NFAT Pathway Research Articles

The PDZ Domain-binding Motif of the Human T Cell Leukemia Virus Type 1 Tax Protein Induces Mislocalization of the Tumor Suppressor hScrib in T cells

Interactions with cellular PDZ domain-containing proteins obviously contribute to the tumorigenic potential of several viral oncoproteins. In this regard, the oncogenic potential of the human T cell leukemia virus type 1 Tax protein correlates with its binding capacity to the tumor suppressor hDlg. Recent results show that hDlg in T cells is associated to a network of scaffolding proteins including another PDZ domain-containing protein termed hScrib. Interestingly, previous studies have revealed complementary activities of both proteins in the control of epithelial cell polarity. Here, we demonstrate that Tax can bind to hScrib and that the resulting Tax/hScrib complex is present in human T cell leukemia virus type 1-infected T cells. By confocal microscopy, we show that Tax modifies the localization of hScrib in transfected COS cells as well as in infected T cell lines and targets hScrib to particular spots exhibiting a granular distribution, mainly distributed in the cytoplasm. Given that Tax sequesters hScrib to these particular structures, we postulate that Tax might inhibit hScrib activity. Providing further support to this idea, we find that transient overexpression of hScrib attenuates T cell receptor-induced NFAT activity but that the presence of Tax counteracts this negative effect on the NFAT pathway. The fact that hDlg and hScrib are both targeted by Tax underlies their importance in T cell function.

Activation of both nuclear factor of activated T cells and inhibitor of nuclear factor‐κB kinase β‐subunit‐/nuclear factor‐κB is critical for cyclooxygenase‐2 induction by benzo[a]pyrene in human bronchial epithelial cells

The carcinogenic effect of benzo[a]pyrene (B[a]P), presenting mainly in cigarette smoke and air pollution, has been well demonstrated both in vitro and in vivo. However, it is still not well understood how B[a]P facilitates pulmonary carcinogenesis. To explore this, we investigated the effect of B[a]P on the induction of cyclooxygenase-2 (COX-2), a critical enzyme implicated in inflammation and cancer development, as well as upstream signaling pathways leading to its expression in human bronchial epithelial cells (Beas-2B). We found that exposure of Beas-2B to B[a]P caused significant COX-2 induction at both the transcriptional and protein levels. B[a]P also switched on the nuclear factor of activated T cells (NFAT) and nuclear factor kappaB (NF-kappaB) signaling pathways. B[a]P-induced COX-2 expression was significantly blocked by inhibition of the NFAT pathway, and impairment of the NF-kappaB signaling pathway by ectopic expression of an inhibitor of nuclear factor-kappaB kinase beta-subunit (IKKbeta) kinase inactive mutant (IKKbeta-KM) also dramatically inhibited COX-2 induction. The IKKbeta/NF-kappaB-dependent COX-2 induction was further confirmed in mouse embryonic fibroblasts with IKKbeta deficiency (IKKbeta(-/-)) and in those that expressed reconstituted IKKbeta. However, activation of the NFAT and NF-kappaB signaling pathways by B[a]P were independent of each other, as blocking one signaling pathway didn't interrupt the activation of the other one. Mutation of either NFAT or NF-kappaB binding sites significantly blocked COX-2 promoter induction by B[a]P. Taken together, these data indicate that exposure of Beas-2B to B[a]P can upregulate COX-2 expression by increasing its transcription, which requires activation of both the NFAT and NF-kappaB signaling pathways.

