Hydrogen peroxide and endothelin‐1 are novel activators of betacellulin ectodomain shedding

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Abstract
The betacellulin precursor (pro‐BTC) is a novel substrate for ADAM10‐mediated ectodomain shedding. In this report, we investigated the ability of novel physiologically relevant stimuli, including G‐protein coupled receptor (GPCR) agonists and reactive oxygen species (ROS), to stimulate pro‐BTC shedding. We found that in breast adenocarcinoma MCF7 cells overexpressing pro‐BTC, hydrogen peroxide (H2O2) was a powerful stimulator of ectodomain shedding. The stimulation of pro‐BTC shedding by H2O2 was blocked by the broad‐spectrum metalloprotease inhibitor TAPI‐0 but was still functional in ADAM17 (TACE)‐deficient stomach epithelial cells indicating the involvement of a distinct metalloprotease. H2O2‐induced pro‐BTC shedding was blocked by co‐culturing cells in the anti‐oxidant N‐acetyl‐L‐cysteine but was unaffected by culture in calcium‐deficient media. By contrast, calcium ionophore, which is a previously characterized activator of pro‐BTC shedding, was sensitive to calcium depletion but was unaffected by co‐culture with the anti‐oxidant, identifying a clear distinction between these stimuli. We found that in vascular smooth muscle cells overexpressing pro‐BTC, the GPCR agonist endothelin‐1 (ET‐1) was a strong inducer of ectodomain shedding. This was blocked by a metalloprotease inhibitor and by overexpression of catalytically inactive E385A ADAM10. However, overexpression of wild‐type ADAM10 or ADAM17 led to an increase in ET‐1‐induced pro‐BTC shedding providing evidence for an involvement of both enzymes in this process. This study identifies ROS and ET‐1 as two novel inducers of pro‐BTC shedding and lends support to the notion of activated shedding occurring under the control of physiologically relevant stimuli. J. Cell. Biochem. 99: 609–623, 2006.

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