Expression, signaling, and function of P2X7 receptors in bone.

Nucleotides released from cells in response to mechanical stimulation or injury may serve as paracrine regulators of bone cell function. Extracellular nucleotides bind to multiple subtypes of P2 receptors on osteoblasts (the cells responsible for bone formation) and osteoclasts (cells with the unique ability to resorb mineralized tissues). Both cell lineages express the P2X7 receptor subtype. The skeletal phenotype of mice with targeted disruption of P2rx7 points to interesting roles for this receptor in the regulation of bone formation and resorption, as well as the response of the skeleton to mechanical stimulation. This paper reviews recent work on the expression of P2X7 receptors in bone, their associated signal transduction mechanisms and roles in regulating bone formation and resorption. Areas for future research in this field are also discussed.

Extracellular acidification enhances osteoclast survival through an NFAT-independent, protein kinase C-dependent pathway.

Systemic acidosis has detrimental effects on the skeleton and local acidosis is associated with bone destruction in inflammatory and neoplastic diseases. However, the mechanisms by which acidosis enhances osteoclastic bone resorption are poorly understood. Our aim was to examine the effects of acid on osteoclast survival and the involvement of cytosolic Ca(2+) in mediating these effects. Osteoclasts were isolated from long bones of newborn rats, and multinucleated osteoclast-like cells were generated from RAW 264.7 cells. Cytosolic free Ca(2+) concentration ([Ca(2+)](i)) was monitored using fura-2. Survival of rat osteoclasts over a period of 18 h was significantly enhanced by acidification of the medium from 40+/-10% at pH 7.6 to 83+/-4% at pH 7.0. Consistent with its effects on survival, acidosis suppressed osteoclast apoptosis at 6 h. We examined the possible involvement of the proton-sensing receptor ovarian cancer G protein-coupled receptor 1 (OGR1) in mediating the effects of acid. Acid-induced rise of [Ca(2+)](i) was inhibited by the OGR1 antagonist Cu(2+) and was suppressed in osteoclast-like cells in which OGR1 transcripts were depleted using RNA interference. These findings support an essential role for OGR1 in acid-induced Ca(2+) signaling in osteoclasts. Addition of Cu(2+) or chelation of cytosolic Ca(2+) with BAPTA abolished the ability of acidification to enhance osteoclast survival. Inhibition of NFAT activation with the cell-permeable peptide 11R-VIVIT did not alter the ability of acid to promote survival; however, it suppressed the increase in survival induced by RANKL. In contrast, inhibition of protein kinase C (PKC) blocked the effect of acid on osteoclast survival. Thus, this study reveals that extracellular acidification enhances osteoclast survival through an NFAT-independent, PKC-dependent pathway. Increased osteoclast survival may contribute to bone loss in systemic and local acidosis.

Activation of transcription factors by extracellular nucleotides in immune and related cell types.

Extracellular nucleotides, acting through P2 receptors, can regulate gene expression via intracellular signaling pathways that control the activity of transcription factors. Relatively little is known about the activation of transcription factors by nucleotides in immune cells. The NF-kappaB family of transcription factors is critical for many immune and inflammatory responses. Nucleotides released from damaged or stressed cells can act alone through certain P2 receptors to alter NF-kappaB activity or they can enhance responses induced by pathogen-associated molecules such as LPS. Nucleotides have also been shown to regulate the activity of other transcription factors (AP-1, NFAT, CREB and STAT) in immune and related cell types. Here, we provide an overview of transcription factors shown to be activated by nucleotides in immune cells, and describe what is known about their mechanisms of activation and potential functions. Furthermore, we propose areas for future work in this new and expanding field.

P2Y6 nucleotide receptors activate NF-kappaB and increase survival of osteoclasts.

