|
1
|
Prakash K, Satishkartik S, Ramalingam S, Gangadaran P, Gnanavel S, Aruljothi KN. Investigating the multifaceted role of nucleolin in cellular function and Cancer: Structure, Regulation, and therapeutic implications. Gene 2025; 957:149479. [PMID: 40210024 DOI: 10.1016/j.gene.2025.149479] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2025] [Revised: 03/20/2025] [Accepted: 04/05/2025] [Indexed: 04/12/2025]
Abstract
Nucleolin (NCL), a highly conserved and multifunctional phosphoprotein, is primarily localized in the nucleolus and participates in various cellular compartments, including the nucleoplasm, cytoplasm, and plasma membrane. Initially discovered in the 1970 s, NCL is integral to ribosome biogenesis through its roles in ribosomal RNA transcription, processing, and assembly. Beyond ribosome synthesis, NCL plays critical roles in cellular processes such as DNA and RNA metabolism, chromatin remodeling, and cell cycle regulation, underscoring its essentiality for cell viability. Structurally, NCL comprises multiple functional domains, which facilitates interaction with various kinases and other proteins. NCL's extensive post-translational modifications influence its localization and function. Importantly, NCL has emerged as a key player in multiple pathologies, particularly cancer, where it contributes to tumor growth, metastasis, and drug resistance. On the cell surface, NCL acts as a co-receptor for growth factors and other ligands, facilitating oncogenic signaling. Additionally, its regulation of non-coding RNAs, stabilization of oncogenic mRNAs, and involvement in immune evasion highlight its potential as a therapeutic target. This review provides an unexplored in-depth overview of NCL's structure, functions, and modifications, with a focus on its role in cancer biology and its therapeutic implications.
Collapse
Affiliation(s)
- Kruthika Prakash
- Department of Genetic Engineering, SRM Institute of Science and Technology, Kattankulathur, Chengalpattu 603203, India
| | - Srisri Satishkartik
- Department of Genetic Engineering, SRM Institute of Science and Technology, Kattankulathur, Chengalpattu 603203, India
| | - Satish Ramalingam
- Department of Genetic Engineering, SRM Institute of Science and Technology, Kattankulathur, Chengalpattu 603203, India
| | - Prakash Gangadaran
- Department of Nuclear Medicine, School of Medicine, Kyungpook National University, Daegu 41944, Republic of Korea; BK21 FOUR KNU Convergence Educational Program of Biomedical Sciences for Creative Future Talents, Department of Biomedical Science, School of Medicine, Kyungpook National University, Daegu 41944, Republic of Korea; Cardiovascular Research Institute, Kyungpook National University, Daegu 41944, Republic of Korea
| | - S Gnanavel
- Biomaterials Laboratory, Department of Biomedical Engineering, SRM Institute of Science and Technology, Kattankulathur, Chengalpattu, Tamil Nadu, 603203, India
| | - K N Aruljothi
- Department of Genetic Engineering, SRM Institute of Science and Technology, Kattankulathur, Chengalpattu 603203, India.
| |
Collapse
|
|
2
|
Said EA, Al-Dughaishi S, Al-Hatmi W, Al-Reesi I, Al-Balushi MS, Al-Bimani A, Al-Busaidi JZ, Al-Riyami M, Al-Khabori M, Al-Kindi S, Procopio FA, Al-Sinawi S, Al-Ansari A, Koh CY, Al-Naamani K, Al-Jabri AA. Differential Production of Midkine and Pleiotrophin by Innate APCs upon Stimulation through Nucleic Acid-Sensing TLRs. J Immunol Res 2023; 2023:7944102. [PMID: 37850119 PMCID: PMC10578979 DOI: 10.1155/2023/7944102] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2023] [Revised: 07/26/2023] [Accepted: 08/26/2023] [Indexed: 10/19/2023] Open
Abstract
Midkine (MK) and pleiotrophin (PTN) belong to the same family of cytokines. They have similar sequences and functions. Both have important roles in cellular proliferation, tumors, and diseases. They regulate and are expressed by some immune cells. We have recently demonstrated MK production by some human innate antigen-presenting cells (iAPCs), i.e., monocyte-derived dendritic cells (MDDCs) and macrophages stimulated through Toll-like receptor (TLR)-4, and plasmacytoid dendritic cells (pDCs) stimulated through TLR 7. While PTN production was only documented in tissue macrophages. TLRs 3, 7, 8, and 9 are nucleic acid sensing (NAS) TLRs that detect nucleic acids from cell damage and infection and induce iAPC responses. We investigated whether NAS TLRs can induce MK and PTN production by human iAPCs, namely monocytes, macrophages, MDDCs, myeloid dendritic cells (mDCs), and pDCs. Our results demonstrated for the first time that PTN is produced by all iAPCs upon TLR triggering (p < 0.01). IAPCs produced more PTN than MK (p < 0.01). NAS TLRs and iAPCs had differential abilities to induce the production of MK, which was induced in monocytes and pDCs by all NAS TLRs (p < 0.05) and in MDDCs by TLRs 7/8 (p < 0.05). TLR4 induced a stronger MK production than NAS TLRs (p ≤ 0.05). Monocytes produced higher levels of PTN after differentiation to macrophages and MDDCs (p < 0.05). The production of MK and PTN differs among iAPCs, with a higher production of PTN and a selective induction of MK production by NAS TLR. This highlights the potentially important role of iAPCs in angiogenesis, tumors, infections, and autoimmunity through the differential production of MK and PTN upon TLR triggering.
Collapse
Affiliation(s)
- Elias A. Said
- Department of Microbiology and Immunology, College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Oman
| | - Sumaya Al-Dughaishi
- Department of Microbiology and Immunology, College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Oman
| | - Wadha Al-Hatmi
- Department of Microbiology and Immunology, College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Oman
| | - Iman Al-Reesi
- Department of Microbiology and Immunology, College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Oman
| | - Mohammed S. Al-Balushi
- Department of Microbiology and Immunology, College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Oman
| | - Atika Al-Bimani
- Department of Microbiology and Immunology, College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Oman
| | - Juma Z. Al-Busaidi
- Department of Microbiology and Immunology, College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Oman
| | - Marwa Al-Riyami
- Department of Pathology, College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Oman
| | - Murtadha Al-Khabori
- Department of Hematology, College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Oman
| | - Salam Al-Kindi
- Department of Hematology, College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Oman
| | - Francesco A. Procopio
- Service of Immunology and Allergy, Lausanne University Hospital (CHUV), Lausanne, Switzerland
| | - Shadia Al-Sinawi
- Department of Pathology, College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Oman
| | - Aliyaa Al-Ansari
- Department of Biology, College of Science, Sultan Qaboos University, Muscat, Oman
| | - Crystal Y. Koh
- Department of Microbiology and Immunology, College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Oman
| | | | - Ali A. Al-Jabri
- Department of Microbiology and Immunology, College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Oman
| |
Collapse
|
|
3
|
Kam NW, Lau CY, Che CM, Lee VHF. Nasopharynx Battlefield: Cellular Immune Responses Mediated by Midkine in Nasopharyngeal Carcinoma and COVID-19. Cancers (Basel) 2023; 15:4850. [PMID: 37835544 PMCID: PMC10571800 DOI: 10.3390/cancers15194850] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2023] [Revised: 09/28/2023] [Accepted: 10/02/2023] [Indexed: 10/15/2023] Open
Abstract
Clinical evidence suggests that the severe respiratory illness coronavirus disease 2019 (COVID-19) is often associated with a cytokine storm that results in dysregulated immune responses. Prolonged COVID-19 positivity is thought to disproportionately affect cancer patients. With COVID-19 disrupting the delivery of cancer care, it is crucial to gain momentum and awareness of the mechanistic intersection between these two diseases. This review discusses the role of the cytokine midkine (MK) as an immunomodulator in patients with COVID-19 and nasopharyngeal carcinoma (NPC), both of which affect the nasal cavity. We conducted a review and analysis of immunocellular similarities and differences based on clinical studies, research articles, and published transcriptomic datasets. We specifically focused on ligand-receptor pairs that could be used to infer intercellular communication, as well as the current medications used for each disease, including NPC patients who have contracted COVID-19. Based on our findings, we recommend close monitoring of the MK axis to maintain the desirable effects of therapeutic regimens in fighting both NPC and COVID-19 infections.
Collapse
Affiliation(s)
- Ngar-Woon Kam
- Department of Clinical Oncology, Centre of Cancer Medicine, School of Clinical Medicine, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong 999077, China; (N.-W.K.); (C.-Y.L.)
- Laboratory for Synthetic Chemistry and Chemical Biology Ltd., Hong Kong Science Park, New Territories, Hong Kong 999077, China;
| | - Cho-Yiu Lau
- Department of Clinical Oncology, Centre of Cancer Medicine, School of Clinical Medicine, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong 999077, China; (N.-W.K.); (C.-Y.L.)
- Laboratory for Synthetic Chemistry and Chemical Biology Ltd., Hong Kong Science Park, New Territories, Hong Kong 999077, China;
| | - Chi-Ming Che
- Laboratory for Synthetic Chemistry and Chemical Biology Ltd., Hong Kong Science Park, New Territories, Hong Kong 999077, China;
- Department of Chemistry, Faculty of Science, The University of Hong Kong, Hong Kong 999077, China
| | - Victor Ho-Fun Lee
- Department of Clinical Oncology, Centre of Cancer Medicine, School of Clinical Medicine, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong 999077, China; (N.-W.K.); (C.-Y.L.)
- Clinical Oncology Center, The University of Hong Kong-Shenzhen Hospital, Shenzhen 518053, China
| |
Collapse
|
|
4
|
Transcriptomic analysis of the innate immune response to in vitro transfection of plasmid DNA. MOLECULAR THERAPY. NUCLEIC ACIDS 2022; 31:43-56. [PMID: 36618265 PMCID: PMC9800263 DOI: 10.1016/j.omtn.2022.11.025] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 03/25/2021] [Accepted: 11/28/2022] [Indexed: 12/12/2022]
Abstract
The innate immune response to cytosolic DNA is intended to protect the host from viral infections, but it can also inhibit the delivery and expression of therapeutic transgenes in gene and cell therapies. The goal of this work was to use mRNA sequencing to identify genes that may influence transfection efficiency in four different cell types (PC-3, Jurkat, HEK-293T, and primary T cells). The highest transfection efficiency was observed in HEK-293T cells, which upregulated only 142 genes with no known antiviral functions after transfection with lipofectamine. Lipofection upregulated 1,057 cytokine-stimulated genes (CSGs) in PC-3 cells, which exhibited a significantly lower transfection efficiency. However, when PC-3 cells were transfected in serum-containing media or electroporated, the observed transfection efficiencies were significantly higher while the expression levels of cytokines and CSGs decreased. In contrast, lipofection of Jurkat and primary T cells only upregulated a few genes, but several of the antiviral CSGs that were absent in HEK-293T cells and upregulated in PC-3 cells were observed to be constitutively expressed in T cells, which may explain the relatively low Lipofection efficiencies observed with T cells (8%-21% GFP+). Indeed, overexpression of one CSG (IFI16) significantly decreased transfection efficiency in HEK-293T cells.
