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

Abstract

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.

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“…One previous study demonstrated that IL-8 production was dependent on early inflammatory gene products, notably TNF>, which activated both the NF-JB and MAPK pathways. 21 Our TNF> blocking test with infliximab treatment demonstrated that infliximab inhibited IL-8 production on MOs at the mRNA and protein levels. Thus, IL-8 not only comes from TF-FVIIaYstimulated MOs but also results from TNF> stimulation from TF-FVIIaY stimulated MOs.…”

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“…31 PP2A can also be activated by ceramide independently of SET/I2PP2A. 33 Thus, as our data in Figure 4 argued that PP2A activity regulated autophagy and cell viability, we next determined whether PTX and SOR treatment acts to promote PP2A activation and cell killing through a ceramide-dependent pathway. Knockdown of ceramide synthase 6 (LASS6) prevented PTX and SOR from activating PP2A (Fig.…”

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“…In fact, previous studies indicated that PP2A is involved in the activation of proinflammatory signaling cascades and participates in the regulation of intricate immune responses. For instance, TNF-a activates PP2A through production of ceramide, which suppresses the JNK, p38, and ERK MAPK pathways governing the termination of cellular inflammatory activation (31,32). On the other hand, it has been reported that PP2A positively regulates the NF-kB pathway; PP2A forms a complex with IkB kinase, which is essential for proper induction of IkB kinase activity (33).…”