Induction of interleukin-2 receptor-alpha gene expression is regulated by post-translational activation of kappa B specific DNA binding proteins

T cell mitogens induce the expression of specific trans-acting DNA binding proteins that in turn regulate the expression of the interleukin-2 receptor-alpha (IL-2R alpha) gene. To investigate whether de novo protein synthesis is required for the activation of these transacting factors and the induced expression of this receptor gene, Jurkat T cells were incubated with various inhibitors of protein synthesis prior to stimulation with phytohemagglutinin and phorbol 12-myristate 13-acetate (PMA). Despite the presence of cycloheximide or anisomycin at concentrations sufficient to block greater than 97% of cellular protein synthesis, phytohemagglutinin and phorbol 12-myristate 13-acetate effectively induced the expression of the IL-2R alpha gene as measured at the mRNA level. Similarly, gel retardation, DNA footprinting, and DNA-protein cross-linking studies revealed that these mitogens induced the activation of two predominant DNA binding proteins (50-55 and 80-90 kDa) in the presence or absence of cycloheximide and anisomycin. Both of these proteins specifically interacted with a kappa B-like binding site present in the IL-2R alpha promoter (-267 to -256) that is requisite for mitogen-induced expression of this receptor gene. These findings support a post-translational mechanism of induction of pre-existing, but inactive, DNA binding proteins which in turn bind to and activate the IL-2R alpha gene.     

OS-2 mitogen activated protein kinase regulates the clock-controlled gene ccg-1 in Neurospora crassa

OS-2 MAP kinase is involved in osmoadaptation in Neurospora crassa. Clock-controlled genes ccg-1, bli-3, and con-10 were induced by osmotic stress in an OS-2 dependent manner. In contrast, osmotic stress did not affect the expression of clock genes frq, wc-1, and wc-2 or of clock-controlled genes ccg-2 and bli-4. These results suggest that OS-2 participates in the regulation of certain circadian-clock output genes.     

The protein tyrosine phosphatase SHP-2 regulates interleukin-1-induced ERK activation in fibroblasts

Focal adhesion complexes are actin-rich, cytoskeletal structures that mediate cell adhesion to the substratum and also selectively regulate signal transduction pathways required for interleukin (IL)-1beta signaling to the MAP kinase, ERK. IL-1-induced ERK activation is markedly diminished in fibroblasts deprived of focal adhesions whereas activation of p38 and JNK is unaffected. While IL-1 signaling is known to involve the activity of protein and lipid kinases including MAP kinases, FAK, and PI3K, little is known about the role of phosphatases in the regulation of IL-1 signal generation and attenuation. Here we demonstrate that SHP-2, a protein tyrosine phosphatase present in focal adhesions, modulates IL-1-induced ERK activation and the transient actin stress fiber disorganization that occurs following IL-1 treatment in human gingival fibroblasts. Using a combination of immunoblotting, immunoprecipitation, and immunostaining we show that SHP-2 is present in nascent focal adhesions and undergoes phosphorylation on tyrosine 542 in response to IL-1 stimulation. Blocking anti-SHP-2 antibodies, electoporated into the cytosol of fibroblasts, inhibited IL-1-induced ERK activation, actin filament assembly, and cell contraction, indicating a role for SHP-2 in these processes. In summary, our data indicate that SHP-2, a focal adhesion-associated protein, participates in IL-1-induced ERK activation likely via an adaptor function.     

Temporal control of NF-kappaB activation by ERK differentially regulates interleukin-1beta-induced gene expression

In cultured rat vascular smooth muscle cells, sustained activation of ERK is required for interleukin-1beta to persistently activate NF-kappaB. Without ERK activation, interleukin-1beta induces only acute and transient NF-kappaB activation. The present study examined whether the temporal control of NF-kappaB activation by ERK could differentially regulate the expression of NF-kappaB-dependent genes, including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), vascular cell adhesion molecule-1 (VCAM-1), and manganese-containing superoxide dismutase (Mn-SOD). Treatment of vascular smooth muscle cells with interleukin-1beta induced the expression of iNOS, COX-2, VCAM-1, and Mn-SOD in a time-dependent manner, but with different patterns. Either PD98059 or U0126, selective inhibitors of MEK, or overexpression of a dominant negative MEK-1 inhibited interleukin-1beta- induced ERK activation and the expression of iNOS and COX-2 but had essentially no effect on the expression of VCAM-1 and Mn-SOD. The expression of these genes was inhibited when NF-kappaB activation was down-regulated by MG132, a proteasome inhibitor, or by overexpression of an I-kappaBalpha mutant that prevented both the transient and the persistent activation of NF-kappaB. Inhibition of ERK did not affect interleukin-1beta-induced I-kappaBalpha phosphorylation and degradation but attenuated I-kappaBbeta degradation. Thus, although NF-kappaB activation was essential for interleukin-1beta induction of each of the proteins studied, gene expression was differentially regulated by ERK and by the duration of NF-kappaB activation. These results reveal a novel functional role for ERK as an important temporal regulator of NF-kappaB activation and NF-kappaB-dependent gene expression.     

