Foscarnet Research Articles

Severe hyponatremia caused by syndrome of inappropriate secretion of antidiuretic hormone developed as initial manifestation of human herpesvirus‐6–associated acute limbic encephalitis after unrelated bone marrow transplantation

Severe hyponatremia is a critical electrolyte abnormality in allogeneic stem cell transplantation (allo-SCT) recipients and >50% of cases of severe hyponatremia are caused by the syndrome of inappropriate secretion of antidiuretic hormone (SIADH). Here, we present a patient with rapidly progressive severe hyponatremia as an initial sign and symptom of human herpesvirus-6-associated post-transplantation acute limbic encephalitis (HHV-6 PALE) after allo-SCT. A 45-year-old woman with acute lymphoblastic leukemia received unrelated bone marrow transplantation from a one locus-mismatched donor at the DR locus. On day 21, she developed a generalized seizure and loss of consciousness with severe hyponatremia, elevated serum antidiuretic hormone (ADH), and decreased serum osmolality. A high titer of HHV-6 DNA was detected in cerebrospinal fluid. Treatment with foscarnet sodium and hypertonic saline was started with improvement of neurological condition within several days. Although an elevated serum ADH, low serum osmolality, and high urinary osmolality persisted for 2months, she had no other recurrent symptoms of encephalitis. Our experience suggests that hyponatremia accompanied by SIADH should be recognized as a prodromal or concomitant manifestation of HHV-6 PALE, and close monitoring of serum sodium levels in high-risk patients for HHV-6 PALE is necessary for immediate diagnosis and treatment initiation.

Dietary phosphate modifies lifespan in Drosophila

Phosphate is required for many important cellular processes and having too little phosphate (hypophosphatemia) or too much (hyperphosphatemia) can cause disease and reduce lifespan in humans. Drosophila melanogaster has been a powerful tool to discover evolutionarily well-conserved nutrient-sensing pathways that are important for the lifespan extension. We have established Drosophila as a model system for studying the effects of dietary phosphate during development and adult life. When absorption of phosphate is blocked by sevelamer or cellular uptake is inhibited by phosphonoformic acid (PFA), larval development is delayed in a phosphate-dependent fashion. Conversely, restriction of phosphate absorption with sevelamer or reduced cellular uptake after treatment with PFA is able to extend the adult lifespan of otherwise normal flies. Gaining an understanding of the specific pathways and mediators that regulate cellular and organismic phosphate levels might ultimately lead to the development of improved dietary and therapeutic approaches to the treatment of human disorders of hypo- and hyperphosphatemia.

Abstract 4715: Activation of lytic cycle of Epstein-Barr virus by suberoylanilide hydroxamic acid affects apoptosis and tumor growth suppression of nasopharyngeal carcinoma

Abstract Nasopharyngeal carcinoma (NPC) is strongly associated with Epstein-Barr virus (EBV). We examined the in vitro and in vivo effects of suberoylanilide hydroxamic acid (SAHA), a FDA-approved histone deacetylase inhibitor, on EBV lytic cycle induction in NPC and investigated the cellular consequences. Micromolar concentrations of SAHA potently induced EBV lytic cycle with replication of EBV DNA, expression of immediate early (Zta and Rta), early (BMRF1) and late (gp350) lytic proteins and production of infectious viral particles in culture supernatants in three of four EBV-positive NPC cell lines. One NPC cell line had abortive EBV lytic cycle (no EBV DNA replication or late lytic protein expression) induction. Same concentrations of SAHA effected enhanced killing of EBV-positive NPC cells in comparison with EBV-negative counterparts, as measured by MTT assay. Increased percentages of annexin V-positive and TUNEL-positive cells and proteolytic cleavage of poly (ADP-ribose) polymerase (PARP), caspase-3, -7 and -9 in EBV-positive versus EBV-negative NPC were also observed. Immunofluorescence staining and flow cytometry showed that the majority of NPC cells (>85%) expressing Zta, BMRF1 or gp350 lytic protein co-expressed cleaved caspase-3. Tracking of expression of EBV lytic protein and cleaved caspase-3 showed that proportion of NPC cells co-expressing EBV lytic protein and cleaved caspase-3 increased over time. Furthermore, knockdown of Zta expression by short-hairpin RNA significantly decreased proteolytic cleavage of caspase-3 in SAHA-treated EBV-positive NPC cells whilst overexpression of Zta induced apoptosis of EBV-positive NPC cells, confirming that lytic cycle induction programmed NPC cells to apoptosis. Interestingly, inhibition of EBV DNA replication and late lytic protein expression by phosphonoformic acid did not impact on SAHA's induced cell death in NPC, suggesting that early rather than late phase of EBV lytic cycle contributed to the apoptotic effect. In vivo effects of SAHA on EBV lytic cycle induction and tumor growth suppression were observed in NPC xenografts established in nude mice. Taken together, our data indicated that activation of lytic cycle from latent cycle of EBV by SAHA effects apoptosis and tumor growth suppression of NPC thereby providing experimental evidence for virus-targeted therapy against EBV-positive cancer. This project is funded by NPC Area of Excellence (AoE/M 06/08 Center for Nasopharyngeal Carcinoma Research) and Epstein-Barr virus research (# 20004525) grants of AKSC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4715. doi:1538-7445.AM2012-4715