Collapse
|
|
5
|
Majaj M, Weckbach LT. Midkine-A novel player in cardiovascular diseases. Front Cardiovasc Med 2022; 9:1003104. [PMID: 36204583 PMCID: PMC9530663 DOI: 10.3389/fcvm.2022.1003104] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2022] [Accepted: 08/30/2022] [Indexed: 11/18/2022] Open
Abstract
Midkine (MK) is a 13-kDa heparin-binding cytokine and growth factor with anti-apoptotic, pro-angiogenic, pro-inflammatory and anti-infective functions, that enable it to partake in a series of physiological and pathophysiological processes. In the past, research revolving around MK has concentrated on its roles in reproduction and development, tissue protection and repair as well as inflammatory and malignant processes. In the recent few years, MK's implication in a wide scope of cardiovascular diseases has been rigorously investigated. Nonetheless, there is still no broadly accepted consensus on whether MK exerts generally detrimental or favorable effects in cardiovascular diseases. The truth probably resides somewhere in-between and depends on the underlying physiological or pathophysiological condition. It is therefore crucial to thoroughly examine and appraise MK's participation in cardiovascular diseases. In this review, we introduce the MK gene and protein, its multiple receptors and signaling pathways along with its expression in the vascular system and its most substantial functions in cardiovascular biology. Further, we recapitulate the current evidence of MK's expression in cardiovascular diseases, addressing the various sources and modes of MK expression. Moreover, we summarize the most significant implications of MK in cardiovascular diseases with particular emphasis on MK's advantageous and injurious functions, highlighting its ample diagnostic and therapeutic potential. Also, we focus on conflicting roles of MK in a number of cardiovascular diseases and try to provide some clarity and guidance to MK's multifaceted roles. In summary, we aim to pave the way for MK-based diagnostics and therapies that could present promising tools in the diagnosis and treatment of cardiovascular diseases.
Collapse
Affiliation(s)
- Marina Majaj
- Walter Brendel Centre for Experimental Medicine, Biomedical Centre, Institute for Cardiovascular Physiology und Pathophysiology, Ludwig-Maximilians-University Munich, Munich, Germany
- Department of Neurology, Heidelberg University Hospital, Heidelberg, Germany
| | - Ludwig T. Weckbach
- Walter Brendel Centre for Experimental Medicine, Biomedical Centre, Institute for Cardiovascular Physiology und Pathophysiology, Ludwig-Maximilians-University Munich, Munich, Germany
- Medizinische Klinik und Poliklinik I, Klinikum der Universität München, Munich, Germany
- Deutsches Zentrum für Herz-Kreislauf-Forschung e. V, Berlin, Germany
| |
Collapse
|
|
6
|
Zheng L, Liu Q, Li R, Chen S, Tan J, Li L, Dong X, Huang C, Wen T, Liu J. Targeting MDK Abrogates IFN-γ-Elicited Metastasis inCancers of Various Origins. Front Oncol 2022; 12:885656. [PMID: 35747815 PMCID: PMC9210922 DOI: 10.3389/fonc.2022.885656] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2022] [Accepted: 05/18/2022] [Indexed: 11/13/2022] Open
Abstract
IFN-γ is a pleiotropic cytokine with immunomodulatory and tumoricidal functions. It has been used as an anti-tumor agent in adjuvant therapies for various cancers. Paradoxically, recent advances have also demonstrated pro-tumorigenic effects of IFN-γ, especially in promoting cancer metastasis, with the mechanism remains unclear. This will undoubtedly hinder the application of IFN-γ in cancer treatment. Here, we verified that IFN-γ treatment led to activation of the epithelial-to-mesenchymal transition (EMT) programme and metastasis in cell lines of various cancers, including the kidney cancer cell line Caki-1, the lung cancer cell line A549, the cervical carcinoma cell line CaSki, the breast cancer cell line BT549 and the colon cancer cell line HCT116. We further disclosed that midkine (MDK), an emerging oncoprotein and EMT inducer, is a common responsive target of IFN-γ in these cell lines. Mechanistically, IFN-γ upregulated MDK via STAT1, a principle downstream effector in the IFN-γ signalling. MDK is elevated in the majority of cancer types in the TCGA database, and its overexpression drove EMT activation and cancer metastasis in all examined cell lines. Targeting MDK using a specific MDK inhibitor (iMDK) broadly reversed IFN-γ-activated EMT, and subsequently abrogated IFN-γ-triggered metastasis. Collectively, our data uncover a MDK-dependent EMT inducing mechanism underlying IFN-γ-driven metastasis across cancers which could be attenuated by pharmacological inhibition of MDK. Based on these findings, we propose that MDK may be used as a potential therapeutic target to eliminate IFN-γ-elicited pro-metastatic adverse effect, and that combined MDK utilization may expand the application of IFN-γ in cancer and improve the clinical benefits from IFN-γ-based therapies.
Collapse
Affiliation(s)
- Luyu Zheng
- Medical Research Center, Beijing Chao-Yang Hospital, Capital Medical University, Beijing, China
| | - Qun Liu
- Department of Obstetrics and Gynaecology, Beijing Anzhen Hospital, Capital Medical University, Beijing, China
| | - Ruijun Li
- Medical Research Center, Beijing Chao-Yang Hospital, Capital Medical University, Beijing, China
| | - Shibin Chen
- Medical Research Center, Beijing Chao-Yang Hospital, Capital Medical University, Beijing, China
| | - Jingyu Tan
- Medical Research Center, Beijing Chao-Yang Hospital, Capital Medical University, Beijing, China
| | - Lina Li
- Medical Research Center, Beijing Chao-Yang Hospital, Capital Medical University, Beijing, China
| | - Xichen Dong
- Medical Research Center, Beijing Chao-Yang Hospital, Capital Medical University, Beijing, China
| | - Changzhi Huang
- State Key Laboratory of Molecular Oncology, Beijing Key Laboratory for Carcinogenesis and Cancer Prevention, Department of Etiology and Carcinogenesis, National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
- *Correspondence: Jian Liu, ; Tao Wen, ; Changzhi Huang,
| | - Tao Wen
- Medical Research Center, Beijing Chao-Yang Hospital, Capital Medical University, Beijing, China
- *Correspondence: Jian Liu, ; Tao Wen, ; Changzhi Huang,
| | - Jian Liu
- Medical Research Center, Beijing Chao-Yang Hospital, Capital Medical University, Beijing, China
- Department of Oncology, Beijing Chao-Yang Hospital Capital Medical University, Beijing, China
- *Correspondence: Jian Liu, ; Tao Wen, ; Changzhi Huang,
| |
Collapse
|
|
7
|
Jia W, Yao Z, Zhao J, Guan Q, Gao L. New perspectives of physiological and pathological functions of nucleolin (NCL). Life Sci 2017; 186:1-10. [PMID: 28751161 DOI: 10.1016/j.lfs.2017.07.025] [Citation(s) in RCA: 158] [Impact Index Per Article: 19.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2017] [Revised: 07/21/2017] [Accepted: 07/23/2017] [Indexed: 12/13/2022]
Abstract
Nucleolin (NCL) is a multifunctional protein that mainly localized in the nucleolus, it is also found in the nucleoplasm, cytoplasm and cell membrane. The three main structural domains allow the interaction of NCL with different proteins and RNA sequences. Moreover, specific post-translational modifications and its shuttling property also contribute to its multifunctionality. NCL has been demonstrated to be involved in a variety of aspects such as ribosome biogenesis, chromatin organization and stability, DNA and RNA metabolism, cytokinesis, cell proliferation, angiogenesis, apoptosis regulation, stress response and microRNA processing. NCL has been increasingly implicated in several pathological processes, especially in tumorigenesis and viral infection, which makes NCL a potential target for the development of anti-tumor and anti-viral strategies. In this review, we present an overview on the structure, localizations and various functions of NCL, and further describe how the multiple functions of NCL are correlated to its multiple cellular distributions.
Collapse
Affiliation(s)
- Wenyu Jia
- Department of Endocrinology, Shandong Provincial Hospital affiliated to Shandong University, Jinan, Shandong Province, PR China; Shandong Provincial Key Laboratory of Endocrinology and Lipid Metabolism, Jinan, Shandong Province, PR China; Institute of Endocrinology and Metabolism, Shandong Academy of Clinical Medicine, Jinan, Shandong Province, PR China
| | - Zhenyu Yao
- Department of Endocrinology, Shandong Provincial Hospital affiliated to Shandong University, Jinan, Shandong Province, PR China; Shandong Provincial Key Laboratory of Endocrinology and Lipid Metabolism, Jinan, Shandong Province, PR China; Institute of Endocrinology and Metabolism, Shandong Academy of Clinical Medicine, Jinan, Shandong Province, PR China
| | - Jiajun Zhao
- Department of Endocrinology, Shandong Provincial Hospital affiliated to Shandong University, Jinan, Shandong Province, PR China; Shandong Provincial Key Laboratory of Endocrinology and Lipid Metabolism, Jinan, Shandong Province, PR China; Institute of Endocrinology and Metabolism, Shandong Academy of Clinical Medicine, Jinan, Shandong Province, PR China
| | - Qingbo Guan
- Department of Endocrinology, Shandong Provincial Hospital affiliated to Shandong University, Jinan, Shandong Province, PR China; Shandong Provincial Key Laboratory of Endocrinology and Lipid Metabolism, Jinan, Shandong Province, PR China; Institute of Endocrinology and Metabolism, Shandong Academy of Clinical Medicine, Jinan, Shandong Province, PR China
| | - Ling Gao
- Department of Endocrinology, Shandong Provincial Hospital affiliated to Shandong University, Jinan, Shandong Province, PR China; Shandong Provincial Key Laboratory of Endocrinology and Lipid Metabolism, Jinan, Shandong Province, PR China; Institute of Endocrinology and Metabolism, Shandong Academy of Clinical Medicine, Jinan, Shandong Province, PR China.
| |
Collapse
|
|
8
|
Sorrelle N, Dominguez ATA, Brekken RA. From top to bottom: midkine and pleiotrophin as emerging players in immune regulation. J Leukoc Biol 2017; 102:277-286. [PMID: 28356350 DOI: 10.1189/jlb.3mr1116-475r] [Citation(s) in RCA: 52] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2016] [Revised: 03/02/2017] [Accepted: 03/06/2017] [Indexed: 01/15/2023] Open
Abstract
Cytokines are pivotal in the generation and resolution of the inflammatory response. The midkine/pleiotrophin (MK/PTN) family of cytokines, composed of just two members, was discovered as heparin-binding neurite outgrowth-promoting factors. Since their discovery, expression of this cytokine family has been reported in a wide array of inflammatory diseases and cancer. In this minireview, we will discuss the emerging appreciation of the functions of the MK/PTN family in the immune system, which include promoting lymphocyte survival, sculpting myeloid cell phenotype, driving immune cell chemotaxis, and maintaining hematopoiesis.
