Obesity and ethnicity alter gene expression in skin.

Obesity is accompanied by dysfunction of many organs, but effects on the skin have received little attention. We studied differences in epithelial thickness by histology and gene expression by Affymetrix gene arrays and PCR in the skin of 10 obese (BMI 35-50) and 10 normal weight (BMI 18.5-26.9) postmenopausal women paired by age and ethnicity. Epidermal thickness did not differ with obesity but the expression of genes encoding proteins associated with skin blood supply and wound healing were altered. In the obese, many gene expression pathways were broadly downregulated and subdermal fat showed pronounced inflammation. There were no changes in skin microbiota or metabolites. African American subjects differed from European Americans with a trend to increased epidermal thickening. In obese African Americans, compared to obese European Americans, we observed altered gene expression that may explain known differences in water content and stress response. African Americans showed markedly lower expression of the gene encoding the cystic fibrosis transmembrane regulator characteristic of the disease cystic fibrosis. The results from this preliminary study may explain the functional changes found in the skin of obese subjects and African Americans.

Effects of Rapid Weight Loss on Systemic and Adipose Tissue Inflammation and Metabolism in Obese Postmenopausal Women.

CONTEXT: Obesity is associated with subclinical white adipose tissue inflammation, as defined by the presence of crown-like structures (CLSs) consisting of dead or dying adipocytes encircled by macrophages. In humans, bariatric surgery-induced weight loss leads to a decrease in CLSs, but the effects of rapid diet-induced weight loss on CLSs and metabolism are unclear. OBJECTIVE: To determine the effects of rapid very-low-calorie diet-induced weight loss on CLS density, systemic biomarkers of inflammation, and metabolism in obese postmenopausal women. DESIGN: Prospective cohort study. SETTING: Rockefeller University Hospital, New York, NY. PARTICIPANTS: Ten obese, postmenopausal women with a mean age of 60.6 years (standard deviation, ±3.6 years). MAIN OUTCOME MEASURES: Effects on CLS density and gene expression in abdominal subcutaneous adipose tissue, cardiometabolic risk factors, white blood count, circulating metabolites, and oxidative stress (urinary isoprostane-M) were measured. RESULTS: Obese subjects lost approximately 10% body weight over a mean of 46 days. CLS density increased in subcutaneous adipose tissue without an associated increase in proinflammatory gene expression. Weight loss was accompanied by decreased fasting blood levels of high-sensitivity C-reactive protein, glucose, lactate, and kynurenine, and increased circulating levels of free fatty acids, glycerol, ß-hydroxybutyrate, and 25 hydroxyvitamin D. Levels of urinary isoprostane-M declined. CONCLUSION: Rapid weight loss stimulated lipolysis and an increase in CLS density in subcutaneous adipose tissue in association with changes in levels of circulating metabolites, and improved systemic biomarkers of inflammation and insulin resistance. The observed change in levels of metabolites (i.e., lactate, ß-hydroxybutyrate, 25 hydroxyvitamin D) may contribute to the anti-inflammatory effect of rapid weight loss.

A randomized clinical trial in vitamin D-deficient adults comparing replenishment with oral vitamin D3 with narrow-band UV type B light: effects on cholesterol and the transcriptional profiles of skin and blood.

