Calcium deposition diseases caused by calcium pyrophosphate dihydrate (CPPD) and basic calcium phosphate (BCP) crystals are a significant source of morbidity in the elderly. We have shown previously that both types of crystals can induce mitogenesis, as well as metalloproteinase synthesis and secretion by fibroblasts and chondrocytes. These responses may promote degradation of articular tissues. We have also shown previously that both CPPD and BCP crystals activate expression of the c-fos and c-jun proto-oncogenes. Phosphocitrate (PC) can specifically block mitogenesis and proto-oncogene expression induced by either BCP or CPPD crystals in 3T3 cells and human fibroblasts, suggesting that PC may be an effective therapy for calcium deposition diseases. To understand how PC inhibits BCP and CPPD-mediated cellular effects, we have investigated the mechanism by which BCP and CPPD transduce signals to the nucleus. Here we demonstrate that BCP and CPPD crystals activate a protein kinase signal transduction pathway involving p42 and p44 mitogen-activated protein (MAP) kinases (ERK 2 and ERK 1). BCP and CPPD also cause phosphorylation of a nuclear transcription factor, cyclic AMP response element-binding protein (CREB), on serine 133, a residue essential for CREB's ability to transactivate. Treatment of cells with PC at 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-dependent fashion. At 10 ؊3 M, a PC analogue, n-sulfo-2-aminotricarballylate and citrate also modulate this signal transduction pathway. Inhibition by PC is specific for BCP-and CPPD-mediated signaling, since all three compounds had no effect on serum-induced p42/P44 or interleukin-1␤ induced p38 MAP kinase activities. Treatment of cells with an inhibitor of MEK1, an upstream activator of MAPKs, significantly inhibited crystal-induced cell proliferation, suggesting that the MAPK pathway is a significant mediator of crystal-induced signals.

Objective. Calcium deposition occurs frequently in osteoarthritic (OA) joints. However, evidence for a causal role of calcification in cartilage degeneration is inferential. The present study was undertaken to examine the role of calcification in OA disease progression and to evaluate a formulation of phosphocitrate (PC) as a potential therapeutic agent.Methods. We have identified a guinea pig OA model in which meniscal calcification appears to correlate with aging and disease progression. We synthesized a new formulation of PC, [CaNa(PC) 2 (H 2 O)] n (CaNaPC), which is a potent antimineralization agent and a specific inhibitor of crystal-induced biologic effects. After weekly treatment of guinea pigs with experimental OA with CaNaPC for 3 months, we examined calcification in menisci and cartilage degeneration. As a control, we examined whether similar CaNaPC treatment had any therapeutic effect in a hemi-meniscectomy model in which there is no known crystal involvement.Results. Meniscal calcification correlated with cartilage degeneration in this animal model. PC treatment led to significant reduction of calcium deposits and arrested OA disease progression. Similar treatment had no effect in the hemi-meniscectomy model.Conclusion. CaNaPC diminishes mineralization in a cutaneous calcergy model and a model of OA in which intraarticular mineralization is a prominent feature. In the OA guinea pig model, inhibition of calcification is accompanied by diminished cartilage degeneration. CaNaPC has no therapeutic effect in the hemimeniscectomy model. We conclude that pathologic calcification may initiate or amplify processes leading to cartilage degeneration and that CaNaPC may interrupt such a pathway.

The inhibition by phosphocitrate of struvite crystal formation and growth has been examined in the present study. Crystal growth in a gel matrix was controlled by phosphocitrate in a dose-dependent manner. The effects of inhibition were followed using scanning electron microscopy, optical microscopy, and single crystal X-ray analysis. The presence of phosphocitrate induced very strong, crystal face specific inhibition of struvite, leading to total cessation of crystal growth when sufficient concentration of the inhibitor was made available. Crystal growth studies and results from molecular modeling indicated strong affinity of phosphocitrate to (101) faces of struvite. This in turn led to an alteration in the expression of these faces and the development of a characteristic arrowhead struvite morphology. Similar changes were not observed in the presence of identical concentrations of citrate, acetohydroxamic acid, and N-sulfo-2 amino tricarballylate (an analog of phosphocitrate), emphasizing the unique interaction of phosphocitrate with the struvite crystal lattice.