Novel nuclear signaling pathway mediates activation of fibroblast growth factor-2 gene by type 1 and type 2 angiotensin II receptors

In bovine adrenal medullary cells synergistically acting type 1 and type 2 angiotensin II (AII) receptors activate the fibroblast growth factor-2 (FGF-2) gene through a unique AII-responsive promoter element. Both the type 1 and type 2 AII receptors and the downstream cyclic adenosine 1',3'-monophosphate- and protein kinase C-dependent signaling pathways activate the FGF-2 promoter through a novel signal-transducing mechanism. This mechanism, which we have named integrative nuclear FGF receptor-1 signaling, involves the nuclear translocation of FGF receptor-1 and its subsequent transactivation of the AII-responsive element in the FGF-2 promoter.     

Phospholipase D regulates myogenic differentiation through the activation of both mTORC1 and mTORC2 complexes

How phospholipase D (PLD) is involved in myogenesis remains unclear. At the onset of myogenic differentiation of L6 cells induced by the PLD agonist vasopressin in the absence of serum, mTORC1 complex was rapidly activated, as reflected by phosphorylation of S6 kinase1 (S6K1). Both the long (p85) and short (p70) S6K1 isoforms were phosphorylated in a PLD1-dependent way. Short rapamycin treatment specifically inhibiting mTORC1 suppressed p70 but not p85 phosphorylation, suggesting that p85 might be directly activated by phosphatidic acid. Vasopressin stimulation also induced phosphorylation of Akt on Ser-473 through PLD1-dependent activation of mTORC2 complex. In this model of myogenesis, mTORC2 had a positive role mostly unrelated to Akt activation, whereas mTORC1 had a negative role, associated with S6K1-induced Rictor phosphorylation. The PLD requirement for differentiation can thus be attributed to its ability to trigger via mTORC2 activation the phosphorylation of an effector that could be PKCα. Moreover, PLD is involved in a counter-regulation loop expected to limit the response. This study thus brings new insights in the intricate way PLD and mTOR cooperate to control myogenesis.     

Cutting edge: inducible costimulator protein regulates both Th1 and Th2 responses to cutaneous leishmaniasis.

The CD28 family member inducible costimulator protein (ICOS) has an important role in T cell differentiation and Ig class switching. To investigate the role of ICOS in vivo, ICOS-/- mice were infected s.c. with Leishmania mexicana. While wild-type mice developed large, cutaneous lesions, the growth of lesions and tissue histopathology was significantly delayed in ICOS-/- mice. ICOS-/- mice exhibited marked decreases in both Th1 and Th2 cytokine production and profound defects in L. mexicana-specific Ig isotype class switching to IgG1 and IgG2a and reduced total IgE levels. Our findings indicate that ICOS is a key regulator of both Th1 and Th2 responses and has a role in controlling cutaneous L. mexicana infection.     

