Caveolin-1 regulates P2X 7 receptor signaling in osteoblasts

Ayala

Journal of Biological Chemistry

et al. 2016

Damage to the CNS can cause a differential spatio-temporal release of multiple factors, such as nucleotides, ATP and UTP. The latter interact with neuronal and glial nucleotide receptors. The P2Y 2 nucleotide receptor (P2Y 2 R) has gained prominence as a modulator of gliotic responses after CNS injury. Still, the molecular mechanisms underlying these responses in glia are not fully understood. Membrane-raft microdomains, such as caveolae, and their constituent caveolins, modulate receptor signaling in astrocytes; yet, their role in P2Y 2 R signaling has not been adequately explored. Hence, this study evaluated the role of caveolin-1 (Cav-1) in modulating P2Y 2 R subcellular distribution and signaling in human 1321N1 astrocytoma cells. Recombinant hP2Y 2 R expressed in 1321N1 cells and Cav-1 were found to co-fractionate in light-density membrane-raft fractions, colocalize via confocal microscopy, and co-immunoprecipitate. Raft localization was dependent on ATP stimulation and Cav-1 expression. This hP2Y 2 R/Cav-1 distribution and interaction was confirmed with various cell model systems differing in the expression of both P2Y 2 R and Cav-1, and shRNA knockdown of Cav-1 expression. Furthermore, shRNA knockdown of Cav-1 expression decreased nucleotide-induced increases in the intracellular Ca 2؉ concentration in 1321N1 and C6 glioma cells without altering TRAP-6 and carbachol Ca 2؉ responses. In addition, Cav-1 shRNA knockdown also decreased AKT phosphorylation and altered the kinetics of ERK1/2 activation in 1321N1 cells. Our findings strongly suggest that P2Y 2 R interaction with Cav-1 in membrane-raft caveolae of 1321N1 cells modulates receptor coupling to its downstream signaling machinery. Thus, P2Y 2 R/Cav-1 interactions represent a novel target for controlling P2Y 2 R function after CNS injury.Neurodegenerative conditions are among the leading causes of death and disability in the United States and have dramatically increased in incidence during the last decade (1, 2). The P2 receptors for extracellular nucleotides have emerged as key modulators of the pathophysiology of neurodegeneration (3-6). G protein-coupled P2Y 2 nucleotide receptors (P2Y 2 Rs) 3 have been identified in both neurons and glia as mediators of pro-inflammatory responses, neurotransmission, apoptosis, proliferation, and cell migration (3-5, 7, 8). In addition, the P2Y 2 Rs have also gained prominence, due to their association with some types of neoplasms, spinal cord injury, and the enhancement of neuronal differentiation (7, 9 -15). Further insight into the spatio-temporal organization of the P2Y 2 R and its signaling cascades in astrocytic cells is required to expand our knowledge of their role in neurodegenerative diseases. In this context, evidence suggests that receptors and associated signaling molecules are not randomly distributed in plasma membranes but are localized in specialized membrane microdomains, namely membrane rafts (MRs), such as caveolae (Cav) (16 -20). MRs are specialized membrane domains enriched in cholesterol and g...

Section: Discussionsupporting

confidence: 91%

Caveolin-1 Regulates the P2Y2 Receptor Signaling in Human 1321N1 Astrocytoma Cells

Ayala

Journal of Biological Chemistry

et al. 2016

“…2b,c arrows). These “clusters” may be located to lipid rafts, as is known to be the case for this receptor 54 , 55 . Furthermore, P2X7R is also found intracellulary in organelles most likely associated with receptor synthesis or endocytosis.…”

Section: Resultsmentioning

confidence: 89%

The P2X7 receptor and pannexin-1 are involved in glucose-induced autocrine regulation in β-cells

Tozzi

Larsen

Lange

et al. 2018

Sci Rep

Extracellular ATP is an important short-range signaling molecule that promotes various physiological responses virtually in all cell types, including pancreatic β-cells. It is well documented that pancreatic β-cells release ATP through exocytosis of insulin granules upon glucose stimulation. We hypothesized that glucose might stimulate ATP release through other non-vesicular mechanisms. Several purinergic receptors are found in β-cells and there is increasing evidence that purinergic signaling regulates β-cell functions and survival. One of the receptors that may be relevant is the P2X7 receptor, but its detailed role in β-cell physiology is unclear. In this study we investigated roles of the P2X7 receptor and pannexin-1 in ATP release, intracellular ATP, Ca2+ signals, insulin release and cell proliferation/survival in β-cells. Results show that glucose induces rapid release of ATP and significant fraction of release involves the P2X7 receptor and pannexin-1, both expressed in INS-1E cells, rat and mouse β-cells. Furthermore, we provide pharmacological evidence that extracellular ATP, via P2X7 receptor, stimulates Ca2+ transients and cell proliferation in INS-1E cells and insulin secretion in INS-1E cells and rat islets. These data indicate that the P2X7 receptor and pannexin-1 have important functions in β-cell physiology, and should be considered in understanding and treatment of diabetes.

Cardiovascular Research

“…We concluded that shear stress regulation of P2X7 is downstream from transcription or mRNA stability because P2X7 mRNA levels were not enhanced by atheroprone flow (data not shown). Interestingly P2X7 receptors are associated with caveolae in osteoblasts, 27 cardiomyocytes, 28 and alveolar epithelial cells, 29 , 30 while post-translational modifications direct P2X7 to lipid microdomains. 31 Thus we speculate that caveolae, which are shear sensitive, 32 may play a role in atheroprone-mediated up-regulation and clustering of P2X7 at the cell surface thereby enhancing calcium influx and inflammatory signalling in response to ATP.…”

Section: Discussionmentioning

confidence: 99%

Atheroprone flow activates inflammation via endothelial ATP-dependent P2X7-p38 signalling

Green

Souilhol

Xanthis

et al. 2017

ObjectiveAtherosclerosis is a focal disease occurring at arterial sites of disturbed blood flow that generates low oscillating shear stress. Endothelial inflammatory signalling is enhanced at sites of disturbed flow via mechanisms that are incompletely understood. The influence of disturbed flow on endothelial adenosine triphosphate (ATP) receptors and downstream signalling was assessed.Methods and resultsCultured human endothelial cells were exposed to atheroprotective (high uniform) or atheroprone (low oscillatory) shear stress for 72 h prior to assessment of ATP responses. Imaging of cells loaded with a calcium-sensitive fluorescent dye revealed that atheroprone flow enhanced extracellular calcium influx in response to 300 µM 2'(3')-O-(4-Benzoylbenzoyl) adenosine-5'-triphosphate. Pre-treatment with pharmacological inhibitors demonstrated that this process required purinergic P2X7 receptors. The mechanism involved altered expression of P2X7, which was induced by atheroprone flow conditions in cultured cells. Similarly, en face staining of the murine aorta revealed enriched P2X7 expression at an atheroprone site. Functional studies in cultured endothelial cells showed that atheroprone flow induced p38 phosphorylation and up-regulation of E-selectin and IL-8 secretion via a P2X7-dependent mechanism. Moreover, genetic deletion of P2X7 significantly reduced E-selectin at atheroprone regions of the murine aorta.ConclusionsThese findings reveal that P2X7 is regulated by shear forces leading to its accumulation at atheroprone sites that are exposed to disturbed patterns of blood flow. P2X7 promotes endothelial inflammation at atheroprone sites by transducing ATP signals into p38 activation. Thus P2X7 integrates vascular mechanical responses with purinergic signalling to promote endothelial dysfunction and may provide an attractive potential therapeutic target to prevent or reduce atherosclerosis.