Flesh and blood: The story of Vav1, a gene that signals in hematopoietic cells but can be transforming in human malignancies

Cancer results from the interaction of multiple aberrations including activation of dominant oncogenes and upregulation of signal transduction pathways. Identification of the genes involved in malignant transformation is a pre-requisite for understanding cancer and improving its diagnosis and treatment. Quite a few of the genes that have been implicated in cancer are mutant or aberrantly expressed versions of genes that are important mediators of the normal growth that occurs during development. An important example of this is Vav1, a cytoplasmic signal transducer protein initially identified as an oncogene. Physiological expression of Vav1 is restricted to the hematopoietic system, where its best-known function is as a GDP/GTP nucleotide exchange factor for Rho/Rac GTPases, an activity strictly controlled by tyrosine phosphorylation. Vav1 was shown to regulate cytoskeletal rearrangement during activation of hematopoietic cells. Vav1 can also mediate other cellular functions including activation of the JNK, ERK, Ras, NF-kB, and NFAT pathways, in addition to association with numerous adapter proteins such as Shc, NCK, SLP-76, GRB2, and Crk. Although the oncogenic form of Vav1 has not been detected in clinical human tumors, its wild-type form has recently been implicated in mammalian malignancies such as neuroblastoma, melanoma, pancreatic tumors and B-cell chronic lymphocytic leukemia. This review addresses the physiological function of wild-type Vav1, its mode of activation as an oncogene, and its emerging role as a transforming protein in human cancer.

Transcription Factor NFAT, Its Role in Cancer Development, and as a Potential Target for Chemoprevention

The nuclear factor of activated T cells (NFAT) family proteins are transcription factors that regulate the expression of a variety of target genes with or without forming complexes with other transcription factors. Although NFAT proteins have been extensively investigated and characterized in immune systems, their role in carcinogenesis are far from being understood. We, to our knowledge, are first to determine the potential involvement of the NFAT pathway in cell responses to carcinogen exposure. Experimental evidence accumulated from our studies indicate the critical role of NFAT3 in some carcinogen-induced cell transformation and tumorigenicity. Moreover, NFAT proteins have been found to be involved in cell cycle regulation, cell differentiation, cell survival, angiogenesis, and tumor cell invasion and metastasis. In the meantime, NFAT inhibitors are being developed with the ultimate aim to specifically switch off NFAT signaling without side effects. This review comprehensively reviews the results from the most recent studies, and also discusses some difficulties in current studies. To validate whether NFAT can be a promising target for chemoprevention, more research has to be done to further detail the roles of NFAT and to differentiate the functions of different members of this protein family in future studies.

678. Cell Permeable, Anti-Inflammatory Peptides Targeting NF-kB and NFAT Pathways Prevent the Onset of Diabetes in NOD Mice

678. Cell Permeable, Anti-Inflammatory Peptides Targeting NF-kB and NFAT Pathways Prevent the Onset of Diabetes in NOD Mice

4-1BB triggers IL-13 production from T cells to limit the polarized, Th1-mediated inflammation

4-1BB (CD137) triggering typically induces Th1 response by increasing IFN-gamma from T cells upon TCR ligation. We found recently that 4-1BB costimulation increased the expression of IL-13 from CD4(+) T cells, as well as CD8(+) T cells. The enhanced IL-13 expression by agonistic anti-4-1BB treatment was mediated via MAPK1/2, PI-3K, JNK, mammalian target of rapamycin, NF-AT, and NF-kappaB signaling pathways. The signaling for IL-13 induction was similar to that of IFN-gamma production by anti-4-1BB treatment in T cells. When the anti-4-1BB-mediated IL-13 expression was tested in an in vivo viral infection model such as HSV-1 and vesicular stomatitis virus, 4-1BB stimulation enhanced IL-13 expression of CD4(+) T, rather than CD8(+) T cells. Although IL-13 was enhanced by anti-4-1BB treatment, the increased IL-13 did not significantly alter the anti-4-1BB-induced Th1 polarization of T cells--increase of T-bet and decrease of GATA-3. Nevertheless, anti-4-1BB treatment polarized T cells excessively in the absence of IL-13 and even became detrimental to the mice by causing liver inflammation. Therefore, we concluded that IL-13 was coinduced following 4-1BB triggering to maintain the Th1/2 balance of immune response.

