposition diseases caused by calcium pyrophosphate dihydrate (CPPD) and basic calcium phosphate (BCP) crystals are a significant so urce of morbidity in the elderly, We have shown previously that both t ypes of crystals can induce mitogenesis, as well as metalloproteinase synthesis and secretion by fibroblasts and chondrocyte, These response s may promote degradation of articular tissues, We have also shown pre viously that both CPPD and BCP crystals activate expression of the c-f os and c-jun proto oncogenes. Phosphocitrate (PC) can specifically blo ck mitogenesis and proto-oncogene expression induced by either BCP or CPPD crystals in 3T3 cells and human fibroblasts, suggesting that PC m ay be an effective therapy for calcium deposition diseases, To underst and how PC inhibits BCP and CPPD-mediated cellular effects, we have in vestigated the mechanism by which BCP and CPPD transduce signals to th e nucleus, Here we demonstrate that BCP and CPPD crystals activate a p rotein kinase signal transduction pathway involving p42 and p44 mitoge n activated protein (MAP) kinases (ERK 2 and ERK 1). BCP and CPPD also cause phosphorylation of a nuclear transcription factor, cyclic AMP r esponse element-binding protein (CREB), on serine 133, a residue essen tial for CREB's ability to transactivate. Treatment of cells with PC a t concentrations of 10(-3) to 10(-5) M blocked both the activation of p42/p44 MAP kinases, and CREB serine 133 phosphorylation, in a dose-de pendent fashion, At 10(-3) M, a PC analogue, n-sulfo-2-aminotricarball ylate and citrate also modulate this signal transduction pathway, Inhi bition by PC is specific for BCP- and CPPD-mediated signaling, since a ll three compounds had no effect on serum-induced p42/P44 or interleuk in-1 beta induced p38 MAP kinase activities. Treatment of cells with a n inhibitor of MEK1, an upstream activator of MAPKs, significantly inh ibited crystal-induced cell proliferation, suggesting that the MAPK pa thway is a significant mediator of crystal-induced signals.