Background: Vitamin D deficiency, defined as a serum 25-hydroxyvitamin D [25(OH)D] concentration <20 ng/mL, is correlated with a more atherogenic lipid profile. However, oral vitamin D supplementation does not lower LDL-cholesterol concentrations or raise HDL-cholesterol concentrations. This uncoupling between association and causation may result from a failure of oral vitamin D to mimic the effect of dermally synthesized vitamin D in response to ultraviolet type B (UVB) light.Objective: We tested the hypothesis that, in vitamin D-deficient adults, the replenishment of vitamin D with UVB exposure would lower LDL-cholesterol concentrations compared with the effect of oral vitamin D3 supplementation.Design: We performed a randomized clinical trial in vitamin D-deficient adults and compared vitamin D replenishment between subjects who received oral vitamin D3 (n = 60) and those who received narrow-band UVB exposure (n = 58) ≤6 mo.Results: There was no difference in the change from baseline LDL-cholesterol concentrations between oral vitamin D3 and UVB groups (difference in median of oral vitamin D3 minus that of UVB: 1.5 mg/dL; 95% CI: -5.0, 7.0 mg/dL). There were also no differences within groups or between groups for changes in total or HDL cholesterol or triglycerides. Transcriptional profiling of skin and blood, however, revealed significant upregulation of immune pathway signaling with oral vitamin D3 but significant downregulation with UVB.Conclusions: Correcting vitamin D deficiency with either oral vitamin D3 or UVB does not improve the lipid profile. Beyond cholesterol, these 2 modalities of raising 25(OH)D have disparate effects on gene transcription. This trial was registered at clinicaltrials.gov as NCT01688102.

Noninvasive Detection of Inflammatory Changes in White Adipose Tissue by Label-Free Raman Spectroscopy.

White adipose tissue inflammation (WATi) has been linked to the pathogenesis of obesity-related diseases, including type 2 diabetes, cardiovascular disease, and cancer. In addition to the obese, a substantial number of normal and overweight individuals harbor WATi, putting them at increased risk for disease. We report the first technique that has the potential to detect WATi noninvasively. Here, we used Raman spectroscopy to detect WATi with excellent accuracy in both murine and human tissues. This is a potentially significant advance over current histopathological techniques for the detection of WATi, which rely on tissue excision and, therefore, are not practical for assessing disease risk in the absence of other identifying factors. Importantly, we show that noninvasive Raman spectroscopy can diagnose WATi in mice. Taken together, these results demonstrate the potential of Raman spectroscopy to provide objective risk assessment for future cardiometabolic complications in both normal weight and overweight/obese individuals.

Altered expression of Raet1e, a major histocompatibility complex class 1-like molecule, underlies the atherosclerosis modifier locus Ath11 10b.

RATIONALE: Quantitative trait locus mapping of an intercross between C57.Apoe⁻/⁻ and FVB.Apoe⁻/⁻ mice revealed an atherosclerosis locus controlling aortic root lesion area on proximal chromosome 10, Ath11. In a previous work, subcongenic analysis showed Ath11 to be complex with proximal (10a) and distal (10b) regions. OBJECTIVE: To identify the causative genetic variation underlying the atherosclerosis modifier locus Ath11 10b. METHODS AND RESULTS: We now report subcongenic J, which narrows the 10b region to 5 genes, Myb, Hbs1L, Aldh8a1, Sgk1, and Raet1e. Sequence analysis of these genes revealed no amino acid coding differences between the parental strains. However, comparing aortic expression of these genes between F1.Apoe⁻/⁻ Chr10SubJ((B/F)) and F1.Apoe⁻/⁻ Chr10SubJ((F/F)) uncovered a consistent difference only for Raet1e, with decreased, virtually background, expression associated with increased atherosclerosis in the latter. The key role of Raet1e was confirmed by showing that transgene-induced aortic overexpression of Raet1e in F1.Apoe⁻/⁻ Chr10SubJ((F/F)) mice decreased atherosclerosis. Promoter reporter constructs comparing C57 and FVB sequences identified an FVB mutation in the core of the major aortic transcription start site abrogating activity. CONCLUSIONS: This nonbiased approach has revealed Raet1e, a major histocompatibility complex class 1-like molecule expressed in lesional aortic endothelial cells and macrophage-rich regions, as a novel atherosclerosis gene and represents one of the few successes of the quantitative trait locus strategy in complex diseases.

STARD4 knockdown in HepG2 cells disrupts cholesterol trafficking associated with the plasma membrane, ER, and ERC.

