FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W3048444324> ?p ?o ?g. }

• W3048444324 endingPage "2236" @default.
• W3048444324 startingPage "2226" @default.
• W3048444324 abstract "In a condition of dysfunctional visceral fat depots, as in the case of obesity, alterations in adipokine levels may be detrimental for the cardiovascular system. The proinflammatory leptin and resistin adipokines have been described as possible links between obesity and atherosclerosis. The present study was aimed at evaluating whether proprotein convertase subtilisin/kexin type 9 (PCSK9), a key regulator of low-density lipoprotein metabolism, is induced by leptin and resistin through the involvement of the inflammatory pathway of STAT3. In HepG2 cells, leptin and resistin up-regulated PCSK9 gene and protein expression, as well as the phosphorylation of STAT3. Upon STAT3 silencing, leptin and resistin lost their ability to activate PCSK9. The knockdown of STAT3 did not affect the expression of leptin and resistin receptors or that of PCSK9. The analysis of the human PCSK9 promoter region showed that the two adipokines raised PCSK9 promoter activity via the involvement of a sterol regulatory element motif. In healthy males, a positive association between circulating leptin and PCSK9 levels was found only when the body mass index was <25 kg/m2. In conclusion, this study identified STAT3 as one of the molecular regulators of leptin- and resistin-mediated transcriptional induction of PCSK9. In a condition of dysfunctional visceral fat depots, as in the case of obesity, alterations in adipokine levels may be detrimental for the cardiovascular system. The proinflammatory leptin and resistin adipokines have been described as possible links between obesity and atherosclerosis. The present study was aimed at evaluating whether proprotein convertase subtilisin/kexin type 9 (PCSK9), a key regulator of low-density lipoprotein metabolism, is induced by leptin and resistin through the involvement of the inflammatory pathway of STAT3. In HepG2 cells, leptin and resistin up-regulated PCSK9 gene and protein expression, as well as the phosphorylation of STAT3. Upon STAT3 silencing, leptin and resistin lost their ability to activate PCSK9. The knockdown of STAT3 did not affect the expression of leptin and resistin receptors or that of PCSK9. The analysis of the human PCSK9 promoter region showed that the two adipokines raised PCSK9 promoter activity via the involvement of a sterol regulatory element motif. In healthy males, a positive association between circulating leptin and PCSK9 levels was found only when the body mass index was <25 kg/m2. In conclusion, this study identified STAT3 as one of the molecular regulators of leptin- and resistin-mediated transcriptional induction of PCSK9. Adipose tissue is an endocrine organ able to secrete active molecules, namely, adipokines, that contribute to regulating appetite and satiety, fat distribution, and energy expenditure.1Perez-Tilve D. 'Et tu, leptin?'.Trends Endocrinol Metab. 2019; 30: 232-233Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar When the metabolic milieu is deranged, as in the case of obesity and adipose tissue metainflammation, adipokines may play a role in cardiovascular (CV) disease, by promoting atherogenesis, plaque progression, and thrombosis.2Barale C. Russo I. Influence of cardiometabolic risk factors on platelet function.Int J Mol Sci. 2020; 21: 623Crossref Scopus (19) Google Scholar Roughly 80% of patients with coronary heart disease are overweight or obese.3Ades P.A. Savage P.D. Obesity in coronary heart disease: an unaddressed behavioral risk factor.Prev Med. 2017; 104: 117-119Crossref PubMed Scopus (29) Google Scholar Among the huge variety of adipokines produced by adipose tissue, the proinflammatory adipokines leptin and resistin have been described as possible links between obesity and atherosclerosis.4Rashid S. Kastelein J.J. PCSK9 and resistin at the crossroads of the atherogenic dyslipidemia.Expert Rev Cardiovasc Ther. 2013; 11: 1567-1577Crossref PubMed Scopus (9) Google Scholar,5Du Y. Li S. Cui C.-J. Zhang Y. Yang S.-H. Li J.