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The cartilage matrix is composed of characteristic components including type II collagen, aggrecan and link protein. In this paper, we report two DNA elements that regulate the link protein gene. Using transient transfection assays with link protein gene constructs in chondrocytes, chloramphenicol acetyl transferase (CAT) assays were used to measure the transcriptional activity of the link protein gene. Previously, we identified an enhancer-like activity within the first intron of the gene. In this paper, we report an active 34 bp (+1390 to +1424) fragment within this region that contains a glucocorticoid-like response element (GRE). Both deletion of, and site-specific mutations within this sequence motif reduced the dexamethasone-inducible activity. The GRE-like sequence from the rat link protein gene, or the homologous sequence from the human link protein gene were included in vectors containing the thymidine kinase promoter linked to the CAT gene (tkCAT). Both human and rat elements transferred the ability to respond to dexamethasone and hydrocortisone with a > 10-fold induction. Deletions through the promoter from -923 to -900 identified a second site required for both glucocorticoid and serum responsiveness. A four base substitution at this site resulted in a loss of serum responsiveness. This region contains an AT-rich element, similar to the AT-rich elements involved in homeotic protein regulation of the growth hormone gene and the muscle creatine kinase gene. Southwestern analysis using oligonucleotides containing the AT-rich element from the link protein gene or the muscle creatine kinase gene, identified a 32 kDa protein band from nuclear extracts of chick chondrocytes. Using these AT-rich oligonucleotides in band-shift analyses, nuclear extracts of chick sternal muscle, rat chondrosarcoma and chick sternal chondrocytes each showed formation of different complexes suggesting cell specificity. AT-rich elements have been identified as binding sites for homeodomain-containing proteins and can contribute to gene regulation by serum response factors. The identification of an AT-rich element in the link protein gene suggests similar functions for this element.

In this issue of Cell, G. Pavlath and coworkers (Horsley et al., 2003) demonstrate a novel role for Interleukin-4 (IL-4) in regulating the fusion of myoblasts with differentiated myotubes. The authors demonstrate that NFATc2 signaling in newly formed myotubes induces IL-4 expression and secretion which promotes myoblast fusion with pre-existing myotubes.

Recently, Li et al. ([1][1]) reported that a fraction of NFATc2 and NFATc1 proteins is constitutively localized in the nuclei of primary regulatory T (Treg) cells. In contrast to our own experience, the authors claimed that this NFAT translocation in resting Treg cells is independent of calcineurin

Pancreatic cancer is the fourth leading cause of cancer-related deaths in the U.S. Current chemotherapeutic agents have limited efficacy and play a minor role in its treatment. We previously determined that the novel agent phosphosulindac (P-S) significantly prevents colon cancer and have now extended our studies into pancreatic cancer. Microarray data demonstrated a 5-fold increase in the gene expression of Nuclear Factor of Activated T-cells (NFAT) in MIA PaCa-2 cells after 2 h of treatment with P-S. NFATc1 and NFATc2 isoforms have been shown to be up-regulated in pancreatic cancer. To determine the role NFATc1 played in response to treatment with our compound, we developed a NFATc1 shRNA model in BxPC-3 and GFP positive MIA PaCa-2 cells, and a NFATc1 overexpression model in GFP labeled MIA PaCa-2 cells. Knockdown of NFATc1 expression by shRNA was confirmed by western blot and qRT-PCR. A 2-fold decrease in IC50 of P-S was observed in NFATc1 shRNA cells. In contrast, overexpression of NFATc1 increased the IC50 of P-S by 1.2-fold. The ability of our compound to reduce cell viability more potently in NFATc1 shRNA cells prompted us to assess the effect of the protein on tumor growth in a MIA PaCa-2 xenograft. 5-6 week old female BALB/c nude mice were subcutaneously injected with NFATc1 wt, knock-down, and overexpression GFP labeled MIA PaCa-2 cells. Animals were treated for 5 weeks, euthanized and tumor volume assessed. We observed a 50% reduction in tumor volume in NFATc1 wt, 63% reduction in NFATC1 knock-down, and a 26% reduction in NFATc1 overexpression xenograft. NFATc1 localization is indicative of its activity, with nuclear localization being the active form. We assessed OCT-embedded sections of our xenograft tissue to determine the localization of NFATc1 in GFP labeled tumor cells. Confocal microscopy data showed that in comparison to control, P-S treated tissue demonstrated decreased nuclear localization of the protein, indicating that P-S may suppress the activation of NFATc1. We observed a similar trend in NFATc1 knock-down and overexpression tissue, though our compound demonstrated a greater effect on tumor reduction in NFATc1 knock-down mice. We conclude that P-S is a potentially important agent for the treatment of pancreatic cancer and that our compound may play a role in NFATc1 localization and activation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4431. doi:1538-7445.AM2012-4431

