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1.
RNA ; 20(6): 899-911, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24751651

ABSTRACT

Macrophage activation by bacterial lipopolysaccharides (LPS) is induced through Toll-like receptor 4 (TLR4). The synthesis and activity of TLR4 downstream signaling molecules modulates the expression of pro- and anti-inflammatory cytokines. To address the impact of post-transcriptional regulation on that process, we performed RIP-Chip analysis. Differential association of mRNAs with heterogeneous nuclear ribonucleoprotein K (hnRNP K), an mRNA-specific translational regulator in differentiating hematopoietic cells, was studied in noninduced and LPS-activated macrophages. Analysis of interactions affected by LPS revealed several mRNAs encoding TLR4 downstream kinases and their modulators. We focused on transforming growth factor-ß-activated kinase 1 (TAK1) a central player in TLR4 signaling. HnRNP K interacts specifically with a sequence in the TAK1 mRNA 3' UTR in vitro. Silencing of hnRNP K does not affect TAK1 mRNA synthesis or stability but enhances TAK1 mRNA translation, resulting in elevated TNF-α, IL-1ß, and IL-10 mRNA expression. Our data suggest that the hnRNP K-3' UTR complex inhibits TAK1 mRNA translation in noninduced macrophages. LPS-dependent TLR4 activation abrogates translational repression and newly synthesized TAK1 boosts macrophage inflammatory response.


Subject(s)
Heterogeneous-Nuclear Ribonucleoprotein K/genetics , Lipopolysaccharides/immunology , MAP Kinase Kinase Kinases/genetics , Macrophage Activation/immunology , Macrophages/immunology , Protein Biosynthesis/genetics , RNA, Messenger/genetics , 3' Untranslated Regions/genetics , 3' Untranslated Regions/immunology , Animals , Cell Line , Gene Expression Regulation/genetics , Gene Expression Regulation/immunology , Heterogeneous-Nuclear Ribonucleoprotein K/immunology , Inflammation/genetics , Inflammation/immunology , Interleukin-10/genetics , Interleukin-10/immunology , Interleukin-1beta/genetics , Interleukin-1beta/immunology , MAP Kinase Kinase Kinases/immunology , Macrophage Activation/genetics , Mice , Protein Biosynthesis/immunology , RNA Processing, Post-Transcriptional/genetics , RNA Processing, Post-Transcriptional/immunology , RNA, Messenger/immunology , Signal Transduction/genetics , Signal Transduction/immunology , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/immunology , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunology
2.
J Heart Lung Transplant ; 30(9): 1051-9, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21489814

ABSTRACT

BACKGROUND: Cardiac allograft vasculopathy (CAV) is the most serious long-term complication after cardiac transplantation. T-cell-mediated immune response has been implicated as the central mechanism for this form of graft rejection, but the role of humoral immunity is still controversial. METHODS: This study investigated whether human leukocyte antigen (HLA) and non-HLA antibodies are associated with CAV and if their presence can be used to identify patients at high risk of developing CAV. Diagnosis of CAV was made by angiography and intravascular ultrasound (IVUS) technology. Sera from 48 heart transplant recipients were assessed for the presence of antibodies. RESULTS: Although anti-HLA or anti-major histocompatibility complex class I chain-related gene A (MICA) antibodies in patients with or without CAV were not statistically different, heterogeneous nuclear ribonucleoprotein K (hnRNP-K) was identified as a new antigenic target after the screening of a human coronary artery smooth muscle cells complementary DNA (cDNA) expression library with a serum sample from a CAV patient. Four years after transplantation, presence of anti-hnRNP-K antibodies was significantly higher in the IVUS-defined CAV group (85.3%) and angiography-defined CAV patients (90.5%) compared with the non-CAV group (p < 0.0001 and p = 0.0023 respectively). CONCLUSIONS: The presence of anti-hnRNP-K antibodies 4 years after the transplant is statistically associated with CAV disease, regardless of the diagnostic technique. Therefore, prospective detection of these antibodies could be proposed as a helpful biomarker in CAV diagnosis.


