Objective-Neural apoptosis-regulated convertase (NARC)-1 is the newest member of the proprotein convertase family implicated in the cleavage of a variety of protein precursors. The NARC-1 gene, PCSK9, has been identified recently as the third locus implicated in autosomal dominant hypercholesterolemia (ADH). The 2 other known genes implicated in ADH encode the low-density lipoprotein receptor and apolipoprotein B. As an approach toward the elucidation of the physiological role(s) of NARC-1, we studied its transcriptional regulation. Methods and Results-Using quantitative RT-PCR, we assessed NARC-1 regulation under conditions known to regulate genes involved in cholesterol metabolism in HepG2 cells and in human primary hepatocytes. We found that NARC-1 expression was strongly induced by statins in a dose-dependent manner and that this induction was efficiently reversed by mevalonate. NARC-1 mRNA level was increased by cholesterol depletion but insensitive to liver X receptor activation. Human, mouse, and rat PCSK9 promoters contain 2 typical conserved motifs for cholesterol regulation: a sterol regulatory element (SRE) and an Sp1 site. Conclusions-PCSK9 regulation is typical of that of the genes implicated in lipoprotein metabolism. In vivo, PCSK9 is probably a target of SRE-binding protein (SREBP)-2.

The gene encoding the proprotein convertase subtilisin/kexin type 9 (PCSK9) is linked to familial hypercholesterolemia, as are those of the low-density lipoprotein receptor (LDLR) and apolipoprotein B. PCSK9 enhances LDLR degradation, resulting in low-density lipoprotein accumulation in plasma. To analyze the role of hepatic PCSK9, total and hepatocyte-specific knockout mice were generated. They exhibit 42% and 27% less circulating cholesterol, respectively, showing that liver PCSK9 was responsible for two thirds of the phenotype. We also demonstrated that, in liver, PCSK9 is exclusively expressed in hepatocytes, representing the main source of circulating PCSK9. The data suggest that local but not circulating PCSK9 regulates cholesterol levels. Although transgenic mice overexpressing high levels of liver and circulating PCSK9 led to the almost complete disappearance of the hepatic LDLR, they did not recapitulate the plasma cholesterol levels observed in LDLRdeficient mice. Single LDLR or double LDLR/PCSK9 knockout mice exhibited similar cholesterol profiles, indicating that PCSK9 regulates cholesterol homeostasis exclusively through the LDLR. Finally, the regenerating liver of PCSK9-deficient mice exhibited necrotic lesions, which were prevented by a high-cholesterol diet. However, lipid accumulation in hepatocytes of these mice was markedly reduced under both chow and high-cholesterol diets, revealing that PCSK9 deficiency confers resistance to liver steatosis. Conclusion: Although PCSK9 is a target for controlling hypercholesterolemia, our data indicate that upon hepatic damage, patients lacking PCSK9 could be at risk. (HEPATOLOGY 2008;48:646-654.) P roprotein convertase subtilisin/kexin type 9 (PCSK9) 1 is the ninth member of the proprotein convertase family. 2 The first seven members, including furin, cleave protein precursors of hormones, growth factors, receptors, or surface glycoproteins at basic sites (after Arg or Lys residues). The eighth member, SKI-1 3 or S1P, 4 is known to cleave membrane-bound transcription factors such as the SREBPs 5 in their luminal domains, resulting in the release of their DNA-binding domain. Proprotein convertases can also inactivate secreted substrates, such as endothelial lipase 6 and PCSK9. 7 PCSK9 is synthesized as a precursor that undergoes autocatalytic cleavage of its N-terminal prosegment in the ER, 1 a step required for its exit from this compartment and its efficient secretion. Secreted PCSK9 remains associated with its prosegment. 1 Different from the other proprotein convertases, this serine protease has no known substrate other than itself. In addition, the tight association of the prosegment with the active site 8 raises the question of the existence of an in trans PCSK9 protease

Objective-Proprotein convertase subtilisin/kexin 9 (PCSK9) promotes the degradation of the low-density lipoprotein receptor (LDLR), and its gene is the third locus implicated in familial hypercholesterolemia. Herein, we investigated the role of PCSK9 in adipose tissue metabolism. Methods and Results-At 6 months of age, Pcsk9Ϫ/Ϫ mice accumulated Ϸ80% more visceral adipose tissue than wild-type mice. This was associated with adipocyte hypertrophy and increased in vivo fatty acid uptake and ex vivo triglyceride synthesis. Moreover, adipocyte hypertrophy was also observed in Pcsk9 Ϫ/Ϫ Ldlr Ϫ/Ϫ mice, indicating that the LDLR is not implicated. Rather, we show here by immunohistochemistry that Pcsk9 Ϫ/Ϫ males and females exhibit 4-and Ϸ40-fold higher cell surface levels of very-low-density lipoprotein receptor (VLDLR) in perigonadal depots, respectively. Expression of PCSK9 in the liver of Pcsk9 Ϫ/Ϫ females reestablished both circulating PCSK9 and normal VLDLR levels. In contrast, specific inactivation of PCSK9 in the liver of wild-type females led to Ϸ50-fold higher levels of perigonadal VLDLR. Key Words: PCSK9 Ⅲ VLDL receptor Ⅲ adipose tissue metabolism Ⅲ fatty acid uptake Ⅲ proprotein convertase P roprotein convertase subtilisin/kexin 9 (PCSK9) is the ninth member of the family of proprotein convertases (encoded by the genes PCSK1 to PCSK9) that share identity with subtilisin and kexin. 1 The first 8 members of the family cleave protein precursors of hormones, growth factors, receptors, and transmembrane transcription factors that transit through or reside in the secretory pathway. 2 In contrast, PCSK9 has no known substrates but itself. It undergoes an autocatalytic cleavage of its N-terminal prosegment, 1,3 which remains trapped in the catalytic pocket. 4 PCSK9 shortens the half-life of the low-density lipoprotein receptor (LDLR), 5 a process independent of its catalytic activity. 6 Gain-of-function mutations in the PCSK9 gene lead to autosomal dominant hypercholesterolemia, 7 as do mutations in the LDLR and APOB genes. Other PCSK9 mutations or polymorphisms responsible for loss of function result in hypocholesterolemia. 8 PCSK9 is highly expressed in the liver, 1 where it binds the LDLR and promotes its internalization and degradation in endosomal/lysosomal compartments. 5 Thus, mice lacking PCSK9 (Pcsk9 Ϫ/Ϫ ) exhibit a 2-to 3-fold increase of the LDLR protein in liver homogenates, and a substantial accumulation of the receptor at the hepatocyte cell surface. 9,10 This leads to hypocholesterolemia (Ϫ40%), with a Ϸ5-fold drop in low-density lipoprotein (LDL) cholesterol levels. In humans, where 70% of cholesterol is associated with LDLs, versus only 30% in mice, the hypocholesterolemia due to PCSK9 deficiency is even more dramatic. Two women lacking functional PCSK9 exhibited LDL cholesterol levels of Ϸ0.4 mmol/L (15 mg/dL). 11,12 PCSK9 is now considered a promising target to treat hypercholesterolemia and prevent coronary heart disease. Conclusion-InHuman PCSK9 is abundant in plasma, 13 and analysis of mice tha...

