Aurélie Fabre

Mutation of the palmitoylation site of estrogen receptor α in vivo reveals tissue-specific roles for membrane versus nuclear actions

Significance The in vivo roles of plasma membrane-associated estrogen receptor (ER)α, including cross-talk with nuclear ERα, are poorly understood. We created a mouse with a point mutation of the palmitoylation site of ERα (C451A-ERα) to obtain membrane-specific loss of function. A complementary mouse lacking the ERα activation function AF-2 (ERα-AF20) provided selective loss of function of nuclear ERα actions. Physiologic studies revealed critical requirements for membrane receptors in ovarian function and thereby in fertility, and in vascular physiology. In contrast, nuclear ERα actions mediate uterine responses to estrogen and genome-wide analysis indicates that membrane-to-nuclear receptor cross-talk in vivo is quite modest in uterus. These findings demonstrate for the first time critical tissue-specific roles for membrane versus nuclear actions of a steroid hormone receptor in vivo. Estrogen receptor alpha (ERα) activation functions AF-1 and AF-2 classically mediate gene transcription in response to estradiol (E2). A fraction of ERα is targeted to plasma membrane and elicits membrane-initiated steroid signaling (MISS), but the physiological roles of MISS in vivo are poorly understood. We therefore generated a mouse with a point mutation of the palmitoylation site of ERα (C451A-ERα) to obtain membrane-specific loss of function of ERα. The abrogation of membrane localization of ERα in vivo was confirmed in primary hepatocytes, and it resulted in female infertility with abnormal ovaries lacking corpora lutea and increase in luteinizing hormone levels. In contrast, E2 action in the uterus was preserved in C451A-ERα mice and endometrial epithelial proliferation was similar to wild type. However, E2 vascular actions such as rapid dilatation, acceleration of endothelial repair, and endothelial NO synthase phosphorylation were abrogated in C451A-ERα mice. A complementary mutant mouse lacking the transactivation function AF-2 of ERα (ERα-AF20) provided selective loss of function of nuclear ERα actions. In ERα-AF20, the acceleration of endothelial repair in response to estrogen–dendrimer conjugate, which is a membrane-selective ER ligand, was unaltered, demonstrating integrity of MISS actions. In genome-wide analysis of uterine gene expression, the vast majority of E2-dependent gene regulation was abrogated in ERα-AF20, whereas in C451A-ERα it was nearly fully preserved, indicating that membrane-to-nuclear receptor cross-talk in vivo is modest in the uterus. Thus, this work genetically segregated membrane versus nuclear actions of a steroid hormone receptor and demonstrated their in vivo tissue-specific roles.

P2Y13 receptor is critical for reverse cholesterol transport.

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 P2Y13 (purinergic receptor P2Y, G protein–coupled, 13) activation is essential for HDL uptake but the potential of P2Y13 as a target to promote RCT has not been documented. Here, we show that P2Y13‐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 P2Y13‐deficient mice. Furthermore, cangrelor, a partial agonist of P2Y13, 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–knockoutliver) but had no effect in P2Y13 knockout mice, which indicate that P2Y13‐mediated HDL uptake pathway is independent of SR‐BI–mediated HDL selective cholesteryl ester uptake. Conclusion: These results establish P2Y13 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. (HEPATOLOGY 2010)

Cell surface adenylate kinase activity regulates the F(1)-ATPase/P2Y (13)-mediated HDL endocytosis pathway on human hepatocytes

Abstract.We have previously demonstrated on human hepatocytes that apolipoprotein A-I binding to an ecto-F1-ATPase stimulates the production of extracellular ADP that activates a P2Y13-mediated high-density lipoprotein (HDL) endocytosis pathway. Therefore, we investigated the mechanisms controlling the extracellular ATP/ADP level in hepatic cell lines and primary cultures to determine their impact on HDL endocytosis. Here we show that addition of ADP to the cell culture medium induced extracellular ATP production that was due to adenylate kinase

Increased atherosclerosis in P2Y(13)/apolipoprotein E double-knockout mice: contribution of P2Y(13) to reverse cholesterol transport

(\hbox{AK}; \hbox{2ADP}\leftrightarrows \hbox{ATP} + \hbox{AMP})

Selective Activation of Estrogen Receptor α Activation Function-1 Is Sufficient to Prevent Obesity, Steatosis, and Insulin Resistance in Mouse

and nucleoside diphosphokinase

L’activation sélective de la fonction de transactivation (AF)-1 du récepteur des estrogènes α hépatiques prévient l’obésité, l’insulino-résistance et la stéatose chez la souris

(\hbox{NDPK}; \hbox{ADP} + \hbox{NTP} \leftrightarrows \hbox{ATP} + \hbox{NDP})