In vivo evidence that furin from hepatocytes inactivates PCSK9

The proprotein convertase PCSK9 plays a key role in cholesterol homeostasis by binding the LDL receptor and targeting it toward degradation. PCSK9 is strongly expressed in the liver and is found in human and mouse plasma as mature (～ 62 kDa) and inactivated (～ 55 kDa) forms. Ex vivo data showed that human PCSK9 is inactivated by cleavage at Arg(218)↓ by the overexpressed convertases furin and PC5/6A. Analysis of the plasma of human heterozygotes for R218S and F216L mutations revealed a ～ 50% reduction in the levels of the ～ 55-kDa form. To identify the convertase(s) responsible for cleavage at Arg(218) in vivo, we inactivated the genes of furin and/or PC5/6 specifically in hepatocytes. The PCSK9-inactivated form was strongly reduced in mice lacking furin in hepatocytes (Fur-hKO) and only slightly reduced in PC5/6-hKO plasma. In agreement with a key role of furin in regulating PCSK9 activity in vivo, we observed an overall 26% drop in the LDL receptor protein levels of Fur-hKO livers, likely due to the compound effects of a 35% increase in PCSK9 mRNA levels and the loss of PCSK9 cleavage, suggesting a higher activity of PCSK9 in these mice. Overexpression of PCSK9 in primary hepatocytes obtained from these mice revealed that only full-length, membrane-bound, but not soluble, furin is the cognate convertase. We conclude that in hepatocytes furin regulates PCSK9 mRNA levels and is the key in vivo-inactivating protease of circulating PCSK9.     

Annexin A2 Is a Natural Extrahepatic Inhibitor of the PCSK9-Induced LDL Receptor Degradation

Proprotein convertase subtilisin/kexin-9 (PCSK9) enhances the degradation of hepatic low-density lipoprotein receptor (LDLR). Deletion of PCSK9, and loss-of-function mutants in humans result in lower levels of circulating LDL-cholesterol and a strong protection against coronary heart disease. Accordingly, the quest for PCSK9 inhibitors has major clinical implications. We have previously identified annexin A2 (AnxA2) as an endogenous binding partner and functional inhibitor of PCSK9. Herein, we studied the relevance of AnxA2 in PCSK9 inhibition and lipid metabolism in vivo. Plasma analyses of AnxA2(-/-) mice revealed: i) a ～1.4-fold increase in LDL-cholesterol without significant changes in VLDLs or HDLs, and ii) a ～2-fold increase in circulating PCSK9 levels. Western blotting and immunohistochemistry of AnxA2(-/-) tissues revealed that the LDLR was decreased by ～50% in extrahepatic tissues, such as adrenals and colon. We also show that AnxA2-derived synthetic peptides block the PCSK9≡LDLR interaction in vitro, and adenoviral overexpression of AnxA2 in mouse liver increases LDLR protein levels in vivo. These results suggest that AnxA2 acts as an endogenous regulator of LDLR degradation, mostly in extrahepatic tissues. Finally, we identified an AnxA2 coding polymorphism, V98L, that correlates with lower circulating levels of PCSK9 thereby extending our results on the physiological role of AnxA2 in humans.     

Variation in vernal species composition in alluvial forests of the Rhine valley,eastern France

Patterns of vernal synusiae in alluvial forests of the upper Rhine valley were analysed phytosociologically. Five synusial types were described. Environmental factors included in the analysis were spring water level, texture, trophic status and soil reaction. A separate environmental study was undertaken in pure stands of six competitive geophytes in the most fertile habitats. In a laboratory experiment effects of water stress and interspecific competition were studied for three competitive geophytes. The vernal species can be grouped into eight groups with respect to species position along the environmental gradients occurring in the floodplain. These groups can be used for bioindication.     

Dissection of the Endogenous Cellular Pathways of PCSK9-induced Low Density Lipoprotein Receptor Degradation: EVIDENCE FOR AN INTRACELLULAR ROUTE*