Mutation of the p53 gene is a common occurrence in human breast cancers but is by no means universal. However, even in tumours where the gene is not mutated altered levels of p53 protein are often detected. This is also observed in cell lines derived from human breast cancers. By transfecting such cell lines containing either wild type or mutant p53 genes with a temperature-sensitive mutant mouse p53 gene we have established that the cellular environment plays a critical role in the regulation of p53 protein expression. The results suggest that tumours that aberrantly express wild-type p53 may have lost the normal growth regulatory response to the protein and thus be functionally similar to those expressing the mutant protein.

p53 protein was localized in the cytoplasm of growing and in the nucleus of growth-arrested MCF-7 cells. While the absolute amount and rate of synthesis of p53 in growing and arrested cells were nearly the same, the protein in growing cells was phosphorylated to a greater extent than in arrested cells. The abilities of the cytoplasmic and nuclear p53 proteins to bind to DNA sequences specific for p53 protein binding did not differ remarkably despite their differential phosphorylation levels. Serum-induced translocation of the p53 protein from the nucleus to the cytoplasm, as well as DNA and protein synthesis, were inhibited by cycloheximide. These results suggest that the DNA synthesis—associated cytoplasmic translocation of p53 protein in response to serum stimulation depends on de novo protein synthesis and not on alteration of the protein's ability to bind to specific DNA sequences. © 1993 Wiley-Liss, Inc.

Clinical benefits of umbilical cord blood (UCB) grafts include lower incidence of acute graft-versus-host disease (aGVHD) following allogeneic transplantation. The dramatically lower expression of the transcription factor Nuclear Factor of Activated T-cells 1 (NFAT1) in UCB-derived CD4+ T-cells compared to adult blood (AB) results in reduced production of inflammatory cytokines following stimulation, in part underlying this advantage. Here we present a novel mechanism by which NFAT1 protein expression is restrained in these cells. Importantly, NFAT1 mRNA (NFATc2) is not significantly reduced in UCB CD4+ T-cells. Gradient separation and cellular fractionation reveal that NFAT1 is indeed translationally repressed in UCB through early stimulation timepoints, which we hypothesized might be due to the activity of microRNA. A search of known microRNA species reveals that miR- 184 is strongly predicted to bind to the NFATc2 3′ untranslated region (UTR). We have confirmed this interaction through luciferase expression and Western Blot analysis. First, luciferase expression vectors containing the short miR-184 binding sequence or the cloned 3′ UTR were constructed and transfected into UCB and AB CD4+ T-cells. In UCB, insertion of the miR-184 binding sequence alone reduced luciferase expression to 38% of control. The cloned 3′ UTR vector exhibited 60% expression compared to control. Both these negative effects were fully reversible by cotransfection with an excess of blocking antisense to miR- 184. Likewise, the miR-184 site did not impact luciferase expression in AB CD4+ T-cells, but expression was reduced by 61% in AB upon cotransfection with miR-184 mimic. miR-184 mimic produced a 23% decrease in luciferase activity from vector containing the NFATc2 3′ UTR. Both these effects were attenuated by cotransfection with an excess of blocking antisense to miR-184. Blocking antisense increased endogenous NFAT1 protein expression in UCB CD4+ T-cells by 86% as measured by quantified Western Blot, without producing a significant change in NFATc2 mRNA quantity. Conversely, miR-184 mimic produced a 31% decrease in NFAT1 protein expression by AB CD4+ T-cells. We confirmed by quantitative RT-PCR (qRT-PCR) that miR-184 exhibited an average of 58.4-times greater expression in UCB than in AB CD4+ T-cells (p=0.005). We have additionally demonstrated via gain-of-function and loss-of-function analyses that the effects on NFAT1 expression by miR-184 are sufficient to affect transcription of the NFAT-dependent pro-inflammatory cytokine IL-2 through 16 hours of in vitro stimulation. This study comprises the first identification of miR-184 activity in primary human lymphocytes and reveals its function in repressing NFAT1 protein expression uniquely in UCB CD4+ T-cells. These findings hold important implications for our further understanding of GVHD, autoimmunity, and neonatal lymphocyte development, and suggest miR-184 as a useful biomarker or target of ex vivo graft analysis.

