IL-8 gene expression is induced by a wide variety of agents including cytokines, growth factors, bacterial and viral products, oxidants and others (Roebuck, 1999
). Induction of IL-8 gene expression is subject to both transcriptional and posttranscriptional regulation in a cell/tissue- and stimulus-specific manner (Roebuck, 1999
) (Hoffmann et al., 2002
). We found that in H441 cells TNF-α induced IL-8 mRNA levels primarily by increasing gene transcription. In A549 lung epithelial cells TNF-α was found to activate IL-8 promoter activity via recruitment of NF-κB to a TNF-α response element consistent with a role for transcriptional mechanisms (Brasier et al., 1998
) in the induction of IL-8 gene expression in lung epithelial cells. A relatively short sequence of DNA spanning −133/+41 bp is necessary and sufficient for the basal and TNF-α induction of IL-8 promoter activity (Yasumoto et al., 1992
; Brasier et al., 1998
). The transcriptional response region contains binding sites for NF-IL-6, NF-κB and AP-1 that act independently and synergistically to activate IL-8 promoter in response to stimulatory agents in a cell type-specific manner [reviewed in (Roebuck, 1999
)]. In this study we found that TNF-α and S1-P induced IL-8 expression by increasing gene transcription and without altering the stability of IL-8 mRNA. However, regulation of IL-8 mRNA stability has been found to play major roles in the control of IL-8 expression in different cells. Nitric oxide, lipopolysaccharide, adenovirus and Shiga toxin increase IL-8 mRNA expression in lung epithelial cells, THP cells, fibroblasts and A549 cells, respectively, by increasing the stability of IL-8 mRNA (Leland Booth and Metcalf, 1999
; Thorpe et al., 2001
; Ma et al., 2004
; Sparkman and Boggaram, 2004
). The half-life of IL-8 mRNA in untreated cells was in the range of 0.5 – 2 h and increased by several fold depending on the cell type and the stimulus.
Sphingomyelin metabolites are increasingly recognized as important mediators of inflammation in the lung, and ceramide has emerged as a putative lipid mediator in TNF-α signaling. Despite the important roles that sphingomyelin metabolites play in TNF-α signaling, little is known about their involvement in the TNF-α induction of IL-8 gene expression in lung cells. Our data showed that among the sphingomyelin metabolites, S1-P but not ceramide or sphingosine induced IL-8 mRNA levels and IL-8 secretion. Consistent with the lack of significant effects of ceramide, inhibition of ceramidase to increase intracellular ceramide levels did not increase IL-8 mRNA levels. We found that the inductive effect of S1-P on IL-8 level in the medium was significantly greater than its effects on IL-8 mRNA levels. Similarly we found that although ceramide did not increase IL-8 mRNA levels it caused a small increase in IL-8 levels. The observed discrepancy between IL-8 mRNA and IL-8 levels in S1-P and ceramide treated cells point to possible translational and/or posttranslational regulation of IL-8 expression. Together our data indicated that elevated intracellular S1-P generated as a result of activation of sphingosine kinase partly mediates TNF-α induction of IL-8 gene expression in H441 cells. Intracellular S1-P levels can also be modulated by the actions of sphingosine-phosphate lyase that catalyzes the irreversible cleavage of S1-P (Reiss et al., 2004
). Whether TNF-α regulates sphingosine-phosphate lyase expression and/or activity to modulate intracellular S1-P levels in H441 cells is not known. In human umbilical vein endothelial cells (HUVEC) TNF-α was found to induce the expression of E-selectin and vascular adhesion molecule-1 (VCAM-1) via increased generation of S1-P by the activation of sphingosine kinase (Xia et al., 1998
). Although TNF-α induced sphingomyelin breakdown and ceramide generation, ceramide failed to mimic the effects of TNF-α to induce E-selectin and VCAM-1 expression.
