Smad3 is acetylated by p300/CBP to regulate its transactivation activity.

Smad proteins are crucial for the intracellular signaling of transforming growth factor-beta (TGF-beta). Upon their receptor-induced activation, Smad proteins are phosphorylated and translocated to the nucleus to activate the transcription of a select set of target genes. Here, we show that the co-activator p300/CBP bound and acetylated Smad3 as well as Smad2 in vivo, and that the acetylation was stimulated by TGF-beta. A major acetylation site of Smad3 by p300/CBP is Lys-378 in the MH2 domain (Smad3C) known to be critical for the regulation of transcriptional activity. Replacement of Lys-378 with Arg decreased the transcriptional activity of GAL4-Smad3C in a luciferase assay. Moreover, p300/CBP potentiated the transcriptional activity of GAL4-Smad3C, but not the acetylation-resistant GAL4-Smad3C(K378R) mutant. These results suggest that acetylation of Smad3 by p300/CBP regulates positively its transcriptional activity.

Synthesis of a set of di- and tri-sulfated galabioses.

Among cell-adhesion molecules, L-selectin recognizes sulfated sLe(x) with relatively low affinity. Here, we aimed at artificial mimics by synthesizing a set of di- and tri-sulfated galabioses, which may surpass the affinity of sulfated sLe(x). As a strategy to obtain 3',6',6-tri-O-sulfogalabioses, regioselective reductive cleavage of 4,6- and 4',6'-di-O-benzylidenegalabioses was employed. Two suitably protected galactose precursors were conjugated to yield alpha and beta anomers (48 and 18%, respectively) by using a pentenyl galactoside donor and iodinium di-sym-collidine perchlorate as the catalyst. For synthesizing the 3',6-di-O-sulfogalabiose, however, a trichloroacetimidate donor was superior (52%) to the pentenyl one (30%).

CHOP, a basic leucine zipper transcriptional factor, contributes to the antiproliferative effect of IL-1 on A375 human melanoma cells through augmenting transcription of IL-6.

Interleukin-1 (IL-1) inhibits the proliferation of A375 human melanoma cells. We have demonstrated previously that p38 mitrogen-activated protein kinase (MAPK) mediated the antiproliferative effect of IL-1 partially through the downregulation of activity and protein level of ornithine decarboxylase (ODC). In this study, we investigated the role of CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), one of the p38 MAPK target transcriptional factors. The mRNA level of CHOP was not affected by IL-1 treatment in A375-6 cells. Unexpectedly, CHOP was constitutively phosphorylated, and IL-1 or p38 MAPK inhibitor, SB203580, did not affect the phosphorylation level. However, A375-6 cells exhibited enhanced sensitivity to IL-1 by transfecting CHOP expression plasmid and reduced sensitivity to IL-1 by antisense CHOP mRNA expression plasmid. Furthermore, CHOP appeared to regulate positively IL-6 production at the transcriptional level. The experiments using CHOP muteins revealed that dimerization ability - but not p38 MAPK-dependent phosphorylation or DNA binding activity - is important for the IL-6 inducing activity of CHOP. These results indicate that CHOP contributes to the IL-1 growth-inhibitory signal through augmenting IL-6 production.

Activation of two distinct anti-proliferative pathways, apoptosis and p38 MAP kinase-dependent cell cycle arrest, by tumor necrosis factor in human melanoma cell line A375.

