To the extent possible under law, AOP-Wiki has waived all copyright and related or neighboring rights to KER:2026
Suppression of STAT5 binding to cytokine gene promoters leads to Suppression of IL-4 production
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding||Point of Contact||Author Status||OECD Status|
|Inhibition of JAK3 leading to impairment of T-Cell Dependent Antibody Response||adjacent||High||High||Brendan Ferreri-Hanberry (send email)||Under development: Not open for comment. Do not cite||Under Development|
Life Stage Applicability
|All life stages||High|
Key Event Relationship Description
A STAT5 binding site (TTCATGGAA) has been identified in intron 2 of the Il4 gene, near HSII (Hural, et al. 2000). Another potential STAT5 binding site (TTCTAAGAA) is conserved between mice and humans, and is located near HSIII. STAT5A binds to the sites near HSII and HSIII, which could provide a mechanism through which STAT5A mediates Il4 gene accessibility and participates in the induction of IL-4 production. Enhanced STAT5 signaling results in a larger proportion of cells producing IL-4. A consensus STAT site that preferentially associates with STAT5 contributes to its enhancer activity in mast cells. The intron element plays a role in acquiring and/or maintaining the IL-4 gene locus in a demethylated state in IL-4-producing cells.
Constitutively active STAT5A (STAT5A1*6) restores the capacity to produce IL-4 in cells primed under Th2 conditions in the absence of IL-2, suggesting that STAT5 activation plays a critical role in Th2 differentiation (Zhu, et al. 2003, Zhu, et al. 2004). Additionally, IL-2 critically regulates Th2 differentiation in a STAT5-dependent manner, acting early at the locus encoding IL-4Ra to induce expression of this receptor (IL-4Rα) (Liao, et al. 2008) and later to open chromatin accessibility at the Th2 locus, which encodes IL-4 and IL-13 (Cote-Sierra, et al. 2004).
The development of Th2 cells was reportedly impaired in STAT5a-/-CD4+ T cells, even in the presence of IL-4. Retrovirus-mediated expression of STAT5A restored Th2 cell differentiation in STAT5a-/-CD4+ T cells. Th2 cell-mediated immune responses were diminished in STAT5a-/- mice. When stimulated with anti-CD3 mAb, CD4+ T cells that produced IL-4, but not IFN-γ (Th2 cells), were significantly decreased in STAT5a-/- mice compared with those in wild-type mice, suggesting that STAT5A plays a regulatory role in T helper cell differentiation (Kagami, et al. 2001).
Evidence Supporting this KER
IL-2 stabilizes the accessibility of the Il4 gene. STAT5, a key transducer of IL-2 function, binds to sites in the second intron of the Il4 gene (Cote-Sierra, et al. 2004).
5C.C7 cells infected with a retrovirus expressing a constitutively active form of STAT5A (STAT5A1*6) were shown to be primed for IL-4 production.
STAT5a/b mutant peripheral T cells in mice are profoundly deficient in proliferation and fail to undergo cell cycle progression or to express genes controlling cell cycle progression. STAT5 proteins are essential mediators of IL-2 signaling in T cells (Willerford, et al. 1995).
IL-2 is one of the earliest cytokines produced by activated T cells and mediates its actions primarily through the activation of STAT5 proteins. A STAT5-chromatin immunoprecipitation assay (ChIP) was performed using chromatin from freshly isolated CD4 T cells to identify in vivo IL-2-activated STAT5 gene targets. The immunoprecipitated chromatin yielded a number of distinct clones based on sequencing. One clone mapped to chromosome 16 152,916 to 153,096 upstream of the C-MAF gene, and contained a consensus GAS motif (Rani, et al. 2011).
Heat map analysis of expression profiles of IL-2 regulated genes (sorted by superenhancer binding scores for STAT5, from strongest to weakest) revealed that STAT5-bound superenhancer-containing genes were highly induced by IL-2 (Li, et al. 2018).