Changes in the levels of sphingolipid metabolites can occur via coordinate activation of the entire cascade of sphingolipid metabolizing enzymes as in the case of oxidized low density lipoprotein induced mitogenesis of smooth muscle cells (Auge et al., 1999
) or via selective activation of one of the enzymes of the pathway as in the case of TNF-α activation of sphingosine kinase to inhibit apoptosis in HUVEC cells (Xia et al., 1999
). In some cells the activation of ceramidase may be so robust that the levels of sphingosine and S1-P are vastly increased in the absence of substantial increases in ceramide levels (Kolesnick, 2002
). Our experiments showed that exogenous ceramide and inhibition of ceramidase to increase intracellular ceramide levels failed to increase IL-8 mRNA and protein levels suggesting that the inability of ceramide to increase IL-8 may not be due to low intracellular ceramide levels.
TNF-α activates sphingomyelin hydrolysis to increase intracellular ceramide levels in lung (Ryan et al., 2003
) and lung cells (Vivekananda et al., 2001
). Sphingolipids generated in response to TNF-α inhibit the expression of CTP:phosphocholine cytidyltransferase (CCTα) (Vivekananda et al., 2001
) the rate-limiting enzyme involved in the synthesis of phosphatidylcholine, an important component of lung surfactant, leading to the perturbation of surfactant lipid composition. These findings suggest that perturbations in surfactant lipid synthesis contribute to lung injury associated with inflammation and that sphingolipids play important roles in mediating these effects. Our findings of the inductive effects of S1-P on IL-8 mRNA levels reveal yet another pathway that potentially contributes to lung injury in inflammation. Increases in IL-8 levels can lead to increased recruitment of neutrophils into the lung contributing to lung injury.
The molecular mechanisms and signal transduction pathways by which S1-P induces the expression of IL-8 in H441 lung epithelial cells remains to be investigated. Our studies showed that S1-P increased AP-1 DNA binding activity in H441 cells suggesting that increase in AP-1 binding may be required for S1-P increase of IL-8 gene transcription. S1-P is known to enhance the DNA binding activity of AP-1 (Su et al., 1994
MAPKs regulate IL-8 expression and secretion in a variety of cells including lung epithelial cells (Hashimoto et al., 1999
) (Matsumoto et al., 1998
) and MAPK regulation of IL-8 expression occurs via transcriptional and mRNA stabilization mechanisms (Holtmann et al., 1999
). Transcription factors NF-κB and AP-1 play central roles in the transcriptional regulation of IL-8 expression. The involvement of ERK, p38 and JNK pathways in the regulation of IL-8 expression appears to be dependent on the cell type and the nature of the stimulus. Induction of IL-8 expression in BEAS-2B bronchial epithelial cells by S1-P (Wang et al., 2002
) and lysophosphatidic acid (Saatian et al., 2006
) required activation of p44/p42 whereas induction by Streptococcus pneumoniae (Schmeck et al., 2006
) and zinc (Kim et al., 2006
) involved activation of JNK and ERK plus JNK respectively. Our studies showed that S1-P activated p44/42, p38 and JNK phosphorylation in H441 cells, however, pharmacological inhibitors of ERK and p38 but not JNK MAPKs inhibited S1-P induction of IL-8 mRNA levels indicating that ERK and p38 signaling pathways are required for S1-P induction. S1-P increased AP-1 DNA binding activity suggesting a role for AP-1 in the induction of IL-8 mRNA expression. It remains to be determined if ERK and p38 MAPK pathways control AP-1 DNA binding activity to increase IL-8 expression. It is known that p38, p44/p42 and JNK MAPKs regulate AP-1 activity (Whitmarsh and Davis, 1996
In summary, our studies have shown that TNF-α induces IL-8 gene expression in H441 lung epithelial cells by increasing gene transcription and that intracellular increases in S1-P levels play important roles in mediating TNF-α induction. S1-P induced IL-8 mRNA expression via activation of p38 and p44/42 MAPK signaling pathways and increase in AP-1 DNA binding activity. Thus TNF-α induces IL-8 gene expression in H441 lung epithelial cells via two pathways – one involving the activation of NF-κB and the other via intracellular elevation of S1-P that results in an increase in AP-1 but not NF-κB binding.