The proliferation of human melanoma cell line A375-6 cells is inhibited by several cytokines, including interleukin-1 (IL-1). A375-R8 cells, a subclone of A375-6, are resistant to IL-1-induced growth inhibition. The proliferation of both cell lines is inhibitable by tumor necrosis factor (TNF). In this study, we characterized the mechanisms of TNF-induced growth inhibition. TNF-induced growth inhibition in both cell lines was partially suppressed by a selective p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580), whereas a combination of SB203580 and Z-VAD-fmk, an inhibitor for a wide range of caspases, completely blocked TNF-induced growth inhibition, indicating that TNF-induced growth inhibition is mediated by both p38 MAPK and caspases. However, Z-VAD-fmk alone suppressed TNF-induced growth inhibition in A375-R8, but not A375-6, cells, suggesting that there may exist a TNF-induced anti-apoptotic mechanism in A375-6 cells which is lost or mutated in A375-R8 cells. Evidence in support of this notion includes (1) TNF-induced apoptosis only in A375-R8, but not A375-6 cells; (2) cycloheximide enabled TNF to induce apoptosis even in A375-6 cells; and (3) somatic hybrid cells between A375-6 and A375-R8 cells are resistant to TNF-induced apoptosis. Since TNF-induced NF-kappa B activation, cell cycle arrest, RB dephosphorylation, and E2F downregulation are indistinguishable in both cell lines, none of these factors is likely to be involved in the TNF-induced anti-apoptotic mechanism in A375-6 cells. Our results indicate that TNF activates two distinct anti-proliferative pathways including p38 MAPK-dependent cell cycle arrest and caspase-mediated apoptosis, as well as an anti-apoptotic mechanism in melanoma cells.

Interleukin-1 or tumor necrosis factor-alpha augmented the cytotoxic effect of mycobacteria on human fibroblasts: application to evaluation of pathogenesis of clinical isolates of Mycobacterium tuberculosis and M. avium complex.

Mycobacteria-induced in vitro events reflecting human tuberculosis can contribute to the evaluation of the pathogenesis of Mycobacterium tuberculosis (MTB). In this study, we propose such an in vitro method based on live mycobacteria-induced cytotoxicity to human cell lines. When human lung-derived normal fibroblast cell line MRC-5 was infected with various strains of mycobacteria (M. tuberculosis H(37)Rv and H(37) Ra, Mycobacterium avium 427S and 2151SmO, and Mycobacterium bovis BCG Pasteur and Tokyo), the fibroblasts were killed by mycobacteria according to the degree of virulence. Other human originated macrophage (U-937, THP-1), myeloid (HL-60), and epithelial carcinoma (A549) cell lines exhibited a similar cytotoxic response to virulent mycobacteria. MRC-5 was most susceptible to virulent mycobacteria among various human cell lines examined. The cytotoxicity was enhanced by the proinflammatory cytokines, interleukin-1 (IL-1) and tumor necrosis factor-a (TNF-alpha), which in the absence of mycobacteria stimulate the growth of normal human fibroblasts. This in vitro evaluation system was applied to clinical isolates of drug-sensitive MTB (DS-MTB), drug-resistant MTB (DR-MTB) including multidrug-resistant (MDR-MTB), and M. avium complex (MAC). MTB strains (n = 24) exhibited strong cytotoxic activity, but MAC strains (n = 5) had only weak activity. Furthermore, there was no significant difference in cytotoxicity between DS-MTB (n = 11) and DR-MTB (n = 13). Collectively, these results suggest that this new in vitro system is useful for evaluating the pathogenesis of mycobacteria and that there was no difference in the pathogenesis between drug-susceptible and drug-resistant clinical isolates.

Endotoxin and cytokine regulation of toll-like receptor (TLR) 2 and TLR4 gene expression in murine liver and hepatocytes.

Toll-like receptor (TLR) 2 and TLR4 are members of the interleukin-1 receptor (IL-1R) family and transduce similar signals as IL-1R in response to bacteria and bacterial components. In this study, we investigated the regulation of their gene expression in murine tissues, especially in the liver and hepatocytes. When mice were administered lipopolysaccharide (LPS), TLR2 mRNA was upregulated in the brain, heart, lung, liver, and kidney. In contrast, it was downregulated in the spleen. TLR4 mRNA was decreased in the brain. In the heart and lung, it increased, and it was not affected in the liver, kidney, and spleen. TLR mRNA was further analyzed in the liver and hepatocytes. Like LPS treatment, administration of IL-1, IL-6, or tumor necrosis factor (TNF) upregulated TLR2 mRNA. However, none of them affected the TLR4 mRNA level. In primary cultured hepatocytes, TLR2 mRNA was upregulated by LPS, IL-1, or TNF but not by IL-6 or dexamethasone. None of them affected TLR4 mRNA expression. Similar responses were observed in the murine hepatoma cell line Hepa 1-6. These results suggest that in infection with gram-negative bacteria, LPS and proinflammatory cytokines differentially regulate gene expression of TLR2 and TLR4 in murine hepatocytes, which may lead to pathologic and host defense reactions in the liver.

