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Barlow, Christy Ann

Cell and Molecular Biology Program

2006

Ph. D.

Lung epithelial cells experience enhanced oxidative stress due to their direct exposure to ambient air and as a consequence of inflammation. Excessive production of oxidants plays a role in the pathogenesis of many pulmonary diseases, including asbestos-induced fibrotic lung disease and cancer. Oxidant stress-mediated phosphorylation of extracellular signal-regulated kinases (ERK1/2) is linked to pathologic signaling in lung epithelium. Although calcium has a clear role in ERK1/2 signaling, there is little information regarding the role of calcium and the calcium-regulated transcription factor, Ca²⁺/cAMP-response element binding protein (CREB), in oxidant and asbestos signaling pathways. Employing both in vitro and in vivo studies utilizing pharmacological inhibitors, dominant negative constructs, siRNA, and transgenic mice, we investigated the role of CREB in oxidant and asbestos-induced signaling in lung epithelial cells. We tested the hypotheses that oxidants induce Ca²⁺-mediated phosphorylation of ERK1/2 and CREB, and that CREB is required for oxidantinduced proliferation and apoptosis. H₂0₂ initiated an influx of extracellular Ca²⁺ that was required for phosphorylation of both ERK1/2 and CREB in C10 lung epithelial cells. H₂0₂-mediated CREB phospholation was sensitive to MEK inhibition, suggesting that crosstalk between Ca²⁺, ERK1/2 and CREB signaling pathways underlies the oxidant-induced response. Because CREs (Ca²⁺/cAMP response elements) are found in the promoters of many genes important for regulation of proliferation and apoptosis, CREB is likely to play an important role in the development of oxidant-mediated lung injury. Reduction of CREB activity, using a dominant-negative CREB construct, inhibited c-fos transcription but unexpectedly enhanced bcl-2 transcription following H₂0₂ exposure. In addition, whereas inhibition of CREB activity had no detectable effect on H₂0₂-stimulation of S-phase entry, loss of CREB activity significantly reduced the number of cells undergoing apoptosis.
Many of the signaling events initiated by H₂0₂ parallel those initiated by exposure of lung cells to asbestos. Because asbestos is known to produce oxidants, we tested the hypothesis that asbestos exposure leads to CREB phosphorylation in lung epithelial cells, and that protein kinase A (PKA) is a central regulator of the CREB activation pathway. Persistent CREB phosphorylation was observed in lung sections from mice following in vivo exposure to oxidant-generating crocidolite asbestos. Crocidolite asbestos exposure of cultured C10 lung epithelial cells also led to rapid CREB phosphorylation that was not sensitive to reduction of oxidants using catalase or N-acetyl cysteine (NAC). Furthermore, the inhibition of PKA, MEK1/2, and EGFR using specific inhibitors, decreased asbestos-induced CREB phosphorylation, suggesting a link between asbestos mediated activation of PKA, ERK1/2, and the CREB transcription factor. These data reveal a novel role for CREB in asbestos-mediated signaling pathways and suggest an important role for PKA in asbestos-induced activation of CREB.