In vivo genome editing using Staphylococcus aureus Cas9.

The RNA-guided endonuclease Cas9 has emerged as a versatile genome-editing platform. However, the size of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for basic research and therapeutic applications that use the highly versatile adeno-associated virus (AAV) delivery vehicle. Here, we characterize six smaller Cas9 orthologues and show that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being more than 1 kilobase shorter. We packaged SaCas9 and its single guide RNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9 in the mouse liver. Within one week of injection, we observed >40% gene modification, accompanied by significant reductions in serum Pcsk9 and total cholesterol levels. We further assess the genome-wide targeting specificity of SaCas9 and SpCas9 using BLESS, and demonstrate that SaCas9-mediated in vivo genome editing has the potential to be efficient and specific.

Effect of K225 residue to the catalytic efficiency of Kex2 protease.

The gene encoding S. cerevisiae Kex2 protease derivative Kex2-667 (encoding the N-terminal 20th to 667th amino acid residues of Kex2 protease, containing the propeptide, catalytic domain, P domain and Ser/Thr enrichment region) and its 225th amino acid residue mutant K225L were overexpressed in Pichia pastoris. Proteases were purified by dialysis and anion exchange chromatography (Q-FF). Their properties were further investigated. For catalysis efficiency, the value of Kcat/Km of Kex2-667-K225L was 3 folds higher than that of Kex2-667. Both were quite stable at 25 °C and 37 °C after 8 h of incubation at pH5.6, while Kex2-667 remained nearly 90% of the total activity while Kex2-667-K225L remained only 80%. The stability of Kex2-667-K225L was lower than that of Kex2-667 from pH4.0 to pH9.0. Due to the mutation site K225 was located at one of the calcium ion binding sites, it resulted in a tighter calcium ion binding region, which may be the reason why the catalytic efficiency of Kex2-667-K225L was improved while the stability was a little decreased.

The Liver Clock Controls Cholesterol Homeostasis through Trib1 Protein-mediated Regulation of PCSK9/Low Density Lipoprotein Receptor (LDLR) Axis.

Disruption of the body clock has been recognized as a risk factor for cardiovascular disease. How the circadian pacemaker interacts with the genetic factors associated with plasma lipid traits remains poorly understood. Recent genome-wide association studies have identified an expanding list of genetic variants that influence plasma cholesterol and triglyceride levels. Here we analyzed circadian regulation of lipid-associated candidate genes in the liver and identified two distinct groups exhibiting rhythmic and non-rhythmic patterns of expression during light-dark cycles. Liver-specific inactivation of Bmal1 led to elevated plasma LDL/VLDL cholesterol levels as a consequence of the disruption of the PCSK9/LDL receptor regulatory axis. Ablation of the liver clock perturbed diurnal regulation of lipid-associated genes in the liver and markedly reduced the expression of the non-rhythmically expressed gene Trib1. Adenovirus-mediated rescue of Trib1 expression lowered plasma PCSK9 levels, increased LDL receptor protein expression, and restored plasma cholesterol homeostasis in mice lacking a functional liver clock. These results illustrate an unexpected mechanism through which the biological clock regulates cholesterol homeostasis through its regulation of non-rhythmic genes in the liver.

Annexin A2 reduces PCSK9 protein levels via a translational mechanism and interacts with the M1 and M2 domains of PCSK9.

Annexin A2 (AnxA2) was reported to be an extracellular endogenous inhibitor of proprotein convertase subtilisin kexin type 9 (PCSK9) activity on cell-surface LDL receptor degradation. In this study, we investigated the effect of silencing the expression of AnxA2 and PCSK9 in HepG2 and Huh7 cells to better define the role of AnxA2 in PCSK9 regulation. AnxA2 knockdown in Huh7 cells significantly increased PCSK9 protein levels as opposed to AnxA2 knockdown in HepG2 cells. However, HepG2 cells overexpressing AnxA2 had lower levels of PCSK9 protein. Overall, our data revealed a plausible new role of AnxA2 in the reduction of PCSK9 protein levels via a translational mechanism. Moreover, the C-terminal Cys/His-rich domain of PCSK9 is crucial in the regulation of PCSK9 activity, and we demonstrated by far-Western blot assay that the M1 and M2 domains are necessary for the specific interaction of PCSK9's C-terminal Cys/His-rich domain and AnxA2. Finally, we produced and purified recombinant PCSK9 from humans and mice, which was characterized and used to perform 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate LDL cell-based assays on the stable knockdown HepG2 and Huh7 cells. We also demonstrated for the first time the equipotency of human and mouse PCSK9 R218S on human cells.

