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P2X7 receptors on osteoblasts couple to production of lysophosphatidic acid: a signaling axis promoting osteogenesis.

ABSTRACT: Nucleotides are released from cells in response to mechanical stimuli and signal in an autocrine/paracrine manner through cell surface P2 receptors. P2rx7-/- mice exhibit diminished appositional growth of long bones and impaired responses to mechanical loading. We find that calvarial sutures are wider in P2rx7-/- mice. Functional P2X7 receptors are expressed on osteoblasts in situ and in vitro. Activation of P2X7 receptors by exogenous nucleotides stimulates expression of osteoblast markers and enhances mineralization in cultures of rat calvarial cells. Moreover, osteogenesis is suppressed in calvarial cell cultures from P2rx7-/- mice compared with the wild type. P2X7 receptors couple to production of the potent lipid mediators lysophosphatidic acid (LPA) and prostaglandin E2. Either an LPA receptor antagonist or cyclooxygenase (COX) inhibitors abolish the stimulatory effects of P2X7 receptor activation on osteogenesis. We conclude that P2X7 receptors enhance osteoblast function through a cell-autonomous mechanism. Furthermore, a novel signaling axis links P2X7 receptors to production of LPA and COX metabolites, which in turn stimulate osteogenesis.

SUBMITTER: Panupinthu N

PROVIDER: S-EPMC2396816 | biostudies-other | 2008 Jun

REPOSITORIES: biostudies-other

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P2X7 receptors on osteoblasts couple to production of lysophosphatidic acid: a signaling axis promoting osteogenesis.

Panupinthu Nattapon N Rogers Joseph T JT Zhao Lin L Solano-Flores Luis Pastor LP Possmayer Fred F Sims Stephen M SM Dixon S Jeffrey SJ

The Journal of cell biology 20080601 5

Nucleotides are released from cells in response to mechanical stimuli and signal in an autocrine/paracrine manner through cell surface P2 receptors. P2rx7-/- mice exhibit diminished appositional growth of long bones and impaired responses to mechanical loading. We find that calvarial sutures are wider in P2rx7-/- mice. Functional P2X7 receptors are expressed on osteoblasts in situ and in vitro. Activation of P2X7 receptors by exogenous nucleotides stimulates expression of osteoblast markers and ...[more]

PMID: 18519738

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Project description:The P2 purinergic receptor family implicated in many physiological processes, including neurotransmission, mechanical adaptation and inflammation, consists of ATP-gated non-specific cation channels P2XRs and G-protein coupled receptors P2YRs. Different cells, including bone forming osteoblasts, express multiple P2 receptors; however, how P2X and P2Y receptors interact in generating cellular responses to various doses of [ATP] remains poorly understood. Using primary bone marrow and compact bone derived osteoblasts and BMP2-expressing C2C12 osteoblastic cells, we demonstrated conserved features in the P2-mediated Ca2+ responses to ATP, including a transition of Ca2+ response signatures from transient at low [ATP] to oscillatory at moderate [ATP], and back to transient at high [ATP], and a non-monotonic changes in the response magnitudes which exhibited two troughs at 10-4 and 10-2 M [ATP]. We identified P2Y2 and P2X7 receptors as predominantly contributing to these responses and constructed a mathematical model of P2Y2R-induced inositol trisphosphate (IP3) mediated Ca2+ release coupled to a Markov model of P2X7R dynamics to study this system. Model predictions were validated using parental and CRISPR/Cas9-generated P2Y2 and P2Y7 knockouts in osteoblastic C2C12-BMP cells. Activation of P2Y2 by progressively increasing [ATP] induced a transition from transient to oscillatory to transient Ca2+ responses due to the biphasic nature of IP3Rs and the interaction of SERCA pumps with IP3Rs. At high [ATP], activation of P2X7R modulated the response magnitudes through an interplay between the biphasic nature of IP3Rs and the desensitization kinetics of P2X7Rs. Moreover, we found that P2Y2 activity may alter the kinetics of P2X7 towards favouring naïve state activation. Finally, we demonstrated the functional consequences of lacking P2Y2 or P2X7 in osteoblast mechanotransduction. This study thus provides important insights into the biophysical mechanisms underlying ATP-dependent Ca2+ response signatures, which are important in mediating bone mechanoadaptation.

Project description:Instability and excessive use of the knee joint can cause osteoarthritis (OA). Reasonable exercise can enhance the stability of the knee joint and prevent and relieve the occurrence and development of OA. As a key switch for inflammation, P2X purinoceptor 7 (P2X7) has attracted much attention in studies of OA. Exercise can regulate P2X7 expression and activation. However, the role of P2X7 in exercise-based prevention and treatment of OA is unknown. We previously showed that moderate-intensity exercise can significantly alleviate OA symptoms. Accordingly, in this study, we evaluated the effects of exercise on P2X7 expression and activation in chondrocytes. Micro-computed tomography, hematoxylin, and eosin staining, Toluidine Blue O staining, immunohistochemistry, and terminal deoxynucleotidyl transferase dUTP nick-end labeling experiments showed that P2X7 expression was lower in the moderate-intensity exercise group than in the inflammation and low- and high-intensity exercise groups. Additionally, chondrocyte death, cartilage destruction, and the degree and severity of pyroptosis were significantly reduced, whereas autophagy levels were significantly increased in the moderate-intensity exercise group. Cell Counting Kit-8 assay, lactate dehydrogenase release, flow cytometry, enzyme-linked immunosorbent assay, cell fluorescence, western blot, reverse transcription-quantitative polymerase chain reaction, and transmission electron microscopy experiments showed that moderate activation of P2X7 promoted autophagy through the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway and promoted autolysosome targeting for degradation of the inflammasome component NLRP3, thereby inhibiting pyroptosis. Additionally, the use of AMPK and mTOR activators and inhibitors indicated that the AMPK-mTOR signaling pathway, as the downstream of P2X7, played a key role in delaying the occurrence and development of OA. We propose that moderate-intensity exercise promoted chondrocyte autophagy through the P2X7/AMPK/mTOR signal axis to alleviate pyroptosis. Our findings provide novel insights into the positive and preventative effects of exercise on OA.

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P2X7 receptors on osteoblasts couple to production of lysophosphatidic acid: a signaling axis promoting osteogenesis.

Publications

P2X7 receptors on osteoblasts couple to production of lysophosphatidic acid: a signaling axis promoting osteogenesis.

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