Jobling, M. F., Mott, J. D., Finnegan, M. T., Jurukovski, V., Erickson, A. C., Walian, P. J., Taylor, S. E., Ledbetter, S., Lawrence, C. M., Rifkin, D. B. and Barcellos-Hoff, M. H. Isoform-Specific Activation of Latent Transforming Growth Factor β (LTGF-β) by Reactive Oxygen Species. Radiat. Res. 166, 839–848 (2006).
The three mammalian transforming growth factor β (TGF-β) isoforms are each secreted in a latent complex in which TGF-β homodimers are non-covalently associated with homodimers of their respective pro-peptide called the latency-associated peptide (LAP). Release of TGF-β from its LAP, called activation, is required for binding of TGF-β to cellular receptors, making extracellular activation a critical regulatory point for TGF-β bioavailability. Our previous work demonstrated that latent TGF-β1 (LTGF-β1) is efficiently activated by ionizing radiation in vivo and by reactive oxygen species (ROS) generated by Fenton chemistry in vitro. In the current study, we determined the specific ROS and protein target that render LTGF-β1 redox sensitive. First, we compared LTGF-β1, LTGF-β2 and LTGF-β3 to determine the generality of this mechanism of activation and found that redox-mediated activation is restricted to the LTGF-β1 isoform. Next, we used scavengers to determine that ROS activation was a function of OH· availability, confirming oxidation as the primary mechanism. To identify which partner of the LTGF-β1 complex was functionally modified, each was exposed to ROS and tested for the ability to form a latent complex. Exposure of TGF-β1 did not alter its ability to associate with LAP, but exposing LAP-β1 to ROS prohibited this phenomenon, while treatment of ROS-exposed LAP-β1 with a mild reducing agent restored its ability to neutralize TGF-β1 activity. Taken together, these results suggest that ROS-induced oxidation in LAP-β1 triggers a conformational change that releases TGF-β1. Using site-specific mutation, we identified a methionine residue at amino acid position 253 unique to LAP-β1 as critical to ROS-mediated activation. We propose that LTGF-β1 contains a redox switch centered at methionine 253, which allows LTGF-β1 to act uniquely as an extracellular sensor of oxidative stress in tissues.