A PP4 Holoenzyme Balances Physiological and Oncogenic Nuclear Factor-Kappa B Signaling in T Lymphocytes

Division of Immunogenetics, Tumor Immunology Program, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
Immunity (Impact Factor: 21.56). 10/2012; 37(4):697-708. DOI: 10.1016/j.immuni.2012.07.014
Source: PubMed


Signal transduction to nuclear factor-kappa B (NF-κB) involves multiple kinases and phosphorylated target proteins, but little is known about signal termination by dephosphorylation. By RNAi screening, we have identified protein phosphatase 4 regulatory subunit 1 (PP4R1) as a negative regulator of NF-κB activity in T lymphocytes. PP4R1 formed part of a distinct PP4 holoenzyme and bridged the inhibitor of NF-κB kinase (IKK) complex and the phosphatase PP4c, thereby directing PP4c activity to dephosphorylate and inactivate the IKK complex. PP4R1 expression was triggered upon activation and proliferation of primary human T lymphocytes and deficiency for PP4R1 caused sustained and increased IKK activity, T cell hyperactivation, and aberrant NF-κB signaling in NF-κB-addicted T cell lymphomas. Collectively, our results unravel PP4R1 as a previously unknown activation-associated negative regulator of IKK activity in lymphocytes whose downregulation promotes oncogenic NF-κB signaling in a subgroup of T cell lymphomas.