Objective. To elucidate the mechanism of basic calcium phosphate (BCP) crystal-induced prostaglandin E 2 (PGE 2 ) production in human foreskin fibroblasts (HFFs), to identify the signaling pathway involved in the induction of cyclooxygenase 2 (COX-2) messenger RNA (mRNA) by BCP crystals, to examine the effect of BCP crystals on interleukin-1␤ (IL-1␤) mRNA expression, and to investigate the potential of phosphocitrate to abrogate the BCP crystal-induced effects.Methods. PGE 2 levels were quantified using a commercial enzyme immunoassay kit. COX-2 and COX-1 transcript levels were quantified using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Induction of IL-1␤ and COX-2 mRNA was examined by end-point RT-PCR. COX-2 protein expression was assessed by Western blotting.Results. PGE 2 production measured 4 and 30 hours after BCP crystal treatment was higher in BCP crystal-treated (mean ؎ SEM 1,891 ؎ 273 pg/ g and 1,792 ؎ 233 pg/ g, respectively) than in untreated (88 ؎ 5 pg/ g and 205 ؎ 93 pg/ g, respectively) HFFs. The PGE 2 produced after 4 hours was sensitive to inhibition with NS398, a selective COX-2 inhibitor, implying that it was COX-2 mediated, whereas the PGE 2 produced at 30 hours could not be completely inhibited by NS398. Real-time RT-PCR demonstrated a 23-fold increase in COX-2 mRNA that was maximal at 4 hours, whereas analysis of mRNA for COX-1 showed up-regulation of transcript peaking at 24 hours poststimulation (1.75-fold increase). The protein kinase C and phosphatidylinositol 3-kinase signal-transduction inhibitors bisindolylmaleimide I and LY294002, respectively, blocked BCP crystal-induced COX-2 mRNA in HFFs. In addition, BCP crystals were found to up-regulate the proinflammatory cytokine IL-1␤ (maximal at 8 hours). The induction of both COX-2 and IL-1␤ by BCP crystals was attenuated when the cells were treated with phosphocitrate.Conclusion. These findings indicate that BCP crystals may be an important amplifier of PGE 2 production through induction of the COX enzymes and the proinflammatory cytokine IL-1␤.Basic calcium phosphate (BCP) crystals include partially carbonate-substituted hydroxyapatite, octacalcium phosphate, and tricalcium phosphate. They form deposits in and around joints and are associated with a number of clinical syndromes, including calcific periarthritis, Milwaukee shoulder syndrome, and osteoarthritis (OA). The prevalence of BCP crystals in synovial fluid from patients with knee OA is between 30% and 60% (1,2). Ample data support the role of BCP crystals in cartilage degeneration. Their presence correlates strongly with the radiographic severity of OA (3), and larger effusions are seen in OA knee joints with crystals

The adsorption of citrate and phosphocitrate ions by hydroxyapatite (HAP) surfaces and their influence on the constant composition growth kinetics of HAP have been investigated. Phosphocitrate was strongly adsorbed to HAP and inhibited crystal growth. When HAP surfaces containing preadsorbed citrate were exposed to phosphocitrate, the uptake of the latter markedly increased. The two additives behaved synergistically in their HAP crystal growth inhibition.

Binding of citrate and phosphocitrate to calcium oxalate monohydrate crystals has been studied using scanning electron microscopy (SEM) and molecular modeling. Phosphocitrate structure has been resolved using low temperature X-ray analysis and ab initio computational methods. The (-1 0 1) crystal surface of calcium oxalate monohydrate is involved in binding of citrate and phosphocitrate, as shown by SEM and molecular modeling. Citrate and phosphocitrate conformations and binding energies to (-1 0 1) faces have been obtained and compared to binding to another set of calcium-rich planes (0 1 0). Difference in inhibitory properties of these compounds has been attributed to better coordination of functional groups of phosphocitrate with calcium ions in (-1 0 1). Relevance of this study to design of new calcium oxalate monohydrate inhibitors is discussed.

Calcium pyrophosphate dihydrate (CPPD) and basic calcium phosphate (BCP) crystal deposition diseases are a group of heterogeneous arthritides which are a significant source of morbidity in the elderly. Both crystals induced mitogenesis and metalloproteinase (MP) synthesis and secretion by fibroblasts and chondrocytes which may promote degradation of intra-articular tissue. We have previously shown that phosphocitrate (PC), an inhibitor of hydroxyapatite crystallization, specifically blocks BCP crystal-induced mitogenesis in 3T3 cells. This led us to examine the effect of PC on BCP and CPPD crystal induction of MP synthesis in human fibroblasts. PC (10(-3) to 10(-4) M) specifically inhibited the crystal-induced collagenase and stromelysin mRNA accumulation while having no effect on epidermal growth factor-induced or basal levels of mRNA for both enzymes. Western blots (collagenase) of conditioned media confirmed that PC blocked crystal-induced proteinase secretion as well. Moreover, PC (10(-3) M) also blocked the crystal induction of c-fos and c-jun. Since FOS and JUN proteins form a transacting activator (AP-1) for expression of collagenase and stromelysin genes, PC may block the synthesis of both enzymes by inhibiting the transcription of c-fos and c-jun.

Objective. Mice with progressive ankylosis, a spontaneous arthropathy, were treated with phosphocitrate (PC) in vivo to determine the effect of PC on disease progression. Methods. Two groups of mice with progressive ankylosis (matched for age, weight, and sex) were treated parenterally for 6 weeks with either PC or saline vehicle. Results. Clinically, histologically, and microradiographically, there were significant differences in disease progression and severity in the PC‐treated and the saline‐treated mice. Conclusion. PC appears to inhibit disease progression in murine progressive ankylosis.