Polycystic kidney disease: genes, proteins, animal models, disease mechanisms and therapeutic opportunities

An increased understanding of the genetic, molecular and cellular mechanisms responsible for the development of polycystic kidney disease has laid out the foundation for the development of rational therapies. Many animal models where these therapies can be tested are currently available. This review summarizes the rationale for these treatments, the results of preclinical trials and the prospects for clinical trials, some already in early phases of implementation.

The Critical Role of Protein Kinase C-θ in Fas/Fas Ligand-Mediated Apoptosis

A functional immune system not only requires rapid expansion of antigenic specific T cells, but also requires efficient deletion of clonally expanded T cells to avoid accumulation of T cells. Fas/Fas ligand (FasL)-mediated apoptosis plays a critical role in the deletion of activated peripheral T cells, which is clearly demonstrated by superantigen-induced expansion and subsequent deletion of T cells. In this study, we show that in the absence of protein kinase C-theta (PKC-theta), superantigen (staphylococcal enterotoxin B)-induced deletion of Vbeta8(+) CD4(+) T cells was defective in PKC-theta(-/-) mice. In response to staphylococcal enterotoxin B challenge, up-regulation of FasL, but not Fas, was significantly reduced in PKC-theta(-/-) mice. PKC-theta is thus required for maximum up-regulation of FasL in vivo. We further show that stimulation of FasL expression depends on PKC-theta-mediated activation of NF-AT pathway. In addition, PKC-theta(-/-) T cells displayed resistance to Fas-mediated apoptosis as well as activation-induced cell death (AICD). In the absence of PKC-theta, Fas-induced activation of apoptotic molecules such as caspase-8, caspase-3, and Bid was not efficient. However, AICD as well as Fas-mediated apoptosis of PKC-theta(-/-) T cells were restored in the presence of high concentration of IL-2, a critical factor required for potentiating T cells for AICD. PKC-theta is thus required for promoting FasL expression and for potentiating Fas-mediated apoptosis.

Protection conferred by myocardial ATP‐sensitive K+ channels in pressure overload‐induced congestive heart failure revealed in KCNJ11 Kir6.2‐null mutant

Ventricular load can precipitate development of the heart failure syndrome, yet the molecular components that control the cardiac adaptive response to imposed demand remain partly understood. Compromised ATP-sensitive K(+) (K(ATP)) channel function renders the heart vulnerable to stress, implicating this metabolic sensor in the homeostatic response that would normally prevent progression of cardiac disease. Here, pressure overload was imposed on the left ventricle by transverse aortic constriction in the wild-type and in mice lacking sarcolemmal K(ATP) channels through Kir6.2 pore knockout (Kir6.2-KO). Despite equivalent haemodynamic loads, within 30 min of aortic constriction, Kir6.2-KO showed an aberrant prolongation of action potentials with intracellular calcium overload and ATP depletion, whereas wild-type maintained ionic and energetic handling. On catheterization, constricted Kir6.2-KO displayed compromised myocardial performance with elevated left ventricular end-diastolic pressure, not seen in the wild-type. Glyburide, a K(ATP) channel inhibitor, reproduced the knockout phenotype in the wild-type, whereas the calcium channel antagonist, verapamil, prevented abnormal outcome in Kir6.2-KO. Within 48 h following aortic constriction, fulminant biventricular congestive heart failure, characterized by exercise intolerance, cardiac contractile dysfunction, hepatopulmonary congestion and ascites, halved the Kir6.2-KO cohort, while no signs of organ failure or mortality were seen in wild-type. Surviving Kir6.2-KO developed premature and exaggerated fibrotic myocardial hypertrophy associated with nuclear up-regulation of calcium-dependent pro-remodelling MEF2 and NF-AT pathways, precipitating chamber dilatation within 3 weeks. Thus, K(ATP) channels appear mandatory in acute and chronic cardiac adaptation to imposed haemodynamic load, protecting against congestive heart failure and death.

