Bmi1 limits dilated cardiomyopathy and heart failure by inhibiting cardiac senescence.

Dilated cardiomyopathy (DCM) is the most frequent cause of heart failure and the leading indication for heart transplantation. Here we show that epigenetic regulator and central transcriptional instructor in adult stem cells, Bmi1, protects against DCM by repressing cardiac senescence. Cardiac-specific Bmi1 deletion induces the development of DCM, which progresses to lung congestion and heart failure. In contrast, Bmi1 overexpression in the heart protects from hypertrophic stimuli. Transcriptome analysis of mouse and human DCM samples indicates that p16(INK4a) derepression, accompanied by a senescence-associated secretory phenotype (SASP), is linked to severely impaired ventricular dimensions and contractility. Genetic reduction of p16(INK4a) levels reverses the pathology of Bmi1-deficient hearts. In parabiosis assays, the paracrine senescence response underlying the DCM phenotype does not transmit to healthy mice. As senescence is implicated in tissue repair and the loss of regenerative potential in aging tissues, these findings suggest a source for cardiac rejuvenation.

A role for the p38 MAP kinase pathway in the nuclear shuttling of NFATp.

Calcium signals lead to the translocation of nuclear factor of activated T cells (NFAT) from the cytoplasm to the nucleus. This process is regulated by the calcium-activated phosphatase calcineurin, which can be cotransported with NFAT to the nucleus to maintain it transcriptionally active for the duration of calcium signaling. When the calcium signal ceases, NFAT is exported to the cytoplasm, and different NFAT kinases have been reported to oppose calcineurin activities and regulate the nuclear export of NFAT. Here we show that p38 MAPK phosphorylates in vitro and interacts in vivo with NFATp. Furthermore, the activation of this pathway in HeLa cells by cotransfection with activated MKK6 and p38 counteracts the calcium-induced nuclear accumulation of NFATp but not that of NFATc. By contrast, activation of JNK or ERK pathways failed to modify the nuclear shuttling of NFATp. Consistently, activation of p38, but not the JNK MAPK pathway, results in the inhibition of NFATp-driven transcription. In addition, the inhibition of the nuclear accumulation of NFATp by p38 appears to be mediated through the activation of NFATp nuclear export and takes place in a Leptomycin B-sensitive fashion, suggesting the involvement of the exportin CRM1 in this process. Thus, the p38 signal transduction pathway appears to play an important role in the regulation of the nuclear shuttling of NFATp and in cellular homeostasis.

Pyrrolidine dithiocarbamate protects mice from lethal shock induced by LPS or TNF-alpha.

Although important advances have been made in the development of antibiotics and medical intensive care technology in recent years, systemic response to infection remains a major health problem, with growing incidence and high mortality rates. Here we demonstrate the ability of the antioxidant agent pyrrolidine dithiocarbamate (PDTC) to inhibit the in vivo activation of NF-kappaB in lung and liver tissues, as well as the systemic release of TNF-alpha in lipopolysaccharide (LPS)-treated mice. The in vivo effect of PDTC on NF-kappaB activation in liver tissues involved the inhibition of both LPS-induced I kappaB-alpha degradation and the translocation of the p50 and p65 NF-kappaB subunits to the nucleus. In addition to protecting mice against lethal LPS doses, PDTC curtailed TNF-alpha-induced lethal shock. This effect was observed even after LPS injection, and when PDTC was administered at a time when TNF-alpha was already at maximum levels in serum. PDTC-treated mice survived despite high IL-1beta and IL-6 levels, induction of VCAM-1 and ICAM-1 expression or leukocyte infiltration in tissues known to be associated with LPS-induced shock, indicating that PDTC does not act by modifying these responses. Taken together, these results indicate that PDTC interferes with the production as well as the action of TNF-alpha, and points to a possible approach toward the treatment of septic shock.

Vascular endothelial growth factor activates nuclear factor of activated T cells in human endothelial cells: a role for tissue factor gene expression.

Vascular endothelial growth factor (VEGF) is a potent angiogenic inducer that stimulates the expression of tissue factor (TF), the major cellular initiator of blood coagulation. Here we show that signaling triggered by VEGF induced DNA-binding and transcriptional activities of nuclear factor of activated T cells (NFAT) and AP-1 in human umbilical vein endothelial cells (HUVECs). VEGF also induced TF mRNA expression and gene promoter activation by a cyclosporin A (CsA)-sensitive mechanism. As in lymphoid cells, NFAT was dephosphorylated and translocated to the nucleus upon activation of HUVECs, and these processes were blocked by CsA. NFAT was involved in the VEGF-mediated TF promoter activation as evidenced by cotransfection experiments with a dominant negative version of NFAT and site-directed mutagenesis of a newly identified NFAT site within the TF promoter that overlaps with a previously identified kappaB-like site. Strikingly, this site bound exclusively NFAT not only from nuclear extracts of HUVECs activated by VEGF, a stimulus that failed to induce NF-kappaB-binding activity, but also from extracts of cells activated with phorbol esters and calcium ionophore, a combination of stimuli that triggered the simultaneous activation of NFAT and NF-kappaB. These results implicate NFAT in the regulation of endothelial genes by physiological means and shed light on the mechanisms that switch on the gene expression program induced by VEGF and those regulating TF gene expression.