-J. Leptin decreases the expression of low-density lipoprotein receptor via PCSK9 pathway: linking dyslipidemia with obesity.J Transl Med. 2016; 14: 276Crossref PubMed Scopus (13) Google Scholar Leptin, a 16-kDa cytokine produced predominantly by adipose tissue, regulates feeding and promotes energy expenditure. This hormone is implicated also in the regulation of the immune system, autonomic, and CV regulation.6Cui H. Lopez M. Rahmouni K. The cellular and molecular bases of leptin and ghrelin resistance in obesity.Nat Rev Endocrinol. 2017; 13: 338-351Crossref PubMed Scopus (163) Google Scholar Leptin exerts its biological actions by binding to a class I cytokine receptor encoded by LEPR in humans. Among the six LepR isoforms (LepRa to LepRf), the LepRb is the only one with a full-length intracellular domain that allows the activation of a cell-signaling pathway. In response to leptin, Janus tyrosine kinase 2 (JAK2) phosphorylates LepR, triggering the recruitment of STAT3 through its SH2 domain. STAT3 is subsequently phosphorylated by JAK2, resulting in its dimerization and nuclear translocation in order to regulate the transcription of STAT3-target genes.7Banks A.S. Davis S.M. Bates S.H. Myers Jr., M.G. Activation of downstream signals by the long form of the leptin receptor.J Biol Chem. 2000; 275: 14563-14572Crossref PubMed Scopus (620) Google Scholar Among the STAT proteins (STAT1, -2, -3, -4, -5a, -5b, and -6), STAT3 contributes to various metabolic processes such as hyperphagia and obesity,8Gao Q. Wolfgang M.J. Neschen S. Morino K. Horvath T.L. Shulman G.I. Fu X.-Y. Disruption of neural signal transducer and activator of transcription 3 causes obesity, diabetes, infertility, and thermal dysregulation.Proc Natl Acad Sci U S A. 2004; 101: 4661-4666Crossref PubMed Scopus (294) Google Scholar leptin being one of the major players in these events.9Bates S.H. Stearns W.H. Dundon T.A. Schubert M. Tso A.W. Wang Y. Banks A.S. Lavery H.J. Haq A.K. Maratos-Flier E. Neel B.G. Schwartz M.W. Myers Jr., M.G. STAT3 signalling is required for leptin regulation of energy balance but not reproduction.Nature. 2003; 421: 856-859Crossref PubMed Scopus (782) Google Scholar Resistin is a 12.5-kDa–sized C-terminal cysteine-rich signaling peptide secreted predominantly by adipose tissue and macrophages in humans and rodents. In humans, resistin adipose tissue expression and circulating levels are increased in overweight and obese subjects, strongly supporting the association with a raised CV risk in obese individuals.10Reilly M.P. Lehrke M. Wolfe M.L. Rohatgi A. Lazar M.A. Rader D.J. Resistin is an inflammatory marker of atherosclerosis in humans.Circulation. 2005; 111: 932-939Crossref PubMed Scopus (735) Google Scholar Among the designated receptors mediating resistin effects, for example, the inflammatory cascade, adenylyl cyclase associated protein 1 (CAP1) has been recently described as a bona fide resistin receptor.11Lee S. Lee H.-C. Kwon Y.-W. Lee S.E. Cho Y. Kim J. Lee S. Kim J.-Y. Lee J. Yang H.-M. Mook-Jung I. Nam K.-Y. Chung J. Lazar M.A. Kim H.-S. Adenylyl cyclase-associated protein 1 is a receptor for human resistin and mediates inflammatory actions of human monocytes.Cell Metab. 2014; 19: 484-497Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar Proprotein convertase subtilisin/kexin type 9 (PCSK9), mainly secreted by the liver and to a lower extent by the intestine, post-translationally regulates the number of cell-surface low-density lipoprotein (LDL) receptors. At the transcriptional level, PCSK9 synthesis is largely controlled by the involvement of transcription factor families sterol regulatory element (SRE)-binding proteins (SREBPs) and by the acute-phase response transcriptional controller hepatocyte nuclear factor-1 (HNF-1) alpha.12Macchi C. Banach M. Corsini A. Sirtori C.R. Ferri N. Ruscica M. Changes in circulating pro-protein convertase subtilisin/kexin type 9 levels - experimental and clinical approaches with lipid-lowering agents.Eur J Prev Cardiol. 2019; 26: 930-949Crossref PubMed Scopus (52) Google Scholar This last evidence and the knowledge that PCSK9 expression is also regulated by the proinflammatory cytokine tumor necrosis factor (TNF)-α, in a suppressor of cytokine signaling (SOCS) 3–dependent manner, suggest a direct link between inflammation and the regulation of lipid metabolism via PCSK9.13Momtazi-Borojeni A.A. Sabouri-Rad S. Gotto A.M. Pirro M. Banach M. Awan Z. Barreto G.E. Sahebkar A. PCSK9 and inflammation: a review of experimental and clinical evidence.Eur Heart J Cardiovasc Pharmacother. 