Activation of NFAT and NFkB Transcription Factors upon B Cell Receptor (BCR) Stimulation Is Restricted to Evolutive Chronic Lymphocytic Leukemia (CLL) Cases.

Chronic lymphocytic leukemia (CLL) is a highly heterogeneous disease in which BCR signaling capacity plays a central role in disease progression. Previously, we have shown that BCR engagement allows the identification of two groups of patients with a strong correlation between the in vitro cell survival response and the biological criteria (IgVH somatic hypermutations, ZAP 70 expression ) or the clinical stage. One group, corresponding to non evolutive patients, showed absence of a substantial BCR signaling (Non responder). In contrast, BCR signaling promoted in vitro cell survival in the group of Responder cases corresponding to evolutive patients. In the latter only, response to BCR ligation was associated with induction of NFkB and NFAT regulated genes, such as Cyclin D2 and CD23.Since NFAT and NFkB transcription factors, regardless of the patients clinical status, have been described as constitutively nuclear in B-CLL cells, the aim of our study was to investigate whether the functional implication of these factors was different between our two groups. First, we confirmed by confocal microscopy and band shift analysis that localization of NFAT and NFkB was uniformely nuclear independently of the CLL status and the BCR response capacity. By RQ-PCR , we also investigated the expression of the various isoforms of NFAT (NFAT1, 2, 3 and 4) and showed that their expression profile was similar to normal B cells. Despite the nuclear localization of NFkB and NFAT, DNA binding capacity was only enhanced in cases responsive to BCR stimulation. Finally, using specific Tat-peptide inhibitors of NFAT and NFkB nuclear transport upregulation of cyclin D2 and CD23 was abrogated and survival advantage was lost in responder cases thereby confirming the crucial role of these factors upon BCR stimulation. However, in non responder cases in which no CD23 or cyclin D2 induction was present, this inhibition had no impact on cell survival upon BCR stimulation . Nonetheless, these cells retained the ability to respond to ionomycin or PMA reflecting functional PKC/calcium-calcineurin signaling pathways. Altogether, our results showed that NFAT and NFkB were activated in responder cells only despite their homogenous expression in all cases. In conclusion, the inability of the cells from stable B-CLL cases to activate the NFAT and NFkB pathways upon BCR signaling is not related to a functional defect or abnormal expression of these factors but likely reflects an early signaling defect. Moreover, our results show that the use of peptide inhibitors may restore a stable profile in B-CLL cells from evolutive patients.

The Role of the Transcription Factor NFAT in the Pathogenesis of Non-Hodgkin's Lymphomas.

NFAT is a family of highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by the Ca2+/calmodulin-dependent serine phosphatase calcineurin, NFAT translocates to the nucleus, where it induces the transcription of a large number of genes necessary for a productive immune response. NFAT signalling has also been implicated in lymphocyte homeostasis and its deregulation has been suggested to be involved in the pathogenesis of different malignancies. A recent immunohistochemical evaluation of approximately 300 Non-Hodgkin's Lymphoma biopsy samples showed overexpression of NFAT2 in the majority of specimens with strong nuclear translocation in certain histologic subtypes (DLBCL, Burkitt's Lymphoma) presumably reflecting activation of the NFAT pathway as part of their pathogenesis. Other recent studies have shown that NFAT activation leads to increased expression of different cell survival factors (CD154, BLyS) in several lymphoma subtypes (DLBCL, MCL).To study the role of constitutive activation of the NFAT pathway on lymphomagenesis, we generated several hyperactivable NFAT mutants which will be used to generate transgenic mice expressing the mutant proteins from the ROSA26 locus. Here, we present the in vitro characterization of these hyperactivable NFAT proteins in cell lines and primary lymphocytes. The major docking site for calcineurin is located at the N terminus of the NFAT regulatory domain and has the consensus sequence PxIxIT (e.g. SPRIEIT in NFAT1 and NFAT2). Substitution of the SPRIEIT sequence of NFAT with HPVIVIT, a higher-affinity version obtained by peptide selection, increases the basal calcineurin sensitivity of the protein significantly. Similarly, a major kinase for NFAT is CK1, and mutation of the CK1 docking site (FSILF to ASILA in NFAT1, FDFEF to ADAEA in NFAT2) also leads to partial nuclear localization of NFAT proteins by decreasing rephosphorylation and nuclear export. In the current analysis we compared the different hyperactivable NFAT proteins with respect to dephosphorylation status and nuclear translocation using western blotting and immunocytochemistry. Whereas the wildtype NFAT protein was entirely localized in the cytoplasm and completely phosphorylated under resting conditions, we observed an increasing degree of nuclear translocation and dephosphorylation for the different mutant proteins (ASILA 20%, VIVIT 30–40%, ASILA-VIVIT 50–70%). This system will allow us to study the impact of different levels of NFAT activation on the pathogenesis of lymphomas in vivo.