400 Ganciclovir-Resistant (GCV-R) CMV Is Common among Lung Transplant Recipients (LTR) Receiving Alemtuzumab Induction and Valganciclovir Prophylaxis (VGC px), and Treatment with Foscarnet (FOS) Is Both Ineffective and Toxic

400 Ganciclovir-Resistant (GCV-R) CMV Is Common among Lung Transplant Recipients (LTR) Receiving Alemtuzumab Induction and Valganciclovir Prophylaxis (VGC px), and Treatment with Foscarnet (FOS) Is Both Ineffective and Toxic

Activation of lytic cycle of Epstein‐Barr virus by suberoylanilide hydroxamic acid leads to apoptosis and tumor growth suppression of nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is strongly associated with Epstein-Barr virus (EBV). We reported that suberoylanilide hydroxamic acid (SAHA) induced EBV lytic cycle in EBV-positive gastric carcinoma cells and mediated enhanced cell death. However, expression of EBV lytic proteins was thought to exert antiapoptotic effect in EBV-infected cells. Here, we examined the in vitro and in vivo effects of SAHA on EBV lytic cycle induction in NPC cells and investigated the cellular consequences. Micromolar concentrations of SAHA significantly induced EBV lytic cycle in EBV-positive NPC cells. Increased apoptosis and proteolytic cleavage of poly(ADP-ribose) polymerase and caspase-3, -7 and -9 in EBV-positive versus EBV-negative NPC cells were observed. More than 85% of NPC cells expressing immediate-early (Zta), early (BMRF1) or late (gp350/220) lytic proteins coexpressed cleaved caspase-3. Tracking of expression of EBV lytic proteins and cleaved caspase-3 over time demonstrated that NPC cells proceeded to apoptosis following EBV lytic cycle induction. Inhibition of EBV DNA replication and late lytic protein expression by phosphonoformic acid did not impact on SAHA's induced cell death in NPC, indicating that early rather than late phase of EBV lytic cycle contributed to the apoptotic effect. In vivo effects of SAHA on EBV lytic cycle induction and tumor growth suppression were also observed in NPC xenografts in nude mice. Taken together, our data indicated that activation of lytic cycle from latent cycle of EBV by SAHA leads to apoptosis and tumor growth suppression of NPC thereby providing experimental evidence for virus-targeted therapy against EBV-positive cancer.

Emergence of cytomegalovirus resistance to foscarnet in a patient receiving foscarnet salvage therapy for multidrug-resistant HIV infection

Foscarnet (FOS) is a pyrophosphate analogue that inhibits both cytomegalovirus (CMV) and human immunodeficiency virus (HIV) replication. We describe herein the emergence of CMV resistance to FOS in a 54-year-old man receiving FOS salvage therapy because of multidrug-resistant HIV-1 infection. Oral valganciclovir (VGCV) treatment allowed subsequently the improvement of FOS-resistant CMV infection.

