Sp1 represses IL-2 receptor alpha chain gene expression

Sp1 is a DNA-binding protein that acts as a positive regulator of eukaryotic gene expression. The interleukin-2 receptor alpha chain (IL2R alpha) gene 5' regulatory region contains a single Sp1 consensus motif that overlaps a CArG box capable of binding serum response factor (SRF). The CArG box has previously been shown to be important for IL2R alpha gene expression. In this study, the results of competition experiments suggest that Sp1 and SRF compete for binding to the CArG region. Site-directed mutagenesis and transient transfection assays indicate that the IL2R alpha gene Sp1 serves the unusual role of repressing gene expression, most likely by competing for binding of nuclear factor(s) to the CArG box.     

Induction of colony stimulating factor in vivo by recombinant interleukin 1 alpha and recombinant tumor necrosis factor alpha 1

In response to a potent inflammatory challenge, such as Gram-negative endotoxin, a number of cytokines are induced that, in turn, mediate many of the pathophysiologic alterations associated with endotoxicity. In this study, we have observed two endotoxin-associated monokines, recombinant interleukin-1 alpha (rIL 1 alpha) and recombinant tumor necrosis factor alpha (rTNF alpha), to induce colony stimulating factor (CSF) in vivo. The CSF activities produced in response to rIL 1 alpha or rTNF alpha gave rise to a mixture of granulocyte-macrophage colonies and were induced in a dose- and time-dependent fashion, peaking within 3 hr of cytokine injection (preceding peak CSF induction by endotoxin by several hours). Combined injection of suboptimal concentrations of rIL 1 alpha and rTNF alpha were additive, and simultaneous injection of optimal concentrations of each failed to increase CSF levels over that observed with either cytokine alone. Unlike endotoxin, neither cytokine induced interferon in vivo. These findings extend our understanding of the cytokine cascade that is operative in an inflammatory response and may account for many of the observed hematopoietic alterations that accompany inflammation.     

Effects of platelet-activating factor, tumor necrosis factor, and interleukin-1alpha on the expression of apolipoprotein M in HepG2 cells

Apolipoprotein M (apoM) is a recently discovered human apolipoprotein predominantly present in high-density lipoprotein (HDL) in plasma, exclusively expressed in liver and in kidney. The function of apoM is yet unknown. The human apoM gene is located in the major histocompatibility complex class III region on chromosome 6. Because many genes located in this region are related to the immune response, we have investigated whether apoM might also be involved in the host inflammatory response. In this study we examined effects of the platelet-activating factor (PAF), tumor necrosis factor (TNF-alpha), and interleukin-1alpha (IL-1alpha) on apoM expression in a hepatoblastoma cell line, HepG2 cells. PAF significantly enhanced the apoM mRNA levels and the secretion of apoM in HepG2 cell cultures. The enhancement of apoM secretion is seen at a low concentration of PAF (2 ng/ml), whereas a high concentration of PAF increases both the apoM mRNA levels and apoM secretion. Neither TNF-alpha nor IL-1alpha influenced apoM mRNA level and secretion. Furthermore, Lexipafant, a PAF-receptor (PAF-R) antagonist significantly suppressed the mRNA level and the secretion of apoM in HepG2 cells in a dose-dependent manner. Neither PAF nor Lexipafant influenced the mRNA levels and the secretion of apoA-I, apoB and apoE in HepG2 cells, indicating that the effects of PAF or Lexipafant on the apoM production on hepatic cells are selective for apoM. The cellular mechanism of the effects of PAF or Lexipafant on apoM metabolism requires further investigations.     

