DOCSLIB.ORG

KLF7 Promotes Macrophage Activation by Activating the NF-Κb Signaling Pathway in Epicardial Adipose Tissue in Patients with Coronary Artery Disease

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
KLF7 Promotes Macrophage Activation by Activating the NF-Κb Signaling Pathway in Epicardial Adipose Tissue in Patients with Coronary Artery Disease European Review for Medical and Pharmacological Sciences 2020; 24: 7002-7014 KLF7 promotes macrophage activation by activating the NF-κB signaling pathway in epicardial adipose tissue in patients with coronary artery disease W.-H. HUANG1, Y.-J. XUE1,2, Y.-J. ZHOU1, X.-G. WU1, X.-X. WU1, X.-X. ZHANG1, X.-X. DONG1, Y.-T. WEI2,3 1Medicine Department, Shihezi University, Shihezi, Xinjiang, China 2Department of Thoracic and Cardiovascular Surgery, The First Affiliated Hospital of Shihezi University School of Medicine, Shihezi, China 3Jining First People’s Hospital, Jining, China Wenhua Huang and Yajun Xue contributed equally to this work Abstract. – OBJECTIVE: Inflammatory ac- potential therapeutic target for cardiovascular cumulation in epicardial adipose tissue (EAT) diseases such as CAD. may influence the formation and development of coronary artery disease (CAD). EAT macro- Key Words: phages exhibit M1 polarization and the secre- KLF7, Macrophage, NF-κB signaling pathway, Coro- tion of a large number of inflammatory factors in nary artery disease. CAD patients. Emerging data demonstrate that Krüppel-like factor-7 (KLF7), contributes to the regulation of adipocyte differentiation and the secretion of adipose tissue inflammation. How- Introduction ever, the function of KLF7 in EAT inflammation still remains to be uncovered. This study aims to Coronary artery disease (CAD) is the leading investigate the role of KLF7 in macrophage acti- vation in EAT. cause of death worldwide, resulting in 9.48 mil- 1 PATIENTS AND METHODS: The levels of in- lion deaths in 2016 . Endothelial dysfunction is terleukin-6 (IL-6) and tumor necrosis factor-α the first sign in the development of atherogenesis (TNF-α) in cell supernatant were measured by (AS)2,3, followed by the invasion of pro-inflamma- enzyme-linked immunosorbent assay (ELISA). tory cytokines, such as interleukin-6 (IL-6) and The mRNA expression levels were measured tumor necrosis factor-α (TNF-α). This occurs in by Real Time-PCR. The protein expression level was detected by Western blot. the epicardial adipose tissue (EAT) and the artery RESULTS: The expression of inflammatory wall around the coronary arteries. factors and KLF7 were markedly increased in EAT evolves from brown adipose tissues, it is CAD EAT than non-CAD EAT. KLF7 is highly ex- in close proximity to the coronary arteries4 and pressed in human THP-1-derived macrophages shares the same origin with omental adipose tis- induced by inflammatory stimuli, such as LPS. sue. As a result, EAT can secrete pro-inflamma- The knockdown of KLF7 inhibited the release tory cytokines and adipokines5. The tissue is also of inflammatory factors and significantly de- creased the expression of KLF7 in human THP- an active endocrine organ that secretes substan- 6 7 1-derived macrophages stimulated by LPS. More- tial TNF-a and IL-6 via paracrine or vasocrine over, transfection with KLF7-siRNA caused the effects. It is able to infiltrate the myocardium and marked inhibition of LPS-induced phosphoryla- coronary artery walls because of the lack of fascia tion of JNK-MAPKs and also suppressed the lev- between the coronary arteries and EAT8-10. els of p-p65 and inhibited the activation of p-IκBα. Macrophage activation is an integral process CONCLUSIONS: Taken together, these results indicate that KLF7 enhances macrophage acti- in the development of atherosclerosis. The best vation, mediated by JNK-NF-κB signaling path- described immunocompetent cells within EAT 11,12 ways in EAT. This suggests that KLF7 may be a are macrophages. Some scholars have shown 7002 Corresponding Author: Yutao Wei, MD; e-mail: [email protected] KLF7 promotes macrophage activation in patients