Comparison of anti-tumor responses against TL positive lymphoma induced by skin grafting and dendritic cell immunization.

When the skin of Tg.Con.3-1 transgenic mice expressing the TL (thymus leukemia) antigen in most tissues is grafted on syngeneic C3H mice, it is rejected, and a cytotoxic T cell (CTL) response against the TL antigen is induced. In this study, we first demonstrated that growth of TL positive lymphoma is suppressed in mice immunized by skin grafting. Immunization with bone marrow derived dendritic cells (DCs) from Tg.Con.3-1, was also found to be associated with an anti-tumor response, but less potent than skin grafting. Relative CTL precursor frequency with DC immunization was also approximately only one third that of skin grafting. The numbers of IFN-gamma producing cells in responder CD8 and CD4 T cell populations were higher with DC immunization than with skin grafting. However, DC immunization seems to induce non-specific immune responses, as re-stimulation with TL negative C3H spleen cells resulted in induction of almost half the number observed with TL positive cells. Thus, the actual number of IFN-gamma producing cells in specific responses to TL is not necessarily larger than with skin grafting immunization. The present results altogether suggest that DC immunization is capable of inducing an anti-tumor reaction, but also possibly unwanted immune responses. In vitro monitoring of specific and non-specific responses in the immune system, thus, is of particular importance for future development of cancer immunotherapy.

Dietary docosahexaenoic acid but not eicosapentaenoic acid suppresses lipopolysaccharide-induced interleukin-1 beta mRNA induction in mouse spleen leukocytes.

Mice were fed a diet supplemented either with beef tallow (BT), BT plus ethyl eicosapentaenoate (EPA) or BT plus ethyl docosahexaenoate (DHA) for 9 weeks. EPA and DHA supplementation increased the content of the respective fatty acid in spleen leukocyte lipids, which was associated with the reduction in the arachidonate content. IL-1beta mRNA induction upon lipopolysaccharide (LPS) stimulation in spleen leukocytes in the DHA diet group was significantly lower than in the BT diet group, but the EPA diet was without any significant effect. The amount of prostaglandin E2 (PGE2) released from LPS-stimulated spleen leukocytes was significantly lower in both the EPA and DHA groups than in the BT group. Thus, dietary EPA and DHA inhibited arachidonate metabolism similarly but had different effects on IL-1beta mRNA induction in mouse spleen leukocytes.

Glycosylated human interleukin-1alpha, neoglyco IL-1alpha, coupled with N-acetylneuraminic acid exhibits selective activities in vivo and altered tissue distribution.

In order to study the effect of glycosylation on its biological activities and to develop IL-1 with less deleterious effects, N-acetylneuraminic acid (NeuAc) with C9 spacer was chemically coupled to human recombinant IL-1alpha. NeuAc-coupled IL-1alpha (NeuAc-IL-1alpha) exhibited reduced activities in vitro and receptor-binding affinities by about ten times compared to IL-1alpha. In this study, we examined a variety of IL-1 activities in vivo. NeuAc-IL-1alpha exhibited a marked reduction in the activity to up-regulate serum IL-6, moderate reduction in the activities to up-regulate serum amyloid A and NOx. However, it exhibited comparable activities as IL-1alpha to down-regulate serum glucose and to improve the recovery of peripheral white blood cells from myelosuppression in 5-fluorouracil-treated mice. In addition, tissue level of NeuAc-IL-1alpha was high compared to IL-1alpha. These results indicate that coupling with NeuAc enabled us to develop neo-IL-1 with selective activities in vivo and enhanced tissue level.

