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.