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Key Event Title
Suppression of STAT5 binding to cytokine gene promoters
|Level of Biological Organization|
Key Event Components
|negative regulation of DNA binding||protein-DNA complex||decreased|
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP||Point of Contact||Author Status||OECD Status|
|Immune dysfunction induced by JAK3 inhibition||KeyEvent||Yasuhiro Yoshida (send email)||Under development: Not open for comment. Do not cite||Under Development|
|All life stages||High|
Key Event Description
IL-2 and other cytokines rapidly activate JAK1 and JAK3 (Beadling, et al. 1994) in peripheral blood lymphocytes. The activation of JAK kinases and STAT proteins by IL-2 and IFN-α does not include the T cell antigen receptor in human T lymphocytes (Beadling, et al. 1994). After activation of JAKs, latent STAT transcription factors induce dimeric STAT proteins (Gaffen, et al. 1995). These proteins then translocate to the nucleus, where they bind to and regulate the transcriptional activation of the promoters of target genes. Dimeric STAT proteins can bind to the palindromic gamma interferon-activated (GAS) sequence TTCNmGAA, where m is 3 for all the STATs, except STAT6. The latter can additionally bind to GAS motifs. The m for STAT6 denotes 4 (Darnell 1997, Decker, et al. 1997, Ihle 1996, Leonard and O'Shea 1998).
How It Is Measured or Detected
EMSA using nuclear extracts and specific oligonucleotides, including transcription factor binding sites, such as cytokine-inducible SH2-containing protein (CIS) gene promoters, are useful to evaluate DNA binding activity (Johnston, et al. 1995). Activated STAT5 binds to specific DNA-probes in splenocytes (Liu, et al. 2010). A cell-permeable non-peptidic nicotinoyl hydrazone compound selectively targets the SH2 domain of STAT5 (IC50 = 47 µM against STAT5b SH2 domain EPO peptide binding activity), with markedly less recognition of the SH2 domain of STAT1, STAT3, or Lck (IC50 > 500 µM). This compound inhibited STAT5/STAT5 DNA binding activity in K562 nuclear extract and inhibited IFN-α-stimulated STAT5 tyrosine phosphorylation in Daudi cells, but not STAT1 or STAT3 (Muller, et al. 2008).
Nuclear extracts were prepared from untreated YT cells or cells treated with recombinant IL-2 (2 nM) for 30 min at 37°C. EMSA was performed using glycerol-containing 5% polyacrylamide gels (29:1) containing 0.5× Tris-borate-EDTA buffer. For supershift assays, nuclear extracts were preincubated for 10 min with antibodies against STAT5. Oligonucleotide sequences from PRRIFV have been used as probes (Maeshima, et al. 2012). Other authors described a supershift ESMA that involved preincubating whole-cell extract with 3 μL of pan-STAT5 antiserum that recognizes both STAT5a and STAT5b. Electrophoresis was carried out at room temperature using 5% or 6% polyacrylamide gels (Heidt, et al. 2010).
Domain of Applicability
STAT5 is expressed in hematopoietic cells, such as T and B cells from humans, rodents, and other mammalian species (Gilmour, et al. 1995).
Beadling C, Guschin D, Witthuhn BA, Ziemiecki A, Ihle JN, Kerr IM, Cantrell DA. 1994. Activation of JAK kinases and STAT proteins by interleukin-2 and interferon alpha, but not the T cell antigen receptor, in human T lymphocytes. EMBO J 13:5605-5615.
Darnell JE, Jr. 1997. STATs and gene regulation. Science 277:1630-1635.
Decker T, Kovarik P, Meinke A. 1997. GAS elements: a few nucleotides with a major impact on cytokine-induced gene expression. J Interferon Cytokine Res 17:121-134. DOI: 10.1089/jir.1997.17.121.
Gaffen SL, Lai SY, Xu W, Gouilleux F, Groner B, Goldsmith MA, Greene WC. 1995. Signaling through the interleukin 2 receptor beta chain activates a STAT-5-like DNA-binding activity. Proc Natl Acad Sci U S A 92:7192-7196.
Gilmour KC, Pine R, Reich NC. 1995. Interleukin 2 activates STAT5 transcription factor (mammary gland factor) and specific gene expression in T lymphocytes. Proc Natl Acad Sci U S A 92:10772-10776. DOI: 10.1073/pnas.92.23.10772.
Heidt S, Roelen DL, Eijsink C, Eikmans M, van Kooten C, Claas FH, Mulder A. 2010. Calcineurin inhibitors affect B cell antibody responses indirectly by interfering with T cell help. Clin Exp Immunol 159:199-207. DOI: 10.1111/j.1365-2249.2009.04051.x.
Ihle JN. 1996. STATs: signal transducers and activators of transcription. Cell 84:331-334.
Johnston JA, Bacon CM, Finbloom DS, Rees RC, Kaplan D, Shibuya K, Ortaldo JR, Gupta S, Chen YQ, Giri JD, et al. 1995. Tyrosine phosphorylation and activation of STAT5, STAT3, and Janus kinases by interleukins 2 and 15. Proc Natl Acad Sci U S A 92:8705-8709.
Leonard WJ, O'Shea JJ. 1998. Jaks and STATs: biological implications. Annu Rev Immunol 16:293-322. DOI: 10.1146/annurev.immunol.16.1.293.
Liu J, Yoshida Y, Kunugita N, Noguchi J, Sugiura T, Ding N, Arashidani K, Fujimaki H, Yamashita U. 2010. Thymocytes are activated by toluene inhalation through the transcription factors NF-kappaB, STAT5 and NF-AT. J Appl Toxicol 30:656-660. DOI: 10.1002/jat.1536.
Maeshima K, Yamaoka K, Kubo S, Nakano K, Iwata S, Saito K, Ohishi M, Miyahara H, Tanaka S, Ishii K, Yoshimatsu H, Tanaka Y. 2012. The JAK inhibitor tofacitinib regulates synovitis through inhibition of interferon-gamma and interleukin-17 production by human CD4+ T cells. Arthritis Rheum 64:1790-1798. DOI: 10.1002/art.34329.
Muller J, Sperl B, Reindl W, Kiessling A, Berg T. 2008. Discovery of chromone-based inhibitors of the transcription factor STAT5. Chembiochem 9:723-727. DOI: 10.1002/cbic.200700701.