Interferon regulatory factor-5 (IRF5), a transcription factor critical for the induction of innate immune responses, contributes to the pathogenesis of the autoimmune disease systemic lupus ...erythematosus (SLE) in humans and mice. Lyn, a Src family kinase, is also implicated in human SLE, and Lyn-deficient mice develop an SLE-like disease. Here, we found that Lyn physically interacted with IRF5 to inhibit ubiquitination and phosphorylation of IRF5 in the TLR-MyD88 pathway, thereby suppressing the transcriptional activity of IRF5 in a manner independent of Lyn’s kinase activity. Conversely, Lyn did not inhibit NF-κB signaling, another major branch downstream of MyD88. Monoallelic deletion of Irf5 alleviated the hyperproduction of cytokines in TLR-stimulated Lyn–/– dendritic cells and the development of SLE-like symptoms in Lyn–/– mice. Our results reveal a role for Lyn as a specific suppressor of the TLR-MyD88-IRF5 pathway and illustrate the importance of fine-tuning IRF5 activity for the maintenance of immune homeostasis.
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•Lyn binds to and inhibits the activity of IRF5 in the TLR-MyD88 pathway•Lyn inhibits post-translational modifications of IRF5 without Lyn’s kinase activity•Lyn deficiency causes IRF5 hyperactivation in DCs•SLE symptoms in Lyn–/– mice are ameliorated by monoallelic deletion of Irf5
How IRF5 activity is negatively regulated remains largely unknown. Tamura and colleagues identify Lyn as an IRF5-binding protein that suppresses the TLR-MyD88-IRF5 pathway. IRF5 is hyperactivated in Lyn-deficient mice suffering from the autoimmune disease SLE, but reducing the abundance of IRF5 ameliorates the disease development.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
The transcription factor interferon regulatory factor 5 (IRF5) is critical for innate immune responses downstream of Toll-like receptors (TLRs). IRF5 is also linked to the pathogenesis of ...systemic lupus erythematosus (SLE) in humans and mice. However, it remains elusive whether and how IRF5 activity can be negatively regulated. The Src family kinase Lyn is also implicated in human SLE, and Lyn−/− mice develop an SLE-like disease. Here we show that Lyn selectively inhibits IRF5 activity, while it does not affect the NF-κB pathway. Lyn physically interacted with IRF5, thereby suppressing the phosphorylation and ubiquitination of IRF5, post-translational modifications important for IRF5 activation. Interestingly, the kinase activity of Lyn was dispensable for the suppression of IRF5 activity. IRF5 was hyper-activated in TLR7/9-stimulated Lyn−/− bone marrow-derived dendritic cells (BMDCs), and these cells hyper-produced IRF5-dependent cytokines especially type-I interferons (IFNs). IRF5 was constitutively activated (phosphorylated and translocated into the nucleus) in splenic DCs isolated from Lyn−/− mice. Importantly, even monoallelic ablation of the Irf5 gene was sufficient to alleviate the hyper-production of type-I IFNs in TLR7/9-stimulated Lyn−/− BMDCs, and to ameliorate the development of SLE-like symptoms, such as autoantibody production and autoimmune glomerulonephritis, in Lyn−/− mice. Our results identify Lyn as a critical suppressor of the TLR-MyD88-IRF5 pathway, and implicate that the selective control of IRF5 activity may contribute to better therapeutics for SLE.
A sensitive two-site enzyme immunoassay (EIA) system was established for human pS2 protein, a small estrogen-inducible secretory protein of unknown function originally identified in MCF-7 human ...breast cancer cells. Our EIA system is based on the sandwiching of antigen between anti-recombinant (r) pS2 antibody IgG coated on a polystyrene plate and biotinylated anti-rpS2 antibody IgG. The amount of pS2 protein was quantified by measurement of the bound enzyme activity of subsequently added streptavidin-linked beta-D-galactosidase (beta-D-galactosidase, EC 3.2.1.23). pS2 protein purified from MCF-7 culture supernatants was detectable at a concentration as low as 3 pg/ml (corresponding to 60 fg/well). This EIA system revealed that the amount of pS2-like immunoreactivity (LI) in human urine was 13.6 ng/mg creatinine (median, n = 416) and that there was no correlation between the pS2-LI concentration in urine and sex or aging. pS2-LI levels in plasma and sera of the normal subjects were 392 pg/ml (median, n = 14) and 494 pg/ml (median, n = 12), respectively. The serum level of the patients with breast cancer (528 pg/ml; median, n = 67) was not statistically different from that of normal subjects, although high levels of pS2 protein in breast cancer tissues had been reported.
By the reverse transcription-polymerase chain reaction we investigated the localization of pS2 mRNA, which encodes a secreted polypeptide of 60 amino acids with unknown function, in the adult mouse. ...This method revealed that the expression of pS2 mRNA was not restricted to the stomach as previously reported but was also found in various other tissues such as brain, heart, spleen, and muscle. The pS2 mRNA was additionally detected in astrocytes cultured from the whole brain of the newborn mouse. The pS2 expression in these astrocytes increased with cell growth and sharply declined after the cells reached the stationary phase. In the cells synchronized by the serum deprivation-refeeding technique, the expression of pS2 mRNA was only found at late G1 and S phase. These results suggest that the transcripts of pS2 gene are widely distributed throughout the entire body of the mouse and that they play some important cell cycle-related role in these tissues.
pS2 is an estrogen‐induced mRNA species that was originally identified in the breast cancer cell line MCF‐7. Exposure of the cells to basic fibroblast growth factor (bFGF) at the concentration of ...10–100 ng/ml for 48–72 h resulted in a marked increase in the concentration of pS2 protein in the medium. The polymerase chain reaction with reverse transcriptase revealed that bFGF increased the amount of intracellular pS2 mRNA; immunocytochemical studies showed that exposure to the factor increased the amount of intracellular pS2 protein. Simultaneous addition of cycloheximide with bFGF completely abolished induction of pS2 protein, although it did not affect the induction of pS2 mRNA. Actinomycin D did not affect the stimulatory effect of bFGF on synthesis/secretion of pS2 protein. bFGF effectively abolished decay of the pS2 mRNA level caused by actinomycin D. These results suggest that the induction of the synthesis/secretion of pS2 protein by bFGF occurs at the post‐transcriptional level, most probably due to the stabilization of pS2 mRNA. Another finding, that bFGF and estradiol have a synergistic effect on induction of pS2 protein, suggests the possibility that these two inducers act by a different but partly overlapping mechanism.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
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