2019; 5: 237-245Crossref PubMed Scopus (32) Google Scholar Considering that PCSK9 circulating levels have been associated with atherosclerosis and to support the hypothesis that the derangement in adipokine secretion is involved in obesity-associated CV risk,14Ruscica M. Baragetti A. Catapano A.L. Norata G.D. Translating the biology of adipokines in atherosclerosis and cardiovascular diseases: gaps and open questions.Nutr Metab Cardiovasc Dis. 2017; 27: 379-395Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar the aim of the present study was to evaluate whether leptin and resistin mediate PCSK9 activation through the inflammatory STAT3 pathway. Indeed, the JAK/STAT signaling pathway, a pillar downstream mediator of a variety of cytokines, is dysregulated in metabolic diseases including obesity.15Dodington D.W. Desai H.R. Woo M. JAK/STAT - emerging players in metabolism.Trends Endocrinol Metab. 2018; 29: 55-65Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar The human hepatocellular carcinoma cell line, HepG2, was routinely cultured in 10% fetal bovine serum (FBS)/Dulbecco’s modified Eagle’s medium (DMEM) supplemented with penicillin, streptomycin, nonessential amino acids, and sodium pyruvate (all from Sigma-Aldrich, Milan, Italy). For the experiments, cells were starved overnight and then incubated with DMEM containing 10% human lipoprotein plasma deprived serum (LPDS). DMEM, trypsin EDTA, penicillin, streptomycin, nonessential amino acid solution, FBS, disposable culture flasks, and petri dishes were from Merck (Milan, Italy). Molecular weight protein standard, precast polyacrylamide gels (4% to 12%), bicinchoninic acid assay for determination of protein concentrations, Maxima First Strand cDNA, and Maxima SYBR Green/Fluorescein qPCR Master Mix were all from Life Technologies (Monza, Italy). Bovine Serum Albumin was from Sigma-Aldrich. Human recombinant leptin (Sigma-Aldrich) and resistin (BioVision, Milpitas, CA) were used at 100 ng/mL. Human recombinant PCSK9 was used at 2.5 μg/mL (item 20631; Cayman Chemicals, Ann Arbor, MI).16Camera M. Rossetti L. Barbieri S.S. Zanotti I. Canciani B. Trabattoni D. Ruscica M. Tremoli E. Ferri N. PCSK9 as a positive modulator of platelet activation.J Am Coll Cardiol. 2018; 71: 952-954Crossref PubMed Scopus (25) Google Scholar ON-TARGET plus SMART pool siRNA directed to STAT3 and PCSK9 or scramble control were from Dharmacon (Carlo Erba Reagents, Milan, Italy). Transfections were performed by using siLentFect Lipid Reagent (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's protocol. The day before the transfection, HepG2 cells were seeded in DMEM/10% FBS at a density of 6 × 105/well (6-well tray). Cells were then transfected with 40 nmol/L siRNA. Forty-eight hours after the transfection, the medium was replaced with DMEM/10% LPDS ± leptin (100 ng/mL) or resistin (100 ng/mL) for an additional 24 hours before performing quantitative PCR analysis. Human, gorilla, mouse, and rat PCSK9 promoter sequences (up to 2000 bp upstream from the start codon) were retrieved from Ensembl Genome Browser17Yates A.D. Achuthan P. Akanni W. Allen J. Allen J. Alvarez-Jarreta J. et al.Ensembl 2020.Nucleic Acids Res. 2020; 48: D682-D688PubMed Google Scholar by using the latest released genome assembly (GRCh38.p13, Kamilah_GGO_v0, GRCm38.p6, and Rnor_6.0 for human, gorilla, mouse, and rat, respectively). JASPAR (http://jaspar.genereg.net, last accessed July 2020), an open-access database of curated, nonredundant transcription factor binding profiles modelled as position-specific weight matrices, was used to scan the selected PCSK9 promoter region for any STAT3 binding site.18Fornes O. Castro-Mondragon J.A. Khan A. van der Lee R. Zhang X. Richmond P.A. Modi B.P. Correard S. Gheorghe M. Baranasic D. Santana-Garcia W. Tan G. Cheneby J. Ballester B. Parcy F. Sandelin A. Lenhard B. Wasserman W.W. Mathelier A. JASPAR 2020: update of the open-access database of transcription factor binding profiles.Nucleic Acids Res. 2020; 48: D87-D92PubMed Google Scholar The relative profile score threshold—defined as the minimum relative score required for reporting a match between the transcription factor binding model and the imputed sequence—was set at 80%. The FIMO tool, from the open-access MEME Suite 5.1.1 (http://meme-suite.org, last accessed July 2020), was used to double-check the results obtained from JASPAR.19Grant C.E. Bailey T.L. Noble W.S. FIMO: scanning for occurrences of a given motif.Bioinformatics. 