3DG Induces ER Stress Pathway Figure 9. Inhibition of RAGE does not alter the induction of ER stress pathway in fibroblasts cultured on 3 DG-collagen. A, Fibroblasts were pretreated with or without the blocking antibody anti-RAGE (10 mg/mL) for 1 h and cultured on native collagen, 1 mM 3 DG- collagen, or 1 mM MG-collagen for 24 h and analyzed for the production of ROS. Fibroblasts were incubated with DCFH-DA for 30 min and the level of intracellular ROS was determined by measuring the fluorescence at 480 nm/530 nm. B, Western blot of GADD153 expression after inhibition of RAGE. The bars correspond to the densitometric value of GADD153 after normalization for b-actin. C, GADD153 localization in the nucleus was analyzed by immunofluorescence using a Cy3-conjugated secondary antibody. Images were taken at 406 magnification on an epi-fluorescence microscope and the MFI of ten nuclei was processed by ImageJ. Arrows indicate nuclei containing GADD153. Scale bar represents 10 mm. D, Caspase- 3 activity analyzed according to the protocol from Caspase-3 Colorimetric Correlate Assay. All samples were performed in triplicate and normalized to the control samples. Data are mean6SD (n = 3), *P,0.001. doi:10.1371/journal.pone.0011093.g009 duction of ROS resulting in increased caspase-3 activation. To verify the role of a1b1 integrin on the ER stress signaling pathway, fibroblasts were pretreated with blocking antibodies against either b1 or a1 integrin and the level of ROS was quantified. a5 integrin, the alpha subunit responsible for binding fibronection, was used as a negative control. Neutralization of both b1 and a1 integrin reduced the production of ROS in fibroblasts cultured on 3DG- collagen to that seen in fibroblasts cultured on native collagen, while neutralization of a5 integrin did not affect the production of ROS (Figure 10A, p,0.001). We next investigated the effect of b1 and a1 integrin neutralization on the expression of GADD153 in fibroblasts cultured on native collagen or 3DG-collagen for 24 h. Blockade of both b1 and a1 integrins suppressed the activation of GADD153 as seen by decreased protein expression and nuclear localization (Figure 10B–C, p,0.001). Moreover, neutralization of b1 and a1 integrin in fibroblasts cultured on 3DG-collagen reduced the activity of caspase-3 to that observed in fibroblasts cultured on native collagen (Figure 10D, p,0.001). These results suggest that the increased binding affinity of a1b1 integrin to 3DG-collagen causes the overproduction of ROS, which in turn leads to increased GADD153 activation and cleavage of caspase-3. Discussion Previous results from our laboratory demonstrated a role for the ER stress pathway in 3DG-collagen signaling; however, the exact signaling mechanism of how 3DG induced ER stress remained elusive [28]. The goal of the present study was to further investigate the induction of caspase-3 activation as an early marker of apoptosis and the signaling events required in fibroblasts cultured on 3DG-collagen. The present study demonstrated that 3DG-collagen induced caspase-3 activation of dermal fibroblasts through activation of GADD153 via induction of ROS and p38 MAPK. Initial studies showed an increase in the apoptotic signaling cascade in fibroblasts cultured on 3DG-collagen, which was confirmed by increases in caspase-3 activity (Figure 1). The level of caspase-3 activity was found to be decreased in fibroblasts cultured on 3DG-collagen and treated with the inhibitors AG and meglumine. Furthermore, we confirmed that 3DG-collagen was inducing caspase-3 activity through induction of ER stress by using an inhibitor of ER stress, salubrinal (Figure 1). In addition to examining the level of caspase-3 activity, we further investigated the role of ER stress and GADD153 in 3DG- collagen signaling. GADD153 is a marker for misfolded proteins in the ER [37,43]. It is well documented that ER stress can induce GADD153, which can lead to apoptosis of the cell [33,34,37,43,46–48]. 3DG-collagen induced a significant increase in the level of GADD153, which could be inhibited with AG and meglumine. We confirmed that 3DG-collagen is activating GADD153 through induction of ER stress as salubrinal abolished the 3DG-collagen-induced expression of GADD153 (Figure 2). To further delineate the signaling mechanism by which 3DG- collagen activates caspase-3, we investigated the expression of ROS. Although ROS are seen as beneficial to the cell during the initial stages of wound healing, chronic activation of ROS have been shown to be stressors on the cell leading to apoptosis [38,54]. ROS have been shown to induce apoptosis through many pathways, one of which includes ER stress [34,35,41]. Addition- ally, circulating AGEs are known to induce the expression of ROS in many different cell types including neuronal and endothelial cells [15–20]. Therefore, we investigated the role of ROS in 3DG- collagen-induced caspase-3 activation in fibroblasts. 3DG-collagen produced ROS, which was attenuated with the addition of the antioxidant ascorbic acid. Inhibitors of 3DG-collagen, AG and meglumine, reduced the level of ROS to that observed in fibroblasts cultured on native collagen suggesting that 3DG is directly responsible for the ROS produced in the fibroblast (Figure 3). To determine the source of increased ROS in fibroblasts cultured on 3DG-collagen we focused our attention to the activation of Nox4. Nox4 has been shown to be highly expressed in fibroblasts [38,39] and Nox4 has been shown to induce ROS through integrin and growth factor signaling [38]. Fibroblasts cultured on 3DG-collagen produced a significant upregulation in the expression of Nox4 both transcriptionally and translationally (Figure 4A–B). This effect could be inhibited with AG and meglumine suggesting that 3DG-collagen specifically induced Nox4 expression. To determine the role of Nox4 in 3DG- collagen-induced ROS, the Nox inhibitor apocynin was used. Pretreatment of fibroblasts with apocynin reduced the level of intracellular ROS to that observed in fibroblasts cultured on native collagen suggesting that Nox4 is responsible for the production of ROS by 3DG-collagen (Figure 4D). During times of cellular stress, ROS can induce apoptosis via p38 MAPK activation [35,37,43,44]. p38 MAPK is a stress- activator kinase and has been shown to be an upstream mediator of GADD153 activation [35,37,43,44]. 3DG-collagen-induced phosphorylation of p38 MAPK was found to be dependent on upstream ROS production by Nox4 as the reversal of p38 MAPK phosphorylation occurred when fibroblasts were pretreated with the antioxidant ascorbic acid and the Nox inhibitor apocynin (Figure 5). Moreover, inhibition of ROS and p38 MAPK reduced the level of GADD153 activation and caspase-3 activity of the cell. These data suggest that 3DG-collagen induces an apoptotic signaling cascade through the ER stress pathway, which is dependent on ROS and p38 MAPK activation via Nox4 (Figures 6–7). It has been well documented that production of intracellular ROS by AGEs induces apoptosis through interaction with its receptor RAGE [19,23–25]. Unlike MG-collagen, a well-studied AGE precursor known to upregulate RAGE, 3DG-collagen did not upregulate the RAGE receptor both transcriptionally or translationally (Figure 8A–B). To provide further evidence that RAGE is not responsible for the ROS-induced caspase-3 activity by 3DG-collagen, a blocking antibody was employed to inhibit the extracellular domain of RAGE. We observed no changes in the levels of ROS, GADD153 expression, or caspase-3 activity when RAGE was blocked suggesting that 3DG-collagen is not signaling through the RAGE receptor (Figure 9A–D). We have previously PLoS ONE | www.plosone.org 11 June 2010 | Volume 5 | Issue 6 | e11093