Role of the ubiquitin-proteasome system in the regulation of P2Y13 receptor expression: impact on hepatic HDL uptake.

The protective effect of high density lipoproteins (HDL) against atherosclerosis is mainly attributed to their capacity to transport excess cholesterol from peripheral tissues back to the liver for further elimination into the bile, a process called reverse cholesterol transport (RCT). Recently, the importance of the P2Y13 receptor (P2Y13-R) was highlighted in HDL metabolism since HDL uptake by the liver was decreased in P2Y13-R deficient mice, which translated into impaired RCT. Here, we investigated for the first time the molecular mechanisms regulating cell surface expression of P2Y13-R. When transiently expressed, P2Y13-R was mainly detected in the endoplasmic reticulum (ER) and strongly subjected to proteasome degradation while its homologous P2Y12 receptor (P2Y12-R) was efficiently targeted to the plasma membrane. We observed an inverse correlation between cell surface expression and ubiquitination level of P2Y13-R in the ER, suggesting a close link between ubiquitination of P2Y13-R and its efficient targeting to the plasma membrane. The C-terminus tail exchange between P2Y13-R and P2Y12-R strongly restored plasma membrane expression of P2Y13-R, suggesting the involvement of the intra-cytoplasmic tail of P2Y13-R in expression defect. Accordingly, proteasomal inhibition increased plasma membrane expression of functionally active P2Y13-R in hepatocytes, and consequently stimulated P2Y13-R-mediated HDL endocytosis. Importantly, proteasomal inhibition strongly potentiated HDL hepatic uptake (>200 %) in wild-type but not in P2Y13-R-deficient mice, thus reinforcing the role of P2Y13-R expression in regulating HDL metabolism. Therefore, specific inhibition of the ubiquitin-proteasome system might be a novel powerful HDL therapy to enhance P2Y13-R expression and consequently promote the overall RCT.

Nicotinic acid inhibits progression of atherosclerosis in mice through its receptor GPR109A expressed by immune cells.

Nicotinic acid (niacin) is a drug used to reduce the progression of atherosclerosis. Its antiatherosclerotic activity is believed to result from lipid-modifying effects, including its ability to decrease LDL cholesterol and increase HDL cholesterol levels in plasma. Here, we report that in a mouse model of atherosclerosis, we found that nicotinic acid inhibited disease progression under conditions that left total cholesterol and HDL cholesterol plasma levels unaffected. The antiatherosclerotic effect was not seen in mice lacking the receptor for nicotinic acid GPR109A. Surprisingly, transplantation of bone marrow from GPR109A-deficient mice into atherosclerosis-prone animals also abrogated the beneficial effect of nicotinic acid. We detected expression of GPR109A in macrophages in atherosclerotic plaques. In macrophages from WT mice, but not from GPR109A-deficient animals, nicotinic acid induced expression of the cholesterol transporter ABCG1 and promoted cholesterol efflux. Furthermore, activation of GPR109A by nicotinic acid inhibited MCP-1-induced recruitment of macrophages into the peritoneal cavity and impaired macrophage recruitment to atherosclerotic plaques. In contrast with current models, our data show that nicotinic acid can reduce the progression of atherosclerosis independently of its lipid-modifying effects through the activation of GPR109A on immune cells. We conclude therefore that GPR109A mediates antiinflammatory effects, which may be useful for treating atherosclerosis and other diseases.

P2Y13 receptor is critical for reverse cholesterol transport.

UNLABELLED: A major atheroprotective functionality of high-density lipoproteins (HDLs) is to promote "reverse cholesterol transport" (RCT). In this process, HDLs mediate the efflux and transport of cholesterol from peripheral cells and its subsequent transport to the liver for further metabolism and biliary excretion. We have previously demonstrated in cultured hepatocytes that P2Y(13) (purinergic receptor P2Y, G protein-coupled, 13) activation is essential for HDL uptake but the potential of P2Y(13) as a target to promote RCT has not been documented. Here, we show that P2Y(13)-deficient mice exhibited a decrease in hepatic HDL cholesterol uptake, hepatic cholesterol content, and biliary cholesterol output, although their plasma HDL and other lipid levels were normal. These changes translated into a substantial decrease in the rate of macrophage-to-feces RCT. Therefore, hallmark features of RCT are impaired in P2Y(13)-deficient mice. Furthermore, cangrelor, a partial agonist of P2Y(13), stimulated hepatic HDL uptake and biliary lipid secretions in normal mice and in mice with a targeted deletion of scavenger receptor class B type I (SR-BI) in liver (hypomSR-BI-knockout(liver)) but had no effect in P2Y(13) knockout mice, which indicate that P2Y(13)-mediated HDL uptake pathway is independent of SR-BI-mediated HDL selective cholesteryl ester uptake. CONCLUSION: These results establish P2Y(13) as an attractive novel target for modulating RCT and support the emerging view that steady-state plasma HDL levels do not necessarily reflect the capacity of HDL to promote RCT.

RhoA/ROCK I signalling downstream of the P2Y13 ADP-receptor controls HDL endocytosis in human hepatocytes.

Cell surface receptors for high-density lipoprotein (HDL) on hepatocytes are major partners in the regulation of cholesterol homeostasis. We have previously demonstrated on human hepatocytes that apolipoprotein A-I binding to an ectopic F(1)-ATPase stimulates the production of extracellular ADP that activates a P2Y(13)-mediated high-density lipoprotein (HDL) endocytosis pathway. However, P2Y(13)-dependent signalling pathway has never been described yet. The current study demonstrates a major role of cytoskeleton reorganization in F(1)-ATPase/P2Y(13)-dependent HDL endocytosis under the control of the small GTPase RhoA and its effector ROCK I. Indeed human hepatocytes (HepG(2) cells) stimulated by ADP or AR-C69931MX (both P2Y(13) agonists) showed a high specific activation of RhoA; in addition, inhibition of Rho proteins by C3 exoenzyme impairs HDL endocytosis whereas a constitutively active form of RhoA stimulates HDL endocytosis at the same level as under F(1)-ATPase/P2Y(13) activation. Pharmacological inhibition of ROCK activity decreased HDL endocytosis following stimulation by apoA-I (F(1)-ATPase ligand), ADP or AR-C69931MX and specific siRNA ROCK I extinction prevented the stimulation of HDL endocytosis without effect of ROCK II extinction. The functional involvement of ROCK I downstream F(1)-ATPase/P2Y(13) was confirmed by the strong enrichment of the membrane fraction in ROCK I and by the requirement of actin polymerization in hepatocyte HDL endocytosis. These results allow the identification of the molecular events downstream P2Y(13) receptor activation for a better understanding of hepatocyte HDL endocytosis, the latest step in reverse cholesterol transport.