Interferon-alpha induces up-regulation and nuclear translocation of the Ro52 autoantigen as detected by a panel of novel Ro52-specific monoclonal antibodies.
Interferon-alpha (IFN-alpha) has been implicated in the pathogenesis of Sjögren's syndrome and systemic lupus erythematosus. Ro52, which was recently identified as an E3 ligase with anti-proliferative and pro-apoptotic properties, is a major autoantigen targeted in both these conditions. Microarray analyses have indicated up-regulation of Ro52 by IFN-alpha, and the objective of the present study was to address the potential link between IFN-alpha and Ro52. To investigate the influence of IFN-alpha on Ro52 protein levels and cellular localization, we generated a panel of monoclonal antibodies to different domains of Ro52. These novel monoclonal antibodies were characterized by immunoprecipitation, Western blot, and enzyme-linked immunosorbent assay using cell lysates, recombinant Ro52 protein, and synthetic peptides. Ro52 was up-regulated in HeLa cells and human B cells at the messenger RNA and protein levels in response to IFN-alpha stimulation as detected by reverse transcriptase polymerase chain reaction and Western blot. After up-regulation, Ro52 translocated from the cytoplasm to the nucleus. The nuclear translocation of Ro52 was observed after staining with generated monoclonal antibodies specific for both the RING, coiled-coil, and B30.2 domains of Ro52 and the nuclear translocation of Ro52 preceded IFN-alpha-induced apoptotic cell death detected by caspase-3 and TUNEL staining in the treated cultures. In conclusion, our data show that IFN-alpha first induces up-regulation of Ro52 protein and then prompts translocation of the up-regulated Ro52 protein in to the nucleus. The translocation precedes apoptosis of the IFN-alpha exposed cells, suggesting a role for Ro52 in mediating the anti-proliferative or pro-apoptotic effects of the autoimmune-related cytokine IFN-alpha.
Strandberg
Linn
L
Rheumatology Unit, Department of Medicine, CMM L8:04, Karolinska Institutet, 171 76 Stockholm, Sweden.
Ambrosi
Aurelie
A
Espinosa
Alexander
A
Ottosson
Lars
L
Eloranta
Maija-Leena
ML
Zhou
Wei
W
Elfving
Ase
A
Greenfield
Edward
E
Kuchroo
Vijay K
VK
Wahren-Herlenius
Marie
M
D021581:Active Transport, Cell Nucleus / Q000276:immunology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000918:Antibody Specificity; D017209:Apoptosis / Q000276:immunology; D001324:Autoantigens / Q000378:metabolism*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000201:enzymology / Q000276:immunology / Q000378:metabolism; D049109:Cell Proliferation; D006367:HeLa Cells; D006801:Humans; D016898:Interferon-alpha / Q000502:physiology*; D051379:Mice; D008815:Mice, Inbred Strains; D012261:Ribonucleoproteins / Q000276:immunology* / Q000378:metabolism* / Q000502:physiology; D015398:Signal Transduction / Q000276:immunology; D044767:Ubiquitin-Protein Ligases / Q000276:immunology / Q000378:metabolism; D015854:Up-Regulation / Q000276:immunology*; D000099207:SS-A Antigen
D000911:Antibodies, Monoclonal; D001324:Autoantigens; D016898:Interferon-alpha; D012261:Ribonucleoproteins; D044767:Ubiquitin-Protein Ligases; D000099207:SS-A Antigen