Nucleotides, released from cells during inflammation and by mechanical stimulation, act through the P2 family of nucleotide receptors. Previous studies have demonstrated the expression of P2Y1 and P2Y2 receptors in osteoclasts. The aim of this study was to determine whether osteoclast P2Y receptors signal through NF-kappaB, a key transcription factor regulating osteoclastogenesis. Immunofluorescence was used to detect the p65 subunit of NF-kappaB, which upon activation translocates from the cytosol to nuclei. Low levels of NF-kappaB activation were observed in untreated rabbit osteoclasts and in those exposed to 2-methylthio ADP (P2Y1 agonist) or ATP or UTP (P2Y2 agonists). In contrast, UDP or INS48823 (P2Y6 agonists) induced a significant increase in the number of cells exhibiting NF-kappaB activation, a process sensitive to the proteasome inhibitor lactacystin. In osteoclasts purified by micromanipulation, reverse transcription-PCR revealed the presence of P2Y1, P2Y2, and P2Y6 receptor transcripts, and application of agonists for these receptors induced the transient rise of cytosolic calcium. Treatment of rat osteoclasts with UDP or INS48823, but not 2-methylthio ADP or UTP, increased osteoclast survival. Osteoprotegerin (a decoy receptor for RANK ligand) did not significantly alter the effects of UDP on NF-kappaB localization or osteoclast survival, consistent with a direct action. Moreover, SN50 (cell-permeable peptide inhibitor of NF-kappaB) suppressed the enhancement of cell survival induced by UDP and INS48823. Our findings demonstrate the presence of functional P2Y6 receptors in osteoclasts. Thus, nucleotides, following their release at sites of inflammation and mechanical stimulation, can act through P2Y6 receptors to initiate NF-kappaB signaling and enhance osteoclast survival.

Extracellular nucleotides act through P2X7 receptors to activate NF-kappaB in osteoclasts.

UNLABELLED: Nucleotides, released in response to mechanical and other stimuli, act on P2 receptors in osteoclasts and other cell types. In vitro studies of osteoclasts from rabbits and P2X7 receptor-deficient mice revealed that P2X7 receptors couple to activation of the key transcription factor NF-kappaB. INTRODUCTION: Osteoclasts express functional P2X4 and P2X7 receptors, which are ATP-gated cation channels. Knockout (KO) of the P2X7 receptor has revealed its role in regulating bone formation and resorption, but the underlying signals are not known. The transcription factor NF-kappaB plays a key role in the response of osteoclasts to RANKL and other cytokines. The aim of this study was to examine whether P2X receptors on osteoclasts signal through NF-kappaB. MATERIALS AND METHODS: Osteoclasts were isolated from neonatal rabbits or wildtype (WT) and P2X7 receptor KO mice. Immunofluorescence was used to detect the p65 subunit of NF-kappaB, which, on activation, translocates from the cytosol to the nuclei. The concentration of cytosolic free Ca2+ ([Ca2+]i) was monitored in single osteoclasts loaded with fura-2. RESULTS: In control samples, few rabbit osteoclasts demonstrated nuclear localization of NF-kappaB. Benzoyl-benzoyl-ATP (BzATP, a P2X7 agonist, 300 microM) induced nuclear translocation of NF-kappaB after 3 h in approximately 45% of rabbit osteoclasts. In contrast, a low concentration of ATP (10 microM, sufficient to activate P2X4 and P2Y2, but not P2X7 receptors) did not induce nuclear translocation of NF-kappaB. Because BzATP activates multiple P2 receptors, we examined responses of osteoclasts derived from WT and P2X7 receptor KO mice. Treatment with BzATP for 30 minutes increased nuclear localization of NF-kappaB in osteoclasts from WT but not KO mice, showing involvement of P2X7 receptors. Both ATP (10 microM) and BzATP (300 microM) caused transient elevation of [Ca2+]i, indicating that rise of calcium alone is not sufficient to activate NF-kappaB. Pretreatment of rabbit osteoclasts with osteoprotegerin inhibited translocation of NF-kappaB induced by RANKL but not by BzATP, establishing that the effects of BzATP are independent of RANKL signaling. CONCLUSION: These findings show that P2X7 nucleotide receptors couple to activation of NF-kappaB in osteoclasts. Thus, nucleotides, released at sites of inflammation or in response to mechanical stimuli, may act through NF-kappaB to regulate osteoclast formation and activity.

Differential effects of glucose on dehydroascorbic acid transport and intracellular ascorbate accumulation in astrocytes and skeletal myocytes.