Elevated levels of plasma low density lipoprotein (LDL)-cholesterol, leading to familial hypercholesterolemia, are enhanced by mutations in at least three major genes, the LDL receptor (LDLR), its ligand apolipoprotein B, and the proprotein convertase PCSK9. Single point mutations in PCSK9 are associated with either hyper- or hypocholesterolemia. Accordingly, PCSK9 is an attractive target for treatment of dyslipidemia. PCSK9 binds the epidermal growth factor domain A (EGF-A) of the LDLR and directs it to endosomes/lysosomes for destruction. Although the mechanism by which PCSK9 regulates LDLR degradation is not fully resolved, it seems to involve both intracellular and extracellular pathways. Here, we show that clathrin light chain small interfering RNAs that block intracellular trafficking from the trans-Golgi network to lysosomes rapidly increased LDLR levels within HepG2 cells in a PCSK9-dependent fashion without affecting the ability of exogenous PCSK9 to enhance LDLR degradation. In contrast, blocking the extracellular LDLR endocytosis/degradation pathway by a 4-, 6-, or 24-h incubation of cells with Dynasore or an EGF-AB peptide or by knockdown of endogenous autosomal recessive hypercholesterolemia did not significantly affect LDLR levels. The present data from HepG2 cells and mouse primary hepatocytes favor a model whereby depending on the dose and/or incubation period, endogenous PCSK9 enhances the degradation of the LDLR both extra- and intracellularly. Therefore, targeting either pathway, or both, would be an effective method to reduce PCSK9 activity in the treatment of hypercholesterolemia and coronary heart disease.High levels of circulating low-density lipoprotein (LDL)³-cholesterol represent a major risk factor that leads to coronary heart disease, the main cause of death and morbidity worldwide (1). LDL particles are cleared mainly from the bloodstream by the hepatic cell surface LDL receptor (LDLR) (2). Genetics studies demonstrated that loss-of-function mutations in either LDLR or apolipoprotein B, the protein component of LDL that binds LDLR, result in familial hypercholesterolemia and premature coronary heart disease (3). More recently, the proprotein convertases subtilisin kexin 9 (PCSK9) gene (4), which is highly expressed in liver and small intestine (5), was identified as the third locus associated with familial hypercholesterolemia (6). It is now clear that PCSK9 binds the LDLR and triggers its intracellular degradation in acidic compartments, resulting in increased circulating plasma cholesterol (7–10).After its autocatalytic cleavage, PCSK9 is secreted as a stable noncovalent complex with its prosegment (pro·PCSK9) (5, 7). This cleavage results in a conformational change (11) that favors the binding of PCSK9 to the epidermal growth factor A domain (EGF-A) of the LDLR (12), with increased affinity at acidic pH values (11). Although the C-terminal Cys-His-rich domain of PCSK9 is a spatially separate domain (11) that does not participate directly in the PCSK9-EGF-A interaction (12), it is a critical determinant for the PCSK9-enhanced cellular degradation of the LDLR (13). In agreement, we recently demonstrated that annexin A2, which binds the Cys-His-rich domain of PCSK9, blocks its effect on LDLR degradation (14).Overexpression studies in liver suggested that both intra- and extracellular PCSK9 target the LDLR (9, 15, 16) toward degradation in late endosomes/lysosomes (LE/L) (7–10). It was shown that the adaptor protein ARH, which interacts with the cytosolic tail of the LDLR, is essential for the endocytosis and degradation of the cell surface PCSK9·LDLR complex in vivo (16). However, hepatic LDLR protein levels were also reduced upon overexpression of PCSK9 in Arh ^−/− mice (9), suggesting the presence of an ARH-independent intracellular pathway. Intriguingly, at endogenous levels of PCSK9, the absence of ARH did not affect hepatic LDLR subcellular localization in LE/L or protein levels (17). This is not the expected result if PCSK9 mostly targets LDLR by the extracellular pathway (18), as one would have expected that in Arh^−/− mice total LDLR levels should have been more elevated.In this study, we focused on the relative contribution of the intra- versus extracellular pathways of endogenous PCSK9-induced LDLR degradation. This information should guide the choice of therapeutic approaches that will best target the site of PCSK9-LDLR interaction to control hypercholesterolemia and coronary heart disease.     

