The transcription factor NF-B plays an important role in the regulated expression of cytokines in human monocytes. A p100 subunit of NF-B has IB-like properties by sequestering the p65 transactivating subunit in the cytosol of cells. In transient transfection assays we demonstrated that p100 has an inhibitory effect on the NF-B-dependent IL-6 promoter activity. In view of this finding, we studied the regulation of the p100 subunit in human monocytes in response to LPS, the inflammatory cytokines IL-1 and TNF-␣ and lymphokines. The results demonstrate that LPS, IL-1, and TNF-␣ induce p100 expression at mRNA and protein level while IFN-␥, IL-3 and IL-4/IL-10 have no effect. The induction of p100 expression was shown to be mediated by a two-fold increase in the p100 transcription rate and a two-fold increase in p100 mRNA stability. Furthermore the p100 mediated upregulation was dependent on a tyrosine kinase dependent pathway rather than the protein kinase C pathway. NF-B is a complex of either p50 homodimers or a p50/p65 heterodimer. The latter is known to strongly autoregulate p100 transcription. We therefore examined the composition of NF-B induced by LPS vs the different lymphokines. LPSinduced NF-B showed a distinct p65 supershift whereas the composition of NF-B induced by different lymphokines did not show a change in p65. We conclude that the p100 subunit of the transcription factor NF-B is induced by different inflammatory mediators while lymphokines fail to induce p100 expression which may be caused by the induction of NF-B predominantly consisting of p50 homodimers.
N-Acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-L-alanyl-D-isoglutam yl-m- diaminopimelyl-D-alanine [G (Anh)MTetra], a naturally occurring breakdown product of peptidoglycan from bacterial cell walls, was studied for its ability to induce granulocyte colony-stimulating factor (G-CSF) mRNA and protein expression in human adherent monocytes. Resting monocytes did not express G-CSF mRNA or secrete G-CSF protein. In contrast, monocytes exposed to G(Anh)MTetra showed a dose-dependent increase in G-CSF mRNA accumulation, which correlates with the secretion of G-CSF protein. Maximal levels of G-CSF mRNA were reached within 2 h of activation. Expression of G-CSF was mediated by an increase in the stability of G-CSF transcripts rather than by an increase in the transcription rate of the G-CSF gene. Experiments with the protein synthesis inhibitor cycloheximide revealed that G(Anh)MTetra-induced G-CSF mRNA expression was independent of new protein synthesis. Furthermore, it was shown that the effect of G(Anh)MTetra was regulated by a protein kinase C-dependent pathway, whereas protein kinase A and tyrosine kinases were not involved. Finally, it was shown that G(Anh)MTetra also induced G-CSF mRNA expression in human endothelial cells. The data indicate that, besides lipopolysaccharide, other naturally occurring bacterial cell wall components are able to induce G-CSF expression in different hematopoietic cells.
Recently it has been demonstrated that in vivo application of interleukin-3 (IL-3) is associated with the release of IL-6. This observation suggests that the transcription factors triggered by IL-3 are in great homology with the transcription factors induced by lipopolysaccharide (LPS). The results of the present study with in vitro activated human monocytes demonstrate that IL-3 alone is incapable of inducing IL-6 mRNA, but primes monocytes to enhance the IL-6 mRNA expression when co-stimulated with LPS. The difference in effect between IL-3 and LPS might be related to our observation that IL-3 induces the p5O subunit of the transcription factor nuclear factor-kappa B (NF-Kappa B), whereas LPS appears to induce both the p5O as well as the p65 subunit of NF-kappa B, as demonstrated with RNA studies and electrophoretic mobility shift assays (EMSA). However, no difference was found with regard to the induction of activator protein-1 (AP-1) and NF-IL6 after treatment with IL-3 or LPS alone. Priming with IL-3 followed by LPS stimulation is associated with a reduced expression of NF-kappa B without changing the composition of the complex. In addition, a reduced expression of c-fos and c-jun mRNA was noticed, combined with a reduced DNA binding activity of AP-1. However, the expression of NF-IL6 was enhanced when priming with IL-3 followed by LPS. Since AP-1 has been suggested as negative regulator of the IL-6 gene expression, it is conceivable that, after priming with IL-3, the reduced DNA binding activity of AP-1, in conjunction with the increased DNA binding of NF-IL6, might result in a synergistic effect on IL-6 mRNA expression, when compared to stimulation with LPS alone.
In the present study we investigated the possible involvement of the mitogen-activated protein kinase family members extracellular-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) in mediating IL-6 gene expression in human monocytes, in particular their role in enhancing NF-κB activity. Freshly isolated monocytes treated with the protein phosphatase inhibitor okadaic acid secreted high levels of IL-6 protein, which coincided with enhanced binding activity of NF-κB as well as with phosphorylation and activation of the ERK1/2 and JNK proteins. The ERK pathway-specific inhibitor PD98059 inhibited IL-6 secretion from monocytes. Transient overexpression of inactive mutants of either Raf-1 or JNK1 showed that both pathways were involved in κB-dependent IL-6 promoter activity. By using PD98059, we demonstrated that the Raf1/MEK1/ERK1/2 pathway did not affect the DNA binding of NF-κB but, rather, acted at the level of transcriptional activity of NF-κB. Interestingly, it was shown that NF-κB-mediated gene transcription, both in the context of the IL-6 promoter as well as on its own, was dependent on both serine kinase activity and interaction with c-Jun protein. We conclude that okadaic acid-induced IL-6 gene expression is at least partly mediated through the ERK1/2 and JNK pathway-dependent activation of NF-κB transcriptional capacity. Our results suggest that the JNK pathway may regulate NF-κB-mediated gene transcription through its phosphorylation and activation of c-Jun.
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