Role of interleukins 1 and 2 on human thymocyte mitogen activation

The effects of d-penicillamine on proliferation and polyclonal activation of lymphocytes were studied in cultures of spleen cells from a variety of murine strains. Inclusion in serum-free or serum-containing medium of optimal concentrations of d-penicillamine resulted in the uptake of tritiated thymidine in a dose-dependent fashion, with both lower and higher doses causing less marked effects. The kinetic peak of these responses was found to occur at Day 2 of culture. Experiments examining the responsiveness of B-cell-enriched and T-cell-enriched populations demonstrated that d-penicillamine, like 2-mercaptoethanol, stimulates both types of cell. The magnitude of the response remained unchanged in populations depleted of adherent cells. In correlation with previous results seen for 2-ME, d-penicillamine was mitogenically active both in reduced and oxidized forms. The oxidized form failed to enhance the response to LPS, in contrast to the marked effect of the reduced form. Additionally, d-penicillamine failed to evoke a mitogenic response from B cells of CBA/N mice, a strain characterized by a deficit in the function of a particular set of mature B cells. Young mice from autoimmune strains responded to d-penicillamine as well as normal mice did. No relationship could be observed between responsiveness to d-penicillamine and the H-2 phenotype of the cell donor, and in A/J mice hyporesponsiveness was shown to be a function of their background rather than H-2. d-Penicillamine was found to function as a polyclonal B-cell activator, and to significantly enhance the primary humoral immune response to sheep erythrocytes in vitro. These immunomodulatory effects of d-penicillamine are discussed in relation to possible pathogenetic mechanisms of its spectrum of autoimmune side effects.     

PAN1/NALP2/PYPAF2, an inducible inflammatory mediator that regulates NF-kappaB and caspase-1 activation in macrophages

Genes encoding proteins with PYRIN/PAAD/DAPIN domains, a nucleotide binding fold (NACHT), and leucine rich repeats have recently been recognized as important mediators in autoimmune inflammatory disorders. Here we characterize the expression and function of a member of the PYRIN and NACHT domain (PAN) family, PAN1 (also known as NALP2 and PYPAF2). PAN1 protein expression is regulated by lipopolysaccharide (LPS) and interferons (IFNbeta and IFNgamma) in THP-1 macrophage cells. In gene transfection studies PAN1 manifests an inhibitory influence on NF-kappaB activation induced by various pro-inflammatory stimuli, including tumor necrosis factor TNFalpha and interleukin-1beta (IL-1beta). Gene transfer-mediated elevations in PAN1 protein also suppressed activation of IkappaB kinases induced by inflammatory cytokines. Conversely, reducing endogenous levels of PAN1 using small interfering RNA enhanced LPS-induced production of ICAM-1 (intercellular adhesion molecule 1), an NF-kappaB-dependent gene. We also show here that PAN1 binds via its PYRIN domain to ASC, an adapter protein involved in caspase-1 activation. This binding is disrupted by mutation of the alpha1 helix of ASC. In gene transfer experiments PAN1 enhances caspase-1 activation and IL-1beta secretion in collaboration with ASC. Conversely, reducing endogenous levels of PAN1 using small interfering RNA significantly reduced LPS-induced secretion of IL-1beta in monocytes. We propose that PAN1 functions as a modulator of the activation of NF-kappaB and pro-caspase-1 in macrophages.     

Differential nuclear localization of the major adenovirus type 2 E1a proteins.

The localization in infected and transformed cells of the two major adenovirus type 2 E1a proteins, of 289 and 243 amino acid residues, was studied with antisera raised against synthetic peptides or a TrpE-E1a fusion protein. Both E1a proteins were detected only in the nucleus of infected cells as determined by immunofluorescence analysis of cells infected with wild-type virus or with the mutants pm975 or dl1500, which produce, respectively, only the 289-residue or only the 243-residue E1a protein. However, the 289-residue protein was more tightly associated with the nucleus than was the 243-residue protein, as determined by the stability of nuclear fluorescence to different fixation procedures and by the use of radioimmunoprecipitation and Western blot analysis to analyze fractions extracted from the nucleus by detergent and other treatments. The latter experiments revealed that only the 289-residue protein, and only a fraction of that protein present in the nucleus, is associated with the nuclear matrix, both in infected HeLa cells and in the transformed human cell line 293.     

Aberrant activation of the interleukin-2 autocrine loop through the nuclear factor of activated T cells by nonleukemogenic human T-cell leukemia virus type 2 but not by leukemogenic type 1 virus

