Abstract 19556: Hypercholesterolemia in a Pcsk5 Mouse Mutant Show P5 Proteolytic Degradation of P9 is Essential for Normal Regulation of Serum Cholesterol
Background: A mouse mutant was recovered with phenotypes comprising all those seen in VACTERL association (vertebral anomalies, anal atresia, cardiac defects, tracheo-esophageal fistula, renal and limb defects). The mutation comprises a deletion spanning part of the catalytic domain of proprotein convertase Pcsk5 (P5), causing loss of proteolytic activity. Given P5 and the related convertase Pcsk9 (P9) both play important roles in cholesterol homeostasis, we analyzed blood lipids in near term (E18.5) mutants.
Methods and Results: Plasma cholesterol increased in P5 mutants (1.10±0.08g/L) vs wildtype (WT; 0.49±0.02g/L) (p<0.001). FPLC showed large increases in plasma LDL-cholesterol. Western blotting revealed apoB100/B48 increased (210±30a.u vs. 100±15a.u in WT, p<0.01). Also increased was the inactive 40 kDa cleaved form of endothelial lipase. ELISA showed increased plasma P9 levels (1015±89 ng/ml vs 476±49 ng/ml in WT, P<0.001), with blotting showing virtual absence of the cleaved form of P9. As P9 is a substrate of P5, this confirmed the loss of P5 proteolytic activity in P5 mutants. LDL receptor (LDLr) in the mutant liver was reduced (60%), consistent with the role of P9 in LDLr turnover. Crossing the P9 knockout (KO) allele into our P5 mutants showed rescue of the cholesterol phenotype in double homozygous KO/mutant mice (p<0.05 for P5/P9 double mutants vs. all other groups, n=15 per group; WT 0.49+/-0.02, homozygous P9KO 0.58 +/-0.06, P5 mutant 1.10 +/- 0.08; P9KO/P5 mutants 0.62 +/- 0.09). P9 was not detected in the plasma of the P9KO or the double mutants, while expressions of LDLr showed a modest increase (15% in P9KO, 19% in P9KO/P5). As P5KO mouse embryos die at E7.5, the term viability of our P5 mutants suggest additional noncatalytic P5 function. Given VACTERL phenotypes overlap those caused by defects in Shh signaling transduced by the cilium, we examined downstream effectors of Shh but found no change. Unexpectedly, immunostaining showed P5 localization to the primary cilium.
Conclusion: These results indicate an essential role for P5 proteolysis of P9 in regulating LDLr and serum levels of LDL-cholesterol and apoB. Further studies are warranted to investigate the potential role of the cilium in P5 modulation of cholesterol.
- © 2010 by American Heart Association, Inc.