Novel muteins of human tumor necrosis factor alpha

For chemical synthesis of a gene coding for human tumor necrosis factor alpha (TNF-alpha), DNA sequence predicted by the amino acid sequence of human TNF molecule was prepared. Codons were chosen according to the codon usage in Escherichia coli (E. coli). The 490 bp gene was assembled by enzymic ligation of 42 oligonucleotides and was cloned into a vector (pKK223-3) for high expression of active TNF-alpha in E. coli. With use of site-directed mutagenesis on this DNA, five different muteins of TNF-alpha were synthesized. TNF-M1 and TNF-M4 have deletions of His-73 and Gln-102, respectively. These deletions didn't cause loss of the cytotoxic activity against L929 cells. TNF-M5, which has a substitution of Asp-10 to Arg, had the similar cytotoxic activity to that of TNF-alpha. The cytotoxic spectra against several tumor cells were not changed by this substitution. TNF-M3 has an amino acid substitution of Glu-116 to His which occupies this position in human TNF-beta. This substitution didn't change the cytotoxicity. In addition, evidence was presented that the change of the carboxyl terminal residue doesn't always influence the cytotoxic activity of TNF-alpha. Many different muteins were also isolated by random mutagenesis with hydroxylamine-HCl. One of the muteins, which carries a mutation of His-15 to Tyr, lost the cytotoxic activity almost completely.     

Induction of human methionine adenosyltransferase 2A expression by tumor necrosis factor alpha. Role of NF-kappa B and AP-1

Two genes (MAT1A and MAT2A) encode for methionine adenosyltransferase (MAT), an essential cellular enzyme responsible for S-adenosylmethionine biosynthesis. MAT1A is expressed mostly in the liver, whereas MAT2A is widely distributed. We showed a switch from MAT1A to MAT2A expression in human hepatocellular carcinoma (HCC), which facilitates cancer cell growth. Using DNase I footprinting analysis, we previously identified a region in the MAT2A promoter protected from DNase I digestion in HCC. This region contains NF-kappa B and AP-1 elements, and the present study examined whether they regulate MAT2A promoter activity. We found nuclear binding of NF-kappa B and AP-1 to the MAT2A promoter increased in HCC. Tumor necrosis factor alpha (TNFalpha), which activates both NF-kappa B and AP-1, increased MAT2A expression in a dose- and time-dependent manner, binding of both NF-kappa B and AP-1 to the MAT2A promoter and MAT2A promoter activity, with the latter effect blocked by site-directed mutagenesis of the NF-kappa B and AP-1 binding sites. Blocking NF-kappa B with I kappa B super-repressor or AP-1 with dominant-negative c-Jun led to decreased basal MAT2A expression and prevented the TNF alpha-induced increase in MAT2A expression. Although blocking NF-kappa B had no influence on the ability of TNF alpha to increase AP-1 nuclear binding, blocking AP-1 with dominant-negative c-Jun prevented the TNF alpha-mediated increase in NF-kappa B binding. In conclusion, both NF-kappa B and AP-1 are required for basal MAT2A expression in HepG2 cells and mediate the increase in MAT2A expression in response to TNF alpha treatment. Increased trans-activation of these two sites also contributes to MAT2A up-regulation in HCC.     

NF-kappaB activation represses tumor necrosis factor-alpha-induced autophagy

Activation of NF-kappaB and autophagy are two processes involved in the regulation of cell death, but the possible cross-talk between these two signaling pathways is largely unknown. Here, we show that NF-kappaB activation mediates repression of autophagy in tumor necrosis factor-alpha (TNFalpha)-treated Ewing sarcoma cells. This repression is associated with an NF-kappaB-dependent activation of the autophagy inhibitor mTOR. In contrast, in cells lacking NF-kappaB activation, TNFalpha treatment up-regulates the expression of the autophagy-promoting protein Beclin 1 and subsequently induces the accumulation of autophagic vacuoles. Both of these responses are dependent on reactive oxygen species (ROS) production and can be mimicked in NF-kappaB-competent cells by the addition of H2O2. Small interfering RNA-mediated knockdown of beclin 1 and atg7 expression, two autophagy-related genes, reduced TNFalpha- and reactive oxygen species-induced apoptosis in cells lacking NF-kappaB activation and in NF-kappaB-competent cells, respectively. These findings demonstrate that autophagy may amplify apoptosis when associated with a death signaling pathway. They are also evidence that inhibition of autophagy is a novel mechanism of the antiapoptotic function of NF-kappaB activation. We suggest that stimulation of autophagy may be a potential way bypassing the resistance of cancer cells to anti-cancer agents that activate NF-kappaB.     