with coronary artery disease that EAT macrophages exhibit M1 polarization the approved guidelines for human experimental in CAD patients, and the percentage of these research. cells is higher in CAD than non-CAD patients. EAT is characterized by the increased infiltra- Study Population tion of macrophages, suggesting that they might Thirty patients with CAD who underwent off- represent an important source of inflammation pump coronary artery bypass grafting (CABG) in adipose tissue. As pattern recognition recep- were enrolled in this study. Other 30 patients who tors, Toll-like receptors (TLRs) associate their underwent atrial septal defect repair or valvular signaling pathways with the infiltration of mac- replacement surgery without coronary artery ste- rophages in the EAT of patients with CAD11. nosis were included as the control group. EAT bi- TLRs can initiate the activation of nuclear fac- opsy samples (average weight 0.5-1.0 g) were col- tor-kB (NF-kB) and induce the expression of in- lected from the anterior wall of the left ventricle, flammatory genes. In CADs, TLR-4 is the main which was adjacent to the diseased segment, one TLR isoform involved in EAT inflammation13. hour after the administration of anesthesia. All Kruppel-like factor 7 (KLF7) is a member of samples were immediately stored in an RNA sta- the 18 KLF family that plays different roles in bilizer and then transferred to -80°C before total gene expression and regulation14,15. This ubiq- RNA extraction. Patients with acute myocardial uitous transcription factor is expressed in car- infarction, severe heart failure, active infections, diovascular, digestive, respiratory, and immune hematologic diseases, rheumatologic diseases, systems and is involved in cardiovascular dis- steroid or immunomodulatory drug use, and his- ease, cancer, and inflammatory conditions16. tories of chemotherapy or radiotherapy were ex- KLF7, which localizes in the nucleus, is a mem- cluded. The baseline characteristics and laborato- ber of the pluripotency transcriptional network ry parameters of the patients are shown in Table I. and plays a vital role in transcriptional activa- tion17,18. There has been little research into the Cell Culture role of KLF7 in causing inflammation. In a re- Human monocytic THP-1 cells were pur- cent study, the expression of KLF7 was remark- chased from the cell bank of the Chinese Acad- ably and positively correlated with TLR4 and emy of Sciences in Shanghai, China. THP-1 IL-6 in adipose tissue samples19. However, the cells were cultured in Roswell Park Memorial functional role of KLF7 in macrophage activa- Institute-1640 (RPMI-1640) medium (HyClone, tion in EAT remains unknown. South Logan, UT, USA) containing 10% fetal This study found that KLF7 had a significant bovine serum (FBS; Gibco, Rockville, MD, positive correlation with inflammatory factors in USA), penicillin (100 U/mL), and streptomycin CAD EAT. Moreover, KLF7 protein levels were (100 μg/mL) at 37°C in a humidified atmosphere markedly increased in CAD EAT compared to containing 5% CO2. The THP-1 cells were seed- non-CAD EAT. Our results indicate that knock- ed in 12-well plates at a density of 2x105 cells out of KLF7 decreases LPS-induced expression per well and differentiated into macrophages by of cytokines, including IL-6 and TNF-α by acti- using 100 μg/mL phorbol-12-myristate 13-ac- vating JNK-NF-κB signaling pathways in human etate (Invitrogen, Carlsbad, CA, USA) for 24 THP-1-derived macrophages. These results sug- hours. The cells were then incubated with 20 gest that KLF7-NF-κB may form a positive feed- ng/mL IFN-γ (Gibco, Rockville, MD, USA) and back loop to drive macrophage activation and the 10 ng/mL LPS (Sigma-Aldrich, St. Louis, MO, production of inflammatory factors in EAT. USA) for 24 hours to induce M1 macrophages before their use in experiments. Patients and Methods Real-Time Polymerase Chain Reaction (PCR) Ethical Note The total RNA from CAD EATs, non-CAD All procedures involving human subjects were EATs, and the experimental groups of cells were approved by the medical Ethics Committee at the extracted using TRIzol (Thermo Fisher Scientif- First Affiliated Hospital of Shihezi University ic, Inc., Waltham, MA, USA) in accordance with School of Medicine. Written informed consent the manufacturer’s instructions. The purity and was obtained from all subjects and all associat- concentration of RNAs were measured using a ed methods were conducted in accordance with NanoDrop 1000 spectrophotometer (Thermo 7003 W.