Human interleukin-1 receptor antagonist: large-scale expression in Bacillus brevis 47-5Q.

Interleukin-1 receptor antagonist (IL-1RA) has been used as a tool to study the biologic activity of IL-1 and as a possible therapeutic substance for inflammatory disease. To perform in vivo study, however, large quantities of IL-1RA are required. Bacillus brevis strains secrete large amounts of protein but little protease into the medium. Using B. brevis 47-5Q, we developed a large-scale expression system of human IL-1RA (HuIL-1RA). The bacteria secreted HuIL-1RA into the culture medium at very high levels, approximately 200 mg/L. The protein was isolated in one-step purification with monoclonal antibody (mAb) against HuIL-1RA. The IL-1RA molecule was determined to be functionally active by the inhibiting assay of HuIL-1-induced cell proliferation in a mouse T cell line, D10N4M.

Alteration of the cellular response to interleukin-1 beta by SV40 large T antigen in rheumatoid synovial fibroblasts.

The large T antigen of SV40 (LT) has been widely used to immortalize primary cells for various studies. In this study, synovial fibroblasts of a patient from rheumatoid arthritis (RA) were transformed with LT gene to analyze the effect of SV40-mediated transformation on the production of cytokines, such as IL-6, IL-8, and GM-CSF, that are under the control of interleukin-1 beta (IL-1 beta), a physiological inducer of nuclear factor kappa B (NF-kappa B). It was noted that the basal levels of GM-CSF and IL-8 were upregulated, whereas that of IL-6 was downregulated. Moreover, the extents of induction of these cytokines in response to IL-1 beta were markedly downregulated in synovial fibroblasts transformed by LT as compared from parental cells. Although IL-1 beta could translocate NF-kappa B to the nucleus in all cells, some of the transformed cells exhibited nuclear translocation of NF-kappa B even before the stimulation with IL-1 beta, suggesting that transformation of LT resulted in the constitutive activation of NF-kappa B, either directly or indirectly. In order to examine whether LT downregulate the kappa B-dependent gene expression, we performed the transient luciferase gene expression assay. We found that cotransfection of LT did not downregulate the kappa B-dependent gene expression that was stimulated with L-1 beta. These observations suggest that the apparent inhibitory effect of LT on the IL-1-induced expression of cytokines may not be through its direct action on the NF-kappa B transactivation.

Migration of exogenous immature hematopoietic cells into adult mouse brain parenchyma under GFP-expressing bone marrow chimera.

Bone marrow transplantation with GFP-expressing cells from GFP-transgenic mice resulted in migration of GFP-positive cells into peripheral tissues and brain parenchyma. Most of these cells were observed as colony-like clusters. GFP-positive clusters in the brain were stained by antibody for ER-MP12, but those in the peripheral tissues were not. Since ER-MP12 antigen has been reported as a marker for murine early-stage myeloid precursor, this might suggest that some parts of phagocytic cells in the brain parenchyma such as microglia are derived from undifferentiated pluripotent hematopoietic cells.

Tyrosine kinase is essential for the constitutive expression of type I interleukin-1 receptor in human fibroblast cells.

The effect of genistein, an inhibitor of tyrosine kinase, on the constitutive expression of type I interleukin-1 receptor (IL-1RI) was examined in the human lung fibroblast cell line TIG-1, which has been shown to express only type I IL-1R. Genistein inhibited the 125I-labeled IL-1alpha binding to TIG-1 cells in both a time and dose dependent manner. Scatchard plot analysis revealed that the number of IL-1RI decreased with no change in binding affinity. Genistein did not affect the level of IL-1RI mRNA, and cycloheximide did not inhibit the down-regulatory effect of genistein. These results indicate that genistein inhibits IL-1RI expression, not at the transcriptional level, but at the post-translational level. IL-1RI expression, IL-1R associated kinase (IRAK) activity and IL-1-induced-IL-6 production were all down-regulated by pretreatment with genistein. These findings indicate that tyrosine kinase activity is essential for the constitutive expression of functional IL-1RI.