NFAT (Nuclear Factor of Activated T cells) is a family of calcium-induced, calcineurin-dependent transcription factors, well characterized as central regulators of inducible gene expression in T lymphocytes but now known to function also in several other cell types in various adaptation and differentiation processes. Activation of NFAT by the phosphatase calcineurin is counteracted by several inhibitory kinases and can be completely blocked by the immunosuppressant Cyclosporin A. The Down syndrome critical region 1 (DSCR1; also termed CSP1, MCIP1 or RCAN1) gene belongs to the calcipressin family of endogenous calcineurin inhibitors and is expressed in several isoforms, one of which (isoform C, coded by exons 4–7) has been described to be a transcriptional target for NFAT in striated muscle, endothelial, and neural cells. The DSCR1 gene is located within the Down syndrome critical region of human chromosome 21 and is, together with 200–300 other genes, overexpressed about 1.5-fold in patients with Down syndrome (DS). Previously, dysregulation of NFAT signaling by overexpression of DSCR1 has been implicated in causing various of the pathophysiological features observed in DS patients. Children with DS also suffer from an about 500-fold increased incidence of acute megakaryocytic leukemia; the respective roles of NFAT or DSCR1 in megakaryocytes of either normal individuals or those with DS, however, has not yet been established. Here we show that DSCR1 is upregulated during megakaryocytic differentiation in a lineage-specific manner, and in mature megakaryocytes is further strongly induced by calcineurin stimulation. DSCR1 expression in megakaryocytes is regulated by NFAT, since overexpression of NFATc2 enhances, while overexpression of the specific inhibitor of NFAT activation, VIVIT, suppresses expression of the gene. We further demonstrate that DSCR1 does not only represent an NFAT target in megakaryocytes, but itself acts an inhibitor of NFAT signaling in these cells. Overexpression of DSCR1 in CMK cells as well as in primary megakaryocytes by retroviral transduction profoundly suppressed ionomycin-induced dephosphorylation and nuclear translocation of NFATc2, as well as transactivation of an NFAT-dependent promoter construct. Finally, overexpression of DSCR1 in megakaryocytes markedly downregulated both the constitutive and induced expression of Fas Ligand, a pro-apoptotic gene recently established as a NFAT target in megakaryocytes. Together, these results suggest that DSCR1 acts as an NFAT-induced NFAT inhibitor in megakaryocytes and, when overexpressed, interferes with the expression of NFAT-dependent megakaryocytic genes.

Protein S, a vitamin K-dependent plasma protein having Gla-residues, increases the rate of inactivation of Factor Va by activated protein C by enhancing the binding of activated protein C to phospholipid [Walker, J.F. (1981) J. Biol. Chem. 256, 11128-11131]. The present study aimed at elucidating the effect of thrombin-modified protein S on Factor Va inactivation by activated protein C. Nondigested protein S consisted 81% of intact form and 19% of modified form, and thrombin-digested protein S had 96% modified form. Protein S, both nondigested and digested, did not show any effects on the amidolytic activity of activated protein C towards synthetic peptide substrate. Nondigested protein S stimulated the Factor Va inactivation by activated protein C, whereas the digested protein appeared to suppress the inactivation. Protein-phospholipid binding experiments showed that although nondigested protein S enhanced the binding of activated protein C to phospholipid stoichiometrically, digested protein S appeared to not only suppress the complex formation, but also dissociate the complex. This evidence suggested that protein S modified by thrombin regulates the action of activated protein C towards Factor Va on phospholipid.

Summary Previous work has shown that a novel protein kinase is induced after infection of cultured cells with herpes simplex virus type 1 (HSV-1). Separately, it has been reported that the protein encoded by HSV-1 gene US3 shows similarity in its amino acid sequence to members of the protein kinase family of eukaryotes. We have investigated the possibility that these two observations are connected by preparing an antiserum to a synthetic oligopeptide corresponding to the carboxy-terminal eight amino acids of the US3 protein. This antiserum reacted on immunoblots with a polypeptide of apparent molecular weight 68000 from extracts of cells which had been infected with HSV-1. The antiserum also reacted strongly with a 68000 molecular weight species from a preparation of the novel HSV-1 protein kinase which had been extensively purified and resolved from other protein kinases. In addition, the purified preparation phosphory-lated a protein species, also of 68000 apparent molecular weight, when incubated with [γ-32P]ATP. These data are consistent with gene US3 encoding the novel protein kinase induced after infection of cells with HSV-1.

