A time- and dose-dependent STAT1 expression system

BACKGROUND: The signal transducer and activator of transcription (STAT) family of transcription factors mediates a variety of cytokine dependent gene regulations. STAT1 has been mainly characterized by its role in interferon (IFN) type I and II signaling and STAT1 deficiency leads to high susceptibility to several pathogens. For fine-tuned analysis of STAT1 function we established a dimerizer-inducible system for STAT1 expression in vitro and in vivo. RESULTS: The functionality of the dimerizer-induced STAT1 system is demonstrated in vitro in mouse embryonic fibroblasts and embryonic stem cells. We show that this two-vector based system is highly inducible and does not show any STAT1 expression in the absence of the inducer. Reconstitution of STAT1 deficient cells with inducible STAT1 restores IFNγ-mediated gene induction, antiviral responses and STAT1 activation remains dependent on cytokine stimulation. STAT1 expression is induced rapidly upon addition of dimerizer and expression levels can be regulated in a dose-dependent manner. Furthermore we show that in transgenic mice STAT1 can be induced upon stimulation with the dimerizer, although only at low levels. CONCLUSION: These results prove that the dimerizer-induced system is a powerful tool for STAT1 analysis in vitro and provide evidence that the system is suitable for the use in transgenic mice. To our knowledge this is the first report for inducible STAT1 expression in a time- and dose-dependent manner

TYK2 Kinase Activity Is Required for Functional Type I Interferon Responses In Vivo

Tyrosine kinase 2 (TYK2) is a member of the Janus kinase (JAK) family and is involved in cytokine signalling. In vitro analyses suggest that TYK2 also has kinase-independent, i.e., non-canonical, functions. We have generated gene-targeted mice harbouring a mutation in the ATP-binding pocket of the kinase domain. The Tyk2 kinase-inactive (Tyk2K923E) mice are viable and show no gross abnormalities. We show that kinase-active TYK2 is required for full-fledged type I interferon- (IFN) induced activation of the transcription factors STAT1-4 and for the in vivo antiviral defence against viruses primarily controlled through type I IFN actions. In addition, TYK2 kinase activity was found to be required for the protein’s stability. An inhibitory function was only observed upon over-expression of TYK2K923E in vitro. Tyk2K923E mice represent the first model for studying the kinase-independent function of a JAK in vivo and for assessing the consequences of side effects of JAK inhibitors

TYK2 is a key regulator of the surveillance of B lymphoid tumors

Aberrant activation of the JAK-STAT pathway has been implicated in tumor formation; for example, constitutive activation of JAK2 kinase or the enforced expression of STAT5 induces leukemia in mice. We show here that the Janus kinase TYK2 serves an opposite function. Mice deficient in TYK2 developed Abelson-induced B lymphoid leukemia/lymphoma as well as TEL-JAK2–induced T lymphoid leukemia with a higher incidence and shortened latency compared with WT controls. The cell-autonomous properties of Abelson murine leukemia virus–transformed (A-MuLV–transformed) TYK2(–/–) cells were unaltered, but the high susceptibility of TYK2(–/–) mice resulted from an impaired tumor surveillance, and accordingly, TYK2(–/–) A-MuLV–induced lymphomas were easily rejected after transplantation into WT hosts. The increased rate of leukemia/lymphoma formation was linked to a decreased in vitro cytotoxic capacity of TYK2(–/–) NK and NKT cells toward tumor-derived cells. RAG2/TYK2 double-knockout mice succumbed to A-MuLV–induced leukemia/lymphoma faster than RAG2(–/–)TYK2(+/–) mice. This defines NK cells as key players in tumor surveillance in Abelson-induced malignancies. Our observations provide compelling evidence that TYK2 is an important regulator of lymphoid tumor surveillance

TYK2^K923E protein level is reduced and TYK2 differs organ-specifically.

<p>A. WT, <i>Tyk2^−/−</i> and <i>Tyk2^K923E</i> mice were used to prepare whole cell extracts from BMMΦs, T cells and various organs (as indicated). Levels of expression of TYK2 and JAK1 were determined by immunoprecipitation and Western blot analysis. NFκB-p65 was used as input control. TYK2^K923E protein levels were quantified using ImageJ software for Mac OS X (open source, <a href="http://rsb.info.nih.gov/ij/index.html" target="_blank">http://rsb.info.nih.gov/ij/index.html</a>) and were between 13% and 30% in BMMΦs and approximately 58% in T cells compared to WT. B. Total RNA was isolated from WT and <i>Tyk2^K923E</i> BMMΦs and cDNA was used to analyse Tyk2 mRNA expression normalized to the housekeeping gene <i>Ube2D2</i>. Results from 4 independent experiments are shown (n = 6 per genotype). C. BMMΦs were treated with the proteasomal inhibitor MG-132 (50 µM), the autophagy-lysosome inhibitor 3-MA (10 mM) or the lysosome-acidification inhibitor bafilomycin A₁ (80 nM) for the indicated period of time (upper panel), for 11 h (middle panel) or 48 h (lower panel). Whole cell extracts were used to determine TYK2 and JAK1 expression levels by immunoprecipitation and Western blot analysis. As a control, a Western blot for HO-1 was performed. D. From day 5 after isolation of WT BMMΦs, cells were treated with JAK inhibitor I (panJAK inhibitor; 15 nM upper panel and 300 nM lower panel) for the indicated period of time. TYK2 and JAK2 expression levels were analysed as described in (A and C); NFκB-p65 was used as input control.</p