Gain-of-function of the p53R172H mutation in a mouse model of Li -Fraumeni syndrome

Missense mutations in the p53 tumor-suppressor gene are the most common alterations of p53 in somatic tumors and in patients with Li-Fraumeni syndrome. p53 missense mutations occur in the DNA binding region and disrupt the ability of p53 to activate transcription. In vitro studies have shown that some p53 missense mutants have a gain-of-function or dominant-negative activity. ^ The p53 175 Arg-to-His (p53 R175H) mutation in humans has been shown to have dominant-negative and gain-of-function properties in vitro. This mutation is observed in the germline of individuals with Li-Fraumeni syndrome. To accurately model Li-Fraumeni syndrome and to examine the mechanistic nature of a gain-of-function missense mutation on in vivo tumorigenesis, we generated and characterized a mouse with the corresponding mutation, p53 R172H. p53R172H homozygous and heterozygous mice developed similar tumor spectra and survival curves as p53 −/− and p53+/− mice, respectively. However, tumors in p53+/R172H mice metastasized to various organs with high frequency, suggesting a gain-of-function phenotype by p53R172H in vivo. Mouse embryonic fibroblasts (MEFs) from p53R172H mice also showed gain-of-function phenotypes in cell proliferation, DNA synthesis, and transformation potential, while cells from p53+/− and p53−/− mice did not. ^ To mechanistically characterize the gain-of-function phenotype of the p53R172H mutant, the role of p53 family members, p63 and p73, was analyzed. Disruption of p63 and p73 by siRNAs in p53 −/− MEFs increased transformation potential and reinitiated DNA synthesis to levels observed in p53R172H/R172H cells. Additionally, p63 and p73 were bound and functionally inactivated by p53R172H in metastatic p53 R172H tumor-derived cell lines, indicating a role for the p53 family members in the gain-of-function phenotype. This study provides in vivo evidence for the gain-of-function effect of p53 missense mutations and more accurately models the Li-Fraumeni syndrome. ^

Potential REST Targets in Neural Stem Cells and Glioblastoma-Derived Stem Cells

Glioblastoma multiforme (GBM) tumors are the most common malignant primary brain tumors in adults. The current theory is that these tumors are caused by self-renewing glioblastoma-derived stem cells (GSCs). At the current time, the mechanisms that regulate self-renewal and other oncogenic properties of GSCs remain unknown. Recently, we found transcriptional repressor REST maintains self-renewal in neural stem cells (NSCs) and in GSCs. REST also regulates other oncogenic properties, such as apoptosis, invasion and proliferation. However, the mechanisms by which REST regulates these oncogenic properties are unknown. In an attempt to determine these mechanisms, we performed loss and gain-of-function experiments and genome-wide mRNA expression analysis in GSCs, and we were able to identify REST-regulated genes in GSCs. This was accomplished, after screening concordantly regulated genes in NSCs and GSCs, utilizing two RE1 databases, and setting two-fold expression as filters on the resulting genes. These results received further validation by qRT-PCR. Ingenuity Pathway Analysis (IPA) analysis further revealed the top REST target genes in GSCs were downstream targets of REST and/or involved in other cancers in other cell lines. IPA also revealed that many of the differentially-regulated genes identified in this study are involved in oncogenic properties seen in GBM, and which we believe are related to REST expression.

A loss-of-function variant of PTPN22 is associated with reduced risk of systemic lupus erythematosus.

A gain-of-function R620W polymorphism in the PTPN22 gene, encoding the lymphoid tyrosine phosphatase LYP, has recently emerged as an important risk factor for human autoimmunity. Here we report that another missense substitution (R263Q) within the catalytic domain of LYP leads to reduced phosphatase activity. High-resolution structural analysis revealed the molecular basis for this loss of function. Furthermore, the Q263 variant conferred protection against human systemic lupus erythematosus, reinforcing the proposal that inhibition of LYP activity could be beneficial in human autoimmunity.

