immunofluorescence observations, Western blot analysis demon- strated an increase to 203%64.1% in GADD153 expression in fibroblasts cultured on 3DG-collagen compared to fibroblasts cultured on native collagen (p,0.0005). AG and meglumine reduced the level of GADD153 expression in fibroblasts cultured on 3DG-collagen to 91%65.2% and 92%64.2% (p,0.0005), respectively and salubrinal prevented the 3DG-collagen-induced increase of GADD153 (p,0.0005) (Figure 2B). These findings suggest that 3DG-collagen is inducing apoptosis through the ER stress-signaling pathway, which is dependent on GADD153 activation. 3DG-collagen stimulates ROS in dermal fibroblasts ROS are known to cause oxidative stress and have been linked to the activation of GADD153-induced apoptosis in cells [35,41,43,46,49,50]. Therefore, we determined if ROS were produced during the culturing of fibroblasts on 3DG-collagen. Fibroblasts cultured on 3DG-collagen produced 376 nM63.4 of intracellular ROS at 24 h in comparison to the 38.7 nM62.2 of ROS produced by fibroblasts grown on native collagen (Figure 3, p,0.001). This increase was comparable to that observed with hydrogen peroxide (H2O2), a free radical involved in ER stress, which produced 458 nM63.2 (Figure 3, p,0.001). Moreover, AG abrogated the rise in ROS only in fibroblasts cultured on 3DG- collagen, and not in cells treated with H2O2 suggesting that 3DG- collagen is specifically producing ROS (Figure 3; 94.3 nM64.0 for 3DG-collagen/AG; 440.6 nM64.0 for H2O2/AG, p,0.001). Meglumine inhibited the production of ROS in cells cultured on 3DG-collagen and partially inhibited ROS in cells treated with H2O2 suggesting that meglumine may prevent ROS induction by 3DG-collagen and/or may be a scavenger of free radicals (92.8 nM64.0 for 3DG-collagen and meglumine; and 210.2 nM64.4 for H2O2 and meglumine, p,0.001). In addition, the induction of ROS by fibroblasts cultured on 3DG-collagen, or treated with H2O2 could be blocked by pretreating fibroblasts with the antioxidant ascorbic acid (Figure 3; 78.2 nM63.6 for 3DG- collagen/ascorbic acid; and 55.6 nM64.8 for H2O2/ascorbic acid, p,0.001). Taken together, these results suggest that ROS could be produced in the fibroblast in response to the modification of collagen by 3DG. 3DG Induces ER Stress Pathway NAD(P)H oxidase 4 is responsible for the 3DG-collagen- dependent production of ROS The NAD(P)H oxidase (Nox) controls the production of ROS through integrin activation, and cytokine and growth factor stimulation [38,40,51,52]. Overexpression of key oxidases such as the non-phagocytic Nox4 has been associated with increased ROS and apoptosis [38,39,53]. Nox4 has been shown to be highly expressed in fibroblasts compared to other Nox homologues [38,39]. Therefore, we determined if 3DG-collagen-induced ROS were mediated by the overexpression of Nox4. Quantitative real- time PCR revealed that Nox4 mRNA expression increased to 880%6200.0% in fibroblasts cultured on 3DG-collagen for 24 h compared to fibroblasts cultured on native collagen (Figure 4A, p,0.02). Moreover, to ensure that Nox4 was the only Nox isoform being overexpressed by 3DG-collagen, quantitative real-time PCR was performed to determine the mRNA transcript levels of the other Nox isoforms, Nox1 and Nox2. Detection of Nox1 and Nox2 mRNA transcripts was not apparent suggesting that dermal fibroblasts overexpress specifically Nox4 (Figure 4A). To show specificity of 3DG, AG and meglumine reduced the transcript levels of Nox4 in fibroblasts cultured on 3DG-collagen to that observed in fibroblasts cultured on native collagen. Additionally, Nox4 protein levels were found to be increased in fibroblasts cultured on 3DG-collagen compared to fibroblasts cultured on native collagen (240%68.6% in 3DG-collagen treated vs. 100%63.3% in native collagen treated cells, Figure 4B, p,0.001). This upregulation was also abrogated by the 3DG inhibitors AG and meglumine. Immunofluorescence demonstrated increased Nox4 localization at the plasma membrane in fibroblasts cultured on 3DG-collagen and that this increase was abrogated by AG and meglumine, suggesting that Nox4 may be activated on the cell surface (Figure 4C). We further investigated the role of Nox4 in the upregulation of ROS in fibroblasts cultured on 3DG-collagen. Fibroblasts were pretreated with apocynin, a broad class Nox inhibitor, cultured on either native collagen, 3DG-collagen, or treated with H2O2 for 24 h, and intracellular ROS was quantified. Apocynin reduced ROS in fibroblasts cultured on 3DG-collagen to that observed in fibroblasts cultured on native collagen (Figure 4D, p,0.001). This further confirms that 3DG-collagen is inducing ROS through Figure 3. 3DG-collagen stimulates intracellular ROS in fibroblasts. Fibroblasts were cultured in 96-well plate coated with either native collagen or 1 mM 3DG-collagen or treated with or without 5 mM AG, 40 mM meglumine, or 100 mg/mL ascorbic acid for 24 h. Treatment of fibroblasts cultured on native collagen with 50 mM H2O2 was used as a positive control. Fibroblasts were loaded with DCFH-DA for 30 min and ROS production was measured by absorbance of fluorescent DCF at a wavelength of 480 nm/530 nm. Comparisons are made to collagen treated with PBS unless otherwise indicated. Data are mean6SD (n = 3), *P,0.001. doi:10.1371/journal.pone.0011093.g003 PLoS ONE | www.plosone.org 4 June 2010 | Volume 5 | Issue 6 | e11093