Collapse
Affiliation(s)
- Noah Sorrelle
- Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, Texas, USA; and
| | - Adrian T A Dominguez
- Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, Texas, USA; and
| | - Rolf A Brekken
- Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, Texas, USA; and .,Division of Surgical Oncology, Departments of Surgery and Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas, USA
| |
Collapse
|
|
9
|
Perrone R, Butovskaya E, Lago S, Garzino-Demo A, Pannecouque C, Palù G, Richter SN. The G-quadruplex-forming aptamer AS1411 potently inhibits HIV-1 attachment to the host cell. Int J Antimicrob Agents 2016; 47:311-6. [PMID: 27032748 PMCID: PMC4840014 DOI: 10.1016/j.ijantimicag.2016.01.016] [Citation(s) in RCA: 45] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2015] [Revised: 01/26/2016] [Accepted: 01/29/2016] [Indexed: 12/31/2022]
Abstract
The G-quadruplex-forming aptamer AS1411 strongly inhibits HIV-1 infection. AS1411 is non-toxic to the host cell at antiviral concentrations. AS1411 blocks viral attachment to the host cell. AS1411 binds cell-surface-expressed nucleolin, a putative HIV-1 co-receptor. AS1411 is a G-rich aptamer that forms a stable G-quadruplex structure and displays antineoplastic properties both in vitro and in vivo. This oligonucleotide has undergone phase 2 clinical trials. The major molecular target of AS1411 is nucleolin (NCL), a multifunctional nucleolar protein also present in the cell membrane where it selectively mediates the binding and uptake of AS1411. Cell-surface NCL has been recognised as a low-affinity co-receptor for human immunodeficiency virus type 1 (HIV-1) anchorage on target cells. Here we assessed the anti-HIV-1 properties and underlying mechanism of action of AS1411. The antiviral activity of AS1411 was determined towards different HIV-1 strains, host cells and at various times post-infection. Acutely, persistently and latently infected cells were tested, including HIV-1-infected peripheral blood mononuclear cells from a healthy donor. Mechanistic studies to exclude modes of action other than virus binding via NCL were performed. AS1411 efficiently inhibited HIV-1 attachment/entry into the host cell. The aptamer displayed antiviral activity in the absence of cytotoxicity at the tested doses, therefore displaying a wide therapeutic window and favourable selectivity indexes. These findings, besides validating cell-surface-expressed NCL as an antiviral target, open the way for the possible use of AS1411 as a new potent and promisingly safe anti-HIV-1 agent.
Collapse
Affiliation(s)
- Rosalba Perrone
- Department of Molecular Medicine, University of Padua, Padua, Italy
| | - Elena Butovskaya
- Department of Molecular Medicine, University of Padua, Padua, Italy
| | - Sara Lago
- Department of Molecular Medicine, University of Padua, Padua, Italy
| | - Alfredo Garzino-Demo
- Department of Molecular Medicine, University of Padua, Padua, Italy; Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD 21201, USA
| | - Christophe Pannecouque
- KU Leuven, Department of Microbiology and Immunology, Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, B-3000 Leuven, Belgium
| | - Giorgio Palù
- Department of Molecular Medicine, University of Padua, Padua, Italy
| | - Sara N Richter
- Department of Molecular Medicine, University of Padua, Padua, Italy.
| |
Collapse
|
|
10
|
Gela A, Jovic S, Nordin SL, Egesten A. Midkine in host defence. Br J Pharmacol 2014; 171:859-69. [PMID: 24024937 PMCID: PMC3925024 DOI: 10.1111/bph.12402] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2013] [Revised: 08/27/2013] [Accepted: 09/02/2013] [Indexed: 12/23/2022] Open
Abstract
UNLABELLED Midkine (MK) shares several features in common with antibacterial proteins of the innate immune system. These include growth factor properties, heparin-binding regions and effects on immune cells, such as recruitment and activation of neutrophils and macrophages. Indeed, recent research has demonstrated potent bactericidal and fungicidal activities of MK. This protein is constitutively expressed at relevant concentrations at barriers of the body, such as the skin and the large airways, where the body first encounters potential pathogens. The antibacterial properties of MK orthologues are preserved during evolution, as exemplified by miple2 of Drosophila. In addition to retinoic acid, promoters of MK gene expression include factors present at sites of infection, reactive oxygen species, activation of the transcription factor NF-κB and hypoxia. In the light of the development of resistance in pathogenic bacteria to conventional antibiotics, MK is an interesting molecule that could serve as a template in developing novel therapeutic strategies against bacterial and fungal infections, either alone or in combination with conventional antibiotics. LINKED ARTICLES This article is part of a themed section on Midkine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-4.
Collapse
Affiliation(s)
- A Gela
- Section for Respiratory Medicine & Allergology, Department of Clinical Sciences Lund Skåne University Hospital, Lund UniversityLund, Sweden
| | - S Jovic
- Section for Respiratory Medicine & Allergology, Department of Clinical Sciences Lund Skåne University Hospital, Lund UniversityLund, Sweden
| | - S L Nordin
- Section for Respiratory Medicine & Allergology, Department of Clinical Sciences Lund Skåne University Hospital, Lund UniversityLund, Sweden
| | - A Egesten
- Section for Respiratory Medicine & Allergology, Department of Clinical Sciences Lund Skåne University Hospital, Lund UniversityLund, Sweden
| |
Collapse
|
|
11
|
Abstract
Midkine (MK) is a heparin-binding growth factor involved in various cellular processes such as cellular proliferation, survival, and migration. In addition to these typical growth factor activities, MK exhibits several other activities related to fibrinolysis, blood pressure, host defense and other processes. Many cell-surface receptors have been identified to account for the multiple biological activities of MK. The expression of MK is frequently upregulated in many types of human carcinoma. Moreover, blood MK levels are closely correlated with patient outcome. Knockdown and blockade of MK suppress tumorigenesis and tumor development. Thus, MK serves as a tumor marker and a molecular target for cancer therapy. Furthermore, there is growing evidence that MK plays pivotal roles in neural and inflammatory diseases. Understanding of the mechanisms of action of MK is expected to create new therapeutic options for several human diseases.
Collapse
Affiliation(s)
- Kazuma Sakamoto
- Department of Biochemistry, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | | |
Collapse
|
|
12
|
Hovanessian AG, Soundaramourty C, El Khoury D, Nondier I, Svab J, Krust B. Surface expressed nucleolin is constantly induced in tumor cells to mediate calcium-dependent ligand internalization. PLoS One 2010; 5:e15787. [PMID: 21203423 PMCID: PMC3009748 DOI: 10.1371/journal.pone.0015787] [Citation(s) in RCA: 170] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2010] [Accepted: 11/24/2010] [Indexed: 12/31/2022] Open
Abstract
BACKGROUND Nucleolin is one of the major proteins of the nucleolus, but it is also expressed on the cell surface where is serves as a binding protein for variety of ligands implicated in tumorigenesis and angiogenesis. Emerging evidence suggests that the cell-surface expressed nucleolin is a strategic target for an effective and nontoxic cancer therapy. METHODOLOGY/PRINCIPAL FINDINGS By monitoring the expression of nucleolin mRNA, and by measuring the level of nucleolin protein recovered from the surface and nucleus of cells, here we show that the presence of nucleolin at the cell surface is dependent on the constant induction of nucleolin mRNA. Indeed, inhibitors of RNA transcription or translation block expression of surface nucleolin while no apparent effect is observed on the level of nucleolin in the nucleus. The estimated half-life of surface nucleolin is less than one hour, whereas that of nuclear nucleolin is more than 8 hours. Nucleolin mRNA induction is reduced markedly in normal fibroblasts that reach confluence, while it occurs continuously even in post-confluent epithelial tumor cells consistent with their capacity to proliferate without contact inhibition. Interestingly, cold and heat shock induce nucleolin mRNA concomitantly to enhanced mRNA expression of the heat shock protein 70, thus suggesting that surface nucleolin induction also occurs in response to an environmental insult. At the cell surface, one of the main functions of nucleolin is to shuttle specific extracellular ligands by an active transport mechanism, which we show here to be calcium dependent. CONCLUSION/SIGNIFICANCE Our results demonstrate that the expression of surface nucleolin is an early metabolic event coupled with tumor cell proliferation and stress response. The fact that surface nucleolin is constantly and abundantly expressed on the surface of tumor cells, makes them a preferential target for the inhibitory action of anticancer agents that target surface nucleolin.
Collapse
Affiliation(s)
- Ara G Hovanessian
- CNRS-Université Paris Descartes, Unité Régulation de la Transcription de Maladies Génétique, Paris, France.
| | | | | | | | | | | |
Collapse
|
|
13
|
Kleinman JG, Sorokina EA, Wesson JA. Induction of apoptosis with cisplatin enhances calcium oxalate crystal adherence to inner medullary collecting duct cells. ACTA ACUST UNITED AC 2010; 38:97-104. [PMID: 20077109 DOI: 10.1007/s00240-009-0250-0] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2009] [Accepted: 12/10/2009] [Indexed: 11/28/2022]
Abstract
Attachment of stone crystals to tubular epithelium may initiate kidney stone formation. We previously reported that apical nucleolin related protein (NRP) expression during mitosis enhance attachment of Ca oxalate monohydrate crystals (COM). Some forms of injury may also increase affinity for crystals. We examined changes in subcellular localization of NRP during the course of cisplatin-induced apoptosis in cultured inner medullary collecting duct cells. Caspase-3 activation and chromatin condensation followed by nuclear fragmentation occurred after 20 h exposure to cisplatin, indicating the development of apoptosis. Cells were fixed without permeabilization and stained for surface NRP. Cells with condensed chromatin showed little or no cytoplasmic or apical NRP. Those at an early stage of nuclear fragmentation had cytoplasmic but not apical NRP and cells with advanced nuclear fragmentation were positively stained for apical NRP. Membrane proteins isolated by apical biotinylation and precipitated with avidin were analyzed by Western blot. Apical NRP was markedly increased after cisplatin compared to control, while expression of the apical marker, GP-135, and other putative attachment protein were unchanged. Hyaluronic acid was decreased. Cultures with apoptotic cells demonstrated increased adherence of COM that was inhibited by the polyanion (poly)aspartic acid. We conclude that pre-existing apoptotic injury may promote calcium oxalate crystals attachment to renal tubular epithelium via apical NRP expression.
Collapse
|
|
14
|
Muramatsu T. Midkine, a heparin-binding cytokine with multiple roles in development, repair and diseases. PROCEEDINGS OF THE JAPAN ACADEMY. SERIES B, PHYSICAL AND BIOLOGICAL SCIENCES 2010; 86:410-425. [PMID: 20431264 PMCID: PMC3417803 DOI: 10.2183/pjab.86.410] [Citation(s) in RCA: 129] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/25/2009] [Accepted: 02/24/2010] [Indexed: 05/29/2023]
Abstract
Midkine is a heparin-binding cytokine or a growth factor with a molecular weight of 13 kDa. Midkine binds to oversulfated structures in heparan sulfate and chondroitin sulfate. The midkine receptor is a molecular complex containing proteoglycans. Midkine promotes migration, survival and other activities of target cells. Midkine has about 50% sequence identity with pleiotrophin. Mice deficient in both factors exhibit severe abnormalities including female infertility. In adults, midkine is expressed in damaged tissues and involved in the reparative process. It is also involved in inflammatory reactions by promoting the migration of leukocytes, induction of chemokines and suppression of regulatory T cells. Midkine is expressed in a variety of malignant tumors and promotes their growth and invasion. Midkine appears to be helpful for the treatment of injuries in the heart, brain, spinal cord and retina. Midkine inhibitors are expected to be effective in the treatment of malignancies, rheumatoid arthritis, multiple sclerosis, renal diseases, restenosis, hypertension and adhesion after surgery.
