Purinergic signaling is required for fluid shear stress-induced NF-κB translocation in osteoblasts

Genetos, Damian C.; Karin, Norman J.; Geist, Derik J; Donahue, Henry J.; Duncan, Randall L.

doi:10.1016/j.yexcr.2011.01.007

Cited by 21 publications

(18 citation statements)

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“…ATP is an important autocrine/paracrine signaling molecule released by osteoblasts and osteocytes in response to mechanical stimulation in vitro (15). Several studies have reported that activation of the purinergic receptors by ATP is required for NF-B translocation, ERK1/2 activation, Cox-2 expression, and PGE 2 release, all major anabolic factors for bone formation (13,14). Although translocation and redistribution of CAV1 have been reported with mechanical stretch in type II epithelial cells (48), our novel findings indicate that CAV1 is redistributed from the lipid microdomain fractions into the denser cytosolic fractions when MC3T3-E1 cells were stimulated with ATP.…”

Section: Discussionmentioning

confidence: 99%

Caveolin-1 regulates P2X₇receptor signaling in osteoblasts

Gangadharan

Nohe

Caplan

et al. 2015

American Journal of Physiology-Cell Physiology

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The synthesis of new bone in response to a novel applied mechanical load requires a complex series of cellular signaling events in osteoblasts and osteocytes. The activation of the purinergic receptor P2X7R is central to this mechanotransduction signaling cascade. Recently, P2X7R have been found to be associated with caveolae, a subset of lipid microdomains found in several cell types. Deletion of caveolin-1 (CAV1), the primary protein constituent of caveolae in osteoblasts, results in increased bone mass, leading us to hypothesize that the P2X7R is scaffolded to caveolae in osteoblasts. Thus, upon activation of the P2X7R, we postulate that caveolae are endocytosed, thereby modulating the downstream signal. Sucrose gradient fractionation of MC3T3-E1 preosteoblasts showed that CAV1 was translocated to the denser cytosolic fractions upon stimulation with ATP. Both ATP and the more specific P2X7R agonist 2=(3=)-O-(4-benzoylbenzoyl)ATP (BzATP) induced endocytosis of CAV1, which was inhibited when MC3T3-E1 cells were pretreated with the specific P2X7R antagonist A-839977. The P2X7R cofractionated with CAV1, but, using superresolution structured illumination microscopy, we found only a subpopulation of P2X7R in these lipid microdomains on the membrane of MC3T3-E1 cells. Suppression of CAV1 enhanced the intracellular Ca 2ϩ response to BzATP, suggesting that caveolae regulate P2X7R signaling. This proposed mechanism is supported by increased mineralization in CAV1 knockdown MC3T3-E1 cells treated with BzATP. These data suggest that caveolae regulate P2X7R signaling upon activation by undergoing endocytosis and potentially carrying with it other signaling proteins, hence controlling the spatiotemporal signaling of P2X7R in osteoblasts.caveolin-1; purinergic signaling; P2X 7R; endocytosis; osteoblasts PURINERGIC SIGNALING PLAYS a critical role in regulation of skeletal mechanotransduction, an intricate process through which a physical stimulus is converted into a biochemical response (7, 23). We and others have shown that ATP and other nucleotides are released in response to fluid shear stress in vitro and activate purinergic (P2) receptors on the membrane of the osteoblasts (13,20,29,38). Although many subtypes of P2 receptors are present in osteoblasts (4), knockout of the purinergic receptor P2X 7 R, a ligand-gated ion channel, resulted in an osteopenic phenotype (22), suggesting that this receptor is important in skeletal maintenance. Further in vivo loading experiments show that load-induced bone formation is significantly abrogated when the P2X 7 R is genetically deleted, demonstrating the importance of this receptor in mechanotransduction (23).Several characteristics distinguish the P2X 7 R from the other P2X receptors. The P2X 7 R is 10 -30 times more specific for 2=(3=)-O-(4-benzoylbenzoyl)ATP (BzATP) than ATP (28), and P2X 7 R activation by BzATP induces dynamic membrane blebbing (39). P2X 7 R activation has been associated with opening of a large pore on the plasma membrane that allows entry of up to 900-Da mol...