Impact of river management history on the community structure,species composition and nutrient status in the Rhine alluvial hardwood forest

The present-day Rhine alluvial hardwood forest (Querco-Ulmetum minoris, Issler 24) in the upper Rhine valley (France/Germany) is comprised of three vegetation units, one still flooded by calm waters (F) and the two others unflooded, one for 30 years (UF30) (after the river canalisation) and the other for about 130 years (UF130) (after river straightening and embankment work in the middle nineteenth century). In the three stands, species composition, structure and diversity of vegetation and nutrient content of mature leaf, leaf litter and soil have been studied. Fungi (Macromycetae) were only studied in two stands (F and UF130). The intensity of nutrient recycling was exemplified by comparing the chemical composition of rainwater, flood, throughfall, mature leaf, leaf litter, soil and groundwater in two of these stands (F and UF30).The elimination of floods has caused a change in floristic composition, tree density and plant diversity. Tree density was higher in the two unflooded stands and was related to a large increase in sapling (< 6 cm dbh) density more than to a change of stem (> 6 cm dbh) density. Sapling density increased 2 times and three times in the UF30 and the UF130 respectively, whereas the stem density increased only 12% in the first stand and decreased 29% in the second one. The saprophytic macromycete communities have been supplemented with mycorrhizal species. Leaf litter production was slightly greater in the flooded (4.44 T ha^-1 yr^-1) than in the two unflooded stands ( 3.72 T ha^-1 yr^-1). Foliar N level is twice as high in the flooded stands in spite of an opposite soil status. P level decreased in soil and leaves with the duration of isolation and was at the same level in the groundwater in two stands (F and UF30). K, Mg and Ca contents of green leaf and leaf litter were high due to the geochemistry of the Rhine substrate (calcareous gravels and pebbles) and similar in all the stands studied, even though there are large inputs of these three elements by floods. Moreover we showed that the groundwater chemistry reflected the variations of nutrient inputs and thus could be a good indicator of the functioning of an alluvial ecosystem and of its change as a result of human activities. The restoration of floods in hardwood forest contributes to the preservation of alluvial vegetational structure and composition, the stimulation of biological processes and a better plant mineral nutrition and water supply.     

The activation and physiological functions of the proprotein convertases

The mammalian secretory proprotein convertases are part of a family of nine serine proteinases of the subtilisin-type. Seven of them cleave after basic amino acids and are called PC1/3, PC2, furin, PC4, PC5/6, PACE4 and PC7. The two other convertases SKI-1/S1P and PCSK9 are implicated in cholesterol and/or fatty acid metabolism. The convertases PC5/6 and PACE4 are activated at the cell surface where they are tethered to heparan sulfate proteoglycans. This activation pathway is unique and differs from that of furin and PC7, which are activated in the trans-Golgi network and from PC1/3 and PC2 that are activated in dense core secretory granules. While some of the basic amino acid-specific convertases may display redundant cleavages of substrates, they uniquely process certain substrates in vivo. Indeed, the conditional knockout of the PC5/6 gene in the embryo proper in mice led to severe malformations, bone morphogenic defects and death at birth. This is likely due to the absence of processing of the growth differentiating factor 11 (Gdf11). Both complete and liver-specific knockout of Pcsk9 revealed that it is a major convertase that regulates the level of circulating low-density lipoproteins (LDL) via the degradation of the hepatic LDL-receptor. This apparently non-enzymatic mechanism implicates the enhanced degradation of the LDLR in endosomes/lysosomes. These data provide evidence that an inhibitor of PCSK9-LDLR interaction is a viable target for the development of a novel cholesterol lowering drug in conjunction with the classical statins.     

Is there a future for wild grapevine (Vitis vinifera subsp. silvestris) in the Rhine Valley?

The wild grapevine, Vitis vinifera L. subsp. silvestris (Gmelin) Hegi, is considered to be an endangered taxon in Europe, mainly as a consequence of the introduction of pathogens from North America and of the destruction of its habitat. In the Rhine Valley, nearly all populations disappeared due to river management, the intensi.cation of forestry, and the introduction of phylloxera. After a growing awareness of the need to preserve endangered forest ecosystems, attempts to reintroduce wild grapevine in the Rhine Valley were performed, particularly in the French nature reserves Erstein and Offendorf since 1992. However, regular surveys of the plants indicate the rapid decline of the populations. In 2002, we proposed to summarise the knowledge accumulated after 10 years of experiments. Results indicate that from the initial 91 individuals planted in 1992, only 14 survived in 2002 (2 in Erstein, 12 in Offendorf). The failure of the experiment may be explained by several factors: unsuitable sites (too shady, absence of support for the young plants), absence of monitoring, vandalism or predation. According to these results and recent knowledge of the ecology of the plant and of vines in general, new transplantation experiments are proposed in which the plants will be monitored during their establishment in the forests. The success of this second transplant (50 plants per reserve) will be enhanced by restoration projects of the Rhine River dynamics, with partial re-.ooding. Floods should help to avoid, or at least to reduce, pest and disease expansion on future adult plants.