Collapse
Affiliation(s)
- Takashi Muramatsu
- Department of Health Science, Faculty of Psychological and Physical Science, Aichi Gakuin University. 12 Araike, Aichi, Japan.
| |
Collapse
|
|
15
|
Krzystek-Korpacka M, Neubauer K, Matusiewicz M. Clinical relevance of circulating midkine in ulcerative colitis. Clin Chem Lab Med 2009; 47:1085-90. [PMID: 19728850 DOI: 10.1515/cclm.2009.248] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
BACKGROUND Non-invasive biochemical markers are needed to support the diagnosis of ulcerative colitis (UC), an incurable disease of unknown pathology. Midkine is an angiogenic cytokine, chemotactic towards neutrophils and macrophages, and a T-regulatory cell suppressor. METHODS Serum midkine was measured immunoenzymatically in 93 UC patients and 108 healthy subjects, and evaluated with respect to disease status, endoscopic, inflammatory and angiogenic activity. The diagnostic value of midkine was compared to C-reactive protein (CRP) using receiver operating characteristics (ROC) analysis. RESULTS Midkine was higher (p<0.0001) in inactive (199 ng/L) and active UC (351 ng/L) compared with controls (93 ng/L), and reflected disease activity (r=0.427, p<0.001). Midkine was correlated with CRP, erythrocyte sedimentation rate (ESR), leukocytes, platelets, interleukin-6, paraoxonase-1, albumin, transferrin, iron, hemoglobin, and hematocrit. Midkine correlated with angiogenic factors: vascular endothelial growth factor-A and platelet-derived growth factor-BB. As a marker of UC, midkine showed a diagnostic accuracy of 85%, sensitivity of 72%, specificity of 82%, whereas CRP showed 83%, 65% and 91%, respectively. As a marker of active UC, midkine showed a diagnostic accuracy of 87%, sensitivity of 84%, specificity of 75%, whereas CRP showed 75%, 63% and 83%, respectively. Combined assessment of midkine and CRP improved sensitivity but substantially decreased specificity. CONCLUSIONS UC is associated with increased circulating midkine, which corresponds with clinical, endoscopic, inflammatory and angiogenic activity, and anemia. Performance of midkine as a marker of UC or active UC was comparable to that of CRP.
Collapse
|
|
16
|
Abstract
Midkine (MK) is a heparin-binding growth factor with its gene first identified in embryonal carcinoma cells at early stages of retinoic acid-induced differentiation. MK is frequently and highly expressed in a variety of human carcinomas. Furthermore, the blood MK level is frequently elevated with advance of human carcinomas, decreased after surgical removal of the tumors. Thus, it is expected to become a promising marker for evaluating the progress of carcinomas. There is mounting evidence that MK plays a significant role in carcinogenesis-related activities, such as proliferation, migration, anti-apoptosis, mitogenesis, transforming, and angiogenesis. In addition, siRNA and anti-sense oligonucleotides for MK have yielded great effects in anti-tumor activities. Therefore, MK appears to be a potential candidate molecular target of therapy for human carcinomas. In this paper, we review MK targeting at nucleoli in different tumor cells and its role in carcinogenesis to deepen our understanding of the mechanism of MK involved in carcinogenesis.
Collapse
|
|
17
|
Losfeld ME, Khoury DE, Mariot P, Carpentier M, Krust B, Briand JP, Mazurier J, Hovanessian AG, Legrand D. The cell surface expressed nucleolin is a glycoprotein that triggers calcium entry into mammalian cells. Exp Cell Res 2009; 315:357-69. [PMID: 19026635 DOI: 10.1016/j.yexcr.2008.10.039] [Citation(s) in RCA: 64] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2008] [Revised: 10/16/2008] [Accepted: 10/23/2008] [Indexed: 11/24/2022]
Abstract
Nucleolin is an ubiquitous nucleolar phosphoprotein involved in fundamental aspects of transcription regulation, cell proliferation and growth. It has also been described as a shuttling molecule between nucleus, cytosol and the cell surface. Several studies have demonstrated that surface nucleolin serves as a receptor for various extracellular ligands implicated in cell proliferation, differentiation, adhesion, mitogenesis and angiogenesis. Previously, we reported that nucleolin in the extranuclear cell compartment is a glycoprotein containing N- and O-glycans. In the present study, we show that glycosylation is an essential requirement for surface nucleolin expression, since it is prevented when cells are cultured in the presence of tunicamycin, an inhibitor of N-glycosylation. Accordingly, surface but not nuclear nucleolin is radioactively labeled upon metabolic labeling of cells with [(3)H]glucosamine. Besides its well-demonstrated role in the internalization of specific ligands, here we show that ligand binding to surface nucleolin could also induce Ca(2+) entry into cells. Indeed, by flow cytometry, microscopy and patch-clamp experiments, we show that the HB-19 pseudopeptide, which binds specifically surface nucleolin, triggers rapid and intense membrane Ca(2+) fluxes in various types of cells. The use of several drugs then indicated that Store-Operated Ca(2+) Entry (SOCE)-like channels are involved in the generation of these fluxes. Taken together, our findings suggest that binding of an extracellular ligand to surface nucleolin could be involved in the activation of signaling pathways by promoting Ca(2+) entry into cells.
Collapse
Affiliation(s)
- Marie-Estelle Losfeld
- Unité de Glycobiologie Structurale et Fonctionnelle, Unité Mixte de Recherche no 8576 du Centre National de la Recherche Scientifique, France
| | | | | | | | | | | | | | | | | |
Collapse
|
|
18
|
Mäkelä AR, Närvänen A, Oker-Blom C. Peptide-mediated interference with baculovirus transduction. J Biotechnol 2008; 134:20-32. [PMID: 18294718 DOI: 10.1016/j.jbiotec.2007.12.010] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2007] [Revised: 12/14/2007] [Accepted: 12/17/2007] [Indexed: 10/22/2022]
Abstract
Baculovirus represents a multifunctional platform with potential for biomedical applications including disease therapies. The importance of F3, a tumor-homing peptide, in baculovirus transduction was previously recognized by the ability of F3 to augment viral binding and gene delivery to human cancer cells following display on the viral envelope. Here, F3 was utilized as a molecular tool to expand understanding of the poorly characterized baculovirus-mammalian cell interactions. Baculovirus-mediated transduction of HepG2 hepatocarcinoma cells was strongly inhibited by coincubating the virus with synthetic F3 or following incorporation of F3 into viral nucleocapsid by genetic engineering, the former suggesting direct interaction of the soluble peptide with the virus particles. Since internalization and nuclear accumulation of the virus were significantly inhibited or delayed, but the kinetics of viral binding, initial uptake, and endosomal release were unaffected, F3 likely interferes with cytoplasmic trafficking and subsequent nuclear transport of the virus. A polyclonal antibody raised against nucleolin, the internalizing receptor of F3, failed to inhibit cellular binding, but considerably reduced viral transduction efficiency, proposing the involvement of nucleolin in baculovirus entry. Together, these results render the F3 peptide a tool for elucidating the mechanism and molecular details conferring to baculovirus-mediated gene transduction in mammalian cells.
Collapse
Affiliation(s)
- Anna R Mäkelä
- NanoScience Center, Department of Biological and Environmental Science, PO Box 35, FIN-40014 University of Jyväskylä, Finland.
| | | | | |
Collapse
|
|
19
|
Inhibition of midkine alleviates experimental autoimmune encephalomyelitis through the expansion of regulatory T cell population. Proc Natl Acad Sci U S A 2008; 105:3915-20. [PMID: 18319343 DOI: 10.1073/pnas.0709592105] [Citation(s) in RCA: 94] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
CD4(+)CD25(+) regulatory T (Treg) cells are crucial mediators of autoimmune tolerance. The factors that regulate Treg cells, however, are largely unknown. Here, we show that deficiency in midkine (MK), a heparin-binding growth factor involved in oncogenesis, inflammation, and tissue repair, attenuated experimental autoimmune encephalomyelitis (EAE) because of an expansion of the Treg cell population in peripheral lymph nodes and decreased numbers of autoreactive T-helper type 1 (T(H)1) and T(H)17 cells. MK decreased the Treg cell population ex vivo in a dose-dependent manner by suppression of STAT5 phosphorylation that is essential for Foxp3 expression. Moreover, administration of anti-MK RNA aptamers significantly expanded the Treg cell population and alleviated EAE symptoms. These observations indicate that MK serves as a critical suppressor of Treg cell expansion, and inhibition of MK using RNA aptamers may provide an effective therapeutic strategy against autoimmune diseases, including multiple sclerosis.
Collapse
|
|
20
|
Nucleolin – Characteristics of Protein and its Role in Biology of Cancers and Viral Infections. ACTA ACUST UNITED AC 2008. [DOI: 10.2478/v10052-008-0003-1] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
|
|
21
|
Storck S, Shukla M, Dimitrov S, Bouvet P. Functions of the histone chaperone nucleolin in diseases. Subcell Biochem 2007; 41:125-44. [PMID: 17484127 DOI: 10.1007/1-4020-5466-1_7] [Citation(s) in RCA: 94] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/03/2022]
Abstract
Alteration of nuclear morphology is often used by pathologist as diagnostic marker for malignancies like cancer. In particular, the staining of cells by the silver staining methods (AgNOR) has been proved to be an important tool for predicting the clinical outcome of some cancer diseases. Two major argyrophilic proteins responsible for the strong staining of cells in interphase are the nucleophosmin (B23) and the nucleolin (C23) nucleolar proteins. Interestingly these two proteins have been described as chromatin associated proteins with histone chaperone activities and also as proteins able to regulate chromatin transcription. Nucleolin seems to be over-expressed in highly proliferative cells and is involved in many aspect of gene expression: chromatin remodeling, DNA recombination and replication, RNA transcription by RNA polymerase I and II, rRNA processing, mRNA stabilisation, cytokinesis and apoptosis. Interestingly, nucleolin is also found on the cell surface in a wide range of cancer cells, a property which is being used as a marker for the diagnosis of cancer and for the development of anti-cancer drugs to inhibit proliferation of cancer cells. In addition to its implication in cancer, nucleolin has been described not only as a marker or as a protein being involved in many diseases like viral infections, autoimmune diseases, Alzheimer's disease pathology but also in drug resistance. In this review we will focus on the chromatin associated functions of nucleolin and discuss the functions of nucleolin or its use as diagnostic marker and as a target for therapy
Collapse
Affiliation(s)
- Sébastien Storck
- Laboratoire Joliot-Curie, Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69007 Lyon, France
| | | | | | | |
Collapse
|
|
22
|
Dai LC, Xu DY, Yao X, Min LS, Zhao N, Xu BY, Xu ZP, Lu YL. Construction of a fusion protein expression vector MK-EGFP and its subcellular localization in different carcinoma cell lines. World J Gastroenterol 2006; 12:7649-53. [PMID: 17171794 PMCID: PMC4088047 DOI: 10.3748/wjg.v12.i47.7649] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To construct an expression plasmid encoding human wild-type midkine (MK) and enhanced green fluorescence protein (EGFP) fusion protein (MK-EGFP), and to analyze the subcellular localization of MK in different carcinoma cell lines.