Cells primed under Th2, but not Th1, conditions showed an association of STAT5A with HSII and HSIII. In addition, cells infected with the STAT5A1*6 retrovirus acquired IL-4-producing capacity, and STAT5 was associated with DNA elements near HSII and HSIII (Zhu, et al. 2003).
CD4+ T cell-mediated allergic inflammation was reportedly diminished in STAT5A-deficient (STAT5a-/-) mice. Furthermore, Th2 cell differentiation was also impaired in STAT5a-/- mice, even when purified CD4+ T cells were stimulated with anti-CD3 and anti-CD28 antibodies in the presence of IL-4 (Kagami, et al. 2001).
Th2 cell differentiation from antigen-stimulated splenocytes was significantly decreased in STAT5a-/- mice as compared with that in wild-type mice. The intrinsic expression of STAT5a in CD4+ T cells is required for Th2 cell differentiation and STAT5a is involved in the development of CD4+CD25+ immunoregulatory T cells that modulate T helper cell differentiation toward Th2 cells (Kagami, et al. 2001).
IL-4 production was reportedly induced by STAT5 phosphorylation. STAT5 phosphorylation facilitates STAT5 dimerization, transport to the nucleus, and gene regulation (56-Levy-2002). PPARs are members of the nuclear hormone receptor superfamily. STAT5 is able to inhibit PPAR-regulated gene transcription. Conversely, ligand-activated PPAR can inhibit STAT5-regulated transcription. STAT5 and PPAR disparate pathways are subject to mutually inhibitory crosstalk. The extent of the inhibitory crosstalk was dependent on the relative expression levels of each transcription factor (Shipley and Waxman 2004).
Uncertainties and Inconsistencies
GAS is a STAT3-target gene, therefore STAT3 could regulate IL-4 production (Campia, et al. 2015). Additionally, Lederer et al. demonstrated that STAT6 binds to a sequence in the IL-4 promoter (Lederer, et al. 1996).
Once STATs are recruited to the activated JAK/receptor complex and are tyrosine phosphorylated within the SH2 domain by JAKs, they form dimers and/or tetramers, translocate to the nucleus, and associate with promoter regions, such as gamma activated sequence (GAS) elements. STAT dimers can bind to GAS DNA sequences (TTCN3GAA) to induce transcription. The STAT5 dimers can also form tetramers through interactions between residues (I28, F81, and L82) in their N-terminal regions. These STAT5 tetramers bind to pairs of GAS motifs separated by a linker of 6–22 nucleotides (Lin, et al. 2012). Mutational studies have demonstrated that STAT5 is important for IL-2-induced gene expression. The interaction of STATs with gene promoters can enhance the expression of its target genes (Able, et al. 2017).
It was reported that while the wild-type construct displayed 4.6-fold IL-2 inducibility in YT cells, selective mutation of GAScI (M1), GASn (M2), and GAScII (M3) motifs modestly lowered IL-2 inducibility (M1 1.7-fold, M2 2.9-fold, M3 1.6-fold, respectively). Double mutation of GAScI and GASn (M4) or GASn and GAScII (M5) more potently decreased IL-2 inducibility, and simultaneous mutation of GAScI and GAScII (M6) or of all the GAS motifs (M7) abrogated IL-2 inducibility (M4 1.2-fold, M5 1.4-fold, M6 1.0-fold, M7 1.0-fold, respectively). These results suggest that all the GAS motifs are required for maximal IL-2 inducibility, including IL-4 induction (Kim, et al. 2001).
A STAT5 binding site (TTCATGGAA) has been identified in intron 2 of the Il4 gene. HS V (also known as CNS2) is a 3’ enhancer in the Il4 locus. HS V is essential for IL-4 production by Tfh cells. Mice lacking HS V display marked defects in Th2 humoral immune responses, as evidenced by abrogated IgE and sharply reduced IgG1 production in vivo. HS V-deficient (ΔV) mice displayed complete abrogation of IgE production despite only mild reduction in Th2 responses. HS V-deficiency affected Il4 transcription in T cells naïve T cells lacking the HS V (CNS2) region were completely unable to produce Il4 transcripts following ex vivo stimulation with anti-CD3 and anti-CD28 antibodies for 180 min. In a similar time course assay (240 min), in vitro differentiated Th2 cells stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin showed only a 50% reduction in Il4 transcription (Vijayanand, et al. 2012).
