Precursor of Advanced Glycation End Products Mediates ER-Stress-Induced Caspase-3 Activation of Human Dermal Fibroblasts through NAD(P)H Oxidase 4 Danielle T. Loughlin, Carol M. Artlett* Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States of America Abstract Background: The precursor for advanced glycation end products, 3-deoxyglucosone (3DG) is highly upregulated in skin explants of diabetic cutaneous wounds, and has been shown to negatively impact dermal fibroblasts, which are crucial in wound remodeling. 3DG induces apoptosis however; the mechanisms involved in the apoptotic action of 3DG in the pathogenesis of diabetic chronic wounds are poorly understood. Therefore, we sought to delineate novel mechanisms involved with the 3DG-collagen induced apoptosis. Methodology/Principal Findings: Using human dermal fibroblasts, we demonstrated that 3DG-modified collagen induces oxidative stress and caspase-3 activation. Oxidative stress was found to be dependent on the upregulation of NAD(P)H oxidase 4 (Nox4), a reactive oxygen species (ROS) Nox homologue, triggering endoplasmic reticulum (ER) stress, as assessed by the ER stress-induced apoptosis marker Growth Arrest and DNA Damage-inducible gene 153 (GADD153). We demonstrated that 3DG-collagen activated GADD153 via phosphorylation of p38 mitogen activated protein kinase (MAPK), and this was dependent on upstream ROS. Inhibition of ROS and/or p38 MAPK abrogated 3DG-collagen induced caspase-3 activation. Our investigations also demonstrated that 3DG-collagen-induced caspase-3 activation did not signal through the canonical receptor for advanced glycation end products (RAGE) but through integrin a1b1. To further verify the role of integrins, neutralization of integrins a1b1 prevented 3DG-collagen-induced upregulation of ROS, GADD153, and caspase-3 activation; suggesting that 3DG-collagen signaling to the fibroblast is dependent on integrins a1b1. Conclusions/Significance: Taken together, these findings demonstrate for the first time that a RAGE independent mechanism is involved in 3DG-collagen-induced apoptosis. Moreover, the ER stress pathway through activation of Nox4 by integrins a1b1 plays a key role in 3DG-collagen-induced caspase-3 activation, which may play an important role in the pathogenesis of diabetic wounds. Citation: Loughlin DT, Artlett CM (2010) Precursor of Advanced Glycation End Products Mediates ER-Stress-Induced Caspase-3 Activation of Human Dermal Fibroblasts through NAD(P)H Oxidase 4. PLoS ONE 5(6): e11093. doi:10.1371/journal.pone.0011093 Editor: Madhuri Kango-Singh, University of Dayton, United States of America Received March 18, 2010; Accepted May 11, 2010; Published June 14, 2010 Copyright: 2010 Loughlin, Artlett. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by grants to C.M. Artlett from the Department of Defense WX81XWh-07-1-0450 and from Dynamis Therapeutics, Inc. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: This work was funded in part by Dynamis Therapeutics, Inc. to C.M.A. C.M.A. is not an employee of Dynamis Therapeutics, Inc. This funding source does not alter the authors’ adherence to all the PLoS ONE policies on the sharing of data and materials. There are no other competing interests regarding this manuscript. * E-mail: carol.artlett@drexelmed.edu Introduction Wound healing is impaired in patients with diabetes as approximately 5–8% of the patients develop chronic foot ulcers [1–3]. Patients suffering from chronic diabetic ulcers have increased levels of apoptosis within their infected tissues and this may interfere with their capacity to efficiently heal wounds [1,3–7]. The high degree of cell death seen in fibroblasts explanted from the skin of diabetic patients may be partly due to the formation of advanced glycation end products (AGEs) [8]. AGEs result from a non- enzymatic reaction between glucose and protein, which can form irreversible cross-links on long-lived proteins such as collagen [6,9–11]. One precursor for AGEs is the highly reactive a-dicarbonyl 3-deoxyglucosone (3DG). 3DG has been shown to play a role in the modification and cross-linking of collagen [9,10,12,13]. One mechanism by which 3DG-modifed collagen could affect wound healing is through increased apoptosis. The role of AGE induction of apoptosis has been investigated extensively, however many of these studies were performed on various cell types including neuronal and endothelial cells [14–20] using soluble AGEs, resulting in differing apoptotic responses [8,20–22]. Soluble AGEs induce apoptosis in endothelial and neuronal cells through engagement of its receptor, receptor for advanced glycation end products (RAGE) [15–20]. This ligand- receptor engagement promotes the upregulation of reactive oxygen species (ROS) resulting in apoptosis [19,23–25]. Moreover, reports utilizing AGEs cross-linked to collagen agree with the current view that AGEs induce apoptosis; however, the mechanisms by which AGE- modifed collagen does so are conflicting and not well understood [8]. More importantly, none of the studies to date have investigated 3DG or its signaling events that induce apoptosis in dermal fibroblasts. One possibility for the conflicting evidence is due to the varying types of precursors responsible for the development of AGEs [9,10,26]. We previously demonstrated that 3DG-collagen signals to PLoS ONE | www.plosone.org 1 June 2010 | Volume 5 | Issue 6 | e11093