Antiproliferative effect of IL-1 is mediated by p38 mitogen-activated protein kinase in human melanoma cell A375.

The role of p38 mitogen-activated protein kinase (MAPK) in IL-1-induced growth inhibition was investigated using IL-1-sensitive human melanoma A375-C2-1 cells and IL-1-resistant A375-R8 cells. In both cells, p38 MAPK was activated by IL-1. A selective inhibitor for p38 MAPK, SB203580, almost completely recovered the IL-1-induced growth inhibition in A375-C2-1 cells. IL-1-induced IL-6 production was also suppressed by SB203580. However, the reversal effect of SB203580 was not due to the suppression of IL-6 production because the SB203580 effect was still observed in the presence of exogenous IL-6. Down-regulation of ornithine decarboxylase (ODC) activity as well as its protein level has been shown to be essential for IL-1-induced growth inhibition. SB203580 also reversed the IL-1-induced down-regulation of ODC activity and intracellular polyamine levels without affecting ODC mRNA levels in A375-C2-1 cells. In IL-1-resistant R8 cells, however, IL-1 only slightly suppressed ODC activity. In A375-C2-1 cells, the mRNA expression level of antizyme (AZ), a regulatory factor of ODC activity, has been shown to be up-regulated by IL-1. IL-1-induced up-regulation of AZ mRNA level was not affected by SB203580. These findings demonstrate that p38 MAPK plays an important role in IL-1-induced growth inhibition in A375 cells through down-regulating ODC activity without affecting the level of ODC mRNA and AZ mRNA. In IL-1-resistant A375-R8 cells, IL-1 signaling pathway is deficient between p38 MAPK activation and down-regulation of ODC activity.

Serotonin derivative, N-(p-Coumaroyl)serotonin, isolated from safflower (Carthamus tinctorius L.) oil cake augments the proliferation of normal human and mouse fibroblasts in synergy with basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF).

N-(p-Coumaroyl)serotonin (CS) with antioxidative activity is present in safflower oil. We have reported that CS inhibits proinflammatory cytokine generation from human monocytes in vitro. As reactive oxygen species (ROS) affect cell proliferation, in this study the effect of CS on the proliferation of various cell types was examined. CS augments the proliferation of normal human and mouse fibroblast cells. The cells continue to proliferate in the presence of CS and form a transformed cell-like focus without transformation. CS, however, does not augment the proliferation of other cell types, either normal or tumor cells. CS augments the proliferation of fibroblasts in synergy with basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF), but not with acidic FGF(aFGF) or platelet-derived growth factor (PDGF). This study using synthesized derivatives of CS reveals that the growth-promoting activity is not due to antioxidative activity. These findings indicate that CS is a natural compound with unique growth-promoting activity for fibroblasts.

Augmentation of type I IL-1 receptor expression and IL-1 signaling by IL-6 and glucocorticoid in murine hepatocytes.

IL-1 signal is transduced through type I receptor (IL-1RI). We have recently reported that LPS augments IL-1RI mRNA expression in the hepatocytes of mice in vivo, and the augmentation is mediated by the interaction of IL-1, IL-6, and glucocorticoid (GC). In this study, we examined whether IL-1RI mRNA expression level in the hepatocytes reflects those of cell surface molecule and IL-1 signaling. When primary cultured murine hepatocytes were treated with dexamethasone (Dex) or IL-6, these two reagents synergistically up-regulated IL-1RI mRNA expression in the cells. 125I-labeled IL-1 binding experiment showed that the level of binding was also up-regulated by the treatment with Dex and IL-6. Scatchard analysis revealed that the number of IL-1R increased. The increased binding of IL-1 was completely inhibited by an Ab against murine IL-1RI, indicating that Dex and IL-6 augmented the expression of cell surface IL-1RI molecule. When hepatocytes were pretreated with Dex and IL-6, the activation of IL-1R-associated kinase was augmented in response to IL-1, indicating that IL-1 signaling was also augmented. In addition, IL-1 treatment following administration of the combination of Dex and IL-6 into mice markedly increased the serum level of serum amyloid A. These results indicate that GC and IL-6 augment the expression of cell surface IL-1RI in hepatocytes, as well as IL-1 signaling and IL-1R-associated kinase activation, through up-regulation of IL-1RI mRNA level, which represents a novel regulatory network between IL-1, GC, and IL-6.