STARD4, a member of the evolutionarily conserved START gene family, has been implicated in the nonvesicular intracellular transport of cholesterol. However, the direction of transport and the membranes with which this protein interacts are not clear. We present studies of STARD4 function using small hairpin RNA knockdown technology to reduce STARD4 expression in HepG2 cells. In a cholesterol-poor environment, we found that a reduction in STARD4 expression leads to retention of cholesterol at the plasma membrane, reduction of endoplasmic reticulum-associated cholesterol, and decreased ACAT synthesized cholesteryl esters. Furthermore, D4 KD cells exhibited a reduced rate of sterol transport to the endocytic recycling compartment after cholesterol repletion. Although these cells displayed normal endocytic trafficking in cholesterol-poor and replete conditions, cell surface low density lipoprotein receptor (LDLR) levels were increased and decreased, respectively. We also observed a decrease in NPC1 protein expression, suggesting the induction of compensatory pathways to maintain cholesterol balance. These data indicate a role for STARD4 in nonvesicular transport of cholesterol from the plasma membrane and the endocytic recycling compartment to the endoplasmic reticulum and perhaps other intracellular compartments as well.

Hyperglycemic Ins2AkitaLdlr⁻/⁻ mice show severely elevated lipid levels and increased atherosclerosis: a model of type 1 diabetic macrovascular disease.

Accelerated atherosclerosis is the leading cause of death in type 1 diabetes, but the mechanism of type 1 diabetes-accelerated atherosclerosis is not well understood, in part due to the lack of a good animal model for the long-term studies required. In an attempt to create a model for studying diabetic macrovascular disease, we have generated type 1 diabetic Akita mice lacking the low density lipoprotein receptor (Ins2(Akita)Ldlr⁻/⁻). Ins2(Akita)Ldlr⁻/⁻ mice were severely hyperglycemic with impaired glucose tolerance. Compared with Ldlr⁻/⁻ mice, 20-week-old Ins2(Akita)Ldlr⁻/⁻ mice fed a 0.02% cholesterol AIN76a diet showed increased plasma triglyceride and cholesterol levels, and increased aortic root cross-sectional atherosclerotic lesion area [224% (P < 0.001) in males and 30% (P < 0.05) in females]. Microarray and quantitative PCR analyses of livers from Ins2(Akita)Ldlr⁻/⁻ mice revealed altered expression of lipid homeostatic genes, including sterol-regulatory element binding protein (Srebp)1, liver X receptor (Lxr)α, Abca1, Cyp7b1, Cyp27a1, and Lpl, along with increased expression of pro-inflammatory cytokine genes, including interleukin (Il)1α, Il1ß, Il2, tumor necrosis factor (Tnf)α, and Mcp1. Immunofluorescence staining showed that the expression levels of Mcp1, Tnfα, and Il1ß were also increased in the atherosclerotic lesions and artery walls of Ins2(Akita)Ldlr⁻/⁻ mice. Thus, the Ins2(Akita)Ldlr⁻/⁻ mouse appears to be a promising model for mechanistic studies of type 1 diabetes-accelerated atherosclerosis.

Trib1 is a lipid- and myocardial infarction-associated gene that regulates hepatic lipogenesis and VLDL production in mice.

Recent genome-wide association studies have identified a genetic locus at human chromosome 8q24 as having minor alleles associated with lower levels of plasma triglyceride (TG) and LDL cholesterol (LDL-C), higher levels of HDL-C, as well as decreased risk for myocardial infarction. This locus contains only one annotated gene, tribbles homolog 1 (TRIB1), which has not previously been implicated in lipoprotein metabolism. Here we demonstrate a role for Trib1 as a regulator of lipoprotein metabolism in mice. Hepatic-specific overexpression of Trib1 reduced levels of plasma TG and cholesterol by reducing VLDL production; conversely, Trib1-knockout mice showed elevated levels of plasma TG and cholesterol due to increased VLDL production. Hepatic Trib1 expression was inversely associated with the expression of key lipogenic genes and measures of lipogenesis. Thus, we provide functional evidence for what we believe to be a novel gene regulating hepatic lipogenesis and VLDL production in mice that influences plasma lipids and risk for myocardial infarction in humans.