METHODS: Two kinds of MK coding sequences with or without signal peptide were cloned into plasmid pEGFP-N2, and the recombinant plasmids constructed were introduced into HepG2, MCF7 and DU145 cells, respectively, by transfection. With the help of laser scanning confocal microscopy, the expression and subcellular localization of MK-GFP fusion protein could be detected.
RESULTS: Compared with the GFP control, in which fluorescence was detected diffusely over the entire cell body except in the nucleolus, both kinds of fusion protein MK-GFP were localized exclusively to the nucleus and accumulated in the nucleolus in the three kinds of cancer cell lines.
CONCLUSION: This study reveals the specific nucleolar translocation independent of signal peptide, which may be involved in the mechanism that MK works. It provides valuable evidence for further study on the functions of MK in nucleus and its possible mechanisms, in which ribosomal RNA transcription and ribosome assembly are involved.
Collapse
Affiliation(s)
- Li-Cheng Dai
- Huzhou Key Laboratory of Molecular Medicine, Huzhou Central Hospital, Huzhou 313000, Zhejiang Province, China.
| | | | | | | | | | | | | | | |
Collapse
|
|
23
|
Tate A, Isotani S, Bradley MJ, Sikes RA, Davis R, Chung LWK, Edlund M. Met-Independent Hepatocyte Growth Factor-mediated regulation of cell adhesion in human prostate cancer cells. BMC Cancer 2006; 6:197. [PMID: 16869958 PMCID: PMC1559714 DOI: 10.1186/1471-2407-6-197] [Citation(s) in RCA: 52] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2006] [Accepted: 07/25/2006] [Indexed: 12/26/2022] Open
Abstract
BACKGROUND Prostate cancer cells communicate reciprocally with the stromal cells surrounding them, inside the prostate, and after metastasis, within the bone. Each tissue secretes factors for interpretation by the other. One stromally-derived factor, Hepatocyte Growth Factor (HGF), was found twenty years ago to regulate invasion and growth of carcinoma cells. Working with the LNCaP prostate cancer progression model, we found that these cells could respond to HGF stimulation, even in the absence of Met, the only known HGF receptor. The new HGF binding partner we find on the cell surface may help to clarify conflicts in the past literature about Met expression and HGF response in cancer cells. METHODS We searched for Met or any HGF binding partner on the cells of the PC3 and LNCaP prostate cancer cell models, using HGF immobilized on agarose beads. By using mass spectrometry analyses and sequencing we have identified nucleolin protein as a novel HGF binding partner. Antibodies against nucleolin (or HGF) were able to ameliorate the stimulatory effects of HGF on met-negative prostate cancer cells. Western blots, RT-PCR, and immunohistochemistry were used to assess nucleolin levels during prostate cancer progression in both LNCaP and PC3 models. RESULTS We have identified HGF as a major signaling component of prostate stromal-conditioned media (SCM) and have implicated the protein nucleolin in HGF signal reception by the LNCaP model prostate cancer cells. Antibodies that silence either HGF (in SCM) or nucleolin (on the cell surfaces) eliminate the adhesion-stimulatory effects of the SCM. Likewise, addition of purified HGF to control media mimics the action of SCM. C4-2, an LNCaP lineage-derived, androgen-independent human prostate cancer cell line, responds to HGF in a concentration-dependent manner by increasing its adhesion and reducing its migration on laminin substratum. These HGF effects are not due to shifts in the expression levels of laminin-binding integrins, nor can they be linked to expression of the known HGF receptor Met, as neither LNCaP nor clonally-derived C4-2 sub-line contain any detectable Met protein. Even in the absence of Met, small GTPases are activated, linking HGF stimulation to membrane protrusion and integrin activation. Membrane-localized nucelolin levels increase during cancer progression, as modeled by both the PC3 and LNCaP prostate cancer progression cell lines. CONCLUSION We propose that cell surface localized nucleolin protein may function in these cells as a novel HGF receptor. Membrane localized nucleolin binds heparin-bound growth factors (including HGF) and appears upregulated during prostate cancer progression. Antibodies against nucleolin are able to ameliorate the stimulatory effects of HGF on met-negative prostate cancer cells. HGF-nucleolin interactions could be partially responsible for the complexity of HGF responses and met expression reported in the literature.
Collapse
Affiliation(s)
- Amanda Tate
- Department of Urology and Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USA
| | - Shuji Isotani
- Department of Urology and Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USA
| | - Michael J Bradley
- Department of Biological Sciences, University of Delaware, Newark, DE, USA
| | - Robert A Sikes
- Department of Biological Sciences, University of Delaware, Newark, DE, USA
| | - Rodney Davis
- Department of Urology, Tulane University Health Sciences Center, New Orleans, LA, USA
| | - Leland WK Chung
- Department of Urology and Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USA
| | - Magnus Edlund
- Department of Urology and Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USA
| |
Collapse
|
|
24
|
Zhang GH, Wang Q, Chen JJ, Zhang XM, Tam SC, Zheng YT. The anti-HIV-1 effect of scutellarin. Biochem Biophys Res Commun 2006; 334:812-6. [PMID: 16023998 DOI: 10.1016/j.bbrc.2005.06.166] [Citation(s) in RCA: 73] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2005] [Accepted: 06/15/2005] [Indexed: 11/19/2022]
Abstract
Scutellarin was purified from the plant Erigeron breviscapus (Vant.) Hand.-Mazz. The activity against 3 strains of human immunodeficiency virus (HIV) was determined in vitro in this study. These were laboratory-derived virus (HIV-1IIIB), drug-resistant virus (HIV-1(74V)), and low-passage clinical isolated virus (HIV-1(KM018)). From syncytia inhibition study, the EC50 of scutellarin against HIV-1IIIB direct infection in C8166 cells was 26 microM with a therapeutic index of 36. When the mode of infection changed from acute infection to cell-to-cell infection, this compound became even more potent and the EC50 reduced to 15 microM. This suggested that cell fusion might be affected by this compound. By comparing the inhibitory effects on p24 antigen, scutellarin was also found to be active against HIV-1(74V) (EC50 253 microM) and HIV-1KM018 (EC50 136 microM) infection with significant difference in potency. The mechanism of its action was also explored in this study. At a concentration of 433 microM, scutellarin inhibited 48% of the cell free recombinant HIV-1 RT activity. It also caused 82% inhibition of HIV-1 particle attachment and 45% inhibition of fusion at the concentrations of 54 microM. In summary, scutellarin was found to inhibit several strains of HIV-1 replication with different potencies. It appeared to inhibit HIV-1 RT activity, HIV-1 particle attachment and cell fusion. These are essential activities for viral transmission and replication.
Collapse
Affiliation(s)
- Gao-Hong Zhang
- Laboratory of Molecular Immunopharmacology, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China; Graduate School of the Chinese Academy of Sciences, Beijing 100039, China
| | | | | | | | | | | |
Collapse
|
|
25
|
Shimakami T, Honda M, Kusakawa T, Murata T, Shimotohno K, Kaneko S, Murakami S. Effect of hepatitis C virus (HCV) NS5B-nucleolin interaction on HCV replication with HCV subgenomic replicon. J Virol 2006; 80:3332-40. [PMID: 16537600 PMCID: PMC1440399 DOI: 10.1128/jvi.80.7.3332-3340.2006] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2005] [Accepted: 01/05/2006] [Indexed: 12/29/2022] Open
Abstract
We previously reported that nucleolin, a representative nucleolar marker, interacts with nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) through two independent regions of NS5B, amino acids 208 to 214 and 500 to 506. We also showed that truncated nucleolin that harbors the NS5B-binding region inhibited the RNA-dependent RNA polymerase activity of NS5B in vitro, suggesting that nucleolin may be involved in HCV replication. To address this question, we focused on NS5B amino acids 208 to 214. We constructed one alanine-substituted clustered mutant (CM) replicon, in which all the amino acids in this region were changed to alanine, as well as seven different point mutant (PM) replicons, each of which harbored an alanine substitution at one of the amino acids in the region. After transfection into Huh7 cells, the CM replicon and the PM replicon containing NS5B W208A could not replicate, whereas the remaining PM replicons were able to replicate. In vivo immunoprecipitation also showed that the W208 residue of NS5B was essential for its interaction with nucleolin, strongly suggesting that this interaction is essential for HCV replication. To gain further insight into the role of nucleolin in HCV replication, we utilized the small interfering RNA (siRNA) technique to investigate the knockdown effect of nucleolin on HCV replication. Cotransfection of replicon RNA and nucleolin siRNA into Huh7 cells moderately inhibited HCV replication, although suppression of nucleolin did not affect cell proliferation. Taken together, our findings strongly suggest that nucleolin is a host component that interacts with HCV NS5B and is indispensable for HCV replication.
Collapse
Affiliation(s)
- Tetsuro Shimakami
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Ishikawa, Japan
| | | | | | | | | | | | | |
Collapse
|
|
26
|
Hovanessian AG. Midkine, a cytokine that inhibits HIV infection by binding to the cell surface expressed nucleolin. Cell Res 2006; 16:174-81. [PMID: 16474431 DOI: 10.1038/sj.cr.7310024] [Citation(s) in RCA: 66] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022] Open
Abstract
The growth factor midkine (MK) is a cytokine that inhibits HIV infection in cell cultures in an autocrine and paracrine manner by blocking the attachment of HIV particles to permissive cells. MK mRNA is systematically expressed in adult peripheral blood lymphocytes from healthy donors, while its expression becomes markedly but transiently increased upon in vitro treatment of lymphocytes with IL-2 or IFN-gamma and activation of T lymphocytes by PHA or through the engagement of the CD28 antigen. The binding of MK to cells occurs specifically at a high and a low affinity binding site. This low affinity-binding site is the cell-surface expressed nucleolin, which is implicated in the mechanism of the initial attachment of HIV particles to cells. Accordingly, the nucleolin-binding HB-19 pseudopeptide has no effect on the MK binding to the high affinity binding site, whereas it prevents the binding of MK to the low affinity binding site, thus suggesting the low affinity receptor of MK is the cell-surface-expressed nucleolin. Confocal immunofluorescence laser microscopy revealed the colocalization of MK and the cell-surface-expressed nucleolin at distinct spots. The use of various deletion constructs of nucleolin then indicates that the extreme C-terminal end of nucleolin, containing repeats of the amino acid motif RGG, as the domain that binds MK. The specific binding of MK to the surface nucleolin is independent of heparan sulfate and chondroitin sulfate proteoglycans. After binding to cells, MK enters cells by an active process in which nucleolin and lipid rafts appear to be implicated. The potent and the distinct anti-HIV action of MK along with its enhanced expression in lymphocytes by various physiological stimuli, point out that MK is a cytokine that could be involved in HIV pathogenesis.