Effects of Phosphonoformic Acid and Renagel on Renal Type IIa Sodium-Dependent Phosphate Cotransporter mRNA Expression in Hyperphosphatemia Rats

Objective: To investigate the effects of phosphonoformic acid (PFA) and sevelamer hydrochloride (Renagel) on renal type IIa sodium-dependent phosphate cotransporter (NaPi-2) mRNA expression in hyperphosphatemia rats. Methods: Thirty rats were randomly divided into five groups based on the diet for 2 weeks after 5/6 nephrectomy (Nx): Nx + high-phosphate (HP; 1.2% P) diet; Nx + low-phosphate (LP; 0.2% P) diet; HP + PFA (injected with 0.15 g/kg PFA daily); HP + Saline (injected with the same amount of saline daily); and HP + Renagel (2%) group. Another 12 rats were sham operated and divided into Sham + HP and Sham + LP groups. Serum ionized calcium, phosphorus (P), and intact parathyroid hormone (iPTH) were measured on days 2, 7, and 14. Serum 1,25(OH)2D3 was measured on day 14 and NaPi-2 mRNA levels were assayed by RT-PCR. Results: PFA decreased iPTH level but had no effect on NaPi-2 mRNA expression. Renagel decreased serum P and iPTH levels, but upregulated renal NaPi-2 mRNA expression. Conclusions: Both PFA and Renagel are effective drugs to decrease iPTH level and they might be potential candidates for treatment of clinical secondary hyperparathyroidism. Renagel can also decrease serum P and upregulate renal NaPi-2 mRNA expression.

Successful treatment of aciclovir and foscarnet resistant Herpes simplex virus lesions with topical imiquimod in patients infected with human immunodeficiency virus type 1

Aciclovir (ACV)-resistant Herpes simplex virus type-2 (HSV-2) infections are observed commonly in patients also infected with HIV-1. The use of foscarnet (FOS) in these patients may also lead to resistance. This situation can become a difficult therapeutic challenge. Four cases of patients infected with HIV and with mucocutaneous HSV-2 resistant to ACV and FOS are reported. These patients were treated successfully with topical 5% imiquimod. Imiquimod treatment also appeared to delay the time to recurrence of HSV lesions.

Cyclopropavir Susceptibility of Cytomegalovirus DNA Polymerase Mutants Selected after Antiviral Drug Exposure

Human cytomegalovirus (CMV) UL54 DNA polymerase (pol) mutants with known patterns of resistance to current antivirals ganciclovir (GCV), foscarnet (FOS), and cidofovir (CDV) were tested for cyclopropavir (CPV) susceptibility by a standardized reporter-based yield reduction assay. Exonuclease and A987G (region V) mutations at codons commonly associated with dual GCV-CDV resistance in clinical isolates paradoxically conferred increased CPV susceptibility. Various polymerase catalytic region mutations conferring FOS resistance with variable low-grade GCV and CDV cross-resistance also conferred CPV resistance, with 50% effective concentration (EC(50)) increases of 3- to 13-fold. CPV EC(50) values against several pol mutants were increased about 2-fold by adding UL97 mutation C592G. Propagation of a CMV exonuclease mutant under CPV selected for pol mutations less often than UL97 mutations. In 21 experiments, one instance each of mutations E756D and M844V, which were shown individually to confer 3- to 4-fold increases in CPV EC(50), was detected. Unlike GCV and CDV, exonuclease mutations are not a preferred mechanism of CPV resistance, but mutations in and near pol region III may confer CPV resistance by affecting its recognition as an incoming base for DNA polymerization.

Valganciclovir as Pre-Emptive Therapy for Cytomegalovirus Infection in Allogeneic Haematopoietic Stem Cell Transplant Recipients