A genome-wide expression quantitative trait loci analysis of proprotein convertase subtilisin/kexin enzymes identifies a novel regulatory gene variant for FURIN expression and blood pressure.

Proprotein convertase subtilisin/kexin (PCSK) enzymes cleave and convert their immature substrates into biologically active forms. Polymorphisms in the PCSK genes have been reported to associate with human diseases and phenotypes, including hypercholesterolemia and blood pressure (BP), and targeting PCSKs is considered a promising future form of drug therapy. PCSK processing is readily induced upon upregulation of the enzyme, but the genetic factors contributing to PCSK expression have not been thoroughly characterized. To gain a comprehensive understanding of the genetic regulation of PCSK expression, we performed, for the first time, a genome-wide expression quantitative trait loci (eQTL) analysis using mRNA expression in >1400 human peripheral blood samples from the Cardiovascular Risk in Young Finns Study and ca. ten million single-nucleotide polymorphisms (SNPs). The expression data showed clear expression for FURIN, PCSK5, PCSK7 and MBTPS1 (membrane-bound transcription factor peptidase, site 1) mRNAs in virtually all tested samples. A discovery analysis demonstrated a genome-wide significant (p < 5 × 10(-8)) association with the selected PCSK probes for 1024 variants, which were located at ten independent loci. Of these loci, 5/10 could be confirmed to regulate PCSK expression in two additional and independent sample sets. Finally, a phenotypic analysis demonstrated that a novel cis-eQTL SNP rs4702 for FURIN is strongly associated with both diastolic (p = 0.012) and systolic (p = 0.035) BP levels, as well as peripheral vascular resistance (p = 0.003). These findings indicate that the expression of the PCSK enzymes is regulated by genetic factors, which have biological roles in health and disease.

Effect of an RNA interference drug on the synthesis of proprotein convertase subtilisin/kexin type 9 (PCSK9) and the concentration of serum LDL cholesterol in healthy volunteers: a randomised, single-blind, placebo-controlled, phase 1 trial.

BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to LDL receptors, leading to their degradation. Genetics studies have shown that loss-of-function mutations in PCSK9 result in reduced plasma LDL cholesterol and decreased risk of coronary heart disease. We aimed to investigate the safety and efficacy of ALN-PCS, a small interfering RNA that inhibits PCSK9 synthesis, in healthy volunteers with raised cholesterol who were not on lipid-lowering treatment. METHODS: We did a randomised, single-blind, placebo-controlled, phase 1 dose-escalation study in healthy adult volunteers with serum LDL cholesterol of 3·00 mmol/L or higher. Participants were randomly assigned in a 3:1 ratio by computer algorithm to receive one dose of intravenous ALN-PCS (with doses ranging from 0·015 to 0·400 mg/kg) or placebo. The primary endpoint was safety and tolerability of ALN-PCS. Secondary endpoints were the pharmacokinetic characteristics of ALN-PCS and its pharmacodynamic effects on PCSK9 and LDL cholesterol. Study participants were masked to treatment assignment. Analysis was per protocol and we used ANCOVA to analyse pharmacodynamic endpoint data. This trial is registered with ClinicalTrials.gov, number NCT01437059. FINDINGS: Of 32 participants, 24 were randomly allocated to receive a single dose of ALN-PCS (0·015 mg/kg [n=3], 0·045 mg/kg [n=3], 0·090 mg/kg [n=3], 0·150 mg/kg [n=3], 0·250 mg/kg [n=6], or 0·400 mg/kg [n=6]) and eight to placebo. The proportions of patients affected by treatment-emergent adverse events were similar in the ALN-PCS and placebo groups (19 [79%] vs seven [88%]). ALN-PCS was rapidly distributed, with peak concentration and area under the curve (0 to last measurement) increasing in a roughly dose-proportional way across the dose range tested. In the group given 0·400 mg/kg of ALN-PCS, treatment resulted in a mean 70% reduction in circulating PCSK9 plasma protein (p<0·0001) and a mean 40% reduction in LDL cholesterol from baseline relative to placebo (p<0·0001). INTERPRETATION: Our results suggest that inhibition of PCSK9 synthesis by RNA interference (RNAi) provides a potentially safe mechanism to reduce LDL cholesterol concentration in healthy individuals with raised cholesterol. These results support the further assessment of ALN-PCS in patients with hypercholesterolaemia, including those being treated with statins. This study is the first to show an RNAi drug being used to affect a clinically validated endpoint (ie, LDL cholesterol) in human beings. FUNDING: Alnylam Pharmaceuticals.