The mouse atherosclerosis locus at chromosome 10 (Ath11) acts early in lesion formation with subcongenic strains delineating 2 narrowed regions.

OBJECTIVE: Ath11, an atherosclerosis susceptibility locus on proximal chromosome 10 (0 to 21 cM) revealed in a cross between apolipoprotein E deficient C57BL/6 (B6) and FVB mice, was recently confirmed in congenic mice. The objectives of this study were to assess how Ath11 affects lesion development and morphology, to determine aortic gene expression in congenics, and to narrow the congenic interval. METHODS AND RESULTS: Assessing lesion area over time in congenic mice showed that homozygosity for the FVB allele increased lesion area at 6 weeks persisting through to 24 weeks of age. Staining of aortic root sections at 16 weeks did not reveal obvious differences between congenics. Aortic expression-array analysis at 6 weeks revealed 97 genes that were >2-fold regulated, including 1 gene in the quantitative trait locus interval, Aldh8a1, and 2 gene clusters regulated by Hnf4alpha and Esr1. Analysis of lesion area in 11 subcongenic strains revealed 2 narrowed regions, 10a (21 genes), acting in females, and 10b (7 genes), acting in both genders. CONCLUSIONS: Ath11 appears to act early in lesion formation, with significant effects on aortic gene expression. This quantitative trait locus is genetically complex, containing a female-specific region 10a from 0 to 7.3 megabases (21 genes) and a gender-independent region 10b from 20.1 to 21.9 megabases (7 genes).

Defective phagocytosis of apoptotic cells by macrophages in atherosclerotic lesions of ob/ob mice and reversal by a fish oil diet.

RATIONALE: The complications of atherosclerosis are a major cause of death and disability in type 2 diabetes. Defective clearance of apoptotic cells by macrophages (efferocytosis) is thought to lead to increased necrotic core formation and inflammation in atherosclerotic lesions. OBJECTIVE: To determine whether there is defective efferocytosis in a mouse model of obesity and atherosclerosis. METHODS AND RESULTS: We quantified efferocytosis in peritoneal macrophages and in atherosclerotic lesions of obese ob/ob or ob/ob;Ldlr(-/-) mice and littermate controls. Peritoneal macrophages from ob/ob and ob/ob;Ldlr(-/-) mice showed impaired efferocytosis, reflecting defective phosphatidylinositol 3-kinase activation during uptake of apoptotic cells. Membrane lipid composition of ob/ob and ob/ob;Ldlr(-/-) macrophages showed an increased content of saturated fatty acids (FAs) and decreased omega-3 FAs (eicosapentaenoic acid and docosahexaenoic acid) compared to controls. A similar defect in efferocytosis was induced by treating control macrophages with saturated free FA/BSA complexes, whereas the defect in ob/ob macrophages was reversed by treatment with eicosapentaenoic acid/BSA or by feeding ob/ob mice a fish oil diet rich in omega-3 FAs. There was also defective macrophage efferocytosis in atherosclerotic lesions of ob/ob;Ldlr(-/-) mice and this was reversed by a fish oil-rich diet. CONCLUSIONS: The findings suggest that in obesity and type 2 diabetes elevated levels of saturated FAs and/or decreased levels of omega-3 FAs contribute to decreased macrophage efferocytosis. Beneficial effects of fish oil diets in atherosclerotic cardiovascular disease may involve improvements in macrophage function related to reversal of defective efferocytosis and could be particularly important in type 2 diabetes and obesity.

Activation of PXR induces hypercholesterolemia in wild-type and accelerates atherosclerosis in apoE deficient mice.