Human T-cell leukemia virus type 1 (HTLV-1) but not HTLV-2 is associated with adult T-cell leukemia. We found that HTLV-2 Tax2 protein stimulated reporter gene expression regulated by the interleukin (IL)-2 promoter through the nuclear factor of activated T cells (NFAT) in a human T-cell line (Jurkat). However, the activity of HTLV-1 Tax1 was minimal in this system. T-cell lines immortalized by HTLV-2 but not HTLV-1 constitutively exhibited activated NFAT in the nucleus and constitutively expressed IL-2 mRNA. Cyclosporine A, an inhibitor of NFAT activation, abrogated the induction of IL-2 mRNA in HTLV-2-immortalized T-cell lines and concomitantly inhibited cell growth. This growth inhibition was rescued by the addition of IL-2 to the culture. Furthermore, anti-IL-2 receptor antibodies significantly reduced the proliferation of HTLV-2-infected T-cell lines but not that of HTLV-1-infected cells. Our results suggest that Tax2 activates an IL-2 autocrine loop mediated through NFAT that supports the growth of HTLV-2-infected cells under low-IL-2 conditions. This mechanism would be especially important in vivo, where this autocrine mechanism establishes a nonleukemogenic life-long HTLV-2 infection. The results also suggest that differences in long-term cytokine production between HTLV-1 and HTLV-2 infection are another factor for the differences in pathogenesis.     

trans activation of granulocyte-macrophage colony-stimulating factor and the interleukin-2 receptor in transgenic mice carrying the human T-lymphotropic virus type 1 tax gene.

Three lines of transgenic mice carrying the human T-cell lymphotropic virus type 1 tax gene have previously been reported to develop neurofibromas composed of perineural fibroblasts (S. H. Hinrichs, M. Nerenberg, R. K. Reynolds, G. Khoury, and G. Jay, Science 237:1340-1343, 1987; M. Nerenberg, S. H. Hinrichs, R. K. Reynolds, G. Khoury, and G. Jay, Science 237:1324-1329, 1987). Tumors from these mice and tumor cell lines derived from them expressed high levels of tax RNA and protein. They also expressed high levels of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene as measured by proliferative responses of FD-CP1 target cells using conditioned media from tumor cells and by Northern (RNA) blot analysis of RNA from tumors and tumor cell lines. Although other tissues, such as salivary glands and muscles, in the transgenic mice also expressed high levels of tax, they did not express the gene for GM-CSF. This indicates that tissue-specific cellular factors, in addition to tax, are required for GM-CSF gene expression. Systemic effects of excessive GM-CSF production were demonstrated by infiltration of polymorphonuclear leukocytes into tumor tissues which are not necrotic, by peripheral granulocytosis, and by splenomegaly resulting from myeloid hyperplasia. The interleukin-2 (IL-2) receptor was also found to be expressed by the tumors and tumor cell lines as measured by IL-2-binding and cross-linking studies. This is the first demonstration that the IL-2 receptor can be activated by tax in a nonlymphoid cell type. These in vivo findings are consistent with other reports which have demonstrated in vitro cis-regulatory elements within the 5'-flanking regions of the genes for GM-CSF and the IL-2 receptor which are responsive to trans activation by the tax gene.     

The immediate-early gene product Egr-1 regulates the human interleukin-2 receptor beta-chain promoter through noncanonical Egr and Sp1 binding sites.

The interleukin-2 IL-2 receptor beta-chain (IL-2Rbeta) is an essential component of the receptors for IL-2 and IL-15. Although IL-2Rbeta is constitutively expressed by lymphocytes, its expression can be further induced by a number of stimuli, including phorbol 12-myristate 13-acetate (PMA). We have now characterized factors that bind to an enhancer region located between nucleotides -170 and -139 of the human IL-2Rbeta promoter. Both Sp1 and Sp3 bound to the 5' portion of this region, whereas a PMA-inducible factor (PIF) mainly bound to its 3' portion and bound to the Sp binding motifs as well. In Jurkat T cells, induction of PIF DNA binding activity was rapidly induced, required de novo protein synthesis, and was sustained at a high level for at least 23 h. Interestingly, PIF was constitutively activated in human T-cell leukemia virus type 1-transformed MT-2 cells. In this paper, we demonstrate that PIF is Egr-1 based on its recognition by anti-Egr-1 antisera in gel mobility shift assays, even though the IL-2Rbeta DNA binding motif differed substantially from the canonical Egr-1 binding site. In addition, Egr-1 bound to the Sp binding site. In Jurkat cells, both sites were required for maximal IL-2Rbeta promoter activity, and in HeLaS3 cells, transfection of Egr-1 could drive activity of a reporter construct containing both sites. Moreover, Sp1 and Egr-1 could form a complex with kinetics that correlated with the production of Egr-1 in Jurkat cells upon PMA stimulation. Thus, Sp1 and Egr-1 physically and functionally cooperate to mediate maximal IL-2Rbeta promoter activity.