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    • "In C. elegans, the PP4 holoenzyme is required for centrosome maturation during mitosis and sperm meiosis [1]. In mammals, PP4 participates in a number of processes essential for normal cellular physiology, including microtubule organization [2], [3], homologous recombination (HR)-mediated DNA repair [4], [5], the DNA damage response [6]–[10], histone modification [11], apoptosis [12], [13], pre-TCR signaling [14], TNF signaling [15], [16], Toll-like receptor (TLR)-4 signaling [17], and NF-κB regulation [18]–[20]. Germline deletion of PP4 in mice is embryonic lethal [14], and conditional deletion of PP4 specifically in murine T cells severely impairs T cell development [14]. We previously ablated PP4 specifically in developing B cells using the mb-1/cre/loxP system and generated mb-1/cre/PP4F/F mice [21]. "
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    ABSTRACT: PP4 is a serine/threonine phosphatase required for immunoglobulin (Ig) VDJ recombination and pro-B/pre-B cell development in mice. To elucidate the role of PP4 in mature B cells, we ablated the catalytic subunit of murine PP4 in vivo utilizing the CD23 promoter and cre-loxP recombination and generated CD23crePP4F/F mice. The development of follicular and marginal zone B cells was unaffected in these mutants, but the proliferation of mature PP4-deficient B cells stimulated by in vitro treatment with either anti-IgM antibody (Ab) or LPS was partially impaired. Interestingly, the induction of CD80 and CD86 expression on these stimulated B cells was normal. Basal levels of serum Igs of all isotypes were strongly reduced in CD23crePP4F/F mice, and their B cells showed a reduced efficiency of class switch recombination (CSR) in vitro upon stimulation by LPS or LPS plus IL-4. When CD23crePP4F/F mice were challenged with either the T cell-dependent antigen TNP-KLH or the T cell-independent antigen TNP-Ficoll, or by H1N1 virus infection, the mutant animals failed to form germinal centers (GCs) in the spleen and the draining mediastinal lymph nodes, and did not efficiently mount antigen-specific humoral responses. In the resting state, PP4-deficient B cells exhibited pre-existing DNA fragmentation. Upon stimulation by DNA-damaging drug etoposide in vitro, mutant B cells showed increased cleavage of caspase 3. In addition, the mutant B cells displayed impaired CD40-mediated MAPK activation, abnormal IgM-mediated NF-κB activation, and reduced S phase entry upon IgM/CD40-stimulation. Taken together, our results establish a novel role for PP4 in CSR, and reveal crucial functions for PP4 in the maintenance of genomic stability, GC formation, and B cell-mediated immune responses.
    PLoS ONE 09/2014; 9(9):e107505. DOI:10.1371/journal.pone.0107505 · 3.23 Impact Factor
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    • "Alternatively, recent reports suggest that Treg cells require proper TCR activation to achieve optimal differentiation and homeostasis [35,36]. Since PP4 is shown to be involved in TCR signaling [2] and NFκB activation [37], altered TCR activation may also contribute to the Treg defects in the CD4cre:PP4f/f mice. We are currently investigating these possibilities. "
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    ABSTRACT: Background Protein phosphates 4 (PP4), encoded by the ppp4c gene, is a ubiquitously expressed phosphatase that has been implicated in the regulation of cytokine signaling and lymphocyte survival; recent reports suggest that PP4 may be involved in pre-TCR signaling and B cell development. However, whether PP4 also modulates the functions of peripheral T cells has not been investigated due to the lack of a suitable in vivo model. Treg cells are a specialized subset of CD4 helper T cells that can suppress the proliferation of activated effector T cells. In the absence of this negative regulation, autoimmune syndromes and inflammatory diseases, such as human Crohn’s disease, will arise. Results In this report, we generated mice with T cell-specific ablation of the ppp4c gene (CD4cre:PP4f/f) and a Foxp3-GFP reporter gene to examine the roles of PP4 in Treg development and function. Characterizations of the CD4cre:PP4f/f mice showed that PP4 deficiency induced partial αβ T lymphopenia and T cell hypo-proliferation. Further analyses revealed significant reductions in the numbers of thymic and peripheral Treg cells, as well as in the efficiency of in vitro Treg polarization. In addition, PP4-deficient Treg cells exhibited reduced suppressor functions that were associated with decreased IL-10, CTLA4, GITR and CD103 expression. More interestingly, the CD4cre:PP4f/f mice developed spontaneous rectal prolapse and colitis with symptoms similar to human Crohn’s disease. The pathogenesis of colitis required the presence of commensal bacteria, and was correlated with reduced Treg cells in the gut. Nevertheless, PP4-deficient Treg cells were still capable of suppressing experimental colitis, suggesting that multiple factors contributed to the onset of the spontaneous colitis. Conclusions While the molecular mechanisms remain to be investigated, our results clearly show that PP4 plays a non-redundant role for the differentiation, suppressor activity and gut homeostasis of Treg cells. The onset of spontaneous colitis in the CD4cre:PP4f/f mice further suggests that PP4 is essential for the maintenance of protective gut immunity. The CD4cre:PP4f/f mice thus may serve as a good model for studying the interactions between Treg cells and gut commensal bacteria for the regulation of mucosal immunity.
    Cell and Bioscience 05/2014; 4(1):25. DOI:10.1186/2045-3701-4-25 · 3.63 Impact Factor
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    • "PP4 is also necessary for DNA repair via the homologous recombination pathway through dephosphorylation of the RPA2 subunit of replication protein A [22], and through dephosphorylation of γH2AX during cell division [18], [23], [24]. Lastly, PP4 has been implicated in multiple signal transduction pathways, including pre-TCR/TCR signaling [25], TNF-α signaling [26], [27], Toll-like receptor 4 signaling [28], and NF-κB signaling [29], [30]. "
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    ABSTRACT: PP4 phosphatase regulates a number of crucial processes but the role of PP4 in B cells has never been reported. We generated B cell-specific pp4 knockout mice and have identified an essential role for PP4 in B cell development. Deficiency of PP4 in B lineage cells leads to a strong reduction in pre-B cell numbers, an absence in immature B cells, and a complete loss of mature B cells. In PP4-deficient pro-B cells, immunoglobulin (Ig) DJH recombination is impaired and Ig µ heavy chain expression is greatly decreased. In addition, PP4-deficient pro-B cells show an increase of DNA double-strand breaks at Ig loci. Consistent with their reduced numbers, residual PP4-deficient pre-B cells accumulate in the G1 phase, exhibit excessive DNA damage, and undergo increased apoptosis. Overexpression of transgenic Ig in PP4-deficient mice rescues the defect in B cell development such that the animals have normal numbers of IgM(+) B cells. Our study therefore reveals a novel function for PP4 in pro-B cell development through its promotion of VHDJH recombination.
    PLoS ONE 07/2013; 8(7):e68804. DOI:10.1371/journal.pone.0068804 · 3.23 Impact Factor
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