Skeletal muscle and brain are major sites of glucose transport and ascorbate (vitamin C) storage. Ascorbate is oxidized to dehydroascorbic acid (DHAA) when used as an enzyme cofactor or free radical scavenger. We evaluated the hypothesis that glucose regulates DHAA uptake and reduction to ascorbate (i.e., recycling) by skeletal muscle cells and cerebral astrocytes. DHAA uptake was inhibited partially by glucose added simultaneously with DHAA. Comparison of wild type L6 skeletal muscle cells with an L6-derived cell line (D23) deficient in facilitative hexose transporter isoform 3 (GLUT3), indicated that both GLUT3 and facilitative hexose transporter isoform 1 (GLUT1) mediate DHAA uptake. Preincubation of muscle cells with glucose inhibited the rates of glucose and DHAA uptake, and decreased the intracellular concentration of ascorbate derived from recycling of DHAA. In contrast, glucose preincubation did not depress GLUT1 protein and activity levels or DHAA recycling in astrocytes. These results establish that glucose downregulates subsequent recycling of DHAA by skeletal muscle cells but not astrocytes.

Accumulation of intracellular ascorbate from dehydroascorbic acid by astrocytes is decreased after oxidative stress and restored by propofol.

Primary rat astrocyte cultures absorbed dehydroascorbic acid from the medium and reduced it to intracellular ascorbate. Uptake of dehydroascorbic acid (5-200 microM) was inhibited only partially by glucose (10 mM). The remaining glucose-insensitive component of dehydroascorbic acid uptake was inhibited reversibly by sulfinpyrazone (IC(50) = 80 microM). Dehydroascorbic acid uptake was not mediated by Na(+)-ascorbate cotransporters or volume-sensitive anion channels because it was neither Na(+)-dependent nor blocked by the channel antagonist, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Oxidative stress, induced in astrocytes by the lipophilic radical generator tert-butyl hydroperoxide, decreased intracellular glutathione concentration and inhibited accumulation of intracellular ascorbate from dehydroascorbic acid. Subsequent administration of either the native antioxidant alpha-tocopherol (200 microM) or anesthetic concentrations of the antioxidant sedative propofol (1-8 microM, administered 30 min after tert-butyl hydroperoxide), did not change glutathione concentration but restored the ability of astrocytes to accumulate intracellular ascorbate from dehydroascorbic acid. These results are consistent with a novel mechanism of astrocytic ascorbate accumulation that is inhibited by lipophilic radicals and protected by lipophilic antioxidants such as propofol.

Sepsis inhibits reduction of dehydroascorbic acid and accumulation of ascorbate in astroglial cultures: intracellular ascorbate depletion increases nitric oxide synthase induction and glutamate uptake inhibition.

Sepsis is associated with oxidative stress and impaired glutamatergic transmission in brain. We investigated whether sepsis impairs accumulation of the antioxidant, ascorbate, and uptake of glutamate by astrocytes. Bacterial endotoxin (Escherichia coli lipopolysaccharide, LPS) and the inflammatory cytokine, interferon-gamma (IFNgamma), were applied to primary astrocyte cultures to model sepsis. In the absence of ascorbate, the combination of LPS and IFNgamma (LPS + IFNgammay) up-regulated inducible nitric oxide synthase (iNOS) and decreased the initial rate of glutamate uptake by 50% within 24 h. Cell viability and facilitated glucose transport activity were not affected at 24 h. Pre-treatment with ascorbate-2-O-phosphate increased intracellular ascorbate concentration and attenuated the induction of iNOS and inhibition of glutamate uptake caused by LPS + IFNgamma. Subsequent experiments examined the mechanisms by which cells accumulate ascorbate. LPS + IFNy decreased slightly the initial rate of uptake of ascorbate and inhibited markedly the rate with which intracellular dehydroascorbic acid (DHAA) was reduced to ascorbate. We conclude that septic insult impairs astrocytic clearance of DHAA from the extracellular fluid and decreases intracellular ascorbate concentration. Furthermore, sepsis induces iNOS and inhibits glutamate uptake by astrocytes through mechanisms that can be modulated by intracellular ascorbate. These results indicate treatments that increase intracellular ascorbate concentration may be beneficial for patients at risk for neurologic complication in sepsis.