Friederike Berberich-Siebelt

The role of NF-AT transcription factors in T cell activation and differentiation.

The family of genuine NF-AT transcription factors consists of four members (NF-AT1 [or NF-ATp], NF-AT2 [or NF-ATc], NF-AT3 and NF-AT4 [or NF-ATx]) which are characterized by a highly conserved DNA binding domain (is designated as Rel similarity domain) and a calcineurin binding domain. The binding of the Ca(2+)-dependent phosphatase calcineurin to this region controls the nuclear import and exit of NF-ATs. This review deals (1) with the structure of NF-AT proteins, (2) the DNA binding of NF-AT factors and their interaction with AP-1, (3) NF-AT target genes, (4) signalling pathways leading to NF-AT activation: the role of protein kinases and calcineurin, (5) the nuclear entry and exit of NF-AT factors, (6) transcriptional transactivation by NF-AT factors, (7) the structure and expression of the chromosomal NF-AT2 gene, and (8) NF-AT factors in Th cell differentiation. The experimental data presented and discussed in the review show that NF-AT factors are major players in the control of T cell activation and differentiation and, in all likelihood, also of the cell cycle and apoptosis of T lymphocytes.

Apoptosis suppression by Raf-1 and MEK1 requires MEK- and phosphatidylinositol 3-kinase-dependent signals.

ABSTRACT Two Ras effector pathways leading to the activation of Raf-1 and phosphatidylinositol 3-kinase (PI3K) have been implicated in the survival signaling by the interleukin 3 (IL-3) receptor. Analysis of apoptosis suppression by Raf-1 demonstrated the requirement for mitochondrial translocation of the kinase in this process. This could be achieved either by overexpression of the antiapoptotic protein Bcl-2 or by targeting Raf-1 to the mitochondria via fusion to the mitochondrial protein Mas p70. Mitochondrially active Raf-1 is unable to activate extracellular signal-related kinase 1 (ERK1) and ERK2 but suppresses cell death by inactivating the proapoptotic Bcl-2 family member BAD. However, genetic and biochemical data also have suggested a role for the Raf-1 effector module MEK-ERK in apoptosis suppression. We thus tested for MEK requirement in cell survival signaling using the interleukin 3 (IL-3)-dependent cell line 32D. MEK is essential for survival and growth in the presence of IL-3. Upon growth factor withdrawal the expression of constitutively active MEK1 mutants significantly delays the onset of apoptosis, whereas the presence of a dominant negative mutant accelerates cell death. Survival signaling by MEK most likely results from the activation of ERKs since expression of a constitutively active form of ERK2 was as effective in protecting NIH 3T3 fibroblasts against doxorubicin-induced cell death as oncogenic MEK. The survival effect of activated MEK in 32D cells is achieved by both MEK- and PI3K-dependent mechanisms and results in the activation of PI3K and in the phosphorylation of AKT. MEK and PI3K dependence is also observed in 32D cells protected from apoptosis by oncogenic Raf-1. Additionally, we also could extend these findings to the IL-3-dependent pro-B-cell line BaF3, suggesting that recruitment of MEK is a common mechanism for survival signaling by activated Raf. Requirement for the PI3K effector AKT in this process is further demonstrated by the inhibitory effect of a dominant negative AKT mutant on Raf-1-induced cell survival. Moreover, a constitutively active form of AKT synergizes with Raf-1 in apoptosis suppression. In summary these data strongly suggest a Raf effector pathway for cell survival that is mediated by MEK and AKT.

Autoregulation of NFATc1/A Expression Facilitates Effector T Cells to Escape from Rapid Apoptosis

Threshold levels of individual NFAT factors appear to be critical for apoptosis induction in effector T cells. In these cells, the short isoform A of NFATc1 is induced to high levels due to the autoregulation of the NFATc1 promoter P1 by NFATs. P1 is located within a CpG island in front of exon 1, represents a DNase I hypersensitive chromatin site, and harbors several sites for binding of inducible transcription factors, including a tandemly arranged NFAT site. A second promoter, P2, before exon 2, is not controlled by NFATs and directs synthesis of the longer NFATc1/B+C isoforms. Contrary to other NFATs, NFATc1/A is unable to promote apoptosis, suggesting that NFATc1/A enhances effector functions without promoting apoptosis of effector T cells.

A1 expression is stimulated by CD40 in B cells and rescues WEHI 231 cells from anti-IgM-induced cell death

Engagement of the antigen receptor on murine immature B cells leads to growth arrest followed by apoptosis. Concomitant signaling through CD40 sustains proliferation and rescues the cells from apoptosis. We show here that cross‐linking CD40 stimulates the expression of A1, a member of the anti‐apoptotic Bcl‐2 family, in primary murine B lymphocytes. CD40‐dependent stimulation of A1 was confirmed in WEHI 231 cells, an immature murine B cell lymphoma line. We transduced WEHI 231 cells with a bicistronic recombinant retroviral vector coding for A1 and a chimeric selection marker comprising the enhanced yellow fluorescent protein and the zeocin resistance protein. A1‐transduced WEHI 231 cells showed a significant higher survival rate after engagement of the antigen receptor. In contrast, constitutive expression of A1 did not abrogate anti‐IgM‐induced c‐myc down‐regulation. Consistant with this, A1 did not release anti‐IgM‐induced cell cycle arrest. Our data indicate that CD40‐stimulated A1 expression permits WEHI 231 cells to survive in the presence of anti‐IgM antibodies and suggests a protective role for A1 in antigen receptor‐mediated apoptosis in B cells.

