Ceramide-dependent PP2A regulation of TNFalpha-induced IL-8 production in respiratory epithelial cells.

IL-8 is a key mediator in the pathophysiology of acute lung injury. TNFalpha stimulates IL-8 production in respiratory epithelial cells by activating both the NF-kappaB and MAP kinase pathways. The precise mechanism by which these pathways are downregulated to terminate IL-8 production remains unclear. We studied the regulatory role of the serine/threonine phosphatase, PP2A, on the signaling pathways involved in IL-8 production from respiratory epithelial cells. Inhibition of PP2A using okadaic acid or gene knockdown using siRNA resulted in an augmentation of TNFalpha-induced IL-8 production. We also found that PP2A inhibition resulted in prolonged activation of JNK, p38, and ERK resulting in both increased transcriptional activation of the IL-8 promoter and posttranscriptional stabilization of IL-8 mRNA. Because TNFalpha had been shown to activate ceramide accumulation, and separate studies had linked ceramide with activation of PP2A, we hypothesized the pathway of TNFalpha-inducing ceramide to activate PP2A comprised an endogenous regulatory pathway. Inhibition of the immediate sphingomyelinase-dependent pathway as well as the de novo synthesis pathway of ceramide production reduced serine/threonine phosphatase activity and augmented IL-8 production. These data suggest that ceramide plays a role in activating PP2A to terminate ongoing IL-8 production. In summary, our data suggest that in respiratory epithelium, TNFalpha induces ceramide accumulation, resulting in subsequent activation of PP2A, which targets those kinases responsible for transcriptional activation of IL-8.