Intestinal lipid handling: evidence and implication of insulin signaling abnormalities in human obese subjects.

OBJECTIVE: Animal models have evidenced the role of intestinal triglyceride-rich lipoprotein overproduction in dyslipidemia. However, few studies have confronted this issue in humans and disclosed the intrinsic mechanisms. This work aimed to establish whether intestinal insulin resistance modifies lipid and lipoprotein homeostasis in the intestine of obese subjects. APPROACH AND RESULTS: Duodenal specimens obtained from 20 obese subjects undergoing bariatric surgery were paired for age, sex, and body mass index with or without insulin resistance, as defined by the homeostasis model assessment of insulin resistance. Insulin signaling, biomarkers of inflammation and oxidative stress, and lipoprotein assembly were assessed. The intestine of insulin-resistant subjects showed defects in insulin signaling as demonstrated by reduced protein kinase B phosphorylation and increased p38 mitogen-activated protein kinase phosphorylation, likely as the result of high oxidative stress (evidenced by malondialdehyde and conjugated dienes) and inflammation (highlighted by nuclear factor-κB, tumor necrosis factor-α, interleukin-6, intercellular adhesion molecule-1, and cyclooxygenase-2). Enhanced de novo lipogenesis rate and apolipoprotein B-48 biogenesis along with exaggerated triglyceride-rich lipoprotein production were observed, concomitantly with the high expression levels of liver and intestinal fatty acid-binding proteins and microsomal transfer protein. The presence of an aberrant intracellular cholesterol transport/metabolism was also suggested by the reduced expression of ATP-binding cassette A1 transporter and proprotein convertase subtilisin/kexin type 9. CONCLUSIONS: According to the present data, the small intestine may be classified as an insulin-sensitive tissue. Dysregulation of intestinal insulin signaling, possibly triggered by oxidative stress and inflammation, was associated with exaggerated lipogenesis and lipoprotein synthesis, which may represent a key mechanism for atherogenic dyslipidemia in patients with metabolic syndrome.

Phenotype of entero-endocrine L cells becomes restricted during development.

BACKGROUND: Glucagon-like peptide (GLP)-1 and glucose-dependent insulinotropic polypeptide (GIP) are hormones secreted by L and K cells, respectively, and by LK cells. To characterize L and K cells during development, we examined ileum from embryonic (e)- 12 to e-17. RESULTS: GLP-1 cells were first seen at e-15 and their number increased at e-17. At e-17, most GLP-1 cells co-expressed GIP. The transcription factors Pax6 and Pdx-1 are required for GIP expression, while Pax6 activates the expression of GLP-1. At e-17, the mucosa has GIP+ Pax6+, GIP+ Pdx-1+, GLP-1+ Pax6+, and GLP-1+ Pdx-1+ cells. Unlike ileal L cells of postnatal and adult mice, a subset of ileal L cells of e-17 embryos co-expressed GLP-1 and glucagon (Glu). Glu-positive cells contain proprotein-convertase 2 (PC2) and PC3/1, the enzymes responsible for Glu and GLP-1 synthesis, respectively. CONCLUSIONS: Our findings indicate that most GLP-1+ cells of ileum of e-17 embryos co-express GIP and, therefore, are LK cells. In addition, a subset of GLP-1+ cells of embryos but not of neonates co-express glucagon, indicating that the expression of Glu in GLP-1+ cells disappears after birth.

Site-1 protease is essential to growth plate maintenance and is a critical regulator of chondrocyte hypertrophic differentiation in postnatal mice.