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Section: Discussionmentioning

confidence: 99%

Caveolin-1 regulates P2X₇receptor signaling in osteoblasts

Gangadharan

Nohe

Caplan

et al. 2015

American Journal of Physiology-Cell Physiology

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“…In addition, Ca 2C influx following P2X7R activation causes sustained proton efflux dependent on glucose and phosphatidylinositol 3-kinase activity (Grol et al 2012) and activation of PKC to mediate phosphorylation of ERK1/2 (Liu et al 2008, Okumura et al 2008, suggestive of more cross talk in osteoblast-like cells. A fluid sheer stress-induced NF-kB nuclear localisation independent of both ERK1/2 LPA signalling has also been demonstrated through P2X7R in osteoblasts (Genetos et al 2011). It seems likely that while the basal/transient activation of P2X7R is osteogenic, sustained stimulation could inhibit the function and activity of these bone-forming cells.…”

Section: Mature Cellsmentioning

confidence: 93%

“…In addition, blockade or absence of P2X7R has been shown to inhibit propagation of intercellular calcium signalling between osteoblasts and osteoclasts in human bone marrow-derived cells (Jorgensen et al 2002), significantly reduce ERK phosphorylation in response to fluid shear stress in mouse primary osteoblasts (Liu et al 2008, Okumura et al 2008) and prevented fluid shear-stress-induced IkBa degradation and nuclear accumulation of nuclear factor kappa B (NFkB) in MC3T3-E1 osteoblasts (Genetos et al 2011).…”

Section: Mature Cellsmentioning

confidence: 99%

“…P2X7R expression has been consistently reported in human (Panupinthu et al 2007, Grol et al 2012, production of lipid mediators in mouse calvarial osteoblasts (Panupinthu et al 2008), substantial induction of transcription factor activating protein-1 (AP-1) in mouse MC3T3-E1 cells (Gavala et al 2010) and reduced bone mineralisation and alkaline phosphatase activity in primary rat osteoblasts . In addition, blockade or absence of P2X7R has been shown to inhibit propagation of intercellular calcium signalling between osteoblasts and osteoclasts in human bone marrow-derived cells (Jorgensen et al 2002), significantly reduce ERK phosphorylation in response to fluid shear stress in mouse primary osteoblasts (Liu et al 2008, Okumura et al 2008) and prevented fluid shear-stress-induced IkBa degradation and nuclear accumulation of nuclear factor kappa B (NFkB) in MC3T3-E1 osteoblasts (Genetos et al 2011). …”

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confidence: 99%

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P2X7 receptors: role in bone cell formation and function

Agrawal¹,

Gartland²

2015

Journal of Molecular Endocrinology

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The role of the P2X7 receptor (P2X7R) is being explored with intensive interest in the context of normal bone physiology, bone-related diseases and, to an extent, bone cancer. In this review, we cover the current understanding of P2X7R regulation of bone cell formation, function and survival. We will discuss how the P2X7R drives lineage commitment of undifferentiated bone cell progenitors, the vital role of P2X7R activation in bone mineralisation and its relatively unexplored role in osteocyte function. We also review how P2X7R activation is imperative for osteoclast formation and its role in bone resorption via orchestrating osteoclast apoptosis. Variations in the gene for the P2X7R (P2RX7) have implications for P2X7R-mediated processes and we review the relevance of these genetic variations in bone physiology. Finally, we highlight how targeting P2X7R may have therapeutic potential in bone disease and cancer. Purinergic signallingIn the last four decades, extensive investigations have led to the recognition of ATP from a 'molecular unit of energy' to an extracellular messenger molecule. ATP-sensitive purinoceptors, omnipresent in vertebrate tissues, are involved in a wide variety of physiological roles (Burnstock 2013) and their function has also been demonstrated in invertebrates (Verkhratsky & Burnstock 2014). While they traditionally act as cell surface sensors (Khakh & North 2006), their participation in signalling within the intracellular compartment in mammals (Qureshi et al. 2007, Kuehnel et al. 2009, Stokes & Surprenant 2009, Toulme et al. 2010) and even protozoans (Fountain et al. 2007, Ludlow et al. 2009, Sivaramakrishnan & Fountain 2012 has also been recognised.Purines (adenine, guanine and uridine) can act as signalling molecules in the form of their 5 0 -nucleotide triphosphates (such as ATP, GTP and UTP), diphosphates (ADP), monophosphate (AMP) or as a nucleoside (adenosine). They can activate one or more of the 19 receptors which are sorted into three classes: P1 (nucleoside) receptors; the metabotropic P2Y receptors and the ionotropic P2X, both of which are nucleotide triggered. Recently, a new family of purine receptors, AdeR or P0-receptors, responsive to adenine has been cloned from rodents (Bender et al. 2002, Gorzalka et al. 2005, von Kugelgen et al. 2008, Thimm et al. 2013, although the human homologue is yet to be identified. Structural and stoichiometrical evidence suggests that all P2X receptor (P2XR) subunits trimerise to form functional receptors (Nicke et al. 1998, Barrera et al. 2005, Mio et al. 2005, Kaczmarek-Hajek et al. 2012 with the subunits forming either homomultimers or heteromultimers depending on the subtypes (Burnstock 2007). Each unit comprises two transmembrane domains (TM1 and TM2) with an intervening large extracellular loop and cytoplasmic N-and C-termini, and the primary agonist of all homomeric and heteromeric P2XR is ATP. The current view holds that ligand binding causes reduction in the Journal of Molecular EndocrinologyReview A AGRAWAL and A GARTLAND P...

