The discovery of autosomal dominant hypercholesterolemic patients with mutations in the PCSK9 gene, encoding the proprotein convertase NARC-1, resulting in the missense mutations suggested a role in low density lipoprotein (LDL) metabolism. We show that the endoplasmic reticulum-localized proNARC-1 to NARC-1 zymogen conversion is Ca 2؉ -independent and that within the zymogen autocatalytic processing site SSVFAQ2SIP Val at P4 and Pro at P3 are critical. The S127R and D374Y mutations result in ϳ50 -60% and >98% decrease in zymogen processing, respectively. In contrast, the double [D374Y ؉ N157K], F216L, and R218S natural mutants resulted in normal zymogen processing. The cell surface LDL receptor (LDLR) levels are reduced by 35% in lymphoblasts of S127R patients. The LDLR levels are also reduced in stable HepG2 cells overexpressing NARC-1 or its natural mutant S127R, and this reduction is abrogated in the presence of 5 mM ammonium chloride, suggesting that overexpression of NARC-1 increases the turnover rate of the LDLR. Adenoviral expression of wild type human NARC-1 in mice resulted in a maximal ϳ9-fold increase in circulating LDL cholesterol, while in LDLR(؊/؊) mice a delayed ϳ2-fold increase in LDL cholesterol was observed. In conclusion, NARC-1 seems to affect both the level of LDLR and that of circulating apoB-containing lipoproteins in an LDLR-dependent and -independent fashion.The mammalian proprotein convertases constitute a family of 9 serine proteinases related to bacterial subtilisin. These include the 7 basic amino acid-specific convertases known as PC1/PC3, PC2, furin, PC4, PACE4, PC5/PC6, PC7/LPS (1, 2) and the two enzymes cleaving at nonbasic residues SKI-1/S1P (3, 4) and NARC-1/PCSK9 (5). These proteases are implicated in the limited proteolysis of precursors of secretory proteins that regulate a variety of cellular functions, including cellular growth, adhesion, differentiation, cell to cell communications, and endocrine/paracrine functions (6, 7). Published gene knockout analyses (reviewed in Ref. 8) revealed that only furin (9) and SKI-1/S1P (10) are embryonic lethal. So far, nothing is known about the phenotype consequences of NARC-1 1 knockout in mice. The cDNA of the enzyme NARC-1 was cloned during pharmaceutical screening of mRNAs up-regulated following induction of neural apoptosis by serum withdrawal, and the encoded protein was called "neural apoptosis regulated convertase 1" (NARC-1) (11). We characterized this enzyme, and we showed that it is highly expressed in liver and small intestine and that specific mutations in the prosegment of NARC-1 completely abrogated its autocatalytic processing (5). We further showed that overexpression of NARC-1 enhances neurogenesis of progenitor brain telencephalic cells. The sustained expression of NARC-1 in liver and small intestine and its transient expression in telencephalon, kidney, and cerebellum beg for the identification of its physiological substrates, which are still unknown.Human genetic point mutations resulting in pathology have been rep...
Scavenger receptor class B, type I (SR-BI), the Scarb1 gene product, is a receptor associated with cholesteryl ester uptake from high-density lipoproteins (HDL), which drives cholesterol movement from peripheral tissues toward the liver for excretion, and, consequently, Scarb1 null mice are prone to atherosclerosis. Because studies have linked atherosclerosis incidence with osteoporosis, we characterized the bone metabolism in these mice. Bone morphometry was assessed through microcomputed tomography and histology. Marrow stromal cells (MSCs) were used to characterize influence of endogenous SR-BI in cell functions. Total and HDL-associated cholesterol in null mice were increased by 32-60%, correlating with its role in lipoprotein metabolism. Distal metaphyses from 2- and 4-mo-old null mice showed correspondingly 46 and 37% higher bone volume fraction associated with a higher number of trabeculae. Histomorphometric analyses in 2-mo-old null male mice revealed 1.42-fold greater osteoblast surface, 1.37-fold higher percent mineralizing surface, and 1.69-fold enhanced bone formation rate. In vitro assays for MSCs from null mice revealed 37% higher proliferation rate, 48% more alkaline phosphatase activity, 70% greater mineralization potential and a 2-fold osterix (Sp7) expression, yet a 0.5-fold decrease in caveolin-1 (Cav1) expression. Selective uptake levels of HDL-associated cholesteryl oleate and estradiol were similar between MSC from wild-type and Scarb1 null mice, suggesting that its contribution to this process is not its main role in these cells. However, Scarb1 knockout stunted the HDL-dependent regulation of Cav1 genic expression. Scarb1 null mice are not prone to osteoporosis but show higher bone mass associated with enhanced bone formation.
Lipoproteins transport many vitamins and hormones that have been shown to be necessary for bone formation. However, the metabolism of LDL and HDL 3 by bone-forming osteoblastic cells remains unknown. Here we report that osteoblastic cells express scavenger receptors of class B that are implicated in the uptake of cholesterol and estradiol from LDL and HDL 3 . Introduction:The bone tissue is continuously remodeled, and its integrity requires a balance between osteoclastic bone resorption and osteoblastic bone formation. Recent studies have reported the importance of triglyceride-rich lipoproteins for the delivery of lipophilic vitamins necessary for normal bone metabolism. However, the ability of osteoblastic cells to process low-and high-density lipoproteins (LDL and HDL 3 ) and the receptors involved remain unknown. Materials and Methods: Binding, competition, degradation, and selective uptake assays with LDL and HDL 3 radiolabeled in their protein and lipid moieties or with [
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