TRANSCRIPTIONAL AND POST-TRANSLATIONAL MECHANISMS CONTRIBUTE TO MAINTENANCE OF REST IN NEURAL TUMORS

The RE-1 silencing transcription factor (REST) is an important regulator of normal nervous system development. It negatively regulates neuronal lineage specification in neural progenitors by binding to its consensus RE-1 element(s) located in the regulatory region of its target neuronal differentiation genes. The developmentally coordinated down-regulation of REST mRNA and protein in neural progenitors triggers terminal neurogenesis. REST is overexpressed in pediatric neural tumors such as medulloblastoma and neuroblastoma and is associated with poor neuronal differentiation. High REST protein correlate with poor prognosis for patients with medulloblastoma, however similar studies have not been done with neuroblastoma patients. Mechanism(s) underlying elevated REST levels medulloblastoma and neuroblastoma are unclear, and is the focus of this thesis project. We discovered that transcriptional and post-translational mechanisms govern REST mis-regulation in medulloblastoma and neuroblastoma. In medulloblastoma, REST transcript is aberrantly elevated in a subset of patient samples. Using loss of function and gain of function experiments, we provide evidence that the Hairy Enhancer of Split (HES1) protein represses REST transcription in medulloblastoma cell lines, modulates the expression of neuronal differentiation genes, and alters the survival potential of these cells in vitro. We also show that REST directly represses its own expression in an auto-regulatory feedback loop. Interestingly, our studies identified a novel interaction between REST and HES1. We also observed their co-occupancy at the RE-1 sites, thereby suggesting potential for co-regulation of REST expression. Our pharmacological studies in neuroblastoma using retinoic acid revealed that REST levels are controlled by transcriptional and post-transcriptional mechanisms. Post-transcriptional mechanisms are mediated by modulation of E3 ligase or REST, SCFβ-TRCP, and contribute to resistance of some cells to retinoic acid treatment.

Functional studies of the homeobox gene {\it Goosecoid\/} during mouse embryogenesis

A fundamental question in developmental biology is to understand the mechanisms that govern the development of an adult individual from a single cell. Goosecoid (Gsc) is an evolutionarily conserved homeobox gene that has been cloned in vertebrates and in Drosophila. In mice, Gsc is first expressed during gastrulation stages where it marks anterior structures of the embryo, this pattern of expression is conserved among vertebrates. Later, expression is observed during organogenesis of the head, limbs and the trunk. The conserved pattern of expression of Gsc during gastrulation and gain of function experiments in Xenopus suggested a function for Gsc in the development of anterior structures in vertebrates. Also, its expression pattern in mouse suggested a role in morphogenesis of the head, limbs and trunk. To determine the functional requirement of Gsc in mice a loss of function mutation was generated by homologous recombination in embryonic stem cells and mice mutant for Gsc were generated.^ Gsc-null mice survived to birth but died hours after delivery. Phenotypic analysis revealed craniofacial and rib cage abnormalities that correlated with the second phase of Gsc expression in the head and trunk but no anomalies were found that correlated with its pattern of expression during gastrulation or limb development.^ To determine the mode of action of Gsc during craniofacial development aggregation chimeras were generated between Gsc-null and wild-type embryos. Chimeras were generated by the aggregation of cleavage stage embryos, taking advantage of two different Gsc-null alleles generated during gene targeting. Chimeras demonstrated a cell-autonomous function for Gsc during craniofacial development and a requirement for Gsc function in cartilage and mesenchymal tissues.^ Thus, during embryogenesis in mice, Gsc is not an essential component of gastrulation as had been suggested in previous experiments. Gsc is required for craniofacial development where it acts cell autonomously in cartilage and mesenchymal tissues. Gsc is also required for proper development of the rib cage but it is dispensable for limb development in mice. ^

The role of coactivator associated arginine methyltransferase 1 (CARM1) in nuclear receptor mediated transcription