The nuclear hormone receptor pregnane X receptor (PXR; also called SXR) functions as a xenobiotic sensor to coordinately regulate xenobiotic metabolism via transcriptional regulation of xenobiotic-detoxifying enzymes and transporters. Although many clinically relevant PXR ligands have been shown to affect cholesterol levels, the role of PXR in cholesterol homeostasis and atherosclerosis has not been thoroughly investigated. Here, we report that activation of PXR by feeding the PXR agonist pregnenolone 16alpha-carbonitrile (0.02%) for 2 weeks to wild-type (WT) mice significantly increased total cholesterol levels and atherogenic lipoproteins VLDL and LDL levels, but had no effect in PXR knockout (PXR(-/-)) mice. Chronic PXR activation in atherosclerosis prone apolipoprotein E deficient (ApoE(-/-)) mice was found to decrease HDL levels and increase atherosclerotic cross-sectional lesion area at both the aortic root and in the brachiocephalic artery by 54% (P < 0.001) and 116% (P < 0.01), respectively. PXR activation significantly regulated genes in the liver involved in lipoprotein transportation and cholesterol metabolism, including CD36, ApoA-IV, and CYP39A1, in both WT and ApoE(-/-) mice. Furthermore, PXR activation can increase CD36 expression and lipid accumulation in peritoneal macrophages of ApoE(-/-) mice. In summary, PXR activation in WT mice increases levels of the atherogenic lipoproteins VLDL and LDL, whereas in ApoE(-/-) mice, PXR increases atherosclerosis, perhaps by diminishing levels of the antiatherogenic ApoA-IV and increasing lipid accumulation in macrophages.

Athsq1 is an atherosclerosis modifier locus with dramatic effects on lesion area and prominent accumulation of versican.

OBJECTIVE: Susceptibility to atherosclerosis is genetically complex, and modifier genes that do not operate via traditional risk factors are largely unknown. A mouse genetics approach can simplify the genetic analysis and provide tools for mechanistic studies. METHODS AND RESULTS: We previously identified atherosclerosis susceptibility QTL (Athsq1) on chromosome 4 acting independently of systemic risk factors. We now report confirmation of this locus in congenic strains carrying the MOLF-derived susceptibility allele in the C57BL/6J-Ldlr(-/-) genetic background. Homozygous congenic mice exhibited up to 4.5-fold greater lesion area compared to noncongenic littermates (P<0.0001). Analysis of extracellular matrix composition revealed prominent accumulation of versican, a presumed proatherogenic matrix component abundant in human lesions but almost absent in the widely-used C57BL/6 murine atherosclerosis model. The results of a bone marrow transplantation experiment suggested that both accelerated lesion development and versican accumulation are mediated, at least in part, by macrophages. Interestingly, comparative mapping revealed that the Athsq1 congenic interval contains the mouse region homologous to a widely-replicated CHD locus on human chromosome 9p21. CONCLUSIONS: These studies confirm the proatherogenic activity of a novel gene(s) in the MOLF-derived Athsq1 locus and provide in vivo evidence for a causative role of versican in lesion development.

Increased ADAM17 mRNA expression and activity is associated with atherosclerosis resistance in LDL-receptor deficient mice.

BACKGROUND: We have previously identified an atherosclerosis quantitative trait locus (QTL) on mouse chromosome (Chr) 12 in an F2-intercross of atherosclerosis-resistant FVB and atherosclerosis-susceptible C57BL/6 (B6) mice on the LDL-receptor deficient (LDL-/-) background. The aim of the present study was to identify potentially causative genes at this locus. METHODS AND RESULTS: Expression QTL (eQTL) analysis of candidate genes in livers of F2-mice revealed that a disintegrin and metalloproteinase 17 (ADAM17) mRNA expression mapped to the physical position of ADAM17 on proximal Chr12 (21.6 Mb, LOD 3.3) and colocalized with the atherosclerosis QTL. The FVB allele was associated with significantly higher ADAM17 mRNA expression (39%) than the B6 allele. Likewise, ADAM17 mRNA levels in the parental strains were significantly elevated in FVB.LDLR-/- compared to B6.LDLR-/- mice in liver, macrophages, and aorta (68%, 58%, and 32%, respectively). Reporter gene assays revealed a genetic variant that might explain these expression differences. Moreover, FVB.LDLR-/- macrophages showed 5-fold increased PMA-induced shedding of tumor necrosis factor (TNF)-alpha and 32% increased release of TNF-receptor I compared to B6.LDLR-/-. The atherosclerosis locus and expression differences were confirmed in Chr12 interval-specific congenic mice. CONCLUSIONS: Our data provide functional evidence for ADAM17 as a candidate gene of atherosclerosis susceptibility at the murine Chr12 QTL.