In haematopoietic stem cell transplant (HSCT) recipients, cytomegalovirus (CMV) infection contributes significantly to morbidity and mortality in both the early and late post-transplant period. Ganciclovir (GCV) is the treatment of choice for CMV, but requires intravenous administration, a fact that complicates its long-term use. Oral valganciclovir (VGCV) and intravenous GCV were recently shown to have similar efficacy for pre-emptive CMV treatment in solid organ transplant recipients, but relatively limited data are available in HSCT recipients. The objectives of this study were to compare the efficacy of VGCV versus intravenous GCV or foscarnet (FOS) for pre-emptive therapy of active CMV infection in allogeneic HSCT and to determine the incidence of adverse effects and relapses. This was a 2-year prospective, comparative cohort study in which 237 episodes of pre-emptive therapy for active CMV infection were collected in 166 allogeneic HSCT recipients out of 717 included in the Spanish Network for Research on Infection in Transplantation (RESITRA/REIPI) database. Intravenous GCV was the first-line treatment in 112 episodes, intravenous FOS in 38 episodes, and oral VGCV in 87 episodes. VGCV was used as pre-emptive therapy for active CMV infection in 87 episodes. Excluding episodes considered as relapse, VGCV was as successful (91.4% [74/81]) as GCV (83.7% [87/14]) or FOS (75.8% [25/33]). In the VGCV arm, 7 (8.6%) cases were considered treatment failures: 4 (4.9%) because of adverse events, 1 (1.2%) due to persistent viral activity and 2 (2.5%) based on clinical decision. There were also 6 (7.4%) cases of recurrent infection. No statistically significant differences were found when compared to GCV or FOS. In allogeneic HSCT recipients, VGCV seemed effective and safe in the pre-emptive therapy of active CMV infection.

Antiviral Treatment of Cytomegalovirus Infection

Cytomegalovirus (CMV) is an opportunistic pathogen associated with significant morbidity and mortality in immunocompromised hosts. Antiviral agents specifically targeting CMV were initially developed during the human immunodeficiency virus (HIV) epidemic to treat end-organ disease in patients with acquired immunodeficiency syndrome (AIDS). There are currently four antiviral drugs licensed for the treatment of CMV infections: ganciclovir (GCV), valganciclovir (VGCV), foscarnet (FOS), and cidofovir (CDV). The role of these agents has evolved from the treatment of disease to include prevention of CMV infection and disease, primarily in solid organ transplant (SOT) and hematopoietic stem cell transplant (HSCT) recipients. The potential use of these drugs is being explored for the treatment of congenital CMV infection, another CMV-associated disease with significant morbidity. The availability of antiviral therapy has provided major advances in the treatment and prevention of CMV infection and has resulted in dramatically improved outcomes for immunocompromised hosts. At the same time, the clinical utility of most of these agents is limited by poor oral bioavailability, associated toxicities, and the potential for development of resistance with extended use. Novel therapeutic agents are needed to address these limitations. In this article, currently available anti-CMV agents will be described. An overview of the clinical syndromes caused by CMV will be provided, with specific reference to the role of antiviral agents in treating and preventing these infections. Antiviral resistance in CMV will be reviewed and novel therapeutic agents that may address resistance will be briefly discussed.

Phosphorus sorption on marine carbonate sediment: Phosphonate as Model Organic Compounds

Organophosphonate, characterized by the presence of a stable, covalent, carbon to phosphorus (C–P) bond, is a group of synthetic or biogenic organophosphorus compounds. The fate of these organic phosphorus compounds in the environment is not well studied. This study presents the first investigation on the sorption of phosphorus (P) in the presence of two model phosphonate compounds, 2-aminothylphosphonoic acid (2-AEP) and phosphonoformic acid (PFA), on marine carbonate sediments. In contrast to other organic P compounds, no significant inorganic phosphate exchange was observed in seawater. P was found to adsorb on the sediment only in the presence of PFA, not 2-AEP. This indicated that sorption of P from phosphonate on marine sediment was compound specific. Compared with inorganic phosphate sorption on the same sediments, P sorption from organic phosphorus is much less in the marine environment. Further study is needed to understand the potential role of the organophosphonate compounds in biogeochemical cycle of phosphorus in the environment.