Phosphorylation of STAT5 was reportedly decreased by nearly two-fold in NOX2-deficient T cells as compared to that in wild-type controls by intracellular staining 12 and 24 h after activation with immobilized anti-CD3 and soluble anti-CD28. PCR analysis also revealed decreases in Il4 and Il4rα mRNA expression in NOX2-deficient T cells (Shatynski, et al. 2012).
Known modulating factors
Adenosine can inhibit IL-2-dependent proliferation of CTLL-2 T cells. This inhibition was reportedly associated with a reduction in tyrosine phosphorylation of STAT5A and STAT5B, which was mediated by the activation of a protein tyrosine phosphatase (PTP). The PTP Src homology region 2 domain-containing phosphatase-2 (SHP-2) was implicated in STAT5A/B dephosphorylation because adenosine strongly increased tyrosine phosphorylation of SHP-2 and the formation of complexes consisting of SHP-2 and STAT5 in IL-2- stimulated CTLL-2 T cells. In contrast, adenosine did not affect the phosphorylation status of the upstream kinases JAK1 or JAK3. The inhibitory effect of adenosine on STAT5A/B phosphorylation was mediated through cell surface A2a and A2b receptors, and involved associated cAMP/protein kinase A (PKA)-dependent signaling pathways (Zhang, et al. 2004).
Known Feedforward/Feedback loops influencing this KER
STAT5 can upregulate a number of molecules, including cytokine-inducible SH2 proteins (CIS family, also referred to as the SOCS or SSI family) (Yasukawa, et al. 2000). Some CIS family proteins might be involved in the cross-regulation of cytokine networks and may regulate Th1 and Th2 cell differentiation (Dickensheets, et al. 1999, Losman, et al. 1999). CIS1, a prototype of CIS family proteins, is induced by STAT5 and inhibits STAT5 activation by blocking the interaction between STAT5 and cytokine receptors (Yasukawa, et al. 2000). Thus, CIS1 seems to function in classical negative feedback of STAT5 signaling.
IL-2 acts on the same cell that secretes the cytokine. For instance, IL-2 produced by T cells operates on the same T cells that make this cytokine or on nearby cells. With the highest levels in secondary lymphoid organs, IL-2 is believed to act in an autocrine or paracrine manner to support effector and memory CD8 T cell differentiation (Kalia and Sarkar 2018). IL-2Rα expression is triggered by antigens, mitogen lectins, or antibodies to the TCR through STAT5. These signals also result in the secretion of IL-2, which in turn can increase and prolong IL-2Rα expression, thus acting as a positive feedback regulator of its own high-affinity receptor (Waldmann 1989). Therefore, STAT5 deficiency disrupted T cell function.
Domain of Applicability
Able AA, Burrell JA, Stephens JM. 2017. STAT5-Interacting Proteins: A Synopsis of Proteins that Regulate STAT5 Activity. Biology (Basel) 6. DOI: 10.3390/biology6010020.
Adamson AS, Collins K, Laurence A, O'Shea JJ. 2009. The Current STATus of lymphocyte signaling: new roles for old players. Curr Opin Immunol 21:161-166. DOI: 10.1016/j.coi.2009.03.013.
Ben-Sasson SZ, Le Gros G, Conrad DH, Finkelman FD, Paul WE. 1990. IL-4 production by T cells from naive donors. IL-2 is required for IL-4 production. J Immunol 145:1127-1136.