The protective effect of A20 on atherosclerosis in apolipoprotein E-deficient mice is associated with reduced expression of NF-kappaB target genes.

Up-regulation of inflammatory responses is considered a driving force of atherosclerotic lesion development. One key regulator of inflammation is the A20 (also called TNF-alpha-induced protein 3 or Tnfaip3) gene, which is responsible for NF-kappaB termination and maps to an atherosclerosis susceptibility locus revealed by quantitative trait locus-mapping studies at mouse proximal chromosome 10. In the current study, we examined the role of A20 in atherosclerotic lesion development. At the aortic root lesion size was found to be increased in C57BL/6 (BG) apolipoprotein E-deficient (ApoE(-/-)) mice haploinsufficient for A20, compared with B6 ApoE(-/-) controls that expressed A20 normally (60% in males and 23% in females; P < 0.001 and P < 0.05, respectively). In contrast, lesion size was found to be decreased in F(1) (B6 x FVB/N) mice overexpressing A20 by virtue of containing an A20 BAC transgene compared with nontransgenic controls (30% in males, P < 0.001, and 17% in females, P = 0.02). The increase in lesions in the A20 haploinsufficient mice correlated with increased expression of proatherosclerotic NF-kappaB target genes, such as vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and macrophage-colony-stimulating factor, and elevated plasma levels of NF-kappaB-driven cytokines. These findings suggest that A20 diminishes atherosclerosis by decreasing NF-kappaB activity, thereby modulating the proinflammatory state associated with lesion development.

Differential gene regulation of StarD4 and StarD5 cholesterol transfer proteins. Activation of StarD4 by sterol regulatory element-binding protein-2 and StarD5 by endoplasmic reticulum stress.

The StarD4 and StarD5 proteins share approximately 30% identity, and each is a steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain. We previously showed StarD4 expression is sterol-repressed, consistent with regulation by sterol regulatory element-binding proteins (SREBPs), whereas StarD5 is not sterol-regulated. Here we further address the regulation and function of StarD4 and StarD5. Unlike StAR, the START family prototype, StarD4 and StarD5 were not induced by steroidogenic stimuli in Leydig cells. However, StarD4 and StarD5 showed StAR-like activity in a cell culture steroidogenesis assay, indicating cholesterol transfer. In transgenic mice expressing active SREBPs, StarD4 was predominantly activated by SREBP-2 rather than SREBP-1a. The mouse and human StarD4 proximal promoters share approximately 70% identity, including several potential sterol regulatory elements (SREs). Reporters driven by the StarD4 promoter from either species were transfected into NIH-3T3 cells, and reporter activity was highly repressed by sterols. Site-directed mutagenesis of potential SREs identified a conserved functional SRE in the mouse (TCGGTCCAT) and human (TCATTCCAT) promoters. StarD5 was not sterol-repressed via SREBPs nor was it sterol-activated via liver X receptors (LXRs). Even though StarD4 and StarD5 were not LXR targets, their overexpression stimulated LXR reporter activity, suggesting roles in cholesterol metabolism. StarD5 expression increased 3-fold in free cholesterol-loaded macrophages, which activate the endoplasmic reticulum (ER) stress response. When NIH-3T3 cells were treated with agents to induce ER stress, StarD5 expression increased 6-8-fold. Because StarD4 is regulated by sterols via SREBP-2, whereas StarD5 is activated by ER stress, they likely serve distinct functions in cholesterol metabolism.

Proprotein convertase subtilisin kexin 9: the third locus implicated in autosomal dominant hypercholesterolemia.