Collapse
Affiliation(s)
- Ara G Hovanessian
- 1UPR 2228 CNRS, UFR Biomedicale-Université René Descartes, 45 rue des Saints Pères, 75270 Paris Cedex 6, France.
| |
Collapse
|
|
27
|
Said EA, Courty J, Svab J, Delbé J, Krust B, Hovanessian AG. Pleiotrophin inhibits HIV infection by binding the cell surface-expressed nucleolin. FEBS J 2005; 272:4646-59. [PMID: 16156786 DOI: 10.1111/j.1742-4658.2005.04870.x] [Citation(s) in RCA: 73] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
The growth factor pleiotrophin (PTN) has been reported to bind heparan sulfate and nucleolin, two components of the cell surface implicated in the attachment of HIV-1 particles to cells. Here we show that PTN inhibits HIV-1 infection by its capacity to inhibit HIV-1 particle attachment to the surface of permissive cells. The beta-sheet domains of PTN appear to be implicated in this inhibitory effect on the HIV infection, in particular the domain containing amino acids 60-110. PTN binding to the cell surface is mediated by high and low affinity binding sites. Other inhibitors of HIV attachment known to bind specifically surface expressed nucleolin, such as the pseudopeptide HB-19 and the cytokine midkine prevent the binding of PTN to its low affinity-binding site. Confocal immunofluorescence laser microscopy revealed that the cross-linking of surface-bound PTN with a specific antibody results in the clustering of cell surface-expressed nucleolin and the colocalization of both PTN and nucleolin signals. Following its binding to surface-nucleolin, PTN is internalized by a temperature sensitive mechanism, a process which is inhibited by HB-19 and is independent of heparan and chondroitin sulfate proteoglycans. Nevertheless, proteoglycans might play a role in the concentration of PTN on the cell surface for a more efficient interaction with nucleolin. Our results demonstrate for the first time that PTN inhibits HIV infection and suggest that the cell surface-expressed nucleolin is a low affinity receptor for PTN binding to cells and it is also implicated in PTN entry into cells by an active process.
Collapse
Affiliation(s)
- Elias A Said
- UPR 2228 CNRS, UFR Biomédicale des Saints-Pères, Paris, France.
| | | | | | | | | | | |
Collapse
|
|
28
|
Dai L, Dai L, Xu D, Yao X, Lu Y, Xu Z. Conformational determinants of the intracellular localization of midkine. Biochem Biophys Res Commun 2005; 330:310-7. [PMID: 15781266 DOI: 10.1016/j.bbrc.2005.02.155] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2005] [Indexed: 11/23/2022]
Abstract
Midkine (MK) is a multifunctional growth factor and has been discovered to play important roles in carcinogenesis. MK has been reported to localize to the nucleus and nucleolus, however, the data are not consistent and the signals responsible for the localization are unknown. Here we reported that human MK exclusively localized to the nucleus and nucleolus in HepG2 cells by using GFP as a tracking molecule. In order to identify the motifs required for the nuclear localization and nucleolar accumulation, point- and deletion-mutations were introduced and the corresponding subcellular localizations were analyzed. Data revealed that (i) K79R81, K86K87, and the C-terminal tail of MK constitute the nuclear localization determinant of MK, and (ii) the C-terminal tail is the key element controlling MK nucleolar accumulation though the N-terminal tail, K79R81, and K86K87 also contribute to this process. Taken together, our results provide the first documentation about the determinants required for MK nuclear and nucleolar localization.
Collapse
Affiliation(s)
- Licheng Dai
- Huzhou Central Hospital, Huzhou 313000 [corrected] China
| | | | | | | | | | | |
Collapse
|
|
29
|
Zahariev S, Guarnaccia C, Zanuttin F, Pintar A, Esposito G, Maravić G, Krust B, Hovanessian AG, Pongor S. Efficient synthesis and comparative studies of the arginine and Nomega,Nomega-dimethylarginine forms of the human nucleolin glycine/arginine rich domain. J Pept Sci 2005; 11:17-28. [PMID: 15635723 DOI: 10.1002/psc.577] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
The Gly- and Arg-rich C-terminal region of human nucleolin is a 61-residue long domain involved in a number of protein-protein and protein-nucleic acid interactions. This domain contains 10 aDma residues in the form of aDma-GG repeats interspersed with Phe residues. The exact role of Arg dimethylation is not known, partly because of the lack of efficient synthetic methods. This work describes an effective synthetic strategy, generally applicable to long RGG peptides, based on side-chain protected aDma and backbone protected dipeptide Fmoc-Gly-(Dmob)Gly-OH. This strategy allowed us to synthesize both the unmodified (N61Arg) and the dimethylated (N61aDma) peptides with high yield ( approximately 26%) and purity. As detected by NMR spectroscopy, N61Arg does not possess any stable secondary or tertiary structure in solution and N(omega),N(omega)-dimethylation of the guanidino group does not alter the overall conformational propensity of this peptide. While both peptides bind single-stranded nucleic acids with similar affinities (K(d) = 1.5 x 10(-7) M), they exhibit a different behaviour in ssDNA affinity chromatography consistent with the difference in pK(a) values. It has been previously shown that N61Arg inhibits HIV infection at the stage of HIV attachment to cells. This study demonstrates that Arg-dimethylated C-terminal domain lacks any inhibition activity, raising the question of whether nucleolin expressed on the cell-surface is indeed dimethylated.
Collapse
Affiliation(s)
- Sotir Zahariev
- Protein Structure and Bioinformatics Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Area Science Park, Padriciano 99, 34012 Trieste, Italy.
| | | | | | | | | | | | | | | | | |
Collapse
|
|
30
|
Joo EJ, ten Dam GB, van Kuppevelt TH, Toida T, Linhardt RJ, Kim YS. Nucleolin: acharan sulfate-binding protein on the surface of cancer cells. Glycobiology 2005; 15:1-9. [PMID: 15329357 PMCID: PMC1237021 DOI: 10.1093/glycob/cwh132] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
Glycosaminoglycans (GAGs) are complex polysaccharides that participate in the regulation of physiological processes through the interactions with a wide variety of proteins. Acharan sulfate (AS), isolated from the giant African snail Achatina fulica, primarily consists of the repeating disaccharide structure alpha-D-N-acetylglucosaminyl (1-->4) 2-sulfoiduronic acid. Exogenous AS was injected subcutaneously near the tumor tissue in C57BL/6 mice that had been implanted with Lewis lung carcinoma cells (LLCs). The location of AS in the tumor was assessed by staining of sectioned tissues with alcian blue and periodic acid-Schiff (PAS) reagent. In vitro assays indicated binding of cells to 50 microg/ml AS (or heparin) after a 5-h incubation. Immunofluorescence assays, using anti-AS antibody, detected AS at the cell surface. The outer-surface of LLCs were next biotinylated to identify the AS-binding proteins. Biotinylated cells were lysed, and the lysates were fractionated on the AS affinity column using a stepwise salt gradient (0, 0.1, 0.3, 0.5, 0.7, 1.0, and 2.0 M). The fractions were analyzed by SDS-PAGE with silver staining and western blotting. We focused on the proteins with high affinity for AS (eluting at 1 M NaCl) and detected only two bands by western blotting. ESI Q-TOF MS analysis of one of these bands, molecular weight approximately 110 kDa, showed it to be nucleolin. A phosphorylated form of nucleolin on the surface of cells acts as a cell surface receptor for a variety of ligands, including growth factors (i.e., basic fibroblast growth factor) and chemokines (i.e., midkine). These results show that nucleolin is one of several AS-binding proteins and suggest that AS might demonstrate its tumor growth inhibitory activity by binding the nucleolin receptor protein on the surface of cancer cells.
Collapse
Key Words
- as-binding protein
- biotinylation
- lewis lung carcinoma
- nucleolin
- as, acharan sulfate
- bsa, bovine serum albumin
- caps, 3-[cyclohexylamino]-1-propanesulfonic acid
- dmem, dulbecco’s modified eagle medium
- d-pbs, dulbecco’s phosphate buffered saline
- edta, ethylenediamine tetraacetic acid
- elisa, enzyme-linked immunosorbent assay
- esi q-tof ms, electrospray ionization quadrupole timeof- flight mass spectrometry
- fgf, fibroblast growth factor
- fitc, fluorescein isothiocyanate
- gag, glycosaminoglycan
- hrp, horseradish peroxidase
- llc, lewis lung carcinoma
- ms/ms, tandem mass spectrometry
- mtt, methylthiazol-2-yl-2,5-diphenyltetrazolium bromide
- pas, periodic acid-schiff
- pvdf, polyvinylidene difluoride
- sds-page, sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- vsv, vesicular stromatitis virus
Collapse
Affiliation(s)
- Eun Ji Joo
- Natural Products Research Institute, College of Pharmacy, Seoul National University, 28 Yeonkun-Dong, Jongno-Ku, Seoul 110-460, Korea
| | - Gerdy B. ten Dam
- Department of Biochemistry, NCMLS, UMC Nijmegen, 6500 HB Nijmegen, The Netherlands
| | | | - Toshihiko Toida
- Graduate School of Pharmaceutical Science, Chiba University, Chiba 263-8522, Japan; and
| | - Robert J. Linhardt
- Department of Chemistry and Chemical Biology, Biology and Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180
| | - Yeong Shik Kim
- Natural Products Research Institute, College of Pharmacy, Seoul National University, 28 Yeonkun-Dong, Jongno-Ku, Seoul 110-460, Korea
| |
Collapse
|
|
31
|
Bose S, Basu M, Banerjee AK. Role of nucleolin in human parainfluenza virus type 3 infection of human lung epithelial cells. J Virol 2004; 78:8146-58. [PMID: 15254186 PMCID: PMC446124 DOI: 10.1128/jvi.78.15.8146-8158.2004] [Citation(s) in RCA: 64] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2003] [Accepted: 03/15/2004] [Indexed: 11/20/2022] Open
Abstract
Human parainfluenza virus type 3 (HPIV-3) is an airborne pathogen that infects human lung epithelial cells from the apical (luminal) plasma membrane domain. In the present study, we have identified cell surface-expressed nucleolin as a cellular cofactor required for the efficient cellular entry of HPIV-3 into human lung epithelial A549 cells. Nucleolin was enriched on the apical cell surface domain of A549 cells, and HPIV-3 interacted with nucleolin during entry. The importance of nucleolin during HPIV-3 replication was borne out by the observation that HPIV-3 replication was significantly inhibited following (i). pretreatment of cells with antinucleolin antibodies and (ii). preincubation of HPIV-3 with purified nucleolin prior to its addition to the cells. Moreover, HPIV-3 cellular internalization and attachment assays performed in the presence of antinucleolin antibodies and purified nucleolin revealed the requirement of nucleolin during HPIV-3 internalization but not during attachment. Thus, these results suggest that nucleolin expressed on the surfaces of human lung epithelial A549 cells plays an important role during HPIV-3 cellular entry.
Collapse
Affiliation(s)
- Santanu Bose
- Department of Virology, The Cleveland Clinic Foundation, Cleveland, OH 44195, USA
| | | | | |
Collapse
|
|
32
|
Maruyama K, Muramatsu H, Ishiguro N, Muramatsu T. Midkine, a heparin-binding growth factor, is fundamentally involved in the pathogenesis of rheumatoid arthritis. ACTA ACUST UNITED AC 2004; 50:1420-9. [PMID: 15146411 DOI: 10.1002/art.20175] [Citation(s) in RCA: 78] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
OBJECTIVE Midkine (MK), a heparin-binding growth factor, promotes growth, survival, and migration of various cells. The essential role of MK in migration of inflammatory cells has been shown using mice deficient in the MK gene (Mdk(-/-) mice). We undertook this study to investigate the role of MK in the pathogenesis of rheumatoid arthritis (RA). METHODS MK levels in specimens from patients were determined by enzyme-linked immunosorbent assay, and localization of MK was revealed by immunohistochemical analysis. Susceptibility to antibody-induced arthritis was compared between Mdk(-/-) and wild-type (WT) mice. Osteoclast differentiation was monitored using macrophage-like cells isolated from human synovial tissue and macrophages from mouse bone marrow. RESULTS MK levels in sera and synovial fluid were increased in most RA patients, indicating a strong correlation between MK expression and RA. MK was expressed in macrophage-like cells and fibroblast-like cells in synovial membranes from the patients. In antibody-induced arthritis, Mdk(-/-) mice seldom developed the disease, while most of the WT mice did. Administration of MK to the Mdk(-/-) mice increased the frequency of antibody-induced arthritis. Migration of inflammatory leukocytes to the synovial membranes in the disease model was suppressed in the Mdk(-/-) mice. Furthermore, MK was found to promote the differentiation of osteoclasts from macrophages. CONCLUSION MK participates in each of the two distinct phases of RA development, namely, migration of inflammatory leukocytes and osteoclast differentiation, and is a key molecule in the pathogenesis of RA.