Arginine methylation has been implicated in the regulation of gene expression. The coactivator-associated arginine methyltransferase 1 (CARMI/PRMT4) binds the p160 family of steroid receptor coactivators (SRCs). This association enhances transcriptional activation by nuclear receptors. Here, we generated and characterized CARM1 knockout mice. Embryos with a targeted disruption of CARM1 are 35% smaller in size than the wild-type littermates and die perinatally. We also generated Carm1-/- and Carm1+/+ mouse embryonic fibroblasts and tested gene expression in response to estrogen. Estrogenresponsive gene expression was aberrant in Carm1-/- fibroblasts and embryos, thus emphasizing the role of arginine methylation as a transcription activation tag. We subsequently studied the role of CARM1 in estrogen signaling in viva in the mammary gland. Conditional knockout of CARM1 in mammary gland and Carml-1-embryonic mammary anlagen transplant experiments did not show any defects in growth and development of the glands. To further dissect the role of CARM1 in estrogen receptor mediated transactivation, we performed cDNA microarray and serial analysis of gene expression on Carm1-/- and Carm1+/+ embryos treated with the estrogen analog, DES. Our results indicate global changes in estrogen regulated genes as well as genes involved in lipid homeostasis. Marker genes for Peroxisome Proliferator Activated Receptor γ (PPARγ) activity, adipsin and aP2, are downregulated in the Carm1-/- embryos. Furthermore, OCT frozen sections of 18.5dpc embryos, processed simultaneously for oil red O staining to look for neutral fat, reveals greatly reduced brown fat accumulation in the Carm1-/- embryos in contrast to wild-type and gain-of-function Carm1 transgenic (ubiquitous) embryo. We used a well-established 3T3-L1 preadipocyte cell line to knockdown CARM1 by short hairpin RNA. 3T3-L1 cells with CARM1 knockdown showed greatly reduced potential to differentiate into mature lipid accumulating adipocytes upon administration of adipogenic stimuli. Ligand-dependent activation of reporter genes by the PPARγ receptor showed that PPRE-luciferase reporter activity was enhanced in the presence of CARM1, additionally, luciferase activity was reduced to background levels when enzyme dead CARM1 (CARM1-VLD) was used. Thus, in this study, we have identified novel pathways that use CARM1 as coactivator and showed that CARM1 functions as a key component of PPARγ receptor mediated gene expression. ^

A review of effective weight loss and/or weight gain prevention for disadvantaged populations

Introduction. The prevalence of overweight and obesity has increased sharply for both adults and children, particularity in disadvantaged populations. Changes in dietary habits are small; however applying behavior-change principles has been associated with weight loss and preventing weight gain. This article will review studies targeting economically disadvantaged and/or communities of color incorporating the Transtheoretical Model of Change (TTM).^ Methods. Inclusion criteria were established. Descriptions of characteristics of the reviewed study interventions are included.^ Results. The search yielded a total of 23 articles identified through the electronic database PubMed that included Transtheoretical Model of Change (TTM) interventions regarding diet and/or nutrition, physical activity and/or exercise in disadvantaged populations. Thirteen study interventions centered solely on diet modification, five focused only on physical activity, and five concentrated on a combination of both. The preponderance of studies targeted WIC and urban recipients.^ Discussion/Conclusion. Although the majority of intervention studies supported the use of the Transtheoretical Model of Change (TTM) for weight loss and preventing weight gain, researchers noted that challenges still exist and further interventions are needed.^

Smooth Muscle Hyperplasia due to ACTA2/MYH11 Mutations: Identification of Novel Pathology and Pathways Leading to Aneurysms and Diverse Vascular Occlusive Diseases

Missense mutations in smooth muscle cell (SMC) specific ACTA2 (á-actin) and MYH11 (â-myosin heavy chain) cause diffuse and diverse vascular diseases, including thoracic aortic aneurysms and dissections (TAAD) and early onset coronary artery disease and stroke. The mechanism by which these mutations lead to dilatation of some arteries but occlusion of others is unknown. We hypothesized that the mutations act through two distinct mechanisms to cause varied vascular diseases: a loss of function, leading to decreased SMC contraction and aneurysms, and a gain of function, leading to increased SMC proliferation and occlusive disease. To test this hypothesis, ACTA2 mutant SMCs and myofibroblasts were assessed and found to not form á-actin filaments whereas control cells did, suggesting a dominant negative effect of ACTA2 mutations on filament formation. A loss of á-actin filaments would be predicted to cause decreased SMC contractility. Histological examination of vascular tissues from patients revealed SMC hyperplasia leading to arterial stenosis and occlusion, supporting a gain of function associated with the mutant gene. Furthermore, ACTA2 mutant SMCs and myofibroblasts proliferated more rapidly in static culture than control cells (p<0.05). We also determined that Acta2-/- mice have ascending aortic aneurysms. Histological examination revealed aortic medial SMC hyperplasia, but minimal features of medial degeneration. Acta2-/- SMCs proliferated more rapidly in culture than wildtype (p<0.05), and microarray analysis of Acta2-/- SMCs revealed increased expression of Actg2, 15 collagen genes, and multiple focal adhesion genes. Acta2-/- SMCs showed altered localization of vinculin and zyxin and increased phosphorylated focal adhesion kinase (FAK) in focal adhesions. A specific FAK inhibitor decreased Acta2-/- SMC proliferation to levels equal to wildtype SMCs (p<0.05), suggesting that FAK activation leads to the increased proliferation. We have described a unique pathology associated with ACTA2 and MYH11 mutations, as well as an aneurysm phenotype in Acta2-/- mice. Additionally, we identified a novel pathogenic pathway for vascular occlusive disease due to loss of SMC contractile filaments, alterations in focal adhesions, and activation of FAK signaling in SMCs with ACTA2 mutations.