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“…Fluid shear stress can also regulate gene expression in osteoblasts, in part by activation of the transcription factor NF-κB, through P2X7 and P2Y 6 receptors [213]. Brief activation of P2X7 receptors on MC3T3-E1 osteoblast-like cells has been shown to trigger a dramatic Ca 2+ -dependent stimulation of metabolic acid production [214].…”

Section: Functional Effects Of P2 Receptor-mediated Signalling In Ostmentioning

confidence: 99%

Purinergic signalling in the musculoskeletal system

Burnstock

Arnett

Orriss

2013

Purinergic Signalling

111

114

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It is now widely recognised that extracellular nucleotides, signalling via purinergic receptors, participate in numerous biological processes in most tissues. It has become evident that extracellular nucleotides have significant regulatory effects in the musculoskeletal system. In early development, ATP released from motor nerves along with acetylcholine acts as a cotransmitter in neuromuscular transmission; in mature animals, ATP functions as a neuromodulator. Purinergic receptors expressed by skeletal muscle and satellite cells play important pathophysiological roles in their development or repair. In many cell types, expression of purinergic receptors is often dependent on differentiation. For example, sequential expression of P2X5, P2Y 1 and P2X2 receptors occurs during muscle regeneration in the mdx model of muscular dystrophy. In bone and cartilage cells, the functional effects of purinergic signalling appear to be largely negative. ATP stimulates the formation and activation of osteoclasts, the bone-destroying cells. Another role appears to be as a potent local inhibitor of mineralisation. In osteoblasts, the bone-forming cells, ATP acts via P2 receptors to limit bone mineralisation by inhibiting alkaline phosphatase expression and activity. Extracellular ATP additionally exerts significant effects on mineralisation via its hydrolysis product, pyrophosphate. Evidence now suggests that purinergic signalling is potentially important in several bone and joint disorders including osteoporosis, rheumatoid arthritis and cancers. Strategies for future musculoskeletal therapies might involve modulation of purinergic receptor function or of the ecto-nucleotidases responsible for ATP breakdown or ATP transport inhibitors.

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Purinergic signaling is required for fluid shear stress-induced NF-κB translocation in osteoblasts

Cited by 21 publications

References 70 publications

Caveolin-1 regulates P2X₇receptor signaling in osteoblasts

Caveolin-1 regulates P2X₇receptor signaling in osteoblasts

P2X7 receptors: role in bone cell formation and function

Purinergic signalling in the musculoskeletal system

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Purinergic signaling is required for fluid shear stress-induced NF-κB translocation in osteoblasts

Cited by 21 publications

References 70 publications

Caveolin-1 regulates P2X7receptor signaling in osteoblasts

Caveolin-1 regulates P2X7receptor signaling in osteoblasts

P2X7 receptors: role in bone cell formation and function

Purinergic signalling in the musculoskeletal system

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Caveolin-1 regulates P2X₇receptor signaling in osteoblasts

Caveolin-1 regulates P2X₇receptor signaling in osteoblasts