Campia I, Buondonno I, Castella B, Rolando B, Kopecka J, Gazzano E, Ghigo D, Riganti C. 2015. An Autocrine Cytokine/JAK/STAT-Signaling Induces Kynurenine Synthesis in Multidrug Resistant Human Cancer Cells. PLoS One 10:e0126159. DOI: 10.1371/journal.pone.0126159
Cote-Sierra J, Foucras G, Guo L, Chiodetti L, Young HA, Hu-Li J, Zhu J, Paul WE. 2004. Interleukin 2 plays a central role in Th2 differentiation. Proc Natl Acad Sci U S A 101:3880-3885. DOI: 10.1073/pnas.0400339101.
Dickensheets HL, Venkataraman C, Schindler U, Donnelly RP. 1999. Interferons inhibit activation of STAT6 by interleukin 4 in human monocytes by inducing SOCS-1 gene expression. Proc Natl Acad Sci U S A 96:10800-10805. DOI: 10.1073/pnas.96.19.10800.
Fang X, Zhang L, Feng Y, Zhao Y, Dai J. 2008. Immunotoxic effects of perfluorononanoic acid on BALB/c mice. Toxicol Sci 105:312-321. DOI: 10.1093/toxsci/kfn127.
He X, Smeets RL, Koenen HJ, Vink PM, Wagenaars J, Boots AM, Joosten I. 2011. Mycophenolic acid-mediated suppression of human CD4+ T cells: more than mere guanine nucleotide deprivation. Am J Transplant 11:439-449. DOI: 10.1111/j.1600-6143.2010.03413.x.
Hural JA, Kwan M, Henkel G, Hock MB, Brown MA. 2000. An intron transcriptional enhancer element regulates IL-4 gene locus accessibility in mast cells. J Immunol 165:3239-3249. DOI: 10.4049/jimmunol.165.6.3239.
Kagami S, Nakajima H, Suto A, Hirose K, Suzuki K, Morita S, Kato I, Saito Y, Kitamura T, Iwamoto I. 2001. Stat5a regulates T helper cell differentiation by several distinct mechanisms. Blood 97:2358-2365. DOI: 10.1182/blood.v97.8.2358.
Kalia V, Sarkar S. 2018. Regulation of Effector and Memory CD8 T Cell Differentiation by IL-2-A Balancing Act. Front Immunol 9:2987. DOI: 10.3389/fimmu.2018.02987.
Kim HP, Kelly J, Leonard WJ. 2001. The basis for IL-2-induced IL-2 receptor alpha chain gene regulation: importance of two widely separated IL-2 response elements. Immunity 15:159-172. DOI: 10.1016/s1074-7613(01)00167-4.
Lederer JA, Perez VL, DesRoches L, Kim SM, Abbas AK, Lichtman AH. 1996. Cytokine transcriptional events during helper T cell subset differentiation. J Exp Med 184:397-406. DOI: 10.1084/jem.184.2.397.
Li Y, Liu X, Wang W, Wang S, Zhang J, Jiang S, Wang Y, Li L, Li J, Zhang Y, Huang H. 2018. Low-dose IL-2 expands CD4(+) regulatory T cells with a suppressive function in vitro via the STAT5-dependent pathway in patients with chronic kidney diseases. Ren Fail 40:280-288. DOI: 10.1080/0886022X.2018.1456462.
Liao W, Schones DE, Oh J, Cui Y, Cui K, Roh TY, Zhao K, Leonard WJ. 2008. Priming for T helper type 2 differentiation by interleukin 2-mediated induction of interleukin 4 receptor alpha-chain expression. Nat Immunol 9:1288-1296. DOI: 10.1038/ni.1656.
Lin JX, Li P, Liu D, Jin HT, He J, Ata Ur Rasheed M, Rochman Y, Wang L, Cui K, Liu C, Kelsall BL, Ahmed R, Leonard WJ. 2012. Critical Role of STAT5 transcription factor tetramerization for cytokine responses and normal immune function. Immunity 36:586-599. DOI: 10.1016/j.immuni.2012.02.017.