-H. Huang, Y.-J. Xue, Y.-J. Zhou, X.-G. Wu, X.-X. Wu, X.-X. Zhang, X.-X. Dong, Y.-T. Wei Fisher Scientific, Waltham, MA, USA). Reverse ed with horseradish peroxidase (HRP) and by transcription was performed using the PrimeS- detection using the Pierce Fast Western Blot cript RT reagent Kit with genomic deoxyribo- Kit (Thermo Fisher Scientific, Waltham, MA, nucleic acid (gDNA) eraser (TaKaRa Bio, Inc., USA). Antibodies used in the study were β-ac- Otsu, Shiga, Japan) with 500 ng of total RNA tin (ZSGBBIO, Beijing, China), KLF7 (Abcam, isolated for each sample. Quantitative Real Cambridge, MA, USA), p-IkBα (S32/S36), Time-PCR (qRT-PCR) was performed with the JNK, p-JNK (T183/ Y185), p-p65 (Cell Sig- QuantiNova SYBR Green PCR kit (Qiagen, naling Technology, Danvers, MA, USA), and Hilden, Germany) using the 7500 Real-Time mouse IgG and rabbit IgG (ZSGBBIO, Beijing, PCR System (Applied Biosystems, Foster City, China). β-actin protein was probed with mice CA, USA). The gene expression levels of beta-ac- antibodies to β-actin (ZSGBBIO, Beijing, Chi- tin, KLF7, TLR4, IL-6, CD68, TNF-α, JNK, and na, TA-09; 1:1000 dilution). KLF7 protein was NF-kB were quantified, and the primer sequenc- probed with rabbit antibodies to KLF7 (Ab- es are listed in Table II. All assays were per- cam, Cambridge, MA, USA, ab129149; 1:1000 formed in triplicate. The relative amounts of the dilution). Phospho-IkBα protein was probed target gene transcripts were calculated using the with mice antibodies to Phospho-IkBα (CST, comparative cycle time method. The relative ex- Danvers, MA, USA, #9246; 1:1000 dilution). pression levels for each tissue and control sam- JNK protein was probed with rabbit antibod- ples were calculated by the comparative critical ies to JNK (CST, Danvers, MA, USA, #9252; threshold method.
Load more

Recommended publications
  • TRANSCRIPTIONAL REGULATION of Hur in RENAL STRESS
    TRANSCRIPTIONAL REGULATION OF HuR IN RENAL STRESS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Sudha Suman Govindaraju Graduate Program in Biochemistry The Ohio State University 2014 Dissertation Committee: Dr. Beth S. Lee, Ph.D., Advisor Dr. Kathleen Boris-Lawrie, Ph.D. Dr. Sissy M. Jhiang, Ph.D. Dr. Arthur R. Strauch, Ph.D Abstract HuR is a ubiquitously expressed RNA-binding protein that affects the post- transcriptional life of thousands of cellular mRNAs by regulating transcript stability and translation. HuR can post-transcriptionally regulate gene expression and modulate cellular responses to stress, differentiation, proliferation, apoptosis, senescence, inflammation, and the immune response. It is an important mediator of survival during cellular stress, but when inappropriately expressed, can promote oncogenic transformation. Not surprisingly, the expression of HuR itself is tightly regulated at multiple transcriptional and post-transcriptional levels. Previous studies demonstrated the existence of two alternate HuR transcripts that differ in their 5’ untranslated regions and have markedly different translatabilities. These forms were also found to be reciprocally expressed following cellular stress in kidney proximal tubule cell lines, and the shorter, more readily translatable variant was shown to be regulated by Smad 1/5/8 pathway and bone morphogenetic protein-7 (BMP-7) signaling. In this study, the factors that promote transcription of the longer alternate form were identified. NF-κB was shown to be important for expression of the long HuR mRNA, as was a newly identified region with potential for binding the Sp/KLF families of transcription factors.