Collapse
Affiliation(s)
- Kiyoko Maruyama
- Department of Biochemistry, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
| | | | | | | |
Collapse
|
|
33
|
Legrand D, Vigié K, Said EA, Elass E, Masson M, Slomianny MC, Carpentier M, Briand JP, Mazurier J, Hovanessian AG. Surface nucleolin participates in both the binding and endocytosis of lactoferrin in target cells. EUROPEAN JOURNAL OF BIOCHEMISTRY 2004; 271:303-17. [PMID: 14717698 DOI: 10.1046/j.1432-1033.2003.03929.x] [Citation(s) in RCA: 167] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Lactoferrin (Lf), a multifunctional molecule present in mammalian secretions and blood, plays important roles in host defense and cancer. Indeed, Lf has been reported to inhibit the proliferation of cancerous mammary gland epithelial cells and manifest a potent antiviral activity against human immunodeficiency virus and human cytomegalovirus. The Lf-binding sites on the cell surface appear to be proteoglycans and other as yet undefined protein(s). Here, we isolated a Lf-binding 105 kDa molecular mass protein from cell extracts and identified it as human nucleolin. Medium-affinity interactions ( approximately 240 nm) between Lf and purified nucleolin were further illustrated by surface plasmon resonance assays. The interaction of Lf with the cell surface-expressed nucleolin was then demonstrated through competitive binding studies between Lf and the anti-human immunodeficiency virus pseudopeptide, HB-19, which binds specifically surface-expressed nucleolin independently of proteoglycans. Interestingly, binding competition studies between HB-19 and various Lf derivatives in proteoglycan-deficient hamster cells suggested that the nucleolin-binding site is located in both the N- and C-terminal lobes of Lf, whereas the basic N-terminal region is dispensable. On intact cells, Lf co-localizes with surface nucleolin and together they become internalized through vesicles of the recycling/degradation pathway by an active process. Morever, a small proportion of Lf appears to translocate in the nucleus of cells. Finally, the observations that endocytosis of Lf is inhibited by the HB-19 pseudopeptide, and the lack of Lf endocytosis in proteoglycan-deficient cells despite Lf binding, point out that both nucleolin and proteoglycans are implicated in the mechanism of Lf endocytosis.
Collapse
Affiliation(s)
- Dominique Legrand
- Institut Fédératif de Recherche n degrees 118, Université des Sciences et Technologies de Lille, Villeneuve d'Ascq, France.
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
34
|
Burrer R, Haessig-Einius S, Aubertin AM, Moog C. Polyclonal immunoglobulin G from patients neutralizes human immunodeficiency virus type 1 primary isolates by binding free virions, but without interfering with an initial CD4-independent attachment of the virus to primary blood mononuclear cells. J Virol 2003; 77:11385-97. [PMID: 14557624 PMCID: PMC229376 DOI: 10.1128/jvi.77.21.11385-11397.2003] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
We investigated the relationship between human immunodeficiency virus type 1 (HIV-1) primary isolate (PI) antibody-mediated neutralization and attachment to primary blood mononuclear cells (PBMC). Incubation of PIs with immunoglobulin G (IgG) purified from infected patients did not inhibit attachment of the viruses with PBMC, but partial to complete neutralization was achieved. Neutralization of PIs already fixed on the cells was achieved by some IgG samples only and was of limited intensity compared to the former neutralization protocol. On the contrary, the binding of IgG to free virions was shown to be sufficient to reach potent neutralization, as the infectivity of IgG-PI complexes purified from the bulk of antibodies before addition to PBMC was strongly diminished compared to mock-treated controls. Monoclonal antibodies to the CDR2 domain of CD4 completely inhibited the infection of PBMC without interfering with the attachment of PIs to the cells, suggesting that, under these experimental conditions, the initial attachment of viruses to PBMC involves alternative cellular receptors. This initial interaction may also involve other components of the viral envelope than gp120, as partial depletion of the surface glycoproteins of primary viral particles that resulted in an almost complete loss of infectivity did not impair attachment to PBMC. A limited inhibition of attachment was observed when interfering with putative interactions with cellular heparan sulfate, whereas no effect was observed for cellular CD147 or nucleolin or for virion-incorporated cyclophilin A. Altogether, our results favor a mechanism of neutralization of HIV-1 PIs by polyclonal IgG where antibodies predominantly bind free virions and neutralize without interfering with the attachment to PBMC, which, in this model, is mainly CD4 independent.
Collapse
Affiliation(s)
- Renaud Burrer
- Inserm U544, Institut de Virologie, Université Louis Pasteur, 67000 Strasbourg, France
| | | | | | | |
Collapse
|
|
35
|
Abstract
Dendritic cells (DCs) are crucial in the defence against pathogens. Invading pathogens are recognized by Toll-like receptors (TLRs) and receptors such as C-type lectins expressed on the surface of DCs. However, it is becoming evident that some pathogens, including viruses, such as HIV-1, and non-viral pathogens, such as Mycobacterium tuberculosis, subvert DC functions to escape immune surveillance by targeting the C-type lectin DC-SIGN (DC-specific intercellular adhesion molecule-grabbing nonintegrin). Notably, these pathogens misuse DC-SIGN by distinct mechanisms that either circumvent antigen processing or alter TLR-mediated signalling, skewing T-cell responses. This implies that adaptation of pathogens to target DC-SIGN might support pathogen survival.
Collapse
Affiliation(s)
- Yvette van Kooyk
- Department of Molecular Cell Biology and Immunology Vrije Universiteit Medical Center Amsterdam, v.d. Boechorststraat 7, 1081 BT Amsterdam, The Netherlands.
| | | |
Collapse
|
|
36
|
Wohlfahrt JG, Kunzmann S, Menz G, Kneist W, Akdis CA, Blaser K, Schmidt-Weber CB. T cell phenotype in allergic asthma and atopic dermatitis. Int Arch Allergy Immunol 2003; 131:272-82. [PMID: 12915770 DOI: 10.1159/000072139] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2002] [Accepted: 04/07/2003] [Indexed: 11/19/2022] Open
Abstract
BACKGROUND T cells are key regulators of immunologic disease parameters. However, their contribution to the process of tissue remodeling is ill defined. In the present study, we investigated gene expression of allergy-characteristic, IL-4-rich T cell cDNAs to monitor expression of genes that might participate in the pathogenesis of allergic diseases. METHODS cDNAs of freshly isolated and restimulated CD4+ T cells from patients with allergic asthma (AA) or atopic dermatitis (AD) and healthy subjects were analyzed on Nylon membrane-based DNA arrays. Three patients were selected for an allergy-characteristic T cell phenotype with high IL-4 expression (AA) or IL-13 expression (AD). RESULTS Several gene families such as the TGF-beta family, chemokines and chemokine receptors were found to be upregulated. Matrix metalloproteinases and their inhibitors were also found to be expressed in an enhanced manner. Furthermore, factors regulating tissue turnover such as fibroblast growth factors and neurotrophic as well as vasoactive factors were found be expressed at a higher level in allergic patient compared to healthy donors. CONCLUSION The present study reveals and confirms genes relevant for allergy and highlights an approach to applying a DNA array technique for diagnostic discrimination of allergic diseases.
Collapse
Affiliation(s)
- Jan G Wohlfahrt
- Swiss Institute of Allergy and Asthma Research (SIAF), Davos, Switzerland
| | | | | | | | | | | | | |
Collapse
|
|
37
|
Bobardt MD, Saphire ACS, Hung HC, Yu X, Van der Schueren B, Zhang Z, David G, Gallay PA. Syndecan captures, protects, and transmits HIV to T lymphocytes. Immunity 2003; 18:27-39. [PMID: 12530973 DOI: 10.1016/s1074-7613(02)00504-6] [Citation(s) in RCA: 169] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
This study demonstrates that syndecan functions as an in trans HIV receptor. We show that syndecan, when expressed in nonpermissive cells, becomes the major mediator for HIV adsorption. This adsorption is mediated by the binding of gp120 to the heparan sulfate chains of syndecan. Although syndecan does not substitute for HIV entry receptors, it enhances the in trans infectivity of a broad range of primate lentiviruses including primary viruses produced from PBMCs. Furthermore, syndecan preserves virus infectivity for a week, whereas unbound virus loses its infectivity in less than a day. Moreover, we obtain evidence suggesting that the vast syndecan-rich endothelial lining of the vasculature can provide a microenvironment which boosts HIV replication in T cells.
Collapse
Affiliation(s)
- Michael D Bobardt
- Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | | | | | | | | | | | | | | |
Collapse
|
|
38
|
Imai K, Iida T, Takano Y, Uozumi N. Membrane-bound heparin binding proteins from HL-60 cells purified in a two-step affinity chromatography differentially eluted with divalent cations. J Chromatogr B Analyt Technol Biomed Life Sci 2002; 780:1-12. [PMID: 12383474 DOI: 10.1016/s1570-0232(02)00404-x] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
Solubilized membrane proteins from HL-60 cells were separated by two-step affinity chromatography. Proteins eluted with MgCl2 in the first heparin-gel were applied to the second heparin-gel and eluted with CaCl2. The eluted proteins were analysed and purified by electrophoresis. N-terminal amino acid sequences of eight proteins on the characteristic bands were determined. Homology search for the sequences indicated that three microsomal proteins, two nuclear proteins and a glycolytic enzyme were eluted with divalent cations, whereas a nuclear ribonucleoprotein and a membrane-cytoskelton linker protein were not dissociated with divalent cations, but with 2 M NaCl. Heparin affinity chromatography combined with differential elution with divalent cations can be a useful method for separation of membrane proteins.