The studies that have been carried out to date suggest that protein S can function as a cofactor protein in the activated protein C catalyzed inactivation of Factor Va. This conclusion is supported by the observation that protein S and activated protein C can form a lipid bound complex that can inactivate Factor Va more rapidly than does soluble activated protein C. Protein S has a number of properties in common with other cofactor proteins. It has been isolated as a distinct entity from plasma, it has no known intrinsic activities, and its effect is maximal when it is bound to a lipid surface. Protein S appears to be important in the regulation of the plasma anticoagulant activity of activated protein C as well. This is supported by the lack of anticoagulant activity of activated protein C in protein S-depleted plasma and by the observation that protein S is important in the species specificity of the anticoagulant activity of the enzyme. Future work in this area is needed to ascertain if there are any clinical manifestations of protein S deficiencies that correlate with the cofactor activity that has been established.

The human ets-2 gene is a homolog of the v-ets oncogene of the E26 virus and codes for a 56-kilodalton nuclear protein. The ets-2 protein is phosphorylated and has a rapid turnover, with a half-life of 20 min. When human lymphocytic CEM cells were treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), the level of the ets-2 protein was quickly elevated 5- to 20-fold. This effect of TPA was mimicked by a synthetic diacylglycerol, 1-oleoyl-2-acetyl glycerol, and was blocked by the protein kinase C inhibitor H7, indicating that protein kinase C is involved in the induction. The increase in the ets-2 protein was due to stabilization of the protein, because the protein had a half-life of more than 2 h in the presence of TPA and the ets-2 mRNA level did not increase significantly upon TPA treatment. The protein synthesis inhibitor cycloheximide enhanced the effect of TPA on the ets-2 protein and could itself slow turnover of the protein. Properties of the ets-2 protein, such as nuclear localization, phosphorylation, rapid turnover, and response to protein kinase C, indicate that this protein belongs to a group of oncogene proteins which are generally thought to have regulatory functions in the nucleus (e.g., myc, fos, myb, and p53). Our results suggest that protein kinase C, either directly or indirectly, regulates the level of the ets-2 protein by posttranslational mechanisms.

Thirteen monoclonal antibodies designated as MFC-1 to MFC-13 were obtained from hybridoma cells cloned after the fusion of mouse myeloma cells with spleen cells of mice immunized with purified human protein C. Studies were made to determine where the antibodies bound to the molecule of protein C and whether they affected the biological actions of protein C. By using the immunoblotting technique, six of these antibodies were shown to bind to the light chain of protein C, and five to the heavy chain of protein C and also activated protein C. The remaining two antibodies bound to neither the light chain nor the heavy chain, though both antibodies bound to the intact protein C. Antibodies specific for the light chain did not bind to the gamma-carboxyglutamic acid-domain. Two of the antibodies specific for the heavy chain (MFC-13 and -1) inhibited the amidolytic activity of activated protein C. The MFC-13 also inhibited the activity of bovine activated protein C, but not that of human Factor IXa, Factor Xa, or thrombin. In addition to these two antibodies, another one for the heavy chain (MFC-10) and two antibodies for the light chain (MFC-9 and -11) inhibited the inactivation of Factor Va by human activated protein C. One of the antibodies which inhibited the enzyme activity (MFC-1) blocked the inhibition of activated protein C by protein C inhibitor. Another one for the heavy chain (MFC-5) inhibited the activation of protein C by thrombin regardless of the presence or absence of thrombomodulin. Based on these results, we have established the positions of some monoclonal antibody-binding sites on the protein C molecule.

Four members of the nuclear factor of activated T cells (NFAT) family (NFATC1, NFATC2, NFATC3, and NFATC4) are Ca2+-regulated transcription factors that regulate several processes in vertebrates, including the development and function of the immune, cardiovascular, musculoskeletal, and nervous systems. Here we describe the structures and alternative splicing of the human and mouse NFAT genes, including novel splice variants for NFATC1, NFATC2, NFATC3, and NFATC4, and show the expression of different NFAT mRNAs in various mouse and human tissues and brain regions by RT-PCR. Our results show that alternatively spliced NFAT mRNAs are expressed differentially and could contribute to the diversity of functions of the NFAT proteins. Since NFAT family members are Ca2+-regulated and have critical roles in neuronal gene transcription in response to electrical activity, we describe the expression of NFATC1, NFATC2, NFATC3, and NFATC4 mRNAs in the adult mouse brain and in the adult human hippocampus using in situ hybridization and show that all NFAT mRNAs are expressed in the neurons of the mouse brain with specific patterns for each NFAT.