Restricted epithelial proliferation by lacritin via PKCα-dependent NFAT and mTOR pathways

Renewal of nongermative epithelia is poorly understood. The novel mitogen “lacritin” is apically secreted by several nongermative epithelia. We tested 17 different cell types and discovered that lacritin is preferentially mitogenic or prosecretory for those types that normally contact lacritin during its glandular outward flow. Mitogenesis is dependent on lacritin's C-terminal domain, which can form an α-helix with a hydrophobic face, as per VEGF's and PTHLP's respective dimerization or receptor-binding domain. Lacritin targets downstream NFATC1 and mTOR. The use of inhibitors or siRNA suggests that lacritin mitogenic signaling involves Gαi or Gαo–PKCα-PLC–Ca2+–calcineurin–NFATC1 and Gαi or Gαo–PKCα-PLC–phospholipase D (PLD)–mTOR in a bell-shaped, dose-dependent manner requiring the Ca2+ sensor STIM1, but not TRPC1. This pathway suggests the placement of transiently dephosphorylated and perinuclear Golgi–translocated PKCα upstream of both Ca2+ mobilization and PLD activation in a complex with PLCγ2. Outward flow of lacritin from secretory cells through ducts may generate a proliferative/secretory field as a different unit of cellular renewal in nongermative epithelia where luminal structures predominate.

Benzo[a]pyrene diol‐epoxide (B[a]PDE) upregulates COX‐2 expression through MAPKs/AP‐1 and IKKβ/NF‐κB in mouse epidermal Cl41 cells

Benzo[alpha]pyrene-7,8-diol-9,10-epoxide (B[a]PDE), the major metabolite of benzo[a]pyrene (B[a]P), shows an ultimate complete carcinogen in various animals and is a causative agent for human cancers. However, its effects on the activation of signal pathways and the expression of genes involved in its carcinogenic effect remain largely unknown. In this study, the effects of B[a]PDE on induction of cyclooxygenase (COX)-2 and the signal pathways leading to the induction were investigated. Treatment of mouse epidermal Cl41 cells with B[a]PDE caused an increase in the expression of COX-2 at both transcription and protein levels, while its parental compound B[a]P did not show significant inductive effect. The COX-2 induction by B[a]PDE was dependent on the activation of mitogen-activated protein kinases (MAPK)s/activation protein (AP)-1 pathway, because inhibition of AP-1 by either overexpression of TAM67 (dominant negative mutant of c-jun), or pretreatment of cells with PD98059 (MEK1/2-ERKs pathway inhibitor) or SB202190 (p38K inhibitor), markedly inhibited B[a]PDE-induced COX-2 expression. In addition, impairment of NF-kappaB pathway by either NEMO-BDBP (an NF-kappaB specific inhibitor) or IkappaB kinase (IKK)beta-KM (dominant negative mutant of IKKbeta) also caused marked reduction of COX-2 induction by B[a]PDE. In contrast, inhibition of nuclear factor of activated T cells (NFAT) with FK506, did not show any effect on B[a]PDE-induced COX-2 expression. Collectively, these data indicate that exposure of Cl41 cells to B[a]PDE can induce COX-2 expression by increasing its transcription, which requires the activation of MAPKs/AP-1 and IKKbeta/NF-kappaB pathways, but not NFAT pathway. In view of the importance of COX-2 in carcinogenesis, we anticipate that the induction of COX-2 by B[a]PDE may coordinate its mutagenic effects to facilitate the development of skin cancer.