2011; 27: 1017-1018Crossref PubMed Scopus (1576) Google Scholar,20Bailey T.L. Boden M. Buske F.A. Frith M. Grant C.E. Clementi L. Ren J. Li W.W. Noble W.S. MEME SUITE: tools for motif discovery and searching.Nucleic Acids Res. 2009; 37: W202-W208Crossref PubMed Scopus (4561) Google Scholar The STAT3 position-specific weight matrix profile was retrieved from JASPAR and imputed in FIMO, and the selected PCSK9 promoter region was scanned. Matches with P ≤ 0.0001 were taken into account. The same analyses were performed for SRE and HNF-1. D1-STAT3-mut plasmid was generated by deleting the STAT3 binding sequence predicted by JASPER in the PCSK9 promoter sequence in plasmid D1. Plasmid D1 is a derivative of the commercial promoter-reporter vector pGL3-Basic, generated by cloning the human PCSK9 promoter fragment spanning −1711 to −94 upstream of the luc reporter gene. Deletion of the STAT3 sequence (CTTCTGGAAAG), spanning −916 to −906 in the PCSK9 promoter, was obtained by two-step PCR according to the following protocol. Two fragments flanking the putative STAT3 sequence were PCR amplified using D1 plasmid as a template and the following primer pairs: AP679 (5′-CGACGAGGTACCGAGCTCGGATCCACTAGTAAC-3′) and AP680 (5′-ATTCAATTTGCAAAGATTC-3′) and generate the upstream fragment, and AP681 (5′-CAAATTGAATCTGAGCTTGTGCCTACCATAG-3′) and AP682 (5′-CCCAAGCTTACTGTGCAGGAGCTGAAGTTC-3′) to generate the downstream fragment. Primer AP679 and AP682 sequences contain the KpnI and HindIII restriction sites, respectively. The upstream and downstream fragments were used as templates for a second round of PCR amplification using AP679 and AP682 primers. The final PCR product was cloned in the D1 plasmid between KpnI and HindIII restriction sites in place of the PCSK9 promoter region, generating the D1-STAT3 plasmid. The deletion of the STAT3 region from the PCSK9 promoter in the D1-STAT3 plasmid was confirmed by DNA sequencing. HepG2 cells were transfected with the plasmid PCSK9 pGL3-PCSK9-D4 containing the 5′ flanking region of the PCSK9 gene from −440 to −94, relative to the ATG start codon in front of the luciferase coding sequence. The promoter constructs contain wild-type, SRE mutated (SRE-mu), and HNF-1 alpha mutated (HNF1-mu) sequences.21Li H. Dong B. Park S.W. Lee H.-S. Chen W. Liu J. Hepatocyte nuclear factor 1alpha plays a critical role in PCSK9 gene transcription and regulation by the natural hypocholesterolemic compound berberine.J Biol Chem. 2009; 284: 28885-28895Crossref PubMed Scopus (212) Google Scholar To measure the human PCSK9 promoter activity, HepG2 cells were seeded in 48-well plates at a density of 4 × 105 cells per well. The day after, cells were transiently transfected with pGL3-PCSK9-D1 (wild-type and STAT3 mutated) and with pGL3-PCSK9-D4 plasmids (wild-type, SRE-mu, and HNF-1-mu), with TurboFect transfection reagent (Thermo Fisher Scientific, Waltham, MA). Forty-eight hours after the transfection, cells were incubated with DMEM/10% LPDS ± leptin (100 ng/mL), resistin (100 ng/mL), or simvastatin (20 μmol/L) for an additional 24 hours. Luciferase activity was measured by using Neolite reagent (Perkin Elmer, Milan, Italy) according to the manufacturer's instructions. Total RNA was extracted with the iScript Sample Preparation Buffer cDNA synthesis preparation reagents (Bio-Rad Laboratories) according to manufacturer's instructions or by spin column (Qiagen, Milan, Italy). Reverse transcription-polymerase first-strand cDNA synthesis was performed by using Maxima First Strand cDNA synthesis kit (Thermo Fisher Scientific). Quantitative PCR was then performed by using the Thermo SYBR Green/ROX qPCR Master Mix kit (Thermo Fisher Scientific) and specific primers for selected genes. The analyses were performed with the 9600 Bio-Rad Real-Time PCR Detection Systems (Bio-Rad Laboratories). The primer sequences are listed in Table 1. PCR cycling conditions were as follows: 94°C for 5 minutes, 40 cycles at 94°C for 15 seconds, and 60°C for 30 seconds. Data are expressed as Ct values and used for the relative quantification of targets with the 2−ΔΔCt calculation.Table 1List and Sequence of Primers Used in