Subject(s)
Antibodies, Anti-Idiotypic/blood , Coronary Disease/epidemiology , Coronary Disease/immunology , Heart Transplantation/immunology , Heterogeneous-Nuclear Ribonucleoprotein K/immunology , Biomarkers/blood , Coronary Angiography , Coronary Disease/complications , Coronary Vessels/diagnostic imaging , Female , Follow-Up Studies , Graft Rejection/etiology , Histocompatibility Antigens Class I/immunology , Humans , Male , Middle Aged , Risk Factors , Transplantation, Homologous , Ultrasonography, Interventional
3.
Hybridoma (Larchmt) ; 30(1): 53-9, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21466286

ABSTRACT

hnRNP K protein is a member of the heterogeneous nuclear protein (hnRNP) complex that, besides its function as a translational regulator of human mRNA, is also considered to be a transcription factor involved in tumorigenesis. PSF is a protein part of the human spliceosome and essential in RNA splicing. Here we report the generation of one monoclonal antibody GG6H9.1C3 that recognized both hnRNP K and PSF proteins using Western blot analysis, flow cytometry, and immunocytochemistry.


Subject(s)
Antibodies, Monoclonal/immunology , Heterogeneous-Nuclear Ribonucleoprotein K/immunology , RNA-Binding Proteins/immunology , Blotting, Western , Cell Nucleus/metabolism , Flow Cytometry , Heterogeneous-Nuclear Ribonucleoprotein K/metabolism , Humans , Immunohistochemistry , PTB-Associated Splicing Factor , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
4.
Ann Hematol ; 89(12): 1255-63, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20623123

ABSTRACT

To identify a new diagnostic marker for the immune pathophysiology of aplastic anemia (AA), we screened sera of immune-mediated AA patients for the presence of antibodies (Abs) specific to proteins derived from a leukemia cell line UT-7 using two-dimensional electrophoresis followed by immunoblotting. The target proteins were identified by peptide mass fingerprinting. Heterogeneous nuclear ribonucleoprotein (hnRNP) K was identified as a novel autoantigen. An enzyme-linked immunosorbent assay revealed high titers of anti-hnRNP K Abs in 85 (31%) of 273 patients with AA. Sixty-four patients received antithymocyte globulin and cyclosporine after undergoing screening for anti-hnRNP K Ab, anti-DRS-1 Ab, anti-moesin Ab, and paroxysmal nocturnal hemoglobinuria (PNH)-type cells. Twenty (87%) of 23 patients with the presence of anti-hnRNP K Abs responded to the immunosuppressive therapy (IST), while 19 (46%) of 41 patients without the presence of anti-hnRNP K Abs responded. A multivariate analysis showed only PNH-type cells and anti-hnRNP K Abs to be significant factors for the prediction of a good response to IST. The detection of anti-hnRNP K Abs as well as PNH-type cells may therefore be useful for diagnosing the immune pathophysiology of AA.


Subject(s)
Anemia, Aplastic/diagnosis , Autoantibodies/blood , Biomarkers/blood , Heterogeneous-Nuclear Ribonucleoprotein K/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Anemia, Aplastic/immunology , Anemia, Aplastic/physiopathology , Autoantibodies/immunology , Blotting, Western , Cell Line, Tumor , Electrophoresis, Gel, Two-Dimensional , Enzyme-Linked Immunosorbent Assay , Female , HL-60 Cells , Heterogeneous-Nuclear Ribonucleoprotein K/metabolism , Humans , K562 Cells , Leukocytes, Mononuclear/metabolism , Male , Middle Aged , Young Adult
5.
Autoimmunity ; 38(1): 25-32, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15804702

ABSTRACT

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are among the most abundant proteins in the eukaryotic cell nucleus and play a direct role in several aspects of the RNA life including splicing, export of the mature RNAs and translation. To date, approximately 30 proteins have been identified. A growing body of evidence points to hnRNPs as an important target of the autoimmune response in rheumatic diseases. Autoantibodies to A and B proteins of the hnRNP complex have been detected in late 1980s in rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD). Beyond their role as diagnostic test in clinical practice, these autoantibodies are starting to be regarded as important tools to obtain deeper insight into the pathogenesis of autoimmune rheumatic diseases. Furthermore, new anti-hnRNP antibodies have been recognized in the last ten years extending the spectrum of anti-hnRNP reactivity in different autoimmune disorders.


Subject(s)
Antibodies, Antinuclear/blood , Heterogeneous-Nuclear Ribonucleoproteins/immunology , Animals , Antibodies, Antinuclear/history , Autoimmune Diseases/immunology , Heterogeneous Nuclear Ribonucleoprotein A1 , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/immunology , Heterogeneous-Nuclear Ribonucleoprotein Group C/immunology , Heterogeneous-Nuclear Ribonucleoprotein K/immunology , History, 20th Century , Humans , Immunoglobulin Isotypes/blood , Rheumatic Diseases/immunology
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