Molecular cloning and expression of human tumor necrosis factor and comparison with mouse tumor necrosis factor

U-937 cells, a monocytic line derived from a human histiocytic lymphoma, were induced for human tumor necrosis factor (TNF) secretion into the medium and were used for the preparation of TNF mRNA. Biological activity of the latter was quantified in a Xenopus laevis oocyte injection system. TNF mRNA was enriched by gradient centrifugation and this size-fractionated mRNA was used for synthesis of cDNA and inserted into the unique PstI site of pAT153. A recombinant plasmid containing human TNF cDNA was selected by colony hybridization using an internal fragment of a mouse TNF cDNA clone [Fransen, L., Mueller, R., Marmenout, A., Tavernier, J., Van der Heyden, J., Kawashima, E., Chollet, A., Tizard, R., Van Heuverswyn, H., Van Vliet, A., Ruysschaert, M. R. & Fiers, W. (1985) Nucleic Acids Res. 13, 4417-4429] as a probe. The sequence of this human TNF cDNA is in agreement with the one published by Pennica et al. [Pennica, D., Nedwin, G. E., Hayflick, J. S., Seeburg, P. H., Derynck, R., Palladino, M. A., Kohr, W. J., Aggarwal, B. B. & Goeddel, D. V. (1984) Nature (Lond.) 312, 724-729]. The 157-amino-acid-long mature sequence is about 80% homologous to mouse TNF and its hydrophilicity plot is also very similar, in spite of the apparent species specificity of TNF. In contrast to mouse TNF, it contains no potential N-glycosylation site. When compared to other cytokines, like IFN-beta, IFN-gamma, or IL-2, there is a remarkably high preference for G X C pairs in the third-letter positions. Expression of the TNF cDNA in monkey COS cells or in Escherichia coli gives rise to a protein having similar biological and serological properties as natural human TNF. A human genomic clone was also identified and sequenced; it was found to be in good agreement with the one recently published by Shirai et al. [Shirai, T., Yamaguchi, H., Ito, H., Todd, C. W. & Wallace, R. B. (1985) Nature (Lond.) 313, 803-806], except for some differences in the introns and 5'-untranslated region.     

Activation of Jun N-terminal kinase/stress-activated protein kinase pathway by tumor necrosis factor alpha leads to intercellular adhesion molecule-1 expression.

Tumor necrosis factor alpha (TNF-alpha) is a cytokine implicated in the pathogenesis of numerous chronic and acute inflammatory conditions. We have previously shown that mouse Sertoli cells respond to TNF-alpha by increasing interleukin-6 production and intercellular adhesion molecule-1 (ICAM-1) expression (1). In this cell type TNF-alpha activates the mitogen-activated protein kinase (MAPK) pathways p42/p44 MAPK, JNK/SAPK, and p38, the last of which is responsible for interleukin-6 production (1). To determine which MAPK signaling pathway is required for TNF-alpha induction of ICAM-1 expression, we have utilized the protein kinase inhibitor dimethylaminopurine, demonstrating that treatment of Sertoli cells with such compound significantly reduced ICAM-1 expression and JNK/SAPK activation. Moreover, dimethylaminopurine treatment increased the expression of MAPK phosphatase-2, providing a possible mechanism of action of this compound. By using agonist antibodies to p55 and to p75 TNF-alpha receptors and both human and mouse TNF-alpha, we demonstrate that both TNF receptors are expressed and that only the p55 receptor is involved in ICAM-1 expression. The p55 receptor activates all of the three pathways, whereas p75 failed to activate any of the MAPKs. Altogether our results demonstrate that TNF-alpha up-regulates ICAM-1 expression through the activation of the JNK/SAPK transduction pathway mediated by the p55 receptor.