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • Supplementary Table S5. Differentially Expressed Gene Lists of PD-1High CD39+ CD8 Tils According to 4-1BB Expression Compared to PD-1+ CD39- CD8 Tils
    BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) J Immunother Cancer Supplementary Table S5. Differentially expressed gene lists of PD-1high CD39+ CD8 TILs according to 4-1BB expression compared to PD-1+ CD39- CD8 TILs Up- or down- regulated genes in Up- or down- regulated genes Up- or down- regulated genes only PD-1high CD39+ CD8 TILs only in 4-1BBneg PD-1high CD39+ in 4-1BBpos PD-1high CD39+ CD8 compared to PD-1+ CD39- CD8 CD8 TILs compared to PD-1+ TILs compared to PD-1+ CD39- TILs CD39- CD8 TILs CD8 TILs IL7R KLRG1 TNFSF4 ENTPD1 DHRS3 LEF1 ITGA5 MKI67 PZP KLF3 RYR2 SIK1B ANK3 LYST PPP1R3B ETV1 ADAM28 H2AC13 CCR7 GFOD1 RASGRP2 ITGAX MAST4 RAD51AP1 MYO1E CLCF1 NEBL S1PR5 VCL MPP7 MS4A6A PHLDB1 GFPT2 TNF RPL3 SPRY4 VCAM1 B4GALT5 TIPARP TNS3 PDCD1 POLQ AKAP5 IL6ST LY9 PLXND1 PLEKHA1 NEU1 DGKH SPRY2 PLEKHG3 IKZF4 MTX3 PARK7 ATP8B4 SYT11 PTGER4 SORL1 RAB11FIP5 BRCA1 MAP4K3 NCR1 CCR4 S1PR1 PDE8A IFIT2 EPHA4 ARHGEF12 PAICS PELI2 LAT2 GPRASP1 TTN RPLP0 IL4I1 AUTS2 RPS3 CDCA3 NHS LONRF2 CDC42EP3 SLCO3A1 RRM2 ADAMTSL4 INPP5F ARHGAP31 ESCO2 ADRB2 CSF1 WDHD1 GOLIM4 CDK5RAP1 CD69 GLUL HJURP SHC4 GNLY TTC9 HELLS DPP4 IL23A PITPNC1 TOX ARHGEF9 EXO1 SLC4A4 CKAP4 CARMIL3 NHSL2 DZIP3 GINS1 FUT8 UBASH3B CDCA5 PDE7B SOGA1 CDC45 NR3C2 TRIB1 KIF14 TRAF5 LIMS1 PPP1R2C TNFRSF9 KLRC2 POLA1 CD80 ATP10D CDCA8 SETD7 IER2 PATL2 CCDC141 CD84 HSPA6 CYB561 MPHOSPH9 CLSPN KLRC1 PTMS SCML4 ZBTB10 CCL3 CA5B PIP5K1B WNT9A CCNH GEM IL18RAP GGH SARDH B3GNT7 C13orf46 SBF2 IKZF3 ZMAT1 TCF7 NECTIN1 H3C7 FOS PAG1 HECA SLC4A10 SLC35G2 PER1 P2RY1 NFKBIA WDR76 PLAUR KDM1A H1-5 TSHZ2 FAM102B HMMR GPR132 CCRL2 PARP8 A2M ST8SIA1 NUF2 IL5RA RBPMS UBE2T USP53 EEF1A1 PLAC8 LGR6 TMEM123 NEK2 SNAP47 PTGIS SH2B3 P2RY8 S100PBP PLEKHA7 CLNK CRIM1 MGAT5 YBX3 TP53INP1 DTL CFH FEZ1 MYB FRMD4B TSPAN5 STIL ITGA2 GOLGA6L10 MYBL2 AHI1 CAND2 GZMB RBPJ PELI1 HSPA1B KCNK5 GOLGA6L9 TICRR TPRG1 UBE2C AURKA Leem G, et al.
