[Frontiers in Bioscience S3, 1038-1046, June 1, 2011]
Bone phenotypes of P2 receptor knockout mice
Isabel Orriss1, Susanne Syberg2, Ning Wang3, Bernard Robaye4, Alison Gartland3, Niklas Jorgensen2, Tim Arnett1, Jean-Marie Boeynaems4
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TABLE OF CONTENTS
1. ABSTRACT
The action of extracellular nucleotides is mediated by ionotropic P2X receptors and G-protein coupled P2Y receptors. The human genome contains 7 P2X and 8 P2Y receptor genes. Knockout mice strains are available for most of them. As their phenotypic analysis is progressing, bone abnormalities have been observed in an impressive number of these mice: distinct abnormalities in P2X7-/- mice, depending on the gene targeting construct and the genetic background, decreased bone mass in P2Y1-/- mice, increased bone mass in P2Y2-/- mice, decreased bone resorption in P2Y6-/- mice, decreased bone formation and bone resorption in P2Y13-/- mice. These findings demonstrate the unexpected importance of extracellular nucleotide signalling in the regulation of bone metabolism via multiple P2 receptors and distinct mechanisms involving both osteoblasts and osteoclasts.
2. INTRODUCTION
Nucleotides, such as ATP and UTP, are mainly intracellular, but they can be released into the extracellular fluids by various mechanisms. One of them is cell damage: both necrotic and apoptotic cells release ATP and other nucleotides that thus constitute "danger signals" (1,2). But they can also be released without cell lysis by specific mechanisms: exocytosis of secretory granules, vesicular transport and membrane channels, such as ABC transporters, pannexins and connexins (3,4). Exocytosis of nucleotides is typically observed during platelet aggregation and synaptic transmission. They are also released in response to various types of stress: hypoxia, pathogen invasion or mechanical stimulation (stretch, shear stress). Once in the extracellular fluid, nucleotides can activate two families of receptors: metabotropic P2Y receptors coupled to G proteins (5) and fast P2X ion channels (6).
In the human genome there are 7 genes encoding P2X receptor subunits (Table 1) and 8 genes encoding P2Y receptors, that can subdivided in 2 subgroups based on structural features and coupling to specific G proteins (Table 2). Knockout mice have been generated for all P2Y receptors except P2Y11, which is not present in murine genome. Phenotypes have also been described for the P2X receptor knockouts, except P2X5 and P2X6. The phenotypic analysis of these mice is revealing an increasing number of functions of these receptors. The most prominent results are listed below.
P2X1-/- mice display male infertility due to reduced vas deferens contraction (7), reduced neurogenic vasoconstriction (8) and reduced autoregulation of renal blood flow (9). P2X2-/- mice have a reduced ventilatory response to hypoxia (10). P2X3-/- mice display urinary bladder hyporeflexia (11) and reduced pain response to formalin (12). A profound deficit of taste responses was found in the taste nerves of P2X2/P2X3 double knockouts, while loss of either P2X2 or P2X3 resulted only in a moderate change (13). P2X4-/- mice are characterized by an impaired blood flow-dependent control of vascular tone (14) and a blunted tactile allodynia following spinal nerve injury (15). The study of P2X7-/- mice revealed a crucial role of this receptor in the function of the inflammasome and the release of IL-1b (16,17).
Both P2Y1-/- and P2Y12-/- mice exhibit a defect in platelet aggregation by ADP and an increased resistance to thromboembolism (18,19). Furthermore atherosclerotic lesions were less in P2Y1-/-/apoE-/- mice, but this protection was independent from platelets and likely involve endothelial cells (20). The chemotactic attraction of both neutrophils (21) and monocytes (2) is decreased in P2Y2-/- mice, while microglia migration and process extension to sites of brain damage is impaired in P2Y12-/- mice (22). The phenotypic analysis of other P2Y knockouts is still at an early phase. The stimulatory effect of ATP on Cl- and water secretion by intestinal epithelial cells was abolished in P2Y4-deficient mice (23). The responses to UDP of vascular endothelial and smooth muscle cells as well as macrophages were impaired in P2Y6-/- mice (24). A reduction in the hepatic clearance of HDL was observed in P2Y13-/- mice (25). The contractile effect of UDP-glucose on the forestomach muscle was decreased in P2Y14-/- mice (26).