Liu Y, Yang T, Li H, Li MH, Liu J, Wang YT, Yang SX, Zheng J, Luo XY, Lai Y, Yang P, Li LM, Zou Q. 2013. BD750, a benzothiazole derivative, inhibits T cell proliferation by affecting the JAK3/STAT5 signalling pathway. Br J Pharmacol 168:632-643. DOI: 10.1111/j.1476-5381.2012.02172.x.
Losman JA, Chen XP, Hilton D, Rothman P. 1999. Cutting edge: SOCS-1 is a potent inhibitor of IL-4 signal transduction. J Immunol 162:3770-3774.
McDyer JF, Li Z, John S, Yu X, Wu CY, Ragheb JA. 2002. IL-2 receptor blockade inhibits late, but not early, IFN-gamma and CD40 ligand expression in human T cells: disruption of both IL-12-dependent and -independent pathways of IFN-gamma production. J Immunol 169:2736-2746. DOI: 10.4049/jimmunol.169.5.2736.
Rani A, Afzali B, Kelly A, Tewolde-Berhan L, Hackett M, Kanhere AS, Pedroza-Pacheco I, Bowen H, Jurcevic S, Jenner RG, Cousins DJ, Ragheb JA, Lavender P, John S. 2011. IL-2 regulates expression of C-MAF in human CD4 T cells. J Immunol 187:3721-3729. DOI: 10.4049/jimmunol.1002354.
Shatynski KE, Chen H, Kwon J, Williams MS. 2012. Decreased STAT5 phosphorylation and GATA-3 expression in NOX2-deficient T cells: role in T helper development. Eur J Immunol 42:3202-3211. DOI: 10.1002/eji.201242659.
Shipley JM, Waxman DJ. 2004. Simultaneous, bidirectional inhibitory crosstalk between PPAR and STAT5b. Toxicol Appl Pharmacol 199:275-284. DOI: 10.1016/j.taap.2003.12.020.
Vijayanand P, Seumois G, Simpson LJ, Abdul-Wajid S, Baumjohann D, Panduro M, Huang X, Interlandi J, Djuretic IM, Brown DR, Sharpe AH, Rao A, Ansel KM. 2012. Interleukin-4 production by follicular helper T cells requires the conserved Il4 enhancer hypersensitivity site V. Immunity 36:175-187. DOI: 10.1016/j.immuni.2011.12.014.
Waldmann TA. 1989. The multi-subunit interleukin-2 receptor. Annu Rev Biochem 58:875-911. DOI: 10.1146/annurev.bi.58.070189.004303.
Willerford DM, Chen J, Ferry JA, Davidson L, Ma A, Alt FW. 1995. Interleukin-2 receptor alpha chain regulates the size and content of the peripheral lymphoid compartment. Immunity 3:521-530.
Yasukawa H, Sasaki A, Yoshimura A. 2000. Negative regulation of cytokine signaling pathways. Annu Rev Immunol 18:143-164. DOI: 10.1146/annurev.immunol.18.1.143.
Zhang H, Conrad DM, Butler JJ, Zhao C, Blay J, Hoskin DW. 2004. Adenosine acts through A2 receptors to inhibit IL-2-induced tyrosine phosphorylation of STAT5 in T lymphocytes: role of cyclic adenosine 3',5'-monophosphate and phosphatases. J Immunol 173:932-944. DOI: 10.4049/jimmunol.173.2.932.
Zhu J, Cote-Sierra J, Guo L, Paul WE. 2003. Stat5 activation plays a critical role in Th2 differentiation. Immunity 19:739-748. DOI: 10.1016/s1074-7613(03)00292-9.
Zhu J, Min B, Hu-Li J, Watson CJ, Grinberg A, Wang Q, Killeen N, Urban JF, Jr., Guo L, Paul WE. 2004. Conditional deletion of Gata3 shows its essential function in T(H)1-T(H)2 responses. Nat Immunol 5:1157-1165. DOI: 10.1038/ni1128.