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 (ϳ62 kDa) and inactivated (ϳ55 kDa) forms. Ex vivo data showed that human PCSK9 is inactivated by cleavage at Arg 218 2 by the overexpressed convertases furin and PC5/6A. Analysis of the plasma of human heterozygotes for R218S and F216L mutations revealed a ϳ50% reduction in the levels of the ϳ55-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 (FurhKO) 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.

The proprotein convertase PC5/6 cleaves protein precursors after basic amino acids and is essential for implantation in CD1/129/Sv/ C57BL/6 mixed-background mice. Conditional inactivation of Pcsk5 in the epiblast but not in the extraembryonic tissue bypassed early embryonic lethality but resulted in death at birth. PC5/6-deficient embryos exhibited Gdf11-related phenotypes such as altered anteroposterior patterning with extra vertebrae and lack of tail and kidney agenesis. They also exhibited Gdf11-independent phenotypes, such as a smaller size, multiple hemorrhages, collapsed alveoli, and retarded ossification. In situ hybridization revealed overlapping PC5/6 and Gdf11 mRNA expression patterns. In vitro and ex vivo analyses showed that the selectivity of PC5/6 for Gdf11 essentially resides in the presence of a P1 Asn in the RSRR2N cleavage motif. This work identifies Gdf11 as a likely in vivo specific substrate of PC5/6 and opens the way to the identification of other key substrates of this convertase.Furin-like ͉ Meox2-cre ͉ Pcsk5

Bone morphogenetic protein 10 (BMP10) is a member of the TGF-␤ superfamily and plays a critical role in heart development. In the postnatal heart, BMP10 is restricted to the right atrium. The inactive pro-BMP10 (ϳ60 kDa) is processed into active BMP10 (ϳ14 kDa) by an unknown protease. Proteolytic cleavage occurs at the RIRR 316 2 site (human), suggesting the involvement of proprotein convertase(s) (PCs). In vitro digestion of a 12-mer peptide encompassing the predicted cleavage site with furin, PACE4, PC5/6, and PC7, showed that furin cleaves the best, whereas PC7 is inactive on this peptide. Ex vivo studies in COS-1 cells, a cell line lacking PC5/6, revealed efficient processing of pro-BMP10 by endogenous PCs other than PC5/6. The lack of processing of overexpressed pro-BMP10 in the furin-and PACE4-deficient cell line, CHO-FD11, and in furin-deficient LoVo cells, was restored by stable (CHO-FD11/ Fur cells) or transient (LoVo cells) expression of furin. Use of cell-permeable and cell surface inhibitors suggested that endogenous PCs process pro-BMP10 mostly intracellularly, but also at the cell surface. Ex vivo experiments in mouse primary hepatocytes (wild type, PC5/6 knock-out, and furin knock-out) corroborated the above findings that pro-BMP10 is a substrate for endogenous furin. Western blot analyses of heart right atria extracts from wild type and PACE4 knock-out adult mice showed no significant difference in the processing of pro-BMP10, implying no in vivo role of PACE4. Overall, our in vitro, ex vivo, and in vivo data suggest that furin is the major convertase responsible for the generation of BMP10.

PC5 belongs to the proprotein convertase family and activates precursor proteins by cleavage at basic sites during their transit through the secretory pathway and/or at the cell surface. These precursors include prohormones, proreceptors, growth factors, adhesion molecules, and viral glycoproteins. The Pcsk5 gene encodes two alternatively spliced isoforms, the soluble PC5A and transmembrane PC5B. We have carefully analyzed the expression of PC5 in the mouse during development and in adulthood by in situ hybridization, as well as in mouse tissues and various cell lines by quantitative reverse transcription-PCR. The data show that adrenal cortex and intestine are the richest sources of PC5A and PC5B, respectively. To better define the specific physiological roles of PC5, we have generated a mouse Pcsk5 ⌬4 -deficient allele missing exon 4 that encodes the catalytic Asp 173 . While ⌬4/؉ heterozygotes were healthy and fertile, genotyping of progeny obtained from ⌬4/؉ interbreeding indicated that ⌬4/⌬4 embryos died between embryonic days 4.5 and 7.5. These data demonstrate that Pcsk5 is an essential gene.

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