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Characterization of a Rac1 Signaling Pathway to Cyclin D1 Expression in Airway Smooth Muscle Cells

We examined the importance of the Rho family GTPase Rac1 for cyclin D1 promoter transcriptional activation in bovine tracheal myocytes. Overexpression of active Rac1 induced transcription from the cyclin D1 promoter, whereas platelet-derived growth factor (PDGF)-induced transcription was inhibited by a dominant-negative allele of Rac1, suggesting that Rac1 functions as an upstream activator of cyclin D1 in this system. Rac1 forms part of the NADPH oxidase complex that generates reactive oxygen species such as H2O2. PDGF stimulated a substantial increase in intracellular reactive oxygen species, as measured by the fluorescence of dichlorofluorescein-loaded cells, and this was blocked by the glutathione peroxidase mimetic ebselen. Pretreatment with ebselen, catalase, and the flavoprotein inhibitor diphenylene iodonium each attenuated PDGF- and Rac1-mediated cyclin D1 promoter activation, while having no effect on the induction of cyclin D1 by mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase-1 (MEK1), the upstream activator of ERKs. Antioxidant treatment also inhibited PDGF-induced cyclin D1 protein expression and DNA synthesis. Overexpression of an N-terminal fragment of p67 phox , a component of NADPH oxidase which interacts with Rac1, attenuated PDGF-induced cyclin D1 promoter activity, whereas overexpression of the wild-type p67 did not. Finally, Rac1 was neither required nor sufficient for ERK activation. Taken together, these data suggest a model by which two distinct signaling pathways, the ERK and Rac1 pathways, positively regulate cyclin D1 and smooth muscle growth.

Interleukin (IL)-1β in tracheal aspirates from premature infants induces airway epithelial cell IL-8 expression via an NF-κB dependent pathway

Tracheal aspirate IL-8 concentration and airway epithelial cell IL-8 expression are each increased in premature infants undergoing mechanical ventilation. We sought to determine the cytokines responsible for IL-8 expression in this context. Tracheal aspirates were collected from 18 mechanically ventilated premature infants. IL-8 protein abundance was high in tracheal aspirates from ventilated premature infants (mean, 5806 ± 4923 pg/mL). IL-1α (mean, 20 ± 6 pg/mL), IL-1β (mean 67 ± 46 pg/mL), and tumor necrosis factor (TNF)-α (mean, 8 ± 2 pg/mL) were also found. Incubation of tracheal aspirates with 16HBE14o- human bronchial epithelial cells increased IL-8 protein in both cell lysates and supernatants, as well as transcription from the IL-8 promoter. Aspirates also induced nuclear factor (NF)-κB activation. Mutation of the IL-8 promoter NF-κB site abolished aspirate-induced IL-8 transcription. Endotoxin concentrations in the tracheal aspirates were negligible and incapable of inducing IL-8 promoter activity. Finally, incubation of tracheal aspirates with a neutralizing antibody against IL-1β reduced epithelial cell IL-8 production, whereas neutralizing antibodies against IL-1α and TNF-α had no effect. We conclude that airway fluid from mechanically ventilated premature infants contains soluble factors capable of inducing airway epithelial cell IL-8 expression via a NF-κB-dependent pathway, and that IL-1β plays a specific role in this process.