This overview of P2 knockout mice phenotypes shows thus an involvement of these receptors in a broad range of functions and cell types : P2Y1, P2Y6 and P2X4 in vascular endothelial cells, P2X1 and P2Y6 in vascular smooth muscle cells, P2Y1 and P2Y12 in platelets, P2X7, P2Y2 and P2Y6 in immune cells, P2X4 and P2Y12 in microglia, P2X2 and P2X3 in neurons and P2Y13 in hepatocytes. In an unexpected way, recent studies of knockout mice described below show that PX7, P2Y1, P2Y2, P2Y6 and P2Y13 play all a role in bone via actions on osteoblasts or osteoclasts or both.
3. BONE PHENOTYPES OF P2X7-/- MICE
Within the last ten years our understanding of the role of the P2X7 receptor in the regulation of bone turnover and in mechanotransduction has progressed significantly. This has to a large extent resulted from the establishment of mouse models with a targeted disruption of the gene encoding the P2X7 receptor. Overall P2X7-/- mice are viable and fertile, and cannot be distinguished from wild type littermates by examination alone. Nevertheless, several defects have been described, including impaired IL-1b production in macrophages stimulated with ATP (27), an attenuated inflammatory response to induced arthritis (28) and enhanced susceptibility to multiple sclerosis (29).
Two P2X7-/- mouse models have been created and both display a bone phenotype. Overall, the P2X7-/- mice produced by the Pfizer group exhibit a reduced bone mass (30). In this study, young (2 months) and old (9 months) mice of both genders were examined. P2X7 knockout animals displayed reduced total and cortical bone mineral content (BMC) and decreased periosteal circumference of the femur (30). These differences tended to be larger in older animals. Interestingly, the effect of P2X7 receptor ablation was more pronounced in male mice than in females. Histomorphometric analyses of the bone tissue demonstrated reduced parameters of bone formation including mineralizing surface (MS/BS), mineral appositional rate (MAR) and bone formation rate (BFR/BS) as well as increased parameters of bone resorption such as osteoclast number per mm bone surface (OCN/BS) and percent osteoclast surface (OCS/BS), supporting the low bone mass phenotype. Moreover calvarial sutures were wider in P2X7-/- mice (31) This phenotype of decreased periosteal bone formation combined with increased trabecular bone resorption resembles the effects of disuse on the skeleton and mimics estrogen deficiency, suggesting that P2X7 might regulate mechanotransduction. This has been confirmed in later studies (see below).
Another murine model, with ablation of the P2X7 gene, has been produced by the group of Gary Buell at GSK (32). As with the Pfizer model, these P2X7-/- mice also displayed a bone phenotype (33). In this study, young (6 weeks) and old (11 months) mice were examined, and no differences between knockout and wild type mice could be detected when looking at bone mineral density (BMD) at either age. Also, no differences were found in trabecular bone volume. However, in contrast to the bone phenotype described for the Pfizer model, Gartland et al showed that cortical thickness was increased in the P2X7-/- animals compared to the wild types (33). Thus, the bone phenotypes described for the two models are conflicting. This contradiction could be related to the method of gene targeting or to the different genetic background of the inbred strains used to generate the knockout mice.
The P2X7-/- construct of the GSK P2X7-/- mice was created by insertion of a LacZ gene into the beginning of exon 1 of the P2X7 gene (32), creating a frameshift mutation. Pfizer's P2X7-/- strain was generated by deletion of the nucleotides 1527-1607 in exon 13 and insertion of a neomycin cassette in the 5' to 3' direction. In the generation of both strains, genomic DNA containing the P2X7 gene was isolated from a genomic library drawn from 129/sv mice (32,34). The Pfizer P2X7-/- mice were generated on 129/Ola x B6 x DBA/2 genetic background, and maintained on the B6 x DBA/2 background and later on the B6 background (27,30). The GSK P2X7-/- mice were maintained on B6 background, but originate from a B6/129 hybrid (32,33). However, the most convincing explanation for the difference between the two models came in 2009, when Nicke et al demonstrated the expression of a functional P2X7 splice variant in some tissues from the GSK P2X7-/- mice (35). Staining by P2X7 antibodies is detectable in brain (36-38) and in lymphocytes (34), at levels identical to wild type littermates. The staining pattern in neurons and lymphocytes is due to the expression of a P2X7-like protein, absent from other tissues with less abundant alternative splicing. This unexpected staining pattern could be due to the expression of the splice variant P2X7-k which escapes gene inactivation in the GSK P2X7-/- mice (35). Although the Pfizer P2X7-/- lymphocytes also express a P2X7-like protein, it is non-functional (34). The P2X7-k splice variant contains an alternative intracellular N terminus and first transmembrane domain, and appears to have higher sensitivity to the P2X7 agonist BzATP when expressed in a HEK293 in vitro expression system. P2X7-k is highly expressed in the spleen and has varying tissue specific expression. Preliminary results indicate that the splice variant is expressed at the mRNA but not protein level in osteoblasts (39), and its expression in bone and other bone cells remains unknown, so that results from studies in the GSK P2X7-/- model should be interpreted with caution. The existence of the splice variant P2X7-k indicates that the role of the P2X7 receptor could have been misinterpreted, especially in tissues expressing the splice variant. For instance, the human P2X7 receptor has been shown to participate in the fusion of multinucleated cells (8), but the murine P2X7 receptor has been reported to be dispensable in formation of murine osteoclasts in the GSK P2X7-/- model, since osteoclasts have been found in these mice (33). If the splice variant is expressed and functional in murine osteoclasts, they would have a high-activity P2X7 receptor instead of a non-functional receptor.