The subcellular localization of the mouse type IV c-abl protein was determined by indirect immunofluorescence of nontransformed NIH 3T3 fibroblasts that overexpress the protein. Unlike the viral transforming protein p160gag/v-abl, which has cytoplasmic and plasma membrane localization, a large fraction of the c-abl (IV) protein is nuclear, with the remainder in the cytoplasm and plasma membrane. Deletion of a small N-terminal regulatory region of the c-abl (IV) protein, sufficient to activate its transforming potential fully, changes the distribution of the protein from the nucleus to the cytoplasm. Mapping of an amino acid sequence responsible for the nuclear localization of the c-abl (IV) protein reveals a nuclear localization signal similar to that of SV40 large T antigen.

A protein kinase, associated with free cytoplasmic ribonucleoprotein particles (free dRNP) has been purified from mouse plasma cell tumours. This protein kinase is able to phosphorylate in vitro endogenous protein from free dRNP. Some characteristics of this protein kinase have been studied. This protein kinase behaves as being cyclic AMP independent. The properties of this protein kinase were compared with other protein kinases: soluble, ribosome-bound, and nuclear protein kinases. Although there are minor differences it is very similar to a ribosome-associated protein kinase from the plasma cell tumours.

In lymphocytes, the three NFAT factors NFATc1 (also designated as NFAT2), NFATc2 (NFAT1) and NFATc3 (NFAT4 or NFATx) are expressed and are targets of immune receptor signals which lead to a rapid rise of intracellular Ca++, the activation of phosphatase calcineurin and to activation of cytosolic NFATc proteins. In addition to rapid activation of NFAT factors, immune receptor signals lead to accumulation of the short NFATc1/aA isoform in lymphocytes which controls their proliferation and survival. In this mini-review we summarize our current knowledge on the structure and transcription of the Nfatc1 gene in lymphocytes which is controlled by two promoters, two poly A addition sites and a remote downstream enhancer. The Nfatc1 gene resembles numerous primary response genes (PRGs) induced by LPS in macrophages. Similar to the PRG promoters, the Nfatc1 promoter region is organized in CpG islands, forms DNase I hypersensitive sites and is marked by histone tail modifications before induction. By studying gene induction in lymphocytes in detail it will be important to elucidate whether the properties of the Nfatc1 induction are not only typical for the Nfatc1 gene but also for other transcription factor genes expressed in lymphocytes.

Background: We recently described increased NFATc1, IRF4, and NIP45 messenger RNA (mRNA) expression in peripheral blood mononuclear cells (PBMCs) of asthmatic children and adults with multiple allergies. Objective: NFATc2 has been described to associate with IRF4 to induce interleukin-4, and to be inhibited by T-bet. Here, we analyzed the role of NFATc2 in asthmatic children and adults. Methods: PBMCs were isolated from the blood of control of asthmatics subjects. Some PBMCs were analyzed untreated and some cultured with and without phytohemagglutinin. Then, RNA was extracted from the cells and cytokines were measured in the supernatants via enzyme-linked immunosorbent assay or multiplex analysis. RNA was then reverse-transcribed and NFATc1, NFATC2, IRF4, and T-bet mRNA were analyzed by real-time polymerase chain reaction. In addition, in peripheral blood cells, NFATc2 expression was analyzed, in a population of asthmatic children and adults from the Asthma BRIDGE study. Results: In addition to NFATc1 and NIP45, also NFATc2 was found upregulated in PBMCs and peripheral blood cells from asthmatic children and adults with allergic asthma. Moreover, NFATc1 directly correlated with lymphocytes number whereas NFATc2 correlated with peripheral eosinophilia in asthma. Conclusions: In addition to NFATc1 and NIP45, NFATc2 was found upregulated in asthma. Moreover, NFATc1 mRNA correlated with lymphocytes both in control and asthma, and NFATC1 and NFATc2 mRNA showed a direct correlation with eosinophils in controls but not in asthma, indicating that NFATc1 is associated with lymphocytes and not eosinophils in asthma. Clinical significance: Targeting NFATc2 in T lymphocytes might ameliorate the allergic phenotype in asthmatic subjects. Keywords: T cells; allergy processes; animals; eosinophils cells; human.