Impact of Primer-induced Conformational Dynamics of HIV-1 Reverse Transcriptase on Polymerase Translocation and Inhibition

The rapid emergence and the prevalence of resistance mutations in HIV-1 reverse transcriptase (RT) underscore the need to identify RT inhibitors with novel binding modes and mechanisms of inhibition. Recently, two structurally distinct inhibitors, phosphonoformic acid (foscarnet) and INDOPY-1 were shown to disrupt the translocational equilibrium of RT during polymerization through trapping of the enzyme in the pre- and the post-translocation states, respectively. Here, we show that foscarnet and INDOPY-1 additionally display a shared novel inhibitory preference with respect to substrate primer identity. In RT-catalyzed reactions using RNA-primed substrates, translocation inhibitors were markedly less potent at blocking DNA polymerization than in equivalent DNA-primed assays; i.e. the inverse pattern observed with marketed non-nucleoside inhibitors that bind the allosteric pocket of RT. This potency profile was shown to correspond with reduced binding on RNA·DNA primer/template substrates versus DNA·DNA substrates. Furthermore, using site-specific footprinting with chimeric RNA·DNA primers, we demonstrate that the negative impact of the RNA primer on translocation inhibitor potency is overcome after 18 deoxyribonucleotide incorporations, where RT transitions primarily into polymerization-competent binding mode. In addition to providing a simple means to identify similarly acting translocation inhibitors, these findings suggest a broader role for the primer-influenced binding mode on RT translocation equilibrium and inhibitor sensitivity.

Pyrovanadolysis, a Pyrophosphorolysis-like Reaction Mediated by Pyrovanadate, Mn2+, and DNA Polymerase of Bacteriophage T7

DNA polymerases catalyze the 3'-5'-pyrophosphorolysis of a DNA primer annealed to a DNA template in the presence of pyrophosphate (PP(i)). In this reversal of the polymerization reaction, deoxynucleotides in DNA are converted to deoxynucleoside 5'-triphosphates. Based on the charge, size, and geometry of the oxygen connecting the two phosphorus atoms of PP(i), a variety of compounds was examined for their ability to carry out a reaction similar to pyrophosphorolysis. We describe a manganese-mediated pyrophosphorolysis-like activity using pyrovanadate (VV) catalyzed by the DNA polymerase of bacteriophage T7. We designate this reaction pyrovanadolysis. X-ray absorption spectroscopy reveals a shorter Mn-V distance of the polymerase-VV complex than the Mn-P distance of the polymerase-PP(i) complex. This structural arrangement at the active site accounts for the enzymatic activation by Mn-VV. We propose that the Mn(2+), larger than Mg(2+), fits the polymerase active site to mediate binding of VV into the active site of the polymerase. Our results may be the first documentation that vanadium can substitute for phosphorus in biological processes.

Safety of pre‐engraftment prophylactic foscarnet administration after allogeneic stem cell transplantation

Human herpesvirus-6 (HHV-6) is a major cause of limbic encephalitis with a dismal prognosis after allogeneic hematopoietic stem cell transplantation (SCT). Because our previous trial of preemptive therapy with foscarnet sodium (phosphonoformic acid; PFA) failed to prevent HHV-6 encephalitis, we conducted a prospective study to examine the safety of prophylactic PFA administration and elucidate the changes in the plasma HHV-6 DNA levels in the early post-SCT period. Plasma HHV-6 DNA was measured thrice weekly from day 6. PFA, 90 mg/kg/day, was administered from days 7 to 21 after bone marrow or peripheral blood SCT and to day 25 after umbilical cord blood transplantation. Of the 10 patients enrolled, 2 dropped out of the study, 1 because of early death, and 1 with a low glomerular filtration rate. Grade 3 or greater adverse events occurred in 9 of the 10 prophylactic PFA patients and in 7 of the 10 control patients who had clinical backgrounds similar to the study subjects and underwent SCT during the same period. Neurological disorders developed in none of the study subjects but in 4 of the 10 control patients, including 2 with HHV-6 encephalitis. HHV-6 reactivation occurred in 3 of the 10 study subjects. The prophylactic PFA regimen was thus safe and it may reduce the risk of limbic encephalitis, but is not considered to be potent enough to prevent HHV-6 reactivation.