A further complexity in the analysis of the bone phenotype of P2X7-/- mice was recently revealed. It is known that several strains of mice including C57Bl/6 have a naturally occurring P451L mutation in the P2X7 gene. This mutation reduces the sensitivity of the P2X7 receptor to nucleotides, which may result in underestimation of the severity of the phenotype exhibited by knockout mice (30).Therefore P2X7-/- mice were recently generated on a BALB/cJ background. Preliminary results indicate that these mice have an increase in BMD compared to wild types, which is related to decreased bone resorption (39).
Physical activity and thus mechanical stimulation is the most powerful anabolic stimulus to bone. As mentioned above, the structural changes in the Pfizer P2X7-null animals resemble the changes observed in immobilized bone, suggesting that the P2X7 receptor might be involved in the mechanotransductory cascade. In a highly interesting study by Li et al (40), the effect of in vivo mechanical loading on the ulnar bone in P2X7-/- and wild type mice was examined. The right forearm of the mouse was loaded at 120 cycles per day for 3 consecutive days and the periosteal bone formation was determined. Interestingly, a marked reduction (73%) in the interlabeling distance (measure of bone formation) was observed in the P2X7-/- animals compared to the wild types (40). Thus, the sensitivity to mechanical loading was significantly reduced indicating an important role of the P2X7 receptor in bone mechanotransduction. In vitro, it was shown that both P2X7 receptor-mediated pore formation and prostaglandin release in response to fluid shear stress were absent in osteoblasts from P2X7-null animals, supporting the role of P2X7 receptors in mechanotransduction in bone cells. A recent study has also investigated the role of the P2X7 receptor in dental mechanotransduction (41). It was found that root resorption was increased and bone formation decreased in P2X7-/- mice, confirming the role of the P2X7 receptor in the mechanotransductory process in bone and related tissues. Fracture repair is another process where loading of the bone is important. Li et al recently examined the fracture healing process in adult P2X7-null mice subjected to osteotomy (42). No differences could be detected in callus formation using standard imaging techniques. Furthermore, mechanical testing at the fracture site showed no statistically significant differences between P2X7-null animals and wild type animals. However, a tendency towards a reduced strength at the fracture site was seen and the lack of statistical significance can be explained by the low power of the study. Histomorphometric analyses also revealed that mineralizing surface and bone formation were significantly decreased in the P2X7-/- animals. Thus, callus remodeling was significantly delayed, indicating a role for the P2X7 receptor in callus formation and fracture repair, probably through effects in mechanotransduction.
4. BONE PHENOTYPE OF P2Y1-/- MICE
Expression of the P2Y1 receptor has been reported on both osteoblasts (43-45) and osteoclasts (43,46). In osteoblasts, activation of the P2Y1 receptor has been reported to modulate cellular responses to systemic factors, such as parathyroid hormone, by increasing c-fos expression (44,47). Stimulation of the P2Y1 receptor by ADP or ATP stimulates the formation and resorptive activity of rodent osteoclasts cultured in vitro (48).