    [Show full text]
  • Supplementary Table 1. Pain and PTSS Associated Genes (N = 604
    Supplementary Table 1. Pain and PTSS associated genes (n = 604) compiled from three established pain gene databases (PainNetworks,[61] Algynomics,[52] and PainGenes[42]) and one PTSS gene database (PTSDgene[88]). These genes were used in in silico analyses aimed at identifying miRNA that are predicted to preferentially target this list genes vs. a random set of genes (of the same length). ABCC4 ACE2 ACHE ACPP ACSL1 ADAM11 ADAMTS5 ADCY5 ADCYAP1 ADCYAP1R1 ADM ADORA2A ADORA2B ADRA1A ADRA1B ADRA1D ADRA2A ADRA2C ADRB1 ADRB2 ADRB3 ADRBK1 ADRBK2 AGTR2 ALOX12 ANO1 ANO3 APOE APP AQP1 AQP4 ARL5B ARRB1 ARRB2 ASIC1 ASIC2 ATF1 ATF3 ATF6B ATP1A1 ATP1B3 ATP2B1 ATP6V1A ATP6V1B2 ATP6V1G2 AVPR1A AVPR2 BACE1 BAMBI BDKRB2 BDNF BHLHE22 BTG2 CA8 CACNA1A CACNA1B CACNA1C CACNA1E CACNA1G CACNA1H CACNA2D1 CACNA2D2 CACNA2D3 CACNB3 CACNG2 CALB1 CALCRL CALM2 CAMK2A CAMK2B CAMK4 CAT CCK CCKAR CCKBR CCL2 CCL3 CCL4 CCR1 CCR7 CD274 CD38 CD4 CD40 CDH11 CDK5 CDK5R1 CDKN1A CHRM1 CHRM2 CHRM3 CHRM5 CHRNA5 CHRNA7 CHRNB2 CHRNB4 CHUK CLCN6 CLOCK CNGA3 CNR1 COL11A2 COL9A1 COMT COQ10A CPN1 CPS1 CREB1 CRH CRHBP CRHR1 CRHR2 CRIP2 CRYAA CSF2 CSF2RB CSK CSMD1 CSNK1A1 CSNK1E CTSB CTSS CX3CL1 CXCL5 CXCR3 CXCR4 CYBB CYP19A1 CYP2D6 CYP3A4 DAB1 DAO DBH DBI DICER1 DISC1 DLG2 DLG4 DPCR1 DPP4 DRD1 DRD2 DRD3 DRD4 DRGX DTNBP1 DUSP6 ECE2 EDN1 EDNRA EDNRB EFNB1 EFNB2 EGF EGFR EGR1 EGR3 ENPP2 EPB41L2 EPHB1 EPHB2 EPHB3 EPHB4 EPHB6 EPHX2 ERBB2 ERBB4 EREG ESR1 ESR2 ETV1 EZR F2R F2RL1 F2RL2 FAAH FAM19A4 FGF2 FKBP5 FLOT1 FMR1 FOS FOSB FOSL2 FOXN1 FRMPD4 FSTL1 FYN GABARAPL1 GABBR1 GABBR2 GABRA2 GABRA4
    [Show full text]
  • Transcriptome Analysis of Complex I-Deficient Patients Reveals Distinct
    van der Lee et al. BMC Genomics (2015) 16:691 DOI 10.1186/s12864-015-1883-8 RESEARCH ARTICLE Open Access Transcriptome analysis of complex I-deficient patients reveals distinct expression programs for subunits and assembly factors of the oxidative phosphorylation system Robin van der Lee1†, Radek Szklarczyk1,2†, Jan Smeitink3,HubertJMSmeets4, Martijn A. Huynen1 and Rutger Vogel3* Abstract Background: Transcriptional control of mitochondrial metabolism is essential for cellular function. A better understanding of this process will aid the elucidation of mitochondrial disorders, in particular of the many genetically unsolved cases of oxidative phosphorylation (OXPHOS) deficiency. Yet, to date only few studies have investigated nuclear gene regulation in the context of OXPHOS deficiency. In this study we performed RNA sequencing of two control and two complex I-deficient patient cell lines cultured in the presence of compounds that perturb mitochondrial metabolism: chloramphenicol, AICAR, or resveratrol. We combined this with a comprehensive analysis of mitochondrial and nuclear gene expression patterns, co-expression calculations and transcription factor binding sites. Results: Our analyses show that subsets of mitochondrial OXPHOS genes respond opposingly to chloramphenicol and AICAR, whereas the response of nuclear OXPHOS genes is less consistent between cell lines and treatments. Across all samples nuclear OXPHOS genes have a significantly higher co-expression with each other than with other genes, including those encoding mitochondrial proteins. We found no evidence for complex-specific mRNA expression regulation: subunits of different OXPHOS complexes are similarly (co-)expressed and regulated by a common set of transcription factors. However, we did observe significant differences between the expression of nuclear genes for OXPHOS subunits versus assembly factors, suggesting divergent transcription programs.