This StudyPrimersForwardReversePCSK95′-CCTGCGCGTGTCAACT-3′5′-GCTGGCTTTTCCGAAACTC-3′STAT35′-CAGCAGCTTGACACACGGTA-3′5′-AAACACCAAAGTGGCATGT-3′ACTB5′-TTCTACAATGAGCTGCGTGTG-3′5′-GGGGTGTTGAAGGTCTCAAA-3′LEPR5′-TACTTTGGAAGCCCCTGATG-3′5′-AAGCACTGAGTGACTGCACG-3′CAP15′-ACTGGCCTGGAGCAAAACG-3′5′-CGGCAGAGGGTCCAGATG-3′IL65′-ACCCCCAGGAGAAGATTCCA-3′5′-GGTTGTTTTCTGCCAGTGCC-3′ACTB, β-actin; CAP1, adenylyl cyclase associated protein 1; LEPR, leptin receptor; PCSK9, proprotein convertase subtilisin/kexin type 9. Open table in a new tab ACTB, β-actin; CAP1, adenylyl cyclase associated protein 1; LEPR, leptin receptor; PCSK9, proprotein convertase subtilisin/kexin type 9. Total cytosolic protein extracts of HepG2 were obtained by collecting cells in 70 μL of Mammalian Protein Extraction Reagents (Thermo Fisher Scientific) containing a cocktail of protease and phosphatase inhibitors (Roche Diagnostics, Basel, Switzerland). Ten micrograms of proteins and a molecular mass marker (Novex Sharp Protein Standard, Invitrogen; Thermo Fisher Scientific) were separated on 4% to 12% SDS-PAGE (Novex NuPAGE 4% to 12% Bis-Tris MiniGels; Invitrogen; Thermo Fisher Scientific) under denaturing and reducing conditions. Proteins were then transferred to a nitrocellulose membrane by using the iBlotTM Gel Transfer Device (Invitrogen; Thermo Fisher Scientific). The membranes were washed with Tris-buffered saline-Tween 20, and nonspecific binding sites were blocked in Tris-buffered saline-Tween 20 containing 5% bovine serum albumin (Sigma-Aldrich) for 90 minutes at room temperature. The blots were incubated overnight at 4°C with a diluted solution (5% bovine serum albumin or nonfat dry milk) of the human primary antibodies (listed below). Membranes were washed with Tris-buffered saline-Tween 2 and then exposed for 90 minutes at room temperature to a diluted solution (5% nonfat dry milk) of the secondary antibodies (anti-mouse and anti-rabbit peroxidase-conjugated secondary antibodies; New England Biolabs, Ipswich, MA). Immunoreactive bands were detected by exposing the membranes to Clarity Western ECL chemiluminescent substrates (Bio-Rad Laboratories) for 5 minutes, and images were acquired with a ChemiDoc XRS System (Bio-Rad Laboratories). Densitometric readings were evaluated using the ImageLab software version 6.0.1 (Bio-Rad Laboratories). The dilution of the human primary antibodies were: PCSK9 (1:1000; Genetex, Irvine, CA), STAT3 (1:1000; Cell Signaling Technology, Danvers, MA), pSTAT3 (1:10,000; Abcam, Cambridge, UK), actin (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), tubulin (1:2000; Sigma-Aldrich), and vinculin (1:1000; Genetex). The association between PCSK9, and leptin and resistin plasma levels was evaluated in 149 healthy male subjects from the Brisighella Heart Study. This study is a longitudinal population study on a randomized sample representative of the entire population of Brisighella, a rural Northern Italian village. The study has been active since 1972 and has been performed in agreement with the Declaration of Helsinki. The protocol was approved by the institutional ethics board of the University Hospital of Bologna.22Ruscica M. Ferri N. Fogacci F. Rosticci M. Botta M. Marchiano S. Magni P. D'Addato S. Giovannini M. Borghi C. Cicero A.F.G. Brisighella Heart Study GroupCirculating levels of proprotein convertase subtilisin/kexin type 9 and arterial stiffness in a large population sample: data from the Brisighella Heart Study.J Am Heart Assoc. 2017; 6: e005764Crossref PubMed Scopus (46) Google Scholar Plasma PCSK9 concentrations were measured by a commercial enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN). The minimum detectable PCSK9 concentration was 0.219 ng/mL.23Ruscica M. Simonelli S. Botta M. Ossoli A. Lupo M.G. Magni P. Corsini A. Arca M. Pisciotta L. Veglia F. Franceschini G. Ferri N. Calabresi L. Plasma PCSK9 levels and lipoprotein distribution are preserved in carriers of genetic HDL disorders.Biochim Biophys Acta Mol Cell Biol Lipids. 