Site-1 protease (S1P) is a proprotein convertase with essential functions in lipid homeostasis and unfolded protein response pathways. We previously studied a mouse model of cartilage-specific knock-out of S1P in chondroprogenitor cells. These mice exhibited a defective cartilage matrix devoid of type II collagen protein (Col II) and displayed chondrodysplasia with no endochondral bone formation even though the molecular program for endochondral bone development appeared intact. To gain insights into S1P function, we generated and studied a mouse model in which S1P is ablated in postnatal chondrocytes. Postnatal ablation of S1P results in chondrodysplasia. However, unlike early embryonic ablations, the growth plates of these mice exhibit a lack of Ihh, PTHrP-R, and Col10 expression indicating a loss of chondrocyte hypertrophic differentiation and thus disruption of the molecular program required for endochondral bone development. S1P ablation results in rapid growth plate disruption due to intracellular Col II entrapment concomitant with loss of chondrocyte hypertrophy suggesting that these two processes are related. Entrapment of Col II in the chondrocytes of the prospective secondary ossification center precludes its development. Trabecular bone formation is dramatically diminished in the primary spongiosa and is eventually lost. The primary growth plate is eradicated by apoptosis but is gradually replaced by a fully functional new growth plate from progenitor stem cells capable of supporting new bone growth. Our study thus demonstrates that S1P has fundamental roles in the preservation of postnatal growth plate through chondrocyte differentiation and Col II deposition and functions to couple growth plate maturation to trabecular bone development in growing mice.

Proprotein convertases in post-menopausal endometrial cancer: distinctive regulation and non-invasive diagnosis.

Proprotein convertases (PCs) play critical roles in cleaving precursor proteins (growth factors, hormones, receptors and adhesion molecules) for activation. PCs are implicated in a number of cellular functions, including oncogenesis. Endometrial cancer is the most common gynecological cancer in the developed world, but the involvement of PCs is unclear. To characterize the role of PCs in endometrial cancer, we assessed expression of seven PCs (PC1/3, PC2, PACE4, PC4, furin, PC5/6 and PC7) by RT-PCR in six well characterized endometrial cancer cell lines. Expression was variable in all lines, with furin being most consistently expressed in all cell lines tested. We next determined the cellular localization and expression levels of four ubiquitously expressed PCs (furin, PACE4, PC5/6 and PC7) in post-menopausal endometrial biopsies from control (n=7) and endometrial cancer patients (n=30) by immunohistochemistry. Furin increased in tumors, whereas PC5/6, PACE4 and PC7 expression was reduced with increasing cancer grades. Uterine lavage is a non-invasive source material for evaluating the endometrium. We thus assessed whether total PC activity was altered in uterine lavage of endometrial cancer patients (n=36) compared to controls (n=10). PC activity was detected in all uterine lavage samples, and significantly elevated in all grades of endometrial cancer. This study demonstrates a complex association between individual PCs and endometrial cancer. Importantly, we show that monitoring the total PC activity in uterine lavage may provide a rapid and non-invasive method for the diagnosis of endometrial cancer in postmenopausal women.

The precursor to the germ cell-specific PCSK4 proteinase is inefficiently activated in transfected somatic cells: evidence of interaction with the BiP chaperone.

Proprotein convertase subtilisin/kexin type 4 (PCSK4), also known as proprotein convertase 4 (PC4), is a serine endoproteinase primarily expressed in testicular germ cells and in sperm. Inactivation of its gene in mouse causes male infertility. From studies of the biosynthesis of PCSK3/furin, its closest relative, it has been inferred that PCSK4 is synthesised in the endoplasmic reticulum as a zymogen; that it is rapidly matured by autocatalytic cleavage between the prodomain and the catalytic domain; that the cleaved prodomain remains attached to the mature enzyme; and that the enzyme is finally activated by the removal of the prodomain peptides following a secondary cleavage within the prodomain. In this study, we used human embryonic kidney 293 (HEK293) cells to study the biosynthesis of rat or human PCSK4. Our results show that the bulk of PCSK4 remains as an intracellular zymogen, presumably trapped in the endoplasmic reticulum, where it interacts with the general molecular chaperone glucose-regulated protein 78/Immunoglobulin heavy-chain binding protein (GRP78/BiP). These data suggest that, unlike other members of the convertase family, proPCSK4 cannot efficiently self-activate in somatic cells. These cells may lack the intracellular environment and the interacting molecules specific to testicular germ cells where this enzyme is normally expressed.

Up-regulation of PCSK6 by lipid oxidation products: A possible role in atherosclerosis.

