Function and distribution of circulating human PCSK9 expressed extrahepatically in transgenic mice - PubMed (original) (raw)

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

Yi Luo et al. J Lipid Res. 2009 Aug.

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 specificity, 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 high-fat, 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 also exhibited heterogeneity and individual variability in the percentage of PCSK9 in free form and in large complexes. We provide strong evidence to support that human PCSK9 proteins secreted from extrahepatic tissue are able to promote LDLR degradation in liver and increase plasma LDL. Our data also suggest that LDLR protein regulation by PCSK9 has tissue specificity, with liver being the most responsive tissue.

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Figures

Fig. 1.

Fig. 1.

Human PCSK9 transgene expressed predominately in kidney in transgenic mice. A: mRNA expression of hPCSK9 transgene and endogenous mouse PCSK9 in liver, kidney, and adrenal. Gene expression was analyzed by quantitative PCR as described in Materials and Methods. The relative gene expression was presented relative to endogenous mPCSK9 of wild-type mice. n = 3 for each group. B: Human PCSK9 protein levels in liver, kidney, and adrenal of transgenic mice. Pooled tissue extracts from wild-type and transgenic mice fed with chow diet were subjected to immunoblot analysis visualized by immunoblotting using antibody against V5-tag (n = 3). PCSK9 precursor, mature form, and a truncated form (*) are detected. C: Plasma levels of human PCSK9 in transgenic mice. Plasma PCSK9 levels were measured by ELISA as described in Materials and Methods. PCSK9, proprotein convertase subtilisin/kexin type 9.

Fig. 2.

Fig. 2.

Effects of overexpressed human PCSK9 on LDLR protein levels in transgenic mice fed with chow diet. A: hPCSK9 expression markedly diminished liver LDLR proteins. Liver extracts from chow fed mice were pooled for immunoblot analysis. n = 3 for each group. B: hPCSK9 expression reduced kidney LDLR protein levels and did not alter adrenal LDLR proteins. Tissue extracts were pooled from chow diet fed wild-type and hPCSK9 trangenic mice as described in Materials and Methods and were subjected to immunoblot analysis. n = 3 for each group. LDLR, LDL receptor; PCSK9, proprotein convertase subtilisin/kexin type 9.

Fig. 3.

Fig. 3.

Effects of human PCSK9 transgene expression on plasma levels of total cholesterol, ApoB, and lipoproteins in transgenic mice. Plasma total cholesterol levels (A) and ApoB levels (B) in wild-type and hPCSK9 transgenic mice fed with either regular chow or HFHC diet for 8 weeks (n = 6 for each group). C: FPLC analysis of plasma lipoprotein profiles in wild-type and hPCSK9 transgenic mice fed with regular chow diet or HFHC diet. Pooled plasma from each group was fractionated by FPLC and total cholesterol levels in each fraction were measured to represent the lipoprotein profile. D: hPCSK9 expression did not alter liver total cholesterol and triglyceride levels. Liver lipid levels were analyzed from liver homogenate of wild-type and hPCSK9 transgenic mice fed with regular chow or HFHC diet. FPLC, fast-protein liquid chromatography; HFHC, high fat, high cholesterol; PCSK9, proprotein convertase subtilisin/kexin type 9.

Fig. 4.

Fig. 4.

Size distribution of plasma PCSK9. A: Distribution profile of plasma hPCSK9 in transgenic mice. Pooled plasma samples from transgenic mice fed with either chow (upper panel) or HFHC diet (lower panel) were analyzed by FPLC fractionation. PCSK9 protein levels in FPLC fractions were determined by ELISA and cographed with the cholesterol profiles. 2ug of purified human PCSK9 protein was fractionated similarly to define the size distribution of free proteins. The size distribution of hPCSK9 was categorized into three forms: LDL-sized large complex, intermediate complex, and free form. (B) Representative size distribution profiles of plasma PCSK9 from two healthy human subjects. Human plasma samples were analyzed by FPLC fractionation, and PCSK9 protein levels were determined by ELISA. FPLC, fast-protein liquid chromatography; HFHC, high fat, high cholesterol; PCSK9, proprotein convertase subtilisin/kexin type 9; TC, total cholesterol.

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