    [Show full text]
  • Using a Seed-Network to Query Multiple Large-Scale Gene
    Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2009 Using a seed-network to query multiple large-scale gene expression datasets from the developing retina in order to identify and prioritize experimental targets Timothy Alcon Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Computer Sciences Commons Recommended Citation Alcon, Timothy, "Using a seed-network to query multiple large-scale gene expression datasets from the developing retina in order to identify and prioritize experimental targets" (2009). Graduate Theses and Dissertations. 11127. https://lib.dr.iastate.edu/etd/11127 This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Using a seed-network to query multiple large-scale gene expression datasets from the developing retina in order to identify and prioritize experimental targets by Timothy C. Alcon A thesis submitted to the graduate faculty in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Major: Bioinformatics and Computational Biology Program of Study Committee: Vasant G. Honavar, Co-major Professor M. Heather West Greenlee, Co-major Professor Jeanne M. Serb Iowa State University Ames, Iowa 2009 Copyright ⃝c Timothy C. Alcon, 2009. All rights reserved. ii DEDICATION I would like to dedicate this thesis to all the family and friends who helped me remain relatively sane during my graduate studies.
    [Show full text]
  • The NANOG Transcription Factor Induces Type 2 Deiodinase Expression and Regulates the Intracellular Activation of Thyroid Hormone in Keratinocyte Carcinomas
    Cancers 2020, 12 S1 of S18 Supplementary Materials: The NANOG Transcription Factor Induces Type 2 Deiodinase Expression and Regulates the Intracellular Activation of Thyroid Hormone in Keratinocyte Carcinomas Annarita Nappi, Emery Di Cicco, Caterina Miro, Annunziata Gaetana Cicatiello, Serena Sagliocchi, Giuseppina Mancino, Raffaele Ambrosio, Cristina Luongo, Daniela Di Girolamo, Maria Angela De Stefano, Tommaso Porcelli, Mariano Stornaiuolo and Monica Dentice Figure S1. Strategy for the mutagenesis of Dio2 promoter. (A) Schematic representation of NANOG Binding Site within the Dio2 promoter region. (B) Schematic diagram for site‐directed mutagenesis of NANOG Binding Site on Dio2 promoter region by Recombinant PCR. (C) Representation of the mutated NANOG Binding Site on Dio2 promoter region. (D) Electropherogram of the NANOG Binding Site mutation within the Dio2 promoter. Cancers 2020, 12 S2 of S18 Figure S2. Strategy for the silencing of NANOG expression. (A) Cloning strategies for the generation of NANOG shRNA expression vectors. (B) Electropherograms of the NANOG shRNA sequences cloned into pcDNA3.1 vector. (C) Validation of effective NANOG down-modulation by two different NANOG shRNA vectors was assessed by Western Blot analysis of NANOG expression in BCC cells. (D) Quantification of NANOG protein levels versus Tubulin levels in the same experiment as in C is represented by histograms. Cancers 2020, 12 S3 of S18 Figure S3. The CD34+ cells are characterized by the expression of typical epithelial stemness genes. The mRNA levels of a panel of indicated stemness markers of epidermis were measured by Real Time PCR in the same experiment indicated in figure 3F and G. Cancers 2020, 12 S4 of S18 Figure S4.
