Cyclosporin A sensitivity of the NF-kappa B site of the IL2R alpha promoter in untransformed murine T cells.

We have investigated the characteristics of IL2R alpha gene induction in untransformed murine T cells. Induction of IL2R alpha mRNA by TCR/CD3 ligands in a murine T cell clone and in short-term splenic T cell cultures was inhibited by protein synthesis inhibitors and by CsA. This result was contrary to previous observations in JURKAT T leukemia cells and human peripheral blood T cells, suggesting a difference in the mechanisms of IL2R alpha gene induction in these different cell types. The CsA sensitivity of IL2R alpha mRNA induction represented a direct effect on the TCR/CD3 response, and was not due to CsA-sensitive release of the lymphokines IL2 or tumour necrosis factor alpha (TNF alpha) and consequent lymphokine-mediated induction of IL2R alpha mRNA. The NF-kappa B site of the IL2R alpha promoter was essential for gene induction through the TCR/CD3 complex, and the induction of reporter plasmids containing multimers of this site was significantly inhibited by CsA. Northern blotting analysis indicated that while the p65 subunit of NF-kappa B was constitutively expressed and not appreciably induced upon T cell activation, mRNA for the p105 precursor of p50 NF-kappa B was induced in response to TCR/CD3 stimulation and this induction was sensitive to CsA. Electrophoretic mobility shift assays and antiserum against the p50 subunit of NF-kappa B indicated that p50 was a component of the inducible nuclear complex that bound to the IL2R alpha kappa B site. Appearance of the kB-binding proteins was insensitive to CsA at early times after activation (approximately 15 min), but was partially sensitive to CsA at later times. Based on these results, we propose that the NF-kappa B site of the IL2R alpha promoter mediates at least part of the CsA sensitivity of IL2R alpha gene induction in untransformed T cells, possibly because de novo synthesis of p105 NF-kappa B is required for sustained IL2R alpha expression.     

Induction of nuclear NF-kappa B DNA binding activity after exposure of lymphoid cells to soluble tax1 protein

We demonstrate that purified HTLV-I Tax1 protein can be taken up by 70Z/3 lymphoid cells and localized in both the nuclear and cytoplasmic compartments. Introduction of the Tax1 protein into the growth medium of 70Z/3 cells resulted in the rapid and transient induction of NF-kappa B binding activity in the nuclear fraction. Tax1 activation of NF-kappa B was not sensitive to either staurosporin or prolonged stimulation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, suggesting that Tax1-dependent NF-kappa B activation did not require the protein kinase C pathway. Purified Tax1 did not directly increase NF-kappa B binding activity in 70Z/3 cytoplasmic extracts, suggesting that NF-kappa B induction may require cellular factors. Western blot and competitive radioimmunoassays demonstrated that Tax1 protein was present in the tissue culture media of HTLV-I-transformed cell lines. These results show that extracellular Tax1 may regulate cellular gene expression in noninfected cells.     

Stimulation of interferon and cytokine gene expression by imiquimod and stimulation by Sendai virus utilize similar signal transduction pathways.

The imidazoquinolineamine derivative 1-(2-methyl propyl)-1H-imidazole [4,5-c]quinoline-4-amine (imiquimod) has been shown to induce alpha interferon (IFN-alpha) synthesis both in vivo and in peripheral blood mononuclear cells in vitro. In this study, we show that, in these cells, imiquimod induces expression of several IFNA genes (IFNA1, IFNA2, IFNA5, IFNA6, and IFNA8) as well as the IFNB gene. Imiquimod also induced the expression of interleukin (IL)-6, IL-8, and tumor necrosis factor alpha genes. Expression of all these genes was transient, independent of cellular protein synthesis, and inhibited in the presence of tyrosine kinase and protein kinase C inhibitors. Infection with Sendai virus led to expression of a similar set of cytokine genes and several of the IFNA genes. Imiquimod stimulates binding of several induction-specific nuclear complexes: (i) the NF-kappa B-specific complexes binding to the kappa B enhancer present in the promoters of all cytokine genes, but not in IFNA genes, and (ii) the complex(es) binding to the A4F1 site, 5'-GTAAAGAAAGT-3', conserved in the inducible element of IFNA genes. These results indicate that imiquimod, similar to viral infection, stimulates expression of a large number of cytokine genes, including IFN-alpha/beta, and that the signal transduction pathway induced by both of these stimuli requires tyrosine kinase and protein kinase activity.