PURPOSE OF REVIEW: Autosomal dominant hypercholesterolemia is a genetic disease in which patients have elevated LDL cholesterol levels and premature atherosclerosis. Mutations in the LDL receptor and its ligand apolipoprotein B are causative for autosomal dominant hypercholesterolemia, and the study of this pathway has been crucial to understanding LDL metabolism and receptor-mediated endocytosis in general. Recently, families were identified with a clinical diagnosis of autosomal dominant hypercholesterolemia, but without linkage to the LDL receptor or apolipoprotein B genes. Identification and study of the causative genes in these families should provide additional insights into LDL metabolism. RECENT FINDINGS: Recent microarray studies and database searches identified a novel member of the proprotein convertase family called proprotein convertase subtilisin kexin 9 (PCSK9). A role for PCSK9 in cholesterol metabolism was proposed from the expression studies and confirmed by the discovery that PCSK9 missense mutations were associated with a form of autosomal dominant hypercholesterolemia, Hchola3. The cellular role for PCSK9 and the mechanism behind its mutations are under study, and a role for PCSK9 in regulating LDL receptor protein levels has been demonstrated. SUMMARY: PCSK9 is the third locus implicated in autosomal dominant hypercholesterolemia (Hchola3), and it appears to play an important role in cellular cholesterol metabolism. Understanding the function of PCSK9 will be important for broadening our knowledge of LDL metabolism and may aid in the development of novel hypocholesterolemic agents.

Overexpression of PCSK9 accelerates the degradation of the LDLR in a post-endoplasmic reticulum compartment.

Proprotein convertase subtilisin kexin 9 (PCSK9) is a member of the subtilisin serine protease family with an important role in cholesterol metabolism. PCSK9 expression is regulated by dietary cholesterol in mice and cellular sterol levels in cell culture via the sterol regulatory element binding protein transcription factors, and mutations in PCSK9 are associated with a form of autosomal dominant hypercholesterolemia. Overexpression of PCSK9 in mice leads to increased total and low-density lipoprotein (LDL) cholesterol levels because of a decrease in hepatic LDL receptor (LDLR) protein with normal mRNA levels. To study the mechanism, PCSK9 was overexpressed in human hepatoma cells, HepG2, by adenovirus. Overexpression of PCSK9 in HepG2 cells caused a decrease in whole-cell and cell-surface LDLR levels. PCSK9 overexpression had no effect on LDLR synthesis but caused a dramatic increase in the degradation of the mature LDLR and a lesser increase in the degradation of the precursor LDLR. In contrast, overexpression of a catalytically inactive mutant PCSK9 prevented the degradation of the mature LDLR; whereas increased degradation of the precursor LDLR still occurred. The PCSK9-induced degradation of the LDLR was not affected by inhibitors of the proteasome, lysosomal cysteine proteases, aspartic acid proteases, or metalloproteases. The PCSK9-induced degradation of the LDLR was shown to require transport out of the endoplasmic reticulum. These results indicate that overexpression of PCSK9 induces the degradation of the LDLR by a nonproteasomal mechanism in a post-endoplasmic reticulum compartment.

Adenoviral-mediated expression of Pcsk9 in mice results in a low-density lipoprotein receptor knockout phenotype.