{\it Hox\/} genes and specification of regional identity in the axial skeleton: Analysis of the individual and combined mutant phenotypes of the paralogous group 4 murine {\it Hox\/} genes; {\it hoxa\/}-4, {\it hoxb\/}-4 and {\it hoxd\/}-4

The Hox gene products are transcription factors involved in specifying regional identity along the anteroposterior body axis. In Drosophila, where these genes are known as HOM-C (Homeotic-complex) genes and where they have been most extensively studied, they are expressed in restricted domains along the anteroposterior axis with different anterior limits. Genetic analysis of a large number of gain- and loss-of-function alleles of these genes has revealed that these genes are important in specifying segmental identity at their anterior limits of expression. Furthermore, there is a functional dominance of posterior genes over anterior genes, such that posterior genes can dominantly specify their developmental programs in spite of the expression of more anterior genes in the same segment. In the mouse, there are four clusters of HOM-C genes, called Hox genes. Thus, there may be up to four genes, called paralogs, that are more highly homologous to each other and to their Drosophila homolog than they are to the other mouse Hox genes. The single mutants for two paralogous genes, hoxa-4 and hoxd-4, presented in this dissertation, are similar to several other mouse Hox mutants in that they show partial, incompletely penetrant homeotic transformations of vertebrae at their anterior limit of expression. These mutants were then bred with hoxb-4 mutants (Ramirez-Solis, et al. 1993) to generate the three possible double mutant combinations as well as the triple mutant. The skeletal phenotypes of these group 4 Hox compound mutants displayed clear alterations in regional identity, such that a nearly complete transformation towards the morphology of the first cervical vertebra occurs. These results suggest a certain degree of functional redundancy among paralogous genes in specifying regional identity. Furthermore, there was a remarkable dose-dependent increase in the number of vertebrae transformed to a first cervical vertebra identity, including the second through the fifth cervical vertebrae in the triple mutant. Thus, these genes are required in a larger anteroposterior domain than is revealed by the single mutant phenotypes alone, such that multiple mutations in these genes result in transformations of vertebrae that are not at their anterior limit of expression. ^

Regulation and function of {\it Xparaxis\/} during the development of paraxial mesoderm in {\it Xenopus laevis\/}

Genes of the basic helix-loop-helix transcription factor family have been implicated in many different developmental processes from neurogenesis to myogenesis. The recently cloned bHLH transcription factor, paraxis, has been found to be expressed in the paraxial mesoderm of the mouse suggesting a role for paraxis in the development of this mesodermal subtype which gives rise to the axial muscle, skeleton, and dermis of the embryo. In order to perform in vivo gain of function assays and obtain a better understanding of the possible roles of paraxis in mesodermal and somitic development, we have successfully identified homologues of paraxis in the frog, Xenopus laevis, where the process of mesodermal induction and development is best understood. The two homologues, Xparaxis-a and Xparaxis-b, are conserved with respect to their murine homologue in structure and expression within the embryo. Xparaxis genes are expressed immediately after gastrulation in the paraxial mesoderm of Xenopus embryos and are down regulated in the myotome of the mature somite with continued expression in the undifferentiated dermatome. Overexpression of Xparaxis-b in Xenopus embryos caused defects in the organization and morphology of the somites. This effect was not dependent on DNA binding of Xparaxis but is likely due to its dimerization with other bHLH factors. Co-injections with XE12 did not diminish the effects indicating that the defects were not the result of limiting amounts of XE12. We also demonstrated that Xparaxis does not cause obvious defects in the cell adhesions and movements required for proper mesoderm patterning during gastrulation. The paraxis proteins also lacked the ability to activate transcription as GAL4 fusion proteins in a GAL4 reporter assay, indicating that the genes may function more as modulators of the activity of dimerization partners than as positively acting cell determination factors. In agreement with this, Xparaxis is regulated in response to other pathways of bHLH gene action, in that XE12 can activate Xparaxis-b, in vivo. In addition we show regulation of Xparaxis in response to mMyoD induced myogenesis pathways, again suggesting Xparaxis plays an important role in the patterning and organization of the paraxial mesoderm. ^

