BACKGROUND: We have shown that serine proteases in German cockroach extract increase TNF-alpha-induced expression of IL-8 in human bronchial epithelial cells. The mechanism by which cockroach proteases regulate cytokine expression is unknown; however, protease-activated receptors (PARs) might play a role. OBJECTIVE: We sought to determine the role of PARs and extracellular-signal-regulated kinase (ERK) in cockroach-induced regulation of IL-8 expression. METHODS: 16HBE14o- human bronchial epithelial cells were treated with the specific PAR-1 and PAR-2 agonists, TFRIFD and SLIGKV, respectively. IL-8 transcription was assessed by transiently transfecting cells with a luciferase-tagged IL-8 promoter construct, and in some cases, dominant-negative expression vectors. To block PAR cleavage, antibodies against the cleavage region of PAR-1 and PAR-2 were used. ERK phosphorylation was determined by Western blot. RESULTS: Although both PAR-1 and PAR-2 were endogenously expressed in 16HBE14o- cells, selective activation of PAR-2 but not PAR-1 mimicked the effect of cockroach extract on IL-8 expression. Using a blocking antibody against cleavage of PAR-2 but not PAR-1 attenuated cockroach-extract-induced responses, suggesting that cockroach proteases cleave PAR-2. Treatment of cells with cockroach extract and SLIGKV each increased phosphorylation of ERK. Chemical or genetic inhibition of Ras and mitogen-activated protein kinase/ERK (MEK), upstream activators of ERK, each attenuated cockroach- and PAR-2-induced IL-8 transcription. CONCLUSION: Cockroach proteases and PAR-2 activation synergistically increase TNF-alpha-induced IL-8 transcription via activation of ERK. These data suggest an important role for PAR-2 and ERK activation in the regulation of cytokine expression in airway epithelium in response to cockroach proteases.
BACKGROUND: We have shown that serine proteases in German cockroach extract increase TNF-alpha-induced expression of IL-8 in human bronchial epithelial cells. The mechanism by which cockroach proteases regulate cytokine expression is unknown; however, protease-activated receptors (PARs) might play a role. OBJECTIVE: We sought to determine the role of PARs and extracellular-signal-regulated kinase (ERK) in cockroach-induced regulation of IL-8 expression. METHODS:16HBE14o- human bronchial epithelial cells were treated with the specific PAR-1 and PAR-2 agonists, TFRIFD and SLIGKV, respectively. IL-8 transcription was assessed by transiently transfecting cells with a luciferase-tagged IL-8 promoter construct, and in some cases, dominant-negative expression vectors. To block PAR cleavage, antibodies against the cleavage region of PAR-1 and PAR-2 were used. ERK phosphorylation was determined by Western blot. RESULTS: Although both PAR-1 and PAR-2 were endogenously expressed in 16HBE14o- cells, selective activation of PAR-2 but not PAR-1 mimicked the effect of cockroach extract on IL-8 expression. Using a blocking antibody against cleavage of PAR-2 but not PAR-1 attenuated cockroach-extract-induced responses, suggesting that cockroach proteases cleave PAR-2. Treatment of cells with cockroach extract and SLIGKV each increased phosphorylation of ERK. Chemical or genetic inhibition of Ras and mitogen-activated protein kinase/ERK (MEK), upstream activators of ERK, each attenuated cockroach- and PAR-2-induced IL-8 transcription. CONCLUSION: Cockroach proteases and PAR-2 activation synergistically increase TNF-alpha-induced IL-8 transcription via activation of ERK. These data suggest an important role for PAR-2 and ERK activation in the regulation of cytokine expression in airway epithelium in response to cockroach proteases.
Authors: Denise W Pallett; Emily Soh; Mary-Lou Edwards; Kathleen Bodey; Laurie C K Lau; J Ian Cooper; Peter H Howarth; Andrew F Walls; Hui Wang Journal: Environ Health Date: 2009-12-21 Impact factor: 5.984