Blockade of T-cell activation by dithiocarbamates involves novel mechanisms of inhibition of nuclear factor of activated T cells.

Dithiocarbamates (DTCs) have recently been reported as powerful inhibitors of NF-kappaB activation in a number of cell types. Given the role of this transcription factor in the regulation of gene expression in the inflammatory response, NF-kappaB inhibitors have been suggested as potential therapeutic drugs for inflammatory diseases. We show here that DTCs inhibited both interleukin 2 (IL-2) synthesis and membrane expression of antigens which are induced during T-cell activation. This inhibition, which occurred with a parallel activation of c-Jun transactivating functions and expression, was reflected by transfection experiments at the IL-2 promoter level, and involved not only the inhibition of NF-kappaB-driven reporter activation but also that of nuclear factor of activated T cells (NFAT). Accordingly, electrophoretic mobility shift assays (EMSAs) indicated that pyrrolidine DTC (PDTC) prevented NF-kappaB, and NFAT DNA-binding activity in T cells stimulated with either phorbol myristate acetate plus ionophore or antibodies against the CD3-T-cell receptor complex and simultaneously activated the binding of AP-1. Furthermore, PDTC differentially targeted both NFATp and NFATc family members, inhibiting the transactivation functions of NFATp and mRNA induction of NFATc. Strikingly, Western blotting and immunocytochemical experiments indicated that PDTC promoted a transient and rapid shuttling of NFATp and NFATc, leading to their accelerated export from the nucleus of activated T cells. We propose that the activation of an NFAT kinase by PDTC could be responsible for the rapid shuttling of the NFAT, therefore transiently converting the sustained transactivation of this transcription factor that occurs during lymphocyte activation, and show that c-Jun NH2-terminal kinase (JNK) can act by directly phosphorylating NFATp. In addition, the combined inhibitory effects on NFAT and NF-KB support a potential use of DTCs as immunosuppressants.

Antioxidants and AP-1 activation: a brief overview.

Activity of the transcription factor AP-1 is controlled by different MAPK cascades that regulate the different AP-1 components at the transcriptional and posttranscriptional level. Recently, AP-1 has been shown to behave as a redox-sensitive transcription factor that can be induced under both pro-oxidative and antioxidative conditions. In this overview we summarize the signaling pathways that converge on the activation of AP-1 and the components of these pathways that have been shown to be targets of antioxidants. The activation of AP-1 by antioxidants may account for the expression of a number of genes that mediate important functions under physiological conditions.

JNK (c-Jun NH2-terminal kinase) is a target for antioxidants in T lymphocytes.

AP-1 has been shown to behave as a redox-sensitive transcription factor that can be activated by both oxidant and antioxidant stimuli. However, the mechanisms involved in the activation of AP-1 by antioxidants are largely unknown. In this study we show that the structurally unrelated antioxidant agents pyrrolidine dithiocarbamate (PDTC), butylated hydroxyanisole, and Nacetylcysteine activated JNK (c-Jun NH2-terminal kinase) in Jurkat T cells. This activation differed substantially from that mediated by phorbol 12-myristate 13-acetate (PMA) and Ca2+ ionophore or produced by costimulation with antibodies against the T cell receptor-CD3 complex and to CD28. The activation of JNK by classical T cell stimuli was transient, whereas that mediated by PDTC and butylated hydroxyanisole (but not N-acetylcysteine) was sustained. The kinetics of JNK activation correlated with the expression of c-jun which was transient after stimulation with PMA plus ionophore and prolonged in response to PDTC, which also transiently induced c-fos. In addition, JNK activation by PMA plus ionophore was sensitive to inhibitors of signaling pathways involving Ca2+, protein kinase C, and tyrosine phosphorylation, which failed to inhibit the activation mediated by PDTC. Transfection of trans-dominant negative expression vectors of ras and raf, together with AP-1-dependent reporter constructs, as well as Western blot analysis using anti-ERK (extracellular signal-regulated kinase) antibodies, indicated that the Ras/Raf/ERK pathway did not appear to mediate the effect of the antioxidant. However, the combined treatment with PDTC and PMA, two agents that synergize on AP-1 activation, resulted in the persistent phosphorylation of ERK-2. In conclusion, our results identify JNK as a target of antioxidant agents which can be regulated differentially under oxidant and antioxidant conditions.