The effect of P2Y1-receptor deletion on skeletal structure was recently investigated using dual energy x-ray absorbtiometry (DEXA) scanning and micro computed tomography (�CT). DEXA analysis performed on whole animals demonstrated a 5% decrease in total BMD and a 7% decrease in total BMC in P2Y1-/- mice (Figure 1). The long bones and spines of these animals exhibited 4-14% decreases in BMC and BMD; weight, lean tissue and fat content were unchanged. More detailed investigation by �CT showed that femoral and tibial trabecular bone volume (BV/TV) were decreased 35% and 23%, respectively, in the P2Y1-/- mice. Trabecular number was also reduced (up to 32%) in the long bones of knockout animals, whereas trabecular thickness was unchanged. The structural model index (SMI) parameter gives an indication of trabecular structure; an increased SMI in the femur and tibia of P2Y1-/- mice indicated that the trabeculae were more rod-like in shape. Cortical bone parameters were unaffected in the knockout animals (49). At present there are no studies detailing changes in the differentiation, survival and function of osteoblasts and osteoclasts derived from P2Y1-/- mice.
5. BONE PHENOTYPE OF P2Y2-/- MICE
The P2Y2 receptor is expressed by both osteoblasts (42,44) and osteoclasts (42,45). Expression of the P2Y2 receptor in osteoblasts increases strongly as differentiation proceeds (45) and this receptor has been implicated in the potent inhibition of bone mineralisation by ATP and UTP observed in osteoblast cultures (50,51). The P2Y2 receptor may also have a role in the propagation of intercellular Ca2+ waves (51) and the stimulation of Erg1 and Runx2 expression in osteoblasts (53,54). Effects of P2Y2 receptor activation on osteoclast formation and resorptive activity have not been reported.
Changes in the bone phenotype of P2Y2-/- mice were first reported in 2007 when a study using DEXA analysis demonstrated increased BMC in the femora (9%) and tibiae/fibulae (17%) of knockout animals (51). Receptor deletion had no effect on weight, lean tissue and fat content (51). A subsequent �CT study of 2-month old P2Y2-/- mice demonstrated large increases in the BV/TV of the long bones (43% and 21% in the femur and tibia, respectively) (Figure 2) (49). Trabecular thickness and trabecular number were also increased in the femora of P2Y2-deficient animals, with decreased femoral SMI, indicating the presence of more plate-like trabeculae. Increased cortical bone volume (up to 25%) was additionally observed in the long bones of P2Y2-/- animals (49). There are no published studies to date on the differentiation, survival and function of osteoblasts and osteoclasts derived from P2Y2-/- mice.
6. PRELIMINARY RESULTS IN P2Y6-/- MICE
The P2Y6 receptor is widely expressed by both osteoblasts (45) and osteoclasts (55). The P2Y6 receptor has been suggested to play a role in osteoclasts survival since receptor activation prevented their spontaneous apoptosis in culture (55). Recently, preliminary studies have reported an altered bone phenotype in the P2Y6 receptor knockout (56). DEXA analysis showed increased bone content in the long bones and spine of the P2Y6R-/- mice. �CT analysis demonstrated increased cortical thickness (up to 25%) and cortical bone volume (up to 18%) in the long bones of P2Y6-/- mice. Additionally, the length of the long bones was increased up to 10% in the P2Y6-/- mice. Cortical bone volume was also increased in the L3 lumbar vertebrae. In both the long bones and spine, no changes in the trabecular bone were observed. Modest reductions in osteoclast formation in M-CSF and RANKL-treated marrow cultures from P2Y6-/- animals were observed, together with striking impairment of resorptive function in the mature cells. Calvarial osteoblasts derived from P2Y6R-/- mice displayed reduced proliferation and mineralisation.
7. PRELIMINARY RESULTS IN P2Y13-/- MICE
The recent availability of the P2Y13-/- mice promises to provide researchers with an invaluable tool to further elucidate the role of this receptor in basic physiology. To that end, initial studies of the P2Y13-/- mice have shown that when maintained under a normal diet these mice had normal weight gain, development, fertility, behaviour, haematological and plasma parameters - although hepatic cholesterol metabolism was impaired (25). The P2Y13-/- mice exhibited normal immune response, and dendritic cells, which have previously been shown to express high levels of P2Y13 receptor mRNA, were not affected by the P2Y13 receptor gene deletion in terms of antigen presentation or lymphocyte activation (25).