2018; 1863: 991-997Crossref PubMed Scopus (8) Google Scholar Concerning the evaluation of PCSK9 in the conditioned medium, HepG2 cells were cultured in 12-well plates (6 × 105 cells/well). The medium (200 μL) was collected after 24-hour treatment with leptin, resistin, and simvastatin. The samples were not diluted, and results were normalized for the total amount of proteins. Plasma leptin concentrations were measured by a commercial ELISA kit (R&D Systems). The minimum detectable dose of human leptin is typically <7.8 pg/mL.24Ruscica M. Macchi C. Gandini S. Morlotti B. Erzegovesi S. Bellodi L. Magni P. Free and bound plasma leptin in anorexia nervosa patients during a refeeding program.Endocrine. 2016; 51: 380-383Crossref PubMed Scopus (11) Google Scholar Statistical analysis was performed using the Prism statistical analysis package version 6.0 (GraphPad Software, San Diego, CA). Data are given as means ± SD of three independent experiments. When possible, P values were determined by t-test. Otherwise, differences between treatment groups were evaluated by one-way analysis of variance. A probability value of P < 0.05 was considered statistically significant. First, a time course experiment (4, 8, 16, and 24 hours) was conducted to evaluate the gene expression of PCSK9 upon leptin (100 ng/mL) and resistin (100 ng/mL) treatment (Supplemental Figure S1). Incubation of HepG2 cells with human recombinant leptin and resistin resulted in a significant rise in PCSK9 gene expression of roughly +50% (Figure 1A) after 24 hours. Compared with medium alone, protein expression, evaluated by Western blot analysis, was increased roughly by 2- and 1.7-fold, upon treatment with leptin and resistin, respectively (Figure 1B). Simvastatin (20 μmol/L)—used as a positive control—raised both PCSK9 gene and protein expression (Figure 1, D and E). To corroborate Western blot analyses, the release of PCSK9 was measured. In the HepG2-conditioned medium, the levels of PCSK9 were increased by 42.6% (P < 0.01) and 36.3% (P < 0.05) following leptin and resistin treatments, respectively (Figure 1C). Simvastatin (20 μmol/L) was used as a positive control (Figure 1F). In this cell-based model, 24-hour treatment with leptin and resistin phosphorylated STAT3 (pSTAT3Y705) without affecting the protein expression of total STAT3 (Figure 2A). To follow up the hypothesis that STAT3 was involved in leptin- and resistin-driven PCSK9 activation, HepG2 cells were knocked down for STAT3. Transfection with specific siRNA anti-STAT3 fully abolished the gene and protein expression of STAT3 (Figure 2, B and C) without affecting that of PCSK9 (Figure 2, D–F). Under these experimental conditions, leptin and resistin did not up-regulate PCSK9 expression (gene and protein). These data suggest that PCSK9 up-regulation by leptin and resistin in hepatocytes requires STAT3. To exclude any effect of PCSK9 on STAT3 expression, it was shown that silencing PCSK9 did not result in any change in STAT3 levels (Figure 2C). Moreover, application of siSTAT3 did not change the gene expression of leptin (LEPR) and resistin (CAP1) receptors (Supplemental Figure S2). The involvement of STAT3 in the PCSK9 pathway was further investigated at the transcriptional level. First, an in silico search of STAT3 binding sites on proximal PCSK9 promoter region was performed by using JASPAR database and FIMO tool from MEME Suite 5.1.1. The analyses revealed a strong STAT3 binding site (JASPAR score: 96%; FIMO threshold: P = 2.07 × 10−5) spanning the region −916 to −906 in the human PCSK9 promoter (Table 2). This region is upstream from the Sp1/HNF1-α/SRE triad already published by Li et al21Li H. Dong B. Park S.W. Lee H.-S. Chen W. Liu J. Hepatocyte nuclear factor 1alpha plays a critical role in PCSK9 gene transcription and regulation by the natural hypocholesterolemic compound berberine.J Biol Chem. 2009; 284: 28885-28895Crossref PubMed Scopus (212) Google Scholar (Figure 3A). To corroborate this finding, a similar analysis was performed on gorilla, mouse, and rat PCSK9 promoters: a multiple alignment of the four sequences was generated thanks to Nucleotide BLAST Alignment tool by NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) (Figure 3A). The mild degeneration in the STAT3 consensus sequence has been already reported in the literature.25Tripathi S.K. Chen Z. Larjo A. Kanduri K. Nousiainen K. Aijo T. Ricano-Ponce I. Hrdlickova B. Tuomela S. Laajala E. Salo V. Kumar V. Wijmenga C. Lahdesmaki H. Lahesmaa R. Genome-wide analysis of STAT3-mediated transcription during early human Th17 cell differentiation.Cell Rep. 2017; 19: 1888-1901Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar After transfection with D1 and D1-STAT3-mut plasmids (Figure 3B), both containing the human PCSK9 promoter region spanning −1711 to −94 nucleotides upstream of the gene coding for the luciferase enzyme, HepG2 cells were treated with leptin, resistin, and simvastatin. A twenty-four–hour treatment enhanced transcriptional activity, an effect that was not counteracted when STAT3 mutation was inserted (Figure 3C). Simvastatin was used as a positive control (Figure 3D). These data suggest that PCSK9 up-regulation by leptin and resistin in hepatocytes is not directly mediated by binding of STAT3 to the PCSK9 promoter.Table 2In Silico Search of STAT3 Binding Sites on the Proximal PCSK9 Promoter RegionPredicted site sequenceStartEndStrandJASPAR score, %FIMO threshold, P valueCTTCTGGAAAG−916−906+95.922.07 × 10−5CTTCCAGAAAG−1082−1072+93.067.44 × 10−4CTTTCTGGAAG−1072−1082−92.421.01 × 10−4ATACTGGGAAG−1091−1101−89.882.93 × 10−4TTGCCTGTAAT−1385−1395−88.454.46 × 10−4CAGCAGGGAAA−1485−1475+87.794.61 × 10−4GTTCAAGAAAT−950−960−87.277.13 × 10−4GTGAAGGGAAA−2184−2174+87.086.22 × 10−4PCSK9, proprotein convertase subtilisin kexin type 9. Open table in a new tab PCSK9, proprotein convertase subtilisin kexin type 9. To further explore the mechanism whereby leptin and resistin up-regulate PCSK9 transcript levels, HepG2 cells were transiently transfected with constructs containing the proximal human PCSK9 promoter region (−440 to −94). Leptin and resistin significantly incremented the luciferase activity by 54% and 15%, respectively (Figure 4A). Similar results were obtained upon treatment with simvastatin (+141%). Because SREBPs and HNF-1 are the major transcription factors regulating PCSK9,26Ruscica M. Ricci C. Macchi C. Magni P. Cristofani R. Liu J. Corsini A. Ferri N. Suppressor of cytokine signaling-3 (SOCS-3) induces proprotein convertase subtilisin kexin type 9 (PCSK9) expression in hepatic HepG2 cell line.J Biol Chem. 2016; 291: 3508-3519Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar luciferase activity was investigated upon the insertion of SRE and HNF-1 mutations in the PCSK9 promoter sequence. The presence of mutation in the SRE sequence completely abolished the PCSK9 transcriptional activity driven by leptin and resistin, as well as that of simvastatin (Figure 4B). Conversely, mutation in HNF-1 did not alter leptin- and resistin-driven luciferase activities, showing an increment of 43% and 58%, respectively (Figure 4C). The circulating levels of leptin and PCSK9 were measured by ELISA in a clinical setting of 149 males (56 ± 4 years of age) free of CV disease at enrollment and belonging to the cohort of Brisighella Heart Study.22Ruscica M. Ferri N. Fogacci F. Rosticci M. Botta M. Marchiano S. Magni P. D'Addato S. Giovannini M. Borghi C. Cicero A.F.G. Brisighella Heart Study GroupCirculating levels of proprotein convertase subtilisin/kexin type 9 and arterial stiffness in a large population sample: data from the Brisighella Heart Study.J Am Heart Assoc. 2017; 6: e005764Crossref PubMed Scopus (46) Google Scholar A positive association between circulating leptin and PCSK9 levels (β = 0.352; P = 0.014) was found only in subjects with a body mass index <25 kg/m2 (Figure 5A). When the body mass index rose, that is, between 25 and 30 kg/m2 (β = 0.147; P = 0.295) (Figure 5B) or >30 kg/m2 (β = −0.030; P = 0.840) (Figure 5C), the association was lost. Concerning resistin, the circulating levels did not associate with PCSK9 (data not shown). The main characteristics of the selected participants have been reported in Supplemental Table S1. Obesity and associated metabolic disorders are becoming major health care concerns worldwide. Obesity is highly associated with chronic low-grade inflammation, and it is believed that this obesity-linked inflammatory state is due to changes in the expression of cytokine" @default.
• W3048444324 created "2020-08-18" @default.
• W3048444324 creator A5005990941 @default.
• W3048444324 creator A5020610956 @default.
• W3048444324 creator A5022387451 @default.
• W3048444324 creator A5027968457 @default.
• W3048444324 creator A5045141716 @default.
• W3048444324 creator A5046547806 @default.
• W3048444324 creator A5048054475 @default.
• W3048444324 creator A5050497878 @default.
• W3048444324 creator A5054743665 @default.
• W3048444324 creator A5055457360 @default.
