Dataset Information

Role of UDP-Sugar Receptor P2Y₁₄ in Murine Osteoblasts.

ABSTRACT: The purinergic (P2) receptor P2Y₁₄ is the only P2 receptor that is stimulated by uridine diphosphate (UDP)-sugars and its role in bone formation is unknown. We confirmed P2Y₁₄ expression in primary murine osteoblasts (CB-Ob) and the C2C12-BMP2 osteoblastic cell line (C2-Ob). UDP-glucose (UDPG) had undiscernible effects on cAMP levels, however, induced dose-dependent elevations in the cytosolic free calcium concentration ([Ca²⁺]_i) in CB-Ob, but not C2-Ob cells. To antagonize the P2Y₁₄ function, we used the P2Y₁₄ inhibitor PPTN or generated CRISPR-Cas9-mediated P2Y₁₄ knockout C2-Ob clones (Y14_KO). P2Y₁₄ inhibition facilitated calcium signalling and altered basal cAMP levels in both models of osteoblasts. Importantly, P2Y₁₄ inhibition augmented Ca²⁺ signalling in response to ATP, ADP and mechanical stimulation. P2Y₁₄ knockout or inhibition reduced osteoblast proliferation and decreased ERK1/2 phosphorylation and increased AMPK? phosphorylation. During in vitro osteogenic differentiation, P2Y₁₄ inhibition modulated the timing of osteogenic gene expression, collagen deposition, and mineralization, but did not significantly affect differentiation status by day 28. Of interest, while P2ry14^-/- mice from the International Mouse Phenotyping Consortium were similar to wild-type controls in bone mineral density, their tibia length was significantly increased. We conclude that P2Y₁₄ in osteoblasts reduces cell responsiveness to mechanical stimulation and mechanotransductive signalling and modulates osteoblast differentiation.

SUBMITTER: Mikolajewicz N

PROVIDER: S-EPMC7216066 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Role of UDP-Sugar Receptor P2Y14 in Murine Osteoblasts.

Mikolajewicz Nicholas N Komarova Svetlana V SV

International journal of molecular sciences 20200415 8

The purinergic (P2) receptor P2Y14 is the only P2 receptor that is stimulated by uridine diphosphate (UDP)-sugars and its role in bone formation is unknown. We confirmed P2Y14 expression in primary murine osteoblasts (CB-Ob) and the C2C12-BMP2 osteoblastic cell line (C2-Ob). UDP-glucose (UDPG) had undiscernible effects on cAMP levels, however, induced dose-dependent elevations in the cytosolic free calcium concentration ([Ca2+]i) in CB-Ob, but not C2-O ...[more]

PMID: 32326617

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Project description:AimsUDP-sugars can act as extracellular signalling molecules, but relatively little is known about their cardiovascular actions. The P2Y14 receptor is a Gi/o-coupled receptor which is activated by UDP-glucose and related sugar nucleotides. In this study we sought to investigate whether P2Y14 receptors are functionally expressed in the porcine coronary artery using a selective P2Y14 receptor agonist, MRS2690, and a novel selective P2Y14 receptor antagonist, PPTN (4,7-disubstituted naphthoic acid derivative).Methods and resultsIsometric tension recordings were used to evaluate the effects of UDP-sugars in porcine isolated coronary artery segments. The effects of the P2 receptor antagonists suramin and PPADS, the P2Y14 receptor antagonist PPTN, and the P2Y6 receptor antagonist MRS2578, were investigated. Measurement of vasodilator-stimulated phosphoprotein (VASP) phosphorylation using flow cytometry was used to assess changes in cAMP levels. UDP-glucose, UDP-glucuronic acid UDP-N-acetylglucosamine (P2Y14 receptor agonists), elicited concentration-dependent contractions of the porcine coronary artery. MRS2690 was a more potent vasoconstrictor than the UDP-sugars. Concentration dependent contractile responses to MRS2690 and UDP-sugars were enhanced in the presence of forskolin (activator of cAMP), where the level of basal tone was maintained by addition of U46619, a thromboxane A2 mimetic. Contractile responses to MRS2690 were blocked by PPTN, but not by MRS2578. Contractile responses to UDP-glucose were also attenuated by PPTN and suramin, but not by MRS2578. Forskolin-induced VASP-phosphorylation was reduced in porcine coronary arteries exposed to UDP-glucose and MRS2690, consistent with P2Y14 receptor coupling to Gi/o proteins and inhibition of adenylyl cyclase activity.ConclusionsOur data support a role of UDP-sugars as extracellular signalling molecules and show for the first time that they mediate contraction of porcine coronary arteries via P2Y14 receptors.

Project description:Diabetes mellitus (DM), an emerging chronic epidemic, contributes to mortality and morbidity around the world. Diabetic cardiac autonomic neuropathy (DCAN) is one of the most common complications associated with DM. Previous studies have shown that satellite glial cells (SGCs) in the superior cervical ganglia (SCG) play an indispensable role in DCAN progression. In addition, it has been shown that purinergic neurotransmitters, as well as metabotropic GPCRs, are involved in the pathophysiological process of DCAN. Furthermore, one traditional Chinese medicine, naringin may potently alleviate the effects of DCAN. Ferroptosis may be involved in DCAN progression. However, the role of naringin in DCAN as well as its detailed mechanism requires further investigation. In this research, we attempted to identify the effect and relevant mechanism of naringin in DCAN mitigation. We observed that compared with those of normal subjects, there were significantly elevated expression levels of P2Y14 and IL-1β in diabetic rats, both of which were remarkably diminished by treatment with either P2Y14 shRNA or naringin. In addition, abnormalities in blood pressure (BP), heart rate (HR), heart rate variability (HRV), sympathetic nerve discharge (SND), and cardiac structure in the diabetic model can also be partially returned to normal through the use of those treatments. Furthermore, a reduced expression of NRF2 and GPX4, as well as an elevated level of ROS, were detected in diabetic cases, which can also be improved with those treatments. Our results showed that naringin can effectively relieve DCAN mediated by the P2Y14 receptor of SGCs in the SCG. Moreover, the NRF2/GPX4 pathway involved in ferroptosis may become one of the principal mechanisms participating in DCAN progression, which can be modulated by P2Y14-targeted naringin and thus relieve DCAN. Hopefully, our research can supply one novel therapeutic target and provide a brilliant perspective for the treatment of DCAN.

Dataset Information

Role of UDP-Sugar Receptor P2Y₁₄ in Murine Osteoblasts.

Publications

Role of UDP-Sugar Receptor P2Y<sub>14</sub> in Murine Osteoblasts.

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