α‐Defensin increase in peripheral blood mononuclear cells from patients with hepatitis C virus chronic infection

The alpha-defensin genes promoter regions contain a putative nuclear factors of activated T cells (NFAT)-binding site and it is known that hepatitis C virus (HCV) core protein activates the interleukin (IL)-2 gene transcription through the NFAT pathway. The aims of this study were to investigate if HCV affects the alpha-defensin expression in peripheral human mononuclear cells (PBMCs) and to evaluate the existence of a correlation between alpha-defensins and liver damage in patients with chronic hepatitis C. Ninety patients with chronic hepatitis C, 30 with chronic hepatitis B and 25 healthy controls were enrolled. Alpha-defensins were identified and quantified in PBMCs by mass spectrometry, enzyme-linked immunosorbent assay, antibacterial activity and mRNA levels. PBMCs from three patients and controls were stimulated with HCV core protein, hepatitis B virus core antigen and the alpha-defensin mRNAs level was quantified. We found that HCV core protein activates in vitro the alpha-defensin transcription. Alpha-defensin levels in patients with chronic hepatitis C (mean +/- SD = 1.103 +/- 0.765 ng/10(6) cells), chronic hepatitis B (0.53 +/- 0.15) and healthy controls (0.217 +/- 0.09) resulted significantly different (P < 0.001). In patients with chronic hepatitis C, the alpha-defensin levels and antibacterial activity correlate with the liver fibrosis. Our data suggest that HCV induces alpha-defensin expression. The high linear correlation of alpha-defensin levels with advancing fibrosis makes the measure of these peptides a reliable marker to evaluate fibrosis stage.

Thrombin-induced Autoinhibitory Factor, Down Syndrome Critical Region-1, Attenuates NFAT-dependent Vascular Cell Adhesion Molecule-1 Expression and Inflammation in the Endothelium

Activation and dysfunction of the endothelium underlie many vascular disorders including atherosclerosis, tumor growth, and inflammation. We recently reported that thrombin and vascular endothelial growth factor, but not tumor necrosis factor-alpha, results in dramatic up-regulation of Down syndrome critical region (DSCR)-1 gene in endothelial cells, a negative feedback regulator of calcineurin-NFAT signaling. Constitutive expression of DSCR-1 in activated endothelial cells markedly impaired NFAT nuclear localization, proliferation, tube formation, and tumor growth. The goal of the present study was to elucidate the relative roles of NFAT/DSCR-1 and NF-kappaB/I-kappaB in mediating thrombin-responsive gene expression in endothelial cells. DNA microarrays of thrombin-treated human umbilical vein endothelial cells overexpressing DSCR-1 or constitutive active IkappaBalpha revealed genes that were dependent on NFAT and/or NF-kappaB activity. Vascular cell adhesion molecule-1 was inhibited both by DSCR-1 and I-kappaB at the level of mRNA, protein, promoter activity, and function (monocyte adhesion). Using a combination of transient transfections, electrophoretic mobility shift assays, and chromatin immunoprecipitation, thrombin was shown to induce time-dependent coordinate binding of RelA and NFATc to a tandem NF-kappaB element in the upstream promoter region of vascular cell adhesion molecule-1. Together, these findings suggest that thrombin-mediated activation of endothelial cells involves an interplay between NFAT and NF-kappaB signaling pathways and their negative feedback inhibitors, DSCR-1 and I-kappaB, respectively. As natural brakes in the inflammatory process, DSCR-1 and I-kappaB may lend themselves to therapeutic manipulation in vasculopathic disease states.