Phosphonoformic Acid Inhibits Viral Replication by Trapping the Closed Form of the DNA Polymerase

Phosphonoformic acid (PFA, foscarnet) belongs to a class of antiviral drugs that inhibit the human cytomegalovirus DNA polymerase (UL54) by mimicking the pyrophosphate leaving group of the nucleotide transfer reaction. Difficulties expressing UL54 have hampered investigation of the precise structural requirements rendering inhibition by this drug. However, a previously engineered chimeric DNA polymerase, constructed by mutating the homologous polymerase from bacteriophage RB69 (gp43) to express several variable elements from UL54, can bypass this obstacle because of its favorable expression and acquired sensitivity to PFA (Tchesnokov, E. P., Obikhod, A., Schinazi, R. F., and Götte, M. (2008) J. Biol. Chem. 283, 34218-34228). Here, we compare two crystal structures that depict the chimeric DNA polymerase with and without PFA bound. PFA is visualized for the first time in the active site of a DNA polymerase, where interactions are resolved between the PP(i) mimic and two basic residues absolutely conserved in the fingers domain of family B polymerases. PFA also chelates metal ion B, the cation that contacts the triphosphate tail of the incoming nucleotide. These DNA complexes utilize a primer-template pair enzymatically chain-terminated by incorporation of acyclo-GMP, the phosphorylated form of the anti-herpes drug acyclovir. We postulate that the V478W mutation present in the chimera is critical in that it pushes the fingers domain to more readily adopt the closed conformation whether or not the drug is bound. The closed state of the fingers domain traps the variant polymerase in the untranslocated state and increases affinity for PFA. This finding provides a model for the mechanism of UL54 stalling by PFA.

Phosphate-Induced Apoptosis in Human Peritoneal Mesothelial Cells in vitro

Background: It has been demonstrated that phosphate uptake through the type III sodium-dependent phosphate co-transporter, Pit-1, induced apoptosis of aortic vascular smooth muscle cells and endothelial cells in vitro. However, the apoptotic effects of high phosphate (HP) level in human peritoneal mesothelial cells (HPMCs) are not known. Methods: To examine whether Pit-1 is expressed in HPMCs, we checked the Western blot assay of immunoreactive Pit-1 and the transcription of Pit-1 by reverse transcriptase PCR. We treated several different phosphate concentrations (1–4 mM) and calcium concentrations (1.8 and 2.8 mM) on HPMCs to assess the effects of concentration. MTT, TUNEL assays, and flow cytometry analysis using Annexin V and propidium iodide were performed to identify cell death and apoptosis. Bax and Bcl-2 by Western blot and caspase-3 activity were evaluated by colorimetric assay. In addition, phosphonoformic acid (PFA) and pan-caspase inhibitor, Z-VAD-FMK, were given to prevent phosphate-induced apoptosis. Results: Pit-1 expression on HPMCs was demonstrated. Apoptosis in HPMCs significantly increased with a high concentration of phosphate in a dose- and time-dependent manner, and was enhanced in the presence of 2.8 mM calcium. HP concentrations significantly decreased the anti-apoptotic Bcl-2/Bax ratio and increased caspase-3 activity. The treatment with PFA and Z-VAD-FMK prevented cell death by HP. Conclusion: Phosphate uptake through Pit-1 induces apoptosis in HPMCs by a caspase-related mechanism.

Inorganic phosphate (Pi) transport by rat Z310 choroid plexus (CP) epithelial cells suggests involvement of type III Na+‐Pi cotransporters (PiT1/2, slc20A1/2)

Pi concentration [Pi] in the cerebrospinal fluid (CSF) is approximately half of that of plasma, suggesting that the [Pi] in the CSF is actively maintained. The membrane proteins responsible for Pi transport by the CP are largely unknown. There may be no type II Na+- Pi cotransporters (NaPiII, slc34A) in the CP (Hisano et al., Brain Research. 772, 1997) and PiT-like transport has been implicated in active Pi transport by spiny dogfish CP (Guerreiro et al., Bull. MDI Biol. Lab. 49:5, 2010). For comparison, we examined mammalian Pi transport properties in Z310 cells, an immortalized rat CP epithelial cell line. The time course and saturation kinetics of radio-labeled Pi uptake revealed a Km similar to those for PiT1/2 in other systems (~0.6 mM). Also consistent with PiT-like transport, Pi uptake was sodium-dependent and the addition of Li+stimulated Pi uptake compared to sodium-free Pi uptake. Arsenate, antimycin A, and depolarization with high K+ significantly inhibited 40–50% of Pi uptake. However, phosphonoformic acid (PFA) had no effect. The ability of Li+ to substitute for Na+ on the transporter and its insensitivity to PFA distinguish PiT1/2-like transport from transport by NaPiII (Virkki et al., Am J Physiol Renal Physiol. 293(3), 2007). mRNA and protein expression of PiT2 is confirmed in the Z310 cells. We tentatively conclude that PiT-like transport is mostly responsible for Pi transport by the CP. Supported by NSF.