The function of math5 and myocilin in mammalian eye development and disease

Complex molecular events underlie vertebrate eye development and disease. The eye is composed of two major tissue types: the anterior and posterior segments. During development, the retinal progenitor cells differentiate into six neuronal and one non-neuronal cell types. These cell types later organize into the distinct laminar structure of the mature retina which occupies the posterior segment. In the developed anterior segment, both the ciliary body and trabecular meshwork regulate intraocular pressure created by the aqueous humor. The disruption in intraocular pressure can lead to a blinding condition called glaucoma. To characterize molecular mechanisms governing retinal development and glaucoma, two separate mouse knockout lines carrying mutations in math5 and myocilin were subjected to a series of in vivo analyses. ^ Math5 is a murine homologue of Drosophila atonal , a bHLH proneural gene essential for the formation of photoreceptor cells. The expression of math5 coincides with the onset of retinal ganglion cell differentiation. The targeted deletion of mouse math5 revealed that a null mutation inhibits the formation of a majority of the retinal ganglion cells. The mutation also interferes with the normal development of other retinal cell types such as amacrine, bipolar and photoreceptor cells. These results suggest that math5 is a proneural gene responsible for differentiation of retinal ganglion cells and may also have a role in normal development of other neuronal cell types within the retina. ^ Myocilin has two unique protein coding regions bearing homology to non-muscle myosin of Dictyostelium discoideum and to olfactomedin, an extracellular matrix molecule first described in the olfactory epithelium of the bullfrog. Recently, autosomal dominant forms of myocilin mutations have been found in individuals with primary open-angle glaucoma. The genetic linkage to glaucoma suggests a role of myocilin in normal intraocular pressure and ocular function. However, the analysis of mice heterozygous and homozygous for a targeted null mutation in myocilin indicates that it is dispensable for normal intraocular pressure or ocular function. Additionally, the lack of a discernable phenotype in both heterozygous and null mice suggests that haploinsufficiency is not a critical mechanism for MYOC-associated glaucoma in humans. Instead, disease-causing mutations likely act by gain of function. ^ In summary, these studies provide novel insights into the embryonic development of the vertebrate retina, and also begin to uncover the molecular mechanisms responsible for the pathogenesis of glaucoma. ^

Function and regulation of anionic phospholipids in mitochondria of Saccharomyces cerevisiae

As the major anionic phospholipids predominantly found in the mitochondrial inner membrane of eukaryotic cells, cardiolipin (CL) and its precursor phosphatidylglycerol (PG) are of great importance in many critical mitochondrial processes. Pgs1Δ cells of Saccharomyces cerevisiae lacking both PG and CL display severe mitochondrial defects. Translation of several proteins including products of four mitochondrial DNA (mtDNA) encoded genes (COX1, COX2, COX3, and COB ) and one nuclear-encoded gene (COX4) is inhibited. The molecular basis of this phenotype was analyzed using a combined biochemical, molecular and genetic approach. ^ Using a mitochondrial targeted green fluorescence protein (mtGFP) fused to the COX4 promoter and its 5′ and 3′ untranslated regions (UTRs), lack of mtGFP expression independent of carbon source and strain background was confirmed to be at the translational level. The translational defect was not due to deficiency of mitochondrial respiratory function but rather caused directly by the lack of PG/CL in the mitochondrial membrane. Re-introduction of a functional PGS1 gene restored PG synthesis and expression of the above mtGFP. Deletional analysis of the 5′ UTR of COX4 mRNA revealed the presence of a 50 nt sequence as a cis-acting element inhibiting COX4 translation. Using similar constructs with HIS3 and lacZ as reporter genes, extragenic spontaneous mutations that allowed expression of His3p and β-galactosidase were isolated, which appeared to be recessive and derived from loss-of-function mutations as determined by mating analysis. Using a tetracycline repressible plasmid-borne PGS1 expression system and an in vivo mitochondrial protein translation method, the translation of mtDNA encoded COX1 and COX3 mRNAs was shown to be significantly inhibited in parallel with reduced levels of PG/CL content. Therefore, the cytoplasmic translation machinery appears to be able to sense the level of PG/CL in mitochondria and regulate COX4 translation coordinately with the mtDNA encoded subunits. ^ The essential requirement of PG and CL in mitochondrial function was further demonstrated in the study of CL synthesis by factors affecting mitochondrial biogenesis such as carbon source, growth phase or mitochondrial mutations at the level of transcription. We have also demonstrated that CL synthesis is dependent on the level of PG and INO2/INO4 regulatory genes. ^