    [Show full text]
  • Evolving Principles Underlying Neural Lineage Conversion
    F1000Research 2019, 8(F1000 Faculty Rev):1548 Last updated: 16 SEP 2019 REVIEW Evolving principles underlying neural lineage conversion and their relevance for biomedical translation [version 1; peer review: 4 approved] Lea Jessica Flitsch, Oliver Brüstle Institute of Reconstructive Neurobiology, University of Bonn School of Medicine & University Hospital Bonn, Bonn, North Rhine Wesphalia, 53127, Germany First published: 30 Aug 2019, 8(F1000 Faculty Rev):1548 ( Open Peer Review v1 https://doi.org/10.12688/f1000research.18926.1) Latest published: 30 Aug 2019, 8(F1000 Faculty Rev):1548 ( https://doi.org/10.12688/f1000research.18926.1) Reviewer Status Abstract Invited Reviewers Scientific and technological advances of the past decade have shed light 1 2 3 4 on the mechanisms underlying cell fate acquisition, including its transcriptional and epigenetic regulation during embryonic development. version 1 This knowledge has enabled us to purposefully engineer cell fates in vitro published by manipulating expression levels of lineage-instructing transcription 30 Aug 2019 factors. Here, we review the state of the art in the cell programming field with a focus on the derivation of neural cells. We reflect on what we know about the mechanisms underlying fate changes in general and on the F1000 Faculty Reviews are written by members of degree of epigenetic remodeling conveyed by the distinct reprogramming the prestigious F1000 Faculty. They are and direct conversion strategies available. Moreover, we discuss the commissioned and are peer reviewed before implications of residual epigenetic memory for biomedical applications such publication to ensure that the final, published version as disease modeling and neuroregeneration. Finally, we cover recent developments approaching cell fate conversion in the living brain and is comprehensive and accessible.
    [Show full text]
  • A Missense Mutation in the KLF7 Gene Is a Potential Candidate Variant for Congenital Deafness in Australian Stumpy Tail Cattle Dogs
    G C A T T A C G G C A T genes Article A Missense Mutation in the KLF7 Gene Is a Potential Candidate Variant for Congenital Deafness in Australian Stumpy Tail Cattle Dogs Fangzheng Xu 1,† , Shuwen Shan 1,†, Susan Sommerlad 2, Jennifer M. Seddon 2 and Bertram Brenig 1,* 1 Institute of Veterinary Medicine, University of Goettingen, 37077 Göttingen, Germany; [email protected] (F.X.); [email protected] (S.S.) 2 School of Veterinary Science, The University of Queensland, Gatton, QLD 4343, Australia; [email protected] (S.S.); [email protected] (J.M.S.) * Correspondence: [email protected]; Tel.: +49-551-3928383; Fax: +49-551-3933392 † These authors have equally contributed. Abstract: Congenital deafness is prevalent among modern dog breeds, including Australian Stumpy Tail Cattle Dogs (ASCD). However, in ASCD, no causative gene has been identified so far. Therefore, we performed a genome-wide association study (GWAS) and whole genome sequencing (WGS) of affected and normal individuals. For GWAS, 3 bilateral deaf ASCDs, 43 herding dogs, and one unaffected ASCD were used, resulting in 13 significantly associated loci on 6 chromosomes, i.e., CFA3, 8, 17, 23, 28, and 37. CFA37 harbored a region with the most significant association −21 (−log10(9.54 × 10 ) = 20.02) as well as 7 of the 13 associated loci. For whole genome sequencing, the same three affected ASCDs and one unaffected ASCD were used. The WGS data were compared with 722 canine controls and filtered for protein coding and non-synonymous variants, resulting in Citation: Xu, F.; Shan, S.; Sommerlad, four missense variants present only in the affected dogs.
    [Show full text]
  • Mutation Analysis of Candidate Genes Within the 2Q33.3 Linkage Area for Familial Early-Onset Generalised Osteoarthritis
    European Journal of Human Genetics (2007) 15, 791–799 & 2007 Nature Publishing Group All rights reserved 1018-4813/07 $30.00 www.nature.com/ejhg ARTICLE Mutation analysis of candidate genes within the 2q33.3 linkage area for familial early-onset generalised osteoarthritis Josine L Min1, Ingrid Meulenbelt*,1, Margreet Kloppenburg2, Cornelia M van Duijn3 and P Eline Slagboom1 1Molecular Epidemiology, Leiden University Medical Centre, Leiden, The Netherlands; 2Department of Rheumatology, Leiden University Medical Centre, Leiden, The Netherlands; 3Department of Epidemiology & Biostatistics, Erasmus Medical Centre, Rotterdam, The Netherlands In a genome-wide linkage scan of seven families with familial early-onset osteoarthritis (FOA), we mapped a FOA locus to a 5 cM region on chromosome 2q33.3–2q34 with a maximum LOD score of 6.05. To identify causal variants, 17 positional candidate genes and FRZB were sequenced for coding, splice sites, and 50 and 30 untranslated regions. The pathogenicity of possible disease-causing variants was evaluated using predicted effects on protein structure and function, splicing enhancers, degree of conservation and frequency in 790 unrelated subjects from the population-based Rotterdam study scored for the presence of radiographic signs of OA (ROA). Nine novel variants, identified in NRP2, XM_371590, ADAM23, IDH1, PIP5K3 and PTHR2, cosegregated with FOA, of which two were promising. IDH1 Y183C cosegregated in one family, involved a conserved amino-acid change and showed a damaging effect predicted by PolyPhen and SIFT. In the Rotterdam sample, carriers of IDH1 Y183C (0.02) had an increased but insignificant risk for generalised ROA. The second variant, NRP2 c.1938-21T4C cosegregated in three families.