Atherosclerosis is a degenerative disease characterized by lesions that develop in the wall of large- and medium-sized arteries due to the accumulation of low-density lipoproteins (LDLs) in the intima. A growing bulk of evidence suggests that cholesterol oxidation products, known as oxysterols, and the aldehyde 4-hydroxy-2-nonenal (HNE), the major pro-atherogenic components of oxidized LDLs, significantly contribute to atherosclerotic plaque progression and destabilization, with eventual plaque rupture. The involvement of certain members of the protein convertase subtilisin/kexin proteases (PCSKs) in atherosclerosis has been recently hypothesized. Among them, PCSK6 has been associated with plaque instability, mainly thanks to its ability to stimulate the activity of matrix metalloproteinases (MMPs) involved in extracellular matrix remodeling and to enhance inflammation. In U937 promonocytic cells and in human umbilical vein endothelial cells, an oxysterol mixture and HNE were able to up-regulate the level and activity of PCSK6, resulting in MMP-9 activation as demonstrated by PCSK6 silencing. Inflammation, enhanced by these lipid oxidation products, plays a key role in the up-regulation of PCSK6 activity as demonstrated by cell pretreatment with NS-398, with epigallocatechin gallate or with acetylsalicylic acid, all with anti-inflammatory effects. For the first time, we demonstrated that both oxysterols and HNE, which substantially accumulate in the atherosclerotic plaque, up-regulate the activity of PCSK6. Of note, we also suggest a potential association between PCSK6 activity and MMP-9 activation, pointing out that PCSK6 could contribute to atherosclerotic plaque development.

Opposing function of the proprotein convertases furin and PACE4 on breast cancer cells' malignant phenotypes: role of tissue inhibitors of metalloproteinase-1.

Proteolytic cleavage of various cancer-related substrates by the proprotein convertases (PC) was reported to be important in the processes of neoplasia. These enzymes are inhibited by their naturally occurring inhibitors, the prosegments (ppPC), and by the engineered general PC inhibitor, the serpin variant alpha1-PDX. In the present study, we sought to compare the effect of these PC inhibitors on malignant phenotypes of breast cancer cells. Overexpression in a stable manner of alpha1-PDX and the prosegment ppPACE4 in MDA-MB-231 breast cancer cells resulted in increased matrix metalloproteinase (MMP)-9 (but not MMP-2) activity and a reduced secretion of tissue inhibitor of metalloproteinase 1 (TIMP-1). This was associated with significant enhancement in cell motility, migration, and invasion of collagen in vitro. In contrast, ppFurin expression in these cells decreased MMP-9 activity and diminished these biological functions, but had no significant effect on TIMP-1 secretion. Taken together, these data showed the specific and opposing roles of Furin and PACE4 in the regulation of MMP-9/TIMP-1-mediated cell motility and invasion.

Xenopus egg extracts: a model system to study proprotein convertases.

The Xenopus egg extract translation system has proved an ideal tool with which to study the biosynthesis of the prohormone convertases. It provides a robust coupled translation/translocation system capable of efficient translocation of any protein containing an N-terminal signal sequence into the lumen of its microsomal membranes, with cotranslational cleavage of the signal peptide. Its main advantage over rival in vitro translation systems is that it will also carry out posttranslational modification of proteins, such as N-glycosylation, and, in the case of the proprotein convertases, support autocatalytic proregion removal. The egg extract also contains an endogenous, acidic pH optimum enzyme activity, suggestive of a proprotein convertase, that can undertake limited proteolysis of precursors containing multibasic processing sites.

Multi-Leu PACE4 Inhibitor Retention within Cells Is PACE4 Dependent and a Prerequisite for Antiproliferative Activity.

The overexpression as well as the critical implication of the proprotein convertase PACE4 in prostate cancer progression has been previously reported and supported the development of peptide inhibitors. The multi-Leu peptide, a PACE4-specific inhibitor, was further generated and its capability to be uptaken by tumor xenograft was demonstrated with regard to its PACE4 expression status. To investigate whether the uptake of this inhibitor was directly dependent of PACE4 levels, uptake and efflux from cancer cells were evaluated and correlations were established with PACE4 contents on both wild type and PACE4-knockdown cell lines. PACE4-knockdown associated growth deficiencies were established on the knockdown HepG2, Huh7, and HT1080 cells as well as the antiproliferative effects of the multi-Leu peptide supporting the growth capabilities of PACE4 in cancer cells.

Two engineered eglin c mutants potently and selectively inhibiting kexin or furin.