Role of telomere dysfunction, DNA damage response and p53 mutations in tumorigenesis and aging

The ends of eukaryotic chromosomes are protected by specialized ribonucleoprotein structures termed telomeres. Telomeres protect chromosomes from end-to-end fusions, inappropriate repair and degradation. Disruption of this complex activates an ATM/ATR DNA damage response (DDR) pathway. One component of the complex is the Protection Of Telomeres 1 (POT1) protein, an evolutionarily conserved protein which binds single-stranded 3' overhang and is required for both chromosomal end protection and telomere length regulation. The mouse contains two POT1 orthologs, Pot1a and Pot1b. Here we show that both proteins colocalize with telomeres through interaction with the adapter protein TPP1. In addition, compared to Pot1a, the OB-folds of Pot1b possess less sequence specificity for telomeres. Disruption of POT1 proteins result in telomere dysfunction and activation of an ATR-dependent DDR at telomeres, suggesting that this response is normally suppressed by POT1 binding to the single-stranded G-overhang. ^ Telomeres are maintained by telomerase, and its absence in somatic cells results in telomere progressive loss that triggers the activation of p53. Telomere dysfunction initiates genomic instability and induces both p53-dependent replicative senescence and apoptosis to suppress tumorigenesis. In the absence of functional p53, this genomic instability promotes cancer. It was previously not known which aspect of the p53 dependent DNA damage response is important to suppress tumorigenesis initiated by dysfunctional telomeres. The p53R172P knock-in mouse, which is unable to induce apoptosis but retains intact cell cycle arrest/cellular senescence pathways, allowed us to examine whether p53-dependent apoptosis is a major tumor suppression pathway initiated in the setting of telomere dysfunction. Spontaneous tumorigenesis remains potently suppressed in late generation telomerase null mice possessing the p53P/P mutation. These results suggest that suppression of spontaneous tumorigenesis initiated by dysfunctional telomeres requires activation of a p53-dependent senescence pathway. In addition, we used another knock-in mouse model with a p53R172H (p53H) point mutation to test the hypothesis that telomere dysfunction promotes chromosomal instability and accelerates the onset of tumorigenesis in vivo in the setting of this most common gain-of-function mutation in the human Li Fraumeni cancer syndrome. We unexpectedly observed that telomerase null mice possessing dysfunctional telomeres in the setting of the p53H/+ mutation develop significantly fewer tumors, die prematurely and exhibit higher level of cellular senescence, apoptosis and elevated genomic instability compared to telomerase intact p53H/+ and telomerase null p53+/+ mice. These contrasting results thus link cancer and aging to the functional status of telomeres and the integrity of the p53 pathway. ^

THE NEW ROLE OF PROPROTEIN CONVERTASE SUBTILISIN/KEXIN TYPE 9: A CONNECTION OF PROPROTEIN CONVERTASE SUBTILISIN/KEXIN TYPE 9, APOLIPOPROTEIN B, and AUTOPHAGY