    [Show full text]
  • NUMB and NUMBL Differences in Gene Regulation
    www.impactjournals.com/oncotarget/ Oncotarget, 2018, Vol. 9, (No. 10), pp: 9219-9234 Research Paper NUMB and NUMBL differences in gene regulation José Manuel García-Heredia1,2,3 and Amancio Carnero1,3 1Instituto de Biomedicina de Sevilla, IBIS, Hospital Universitario Virgen del Rocío, Universidad de Sevilla, Consejo Superior de Investigaciones Científicas, Seville, Spain 2Department of Vegetal Biochemistry and Molecular Biology, University of Seville, Seville, Spain 3CIBER de Cáncer, Instituto de Salud Carlos III, Pabellón 11, Planta 0, Madrid, Spain Correspondence to: Amancio Carnero, email: [email protected] Keywords: NUMB; NUMBL; cancer; Notch pathway; WNT pathway Received: September 06, 2017 Accepted: January 03, 2018 Published: January 11, 2018 Copyright: García-Heredia et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT NUMB, and its close homologue NUMBL, behave as tumor suppressor genes by regulating the Notch pathway. The downregulation of these genes in tumors is common, allowing aberrant Notch pathway activation and tumor progression. However, some known differences between NUMB and NUMBL have raised unanswered questions regarding the redundancy and/or combined regulation of the Notch pathway by these genes during the tumorigenic process. We have found that NUMB and NUMBL exhibit mutual exclusivity in human tumors, suggesting that the associated tumor suppressor role is regulated by only one of the two proteins in a specific cell, avoiding duplicate signaling and simplifying the regulatory network. We have also found differences in gene expression due to NUMB or NUMBL downregulation.
    [Show full text]
  • Krüppel-Like Transcription Factors in the Nervous System: Novel Players in Neurite Outgrowth and Axon Regeneration
    Molecular and Cellular Neuroscience 47 (2011) 233–243 Contents lists available at ScienceDirect Molecular and Cellular Neuroscience journal homepage: www.elsevier.com/locate/ymcne Review Krüppel-like transcription factors in the nervous system: Novel players in neurite outgrowth and axon regeneration Darcie L. Moore 1, Akintomide Apara, Jeffrey L. Goldberg ⁎ Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL, USA article info abstract Article history: The Krüppel-like family of transcription factors (KLFs) have been widely studied in proliferating cells, though Received 13 May 2011 very little is known about their role in post-mitotic cells, such as neurons. We have recently found that the Accepted 16 May 2011 KLFs play a role in regulating intrinsic axon growth ability in retinal ganglion cells (RGCs), a type of central Available online 24 May 2011 nervous system (CNS) neuron. Previous KLF studies in other cell types suggest that there may be cell-type fi Keywords: speci c KLF expression patterns, and that their relative expression allows them to compete for binding sites, or to act redundantly to compensate for another's function. With at least 15 of 17 KLF family members Axon Regeneration expressed in neurons, it will be important for us to determine how this complex family functions to regulate Krüppel-like factor (KLF) the intricate gene programs of axon growth and regeneration. By further characterizing the mechanisms of CNS the KLF family in the nervous system, we may better understand how they regulate neurite growth and axon Growth regeneration. Transcription factor © 2011 Elsevier Inc. All rights reserved.
    [Show full text]