BTNL2, a Butyrophilin-Like Molecule That Functions to Inhibit T Cell Activation

B7 family members regulate T cell activation and tolerance. Although butyrophilin proteins share sequence homology with the B7 molecules, it is unclear whether they have any function in immune responses. In the present study, we characterize an MHC class II gene-linked butyrophilin family member, butyrophilin-like 2 (BTNL2), the mutation of which has been recently associated with the inflammatory autoimmune diseases sarcoidosis and myositis. Mouse BTNL2 is a type I transmembrane protein with two pairs of Ig-like domains separated by a heptad peptide sequence. BTNL2 mRNA is highly expressed in lymphoid tissues as well as in intestine. To characterize the function of BTNL2, we produced a BTNL2-Ig fusion protein. It recognized a putative receptor whose expression on B and T cells was significantly enhanced after activation. BTNL2-Ig inhibited T cell proliferation and TCR activation of NFAT, NF-kappaB, and AP-1 signaling pathways. BTNL2 is thus the first member of the butyrophilin family that regulates T cell activation, which has implications in immune diseases and immunotherapy.

Tacrolimus and cyclosporine A inhibit human osteoclast formation via targeting the calcineurin-dependent NFAT pathway and an activation pathway for c-Jun or MITF in rheumatoid arthritis

In the present study, we aimed to determine whether tacrolimus (FK506) and cyclosporine A act directly on human osteoclast precursors obtained from patients with rheumatoid arthritis (RA) and influence monocyte-osteoclast differentiation induced by receptor activator of NF-kappaB ligand (RANKL) in vitro, the stage at which differentiation was affected and the manner in which tacrolimus or cyclosporine A affected the osteoclast signaling pathway. Peripheral blood mononuclear cells (PBMCs) were isolated from RA patients and cultured in the presence of RANKL and macrophage-colony stimulating factor (M-CSF). Tacrolimus or cyclosporine A was added to these cultures to determine the effect on the osteoclast differentiation. Osteoclast formation was determined by assessing the number of tartrate resistant acid phosphatase (TRAP) staining cells and measuring the extent of lacunar resorption. The expression of osteoclast transcription factors, such as TNF receptor-associated factor 6 (TRAF6), nuclear factor of activated T cells c1 (NFATc1), c-Fos, c-Jun, microphthalmia transcription factor (MITF) and PU.1 in mononuclear cells (MNCs) was assayed by quantitative reverse transcription-polymerase chain reaction. Addition of tacrolimus or cyclosporine A resulted in a decrease in the number of TRAP-positive multinucleated cells (TRAP+ MNCs) and a decrease in the extent of lacunar resorption pit formation as compared to the control cultures; thus, human monocyte-osteoclast differentiation was more effectively inhibited at the late stage and addition of tacrolimus or cyclosporine A resulted in a decrease in the mRNA expression of NFATc1, c-Jun, and MITF at the late stage. Our results suggest that tacrolimus or cyclosporine A acts directly on human osteoclast precursors in RA patients and exerts their immunosuppressive effects on human monocyte-osteoclast formation via targeting both the calcineurin-dependent NFAT pathway and activation pathway for c-Jun or MITF.

