acid and 82%64.6% treated with apocynin, and 75%62.2% H2O2 treated with ascorbic acid and 188%63.6% treated with apocynin, p,0.0001). These results suggest that the generation of ROS by Nox4 lies upstream of GADD153. Next, the role of p38 MAPK in GADD153 activation was assessed in fibroblasts cultured on native collagen or 3DG- collagen. Fibroblasts were pretreated with the p38 MAPK inhibitor SB202190 and cultured on native collagen, 3DG- collagen, or native collagen and treated with H2O2 for 24 h. Inhibition of p38 MAPK by SB202190 reduced the localization of GADD153 in the nucleus to 7.23MFI61.13% in fibroblasts cultured on 3DG-collagen (Figure 6A, p,0.007), and reduced the expression of GADD153 to 40%64.0% (Figure 6A, p,0.0001). In addition, inhibition of p38 MAPK reduced the level of GADD153 expression in fibroblasts grown on native collagen treated with H2O2 to 51%63.6% (Figure 6B, p,0.0001), and its nuclear localization to 8.21MFI62.4 (Figure 6A, p,0.007). These results suggest that the induction of p38 MAPK by upstream ROS is responsible for the activation of GADD153 by 3DG-collagen. 3DG-collagen-induced caspase-3 activation is dependent on upstream ROS and p38 MAPK activation A caspase-3 assay was performed to determine if ROS and p38 MAPK are responsible for the increased caspase-3 activation observed in fibroblasts cultured on 3DG-collagen. Fibroblasts were pretreated with ascorbic acid, the Nox inhibitor apocynin, or the p38 MAPK inhibitor SB202190; and cultured on native collagen or 3DG-collagen for 24 h, or cultured on native collagen and treated with H2O2 as a positive control. Fibroblasts treated with ascorbic acid, apocynin, or SB202190 and cultured on 3DG- collagen reduced the activation of caspase-3 to 116%64.7%, 115%64.5%, and 105%62.5% respectively. This expression was comparable to the level of caspase-3 cleavage observed in fibroblasts cultured on native collagen and treated with H2O2 in 3DG Induces ER Stress Pathway the presence of ascorbic acid (112%67.2%) or SB202190 (104.2%66.6%), and fibroblasts cultured on native collagen (100%61.4%; Figure 7, p,0.0002). This data suggests that 3DG-collagen is inducing caspase-3 activation through ER stress, which is dependent on upstream activation of ROS and p38 MAPK through upregulation of Nox4. 3DG-collagen induces ROS and apoptosis independent of RAGE signaling When AGEs bind to their receptor, RAGE, ROS are released as a downstream byproduct [25]. To determine if 3DG-collagen is signaling via its interaction with RAGE, we investigated transcript levels of total RAGE. To our surprise, 3DG-collagen did not upregulate RAGE transcript levels (Figure 8A). As a control for the induction of RAGE we cross-linked collagen with MG, which is a well studied AGE precursor known to signal via RAGE [30,31]. We observed a significant induction of RAGE transcript levels in fibroblasts cultured on MG-collagen; 400%612% upregulation of RAGE (Figure 8A, p,0.002). Additionally, treatment of MG- collagen with AG reduced the transcript levels of RAGE to 119%613.3%, which confirms that MG can upregulate RAGE mRNA expression. To ensure that 3DG-collagen is not regulating the RAGE receptor post-transcriptionally we measured RAGE protein levels by Western blot. In contrast to the 238%611.8% upregulation of RAGE protein in fibroblasts cultured on MG- collagen, 3DG-collagen did not upregulate RAGE protein expression in fibroblasts (107%610.5%; Figure 8B). To further confirm the specificity of MG, AG abrogated the increase in RAGE protein expression (104%68.2%) in fibroblasts cultured on MG-collagen (Figure 8B, p,0.002). This data suggests that 3DG- collagen is not upregulating RAGE at both the level of transcription or translation. To further verify the absence of RAGE expression in 3DG- collagen signaling, RAGE was blocked using a blocking antibody Figure 7. Inhibition of ROS, and p38 MAPK reduces caspase-3 cleavage induced by 3DG-collagen. Fibroblasts were pretreated with 100 mg/mL of ascorbic acid, 1 mM apocynin, or 10 mM SB202190 for 1 h and cultured on native collagen, 1 mM 3 DG-collagen, or treated with 50 mM H2O2 for 24 h. Treatment of fibroblasts with 50 mM H2O2 for 24 h was used as a positive control. 100 mg of whole cell lysate was assayed for caspase-3 activity according to the protocol from Caspase-3 Colorimetric Correlate Assay. All samples were performed in triplicate and normalized to the control samples. All comparisons are made against collagen treated with PBS unless otherwise indicated. Data are mean6SD (n = 3), **P,0.0002, *P,0.001. doi:10.1371/journal.pone.0011093.g007 PLoS ONE | www.plosone.org 8 June 2010 | Volume 5 | Issue 6 | e11093