Synthesis of glycosylated human interleukin-1alpha, neoglyco IL-1alpha, coupled with N-acetylneuraminic acid.

In order to develop glycosylated cytokine, recombinant human IL-1alpha was chemically modified with N-acetylneuraminic acid (NANA). NANA with C9 spacer, 8-(hydrazinocarbonyl)octyl 5-acetamido-3, 5-dideoxy-D-glycero-alpha-D-galacto-2-nonulo-pyranosidonic acid potassium salt (6), was synthesized by glycosylation of C9 spacer, 8-[2-N-(benzyloxycarbonyl)hydrazinocarbonyl]octanol, with methylthio glycoside derivatives of NANA in the presence of molecular sieves 3A and methyl (methylthio)sulfonium trifrate in propionitrile, followed by separation of a and beta anomers with a column chromatography and deprotection. Compound 6 was coupled to IL-1alpha by the acyl azide method. The glycosylated IL-1 was purified by anion-exchange chromatography, and NANA coupled to IL-1 was confirmed by oxidation with NaIO4. Based on the molecular weight average number of carbohydrate molecules introduced per molecule of IL-1alpha was estimated to be 2.9.

In vitro biological activities of glycosylated human interleukin-1alpha, neoglyco IL-1alpha, coupled with N-acetylneuraminic acid.

In the previous study, N-acetylneuraminic acid (NANA) with C9 spacer was chemically coupled to human recombinant (rh) IL-1alpha in order to study the effect of glycosylation on its biological activities, and to develop IL-1 with less deleterious effects. In this study we examined a variety of IL-1 activities in vitro, including proliferative effect on T cells, antiproliferative effect on myeloid leukemic cells and melanoma cells, stimulatory effects on IL-6 synthesis by melanoma cells and PGE2 synthesis by fibroblast cells. NANA-introduced IL-1alpha (NANA-IL-1alpha) exhibited reduced activities about ten times compared with original IL-1alpha in all the activities performed in vitro. The competitive binding of 125I-IL-1alpha to mouse T cells and pre-B cells with unlabeled IL-1alphas suggests the decrease in binding affinities of NANA-IL-1alpha to both type I and type II IL-1 receptors. Therefore, reduced activities of NANA-IL-1alpha well correlated with the decrease in its receptor binding affinities.

Serotonin derivative, N-(p-coumaroyl) serotonin, inhibits the production of TNF-alpha, IL-1alpha, IL-1beta, and IL-6 by endotoxin-stimulated human blood monocytes.

We have reported that N-(p-coumaroyl) serotonin (CS) and its derivatives with antioxidative activity are present in safflower seeds. As reactive oxygen species (ROS) are implicated in the signaling of lipopolysaccharide (LPS), we examined whether CS has a suppressive effect on inflammatory cytokine generation from human monocytes in vitro. CS at 50-200 microM reduced tumor necrosis factor (TNF), interleukin-1 (IL-1), and IL-6 activities in the culture supernatants from LPS-stimulated human blood monocytes without cytotoxicity. ELISA assay revealed that the production of TNF-alpha, IL-1alpha, IL-1beta, and IL-6 was inhibited by CS. Northern blot analysis showed that LPS-induced expression of these cytokine mRNA in monocytes was suppressed by CS. NF-kappaB activation was also inhibited by CS. These findings indicate that CS has a suppressive effect on proinflammatory cytokine production from monocytes, and this effect is based in part on the suppression of cytokine mRNA expression through inhibition of NF-kappaB activation.