In view of previous data suggesting that the cells responsible for maintaining skeletal health, especially osteoclasts (bone resorbing) and osteoblasts (bone building), would express the P2Y13 receptor (57-59), we have made a detailed investigation of the skeletal phenotype of the P2Y13-/- mice (60). Analysis of the bone micro-architecture of the P2Y13-/- mice was performed using a Skyscan 1172 MicroCT machine at the scan resolution of 4.3μm. Results showed that P2Y13-/- mice had significantly lower trabecular bone volume/tissue volume, significantly lower trabecular number and an increased cortical bone thickness. Histology of the tibia revealed a significant decrease in both osteoblast and osteoclast numbers on the endocortical surface of the P2Y13-/- mice. We also performed in vitro cultures to assess the function of the bone cells. Primary osteoblasts derived from the calvaria of neonatal P2Y13-/- mice showed reduced alkaline phosphatase activity, whilst there was also a reduction in the number and function of multinucleated osteoclasts derived from the mononuclear hematopoietic cell population of 10 weeks old female P2Y13-/- mice. The molecular basis for the observed effect of P2Y13 receptor deletion in osteoblasts and osteoclasts is currently under investigation. In summary, deletion of the P2Y13 receptor in mice leads to an abnormal bone phenotype, including less trabecular bone but thicker cortical bone. This is a consequence of reduced rates of bone turnover caused by decreased number and function of both osteoblasts and osteoclasts.
8. SUMMARY AND PERSPECTIVE
Unexpectedly all strains of P2-deficient mice studied so far have displayed a bone phenotype. No information is available on mice heterozygous for these deletions. Interestingly each of these phenotypes is unique. These studies emphasize thus the importance of nucleotide signalling in the control of bone metabolism and the unique role of each receptor.
Studies of the bone phenotype of P2X7-/- mice have provided conflicting results between two distinct models generated at Pfizer and GSK respectively. Mice generated at Pfizer exhibited a decreased bone mass due to decreased bone formation and increased bone resorption (30). The GSK mice had a mild phenotype with an increased cortical thickness (33). This apparent contradiction can probably be explained by the expression of a functional splice variant of P2X7 in the GSK mice (35). But these mice were generated in the B6 background which harbors a natural mutation of the P2X7 gene. Preliminary results show an increased bone mass and decreased resorption in P2X7-/- mice in the BALB/cJ background (39).
Studies have shown that ATP and ADP, acting via the P2Y1 receptor, increase osteoclast formation and activity in vitro (48). Given this observation, the reduced trabecular bone observed in the long bones of P2Y1-/- mice was somewhat unexpected. Thus, the effects of receptor deletion may predominantly affect osteoblasts or, given the defects in platelet aggregation and blood clotting seen in P2Y1-/- mice (18), other systemic perturbations may indirectly influence bone cell function. Indeed there is growing evidence that hematopoietic cells such as megakaryocytes, which are known to express P2Y1 (61), can influence osteoblasts and osteoclasts (62).
The P2Y2 receptor is thought to mediate the inhibitory effects of ATP and UTP on bone mineralisation in vitro (50,51). Consistent with these observations, trabecular and cortical bone mass was increased in the P2Y2-/- mice; thus, deletion of the P2Y2 receptor could potentially limit the negative actions of extracellular nucleotides on bone.
Preliminary results indicate that P2Y6-/- mice have an increased bone mass that can be explained by the decreased resorptive function of osteoclasts (56). Bone resorption was also decreased in P2Y13-/- mice, but this was explained by a decrease in the osteoclast number rather than osteoclast function (60). In these mice, decreased bone resorption was associated with decreased bone formation : this decreased bone turnover resulted in opposite effects on trabecular (decreased volume) and cortical (increased thickness) bone. A P2Y13 antagonist might thus be beneficial in osteoporosis which is characterized by an increased bone turnover.
These studies of knockout mice demonstrate thus that not less than five P2 receptors play a role in bone formation and/or resorption. This confirms the prediction that nucleotides acting via both P2X and P2Y receptors could play important roles in the regulation of bone metabolism (63,64). These important roles can be related to the fact that mechanical stimulation, which is an important factor in bone remodelling, stimulates the release of nucleotides.
9. ACKNOWLEDGEMENTS
The work was kindly supported by the European Commission under the 7th Framework Programme (proposal #202231) performed as a collaborative project among the members of the ATPBone Consortium (Copenhagen University, University College London, University of Maastricht, University of Ferrara, University of Liverpool, University of Sheffield, and Université Libre de Bruxelles), and is a sub study under the main study "Fighting osteoporosis by blocking nucleotides: purinergic signalling in bone formation and homeostasis".
Part of this work was performed by the Bone Analysis Lab at the Mellanby Centre for Bone Research.
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Key Words : Nucleotides, P2 receptors, Gene Targeting, Bone, Review
Send correspondence to: Jean-Marie Boeynaems, 808 Route de Lennik 1070 Brussels Belgium, Tel : 32-2-5553922, Fax: 32-2-5556655 , E-mail:jmboeyna@ulb.ac.be