• W3048444324 creator A5061184420 @default.
• W3048444324 creator A5064742409 @default.
• W3048444324 creator A5065069460 @default.
• W3048444324 creator A5067684824 @default.
• W3048444324 date "2020-11-01" @default.
• W3048444324 modified "2023-10-16" @default.
• W3048444324 title "Leptin, Resistin, and Proprotein Convertase Subtilisin/Kexin Type 9" @default.
• W3048444324 cites W1986029658 @default.
• W3048444324 cites W1993940662 @default.
• W3048444324 cites W1994045865 @default.
• W3048444324 cites W1994137263 @default.
• W3048444324 cites W2011430620 @default.
• W3048444324 cites W2024802365 @default.
• W3048444324 cites W2042893069 @default.
• W3048444324 cites W2045686436 @default.
• W3048444324 cites W2047695409 @default.
• W3048444324 cites W2050720508 @default.
• W3048444324 cites W2062570379 @default.
• W3048444324 cites W2064877960 @default.
• W3048444324 cites W2067539811 @default.
• W3048444324 cites W2082817623 @default.
• W3048444324 cites W2084411358 @default.
• W3048444324 cites W2101667330 @default.
• W3048444324 cites W2103270369 @default.
• W3048444324 cites W2106759269 @default.
• W3048444324 cites W2126393987 @default.
• W3048444324 cites W2127596176 @default.
• W3048444324 cites W2134414477 @default.
• W3048444324 cites W2140952049 @default.
• W3048444324 cites W2149423081 @default.
• W3048444324 cites W2150102205 @default.
• W3048444324 cites W2157009395 @default.
• W3048444324 cites W216528586 @default.
• W3048444324 cites W2212216121 @default.
• W3048444324 cites W224912761 @default.
• W3048444324 cites W2273429777 @default.
• W3048444324 cites W2476206325 @default.
• W3048444324 cites W2522376836 @default.
• W3048444324 cites W2565437824 @default.
• W3048444324 cites W2587148203 @default.
• W3048444324 cites W2590097571 @default.
• W3048444324 cites W2594716767 @default.
• W3048444324 cites W2606128734 @default.
• W3048444324 cites W2610355229 @default.
• W3048444324 cites W2617891139 @default.
• W3048444324 cites W2619053053 @default.
• W3048444324 cites W2768516807 @default.
• W3048444324 cites W2790143410 @default.
• W3048444324 cites W2806952322 @default.
• W3048444324 cites W2807631284 @default.
• W3048444324 cites W2902133661 @default.
• W3048444324 cites W2917268512 @default.
• W3048444324 cites W2917273626 @default.
• W3048444324 cites W2955932804 @default.
• W3048444324 cites W2960913366 @default.
• W3048444324 cites W2961683851 @default.
• W3048444324 cites W2981286566 @default.
• W3048444324 cites W2996613943 @default.
• W3048444324 cites W2998838475 @default.
• W3048444324 doi "https://doi.org/10.1016/j.ajpath.2020.07.016" @default.
• W3048444324 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/32798443" @default.
• W3048444324 hasPublicationYear "2020" @default.
• W3048444324 type Work @default.
• W3048444324 sameAs 3048444324 @default.
• W3048444324 citedByCount "25" @default.
• W3048444324 countsByYear W30484443242020 @default.
• W3048444324 countsByYear W30484443242021 @default.
• W3048444324 countsByYear W30484443242022 @default.
• W3048444324 countsByYear W30484443242023 @default.
• W3048444324 crossrefType "journal-article" @default.
• W3048444324 hasAuthorship W3048444324A5005990941 @default.
• W3048444324 hasAuthorship W3048444324A5020610956 @default.
• W3048444324 hasAuthorship W3048444324A5022387451 @default.
• W3048444324 hasAuthorship W3048444324A5027968457 @default.
• W3048444324 hasAuthorship W3048444324A5045141716 @default.
• W3048444324 hasAuthorship W3048444324A5046547806 @default.
• W3048444324 hasAuthorship W3048444324A5048054475 @default.
• W3048444324 hasAuthorship W3048444324A5050497878 @default.
• W3048444324 hasAuthorship W3048444324A5054743665 @default.
• W3048444324 hasAuthorship W3048444324A5055457360 @default.
• W3048444324 hasAuthorship W3048444324A5061184420 @default.
• W3048444324 hasAuthorship W3048444324A5064742409 @default.
• W3048444324 hasAuthorship W3048444324A5065069460 @default.
• W3048444324 hasAuthorship W3048444324A5067684824 @default.
• W3048444324 hasBestOaLocation W30484443241 @default.
• W3048444324 hasConcept C126322002 @default.

• next