Follicular regulatory T cells control humoral autoimmunity via NFAT2-regulated CXCR5 expression

T cell–specific NFAT2 deletion results in reduced CXCR5+ follicular regulatory T cells, leading to uncontrolled germinal center responses and humoral autoimmunity.

Dependence on nuclear factor of activated T-cells (NFAT) levels discriminates conventional T cells from Foxp3+ regulatory T cells.

Several lines of evidence suggest nuclear factor of activated T-cells (NFAT) to control regulatory T cells: thymus-derived naturally occurring regulatory T cells (nTreg) depend on calcium signals, the Foxp3 gene harbors several NFAT binding sites, and the Foxp3 (Fork head box P3) protein interacts with NFAT. Therefore, we investigated the impact of NFAT on Foxp3 expression. Indeed, the generation of peripherally induced Treg (iTreg) by TGF-β was highly dependent on NFAT expression because the ability of CD4+ T cells to differentiate into iTreg diminished markedly with the number of NFAT family members missing. It can be concluded that the expression of Foxp3 in TGF-β–induced iTreg depends on the threshold value of NFAT rather than on an individual member present. This is specific for iTreg development, because frequency of nTreg remained unaltered in mice lacking NFAT1, NFAT2, or NFAT4 alone or in combination. Different from expectation, however, the function of both nTreg and iTreg was independent on robust NFAT levels, reflected by less nuclear NFAT in nTreg and iTreg. Accordingly, absence of one or two NFAT members did not alter suppressor activity in vitro or during colitis and transplantation in vivo. This scenario emphasizes an inhibition of high NFAT activity as treatment for autoimmune diseases and in transplantation, selectively targeting the proinflammatory conventional T cells, while keeping Treg functional.

NFATc1 affects mouse splenic B cell function by controlling the calcineurin--NFAT signaling network.

Mouse B cells lacking NFATc1 exhibit defective proliferation, survival, isotype class switching, cytokine production, and T cell help.

Signal-specific and phosphorylation-dependent RelB degradation: a potential mechanism of NF-κB control

RelB is an unusual member of the Rel/NF-κB family of transcription factors which are involved in oncogenic processes. Due to a relaxed control by the IκBs, the cytosolic NF-κB inhibitors, RelB is constitutively expressed in the nuclei of lymphoid cells. We show here that RelB is inducibly degraded upon activation of T cells in a fashion similar to the IκBs. However, RelB degradation differs from that of IκBs since it is not induced by TNFα but only by T cell receptor or TPA/ionomycin stimulation. Moreover, RelB degradation occurs in three steps: (i) after stimulation RelB is rapidly phosphorylated at amino acids Thr84 and Ser552 followed by (ii) an N-terminal cut and, finally, (iii) the complete degradation in the proteasomes. Since mutation of the two phosphoacceptor sites to non-acceptor sites abolished RelB phosphorylation in vivo and led to the stabilization of the mutated RelBDM, site-specific phosphorylation appears to be a necessary prerequisite for RelB degradation. RelB is a crucial regulator of NF-κB-dependent gene expression. Thus, the signal-induced degradation of RelB should be an important control mechanism of NF-κB activity.

Sumoylation of the Transcription Factor NFATc1 Leads to Its Subnuclear Relocalization and Interleukin-2 Repression by Histone Deacetylase

The family of NFAT (nuclear factor of activated T-cells) transcription factors plays an important role in cytokine gene regulation. In peripheral T-cells NFATc1 and -c2 are predominantly expressed. Because of different promoter and poly(A) site usage as well as alternative splicing events, NFATc1 is synthesized in multiple isoforms. The highly inducible NFATc1/A contains a relatively short C terminus, whereas the longer, constitutively expressed isoform NFATc1/C spans an extra C-terminal peptide of 246 amino acids. Interestingly, this NFATc1/C-specific terminus can be highly sumoylated. Upon sumoylation, NFATc1/C, but not the unsumoylated NFATc1/A, translocates to promyelocytic leukemia nuclear bodies. This leads to interaction with histone deacetylases followed by deacetylation of histones, which in turn induces transcriptionally inactive chromatin. As a consequence, expression of the NFATc1 target gene interleukin-2 is suppressed. These findings demonstrate that the modification by SUMO (small ubiquitin-like modifier) converts NFATc1 from an activator to a site-specific transcriptional repressor, revealing a novel regulatory mechanism for NFATc1 function.

Steady state migratory RelB+ langerin+ dermal dendritic cells mediate peripheral induction of antigen-specific CD4+CD25+Foxp3+ regulatory T cells

Tolerance to self‐antigens expressed in peripheral organs is maintained by CD4+ CD25+ Foxp3+ Treg cells, which are generated as a result of thymic selection or peripheral induction. Here, we demonstrate that steady‐state migratory DCs from the skin mediated Treg conversion in draining lymph nodes of mice. These DCs displayed a partially mature MHC IIint CD86int CD40hi CCR7+ phenotype, used endogenous TGF‐β for conversion and showed nuclear RelB translocation. Deficiency of the alternative NF‐κB signaling pathway (RelB/p52) reduced steady‐state migration of DCs. These DCs transported and directly presented soluble OVA provided by s.c. implanted osmotic minipumps, as well as cell‐associated epidermal OVA in transgenic K5‐mOVA mice to CD4+ OVA‐specific TCR‐transgenic OT‐II T cells. The langerin+ dermal DC subset, but not epidermal Langerhans cells, mediated conversion of naive OT‐II×RAG‐1−/− T cells into proliferating CD4+ CD25+ Foxp3+ Tregs. Thus, our data suggest that steady‐state migratory RelB+ TGF‐β+ langerin+ dermal DCs mediate peripheral Treg conversion in response to epidermal antigen in skin‐draining lymph nodes.