Collapse
Affiliation(s)
- Katsuyuki Imai
- Department of Science of Human Life, City College of Mie, Issinden-Nakano, Tsu-shi, Mie 514-0112, Japan. imai_
| | | | | | | |
Collapse
|
|
39
|
Said EA, Krust B, Nisole S, Svab J, Briand JP, Hovanessian AG. The anti-HIV cytokine midkine binds the cell surface-expressed nucleolin as a low affinity receptor. J Biol Chem 2002; 277:37492-502. [PMID: 12147681 DOI: 10.1074/jbc.m201194200] [Citation(s) in RCA: 110] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The growth factor midkine (MK) is a cytokine that inhibits the attachment of human immunodeficiency virus particles by a mechanism similar to the nucleolin binding HB-19 pseudopeptide. Here we show that the binding of MK to cells occurs specifically at a high and a low affinity binding site. HB-19 prevents the binding of MK to the low affinity binding site only. Confocal immunofluorescence laser microscopy revealed the colocalization of MK and the cell-surface-expressed nucleolin at distinct spots. The use of various deletion constructs of nucleolin then indicated that the extreme C-terminal end of nucleolin, containing repeats of the amino acid motif RGG, is the domain that binds MK. The specific binding of MK to cells is independent of heparan sulfate and chondroitin sulfate expression. After binding to cells, MK enters cells by an active process. Interestingly, the cross-linking of surface-bound MK with a specific antibody results in the clustering of surface nucleolin along with glycosylphosphatidylinositol-linked proteins CD90 and CD59, thus, pointing out that MK binding induces lateral assemblies of nucleolin with specific membrane components of lipid rafts. Our results suggest that the cell surface-expressed nucleolin serves as a low affinity receptor for MK and could be implicated in its entry process.
Collapse
Affiliation(s)
- Elias A Said
- Unité de Virologie et Immunologie Cellulaire (URA 1930 CNRS), Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France
| | | | | | | | | | | |
Collapse
|
|
40
|
Shibata Y, Muramatsu T, Hirai M, Inui T, Kimura T, Saito H, McCormick LM, Bu G, Kadomatsu K. Nuclear targeting by the growth factor midkine. Mol Cell Biol 2002; 22:6788-96. [PMID: 12215536 PMCID: PMC134045 DOI: 10.1128/mcb.22.19.6788-6796.2002] [Citation(s) in RCA: 108] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2002] [Accepted: 06/17/2002] [Indexed: 01/08/2023] Open
Abstract
Ligand-receptor internalization has been traditionally regarded as part of the cellular desensitization system. Low-density lipoprotein receptor-related protein (LRP) is a large endocytosis receptor with a diverse array of ligands. We recently showed that LRP binds heparin-binding growth factor midkine. Here we demonstrate that LRP mediates nuclear targeting by midkine and that the nuclear targeting is biologically important. Exogenous midkine reached the nucleus, where intact midkine was detected, within 20 min. Midkine was not internalized in LRP-deficient cells, whereas transfection of an LRP expression vector restored midkine internalization and subsequent nuclear translocation. Internalized midkine in the cytoplasm bound to nucleolin, a nucleocytoplasmic shuttle protein. The midkine-binding sites were mapped to acidic stretches in the N-terminal domain of nucleolin. When the nuclear localization signal located next to the acidic stretches was deleted, we found that the mutant nucleolin not only accumulated in the cytoplasm but also suppressed the nuclear translocation of midkine. By using cells that overexpressed the mutant nucleolin, we further demonstrated that the nuclear targeting was necessary for the full activity of midkine in the promotion of cell survival. This study therefore reveals a novel role of LRP in intracellular signaling by its ligand and the importance of nucleolin in this process.
Collapse
Affiliation(s)
- Yoshihisa Shibata
- Department of Biochemistry, Nagoya University School of Medicine, Showa-ku, Japan
| | | | | | | | | | | | | | | | | |
Collapse
|
|
41
|
Nisole S, Krust B, Hovanessian AG. Anchorage of HIV on permissive cells leads to coaggregation of viral particles with surface nucleolin at membrane raft microdomains. Exp Cell Res 2002; 276:155-73. [PMID: 12027446 DOI: 10.1006/excr.2002.5522] [Citation(s) in RCA: 62] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The cross-linking of HIV on permissive cells results aggregation of HIV particles with surface nucleolin, CD4, and CXCR4, but without affecting the organization of CD45. In addition, HIV particles and nucleolin coaggregate with glycolipid-enriched membrane microdomains (GEMs) containing ganglioside, and glycosylphosphatidylinositol-linked proteins CD90 and CD59, pointing out that HIV anchorage induces lateral assemblies of specific membrane components into lipid rafts in which surface nucleolin is also incorporated. Consequently, equilibrium density fractionation of extracts from infected cells revealed that HIV proteins and nucleolin copurify with Triton X-100-resistant GEM-associated proteins. After HIV entry, nucleolin is recovered also in fractions containing HIV DNA, viral matrix, and reverse transcriptase, thus suggesting that it could accompany viral entry. We show that surface nucleolin is markedly down-regulated a few hours following HIV entry into permissive cells; an effect that appears to be the consequence of its translocation into the cytoplasm. Our findings demonstrate that anchorage of HIV particles on permissive cells induces aggegation of surface nucleolin and its association with detergent-insoluble lipid raft components. Moreover, they support the suggestion that surface nucleolin and lipid rafts are implicated in early events in the HIV entry process.
Collapse
Affiliation(s)
- Sébastien Nisole
- Unité de Virologie et Immunologie Cellulaire, URA 1930 CNRS, Institut Pasteur, Paris, France
| | | | | |
Collapse
|
|
42
|
Nisole S, Said EA, Mische C, Prevost MC, Krust B, Bouvet P, Bianco A, Briand JP, Hovanessian AG. The anti-HIV pentameric pseudopeptide HB-19 binds the C-terminal end of nucleolin and prevents anchorage of virus particles in the plasma membrane of target cells. J Biol Chem 2002; 277:20877-86. [PMID: 11919179 DOI: 10.1074/jbc.m110024200] [Citation(s) in RCA: 75] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The multivalent pseudopeptide HB-19 that binds the cell-surface-expressed nucleolin is a potent inhibitor of human immunodeficiency virus (HIV) infection by blocking virus particle attachment and thus anchorage in the plasma membrane. We show that cross-linking of surface-bound HB-19A (like HB-19 but with a modified template) results in aggregation of HB-19A with surface nucleolin. Consistent with its specific action, HB-19A binding to different types of cells reaches saturation at concentrations that have been reported to result in inhibition of HIV infection. By using Chinese hamster ovary mutant cell lines, we confirm that the binding of HB-19A to surface nucleolin is independent of heparan and chondroitin sulfate proteoglycans. In vitro generated full-length nucleolin was found to bind HB-19A, whereas the N-terminal part containing the acidic amino acid stretches of nucleolin did not. The use of various deletion constructs of the C-terminal part of nucleolin then permitted the identification of the extreme C-terminal end of nucleolin, containing repeats of the amino acid motif, RGG, as the domain that binds HB-19A. Finally, a synthetic peptide corresponding to the last C-terminal 63 amino acids was able to inhibit HIV infection at the stage of HIV attachment to cells, thus suggesting that this domain could be functional in the HIV anchorage process.
Collapse
Affiliation(s)
- Sébastien Nisole
- Unité de Virologie et Immunologie Cellulaire (URA 1930 CNRS), Plateau Technique, Institut Pasteur, 28 Rue du Dr. Roux, 75724 Paris Cedex 15, France
| | | | | | | | | | | | | | | | | |
Collapse
|
|
43
|
Rus V, Atamas SP, Shustova V, Luzina IG, Selaru F, Magder LS, Via CS. Expression of cytokine- and chemokine-related genes in peripheral blood mononuclear cells from lupus patients by cDNA array. Clin Immunol 2002; 102:283-90. [PMID: 11890715 DOI: 10.1006/clim.2001.5182] [Citation(s) in RCA: 87] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Systemic lupus erythematosus (SLE) is characterized by diverse and complex immune abnormalities. In an effort to begin to characterize the full complexity of immune abnormalities, the expression pattern of 375 potentially relevant genes was analyzed using peripheral blood mononuclear cells (PBMC) from 21 SLE patients and 12 controls by cDNA arrays. When mean gene expression for patients was compared to controls, 50 genes were identified that exhibited more than 2.5-fold difference in expression level. By the Mann-Whitney U test, 20 genes were significantly different (P < 0.05) between patients and controls. Most of these genes have not been previously associated with SLE and belong to a variety of families such as TNF/death receptor, IL-1 cytokine family, and IL-8 and its receptors. Hierarchical clustering of samples and differentially expressed genes revealed that with few exceptions, patients clustered separately from controls. These results highlight the potential use of the microarray data in identifying genes associated with SLE, which could become candidate molecular markers or future therapeutic targets.
Collapse
Affiliation(s)
- Violeta Rus
- Division of Rheumatology and Clinical Immunology, Department of Medicine, University of Maryland Medical School, Baltimore, Maryland 21201, USA.
| | | | | | | | | | | | | |
Collapse
|
|
44
|
Abstract
A growing number of peptide hormones and growth factors have been shown to operate in the intracellular space after either internalization or retention in their cells of synthesis. These factors, called intracrines, in many cases are expressed as multiple isoforms, traffic to nucleus or nucleolus, and regulate gene transcription. Some intracrines are angiogenic. It is here argued that intracrine action is the modern analogue of a biologically ancient mechanism for regulating message translation and ribosome synthesis. The implications of this view for research and therapeutics are discussed.
Collapse
Affiliation(s)
- Richard N Re
- Division of Research, Alton Ochsner Medical Foundation, New Orleans, Louisiana 70121, USA.
| |
Collapse
|
|
45
|
Krust B, Vienet R, Cardona A, Rougeot C, Jacotot E, Callebaut C, Guichard G, Briand JP, Grognet JM, Hovanessian AG, Edelman L. The anti-HIV pentameric pseudopeptide HB-19 is preferentially taken up in vivo by lymphoid organs where it forms a complex with nucleolin. Proc Natl Acad Sci U S A 2001; 98:14090-5. [PMID: 11698640 PMCID: PMC61173 DOI: 10.1073/pnas.221467298] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2001] [Accepted: 09/04/2001] [Indexed: 11/18/2022] Open
Abstract
The HB-19 pseudopeptide 5[Kpsi(CH(2)N)PR]-TASP, psi(CH(2)N) for reduced peptide bond, is a specific inhibitor of HIV infection in different CD4(+) cell lines and in primary T-lymphocytes and macrophages. It blocks virus-particle attachment to permissive cells by binding and forming a stable complex with nucleolin expressed on the cell surface. Here, we have investigated the tissue distribution of the tritiated HB-19 by using beta-radio imager whole-body mapping in rats. A rapid, selective, and stable distribution and accumulation of the systematically administered HB-19 was demonstrated within the spleen, liver, bone, and kidney as soon as 5 min following its administration. No apparent uptake of HB-19 occurred in the brain and the muscle tissue. Interestingly and despite its rapid clearance from the blood, at 24 h postexposure a significant proportion of HB-19 was still recovered from target organs, of which 16-37% could be accounted for intact pseudopeptide. The elimination of HB-19 mainly occurred by renal glomerular filtration and most of the excreted radioactivity appeared to be HB-19 metabolites. Finally, injection of the biotin-labeled HB-19 pseudopeptide but not its control counterpart allowed the recovery of the HB-19-nucleolin complex from the liver, spleen, thymus, and bone marrow, thus indicating that the in vivo molecular target of HB-19 is surface nucleolin. Our results demonstrate the preferential uptake and stability of HB-19 in lymphoid organs that are the site of HIV propagation.
Collapse
Affiliation(s)
- B Krust
- Unité de Virologie et Immunologie Cellulaire, Unité de Recherche Associée 1930 Centre National de Recherche Scientifique (CNRS), 28 Rue du Dr Roux, 75714 Paris Cedex 15, France
| | | | | | | | | | | | | | | | | | | | | |
Collapse
|