Proprotein convertase subtilisin kexin 9 (Pcsk9) is a subtilisin serine protease with a putative role in cholesterol metabolism. Pcsk9 expression is down-regulated by dietary cholesterol, and mutations in Pcsk9 have been associated with a form of autosomal dominant hypercholesterolemia. To study the function of Pcsk9 in mice, an adenovirus constitutively expressing murine Pcsk9 (Pcsk9-Ad) was used. Pcsk9 overexpression in wild-type mice caused a 2-fold increase in plasma total cholesterol and a 5-fold increase in non-high-density lipoprotein (HDL) cholesterol, with no increase in HDL cholesterol, as compared with mice infected with a control adenovirus. Fast protein liquid chromatography analysis showed that the increase in non-HDL cholesterol was due to an increase in low-density lipoprotein (LDL) cholesterol. This effect appeared to depend on the LDL receptor (LDLR) because LDLR knockout mice infected with Pcsk9-Ad had no change in plasma cholesterol levels as compared with knockout mice infected with a control adenovirus. Furthermore, whereas overexpression of Pcsk9 had no effect on LDLR mRNA levels, there was a near absence of LDLR protein in animals overexpressing Pcsk9. These results were confirmed in vitro by the demonstration that transfection of Pcsk9 in McA-RH7777 cells caused a reduction in LDLR protein and LDL binding. In summary, these results indicate that overexpression of Pcsk9 interferes with LDLR-mediated LDL cholesterol uptake. Because Pcsk9 and LDLR are coordinately regulated by cholesterol, Pcsk9 may be involved in a novel mechanism to modulate LDLR function by an alternative pathway than classic cholesterol inhibition of sterol regulatory element binding protein-mediated transcription.

A20, a regulator of NFkappaB, maps to an atherosclerosis locus and differs between parental sensitive C57BL/6J and resistant FVB/N strains.

An intercross between atherosclerosis susceptible (C57BL/6J ApoE0) and resistant (FVB/N ApoE0) mice revealed a susceptibility locus on chromosome 10 (11 cM, logarithm of odds 7.8). Surprisingly, the genotypic means for this locus revealed that heterozygosity or homozygosity for the C57BL/6J allele was associated with decreased atherosclerosis. A candidate gene in this region is A20, which is involved in the feedback suppression of NFkappaB activation induced by tumor necrosis factor alpha (TNFalpha). We sequenced the A20 gene coding region from the parental strains and found a single-nucleotide polymorphism resulting in a single amino acid exchange, Glu627Ala (C57BL/6J vs. FVB/N). This mutation introduces a putative casein kinase 2 phosphorylation site in C57BL/6J-A20 not present in FVB/N-A20. NFkappaB reporter gene assays showed that this amino acid change results in less effective termination of TNFalpha-stimulated NFkappaB activation by C57BL/6J-A20. In accordance, the TNFalpha-induced expression of NFkappaB target genes (A20, IkappaBalpha) in vascular smooth muscle cells was prolonged in cells isolated from C57BL/6J compared with FVB/N mice. In light of the genotypic means for atherosclerosis at the chromosome 10 locus in F2 mice from this intercross, the observations now reported suggest that prolonged expression of genes induced by NFkappaB might be antirather than proatherogenic.

Localization of the PE methylation pathway and SR-BI to the canalicular membrane: evidence for apical PC biosynthesis that may promote biliary excretion of phospholipid and cholesterol.

To better understand the regulation of biliary phospholipid and cholesterol excretion, canalicular membranes were isolated from the livers of C57BL/6J mice and abundant proteins separated by SDS-PAGE and identified by matrix-assisted laser desorption/ionization mass spectrometry. A prominent protein revealed by this analysis was betaine homocysteine methyltransferase (BHMT). This enzyme catalyzes the first step in a three-enzyme pathway that promotes the methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC). Immunoblotting confirmed the presence of BHMT on the canalicular membrane, failed to reveal the presence of the second enzyme in this pathway, methionine adenosyltransferase, and localized the third enzyme of the pathway, PE N-methyltransferase (PEMT). Furthermore, immunfluorescence microscopy unambiguously confirmed the localization of PEMT to the canalicular membrane. These findings indicate that a local mechanism exists in or around hepatocyte canalicular membranes to promote phosphatidylethnolamine methylation and PC biosynthesis. Finally, immunoblotting revealed the presence and immunofluorescence microscopy unambiguously localized the scavenger receptor class B type I (SR-BI) to the canalicular membrane. Therefore, SR-BI, which is known to play a role in cholesterol uptake at the hepatocyte basolateral membrane, may also be involved in biliary cholesterol excretion. Based on these findings, a model is proposed in which local canalicular membrane PC biosynthesis in concert with the phospholipid transporter mdr2 and SR-BI, promotes the excretion of phospholipid and cholesterol into the bile.