Function and distribution of circulating human PCSK9 expressed extrahepatically in transgenic mice

Abstract

tions in PCSK9 cause hypercholesterolemia in humans ( 1-4 ), whereas loss-of-function mutations in humans lead to a reduction in LDL cholesterol and a marked decrease in the risk of coronary heart disease ( 5-7 ). PCSK9 is a member of the subtilisin serine protease family and the proteinase K subfamily. The protein is synthesized as a proprotein that autocatalytically cleaves to generate mature protein ( 8 ). PCSK9 protein is secreted as a complex containing cleaved N-terminal prodomain, which remains associated with the catalytic domain ( 8 ). The catalytic activity is required for PCSK9 maturation and secretion ( 9 ) but appears not to be essential for the reduction of LDLR (LDL receptor) by secreted PCSK9 ( 10 ).PCSK9 is predominantly expressed in liver, intestine, and to a lesser extent, in kidney ( 8 ). PCSK9 gene expression is regulated by cholesterol via sterol regulatory element-binding protein (SREBP) pathways ( 11,12 ). In mice, PCSK9 gene expression is downregulated by dietary cholesterol and dramatically upregulated by hepatic overexpression of the transactivation domain of SREBP-1a and SREBP-2 ( 12 ). Deletion of PCSK9 in mice leads to elevated liver LDLR protein (but not mRNA) and to reduced plasma total cholesterol, mainly HDL cholesterol ( 13 ). Furthermore, PCSK9 defi ciency increases LDL clearance and enhances the response to statin ( 13 ). Knockdown of PCSK9 by antisense approach also decreases total cholesterol in high-fat-fed mice in an LDLR dependent manner ( 14 ). Hepatic overexpression of murine or human PCSK9 by adenovirus ( 9, 15, 16 ) or transgene ( 17 ) results in the reduction of liver LDLR protein and elevation of LDL cholesterol. Transgenic mice overexpressing PCSK9 in liver accumulated PCSK9 to a level of 100-400 g/ml in plasma and reduced liver LDLR protein in wild-type mice that are parabiotically Abstract Proprotein convertase subtilisin/kexin type 9 (PCSK9) is predominantly expressed in liver and regulates cholesterol metabolism by down regulating liver LDL receptor (LDLR) proteins. Here we report transgenic overexpression of human PCSK9 in kidney increased plasma levels of PCSK9 and subsequently led to a dramatic reduction in liver LDLR proteins. The regulation of LDLR by PCSK9 displayed tissue specifi city, with liver being the most responsive tissue. Even though the PCSK9 transgene was highly expressed in kidney, LDLR proteins were suppressed to a lower extent in this tissue than in liver. Adrenal LDLR proteins were not regulated by elevated plasma PCSK9. hPCSK9 transgene expression and subsequent reduction of liver LDLR led to increases in plasma total cholesterol, LDL cholesterol, and ApoB, which were further increased by a highfat, high-cholesterol diet. We also observed that the size distribution of hPCSK9 in transgenic mouse plasma was heterogeneous. In chow-fed mice, the majority of PCSK9 proteins were in free forms; however, feeding a high-fat, high-cholesterol diet resulted in a shift of hPCSK9 distribution toward larger complexes. PCSK9 distribution in human plasma...

Dissection of the Endogenous Cellular Pathways of PCSK9-induced Low Density Lipoprotein Receptor Degradation

“…Although hepatocyte-specific transgenic mice weakly overexpressing PCSK9 (3-fold) exhibit no significant change in circulating LDL-cholesterol, a ϳ30-fold overexpression increased it by ϳ5-fold (20). Data from transgenic mice expressing very high levels of human PCSK9 in mouse kidney (35) or liver (16), or continuous infusions of recipient WT mice with recombinant PCSK9 (36) indicated that Ͼ100 nM amounts of circulating PCSK9 are required to affect liver LDLR protein levels significantly, without affecting extrahepatic LDLR. In that context, we recently showed that annexin A2 inhibits the effect of extracellular PCSK9 on LDLR, and in view of its absence from hepatocytes in vivo, it may exert its inhibitory effect in extrahepatic tissues (14).…”

Pcsk9

“…111 In addition, transgenic mice expressing human PCSK9 mainly in the kidney resulted in LDLR degradation mostly in the liver but not in adrenals. 112,113 Thus, it is probable that the high levels of annexin A2 in adrenals are responsible for the refractoriness of the LDLR in this tissue to the action of PCSK9. 110,111 Another, not mutually exclusive, possibility is that in refractory tissues, such as the adrenals, the complex [PCSK9≡LDLR] enters endosomes but does not traffic to lysosomes and may actually recycle back to the cell surface.…”

PCSK9 reduces the protein levels of the LDL receptor in mouse brain during development and after ischemic stroke

“…Injection of purifi ed PCSK9 or a recombinant adenovirus overexpressing PCSK9 in mice reduced the protein levels of the LDLR in liver, lung, kidney, and small intestine, but not in the adrenal glands ( 11,12 ). In addition, upon overexpression in kidney, the secreted plasma PCSK9 promotes LDLR degradation mostly in the liver and raises plasma LDL ( 49 ). Altogether, our data and those in the literature show that not all tissues respond equally to local or circulating PCSK9 and that in adult brain LDLR does not respond to circulating PCSK9.…”