Epithelial-to-mesenchymal transitions (EMTs) underlie cell plasticity required in embryonic development and frequently observed in advanced carcinogenesis. Transforming growth factor-b (TGF-b) induces EMT phenotypes in epithelial cells in vitro and has been associated with EMT in vivo. Here we report that expression of the hairy/enhancer-of-split-related transcriptional repressor Hey1, and the Notch-ligand Jagged1 (Jag1), was induced by TGF-b at the onset of EMT in epithelial cells from mammary gland, kidney tubules, and epidermis. The HEY1 expression profile was biphasic, consisting of immediate-early Smad3-dependent, Jagged1/Notch-independent activation, followed by delayed, indirect Jagged1/ Notch-dependent activation. TGF-b-induced EMT was blocked by RNA silencing of HEY1 or JAG1, and by chemical inactivation of Notch. The EMT phenotype, biphasic activation of Hey1, and delayed expression of Jag1 were induced by TGF-b in wild-type, but not in Smad3-deficient, primary mouse kidney tubular epithelial cells. Our findings identify a new mechanism for functional integration of Jagged1/Notch signalling and coordinated activation of the Hey1 transcriptional repressor controlled by TGF-b/ Smad3, and demonstrate functional roles for Smad3, Hey1, and Jagged1/Notch in mediating TGF-b-induced EMT.

In T cells anergy may be evoked by an unbalanced stimulation of the T-cell receptor in the absence of costimulation. Anergic T cells are unresponsive to new antigen receptor engagement and do not produce interleukin 2. We present evidence that anergizing stimuli induce changes in histone acetylation, which mediates transcriptional repression of interleukin 2 expression. In response to calcium signaling, anergic T cells up-regulate the expression of Ikaros, a zinc finger transcription factor essential for lymphoid lineage determination. Ikaros binds to the interleukin 2 promoter where it induces histone deacetylation. Confirming the role of Ikaros in the induction of T-cell anergy, cells with reduced Ikaros activity show defective inactivation in response to an anergizing stimulus. We propose a model in which tolerizing stimuli induce epigenetic changes on the interleukin 2 locus that are responsible for the stable inhibition of the expression of this cytokine in anergic T cells.

ADP-ribosylation is a reversible posttranslational modification mediated by poly-ADP-ribose polymerase (PARP). The results of recent studies demonstrate that ADP-ribosylation contributes to transcription regulation. Here, we report that transcription factor NFAT binds to and is ADP-ribosylated by PARP-1 in an activation-dependent manner. Mechanistically, ADP-ribosylation increases NFAT DNA binding. Functionally, NFAT-mediated interleukin-2 (IL-2) expression was reduced in T cells upon genetic ablation or pharmacological inhibition of PARP-1. Parp-1 ؊/؊ T cells also exhibit reduced expression of other NFAT-dependent cytokines, such as IL-4. Together, these results demonstrate that ADP-ribosylation mediated by PARP-1 provides a molecular switch to positively regulate NFAT-dependent cytokine gene transcription. These results also imply that, similar to the effect of calcineurin inhibition, PARP-1 inhibition may be beneficial in modulating immune functions.ADP-ribosylation is a reversible posttranslational modification that transfers ADP-ribose from NAD ϩ to Glu, Asp, and/or Arg amino acids of target proteins (18). Similar to ubiquitination, ADP-ribosylation modifies target proteins to various masses due to the assorted chain lengths of the ADPribose. ADP-ribosylation is inhibited by the NAD ϩ analog 3-aminobenzamide and, more specifically, by PJ-34 (45). Poly-ADP-ribose polymerase-1 (PARP-1) is a nuclear enzyme that accounts for the bulk of ADP-ribosylation in vivo (43). Indeed, only ϳ10% of PARP activity remains in Parp-1 Ϫ/Ϫ cells upon DNA damage. In addition to its role in DNA damage repair, the results of recent studies demonstrate that PARP-1 contributes to gene transcription regulation (26,40).Transcription factor NFAT is the master regulator of interleukin-2 (IL-2) gene transcription (24,42). In resting cells, NFAT resides in the cytosol. The nuclear accumulation of NFAT is regulated by calcineurin-mediated dephosphorylation (9, 15, 23). The immunosuppressant drugs cyclosporine A (CsA) and tacrolimus (FK506) inhibit calcineurin and abrogate NFAT activation. Indeed, understanding the mechanism of NFAT activation has contributed to the great advances in transplantation surgery (27). Given that immunosuppressant therapy using CsA or FK506 causes neuro-and nephrotoxicity (1, 19), further understanding of the molecular basis of NFAT activation will provide alternate therapeutic targets for the treatment of transplant patients.Once in the nucleus, NFAT interacts with coregulators to achieve optimal NFAT activation (11,21,28). These NFAT coregulators include Fos-Jun, C/EBPs, and Fox3p, which form a composite transcription complex to regulate NFAT-mediated gene transcription. In addition, transcription coactivator CREBbinding protein/p300 and class II histone deacetylases are recruited to modulate NFAT-mediated transcription (3,12,16,48). Here, we report that PARP-1 binds to and ADP-ribosylates NFAT. The ADP-ribosylation mediated by PARP-1 provides a molecular switch to positively regulate NFAT-dependent cytokine ge...

In T cells, anergy can be induced after T cell receptor engagement in the absence of costimulation. Under these conditions, the expression of a specific set of anergy-associated genes is activated. Several lines of evidence suggest that nuclear factor of activated T cells (NFAT) proteins may regulate the expression of many of those genes; however, the nature of the complexes responsible for the induction of this new program of gene expression is unknown. Here, we show that transcriptional complexes formed by NFAT homodimers are directly responsible for the activation of at least two anergy-inducing genes, Grail and Caspase3. Our data shows that Grail expression is activated by direct binding of NFAT dimers to the Grail promoter at two different sites. Consequently, a mutant NFAT protein with impaired ability to dimerize is not able to induce an unresponsive state in T cells. Our results not only identify a new biological function for NFAT dimers but also reveal the different nature of NFAT-containing complexes that induce anergy versus those that are activated during a productive immune response. These data also establish a basis for the design of immunomodulatory strategies that specifically target each type of complex.

Self-reactive T cells that escape negative selection in the thymus must be kept under control in the periphery. Mechanisms of peripheral tolerance include deletion or functional inactivation of self-reactive T cells and mechanisms of dominant tolerance mediated by regulatory T cells. In the absence of costimulation, T cell receptor (TCR) engagement results in unopposed calcium signaling that leads to the activation of a cell-intrinsic program of inactivation, which makes T cells hyporesponsive to subsequent stimulations. The activation of this program in anergic T cells is a consequence of the induction of a nuclear factor of activated T cells (NFAT)-dependent program of gene expression. Recent studies have offered new insights into the mechanisms responsible for the implementation and maintenance of T cell anergy and have provided evidence that the proteins encoded by the genes upregulated in anergic T cells are responsible for the implementation of anergy by interfering with TCR signaling or directly inhibiting cytokine gene transcription.