Coordination of NF-κB and NFAT Antagonism by the Forkhead Transcription Factor Foxd1

Forkhead transcription factors play critical roles in the maintenance of immune homeostasis. In this study, we demonstrate that this regulation most likely involves intricate interactions between the forkhead family members and inflammatory transcription factors: the forkhead member Foxd1 coordinates the regulation of the activity of two key inflammatory transcription factors, NF-AT and NF-kappaB, with Foxd1 deficiency resulting in multiorgan, systemic inflammation, exaggerated Th cell-derived cytokine production, and T cell proliferation in autologous MLRs. Foxd1-deficient T cells possess increased activity of both NF-AT and NF-kappaB: the former correlates with the ability of Foxd1 to regulate casein kinase 1, an NF-AT inhibitory kinase; the latter with the ability of Foxd1 to regulate Foxj1, which regulates the NF-kappaB inhibitory subunit IkappaB beta. Thus, Foxd1 modulates inflammatory reactions and prevents autoimmunity by directly regulating anti-inflammatory regulators of the NF-AT pathway, and by coordinating the suppression of the NF-kappaB pathway via Foxj1. These findings indicate the presence of a general network of forkhead proteins that enforce T cell quiescence.

14‐3‐3 Proteins regulate glycogen synthase 3β phosphorylation and inhibit cardiomyocyte hypertrophy

14-3-3 proteins are dimeric phophoserine-binding molecules that participate in important cellular processes such as cell proliferation, cell-cycle control and the stress response. In this work, we report that several isoforms of 14-3-3s are expressed in neonatal rat cardiomyocytes. To understand their function, we utilized a general 14-3-3 peptide inhibitor, R18, to disrupt 14-3-3 functions in cardiomyocytes. Cardiomyocytes infected with adenovirus-expressing YFP-R18 (AdR18) exhibited markedly increased protein synthesis and atrial natriuretic peptide production and potentiated the responses to norepinephrine stimulation. This response was blocked by the pretreatment with LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor. Consistent with a role of PI3K in the R18 effect, R18 induced phosphorylation of a protein cloned from the vakt oncogene of retrovirus AKT8 (Akt - also called protein kinase B, PKB) at Ser473 and glycogen synthase 3beta (GSK3beta) at Ser9, but not extracellular signal-regulated kinase 1/2 (ERK1/2). AdR18-induced PKB and GSK3beta phosphorylation was completely blocked by LY294002. In addition, a member of the nuclear factor of activated T cells (NFAT) family, NFAT3, was converted into faster mobility forms and translocated into the nucleus upon the treatment of AdR18. These results suggest that 14-3-3s inhibits cardiomyocytes hypertrophy through regulation of the PI3K/PKB/GSK3beta and NFAT pathway.

Tetraspanin CD81 Provides a Costimulatory Signal Resulting in Increased Human Immunodeficiency Virus Type 1 Gene Expression in Primary CD4+T Lymphocytes through NF-κB, NFAT, and AP-1 Transduction Pathways

The tetraspanin superfamily member CD81 has been shown to form microdomains in the plasma membrane and to participate in the recruitment of numerous adhesion molecules, receptors, and signaling proteins in the central zone of the immune synapse. Beside its structural role, CD81 also delivers a cosignal for T cells to trigger cytokine production and cellular proliferation, thus suggesting a key role in some fundamental biological functions. It has been shown that signaling events initiated through the T-cell receptor (TCR)/CD3 complex and the coactivator CD28 positively affect human immunodeficiency virus type 1 (HIV-1) gene expression, but no study had investigated the putative costimulatory activity of CD81 on HIV-1 transcriptional activity. We observed that CD81 engagement potentiates TCR/CD3-mediated signaling, resulting in an enhancement of HIV-1 transcription and de novo virus production in both established Jurkat cells and primary CD4+ T lymphocytes at a magnitude that approximates that with CD28. These observations were made by using transiently transfected plasmids (i.e., nonintegrated viral DNA) and fully competent viruses (i.e., stably integrated provirus). Moreover, the CD81-mediated enhancement of HIV-1 gene expression is linked with increased nuclear translocation of transcription factors known to positively regulate virus transcription, i.e., NF-kappaB, NFAT, and AP-1. These findings suggest that engagement of CD81 decreases the signaling threshold required to initiate TCR/CD3-mediated induction of integrated HIV-1 proviral DNA in primary CD4+ T cells.