Phosphate increases bone morphogenetic protein-2 expression through cAMP-dependent protein kinase and ERK1/2 pathways in human dental pulp cells

Extracellular phosphate (Pi) is known to play a key role in promoting osteoblastic differentiation by altering gene expression and cellular function. Importantly, it may be possible to use this knowledge as a means to deliver Pi to local sites to regenerate mineralized tissues associated with the oral cavity. Therefore, we determined the ability of Pi to regulate differentiation of pulp cells toward an odontoblast phenotype and further determined if this was in part due to an increase in the expression of bone morphogenetic protein (BMP)-2, a crucial regulator of mineralization. Results showed that Pi increased BMP-2 expression at both mRNA and protein level and BMP-2 promoter activity. Signaling inhibitors revealed that increased BMP-2 expression was dependent on cAMP/protein kinase A but not the protein kinase C signaling pathway. Treatment with 8-Br-cAMP, a cell-permeable analog of cAMP, enhanced Pi-mediated BMP-2 expression, but treatment with 8-Br-cAMP alone did not increase BMP-2, suggesting that cAMP is indispensable but not sufficient for Pi-mediated BMP-2 expression. Pi activated ERK1/2, and treatment with PD98059, an ERK1/2 inhibitor, suppressed Pi-mediated BMP-2 increase, indicating a requirement for activation of ERK1/2. ERK1/2 pathway may operate independently of cAMP-dependent signaling because MDL12,330A, an adenylate cyclase inhibitor, did not inhibit phosphorylation of ERK1/2 in response to Pi. Pulp cells expressed the sodium-dependent Pi transporter (NaPi) III type, but not NaPi-I type or NaPi-II type. Pi-mediated BMP-2 increase was inhibited in the presence of phosphonoformic acid, an inhibitor not only of NaPi transport but also of crystal nucleation. Furthermore, a similar inhibition was observed in the presence of pyrophosphate, a mineralization inhibitor. These findings demonstrate, for the first time, that Pi regulates BMP-2 expression via cAMP/protein kinase A and ERK1/2 pathways in human dental pulp cells.

Cellular ATP Synthesis Mediated by Type III Sodium-dependent Phosphate Transporter Pit-1 Is Critical to Chondrogenesis

Disturbed endochondral ossification in X-linked hypophosphatemia indicates an involvement of P(i) in chondrogenesis. We studied the role of the sodium-dependent P(i) cotransporters (NPT), which are a widely recognized regulator of cellular P(i) homeostasis, and the downstream events in chondrogenesis using Hyp mice, the murine homolog of human X-linked hypophosphatemia. Hyp mice showed reduced apoptosis and mineralization in hypertrophic cartilage. Hyp chondrocytes in culture displayed decreased apoptosis and mineralization compared with WT chondrocytes, whereas glycosaminoglycan synthesis, an early event in chondrogenesis, was not altered. Expression of the type III NPT Pit-1 and P(i) uptake were diminished, and intracellular ATP levels were also reduced in parallel with decreased caspase-9 and caspase-3 activity in Hyp chondrocytes. The competitive NPT inhibitor phosphonoformic acid and ATP synthesis inhibitor 3-bromopyruvate disturbed endochondral ossification with reduced apoptosis in vivo and suppressed apoptosis and mineralization in conjunction with reduced P(i) uptake and ATP synthesis in WT chondrocytes. Overexpression of Pit-1 in Hyp chondrocytes reversed P(i) uptake and ATP synthesis and restored apoptosis and mineralization. Our results suggest that cellular ATP synthesis consequent to P(i) uptake via Pit-1 plays an important role in chondrocyte apoptosis and mineralization, and that chondrogenesis is ATP-dependent.