Plasma low-density lipoprotein (LDL) levels are positively correlated with the incidence of coronary artery disease. In the circulation, the plasma LDL clearance is mainly achieved by the uptake via LDL receptor (LDLR). Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a newly discovered gene, playing an important role in LDL metabolism. Gain-of-function mutations of PCSK9 lead to hypercholesterolemia and loss-of-function mutations of PCSK9 are associated with decrease of LDL cholesterol. The effects of PCSK9 on cholesterol levels are the consequence of a strong interaction between the catalytic domain of PCSK9 and epidermal growth factor-like repeat A (EGF-A) domain of LDLR on the cell surface of hepatocytes. This PCSK9/LDLR complex enters the cell via endocytosis, where both PCSK9 and LDLR are removed via the lysosome pathway, resulting in decreased levels of LDLR and accumulation of LDL in the plasma. However, whether this is the exclusive function of PCSK9 on LDL metabolism was challenged by us; we observed PCSK9 interacted with apolipoprotein B (apoB) and increased apoB production, irrespective of the LDLR. ApoB is the primary structure protein of LDL particle and it also serves as the ligand for the LDL receptor. There is ample evidence showing that the levels of apoB are a better indicator for heart disease than either total cholesterol or LDL cholesterol levels. We used a second-generation adenoviral vector to overexpress PCSK9 (Ad-PCSK9) in wild-type C57BL/6 and LDLR deficient mice (Ldlr-/- and Ldlr-/-Apobec1-/-). Our study revealed that overexpression of PCSK9 promoted the production and secretion of apoB in the form of very-low density lipoprotein (VLDL), which is the precursor of LDL, in the 3 mouse models studied (C57BL/6J, Ldlr-/-, and Ldlr-/-Apobec1-/-). The increased apoB production in mice was regulated at post-transcriptional levels, since there was no difference in apoB mRNA levels between mice treated with Ad-PCSK9 and control vector Ad-Null. By using pulse-chase experiment on primary hepatocytes, we showed that overexpression of PCSK9 increased the secretion of apoB, independent of LDLR. In the circulation, we showed that PCSK9 was associated with LDL particles. By using 3 different protein–protein interaction assays of co-immunoprecipitation, mammalian two-hybrid system, and in situ proximity ligation assay, we demonstrated a direct protein–protein interaction between PCSK9 and apoB. The impact of this interaction inhibited the physiological removal process of apoB via autophagosome/lysosome pathway in an LDLR-independent fashion, resulting in increased production and secretion of apoB-containing lipoproteins. The significance of this process was shown in the Pcsk9 knockout mice in the background of Ldlr-/-Apobec1-/- mice (triple knockout mice); in the absence of Pcsk9 (triple knockout mice) the levels of cholesterol, triacylglycerol, and apoB decreased significantly in comparison to that of Ldlr-/-Apobec1-/- mice. Taken together, our study demonstrated a direct intracellular interaction of PCSK9 with apoB, resulting in the inhibition of apoB degradation via the autophagosome/lysosome pathway independent of LDLR. This discovery provides a new concept of the importance of PCSK9 and suggests new approaches for the therapeutic intervention of hyperlipidemia.

Body weight, insulin resistance, and serum adipokine levels 2 years after 2 types of bariatric surgery.

OBJECTIVE: Bariatric surgery reverses obesity-related comorbidities, including type 2 diabetes mellitus. Several studies have already described differences in anthropometrics and body composition in patients undergoing Roux-en-Y gastric bypass compared with laparoscopic adjustable gastric banding, but the role of adipokines in the outcomes after the different types of surgery is not known. Differences in weight loss and reversal of insulin resistance exist between the 2 groups and correlate with changes in adipokines. METHODS: Fifteen severely obese women (mean body mass index [BMI]: 46.7 kg/m(2)) underwent 2 types of laparoscopic weight loss surgery (Roux-en-Y gastric bypass=10, adjustable gastric banding=5). Weight, waist and hip circumference, body composition, plasma metabolic markers, and lipids were measured at set intervals during a 24-month period after surgery. RESULTS: At 24 months, patients who underwent Roux-en-Y were overweight (BMI 29.7 kg/m(2)), whereas patients who underwent gastric banding remained obese (BMI 36.3 kg/m(2)). Patients who underwent Roux-en-Y lost significantly more fat mass than patients who underwent gastric banding (mean difference 16.8 kg, P<.05). Likewise, leptin levels were lower in the patients who underwent Roux-en-Y (P=.003), and levels correlated with weight loss, loss of fat mass, insulin levels, and Homeostasis Model of Assessment 2. Adiponectin correlated with insulin levels and Homeostasis Model of Assessment 2 (r=-0.653, P=.04 and r=-0.674, P=.032, respectively) in the patients who underwent Roux-en-Y at 24 months. CONCLUSION: After 2 years, weight loss and normalization of metabolic parameters were less pronounced in patients who underwent gastric banding compared with patients who underwent Roux-en-Y gastric bypass. Our findings require confirmation in a prospective randomized trial.