Eglin c with mutants L45R and D42R at the P(1) and P(4) positions has been reported to become a stable inhibitor toward the proprotein convertases (PC), furin and kexin, with a K(i) of 2.3x10(-8) and 1.3x10(-10) M, respectively. The mutant was further engineered at the P(2)'-P(4)' positions to create a more potent and selective inhibitor for each enzyme. The residue Asp at P(1)' which is crucial for stabilizing the conformation of eglin c remained unchanged. The eglin c mutants cloned into the vector pGEX-2T and expressed in Escherichia coli (DH5alpha) were purified to homogeneity, and their inhibitory activities toward the purified recombinant furin and kexin were examined. The results showed that (1) Leu47 at P(2)' replaced with either a positively or negatively charged residue resulted in a decrease in inhibitory activities to both enzymes; (2) the replacement of Arg with Asp at P(3)' was favorable for inhibiting furin with a K(i) of 7.8 x 10(-9) M, but not for inhibiting kexin; (3) the replacement of Tyr with Glu at P(4)' increased the inhibitory activity to kexin with a K(i) of 3 x 10(-11) M, but was almost without any influence on furin inhibition. It was indicated that the inhibitory specificity of eglin c could be changed from inhibiting elastase to inhibiting PCs by site-directed mutation at the P positions, while the inhibitory selectivity to furin or kexin could be optimized by mutation at the P' positions.

Gene expression patterns of pro-opiomelanocortin-processing enzymes PC1 and PC2 during postnatal development of rat corticotrophs.

We examined the expression and localization of the prohormone convertases, PC1 and PC2, in the anterior pituitary cells of developing rats by a double staining procedure using in situ RT-PCR and an immunofluorescence technique. In the adult, both PC1 mRNA and PC2 mRNA were expressed in corticotrophs, gonadotrophs, thyrotrophs, and mammotrophs. These cells, except for corticotrophs, had previously been considered to be ones in which proprotein processing does not take place, but both PC1 and PC2 may be necessary to process other proteins, such as granin family proteins, having proteolytic cleavage sites and located in secretory granules of the above trophs. In addition, no PC1 or PC2 mRNA was expressed in somatotrophs, which is consistent with the fact that somatotrophs do not contain these granins. In addition, 7B2 mRNA was expressed in these PC2-positive trophs, suggesting that there is a functional relationship between PC2 and 7B2 proteins. We found that alpha-MSH was expressed in the corticotrophs of the postnatal rat and that the number of alpha-MSH-immunopositive corticotrophs decreased as development proceeded. Because the changes in the pattern of POMC processing are considered to depend on the relative expression levels of PC1 and PC2, PC1 and PC2 mRNAs were examined in corticotrophs during postnatal development. We found a decrease in the number of PC2 mRNA-positive cells, which coincided with one in the number of alpha-MSH-immunopositive corticotrophs, as postnatal development proceeded. Our present data demonstrate that the alpha-MSH production varies directly in accordance with the expression of PC2. We also discuss the possible significance of alpha-MSH production during the postnatal period.

CETP inhibitors downregulate hepatic LDL receptor and PCSK9 expression in vitro and in vivo through a SREBP2 dependent mechanism.

BACKGROUND: CETP inhibitors block the transfer of cholesteryl ester from HDL-C to VLDL-C and LDL-C, thereby raising HDL-C and lowering LDL-C. In this study, we explored the effect of CETP inhibitors on hepatic LDL receptor (LDLR) and PCSK9 expression and further elucidated the underlying regulatory mechanism. RESULTS: We first examined the effect of anacetrapib (ANA) and dalcetrapib (DAL) on LDLR and PCSK9 expression in hepatic cells in vitro. ANA exhibited a dose-dependent inhibition on both LDLR and PCSK9 expression in CETP-positive HepG2 cells and human primary hepatocytes as well as CETP-negative mouse primary hepatocytes (MPH). Moreover, the induction of LDLR protein expression by rosuvastatin in MPH was blunted by cotreatment with ANA. In both HepG2 and MPH ANA treatment reduced the amount of mature form of SREBP2 (SREBP2-M). In vivo, oral administration of ANA to dyslipidemic C57BL/6J mice at a daily dose of 50 mg/kg for 1 week elevated serum total cholesterol by approximately 24.5% (p < 0.05%) and VLDL-C by 70% (p < 0.05%) with concomitant reductions of serum PCSK9 and liver LDLR/SREBP2-M protein. Finally, we examined the in vitro effect of two other strong CETP inhibitors evacetrapib and torcetrapib on LDLR/PCSK9 expression and observed a similar inhibitory effect as ANA in a concentration range of 1-10 µM. CONCLUSION: Our study revealed an unexpected off-target effect of CETP inhibitors that reduce the mature form of SREBP2, leading to attenuated transcription of hepatic LDLR and PCSK9. This negative regulation of SREBP pathway by ANA manifested in mice where CETP activity was absent and affected serum cholesterol metabolism.