3DG Induces ER Stress Pathway Figure 8. 3 DG-collagen does not induce the expression of RAGE. A, Fibroblasts were cultured on native collagen, 1 mM 3 DG-collagen, or 1 mM MG-collagen with or without 5 mM AG for 24 h. mRNA was analyzed for the expression of RAGE by real-time PCR. All transcripts were normalized to b-actin. B, Fibroblasts were treated as in A and analyzed for the expression of RAGE by Western blot. The bars correspond to the densitometric value of RAGE after normalization for b-actin. All comparisons are made against collagen treated with PBS. Data are mean6SD (n = 3), *P,0.002. doi:10.1371/journal.pone.0011093.g008 specific for the extracellular domain of RAGE, and the levels of ROS were quantified. Intriguingly, the levels of ROS in fibroblasts cultured on 3DG-collagen did not alter after blockade of RAGE. However, we observed the downregulation of ROS with the inhibition of RAGE binding in fibroblasts cultured on MG- collagen (Figure 9A, p,0.001). To further demonstrate that 3DG- collagen signaling was independent of RAGE, we investigated the expression of GADD153. Fibroblasts were pretreated with the RAGE blocking antibody and cultured on native collagen, 3DG- collagen, or MG-collagen; and the level of activated GADD153 was quantified. Blockade of RAGE in fibroblasts cultured on 3DG-collagen did not suppress the activation of GADD153, while GADD153 was suppressed after blockade of RAGE in fibroblasts cultured on MG-collagen (Figure 9B–C, p,0.001). Blockade of RAGE did not decrease the level of caspase-3 activity in fibroblasts cultured on 3DG-collagen, while suppression of caspase-3 activity was observed in fibroblasts pretreated with the RAGE antibody and cultured on MG-collagen (Figure 9D, p,0.001). These results suggest that 3DG-collagen is not signaling through the RAGE receptor as is observed with MG. 3DG-collagen activates the ER stress signaling cascade through a1b1 integrin To delineate the receptor involved in activating the ER stress pathway by 3DG-collagen, we investigated a1b1 integrin collagen receptor. Previous data has demonstrated that fibroblasts have an increased adherence to 3DG-collagen, which is dependent on binding by a1b1 integrin [28]. Fibroblasts can change their binding affinity for 3DG-collagen, which may cause an overpro- PLoS ONE | www.plosone.org 9 June 2010 | Volume 5 | Issue 6 | e11093